Queen Essays

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Abdominal Radiology (2022) 47:288–296 https://doi.org/10.1007/s00261-021-03296-1

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KIDNEYS, URETERS, BLADDER, RETROPERITONEUM

Evaluation of renal fibrosis in various causes of glomerulonephritis by MR elastography: a clinicopathologic comparative analysis

Alper Tuna Güven1  · Ilkay S. Idilman2 · Cebrayil Cebrayilov3 · Ceren Önal3 · Müge Üzerk Kibar3 · Arzu Sağlam4 · Tolga Yıldırım3 · Rahmi Yılmaz3 · Bülent Altun3 · Yunus Erdem3 · Muşturay Karçaaltıncaba2 · Mustafa Arıcı3

Received: 8 July 2021 / Revised: 22 September 2021 / Accepted: 27 September 2021 / Published online: 11 October 2021 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract Background Renal parenchymal fibrosis is the most important determinant of kidney disease progression and it is determined via biopsy. The aim of this study is to evaluate the renal stiffness noninvasively by magnetic resonance elastography (MRE) and to compare it with clinicopathologic parameters in glomerulonephritis and AA amyloidosis patients. Methods Thirty-four patients with glomerular filtration rate (GFR) over 20 ml/min/1.73m2 had non-contrast MRE prospec- tively. Kidney stiffness values were obtained from whole kidney, cortex, and medulla. Values were correlated with GFR, albuminuria, proteinuria, and degree of fibrosis that are assessed via renal biopsy. Patients were grouped clinicopathologi- cally to assess the relation between stiffness and chronicity. Results Mean whole kidney, cortex, and medulla stiffnesses were 3.78 (± 1.26), 3.63 (± 1.25), and 4.77 (± 2.03) kPa, respectively. Mean global glomerulosclerosis was 22% (± 18%) and median segmental glomerulosclerosis was 4% (min– max: 0%–100%). Extent of tubulointerstitial fibrosis was less than 25% in 26 of the patients (76.5%), 25%–50% in 6 of the patients (17.6%), and higher than 50% in 2 of the patients (5.9%). Fourteen patients were defined to have chronic renal parenchymal injury. MRE-derived stiffness values correlated negatively with parameters of fibrosis. Lower stiffness values were observed in patients with chronic renal injury compared to those without (P < 0.05 for whole kidney and medulla MRE-derived stiffness). Conclusion MRE-derived stiffness values were lower in patients with chronic injury. Stiffness decreases as glomeruloscle- rosis and tubulointerstitial fibrosis progresses in patients with primary glomerulonephritis and AA amyloidosis. With future studies, there may be a role for MRE to assess renal function in concert with conventional markers.

Keywords Magnetic resonance imaging · Glomerulonephritis · Amyloidosis · Fibrosis · Kidney biopsy

Introduction

Kidney fibrosis is the manifestation of chronic paren- chymal injury to most glomerular and tubulointerstitial insults [1, 2]. As fibrosis is one of the major determinants

of outcome, it is crucial to determine its extent and sever- ity for diagnostic and therapeutic purposes [3, 4]. Firstly, GFR may not decrease despite presence of renal fibro- sis, sometimes not until the point where fibrotic damage is extensive, due to the kidney’s inherent compensatory capacity. Secondly, decreases in GFR may not only be related with the chronic damage/parenchymal fibrosis [5]. Hence estimating GFR via serum markers provides only rough and approximate estimations of kidney fibrosis and may in fact even be misguiding. The most accurate way of assessing kidney fibrosis is to obtain a kidney biopsy [6]. Biopsy is not only an invasive procedure with com- plications and contraindications, but also prone to sam- pling errors by sampling < 1% of the kidney parenchyma. Taking into account the heterogeneous and patchy dis- tribution of fibrosis within kidneys, the value of kidney

* Alper Tuna Güven [email protected]

1 Department of Internal Medicine, Faculty of Medicine, Hacettepe University, Ankara, Turkey

2 Department of Radiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

3 Department of Nephrology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

4 Department of Pathology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

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biopsy may be further impaired [6–9]. It is also unrealis- tic to obtain serial biopsies over time to measure degree of fibrosis. The necessity to assess fibrosis noninvasively and accurately led to studies involving various imaging techniques, including ultrasound and magnetic resonance imaging (MRI) [10]. While multiple MRI techniques have been studied in order to assess fibrosis [11–21], magnetic resonance elastography (MRE) seems to hold promise [5, 22–27]. MRE combines MRI with the assessment of acoustic waves for the quantitative determination of vis- coelastic properties of tissues based on their response to external mechanical vibration and was originally devel- oped to assess liver fibrosis [28]. Studies in kidneys dem- onstrated that MRE correlates with the level of fibrosis in renal allografts and diabetic kidneys. These studies have shown that while kidney stiffness increases with increasing levels of fibrosis in cirrhotic liver and renal allografts [24, 25, 27], kidney stiffness decreases in diabetic nephropathy [5]. To date, no study has evaluated the relation of kid- ney fibrosis and stiffness in various primary glomerular diseases and AA amyloidosis patients. The aim of this study is to assess kidney parenchymal fibrosis in glomeru- lonephritis and AA amyloidosis patients using 2D MRE- derived stiffness as a surrogate marker and compare MRE findings with clinicopathological correlates of glomerular diseases.

Materials and methods

Patients

Patients with primary glomerular diseases with GFR > 20 ml/min/1.73m2 who had undergone renal biopsy were recruited. Exclusion criteria were acute kidney injury, pregnancy, renal transplantation, prior corticos- teroids or immunosuppressive use, secondary glomeru- lar diseases, presence of hydronephrosis and renal vein thrombosis on conventional MRI sequences and contrain- dications to MRI, and declining the informed consent. Patients underwent MRE prior to kidney biopsy. MRE acquisition and kidney biopsy were both performed on the same (left) kidney. The study was approved by Hacettepe University Non-Invasive Clinical Studies Ethical Commit- tee (GO 18/1147). Written informed consent was obtained from each patient. No adverse event occurred related to the study. Clinical data regarding age, gender, serum creatinine (mg/dl), GFR (using CKD-EPI equation, ml/ min/1.73m2), serum albumin (gr/dl), serum protein (gr/dl), urine protein to creatinine ratio (mg/gr creatinine), urine albumin to creatinine ratio (mg/gr creatinine) 24-h albu- min, and protein in urine (mg/24 h) before biopsy were

collected from each patient’s electronic health records at the study entry.

Kidney biopsy and histopathological examination

All patients underwent ultrasound-guided percutaneous kidney biopsy after their MRE acquisitions. All biopsies were reported by a blinded nephropathologist with substan- tial expertise who was unaware of the MRE results. Jones’ methenamine silver (JMS), Masson’s trichrome stain, peri- odic acid methenamine silver (PAMS), periodic acid–Schiff (PAS), and Congo red stains were performed on biopsy specimens as part of the routine kidney biopsy workup. Immunofluorescence stains for IgA, IgG, IgM, C3, C4, C1q, kappa, and lambda were performed as part of the routine kidney biopsy workup as well. Histopathological parameters assessed as markers of chronic renal injury (renal fibrosis) were extent of global glomerulosclerosis, segmental glo- merulosclerosis, and tubulointerstitial fibrosis. Global and segmental glomerulosclerosis were reported in percentages and tubulointerstitial fibrosis was reported categorically as comprising < 25%, 25%–50%, and > 50 percent of the corti- cal renal parenchyma.

Determination of the chronicity

The patients were grouped according to evidence of chronic renal injury. This grouping was based on both clinical and pathologic parameters, taking into consideration of the biopsy findings, serum parameters and their course over time, and response to treatment. More than 50% of global sclerosis and > 50% cortical tubulointerstitial scarring were considered as histopathologic parameters of chronic renal injury. Persistently elevated serum creatinine, development of unremitting proteinuria, and persistently low GFR were accepted as the clinical parameters of chronicity. If clinical follow-up (i.e., trends in serum creatinine, level of protein- uria) suggested chronic renal injury despite less extensive global glomerular sclerosis and tubular atrophy/interstitial fibrosis, the patient was included in the group with chronic renal parenchymal injury.

MR elastography and analysis

MR imaging was performed with a 1.5-T MR system (Mag- netom Aera, Siemens Healthcare, Erlangen, Germany). A 30-channel phased array body coil was used for this acqui- sition. The subjects were examined in supine position. The placement of passive driver is shown in Fig. 1a and three- plane localization imaging gradient echo sequence was per- formed at the beginning of the examination. The parameters of MRE were as follows: TR/TE, 50/21.41 ms; flip angle 25°; section thickness 50 mm; field-of-view FOV 350 × 350

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mm2. Using a workstation, regions of interest (ROIs) were drawn as geographic areas guided by the magnitude image to include kidney parenchyma, medulla, and cortex of the kidney by excluding areas close to the kidney margins and collecting system.

Statistical analysis

Shapiro–Wilk test of normality was used to determine dis- tribution of variables. Normally distributed variables were reported as mean (± standard deviation) and non-normally distributed variables were reported as median (mini- mum–maximum). Spearman’s correlation analysis was used to assess correlation between MRE-derived stiffness values, clinical data, and histopathological data. Mann–Whitney U test was used as non-parametric test to assess distribution of independent samples. IBM SPSS statistics version 22.0 was used for statistical analysis. For all tests, a two-tailed P

value of less than 0.05 was considered statistically signifi- cant (Figs. 2, 3).

Results

Clinical data

A total of 39 patients were enrolled in the study between December 2018 and January 2020. Five patients were excluded as their biopsy results did not meet inclusion cri- teria for the study. Four patients did not have urine albumin to creatinine ratio data. Only 16 patients had 24-h protein in urine data. Median serum creatinine levels were 0.95 mg/ dl (range, 0.2–3.7), median serum albumin and protein lev- els were 3.18 gr/dl (range, 1.3–4.4) and 6.04 gr/dl (range, 3.9–7.6), respectively, median urine albumin to creatinine and median protein to creatinine ratios were 2518 mg/gr

Fig. 1 MRE set-up and optimal passive driver placement are seen on both illustration a and localizer image (b)

Fig. 2 MR stiffness measure- ment for kidney. A freehand ROI was drawn on the magni- tude image a and then copied to the stiffness map (b)

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creatinine (range, 78–9016) and 3656 mg/gr creatinine (range, 475–18,193), respectively. Fourteen patients were assessed as displaying evidence of chronic renal parenchy- mal injury (CRPI), whereas 20 did not display evidence of CRPI. Patients with evidence of CRPI had lower median cre- atinine, higher albumin and protein levels, and lower urine albumin to creatinine and urine protein to creatinine ratios than those lacking evidence of CRPI (all P < 0.05). Patient’s clinical data are shown in detail in Table 1.

Histopathological characteristics

Of the 34 patients, 12 had membranous nephropathy, 10 had focal segmental glomerulosclerosis, 5 had AA

amyloidosis, 4 had IgA nephropathy, and the remaining 3 patients had membranoproliferative glomerulonephritis, focal segmental glomerulosclerosis plus IgA nephropa- thy, and focal segmental glomerulosclerosis plus Alport syndrome. Ratio of mean global glomerulosclerosis was 22% (± 18%) and median segmental sclerosis score was 4% (range, 0%–100%). Extent of tubulointerstitial fibro- sis was less than 25% in 26 of the patients (76.5%), 25 to 50% in 6 of the patients (17.6%), and higher than 50% in 2 of the patients (5.9%). Patients with evidence of CRPI had higher global and segmental glomerulosclerosis per- centages compared to those lacking evidence of CRPI (all P < 0.05). Patients’ histopathological characteristics are shown in detail in Table 2.

Fig. 3 MR stiffness measurements for renal cortex and medulla. Freehand ROI was used for delineation of medulla a and cortex d on T1W image and copied to the magnitude image b and e and stiffness map (c and f)

Table 1 Clinical characteristics of all patients and their subgroups as CRPI and Non-CRPI patients

BUN Blood urea nitrogen, CRPI Chronic renal parenchymal injury

Parameters Results mean (± SD) or median (Min–Max) Distribution

All Patients CRPI Non-CRPI P

Serum albumin (gr/dl) 3.1 (1.3–4.4) 3.77 (2.7–4.4) 2.75 (1.3–4.1) P < 0.01 Serum protein (gr/dl) 6.0 (3.9–7.6) 6.80 (5.4–7.6) 5.51 (3.9–7.4) P < 0.01 BUN (mg/dl) 17 (6–95) 25 (17–59) 12 (6–95) P < 0.01 Serum creatinine (mg/dl) 0.9 (0.2–3.7) 1.5 (1.0–3.7) 0.9 (0.2–2.5) P < 0.01 Urine albumin to creatinine (mg/gr creatinine) 2518 (78–9016) 1816 (78–4050) 3254 (419–9016) P < 0.05 Urine protein to creatinine (mg/gr creatinine) 3656 (475–18,193) 2670 (475–5406) 4300 (984–18,193) P < 0.05 24-h urine albumin (mg/24 h) 2628 (422–3354) 2452 2772 (422–3354) P > 0.05 24-h urine protein (mg/24 h) 3768 (1122–20,284) 2580 (1874–7539) 4224 (1122–20,284) P > 0.05

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MR elastography data

Regarding all patients, mean whole kidney stiffness was 3.78 kPa (± 1.26), cortex stiffness was 3.63 kPa (± 1.25), and medulla stiffness was 4.77 kPa (± 2.04). Whole kidney, cortex, and medulla stiffness values were lower across the group of patients with evidence of CRPI compared to those lacking evidence of CRPI (all P < 0.05, except for the cortex stiffness, P = 0.112) (Table 3).

Relationship between MRE‑derived stiffness and clinical parameters

Whole kidney, cortex, and medulla MRE-derived stiffness values correlated positively with urine albumin to creati- nine, urine protein to creatinine, and 24-h protein in urine. Whole kidney, cortex, and medulla MRE-derived stiffness values correlated negatively with serum albumin and serum protein levels. Whole kidney and medulla MRE-derived stiffness also correlated negatively with creatinine levels. Regarding the group of patients with evidence of CRPI, no correlation was observed between serum albumin, pro- tein; urine albumin, protein levels, and stiffness values. The group of patients lacking evidence of CRPI showed no cor- relation between BUN, creatinine, and stiffness, but whole

kidney and cortex stiffness values correlated negatively with serum albumin (r = − 0.483 and -0.540, P < 0.05) and protein (r = − 0.562 and − 0.571, P < 0.05). Correlation data regard- ing MRE-derived stiffness and clinical parameters are shown in detail in Table 4.

Relationship between MRE‑derived stiffness and histopathological parameters

Whole kidney and medulla MRE-derived stiffness corre- lated negatively with extent of global glomerulosclerosis, segmental glomerulosclerosis, and tubulointerstitial fibro- sis. In contrast to these correlations, cortex MRE-derived stiffness did not correlate with global glomerulosclerosis

Table 2 Histopathological characteristics of all patients and their subgroups as CRPI and Non-CRPI patients

CRPI Chronic renal parenchymal injury

Parameters Results mean (± SD) or median (Min–Max)

All Patients CRPI Non-CRPI

Global glomerulosclerosis 22% (± 18%) 39% (± 12%) 6% (0–32%) Segmental glomerulosclerosis 4% (0–100%) 43% (4–100%) 5% Tubulointerstitial fibrosis   < 25% 26 (76.5%)  25–50% 6 (17.6%)   > 50% 2 (5.9%)

Table 3 Magnetic resonance elastography characteristics of all patients and their subgroups as CRPI and Non-CRPI patients

MRE Magnetic resonance elastography, kPa Kilopascal, CRPI Chronic renal parenchymal injury

Sequences Results mean (± SD) or median (Min– Max)

Distribution

All Patients CRPI Non-CRPI P

MRE (kPa)  Whole

Kidney 3.78

(± 1.26) 3.13

(± 0.89) 4.23

(± 1.30) P < 0.05

 Cortex 3.63 (± 1.25)

3.20 (± 1.15)

3.94 (± 1.26)

P > 0.05

 Medulla 4.77 (± 2.03)

3.73 (± 1.54)

5.50 (± 2.04)

P < 0.01

Table 4 Correlation analysis of MRE-derived stiffness values and clinical parameters

MRE Magnetic resonance elastography

Groups of Correlation Spearman Rho P Value

Whole kidney MRE-derived stiffness and  Urine albumin to creatinine 0.418 0.02  Urine protein to creatinine 0.396 0.02  24-h protein in urine 0.620 0.01  Serum albumin − 0.518 < 0.01  Serum protein − 0.610 < 0.01  Serum creatinine − 0.357 0.03

Cortex MRE-derived stiffness and  Urine albumin to creatinine 0.378 0.03  Urine protein to creatinine 0.343 0.04  24-h protein in urine 0.576 0.01  Serum albumin − 0.458 < 0.01  Serum protein − 0.568 < 0.01

Medulla MRE-derived stiffness and  Urine albumin to creatinine 0.380 0.03  Urine protein to creatinine 0.384 0.03  24-h protein in urine 0.644 < 0.01  Serum albumin 0.427 0.01  Serum protein − 0.530 < 0.01  Serum creatinine − 0.349 0.03

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(r = − 0.295; P = 0.09), segmental glomerulosclero- sis (r = − 0.270; P = 0.12), nor tubulointerstitial fibrosis (r = − 0.93; P = 0.603). Regarding the group of patients with evidence of CRPI, there was no correlation observed

between any stiffness value and histopathological parameter except for the negative correlation between confidence map MRE-derived stiffness and segmental glomerulosclerosis (r = − 0.630, P = 0.016). No correlation between MRE- derived stiffness values and histopathological parameters was seen in the group of patients lacking evidence of CRPI. The relationship between MRE-derived stiffness and fibrosis is shown in Table 5 and Figs. 4, 5, and 6, 7.

Discussion

In this study, we have shown that kidney stiffness measured via MRE decreases with increasing extent of glomerulo- sclerosis and tubulointerstitial fibrosis as well as with clin- icopathological evidence of CRPI. To our knowledge, this is the first study to show a negative correlation between

Table 5 Correlation analysis of MRE-derived stiffness values and histopathological parameters

MRE Magnetic resonance elastography

Groups of Correlation Spearman Rho P Value

Whole kidney MRE-derived stiffness and  Global glomerulosclerosis − 0.391 0.022

Medulla MRE-derived stiffness and  Global glomerulosclerosis − 0.412 0.015  Segmental glomerulosclerosis − 0.361 0.036  Tubulointerstitial fibrosis − 0.401 0.019

Fig. 4 Whole Kidney MRE- Derived Stiffness vs. Global Glomerulosclerosis

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Fig. 5 Medulla MRE-Derived Stiffness vs. Global Glomerulo- sclerosis

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MRE-derived stiffness and evidence of CRPI in patients with various causes of glomerulonephritis and AA amyloi- dosis. Regarding all 34 patients, MRE-derived stiffness val- ues correlated negatively with histopathological parameters of fibrosis consistent with decreased stiffness of kidneys with worsening renal functions. Our findings are similar to Brown et al.’s [5] study in which MRE-derived stiffness val- ues decrease as kidney fibrosis worsens in diabetic nephro- pathic kidneys. Contrary to ours and Brown et al.’s findings, several studies [22, 24, 25] demonstrated that MRE-derived stiffness increases with worsening renal fibrosis in renal allo- grafts, similar to liver fibrosis [28]. Lastly, Han et al. [29] using MR elastography showed that renal tissue stiffness in patients with CKD significantly increases as CKD stage progresses although it decreases in stage 5 CKD.

Tissue stiffness is not only affected from fibrosis but is also affected from hydrostatic pressures as well. Brown et al. used arterial spin labeling MRI to assess cortical blood flow contributing to kidney stiffness. They discovered that decrease in stiffness with progression of diabetic nephropa- thy was caused by reduced turgor stiffness due to decrease in cortical blood flow. Han et al. [29] also linked their finding to the reduction in renal blood flow that occurs in patients with stage 5 CKD. Since we did not perform arterial spin labeling MRI, we could not assess whether our findings were also caused by diminished cortical blood flow.

Considering that the kidney receives up to 20% of the cardiac output [30], effect of hydrostatic forces on kidney stiffness is more prominent than it is in liver. Alongside these factors, the deeper localization of the kidneys brings

Fig. 6 Medulla MRE-Derived Stiffness vs. Segmental Glo- merulosclerosis

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Medulla MRE Derived S�ffness vs. Segmental Glomerulosclerosis

Fig. 7 Mean Medulla MRE- Derived Stiffness vs Tubuloint- erstitial Fibrosis

<25%, 5.17

25-50%, 3.66 >50%, 2.99

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different properties in comparison to the liver when mechan- ical vibrations are applied.

We have shown statistically significant negative correla- tions between fibrosis and MRE-derived stiffness acquired from whole kidney and medulla. In contrast to this finding, correlation between cortex-derived MRE stiffness and extent of global glomerulosclerosis was weak and statistically insignificant; moreover, there was no relation with extent of segmental glomerulosclerosis and tubulointerstitial fibrosis. Considering that the cortex constitutes a thin part of the kidney, it is hard to differentiate the wave propagation and mechanical properties of the cortex from the medulla. This problem was also underlined by Brown et al. [5] and Lee et al. [22], stating that using 3D MRE or higher frequency vibration could overcome this problem. We attributed the lack of correlation in our study to the fact that we used 2D and relatively low frequency MRE.

Our study is the first to compare MRE stiffness of kidneys with and without evidence of CRPI in glomerulonephritis patients. For the first time, we have revealed that chronically injured kidneys became significantly softer in the glomeru- lonephritis and AA amyloidosis patient cohort.

Although creatinine is not an accurate marker of fibro- sis, all three histopathological parameters of fibrosis cor- related positively with creatinine in our patient cohort. This is expected given the fact that our cohort was devoid of patients with acute tubulointerstitial injury or severe glo- merular injury characterized by extracapillary proliferation. Similar to the histopathological correlations, whole kidney and medulla MRE-derived stiffness values correlated nega- tively with creatinine, further suggesting kidney stiffness decreases as kidney functions deteriorate in patients with glomerulonephritis and AA amyloidosis. Lack of correlation between cortex MRE-derived stiffness and creatinine may be caused by the limited capability of MRE to distinguish mechanical properties of cortex and medulla.

Our study has many novel findings and strengths. Most important aspect of this study is the fact that all 34 patients had histopathological scores matched with their MRE- derived stiffness values and clinical parameters. Another strength of this study is that we grouped the patients according to chronicity and assessed relation between chronicity and stiffness. We also acknowledge some limi- tations. Most important limitation of this study is that it only consisted of 34 patients with unequal distribution of patient subgroups, leading to lack of generalizability to each subgroup. While 2D MRE is used in clinical practice to assess liver fibrosis and previous studies conducted in kidneys also demonstrated the feasibility of 2D MRE, it is acknowledged that 3D MRE is superior to 2D MRE by minimizing quantitative artifacts related to varying direc- tionality of wave propagation, which is not commercially available. We also did not weight each patient thus could

not report the patients’ BMIs. Although we did not have any marginally weighted (i.e., underweight or morbid obese) patient, differences in body fat could bring expla- nation to different wave propagation characteristics.

As determinants of fibrosis (i.e., fibrosis, hydrostatic pressure, and blood flow) vary between various disease processes of different organs, findings of this study are not generalizable to other diseases and organs and only applies to primary glomerulopathy/glomerulonephritis and AA amyloidosis of the kidney. However, future studies may bring further insight to the use of MRE measurements with specific cut-offs according to the different causes of glomerular diseases. This may be studied in the future with evolving MR technology.

Since fibrosis is the end result of various insults to kid- ney [2], quantification of fibrosis could potentially help identify high-risk patients whose routine kidney function markers such as creatinine are not markedly impaired yet. Considering evolving novel anti-fibrotic therapies [1, 31, 32], importance of detecting fibrotic burden becomes more remarkable. Kidney biopsy is the gold standard to assess fibrosis [6]. Alongside its disadvantages related to inva- siveness, patchy distribution of fibrosis within kidneys and small sample amounts lead to flawed fibrosis burden assessment, thus necessitating development of non-inva- sive and more accurate means. We believe that future stud- ies will elucidate and quantify factors associated with stiff- ness measured via MR elastography and increase its utility as well as quality. Alternative kidney function assessment methods such as MR elastography will reveal chronicity and extent of renal fibrosis superior to current methods and will replace kidney biopsy. This will help eliminate controversies regarding biopsy need and immunosuppres- sive drug use by better defining patients that will benefit from interventions and drugs.

In conclusion, we have demonstrated the feasibility of MRE to assess fibrosis and chronicity in patients with primary glomerulonephritis and AA amyloidosis. Kidney stiffness decreases with chronicity and worsening kidney functions as indicated both by histopathological markers and creatinine levels.

Acknowledgements This study was supported by hacettepe üniversi- tesi with Grant No. TTU 2019-17871.

Author contributions İ.S.İ, A.S., M.K., and M.A. designed the study, A.T.G., C.C., C.Ö. M.Ü.K. T.Y., R.Y., B.A, and Y.E collected the data, A.T.G., İ.S.İ, A.S., M.K., and M.A. analyzed the data; A.T.G., İ.S.İ, A.S., M.K., and M.A. wrote the paper.

Declarations

Conflict of interest The authors declare that they have no conflict of interest.

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References

1. Boor P, Ostendorf T, Floege J. Renal fibrosis: novel insights into mechanisms and therapeutic targets. Nat Rev Nephrol. 2010 Nov;6(11):643–56.

2. Rockey DC, Bell PD, Hill JA. Fibrosis–a common path- way to organ injury and failure. N Engl J Med. 2015 Mar;372(12):1138–49.

3. Roberts ISD, Cook HT, Troyanov S, Alpers CE, Amore A, Barratt J, et al. The Oxford classification of IgA nephropathy: pathology definitions, correlations, and reproducibility. Kidney Int. 2009 Sep;76(5):546–56.

4. Risdon RA, Sloper JC, De Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet (London, England). 1968 Aug;2(7564):363–6.

5. Brown RS, Sun MRM, Stillman IE, Russell TL, Rosas SE, Wei JL. The utility of magnetic resonance imaging for noninvasive evalu- ation of diabetic nephropathy. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc – Eur Ren Assoc. 2020 Jun;35(6):970–8.

6. Luciano RL, Moeckel GW. Update on the Native Kidney Biopsy: Core Curriculum 2019. Am J kidney Dis Off J Natl Kidney Found. 2019 Mar;73(3):404–15.

7. Lees JS, McQuarrie EP, Mordi N, Geddes CC, Fox JG, Mackinnon B. Risk factors for bleeding complications after nephrologist-per- formed native renal biopsy. Clin Kidney J. 2017 Aug;10(4):573–7.

8. Roccatello D, Sciascia S, Rossi D, Naretto C, Bazzan M, Solfietti L, et al. Outpatient percutaneous native renal biopsy: safety profile in a large monocentric cohort. BMJ Open. 2017 Jun;7(6):e015243.

9. Fiorentino M, Bolignano D, Tesar V, Pisano A, Van Biesen W, D’’Arrigo G, et al. Renal Biopsy in 2015 – From Epidemiol- ogy to Evidence-Based Indications. Am J Nephrol [Internet]. 2016;43(1):1–19. Available from: https://www.karger.com/ DOI/https:// doi. org/ 10. 1159/ 00044 4026

10. Jiang K, Ferguson CM, Lerman LO. Noninvasive assessment of renal fibrosis by magnetic resonance imaging and ultrasound tech- niques. Transl Res. 2019 Jul;209:105–20.

11. Kaimori J-Y, Isaka Y, Hatanaka M, Yamamoto S, Ichimaru N, Fujikawa A, et al. Visualization of kidney fibrosis in diabetic nephropathy by long diffusion tensor imaging MRI with spin- echo sequence. Sci Rep. 2017 Jul;7(1):5731.

12. Hueper K, Khalifa AA, Bräsen JH, Vo Chieu VD, Gutberlet M, Wintterle S, et al. Diffusion-Weighted imaging and diffusion ten- sor imaging detect delayed graft function and correlate with allo- graft fibrosis in patients early after kidney transplantation. J Magn Reson Imaging. 2016 Jul;44(1):112–21.

13. Xu X, Palmer SL, Lin X, Li W, Chen K, Yan F, et al. Diffusion- weighted imaging and pathology of chronic kidney disease: initial study. Abdom Radiol (New York). 2018 Jul;43(7):1749–55.

14. Feng Q, Ma Z, Wu J, Fang W. DTI for the assessment of disease stage in patients with glomerulonephritis–correlation with renal histology. Eur Radiol. 2015 Jan;25(1):92–8.

15. Friedli I, Crowe LA, Berchtold L, Moll S, Hadaya K, de Perrot T, et al. New Magnetic Resonance Imaging Index for Renal Fibrosis Assessment: A Comparison between Diffusion-Weighted Imag- ing and T1 Mapping with Histological Validation. Sci Rep. 2016 Jul;6:30088.

16. Zhao J, Wang ZJ, Liu M, Zhu J, Zhang X, Zhang T, et al. Assess- ment of renal fibrosis in chronic kidney disease using diffusion- weighted MRI. Clin Radiol. 2014 Nov;69(11):1117–22.

17. Leung G, Kirpalani A, Szeto SG, Deeb M, Foltz W, Simmons CA, et al. Could MRI Be Used To Image Kidney Fibrosis? A Review of Recent Advances and Remaining Barriers. Clin J Am Soc Nephrol. 2017 Jun;12(6):1019–28.

18. Kline TL, Edwards ME, Garg I, Irazabal M V, Korfiatis P, Harris PC, et al. Quantitative MRI of kidneys in renal disease. Abdom Radiol (New York). 2018 Mar;43(3):629–38.

19. Thiravit S, Suwanchatree P, Skulratanasak P, Thiravit P, Suvan- narerg V. Correlation Between Apparent Diffusion Coefficient Values of the Renal Parenchyma and Estimated Glomerular Fil- tration Rates on 3-T Diffusion-Weighted Echo-Planar Magnetic Resonance Imaging. J Comput Assist Tomogr. 2019;43(5):780–5.

20. Toya R, Naganawa S, Kawai H, Ikeda M. Correlation between estimated glomerular filtration rate (eGFR) and apparent diffusion coefficient (ADC) values of the kidneys. Magn Reson Med Sci MRMS an Off J Japan Soc Magn Reson Med. 2010;9(2):59–64.

21. Namimoto T, Yamashita Y, Mitsuzaki K, Nakayama Y, Tang Y, Takahashi M. Measurement of the apparent diffusion coefficient in diffuse renal disease by diffusion-weighted echo-planar MR imaging. J Magn Reson Imaging. 1999 Jun;9(6):832–7.

22. Lee CU, Glockner JF, Glaser KJ, Yin M, Chen J, Kawashima A, et al. MR elastography in renal transplant patients and correlation with renal allograft biopsy: a feasibility study. Acad Radiol. 2012 Jul;19(7):834–41.

23. Marticorena Garcia SR, Fischer T, Dürr M, Gültekin E, Braun J, Sack I, et al. Multifrequency Magnetic Resonance Elastography for the Assessment of Renal Allograft Function. Invest Radiol. 2016 Sep;51(9):591–5.

24. Kim JK, Yuen DA, Leung G, Jothy S, Zaltzman J, Ramesh Prasad G V, et al. Role of Magnetic Resonance Elastography as a Non- invasive Measurement Tool of Fibrosis in a Renal Allograft: A Case Report. Transplant Proc. 2017 Sep;49(7):1555–9.

25. Kirpalani A, Hashim E, Leung G, Kim JK, Krizova A, Jothy S, et al. Magnetic Resonance Elastography to Assess Fibrosis in Kid- ney Allografts. Clin J Am Soc Nephrol. 2017 Oct;12(10):1671–9.

26. Samir AE, Allegretti AS, Zhu Q, Dhyani M, Anvari A, Sullivan DA, et al. Shear wave elastography in chronic kidney disease : a pilot experience in native kidneys. 2015;1–9.

27. Chen J, Kawashima A, Kim B, Kremers WK, Ehman RL, Gloor JM. MR Elastography in Renal Transplant Patients and Corre- lation with Renal Allograft Biopsy : A Feasibility Study. Acad Radiol [Internet]. 19(7):834–41. Available from: http://dx.doi. org/https:// doi. org/ 10. 1016/j. acra. 2012. 03. 003

28. Toguchi M, Tsurusaki M, Yada N, Sofue K, Hyodo T, Onoda M, et al. Magnetic resonance elastography in the assessment of hepatic fibrosis: a study comparing transient elastography and his- tological data in the same patients. Abdom Radiol (New York). 2017 Jun;42(6):1659–66.

29. Han JH, Ahn J-H, Kim J-S. Magnetic resonance elastography for evaluation of renal parenchyma in chronic kidney disease: a pilot study. Radiol Med. 2020 May;

30. Schiller AM, Pellegrino PR, Zucker IH. Eppur Si Muove: The dynamic nature of physiological control of renal blood flow by the renal sympathetic nerves. Auton Neurosci. 2017 May;204:17–24.

31. Liu R, Das B, Xiao W, Li Z, Li H, Lee K, et al. A Novel Inhibitor of Homeodomain Interacting Protein Kinase 2 Mitigates Kidney Fibrosis through Inhibition of the TGF-β1/Smad3 Pathway. J Am Soc Nephrol. 2017 Jul;28(7):2133–43.

32. Karihaloo A. Anti-fibrosis therapy and diabetic nephropathy. Curr Diab Rep. 2012 Aug;12(4):414–22.

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ORIGINAL ARTICLE

Therapy and outcomes of C3 glomerulopathy and immune-complex membranoproliferative glomerulonephritis

Priyanka Khandelwal1 & Swati Bhardwaj1 & Geetika Singh2 & Aditi Sinha1 & Pankaj Hari1 & Arvind Bagga1

Received: 30 April 2020 /Revised: 13 July 2020 /Accepted: 31 July 2020 # IPNA 2020

Abstract Background Data on therapy and outcome of dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune- complex MPGN (IC-MPGN) in children are limited. Methods In this retrospective single-center study from 2007 to 2019, kidney biopsies were reviewed to include patients aged <18-years with C3 glomerulopathy and IC-MPGN. Initial immunosuppression comprised prednisolone, mycophenolate mofetil (n = 51), tacrolimus (n = 11), and/or IV cyclophosphamide (n = 20). Clinicopathological features, response to therapy, and adverse outcome (eGFRcr < 15 mL/min/1.73 m2 or death) were evaluated. Results A total of 92 patients were classified as DDD (n = 48, 52.2%), C3GN (n = 26, 28.3%), and IC-MPGN (n = 18, 19.6%) by immunohistochemistry and electron microscopy; 8 patients with DDDwere misclassified as IC-MPGN on immunofluorescence. At last follow-up (median 4.3 years), complete or partial remission occurred in 28.5, 36.1, and 16.7% patients with DDD, C3GN, and IC-MPGN, respectively. Serum albumin at onset < 2.5 g/dL (HR = 0.29, P = 0.005) and persistently low serum C3 (HR = 0.34, P = 0.02) were associated with lack of remission. The 5-year kidney survival was 62.6, 85.5, and 88.5% in patients with DDD, C3GN, and IC-MPGN, respectively (log-rank, P = 0.006). Presentation as rapidly progressive GN (HR = 11.2, P < 0.001), age > 10 years at onset (HR = 4.0, P = 0.004), and DDD (HR = 4.2, P = 0.02) were independently associated with adverse outcome; achieving remission was protective (HR = 0.04; P < 0.001). Conclusion Outcome in patients with C3 glomerulopathy and IC-MPGN was unsatisfactory, and only a small proportion of patients achieved complete or partial remission. Patients with DDDwere more likely to present with rapidly progressive GN and were at higher risk of adverse outcomes, including kidney failure.

Keywords Dense deposit disease . Rapidly progressive glomerulonephritis . Calcineurin inhibitor . Mycophenolate mofetil .

Cyclophosphamide . Children

Introduction

C 3 g l o m e r u l o p a t h y a n d i mm u n e – c o m p l e x membranoproliferative glomerulonephritis (IC-MPGN) are rare

glomerular diseases with high risk of progressive kidney failure [1, 2]. C3 glomerulopathy is classified as C3 deposition that is ≥ 2 orders ofmagnitude higher than accompanying immunoglobulin, while IC-MPGN shows predominant IgG staining on immuno- fluorescence microscopy [3–5]. C3 glomerulopathy is further classified into dense deposit disease (DDD) and C3 glomerulo- nephritis (C3GN) based on the presence or absence, respectively, of ribbon-like intramembranous electron dense deposits [4, 5]. The pathogenetic process underlying C3 glomerulopathy is dys- regulation of the alternative complement pathway [6]. C3 nephrit- ic factor (C3Nef), autoantibodies to factors H (FH), B, and C3b, and variants or copy number variations in genes regulating the alternative complement pathway may be found in patients with C3 glomerulopathy and IC-MPGN [7–9]. Therapy, especially in children, is based on case series and expert opinion and often influenced by the degree of proteinuria or kidney dysfunction

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00467-020-04736-8) contains supplementary material, which is available to authorized users.

* Pankaj Hari [email protected]

1 Division of Nephrology, ICMR Center for Advanced Research in Nephrology, Department of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

2 Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

https://doi.org/10.1007/s00467-020-04736-8

/ Published online: 4 September 2020

Pediatric Nephrology (2021) 36:591–600

[9–13]. Immunosuppression with steroids, mycophenolate mofe- til, cyclophosphamide, calcineurin inhibitor, and rituximab has not shown consistent benefit; plasmapheresis has been used an- ecdotally [1, 2, 9–12]. While complement blockade with eculizumab might hold promise [14], availability and cost limit its use in developing countries. In the absence of therapy, long- term outcome is unsatisfactory with progression to chronic kid- ney disease (CKD) stage 5 [15]. We aimed to report the clinico- pathological features, response to therapy, and outcome in chil- dren diagnosed with C3 glomerulopathy and IC-MPGN.

Methods

A retrospective chart review of patients managed in the divi- sion of Pediatric Nephrology at this hospital fromMarch 2007 to September 2019 was performed. Patients aged < 18 years, diagnosed as types I–III MPGN, C3 glomerulopathy, or IC- MPGN on initial or re-biopsy were included following insti- tutional ethics committee approval. In addition, patients with diffuse proliferative GN with immunofluorescence (IF) show- ing glomerular staining for C3 with or without co-dominant IgG were included if complete clinical recovery did not occur within 3 months. Patients with inadequate tissue for electron microscopy (EM) or IC-MPGN secondary to hepatitis B and C or human immunodeficiency virus (HIV) were excluded. All biopsies, including those with an initial diagnosis of MPGN types I–III, were retrospectively analyzed by a renal pathologist (GS) to classify into IC-MPGN, DDD, and C3GN based on IF and EM findings (Supplementary Figs. 1 and 2). C3 glomerulopathy, defined as dominant C3 staining (inten- sity ≥ 2 orders of magnitude more than any other immunoreactant), was further classified as DDD (dense osmiophilic intramembranous deposits) or C3GN (light dense, amorphous mesangial, paramesangial, subendothelial, and subepithelial deposits) based on EM; biopsies with co- dominant or predominant IgG staining were classified as IC- MPGN [3, 13].

Clinical details

Clinical features, therapy, and outcome were recorded. Investigations included blood levels of creatinine, albumin, electrolytes, cholesterol, C3, antinuclear antibody (ANA), antineutrophil cytoplasmic antibody (ANCA), serology for hepatitis B and C and HIV, and urinalysis. The 24-h urine protein excretion was estimated at baseline and follow-up. Hypertension was defined as blood pressure > 95th centile for age, height, and sex [16]. Glomerular filtration rate (eGFRcr) was estimated using modified Schwartz equation [17]. Microscopic hematuria was defined as > 5 red blood cells per high-power field of centrifuged urine specimen. Nephrotic-range proteinuria was > 1 g/m2/day or urine-

protein-to-creatinine ratio (Up/Uc) > 2mg/mg. Nephrotic syn- drome was the presence of nephrotic range proteinuria, hypo- albuminemia (< 2.5 g/dL), and edema. Acute glomerulone- phritis (GN) was the presence of hematuria and proteinuria, with variable degrees of hypertension and acute kidney injury. Rapidly progressive GN was defined in patients with acute GN who showed rapid decline in kidney function within 7– 10 days. Chronic GN was eGFRcr < 60 mL/min/1.73 m2 with variable degrees of hematuria and proteinuria persisting for > 3 months.

Patients were followed-up every 3–6 months. Patients with eGFRcr > 30 mL/min/1.73 m2 received an angiotensin- converting enzyme inhibitor or angiotensin II receptor blocker. Immunosuppression consisted of prednisolone (60 mg/m2/day for 4 weeks, followed by 40 mg/m2 on alter- nate days for 4 weeks and tapered by 0.1–0.2 mg/kg every 2 weeks to 5 mg on alternate days) with or without mycophe- nolate mofetil (MMF; 800–1000 mg/m2/day in two divided doses) or tacrolimus (0.12–0.15 mg/kg/day; trough levels 4– 8 ng/dL). Patients with rapidly progressive GN, crescents on biopsy, or persistently deranged kidney function at onset were treated with intravenous (IV) methylprednisolone (3–6 doses of 30 mg/kg) and IV cyclophosphamide (6 doses of 500 mg/ m2 every 3–4 weeks), followed by MMF and prednisolone.

Complete remission was defined as absence of proteinuria (< 100 mg/m2/day or Up/Uc < 0.2 mg/mg), serum albumin > 3.0 g/dL and eGFRcr > 90 mL/min/1.73 m2. Partial remission was proteinuria 100–1000 mg/m2/day or Up/Uc 0.2–2.0 mg/ mg, serum albumin > 3.0 g/dL, and improved or stable eGFRcr

(± 15 mL/min/1.73 m2). Non-response was defined as lack of complete or partial remission after 6 months of therapy.

Histopathology

Biopsy specimens with at least 10 glomeruli were considered adequate. The extent of mesangial and endocapillary prolifer- ation, basement membrane thickening and splitting, propor- tion of cellular, fibrocellular, or fibrous crescents, and extent of global or focal sclerosis were noted. Interstitial fibrosis and tubular atrophy were graded (grade 0: none; grade 1: 0–25%; grade 2: 25–50%; and grade 3: > 50%). Intensity of mesangial and capillary wall staining for IgG, IgA, IgM, C3, and C1q was graded from 0 to 3+ on IF. Location and type of deposits and extent of podocyte effacement were recorded on EM.

Statistics

Data are presented as proportions and median (1st and 3rd quartiles, Q1 and Q3) and analyzed using Stata version 14.0 (Stata Corp., College Station, TX). Tests for significance in- cluded Wilcoxon signed-rank and rank-sum tests and chi- square test. Adverse outcome was defined as eGFRcr < 15 mL/min/1.73 m2 or death. Probability of kidney survival

592 Pediatr Nephrol (2021) 36:591–600

free of adverse outcome, determined using the Kaplan-Meier method, was compared in patients with DDD, C3GN, and IC- MPGN. Determinants of complete or partial remission and adverse outcome were estimated as hazards ratios, by univar- iate and multivariable analyses, using Cox proportional haz- ards model. Variables withP < 0.1 on univariate analysis were included in the multivariable models. The ratio of number of outcome events to the number of independent variables

(events per variable) in the multivariable models was ≥ 5. Two-tailed P < 0.05 was considered significant.

Results

Of 2215 initial and re-biopsies performed from 2007 to 2019, 92 biopsies were classified as C3 glomerulopathy and IC-MPGN.

Fig. 1 Flow of study showing enrollment of 92 patients from 2007 to 2019. Patients aged < 18 years, with either biopsy suggestive of membranoproliferative glomerulonephritis (MPGN) or diffuse prolifera- tive GN with dominant C3 or co-dominant IgG and persistent disease > 3 months were included. Kidney biopsies, including 18 initially diag- nosed as MPGN types I–III (7 type I, 11 type II), were reclassified as C3 glomerulopathy (n = 74) and immune-complex MPGN (n = 18). Nine patients classified as immune-complex MPGN on immunofluorescence (IF) were classified as dense deposit disease (DDD; n = 8) on electron

microscopy (EM) and C3 glomerulonephritis (C3GN; n = 1) on re-biop- sy. Four of nine misclassified patients showed predominant C3 staining on subsequent re-biopsy. aLack of enough tissue or advanced glomerulosclerosis. bInitial IF showed C3 1+ and IgG 3+ after pronase digestion; C3 3+ and absence of IgG on re-biopsy. cInitial IF after pronase digestion showed 1–2+ C3 (n = 2), co-dominant 2+ IgG (n = 2), or bright 3+ IgG (n = 4); re-biopsy in 3 patients showed C3 staining 3–4+ in all with or without absent IgG staining

593Pediatr Nephrol (2021) 36:591–600

These included 18 biopsies from 2007 to 2013 initially diag- nosed as MPGN types I–III that were re-classified. Figure 1 shows flow of patient enrolment and classification. Of 92 pa- tients, 48 (52.2%) were classified as DDD, 26 (28.3%) as C3GN, and 18 (19.6%) as IC-MPGN. One patient with DDD, mimicking necrotizing vasculitis, has been reported previously [18]. Clinical and histological features of patients are shown in Table 1. Patients presented at all ages (range 4.1–18 years); nine patients were < 6 years old. A minor prodromal illness, most frequently fever and upper respiratory tract infection, was noted in 29 (31.5%). Chief presenting features were nephrotic syn- drome (65.2%), acute GN (15.2%), and rapidly progressive GN (15.2%). At presentation, 14 (15.2%) patients required dial- ysis. Most had significant hypertension and eight had hyperten- sive encephalopathy with seizures.

Rapidly progressive GN (with 40–100% crescents on bi- opsy), gross hematuria, and dialysis dependence were signif- icantly higher in patients with DDD compared with C3GN (Table 1). As compared with IC-MPGN, more patients with C3 glomerulopathy (DDD and C3GN) showed low C3 at on- set (< 90 mg/dL; 85.7% versus 41.1%, P < 0.001), which persisted during follow-up (Table 1). No patient had family history of similar illness or any other autoimmune disease.

Biopsy characteristics

Histopathological findings are shown in Table 2. Of 33 biop- sies showing any crescents, 27 (81.8%) were classified as DDD. Classification based on IF and EM is shown in Fig. 1. In patients classified by IF alone, 8 (18.2%) with DDD and

Table 1 Baseline clinical and biochemical parameters in patients with dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune-complex membranoproliferative glomeru- lonephritis (IC-MPGN)

C3 glomerulopathy (N = 74) IC-MPGN (N = 18) P† P‡

DDD (N = 48) C3GN (N = 26)

Age at onset, yr 9.6 (7.6, 11.4) 10.0 (8.2, 13.1) 9.5 (7.3, 10.3) 0.13 0.28

Age at diagnosis, yr 9.9 (8.3, 11.6) 10.5 (8.7, 13) 9.6 (7.9, 11.0) 0.21 0.39

Boys 24 (50) 19 (73.1) 13 (72.2) 0.05 0.27

Prodromal symptoms 20 (41.7) 5 (19.2) 4 (22.2) 0.05 0.34

First clinical manifestationa 0.05 0.45 Nephrotic syndrome 26 (54.2) 20 (76.9) 14 (77.8)

Rapidly progressive GN 12 (25.0) 1 (3.8) 1 (5.6)

Acute GN 6 (12.5) 5 (19.2) 3 (16.7)

Chronic GN 3 (6.3) – –

Hematuria 0.05 0.37 None 7 (14.6) 4 (15.4) 1 (5.6)

Microscopic 26 (54.2) 20 (76.9) 15 (83.3)

Gross 15 (31.3) 2 (7.7) 2 (11.1)

Hypertension 0.24 0.54 None 9 (18.8) 7 (26.9) 2 (11.1)

Stage 1 16 (33.3) 13 (50.0) 7 (38.9)

Stage 2 23 (47.9) 6 (23.1) 9 (50)

eGFRcr, mL/min/1.73 m2 b 79 (60, 94) 101 (65, 133) 89 (76, 121) 0.06 0.60

eGFRcr < 60 mL/min/1.73 m2 19 (39.6) 6 (23.1) 3 (16.7) 0.15 0.16

Serum albumin, g/dL 1.9 (1.7, 2.6) 2.4 (1.8, 3.0) 2.1 (1.8, 2.5) 0.13 0.66

24-h urine protein, g/dayc 2.2 (1.5, 3.9) 2.0 (1.5, 3.3) 2.4 (1.9, 4.5) 0.68 0.16

Serum C3, mg/dLd 29 (15, 56) 27.5 (17, 61.5) 91 (32.5, 109.5) 0.86 <0.001

Persistently low C3e 25 (53.2) 14 (53.8) 3 (16.7) 0.60 0.001

Numbers are N (%) or median (first, third quartiles)

eGFRcr estimated glomerular filtration rate; GN glomerulonephritis †Comparison between DDD and C3GN ‡Comparison between C3 glomerulopathy and IC-MPGN aOne patient with DDD presented with isolated gross hematuria b Computed in non-dialysis dependent patients c Proteinuria quantified in 33 DDD, 18 C3GN, and 11 IC-MPGN at baseline d Two patients with DDD and one with C3GN had serum C3 levels < 15 mg/dL; not available in 5 patients e Persistently low C3 defined as ≥ 2 low values (< 90 mg/dL) over ≥ 6 months; not available for one patient with DDD

594 Pediatr Nephrol (2021) 36:591–600

one (3.8%) with C3GN were misclassified as IC-MPGN due to lower intensity of C3 staining compared with IgG. Four of these 9 patients underwent a re-biopsy, 12–51.4 months later, which showed increased C3 staining to 3–4+ in all and two- order decrease in intensity of IgG staining in three patients (Fig. 1). Therefore, IF alone correctly classified 87.1% pa- tients with DDD (81.8% sensitivity, 100% specificity).

DDD was characterized by dense osmiophilic deposits along the lamina densa that were continuous in 38 or discon- tinuous in 10 patients; additional subepithelial and subendothelial deposits were noted in 7 patients, each. Electron dense deposits in C3GN were mesangial in 20, subendothelial 19, light intramembranous 7, paramesangial 6, and subepithelial in 4 biopsies. Deposits in IC-MPGNwere subendothelial in 10, mesangial 9, subepithelial 3, and paramesangial in 2 biopsies.

A total of 26 patients underwent a second kidney biopsy after median of 36.1 (16.8, 61.2) months. Indications included unsatisfactory EM on initial biopsy in 10 patients (5 DDD, 4 C3GN, and 1 IC-MPGN), for assessing disease progression in

15, and post-transplant recurrence in one. Original pattern on light microscopy was preserved in all; one patient showed recurrence of crescentic pattern of kidney injury. Re-biopsies showed persistence of neutrophilic infiltration and endocapillary proliferation in 12 and 8 patients, respectively. There was an increase in grade of interstitial fibrosis and/or tubular atrophy in 11 biopsies, and median global sclerosis increased from 0 to 10%. The IF pattern was consistent with the initial biopsy in four and seven patients with IC-MPGN and C3GN, respectively, but altered in four patients initially classified as IC-MPGN (described previously).

Therapy

Initial therapy comprisedMMF in 51, tacrolimus in 11, and IV cyclophosphamide in 20 patients with additional IV methyl- prednisolone in 15 and plasma exchanges in 6 patients (Table 3). Induction with IV cyclophosphamide was followed by therapy with MMF in 11, tacrolimus in 2, and azathioprine in one patient. Patients with non-response were switched to

Table 2 Light microscopic findings in patients with dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune-complex membranoproliferative glomeru- lonephritis (IC-MPGN)

C3 glomerulopathy (N = 74) IC-MPGN (N = 18) P† P‡

DDD (N = 48) C3GN (N = 26)

No. of glomeruli 19 (13.5, 27) 17.5 (13.5, 28.5) 11 (15, 23) 0.47 0.36

Patterna

Membranoproliferative GN 35 (72.9) 23 (88.5) 17 (94.4) 0.12 0.16

Diffuse proliferative GN 5 (10.4) 2 (7.7) 1 (5.6) 0.70 0.63

Crescentic GN 7 (14.6) 1 (3.8) – 0.16 –

Crescents (% glomeruli) <0.001 0.33

None 21 (43.8) 25 (96.2) 13 (72.2)

< 50 20 (41.7) – 5 (27.8)

≥ 50 7 (14.6) 1 (3.8) –

Endocapillary proliferation 27 (56.3) 15 (57.7) 12 (66.7) 0.76 0.60

Neutrophilic infiltration 38 (79.2) 18 (69.2) 8 (44.4) 0.49 0.02

Focal sclerosis 8 (16.7) 5 (19.2) – 0.75 –

Global sclerosis (% glomeruli) 0.36 0.09

None 28 (58.3) 18 (69.2) 7 (38.9)

< 50 17 (35.4) 8 (30.8) 11 (61.1)

≥ 50 3 (6.3) – –

Interstitial fibrosis 0.21 0.54

None 28 (58.3) 19 (73.1) 9 (50.0)

Grade 1 13 (27.1) 7 (26.9) 7 (38.9)

Grade 2 5 (10.4) – 2 (11.1)

Grade 3 2 (4.2) – –

GN glomerulonephritis

Numbers are median (first, third quartiles) or N (%) †Comparison between DDD and C3GN ‡Comparison between C3 glomerulopathy and IC-MPGN a Sclerosed glomeruli in one patient with DDD not allowing interpretation

595Pediatr Nephrol (2021) 36:591–600

therapy with MMF in 2, tacrolimus 26, IV cyclophosphamide 7, rituximab 6, and azathioprine in 3 patients.

In patients with C3 glomerulopathy, initial therapy with MMF and tacrolimus were administered for 19.7 (12.0, 35.9) and 20.3 (9.9, 25.4) months, respectively. Complete or partial remission was achieved in 11/40 (27.5%) patients with MMF and 5/10 (50%) patients with tacrolimus. IV cyclophos- phamide was preferentially used in patients with C3 glomer- ulopathy (n = 16) and induced remission in 6 (37.5%) patients.

Of 11 patients with IC-MPGN initially treated with MMF, 3 (27.3%) achieved complete or partial remission. One of four patients administered IV cyclophosphamide achieved partial remission and one receiving tacrolimus remained non- responsive.

Outcome

Median duration of follow-up was 4.3 (2.3, 7.2) years. Proportion of patients with DDD, C3GN, and IC-MPGN in complete or partial remission at last follow-up were 28.5,

36.1, and 16.7%, respectively [log-rank P = 0.54, Fig. 2(a)]. Parameters associated with complete or partial remission at last follow-up are shown in Table 4. On multivariable analy- sis, albumin < 2.5 g/dL at onset (HR 0.29; P = 0.005) and persistently low serum C3 (HR 0.34; P = 0.02) were indepen- dently associated with non-response. Of 9 patients (5 DDD, 3 C3GN, and 1 IC-MPGN) who were lost to follow-up after median of 2.3 (2.0, 2.5) years, 5 had achieved complete or partial remission. Proteinuria recurred following remission in 15 patients after median of 23.8 (13.2–42.5) months; 13 remained non-responsive.

Median kidney survival in patients with DDD was 6.9 years. The 5-year kidney survival was 62.6, 85.5, and 88.5% in patients with DDD, C3GN, and IC-MPGN, respec- tively [log-rank P = 0.006, Fig. 2(b)]. Five patients (3 DDD, 2 IC-MPGN), all with CKD stage 5, died after median of 3.5 (3.4, 6.1) years due to sepsis or complications of kidney fail- ure. On multivariable analysis, presentation as rapidly pro- gressive GN (HR = 11.2; P < 0.001), age > 10 years at onset (HR = 4.0; P = 0.004), and DDD (HR = 4.2; P = 0.02) were independently associated with adverse outcome (Table 4);

Table 3 Therapy and outcome in patients with dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune-complex membranoproliferative glomeru- lonephritis (IC-MPGN)

C3 glomerulopathy IC-MPGN P

DDD C3GN

Therapya N = 48 N = 26 N = 18

Mycophenolate mofetil 22 (45.8) 18 (69.2) 11 (61.1)

Tacrolimus 4 (8.3) 6 (23.1) 1 (5.6)

IV cyclophosphamideb +maintenance immunosuppressionc 14 (29.2) 2 (7.7) 4 (22.2)

Prednisolone alone 4 (8.3) – 2 (11.1)

6 months follow-up N = 48 N = 26 N = 18 0.95

Complete remission 6 (12.5) 3 (11.5) 1 (5.6)

Partial remission 8 (16.7) 4 (15.4) 3 (16.7)

Non-response 34 (70.8) 17 (73.1) 14 (77.8)

eGFRcr < 15 mL/min/1.73 m2 or death 6 (12.5) 1 (3.9) – 0.16

12-months follow-up N = 48 N = 25 N = 17 0.49

Complete remission 8 (16.7) 2 (8.0) 2 (11.8)

Partial remission 10 (20.8) 5 (20.0) 1 (5.9)

Non-response 30 (62.5) 18 (72.0) 14 (82.3)

eGFRcr < 15 mL/min/1.73 m2 or death 7 (14.6) 1 (4.0) – 0.12

Last follow-up N = 48 N = 26 N = 18 0.78

Complete remission 5 (10.4) 3 (11.5) 2 (11.1)

Partial remission 8 (16.7) 5 (19.2) 1 (5.6)

Non-response 35 (72.9) 18 (69.2) 15 (83.3)

eGFRcr < 15 mL/min/1.73 m2 or death 19 (39.6) 2 (7.7) 2 (11.1) 0.003

Numbers are N (%) or median (first, third quartiles)

eGFRcr, estimated glomerular filtration rate a Other therapies were IV rituximab (n = 1, DDD) or no immunosuppression (n = 2 DDD, n = 1 C3GN) bAdditional 3–6 doses of IV methylprednisolone (n = 15) or plasma exchanges (n = 6) cMaintenance immunosuppression, begun if eGFRcr > 30 mL/min/1.73 m2 at 6 months, comprised mycopheno- late mofetil (n = 11), tacrolimus (n = 2), azathioprine (n = 1), and prednisolone (n = 1)

596 Pediatr Nephrol (2021) 36:591–600

achieving complete or partial remission was protective (HR = 0.04; P < 0.001).

Discussion

We report a single center experience of 92 children with C3 glomerulopathy and IC-MPGN. Patients with DDD com- prised almost half of the cohort and were more likely to

present with rapidly progressive GN. Immunofluorescence misclassified 18.2% patients with DDD as IC-MPGN, highlighting the histological overlap between C3 glomerulop- athy and IC-MPGN. One-fourth of the patients achieved com- plete or partial remission following immunosuppressive ther- apy; rate of remission was similar in patients with C3 glomer- ulopathy and IC-MPGN. Rapidly progressive GN, older age (> 10 years), DDD, and lack of remission were independently associated with progression to CKD stage 5 or death.

a

b

Fig. 2 Probability of complete or partial remission at last follow-up and kidney survival free of ad- verse outcomes. (a) Complete or partial remission at 6 months, 12 months, and last follow-up was achieved in 18.9, 23.3, and 28.5% patients with DDD, re- spectively (continuous line). Corresponding proportions in pa- tients with C3GN and IC-MPGN were 15.4, 19.6, and 36.1% and 11.1, 16.7, and 16.7%, respec- tively (interrupted lines, log-rank P = 0.54). (b) The 1- and 5-year kidney survival was 81.3 and 62.6% in patients with DDD (continuous line), 96.2 and 85.5% in C3GN, and 100 and 88.5% in IC-MPGN (interrupted lines), re- spectively (log-rank P = 0.006). Kidney survival in patients with C3GN and IC-MPGNwas similar (log-rank, P = 0.93)

597Pediatr Nephrol (2021) 36:591–600

Clinical features of patients in the current study showed similarities with previously reported pediatric and adult series. Nephrotic syndrome was the chief presentation in 65.2% of our patients similar to other pediatric reports (22–69.7%) [19–21]. We observed similar frequency of nephrotic syn- drome in C3 glomerulopathy (62%) and IC-MPGN (78%). In mixed pediatric and adult cohorts, nephrotic syndrome was more frequent in IC-MPGN (43–70%) compared with C3 glomerulopathy (26–52%) [1, 9, 22]. One-fourth of pa- tients with DDD presented with rapidly progressive GN, and 14.6% showed more than 50% crescents. The prevalence of crescentic GN in DDD is reported as 6–21% in adults and children [11, 19, 21, 23]. Majority had hematuria (86.9%) and significant hypertension (41.3%), comparable to 38– 100% and 55–79%, respectively, in other pediatric series [7, 19, 21, 23]. Acute GN, variably reported as 17–61% at onset [20, 21, 23], occurred in 15% of patients in this study. Levels of serum C3 were lower in patients with C3 glomerulopathy compared with IC-MPGN but similar in DDD and C3GN. Other reports suggest that C3 levels in DDD are lower than C3GN [9, 11]. About 40% of patients with IC-MPGN had low C3 at onset, similar to previous studies [9, 24, 25].

An infectious trigger at onset occurred in 31.5% of the present patients, similar to 28–57% in pediatric cohorts from

Europe and North America [7, 20, 21]. It is suggested that an infectious trigger might lead to sustained activation of the alternative pathway and result in persistent glomerular inflam- mation even after the infection is controlled. Infectious trig- gers activate the classical or lectin pathways which may ex- plain the bright IgG staining in comparison to C3 deposits on eight biopsies of patients with DDD. In support of this hy- pothesis, we previously reported bright glomerular C4d stain- ing in biopsies with C3 glomerulopathy [18]. Thus, the IF criteria of C3 dominance at least two orders of magnitude stronger than other immune deposits had a sensitivity of 81.8% for DDD in the present cohort, similar to 88% reported by Hou et al. [3]. In addition, as reported previously, three patients with C3 glomerulopathy had transient IgG staining at onset that disappeared on re-biopsy [24]. Due to the evolu- tion of IF findings over time, the consensus guidelines on C3 glomerulopathy suggest using EM to distinguish DDD from C3GN [13].

The treatment of these conditions is challenging. While MMF was shown to induce remission in 45–86% adults with C3 glomerulopathy in retrospective series [10, 26, 27], its efficacy was not replicated in another cohort where 25% of patients with more severe disease progressed to kidney failure [28]. MMF was the initial therapy in 62% of the present

Table 4 Association of remission and progression to chronic kidney disease (CKD) stage 5 or death

Parameter Complete or partial remission at last follow-up CKD stage 5 or death

Univariate analysis Multivariable analysis Univariate analysis

Multivariable analysis

Baseline Hazards ratio (95% CI)

P Hazards ratio (95% CI)

P Hazards ratio (95% CI)

P Hazards ratio (95% CI)

P

Age > 10 years 1.27 (0.57, 2.83) 0.56 3.77 (1.57, 9.03) 0.003 3.98 (1.55, 10.25) 0.004 Gross hematuria 2.26 (0.63, 8.16) 0.21 1.25 (0.49, 3.18) 0.65 Onset serum albumin < 2.5 g/dL 0.40 (0.18, 0.89) 0.03 0.29 (0.12, 0.69) 0.005 0.63 (0.33, 1.25) 0.19 24-h urine protein at onset 1.00 (0.99, 1.00) 0.58 1.00 (0.99, 1.00) 0.85 Low C3 at onset (< 90 mg/dL) 0.66 (0.28, 1.60) 0.36 1.00 (0.39, 2.56) 0.99 Rapidly progressive GNa 1.83 (0.68, 4.92) 0.23 3.23 (1.26, 8.29) 0.02 11.19 (3.21, 39.01) < 0.001 Onset eGFRcr < 30 mL/min/1.73 m2 1.30 (0.44, 3.81) 0.64 6.14 (2.40, 15.74) < 0.001 1.76 (0.48, 6.53) 0.40b

Histopathology Dense deposit diseasec 1.84 (0.52, 6.46) 0.34 4.67 (1.09, 20.1) 0.04 4.21 (1.29, 13.69) 0.02 C3 glomerulonephritisc 2.05 (0.54, 7.74) 0.29 0.93 (0.13, 6.64) 0.94 Neutrophilic infiltration 1.65 (0.62, 4.42) 0.32 1.43 (0.53, 3.89) 0.48 Endocapillary proliferation 0.81 (0.36, 1.81) 0.61 1.19 (0.51, 2.78) 0.68 Interstitial fibrosis, tubular atrophy stages 2–3

0.67 (0.27, 1.69) 0.40 5.83 (2.37, 14.39) < 0.001 2.76 (0.99, 7.64) 0.05

Therapy and follow-upd

Persistently low C3 0.36 (0.15, 0.90) 0.03 0.34 (0.13, 0.84) 0.02 1.43 (0.53, 3.89) 0.48 Therapy with MMF 0.66 (0.30, 1.47) 0.31 0.51 (0.22, 1.19) 0.12 Therapy with tacrolimus 1.49 (0.51, 4.37) 0.47 0.55 (0.23, 1.32) 0.18 Complete or partial remission 0.12 (0.03, 0.49) 0.004 0.04 (0.01, 0.23) < 0.001

eGFRcr, estimated glomerular filtration rate; GN, glomerulonephritis; MMF, mycophenolate mofetil a All patients had > 40% crescents b eGFRcr < 30 mL/min/1.73 m2 was not significant in the multivariable model and therefore excluded c Compared with IC-MPGN as reference d Therapy with IV cyclophosphamide was not considered due to preferential use in patients with rapidly progressive GN

598 Pediatr Nephrol (2021) 36:591–600

patients among whom 27.5% attained complete or partial re- mission; response rates in C3 glomerulopathy and IC-MPGN were similar. Therapy with tacrolimus showed remission in 5 of 11 (45.5%) patients; variable remission rates of 11–94% are reported in adults [12, 26, 29]. Cyclophosphamide has shown conflicting results in adults with up to 78% complete remis- sion [30], but without improvement in kidney survival [31]. We used IV cyclophosphamide, chiefly in patients with severe disease, which induced complete or partial remission in 35% of patients. While complement blockade with eculizumab ap- pears to be promising in patients with severe disease, especial- ly crescentic GN [14], this is not available in our country.

Complete or partial remission occurred in 28.5% patients with DDD, 36.1% in C3GN, and 16.7% in IC-MPGN at last follow-up in the present cohort. In other pediatric studies, rate of remission following immunosuppressive therapy was re- ported as 22–78% [7, 19–21, 23, 32]. In a multivariable Cox proportional hazard model, we showed that serum albumin < 2.5 g/dL at onset and persistently low levels of C3 were inde- pendent factors associated with non-response to therapy; such patients may require aggressive management. In addition to the histological overlap, we showed that kidney survival of patients with IC-MPGN and C3GN was similar. In contrast, 67–88% of patients with C3GN in Japanese pediatric cohorts progressed to CKD stage 5 as compared to 32–50% of patients with IC-MPGN [24, 25]. While DDD was associated with a poor kidney survival in the present study, similar to previous observations [11, 33], others show no difference in survival between C3GN and DDD [4, 9, 10, 21]. Progressive kidney failure occurred in 29–50% patients by 2–11 years in other cohorts [9–12, 34]. Similar to previous studies, we observed rapidly progressive GN, older age, and non-response to ther- apy were other independent factors associated with progres- sion to CKD stage 5 or death [9–11, 19, 21, 28].

This study was limited by its retrospective and single- center design. While EM-based classification was done in all 92 patients, biopsies that were inadequate for EM were not included and may have led to selection bias. Approximately 40% of patients were enrolled in the last four years and 9.8% were lost to follow-up; therefore, the median duration of follow-up was rather short (4.3 years). We did not perform assays for C3Nef, sC5b-9, autoantibodies to factor B, and genetic testing for complement regulatory genes or copy num- ber variations in CFHR1–5. Clinical utility of results of such testing in making therapeutic decisions is as yet unclear.

In summary, we present clinicopathological phenotype and outcome of children with C3 glomerulopathy and IC-MPGN. We found that immunofluorescence misclassified one fifth of patients with DDD as IC-MPGN, highlighting the histological overlap of C3 glomerulopathy and IC-MPGN. While DDD was independently associated with progressive kidney failure, outcomes were similar in IC-MPGN and C3GN. Rapidly pro- gressive GN, older age, and non-response to therapy were

additionally associated with adverse outcome. Response to immunosuppression was unsatisfactory irrespective of histo- logical classification or the type of immunosuppressive thera- py, therefore, emphasizing the need for disease-specific ther- apies, complement inhibitors or modulators.

Acknowledgments The study received funding support from the Department of Biotechnology, Government of India [BT/PR11030/ MED/30/1644/2016].

Compliance with ethical standards The study was conduct- ed following the Institute ethical clearance (IEC/NP-353/08-10-2014).

Conflict of interest The authors declare that they have no conflict of interest.

References

1. Fakhouri F, Le Quintrec M, Fremeaux-Bacchi V (2020) Practical management of C3 glomerulopathy and immunoglobulin-mediated MPGN: facts and uncertainties. Kidney Int. https://doi.org/10.1016/ j.kint.2020.05.053

2. Smith RJH, Appel GB, Blom AM, Cook HT, D'Agati VD, Fakhouri F, Fremeaux-Bacchi V, Jozsi M, Kavanagh D, Lambris JD, Noris M, Pickering MC, Remuzzi G, de Cordoba SR, Sethi S, Van der Vlag J, Zipfel PF, Nester CM (2019) C3 glomerulopathy— understanding a rare complement-driven renal disease. Nat Rev Nephrol 15:129–143

3. Hou J, Markowitz GS, Bomback AS, Appel GB, Herlitz LC, Barry Stokes M, D'Agati VD (2014) Toward a working definition of C3 glomerulopathy by immunofluorescence. Kidney Int 85:450–456

4. Bomback AS, Appel GB (2012) Pathogenesis of the C3 glomeru- lopathies and reclassification of MPGN. Nat Rev Nephrol 8:634– 642

5. Pickering MC, D'Agati VD, Nester CM, Smith RJ, Haas M, Appel GB, Alpers CE, Bajema IM, Bedrosian C, Braun M, Doyle M, Fakhouri F, Fervenza FC, Fogo AB, Fremeaux-Bacchi V, Gale DP, Goicoechea de Jorge E, Griffin G, Harris CL, Holers VM, Johnson S, Lavin PJ, Medjeral-Thomas N, Paul Morgan B, Nast CC, Noel LH, Peters DK, Rodriguez de Cordoba S, Servais A, Sethi S, Song WC, Tamburini P, Thurman JM, Zavros M, Cook HT (2013) C3 glomerulopathy: consensus report. Kidney Int 84: 1079–1089

6. Sethi S, Fervenza FC (2011) Membranoproliferative glomerulone- phritis: pathogenetic heterogeneity and proposal for a new classifi- cation. Semin Nephrol 31:341–348

7. Holle J, Berenberg-Gossler L, Wu K, Beringer O, Kropp F, Muller D, Thumfart J (2018) Outcome of membranoproliferative glomer- ulonephritis and C3-glomerulopathy in children and adolescents. Pediatr Nephrol 33:2289–2298

8. Iatropoulos P, Noris M,Mele C, Piras R, Valoti E, Bresin E, Curreri M, Mondo E, Zito A, Gamba S, Bettoni S, Murer L, Fremeaux- Bacchi V, Vivarelli M, Emma F, Daina E, Remuzzi G (2016) Complement gene variants determine the risk of immunoglobulin- associated MPGN and C3 glomerulopathy and predict long-term renal outcome. Mol Immunol 71:131–142

9. Servais A, Noel LH, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey MA, Macher MA, Zuber J, Karras A, Provot F, Moulin B, Grunfeld JP, Niaudet P, Lesavre P, Fremeaux-Bacchi V (2012) Acquired and genetic complement abnormalities play a

599Pediatr Nephrol (2021) 36:591–600

critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 82:454–464

10. Bomback AS, Santoriello D, Avasare RS, Regunathan-Shenk R, Canetta PA, Ahn W, Radhakrishnan J, Marasa M, Rosenstiel PE, Herlitz LC, Markowitz GS, D'Agati VD, Appel GB (2018) C3 glomerulonephritis and dense deposit disease share a similar dis- ease course in a large United States cohort of patients with C3 glomerulopathy. Kidney Int 93:977–985

11. Medjeral-Thomas NR, O'Shaughnessy MM, O'Regan JA, Traynor C, Flanagan M, Wong L, Teoh CW, Awan A, Waldron M, Cairns T, O'Kelly P, Dorman AM, Pickering MC, Conlon PJ, Cook HT (2014) C3 glomerulopathy: clinicopathologic features and predic- tors of outcome. Clin J Am Soc Nephrol 9:46–53

12. Ravindran A, Fervenza FC, Smith RJH, De Vriese AS, Sethi S (2018) C3 glomerulopathy: ten years' experience at Mayo Clinic. Mayo Clin Proc 93:991–1008

13. Goodship TH, Cook HT, Fakhouri F, Fervenza FC, Fremeaux- Bacchi V, Kavanagh D, Nester CM, Noris M, Pickering MC, Rodriguez de Cordoba S, Roumenina LT, Sethi S, Smith RJ, Conference Participants (2017) Atypical hemolytic uremic syn- drome and C3 glomerulopathy: conclusions from a “Kidney dis- ease: improving global outcomes” (KDIGO) controversies confer- ence. Kidney Int 91:539–551

14. Le Quintrec M, Lapeyraque AL, Lionet A, Sellier-Leclerc AL, Delmas Y, Baudouin V, Daugas E, Decramer S, Tricot L, Cailliez M, Dubot P, Servais A, Mourey-Epron C, Pourcine F, Loirat C, Fremeaux-Bacchi V, Fakhouri F (2018) Patterns of clinical re- sponse to eculizumab in patients with C3 glomerulopathy. Am J Kidney Dis 72:84–92

15. Habib R, Kleinknecht C, Gubler MC, Levy M (1973) Idiopathic membranoproliferative glomerulonephritis in children. Report of 105 cases. Clin Nephrol 1:194–214

16. Flynn JT, Kaelber DC, Baker-Smith CM, Blowey D, Carroll AE, Daniels SR, de Ferranti SD, Dionne JM, Falkner B, Flinn SK, Gidding SS, Goodwin C, Leu MG, Powers ME, Rea C, Samuels J, Simasek M, Thaker VV, Urbina EM, Subcommittee on Screening and Management of High Blood Pressure in Children (2017) Clinical practice guideline for screening and management of high blood pressure in children and adolescents. Pediatrics 140: e20171904

17. Schwartz GJ, Munoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, Furth SL (2009) New equations to estimate GFR in children with CKD. J Am Soc Nephrol 20:629–637

18. Singh G, Singh SK, Nalwa A, Singh L, Pradeep I, Barwad A, Sinha A, Hari P, Bagga A, Bagchi S, Agarwal SK, Dinda AK (2019) Glomerular C4d staining does not exclude a C3 glomerulopathy. Kidney Int Rep 4:698–709

19. Caltik Yilmaz A, Aydog O, Akyuz SG, Bulbul M, Demircin G, Oner A (2014) The relation between treatment and prognosis of childhood membranoproliferative glomerulonephritis. Ren Fail 36:1221–1225

20. Nicolas C, Vuiblet V, Baudouin V, Macher MA, Vrillon I, Biebuyck- GougeN,DehennaultM,Gie S,MorinD,NivetH,Nobili F,Ulinski T, Ranchin B, Marinozzi MC, Ngo S, Fremeaux-Bacchi V, Pietrement C (2014) C3 nephritic factor associated with C3 glomerulopathy in chil- dren. Pediatr Nephrol 29:85–94

21. Nasr SH, Valeri AM, Appel GB, Sherwinter J, Stokes MB, Said SM, Markowitz GS, D'Agati VD (2009) Dense deposit disease: clinicopathologic study of 32 pediatric and adult patients. Clin J Am Soc Nephrol 4:22–32

22. Iatropoulos P, Daina E, Curreri M, Piras R, Valoti E, Mele C, Bresin E, Gamba S, Alberti M, Breno M, Perna A, Bettoni S, Sabadini E, Murer L, Vivarelli M, Noris M, Remuzzi G, Registry of Membranoproliferative Glomerulonephritis CG, Nastasi (2018) Cluster analysis identifies distinct pathogenetic patterns in C3 g l o m e r u l o p a t h i e s / i m m u n e c o m p l e x – m e d i a t e d membranoproliferative GN. J Am Soc Nephrol 29:283–294

23. Yazilitas F, Kargin Cakici E, Kurt Sukur ED, Can G, Gungor T, Orhan D, Bulbul M (2020) C3 glomerulopathy: experience of a pediatric nephrology center. Acta Clin Belg. https://doi.org/10. 1080/17843286.2020.1713450

24. Kawasaki Y, Kanno S, OnoA, Suzuki Y, Ohara S, SatoM, Suyama K, Hashimoto K, HosoyaM (2016) Differences in clinical findings, pathology, and outcomes between C3 glomerulonephritis and membranoproliferative glomerulonephritis. Pediatr Nephrol 31: 1091–1099

25. Okuda Y, Ishikura K, Hamada R, Harada R, Sakai T, Hamasaki Y, Hataya H, Fukuzawa R, Ogata K, Honda M (2015) Membranoproliferative glomerulonephritis and C3 glomerulone- phritis: frequency, clinical features, and outcome in children. Nephrology (Carlton) 20:286–292

26. Avasare RS, Canetta PA, Bomback AS, Marasa M, Caliskan Y, Ozluk Y, Li Y, Gharavi AG, Appel GB (2018) Mycophenolate mofetil in combination with steroids for treatment of C3 glomeru- lopathy: a case series. Clin J Am Soc Nephrol 13:406–413

27. Rabasco C, Cavero T, Roman E, Rojas-Rivera J, Olea T, Espinosa M, Cabello V, Fernandez-Juarez G, Gonzalez F, Avila A, Baltar JM, Diaz M, Alegre R, Elias S, Anton M, Frutos MA, Pobes A, BlascoM,Martin F, Bernis C,MaciasM, Barroso S, de Lorenzo A, Ariceta G, Lopez-Mendoza M, Rivas B, Lopez-Revuelta K, Campistol JM, Mendizabal S, de Cordoba SR, Praga M, Spanish Group for the Study of Glomerular Diseases (GLOSEN) (2015) Effectiveness of mycophenolate mofetil in C3 glomerulonephritis. Kidney Int 88:1153–1160

28. Caliskan Y, Torun ES, Tiryaki TO, Oruc A, Ozluk Y, Akgul SU, Temurhan S, Oztop N, Kilicaslan I, Sever MS (2017) Immunosuppressive treatment in C3 glomerulopathy: is it really effective? Am J Nephrol 46:96–107

29. Bagheri N, Nemati E, Rahbar K, Nobakht A, Einollahi B, Taheri S (2008) Cyclosporine in the treatment of membranoproliferative glo- merulonephritis. Arch Iran Med 11:26–29

30. Faedda R, Satta A, Tanda F, Pirisi M, Bartoli E (1994) Immunosuppressive treatment of membranoproliferative glomeru- lonephritis. Nephron 67:59–65

31. Cattran DC, Cardella CJ, Roscoe JM, Charron RC, Rance PC, Ritchie SM, Corey PN (1985) Results of a controlled drug trial in membranoproliferative glomerulonephritis. Kidney Int 27:436–441

32. Kojc N, Bahovec A, Levart TK (2019) C3 glomerulopathy in chil- dren: is there still a place for anti-cellular immunosuppression? Nephrology (Carlton) 24:188–194

33. Viswanathan GK, Nada R, Kumar A, Ramachandran R, Rayat CS, Jha V, Sakhuja V, Joshi K (2015) Clinico-pathologic spectrum of C3 glomerulopathy—an Indian experience. Diagn Pathol 10:6

34. Tarshish P, Bernstein J, Tobin JN, Edelmann CM Jr (1992) Treatment of mesangiocapillary glomerulonephritis with alternate-day prednisone—a report of the International Study of Kidney Disease in Children. Pediatr Nephrol 6:123–130

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ORIGINAL ARTICLE

Crescentic glomerulonephritis in children

Ulrike Mayer1 & Jessica Schmitz2 & Jan Hinrich Bräsen2 & Lars Pape1

Received: 12 October 2019 /Revised: 21 November 2019 /Accepted: 25 November 2019 # The Author(s) 2020

Abstract Background To date, there is insufficient knowledge about crescentic glomerulonephritis (cGN), the most frequent immunologic cause of acute kidney injury in children. Methods Over a period of 16 years, we retrospectively analyzed kidney biopsy results, the clinical course, and laboratory data in 60 pediatric patients diagnosed with cGN. Results The underlying diseases were immune complex GN (n = 45/60, 75%), including IgA nephropathy (n = 19/45, 42%), lupus nephritis (n = 10/45, 22%), Henoch-Schoenlein purpura nephritis (n = 7/45, 16%) and post-infectious GN (n = 7/45, 16%), ANCA-associated pauci-immune GN (n = 10/60, 17%), and anti-glomerular basement-membrane GN (n = 1/60, 2%). Patient CKD stages at time of diagnosis and at a median of 362 days (range 237–425) were CKD I: n = 13/n = 29, CKD II: n = 15/n = 9, CKD III: n = 16/n = 7, CKD IV: n = 3/n = 3, CKD V: n = 13/n = 5. Course of cGN was different according to class of cGN, duration of disease from first clinical signs to diagnosis of cGN by biopsy, percentage of crescentic glomeruli, amount of tubular atrophy/interstitial fibrosis and necrosis on renal biopsy, gender, age, nephrotic syndrome, arterial hypertension, dialysis at presentation, and relapse. Forty-eight/60 children were treated with ≥ 5 (methyl-) prednisolone pulses and 53 patients received oral prednis(ol)one in combination with mycophenolate mofetil (n = 20), cyclosporine A (n = 20), and/or cyclophosphamide (n = 6), rituximab (n = 5), azathioprine (n = 2), tacrolimus (n = 1), and plasmapheresis/immunoadsorption (n = 5). Conclusions The treatment success of cGN is dependent on early diagnosis and aggressive therapy, as well as on the percentage of crescentic glomeruli on renal biopsy and on the underlying type of cGN. CsA and MMF seem to be effective alternatives to cyclophosphamide.

Keywords Glomerulonephritis . Acute kidney injury . Prednisolone . Dialysis . Kidney biopsy . Children

Introduction

Crescentic glomerulonephritis (cGN) is not a single disease entity but a pattern that can occur in a variety of glomerular diseases [1]. Caused by different pathomechanisms lesions and necrosis develop in glomerular capillaries in the case of systemic and kidney-restricted diseases. Ruptures of the glo- merular basement membrane lead to fibrin exudation as well as cellular and humoral components of inflammation in the Bowman’s capsule. Parietal epithelial cells proliferate. This

leads to the so-called extracapillary proliferations that narrow the remaining space in the capsule and appear as crescents on renal biopsy [1–3] (Fig. 1). Because of the ongoing inflamma- tion, this process can lead to renal scarring. cGN is frequently associated with fast deterioration of kidney function and there- fore often referred to as rapidly progressive glomerulonephri- tis (RPGN) [4]. Depending on the clinical context, the wide- spread definition of “crescentic GN” as a process involving > 50% of glomeruli [3] can be misleading, as there may be major diagnostic and clinical significance in the finding of even one fresh crescent.

The clinical course of cGN is dependent on the severity of the histopathological findings, i.e., by the percentage of glo- meruli with crescents [5], but in adult series also on the un- derlying disease. In proliferative lupus nephritis, for example, the outcome is much worse than in post-streptococcal GN even if the percentage of glomeruli with crescents is similar at 25% [1]. An additional parameter for the prognosis is the severity of acute kidney injury at the time of diagnosis [2, 5].

* Lars Pape [email protected]

1 Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

2 Department of Pathology, Nephropathology Unit, Hannover Medical School, Hannover, Germany

https://doi.org/10.1007/s00467-019-04436-y Pediatric Nephrology (2020) 35:829–842

/Published online: 12 2020February

The primary signs of cGN are hematuria, albuminuria, ne- phritic sediment, decreasing glomerular filtration rate (GFR), and oliguria. These renal symptoms might be associated with other organ manifestations in the case of an underlying sys- temic disease [3]. cGN is classified by different subtypes: Type I: anti-glomerular basement-membrane (anti-GBM) dis- ease; Type II: GN caused by deposition of immune complexes (i.e., in IgA nephropathy (IgAN), lupus nephritis, post- infectious GN (PIGN), Henoch-Schoenlein purpura nephritis (HSPN)) and Type III: pauci-immune GN (i.e., caused by ANCA-vasculitis) [3]. In contrast to older patients in whom Type III predominates, Type II is the most common disease in the pediatric setting. Early induction therapy with intravenous (IV) steroids and/or cyclophosphamide is recommended in cGN, and in adults, plasmapheresis is sometimes used in ad- dition [2]. In children, no such recommendations exist and adult guidelines are therefore used. However, the use of cy- clophosphamide is declining, especially in children, because of long-term side effects such as infertility and carcinogenic- ity. Therefore, newer immunosuppressants such as cyclospor- ine A (CsA), mycophenolate mofetil (MMF), or rituximab have been evaluated [3]. Despite these general recommenda- tions, the underlying disease has to be treated according to the disease-specific guidelines. As the 2012 Kidney Disease: Improving Global Outcomes (KIDGO) Guidelines are partly outdated and not child-specific, other published pediatric con- sensuses have to be followed.

Unfortunately, to date, only one publication on the clinical course and treatment of cGN has been published, nearly 30 years ago [6]. A more recent review of newer treatments and outcomes is therefore warranted. Consequently, we assessed the clinical course and morphological parameters of pediatric patients with biopsy-proven cGN in our center in order to determine diagnostic parameters for development of estimated glomerular filtration rate (eGFR) and dialysis-free survival.

Patients and methods

Eight hundred eighty kidney biopsies performed in native and transplanted kidneys of children at Hannover Medical School between 1999 and 2015 were investigated, with crescents de- tected in 61/808 patients. Disease duration was defined as the time between first symptoms of the underlying disease and diagnoses of cGN on renal biopsy. For definition of crescents (cellular, fibrocellular and fibrous), the Oxford IgA classifica- tion was used [7]. A crescent was defined as extracapillary proliferation of more than two cell layers of any size (regard- ing the glomerular circumference); a cellular crescent was defined as > 50% of the proliferation occupied by cells, a fibrocellular crescent by < 50% of the lesion occupied by cells, and < 90% by matrix. Fibrous crescents (defined as > 90% of the lesion occupied by matrix) were not taken into

a

c d

b

Fig. 1 Histology of pediatric crescentic glomerulonephritis (cGN): a Granulomatosis with polyangiitis (GPA), female 16 years, arrow depicts cellular crescent. b ANCA-negative pauci-immune glomerulonephritis (GN), male 7 years; arrow points out cellular crescent and arrowhead fibrinoid necrosis. c Lupus nephritis revealing mesangiocapillary (arrowhead) and crescentic (arrow) proliferation (insert illustrates positive

IgG immunohistochemistry (brown, DAB)), female 16 years. d Mesangioproliferative IgA nephropathy (insert shows glomerular positiv- ity for IgA (brown, DAB), male 12 years. a, d H&E, b, c Jones methe- namine counterstained with H&E. Bars represent 50 μm in a, b, and 100 μm in c, d.

Pediatr Nephrol (2020) 35:829–842830

account. Each patient with a minimum of one crescent in a biopsy was included in this analysis. A median of 20 glo- meruli per biopsy (range 5 to 107) was evaluated. One 17- year-old patient was excluded because no follow-up data were available as he was treated in an adult nephrology unit, leaving 60 patients for analyses. All 60 kidney biop- sies were re-evaluated by the same experienced nephropathologist (JHB) for this work using light, immu- nohistochemical, and electron microscopy. The pathologist was blinded to the patient’s data 12-month outcome. Clinical signs of GN, such as macrohematuria, edema, oliguria, arterial hypertension (defined by the Kinder- und Jugendgesundheitssurvey [Health Interview and Examination Survey for Children and Adolescents] (KiGGS) criteria [8]), non-renal signs and demographic parameters including age, gender, and duration of symp- toms at presentation, were evaluated. Renal volume was measured by ultrasound (Ellipsoid formula) at time of di- agnosis. At time of disease onset and at 1 week and after 1, 3, 6, and 12 months, the following laboratory values were documented: serum levels for creatinine, urea, albumin, electrolytes, hemoglobin, WBC, and eGFR as determined by the 2009 Schwartz bedside formula [9]. Arterial hyper- tension was defined as office blood pressures above the gender- and height-matched 95th percentile. End-stage re- nal disease (ESRD) was defined as need for renal- replacement therapy (dialysis, transplantation).

At time of diagnosis (time of renal biopsy), the following examinations and immunological parameters were deter- mined: p-ANCA, c-ANCA, MPO-antibody (Ab) PR3-Ab,

ANA, double-stranded-DNA Ab (anti-DNS), C3, C4, anti- streptolysin, IgA serum-level and glomerular basement- membrane Ab (anti-GMB) (Table 1(a)). As data evaluation was performed retrospectively, time ranges were used instead of exact time points and data was not available for each patient at each timepoint (Table 1(b)).

Data were documented using Microsoft Excel 365 (Microsoft Cooperation, Seattle, WA, USA). Time of disease duration was defined as the time between first clinical symp- toms and kidney biopsy. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Exploratory data analyses were primarily performed. All data was negatively tested for normal distribution. Therefore, me- dian values between different groups were compared using the Mann-Whitney U test for pre-defined subgroups as gender, age, nephrotic syndrome, and arterial hypertension. Paired data was compared by Wilcoxon signed-rank test. Logistic regression analyses were performed to evaluate the relation- ship between one dependent binary variable and one or more nominal or ordinal independent variables. Kaplan Meier anal- yses were done to determine survival. p < 0.05 was considered as statistically significant. We have not based the analyses on the three categories of cGN, as there was only one patient in group I and as we could observe large differences between the underlying diagnoses in groups II and III in relation to outcome.

All patients have agreed with their hospital treatment con- tract that their data can be used for research in anonymized matter. The Ethics Committee of Hannover Medical School has agreed to this policy.

Table 1 Clinical tests performed and time points of evaluation (a)

Laboratory chemical tests

Serological investigations: Creatinine, urea, albumin, protein

Immunological investigations: P-ANCA, c-ANCA, MPO-Antibody (Ab) PR3-Ab, ANA, double-stranded-DNA Ab, C3, C4, anti-streptolysin, IgA serum-level and glomerular basement-membrane Ab.

Urine analysis: Red blood cells and protein in urinary dipstick investigation, creatinine and albumin in spot urine.

Clinical investigations

Gender, age, weight, body length, blood pressure, disease duration, edema, oliguria, macrohematuria, treatment, relapse of disease during observation time..

Renal ultrasound

Measurement of kidney volume.

Renal biopsy

Light, immunohistochemical and electron microscopy.

(b)

Time point T1 T2 T3 T4 T5 T6

Median time after kidney biopsy [days] 0 7 38 96 187 362

Range [days] 4–15 27–57 74–135 140–255 237–425

N 60 54 54 55 52 53

Pediatr Nephrol (2020) 35:829–842 831

Results

The 60 patients (median age 13 years, range 3–18, 31 male) could be subclassified into three groups of cGN and, further, by the underlying disease as demonstrated in the flow chart in Table 2 (which also gives the number of patients with data available 1 year after diagnosis). Interestingly, there was no gender difference overall. Figure 2 a shows the number of newly diagnosed diseases associated with cGN within the time period of the study. eGFR at time of diagnosis and courses of s-creatinine (serum-creatinine) in the disease groups are shown in Fig. 2b, c.

Glomerular filtration rate

The median eGFR increased from 55 (range 4–161) from time of diagnosis to 92 ml/min/1.73m2 (range 5–175), p < 0.001, 1 year later, with differences in the three subgroups.

The patient with cGN type I presented with terminal renal failure which did not significantly improve (eGFR 8 to 14 ml/ min/1.73m2 at presentation and 1 year later, respectively). In children with cGN type II, median eGFR increased from 65 ml/min/1.73m2 (range 9–161) to 100 ml/min/1.73m2

(range 5–175), p < 0.001. In patients with cGN type III, me- dian eGFR increased from 28 ml/min/1.73m2 (range 8–94) to 60 ml/min/1.73m2 (range 37–113), p = 0.013. Patients with a renal disease not classifiable by clinical and histopathological techniques had an initial median eGFR of 9 ml/min/1.73m2

(range 6–46) and a final median eGFR of 10 ml/min/1.73 m2

(range 5–54), p > 0.999 (Fig. 3a, b). Patients with a fulminant progressive course of disease,

defined as less than 30 days between time of first clinical symptoms and kidney biopsy (short disease duration) experi- enced a fast deterioration of kidney function (median eGFR ≤ 30 days 20 ml/min/1.73 m2 [range 8–115] and > 30 days 64 ml/min/1.73 m2 [range 6–161], p = 0.001) (Fig. 2d). They also presented with the highest urea values (median urea ≤ 30 days 20 mmol/l [range 4–40] and > 30 days 8 mmol/l [range 3–40], p < 0.001). GFR increased significantly during the observation time only in children with a disease course > 30 days (median eGFR disease course ≤ 30 days 57 ml/min/ 1.73m2 [range 5–151], p = 0.07 and disease course >30 days 94 ml/min/1.73 m2 [range 6–175], p = 0.003).

Disease duration

The median duration time of the underlying disease before cGN was diagnosed in renal biopsy and therapy started was 60 days (range 3–1806). Patients with ANCA-negative pauci- immune GN demonstrated the shortest periods between diag- nosis and therapy (3 to 5 days) as did the patients with micro- scopic polyangiitis and renal-limited vasculitis (MPA/RLV), PIGN, and dense-deposit disease (DDD). Patients with IgAN presented with a large variety in time between first symptoms and when biopsy was performed (range 22–1806 days) (Fig. 2e).

Table 2 Subtyping and number of follow up of the patients with crescentic glomerulonephritis (cGN)

Median observation time 362 days (R 237-425)

n = 53

ANCA neg. GN

n = 2 m

Age: 8 and 17

Anti-GBM GN

n = 1

Unknown

n = 4

MPA/RLV

n = 3; 1 child was kidney transplantated

DDD

n = 1 m

Age: 4

IgAN

n = 16

PSHN

n = 6

LN

n = 10

DDD

n = 1

MWS

n = 1

ANCA neg. GN

n = 2

GPA

n = 4

Crescentic Glomerulonephritis At time of diagnosis: n = 60 (29 f, 31m)

Median age 13 years (R 3–18)

Anti-GBM GN

n = 1 m

Age: 17

LN

n = 10 (9 f, 1 m)

Median age: 13 (R 10-17)

IgAN

n = 19 (5 f, 14 m)

Median age: 13 (R 6–17)

PSHN

n = 7 (3 f, 4 m)

Median age: 15 (R 7-18)

PIGN

n = 7 (3 f, 4 m)

Median age: 7 (R 3–12)

GPA

n = 4 f

Median age: 11 (R 9-17)

MPA/RLV

n = 4 (3 f, 1 m)

Median age: 15 (R 9–17)

MWS

n = 1 f

Age: 9

Unknown

n = 4 (1 f, 3 m)

Median age: 13 (R 7-17)

Undetermined

n = 4 (1 f , 3 m)

PIGN

n = 5

Type II

n = 45 (21 f, 24 m)

Type I

n = 1 m

Type III

n = 10 (7 f, 3 m)

DDD dense-deposit disease, GBM glomerular basement-membrane, GPA granulomatosis with polyangiitis, IgAN IgA nephropathy, LN lupus nephritis, MPA/RLV microscopic polyangiitis and renal-limited vasculitis, MWS Muckle-Wells syndrome, PIGN post-infectious glomerulonephritis, PSHN Henoch-Schoenlein purpura nephritis

Pediatr Nephrol (2020) 35:829–842832

Dialysis

Twelve children received initial dialysis (Table 3), with five remaining on dialysis during the observation period, one of whom received a kidney transplant. One patient required di- alysis during the observation time without the need of dialysis at presentation. The need for primary dialysis was associated with a significantly worse outcome of kidney function: medi- an eGFR primary dialysis 16 ml/min/1.73 m2 (range 5–134) versus no primary dialysis 93ml/min/1.73 m2 (range 21–175), p < 0.001 (Fig. 4a).

Clinical factors

There was no difference between initial eGFR between chil- dren > 12 years and younger patients. However, gain of func- tion (eGFR) was lower in the older children: median GFR > 12 years 72 ml/min/1.73 m2 (range 6–151) compared to < 12 years 107 ml/min/1.73m2 (range 5–175), p = 0.020 (Fig. 4b).

Male gender was associated with worse outcome despite gender-independent eGFR at timepoint of diagnosis: median eGFR at last visit was 66 ml/min/1.73m2 (range 5–175) in

Fig. 2 Number of crescentic glomerulonephritis (cGN) diagnosis per year and subclassification (a). Initial estimated glomerular filtration rate (eGFR) in the subgroups (b). Course of s-creatinine over the observation time (c). Initial eGFR depending on time of disease duration (d). Time of disease duration in the subgroups (e). Abbreviations: DDD, dense-deposit

disease; GBM, glomerular basement-membrane; GPA, granulomatosis with polyangiitis; IgAN, IgA nephropathy; LN, lupus nephritis; MPA/ RLV, microscopic polyangiitis and renal-limited vasculitis; MWS, Muckle-Wells Syndrome; PIGN, post-infectious glomerulonephritis; PSHN, Henoch-Schoenlein purpura nephritis

Pediatr Nephrol (2020) 35:829–842 833

19 99 20

00 20

01 20

02 20

03 20

04 20

05 20

06 20

07 20

08 20

09 20

10 20

11 20

12 20

13 20

14 20

15 0 1 2 3 4 5 6 7 8 9

10

Year

N um

be r o

f p at

ie nt

s (n

=6 0)

a

0 – 15

16 –

30

31 –

91

92 –

36 5

> 3 65

0 2 4 6 8

10 12 14 16 18 20

N um

be r o

f p at

ie nt

s (n

= 60

)

Time of disease duration [days]

e

0 4

– 1 5

27 –

57

74 –

13 6

14 0 –

25 6

23 7 –

42 5

100

20

250

500 750

50

1103

S -C

re at

in in

e [µ

m ol

/l]

Time points of evaluation after diagnosis [days]

c

< 15

15 –

29

30 –

59

60 –

89 >

90 0 2 4 6 8

10 12 14 16 18 20

Initial eGFR [ml/min/1.73 m 2]

N um

be r

of p

at ie

nt s

(n =6

0)

b

0 – 1

5

16 –

30

31 –

91

92 – 3

65 >

36 5

0

25

50

75

100

125

150

175

In iti

al e

G F

R [

m l/m

in /1

.7 3m

2 ]

Time of disease duration [days]

d p=0.001

Anti-GBM GN IgAN PSHN

PIGN LN DDD

MWS

ANCA-neg. GN

GPA MPA/RLV

Main diseases highlighted:

Undetermined

boys and 100 ml/min/1.73 m2 (range 37–151) in girls, respec- tively p = 0.039 (Fig. 4b).

We also observed that initially nephrotic children showed reduced recovery of kidney function compared to the remain- der of the cohort although the initial eGFR of both groups was not significantly different: median eGFR of initially nephrotic and initially non-nephrotic children at last visit was 63 ml/ min/1.73 m2 (range 5–151) and 92 ml/min/1.73 m2 (range 6–151), respectively, p = 0.045 (Fig. 4c).

Forty-five percent of patients had arterial hypertension at the time of diagnosis. Arterial hypertension at the time of diagnosis was another independent risk factor of having poorer kidney function after 1 year: median eGFR of patients having initial arterial hypertension was 62 ml/min/1.73 m2

(range 5–128) compared to children with no initial arterial hypertension, 100 ml/min/1.73 m2 (range 5–175), p = 0.006 (Fig. 4c).

Recurrence

Eleven patients developed a recurrence of underlying disease during the observation period. The increase of eGFR was significantly lower in this group with a me- dian eGFR of 63 ml/min/1.73m2 (range 25–163) com- pared to the non-relapsing children 92 ml/min/1.73m2

(range 5–175), p = 0.298 (Fig. 4a).

Clinical signs

Dialysis-free survival of the native kidney was different in Kaplan Meier analysis between those patients with a short (≤ 30 days) or longer period (> 30 days) of time from first documented symptom to biopsy (p = 0.006, Fig. 4d), between the different underlying diagnoses (p < 0.001, Fig. 4e), as well

as between patients with greater or fewer than 50% of glomer- uli with crescents (p = 0.002, Fig. 4f). At time of diagnosis, all children presented with hematuria, 55%with macrohematuria. Fifty/52 patients had proteinuria, with 58% being nephrotic. The albumin-to-creatinine-ratio (ACR) varied significantly between the patients regardless of the underlying disease (Table 3) and decreased from 266 g/mol (range 8–6541) at time of diagnosis to 8 g/mol (range 1–679), p < 0.001, 1 year later. At time of diagnosis, the patients with an eGFR less than 90 ml/min/1.73 m2 (median ACR 277 g/mol, range 15–6541) had a higher ACR than those with an eGFR > 90 ml/min/ 1.73 m2 (median ACR 83 g/mol, range 8–601), p = 0.045.

Serum IgA was determined in 47 of the patients. Twenty- nine children presented with normal IgA values, 12 of them with IgAN and two with PSHN. From 18 children with in- creased IgA, two suffered from HSPN, six from IgAN and ten from other underlying diseases. Complement factor C3 was normal in 68% of cases. All children with lupus GN and DDD had decreased C3. P-ANCA and c-ANCA could be detected in ten and two children, respectively.

ANAs were negative in 27 patients and positive in all lupus nephritis children, whereas all lupus patients were also posi- tive for anti-DNS. Anti-GBM-Ab was only positive in the single patient with anti-GBM GN. The anti-streptolysin titer was positive in 4/6 children with PIGN and in 11 children with other underlying diseases.

At time of diagnosis, renal ultrasound was only document- ed or available in 33/60 children. This is possibly due to the fact that many patients were not primarily seen at our center but transferred. Seventy percent presented with an elevated (> 95th percentile) and 18% with a borderline elevated (90-95th percentile) renal volume as compared to weight-matched nor- mal values. Nephrotic range proteinuria was defined as a uri- nary albumin/creatinine ratio > 220 mg/mmol in spot urine.

All

An ti-G

BM GN IgA

N PS HN

PIG N LN DD

D MW

S

AN CA -ne

g. GN GP

A

MP A/R

LV

Un de ter mi ne d

25

50

75

100

125

150

175

15

eG FR

[m l/m

in /1 .7 3m

2 ]

a

All

An ti-G

BM GN IgA

N PS HN

PIG N LN DD

D MW

S

AN CA -ne

g. GN GP

A

MP A/R

LV

Un de ter mi ne d

25

50

75

100

125

150

175

15

eG FR

[m l/m

in /1 .7 3 m

2 ]

b p<0.001

Fig. 3 Course of estimated glomerular filtration rate (eGFR) during ob- servation period dependent on underlying disease at time of diagnosis (a) and 1 year later (b). Abbreviations: DDD, dense-deposit disease; GBM GN, glomerular basement-membrane glomerulonephritis; GPA,

granulomatosis with polyangiitis; IgAN, IgA nephropathy; LN, lupus nephritis; MPA/RLV, microscopic polyangiitis and renal-limited vasculi- tis; MWS,Muckle-Wells Syndrome; PIGN, post-infectious glomerulone- phritis; PSHN, Henoch-Schoenlein purpura nephritis

Pediatr Nephrol (2020) 35:829–842834

Ta bl e 3

C lin

ic al da ta of

th e to ta lc oh or t

P at .I D

eG FR

[m l/m

in /

1. 73

m 2 ]

U ri na ry

di ps tic k

an al ys is

A lb um

in -t o-

cr ea tin

in e

ra tio

[g /m

ol ]

G lo m er ul i

w ith

cr es ce nt s

[% of

al lg lo m er ul i]

D is ea se

du ra tio

n [d ay s]

M et hy l- /p re dn is ol on e

pu ls e th er ap y

(n :i ni tia l+

du ri ng

th e

co ur se

of ob se rv at io n)

P re dn is o- (l o) ne

(n :i ni tia l+

du ri ng

th e co ur se

of ob se rv at io n)

A dd iti on al

im m un os up pr es si ve

th er ap y (i ni tia l+

du ri ng

th e co ur se

of ob se rv at io n)

H em

od ia ly si s (H

D ),

pe ri to ne al di al ys is (P D ),

pl as m a ex ch an ge

(P E ),

im m un oa ds or pt io n (I A )

(i ni tia l+

du ri ng

th e

co ur se

of ob se rv at io n)

Fi rs t

vi si t

L as t

vi si t

R B C

[/ μ l]

Pr ot ei n

[m g/ dl ]

F ir st

vi si t

L as t

vi si t

A nt i- G B M

gl om

er ul on ep hr iti s

1 8

H D

14 25 0

N eg .

15 A nu ri a

10 0

48 Y es

(5 )

Y es

(c a. 50 )

C Y C 14

da ys

p. o. + 1×

R T X

H D ,P

E :3

+ 6

Ig A ne ph ro pa th y

10 38

58 25 0

50 0

39 2

4 42

41 Y es

(6 + 2)

Y es

(2 19 )

M M F

N o

13 47

95 25 0

10 0

21 1

23 89

70 Y es

(6 )

Y es

(4 2)

N o

N o

14 11 5

15 1

20 0

30 0

59 9

1 42

12 Y es

(6 )

Y es

(7 1)

C sA

N o

18 68

a 4 9

N B D

N B D

70 9

a 3 13

17 Y es

(6 )

Y es

(> 18 6)

M M F + C sA

on to p

N o

25 91

95 20 0

10 0

86 3

22 42

N o

Y es

(1 84 )

+ C sA

N o

27 10 0

15 1

20 0

> 30 0

80 18

8 24 4

N o

N o

N o

N o

28 71

13 0

20 0

> 30 0

25 16

10 17 66

Y es

(6 )

Y es

(5 6)

N o

N o

30 11

14 0

25 0

N eg .

N B D

1 8

42 3

Y es

(6 )

Y es

(6 )

N o

N o

36 66

64 20 0

> 30 0

52 1

14 50

18 06

Y es

(6 )

N o

M M F

N o

40 47

67 20 0

> 30 0

16 2

7 36 2

Y es

(6 )

Y es

(2 78 )

M M F

N o

44 10 4

N B D

80 > 30 0

60 1

N B D

6 19 4

Y es

(6 )

Y es

(u nk no w n)

C sA

N o

47 29

92 20 0

> 30 0

77 4

31 30 9

Y es

(5 )

Y es

(5 6)

C sA

N o

50 10 7

12 3

25 0

30 67

23 20

97 N o

N o

N o

N o

52 63

a 6 6

20 0

> 30 0

61 1

a 3 77

28 33

Y es

(6 )

Y es

(u nk no w n)

C sA

N o

59 79

92 25 0

10 0

84 1

18 34

Y es

(6 )

Y es

(6 9)

C sA

N o

63 13 5

12 7

10 10 0

N B D

9 4

11 6

N o

Y es

(1 32 )

N o

N o

67 61

21 20 0

> 30 0

27 6

67 9

25 13 19

Y es

(6 + 5 be fo re

N B x)

Y es

(4 3)

C sA

(f or

3 ye ar s) + M M F

on to p

N o

68 62

56 50

50 0

91 8

81 71

20 3

Y es

(6 )

Y es

(5 5)

TA C (L iv er -T x)

N o

69 10 3

13 6

20 0

> 30 0

36 7

14 4

6 30 5

N o

N o

N o

N o

M ed ia n ra ng e

68 11 –1 35

95 21 –1 51

20 0

> 30 0

27 6

12 20 4– 89

19 4

12 –1 80 6

Y: 14 ; N : 5

Y: 15 ; N : 4

C sA : 6 + 2;

M M F :4

+ 1;

TA C : 1;

N on e:

7

N o

H en oc h- Sc ho en le in

Pu rp ur a ne ph ri tis

24 55

61 20 0

> 30 0

21 93

1 10

22 Y es

(6 )

Y es

(1 22 )

C sA

W ith

re cu rr en ce 😛

E :5

an d H D :6

29 92

N B D

> 20 0

10 0

13 6

N B D

12 83 6

Y es

(5 be fo re

N B x)

Y es

(u nk no w n)

C sA

N o

33 74

10 8

20 0

> 30 0

27 7

15 42

42 Y es

(3 )

Y es

(1 13 )

N o

N o

48 70

63 N B D

N B D

21 4

8 31

36 Y es

(6 + 6)

Y es

(u nk no w n)

C Y C 3 M o p. o. + C sA

N o

53 73

17 5

20 0

> 30 0

57 8

N B D

33 70

Y es

(3 )

Y es

(1 08 )

N o

N o

57 14

17 20 0

> 30 0

32 8

7 83

78 4

Y es

(6 )

Y es

(8 5)

C sA

H D fo r 2 m on th s

62 47

72 20 0

10 0

16 7

5 65

11 7

Y es

(6 )

Y es

(1 07 )

C sA

N o

M ed ia n ra ng e

70 14 –9 2

68 17 –1 75

20 0

> 30 0

27 7

7 33 10 –8 3

70 22 –8 36

Y: 7

Y: 7

C sA : 4 + 1,

C YC

:1 N on e:

2

P E :1

; H D :2

Po st -i nf ec tio

us gl om

er ul on ep hr iti s

8 52

12 9

20 0

> 30 0

24 6

4 13

8 N o

N o

N o

N o

11 49

94 20 0

> 30 0

24 2

2 50

10 1

N o

Y es

(1 49 )

W ith

re cu rr en ce C sA

N o

Pediatr Nephrol (2020) 35:829–842 835

T ab

le 3

(c on tin

ue d)

P at .I D

eG FR

[m l/m

in /

1. 73

m 2 ]

U ri na ry

di ps tic k

an al ys is

A lb um

in -t o-

cr ea tin

in e

ra tio

[g /m

ol ]

G lo m er ul i

w ith

cr es ce nt s

[% of

al lg lo m er ul i]

D is ea se

du ra tio

n [d ay s]

M et hy l- /p re dn is ol on e

pu ls e th er ap y

(n :i ni tia l+

du ri ng

th e

co ur se

of ob se rv at io n)

P re dn is o- (l o) ne

(n :i ni tia l+

du ri ng

th e co ur se

of ob se rv at io n)

A dd iti on al

im m un os up pr es si ve

th er ap y (i ni tia l+

du ri ng

th e co ur se

of ob se rv at io n)

H em

od ia ly si s (H

D ),

pe ri to ne al di al ys is (P D ),

pl as m a ex ch an ge

(P E ),

im m un oa ds or pt io n (I A )

(i ni tia l+

du ri ng

th e

co ur se

of ob se rv at io n)

Fi rs t

vi si t

L as t

vi si t

R B C

[/ μ l]

Pr ot ei n

[m g/ dl ]

F ir st

vi si t

L as t

vi si t

16 9

13 4

N B D

N B D

88 2

74 9

Y es

(6 )

Y es

(6 0)

M M F

PD 9 d

22 33

12 2

20 0

> 30 0

36 1

3 50

25 Y es

(3 + 2)

Y es

(7 8)

N o

N o

23 59

12 3

20 0

> 30 0

73 6

12 8

67 32

Y es

(6 + 11 –1 3)

Y es

(u nk no w n)

N o

N o

32 24

92 50

50 0

96 9

N B D

21 20

Y es

(6 )

Y es

(u nk no w n)

N o

PD 4 d

58 11

PD 5

25 0

50 0

N B D

A nu ri a

10 0

17 Y es

(5 + 6)

Y es

(6 3)

N o

PD

M ed ia n ra ng e

33 9– 59

12 2

5– 12 9

20 0

> 30 0

24 6

3 50 13 –1 00

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Y: 5;

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ne ph ri tis

7 65

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(6 )

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26 61

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(+ 6)

Y es

(2 48 )

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34 91

12 8

80 > 30 0

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N o

41 9

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77 97

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46 82

91 80

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49 16 1

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66 38

10 6

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91 5

40 63

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C Y C 6x

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M ed ia n ra ng e

74 9– 16 1

10 0

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20 0

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28 2

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M M F :5

; C YC

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N o

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(u nk no w n)

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Pediatr Nephrol (2020) 35:829–842836

T ab

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(c on tin

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G lo m er ul i

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D is ea se

du ra tio

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M et hy l- /p re dn is ol on e

pu ls e th er ap y

(n :i ni tia l+

du ri ng

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co ur se

of ob se rv at io n)

P re dn is o- (l o) ne

(n :i ni tia l+

du ri ng

th e co ur se

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A dd iti on al

im m un os up pr es si ve

th er ap y (i ni tia l+

du ri ng

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H em

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pe ri to ne al di al ys is (P D ),

pl as m a ex ch an ge

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vi si t

L as t

vi si t

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Pr ot ei n

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vi si t

L as t

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M ed ia n ra ng e

32 8– 94

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95 63 7– 77

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> 30 0

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no 1 ye ar

da ta w as

av ai la bl e

Pediatr Nephrol (2020) 35:829–842 837

Pathology

Table 3 shows the results of the proportion of glomeruli with crescents (extracapillary proliferation) relating to the number of non-sclerotic glomeruli (median 38%, range 4 to 100). Fibrocellular crescents were found in only 21 patients, and they accounted for 19% of the total amount (80 fibrocellular versus 333 cellular crescents). In 26/60 patients, crescents were detected in ≥ 50% of the glomeruli. This criterion was fulfilled by 100% of the children with anti-GBM GN, DDD, and ANCA-negative pauci-immune GN, as well as in 75% of patients with systemic granulomatosis with polyangiitis (GPA), MPA/RLV, or unknown GN, and in 71% of children with PIGN. Only 16% of children with IgAN and 29% with HSPN had more than 50% crescents. This subgroup presented with a lower initial median eGFR (24 ml/min/1.73m2, range

6–79) compared to the patients with crescents in < 50% of glomeruli (74 ml/min/1.73 m2, range 11–161), p < 0.001. In addition, the improvement of eGFR was better for children with crescents in > 50% of the glomeruli (24 to 62 ml/min/ 1.73m2, range 5–123) compared to the other patients (74 to 100 ml/min/1.73m2, range 21–175), p < 0.001. In this context, it is important to refer to the 12 children with extracapillary proliferations in > 80% of glomeruli, as seven of these chil- dren developed ESRD, with initial median eGFR 11 ml/min/ 1.73 m2 (range 6–79) and final eGFR 17 ml/min/1.73 m2

(range 5–113), respectively, p = 0.3875. Nevertheless, three of the patients with extracapillary proliferations in > 80% of glomeruli, who initially experienced moderately impaired kid- ney function or kidney failure, had a normalization of eGFR within the observation period. It is also noticeable in this group that median duration of disease was 25 days (range 5–

0

50

100

150

15

75

125

175

eG F

R [

m l/m

in /1

.7 3m

2 ]

firs t v

isi t

p=0.129 p=0.039 p=0.465 p=0.020

Female

Male

Younger than 12 years

Older than 12 years

las t v

isi t

firs t v

isi t

las t v

isi t

0

50

100

150

15

75

125

175

eG F

R [

m l/m

in /1

.7 3m

2 ]

p=0.622 p=0.045 p=0.624 p=0.007

Nephrotic syndrome

No nephrotic syndrome

Arterial hypertension

No arterial hypertension

firs t v

isi t

las t v

isi t

firs t v

isi t

las t v

isi t

0 4-

15 27

-57

74 -1

35

14 0-

25 5

23 7-

42 5

0

20

40

60

80

100

Time [days]

D ia

ly si

s- fre

e su

rv iv

al [%

] < 50% (n=29)

Extracapillary proliferations:

50% (n=25)

Log-rank p-value = 0.002

0

50

100

150

15

75

125

175

eG F

R [

m l/m

in /1

.7 3m

2 ] p=0.767 p = 0.298 p<0.001 p<0.001

Relapse of disease during observation time

No relapse

Dialysis at presentation

No dialysis at presentation

firs t v

isi t

las t v

isi t

firs t v

isi t

las t v

isi t

0 4-

15 27

-5 7

74 -1

35

14 0-

25 5

23 7-

42 5

0

20

40

60

80

100

Time [days]

D ia

ly si

s- fre

e su

rv iv

al [%

] cGN Typ I (n=1)

cGN Typ II (n=39)

cGN Typ III (n=10)

Undetermined (n=4)

Log-rank p-value < 0.001

Classification:

0 4-

15 27

-57

74 -1

35

14 0-

25 5

23 7-

42 5

0

20

40

60

80

100

Time [days] D

ia ly

si s-

fre e

su rv

iv al

[% ] 30 d (n=15)

> 30 d (n=39)

Disease Duration:

Log-rank p-value = 0.006

b

c

f

a

e

d

Fig. 4 Factors influencing the course of estimated glomerular filtration rate (eGFR) during observation time. Disease relapse and dialysis at first visit (a), gender and age at time of disease onset (b), and nephrotic syn- drome and arterial hypertension at time of disease onset (c). Kaplan-

Meier curves of dialysis-free survival depending on disease duration (d), the classification of crescentic glomerulonephritis (cGN) (e), and the percentage of crescents (f)

Pediatr Nephrol (2020) 35:829–842838

Table 4 Additional renal biopsy findings of patients

Median eGFR [ml/min/1.73 m2] (range)

First visit Last visit

Tubular atrophy/interstitial fibrosis < 20% (n = 50) 55 (8–161) 94 (5–175)

≥ 20% (n = 10) 56 (6–91) 54 (5–128)

p value p = 0.425 p = 0.021

Glomeruli with necrosis < 20% (n = 49) 62 (6–161) 95 (5–175)

≥ 20% (n = 11) 11 (8–70) 62 (5–113)

p value p < 0.001 p = 0.022

Tubulointerstitial inflammation < 50% (n = 41) 65 (8–161) 95 (14–175)

≥ 50% (n = 18) 24 (6–115) 58 (5–151)

p value p < 0.001 p = 0.009

Tubulointerstitial inflammation [Intensity 0–3]

0 (n = 1) 135 125

1 (n = 20) 69 (9–161) 92 (21–151)

2 (n = 28) 54 (8–115) 95 (5–175)

3 (n = 10) 26 (6–82) 66 (5–123)

RBC casts [Intensity 0–3]

0 (n = 13) 50 (6–161) 85 (5–151)

1 (n = 26) 64 (10–135) 100 (14–175)

2 (n = 9) 70 (13–115) 67 (25–130)

3 (n = 10) 11 (8–47) 92 (5–140)

Thrombi within glomeruli Yes (n = 7) 13 (8–114) 78 (5–140)

No (n = 51) 55 (6–161) 92 (5–175)

p value p = 0.486 p = 0.62

ATI severity score [Intensity 1–3]

1 (n = 7) 73 (47–161) 99 (52–175)

2 (n = 32) 65 (6–115) 100 (6–151)

3 (n = 21) 38 (8–82) 56 (5–113)

IgA [Intensity 0–3]

< 1 (n = 15) 50 (9–94) 66 (5–136)

1 (n = 13) 63 (8–115) 92 (14–175)

2 (n = 9) 59 (6–161) 99 (6–151)

3 (n = 18) 70 (11–135) 100 (56–140)

IgG [Intensity 0–3]

< 1 (n = 15) 47 (6–135) 81 (6–151)

1 (n = 23) 51 (9–115) 67 (5–175)

2 (n = 12) 68 (9–114) 104 (21–129)

3 (n = 5) 65 (47–161) 100 (92–105)

IgM [Intensity 0–3]

0 (n = 4) 32 (14–89) 59 (17–136)

1 (n = 24) 67 (9–115) 98 (5–151)

2 (n = 22) 57 (6–161) 60 (6–175)

3 (n = 4) 47 (15–61) 100 (72–100)

C1q [Intensity 0–3]

0 (n = 5) 55 (13–94) 76 (60–136)

1 (n = 15) 50 (9–115) 95 (5–151)

2 (n = 19) 55 (8–107) 60 (17–175)

3 (n = 18) 67 (6–161) 100 (6–128)

C3c [Intensity 0–3]

< 1 (n = 6) 53 (13–103) 71 (52–136)

1 (n = 10) 70 (14–135) 92 (17–127)

2 (n = 24) 64 (9–161) 92 (14–175)

3 (n = 16) 50 (6–100) 107 (5–151)

Mesangial hypercellularity overall ≤ 50% of glomeruli (n = 23) 51 (8–103) 76 (5–136)

> 50% of glomeruli (n = 32) 64 (6–161) 100 (6–175)

p value p = 0.05 p = 0.09

Pediatr Nephrol (2020) 35:829–842 839

784) shorter than in the patients with extracapillary prolifera- tions in < 80% of glomeruli. In regression analysis, the per- centage of crescents correlated negatively with the eGFR at the end of observation time (R2 = 31%, p < 0.001) with more crescents leading to a lower eGFR.

Table 4 shows the additional renal biopsy findings from patients. Tubular atrophy and interstitial fibrosis of ≥ 20% and tubulointerstitial inflammation in ≥ 50% of the tubulointerstitium, as well as necrosis in ≥ 20% of glomeruli, were associated with a worse eGFR at last visit.

Treatment

Eighty percent of patients were treated with 5–6 (methyl-)- prednisolone pulses with a median prednisolone dose of 309 mg/m2 body surface area (BSA) (range 281–513) if BSAwas < 1.67 m2. The remaining patients were treated with 500 mg per day. The median dose for methylprednisolone was 561 mg/m2 BSA (range 275–708). In addition, 53 patients were treated with oral prednis(ol)one with a median duration of 151 days (range 6–> 365). Seventy-two percent of the total cohort was additionally treated with other immunosuppres- sants: MMF (n = 20), CsA (n = 20), cyclophosphamide (n = 6), azathioprine (n = 2), and rituximab (n = 5). One liver- transplanted patient continued with tacrolimus, and the patient the Muckle-Wells syndrome was additionally treated with canakinumab. Table 3 shows the mono- or combination ther- apy immunosuppressive treatments used in association with the underlying disease. Depending on the underlying disease, four patients received therapeutic plasmapheresis or immunoadsorption at time of disease manifestation. One pa- tient with a relapse was treated with plasmapheresis as rescue therapy (Table 3).

Discussion

This analysis of pediatric patients with cGN in our cohort revealed a wide inter-individual variability in initial kidney function which was independent of the underlying disease. An early treatment with IV(methyl-)prednisolone followed by oral steroids in combination with other immunosuppres- sants was most often successful. Outcome was dependent on percentage of glomerular crescents, disease duration, and the underlying type of cGN. No patient died during the observa- tion time.

The frequency of underlying diagnoses is in accordance with the published literature [10], with the highest prevalence of immune complex crescentic GN. Regarding the percentage of crescents, the highest incidence in the total cohort was for IgAN. PIGN was most frequently associated with crescents in more than 50% of glomeruli.

Our results confirm other studies demonstrating that the percentage of glomeruli with crescents correlates with the se- verity of cGN [3, 5]. All of our seven patients who progressed to ESRD had crescents in more than 80% of the glomeruli, in most cases, a short disease duration and nearly all presented with acute kidney failure. However, even in the majority of patients with a high number of crescents, our results suggest that intensive immunosuppressive treatment has a good chance of success. However, our retrospective data analysis does not allow us to build a predictive model based on these factors. Only one patient required dialysis during the observation time. Another prognostic factor is the time of disease duration: those patients with a faster, more fulminant course of disease showed minor improvement in renal function and resulted in a higher percentage of ESRD. The term “rapid progressive glomerulo- nephritis”—though not clearly defined in the literature—can be used to describe this latter group. On the other hand, some

Table 4 (continued)

Median eGFR [ml/min/1.73 m2] (range)

First visit Last visit

Mesangial hypercellularity global ≤ 50% of glomeruli (n = 29) 52 (8–161) 85 (5–136)

> 50% of glomeruli (n = 26) 62 (6–135) 100 (6–175)

p value p = 0.27 p = 0.18

Mesangial hypercellularity segmental ≤ 50% of glomeruli (n = 53) 59 (6–135) 92 (5–175)

> 50% of glomeruli (n = 2) 92 + 161 105

Intracapillary hypercellularity Yes (n = 54) 57 (6–161) 92 (5–175)

No (n = 2) 9 + 94 92 + 85

Intracapillary hypercellularity ≤ 50% of glomeruli (n = 35) 61 (8–161) 74 (5–175)

> 50% of glomeruli (n = 21) 55 (6–115) 103 (6–151)

p value p = 0.93 p = 0.16

eGFR estimated glomerular filtration rate, RBC red blood cells, ATI acute tubular injury

Pediatr Nephrol (2020) 35:829–842840

patients (i.e., with IgA Nephropathy) had a long time between primary diagnosis of their underlying disease and detection of cGNon renal biopsy. For those patients, continuous, closemon- itoring of renal function seems to be important so that a sudden deterioration of their kidney function can be diagnosed early enough for successful intervention.

Interestingly, in addition to children with initial dialysis or disease relapse during observation, male patients, children with arterial hypertension or nephrotic syndrome at presenta- tion, and those older than 12 years had a worse outcome. Further renal damage factors, such as tubular atrophy and interstitial fibrosis, glomerular fibrinoid necrosis, or tubulointerstitial inflammation, were associated with worse kidney function outcome.

Our results show that high kidney volume seems to be a good non-invasive surrogate parameter for the diagnosis of cGN, especially in combination with high blood pressure and urinary dipstick analysis positive for erythrocytes and protein.

Serum IgA did not prove itself as a marker for IgAN in our cohort, due to its low positive predictive value [11], which contradicts results published elsewhere [12]. The same is true for ASL-titer. In contrast, a decreased serum C3 was a good marker for lupus GN or DDD. On the other hand, as shown before, the anti-DNS titer did not correlate with disease sever- ity of lupus GN [13].

MPO antibodies (p-ANCA) were detectable in all children with MPA/RLV, whereas in children with GPA, the detection of PR3 antibodies (c-ANCA) dominated, which broadly cor- responds to the literature [14].

Historically, despite steroids, the primary immunosuppres- sant administered in cGN has been cyclophosphamide [13, 15–17]. As cyclophosphamide therapy is associated with a long-term risk for infertility and has a pro-oncogenic charac- ter, other immunosuppressive therapies such as CsA, MMF, and rituximab have been administered in many patients with good results (although treatment is obviously dependent on the underlying disease). This is especially important in chil- dren who have a long life expectancy. However, a detailed disease-based analysis of the efficacy of different immunosup- pressive therapies is not possible in our cohort because of its heterogeneous, retrospective nature and the small number of patients in each group. The CD20 antibody rituximab has been shown to be effective in children with recurrence; how- ever, we could not analyze the effect of rituximab in detail because of the large differences between the five rituximab patients in underlying diagnosis and disease course. In severe, antibody-based cGN, plasma exchange, or immunoadsorption were effectively used for induction therapy or in the case of a relapse.

Our analysis has several limitations. Because of the retro- spective character of the analysis, patient clinical data were not documented in a standardized fashion. There are only a

few uniformly accepted treatment recommendations for subdiagnoses of cGN in children, and over the long inclusion time of our analysis, these have changed. According to the high number of different underlying diagnoses, an analysis for differences in the relationship of all subdiagnoses and eGFR development was not statistically possible. Also, the influence of additional treatment strategies for the underlying diseases could not completely be analyzed. The same is true for side effects of medications, as the retrospective nature of this study did not allow a complete documentation of adverse events, especially when patients were seen by other physi- cians. The initial pathological evaluations on which therapeu- tic interventions were based were performed by different pa- thologists. Moreover, classifications and classification criteria have changed during the study period. Aiming at harmoniza- tion of morphological analyses, all histologies have been re- evaluated using accepted criteria by the same pathologist. Some patients had biopsies with fewer glomeruli than required by the Oxford Classification (8 glomeruli) or the HSPN criteria (10 glomeruli) [7, 17]. Interestingly, treatment of cGN in our center did not really change during the long ob- servation time of our study. Steroid pulse therapy was conse- quently used over the whole period. Rituximab was intro- duced in our center for children as early as 2005. An exception was the use of cyclophosphamide, only administered in the earlier period until 2006 for some cases of lupus nephritis, GPA, and anti-GBM nephritis, according to older recommen- dations. Despite these mentioned limitations, this is the first analysis of a large group of children with cGN using a stan- dardized pathological assessment by the same pathologist. Although some of the morphological data confirm published results of smaller collectives, a comprehensive morphological workup proves to be valuable in assessment of disease sever- ity and therapeutic strategy planning.

We conclude that early detection and immediate aggressive treatment of cGN in children leads to stable remissions in the majority of patients and should therefore be implemented in most cases. To further determine more detailed therapy rec- ommendations (including new, emerging therapies for cGN, such as mifepristone, budesonide or erlotinib [18–20] not yet used in children), improved cGN classification should be de- veloped, including documentation of comprehensive clinical data in an international registry. Due to the small, heteroge- neous group of patients, randomized, controlled trials do not appear to be feasible.

Acknowledgments None.

Authors’ contributions L.P. and U.M. initiated the study, collected clini- cal data, and analyzed data. J.H.B. and J.S. conducted the pathological analyses. U.M. performed statistical analyses. The original manuscript draft was prepared by U.M. and L.P., J.H.B. and J.S. prepared critical revisions. All the authors contributed to the interpretation and discussion of results and reviewed and edited the manuscript.

Pediatr Nephrol (2020) 35:829–842 841

Funding Information Open Access funding provided by Projekt DEAL.

Compliance with ethical standards The Ethics Committee of Hannover Medical School has agreed to this policy.

Conflict of interest The authors declare that they have no conflict of interest.

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References

1. Jennette JC, Thomas DB (2001) Crescentic glomerulonephritis. Nephrol Dial Transplant 16(Suppl 6):80–82

2. Moroni G, Ponticelli C (2014) Rapidly progressive crescentic glo- merulonephritis: early treatment is a must. Autoimmun Rev 13: 723–729

3. Greenhall GH, Salama AD (2015) What is new in the management of rapidly progressive glomerulonephritis? Clin Kidney J 8:143– 150

4. Jennette JC (2003) Rapidly progressive crescentic glomerulone- phritis. Kidney Int 63:1164–1177

5. Baldwin DS, Neugarten J, Feiner HD, Gluck M, Spinowitz B (1987) The existence of a protracted course in crescentic glomeru- lonephritis. Kidney Int 31:790–794

6. Jardim HM, Leake J, Risdon RA, Barratt TM, Dillon MJ (1992) Crescentic glomerulonephritis in children. Pediatr Nephrol 6:231– 235

7. Trimarchi H, Barratt J, Cattran DC, Cook HT, Coppo R, Haas M, Liu ZH, Roberts IS, Yuzawa Y, Zhang H, Feehally J, IgAN Classification Working Group of the International IgA Nephropathy Network and the Renal Pathology Society, Conference Participants (2017) Oxford classification of IgA ne- phropathy 2016: an update from the IgA nephropathy classification working group. Kidney Int 91:1014–1021

8. Neuhauser H, Thamm M (2007) Blood pressure measurement in the german health interview and examination survey for children

and adolescents (KiGGS). Methodology and initial results. Bundesgesundheitsbl Gesundheitsforsch Gesundheitsschutz 50: 728–735

9. Schwartz GJ, Work DF (2009) Measurement and estimation of GFR in children and adolescents. Clin J Am Soc Nephrol 4: 1832–1843

10. Rauen T, Floege J (2017) Inflammation in IgA nephropathy. Pediatr Nephrol 32:2215–2224

11. Floege J (2015) Glomerulonephritides. Internist (Berl) 56:1277– 1285 quiz 1286

12. Kemper M (2004) Primaere IgA-nephropathie und purpura schoenlein-henoch-nephritis. Monatsschrift Kinderheilkd 152: 257–264

13. Arbeitsgemeinschaft fuer Paediatrische Nephrologie, (A P N ) (2013) Therapieempfehlung zur lupusnephritis bei kindern und jugendlichen. Monatsschr Kinderheilkd 161:543–553

14. Rowaiye OO, Kusztal M, Klinger M (2015) The kidneys and ANCA-associated vasculitis: from pathogenesis to diagnosis. Clin Kidney J 8:343–350

15. Haubitz M (2018) Nierenbeteiligung bei ANCA-assoziierten vaskulitiden. Dtsch Med Wochenschr 143:79–88

16. Noone D, Hebert D, Licht C (2018) Pathogenesis and treatment of ANCA-associated vasculitis-a role for complement. Pediatr Nephrol 33:1–11

17. Pohl M, Dittrich K, Ehrich JHH, Hoppe B, Kemper MJ, Klaus G, Schmitt CP, Hoyer PF, Gesellschaft fuer Paediatrische Nephrologie (G P N) (2013) Behandlung der purpura-schoenlein-henoch- nephritis bei kindern und jugendlichen. Monatsschrift Kinderheilkd 161:554–553

18. Bollee G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, Schordan E, Sabaa N, Vandermeersch S, Galaup A, Rodenas A, Casal I, Sunnarborg SW, Salant DJ, Kopp JB, Threadgill DW, Quaggin SE, Dussaule JC, Germain S, Mesnard L, Endlich K, Boucheix C, Belenfant X, Callard P, Endlich N, Tharaux PL (2011) Epidermal growth factor receptor promotes glomerular inju- ry and renal failure in rapidly progressive crescentic glomerulone- phritis. Nat Med 17:1242–1250

19. Fellstrom BC, Barratt J, Cook H, Coppo R, Feehally J, de Fijter JW, Floege J, Hetzel G, Jardine AG, Locatelli F, Maes BD, Mercer A, Ortiz F, Praga M, Sorensen SS, Tesar V, Del Vecchio L, Trial Investigators NEFIGAN (2017) Targeted-release budesonide ver- sus placebo in patients with IgA nephropathy (NEFIGAN): a dou- ble-blind, randomized, placebo-controlled phase 2b trial. Lancet 389:2117–2127

20. Kuppe C, van Roeyen C, Leuchtle K, Kabgani N, Vogt M, Van Zandvoort M, Smeets B, Floege J, Grone HJ, Moeller MJ (2017) Investigations of glucocorticoid action in GN. J Am Soc Nephrol 28:1408–1420

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REVIEW

Crescents in primary glomerulonephritis: a pattern of injury with dissimilar actors. A pathophysiologic perspective

Hernán Trimarchi1

Received: 4 May 2021 /Revised: 2 June 2021 /Accepted: 18 June 2021 # IPNA 2021

Abstract Cellular crescents are defined as two or more layers of proliferating cells in Bowman’s space and are a hallmark of inflammatory active glomerulonephritis and a histologic marker of severe glomerular injury. In general, the percentage of glomeruli that exhibit crescents correlates with the severity of kidney failure and other clinical manifestations of nephritic syndrome. In general, a predominance of active crescents is associated with rapidly progressive glomerulonephritis and a poor outcome. The duration and potential reversibility of the underlying disease correspond with the relative predominance of cellular or fibrous components in the crescents, the initial location of the immunologic insult inside the glomerulus, and the sort of involved cells and inflammatory mediators. However, the presence of active crescents may not have the same degree of significance in the different types of glomerulopathies. The pathophysiology of parietal cell proliferation may have dissimilar origins, underscoring the fact that the resultant crescents are a non-specific morphological pattern of glomerular injury with different implications in clinical prognosis in the scope of glomerular diseases.

Keywords Crescents . Proteinuria . Glomerulonephritis . Microhematuria . Glomerulus

Introduction

Cellular crescents are defined as two or more layers of prolif- erating cells in Bowman’s space, affecting 10% or more of the glomerular circumference. In general, the presence of cres- cents in a kidney biopsy represents a matter of concern. Many aspects are to be taken into consideration when they are diagnosed: the number of crescents with respect to the number of obtained glomeruli in a kidney biopsy, the subtypes of crescents according to the degree of proliferation and fibro- sis, the relationship between crescents and the underlying glo- merulopathy, the pathophysiological implications of crescents with other concomitant primary glomerular lesions such as endothelial and/or mesangial proliferation, fibrinoid necrosis, vascular infiltration by inflammatory cells, glomerular base- ment membrane (GBM) insults, and podocyte damage.

Cellular crescents can be reversible, but when the growth of parietal epithelial cells (PECs) associate with an epithelial–

mesenchymal transition-like change in cell phenotype, fibrous crescents form, and crescents become irreversible. Different molecular pathways trigger the activation of PECs. Crescent formation requires vascular injury causing ruptures in the GBM that trigger plasmatic coagulation within Bowman’s space (Fig. 1). This vascular necrosis can be triggered by different mechanisms, as small vessel vasculitis, immune complex glomerulonephritis, anti-GBM disease, and C3 glo- merulonephritis that all share complement activation but com- prise different upstream immunologic pathways outside the kidney [1].

Moreover, the burden crescents may be playing in the clin- ical scene are of utmost importance. As proliferative lesions, the presence of hematuria is a hallmark [2]. The decrease in the glomerular filtration area is accompanied by a concomitant fall in the glomerular filtration rate (GFR) that, if not solved, will render the compromised glomeruli to obliteration and a permanent decrease in kidney function. Varying degrees of proteinuria are always present due to the damage caused to the glomerular filtration barrier. The functional consequence of crescent formation is a decline in single-nephron GFR for two reasons. First, the increasing intra-glomerular mass in- creases the counter-pressure that together with oncotic pres- sure counterbalances arterial filtration pressure and may lead

* Hernán Trimarchi [email protected]

1 Nephrology Service, Hospital Británico de Buenos Aires, Perdriel 74 (1280), Buenos Aires, Argentina

https://doi.org/10.1007/s00467-021-05199-1

/ Published online: 27 July 2021

Pediatric Nephrology (2022) 37:1205–1214

to the collapse of the glomerular tuft [3]. Second, once a glo- merular crescent involves and obstructs the urinary pole, that single nephron will not contribute to the total GFR, rendering atubular glomeruli [1, 4].

In this review, the pathophysiology of crescents is discussed, as well as the impact crescents may play in the main primary glomerular diseases.

The origin of a crescent

The formation of a crescent appears to represent a non-specific response to a severe injury against the glomerular capillary wall (Fig. 1) [5]. The initiating event is the development of

physical gaps (also called rents or holes) in the glomerular capillary wall, then in the GBM, and finally in Bowman’s capsule, first in the visceral layer where podocytes lie and then the parietal layer [5]. This histological direction of events pre- sents relevant considerations to be made. Due to a primary insult to the endothelial compartment of the filtration barrier and the subsequent proliferation of cells, dysmorphic microhematuria is a frequent finding in the urinary sediment. As podocytes are both focally and secondarily damaged, the damage at the filtration barrier is lesser, and consequently proteinuria is not as severe as that found in podocytopathies or in subepithelial deposition of immune complexes. The pres- ence of gaps in the glomerular filtration barrier allows the leak of coagulation factors and leukocytes to begin the repair

Activated fibroblast

Quiescent fibroblast

Macrophages

CD11b+ dendritic cell

Lymphocyte

Healthy

podocyte

Quiescent PEC

Proliferative PECs

Endothelial cell

Stressed

podocyte

Fig. 1 Leading cells and molecules involved in the development of a crescent. Inside the glomerulus, the development of a crescent is due to many factors: An initial vascular injury causing ruptures (rents ) in the GBM causes the leakage of plasmatic pro-coagulant molecules (tissue factor, fibrinogen, and fibrin ) secreted by podocytes, the endothelium, and macrophages, within Bowman’s space. Involved stressed podocytes start to efface their processes, can migrate to the forming crescent, or

finally detach, and endothelial cells tend to duplicate. PECs start to pro- liferate, stimulated by several actors: influx of macrophages and lympho- cytes from interstitium and circulation and fibroblasts from neighboring parenchyma, infiltrating CD11b+ dendritic and T cells. Involved glomer- ular filtration barrier is in bold; activated cells (macrophages, lympho- cytes, PECs, podocytes, fibroblasts) present a deeper hue than non- activated ones

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mechanisms. Fibrinogen is converted to fibrin, while macro- phages and lymphocytes promote crescent formation [6]. In addition, crescent formation is initially mediated by T helper 1 CD4+ T cells, while macrophages and fibrin may act as effec- tors of cellular immunity [7]. Nephritogenic Th17 CD4 effec- tor cells, controlled by STAT3-dependent Treg17 cells ex- pressing the chemokine receptor CCR6, also play a role [8, 9].

The leakage of these molecules and cells to Bowman’s space through the capillary wall rents represents the first step that drives the formation of cellular crescents. The detachment of podocytes and podocyturia, and the loss of parietal cells due to the inflammatory process render both the GBM and Bowman’s capsule basement membrane denuded, promoting the adherence of both basement membranes. Neighboring pa- rietal cells respond to this insult with proliferation, favored by the subsequent participation of coagulation factors, particular- ly fibrin; tissue factor, which promotes fibrin deposition; and several different cell types, including macrophages, dendritic cells, renal progenitor cells, and interstitial fibroblasts. The contents in Bowman’s space pass to the interstitium and con- tribute to the periglomerular inflammation and the migration of fibroblasts. Stimulation of toll-like receptor 4 (TLR4) or 9 (TLR9) also takes part in the development of crescents [10] (Fig. 1).

As mentioned above, thrombogenesis is critical in the for- mation of crescents. Coagulation factors lead to the cross- linking of fibrin [11]. The primary stimulus to fibrin deposi- tion in crescent formation appears to be tissue factor, derived from endothelial cells, podocytes, and macrophages [12]. Finally, injured glomerular endothelial cells secrete tissue fac- tor due to macrophage-derived interleukin-1 and tumor necro- sis factor stimulation [13].

It has been reported that in crescentic glomerulonephritis, there exists a decreased fibrinolytic activity due to both a reduction in tissue-type plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1) [14]. This results in extraglomerular fibrin cross-linking in Bowman’s space. As a potent chemoattractant, fibrin contributes to the recruitment of macrophages into the glomeruli [15]. Protease-activated receptor-2 (PAR-2), a cellular receptor in glomerular cells and macrophages, worsens crescentic glomerulonephritis in- creasing PAI-1 expression and inhibit ing matrix metalloproteinase-9 activity [16]. By contrast, mice lacking PAR-2 have reductions in PAI-1 activity, fibrin deposition, and crescent formation [16].

Macrophages and crescents

Circulating macrophages or those close to the periglomerular area are critical in the formation of crescents as tissue factor expression and fibrin deposition derive from macrophages and may comprise from 40 to 70 percent of the cellular com- ponent of early crescents in some sorts of glomerulonephritis

[17] (Fig. 1). The migration of macrophages is a result of the action taken by macrophage chemoattractant protein-1 (MCP- 1), macrophage migration inhibitory factor (MIF), macro- phage inflammatory protein-1-alpha (MIP-1-alpha), osteo- pontin, and chemokine receptor 2B (CCR2B), the receptor for MCP-1, which are all essential actors [18, 19]. Moreover, vascular cell adhesion molecule (VCAM)-1, inter- cellular adhesion molecule (ICAM)-1, and CD44 (a marker of PECs) may also participate in the migration of macrophages [20]. Finally, granulocyte–macrophage colony-stimulating factor (GM-CSF) may increase expression of VCAM-1, MCP-1, and IL-1 beta and play a role in the genesis of cres- cents [21]. Among the macrophage-secreted molecules, IL-1, TNF, and TGF-beta are the most relevant. TGF-beta appears to play an important role in the progression of crescentic glo- merulonephritis and a lower response to immunosuppression [22]. Macrophages contribute to kidney dysfunction and tis- sue damage in established crescentic glomerulonephritis as it progresses from the acute inflammatory to a chronic fibrotic phase [23].

Several studies in patients with crescentic glomerulone- phritis found increased glomerular expression of matrix me- talloproteinases (MMP) -2, -3, -9, and -11 and tissue inhibitor of metalloproteinases (TIMP)-1, which correlated with cellu- lar crescents and disease activity [24]. In experimental studies, MMP-9 protects against experimental crescentic glomerulo- nephritis through its fibrinolytic activity [16].

Dendritic cells, T cells, and crescents

Dendritic cell depletion at early stages appears to flare glo- merulonephritis, while their depletion at a later stage leads to attenuation [25]. The CD11b+ subset of dendritic cells pro- motes crescentic glomerulonephritis, whereas the smaller pop- ulation of CD103+ dendritic cells protects from glomerulone- phritis by promoting Treg accumulation [26] (Fig. 1). T cells are found in Bowman’s space as well as in crescents [27]. T helper cells in the glomeruli are involved in the secretion of chemoattractants such asMCP andMIP, cytokines such as IL- 12 and IL-18, mast cells, and costimulatory ligands on mac- rophages (CD80 and CD86) [28]. The role of T cells in glo- merular injury may be related to antigen recognition and mac- rophage recruitment [7].

Glomerular parietal epithelial cells and crescents

Glomerular PECs are significant constituents of crescents [29] (Fig. 1). PECs have a high capacity to proliferate, presumably in response to growth factors, such as platelet-derived growth factor and fibroblast growth factor-2 [15]. Mice deficient in CD44 are partly protected from crescentic glomerulonephritis [2]. Since PECs are not major sources of procoagulant mole- cules or growth factors, it is unlikely that they are as important

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as macrophages and interstitial fibroblasts in determining the course and consequences of crescent formation. However, glomerular parietal epithelial cells can undergo dedifferentia- tion and become macrophage-like inflammatory effector cells and may be the primary cells producing type I collagen [30]. Macrophages and the coagulation system modulate PECs and are the main driving molecules in this respect. PECs may also proliferate and/or trans-differentiate into myofibroblasts pos- sibly by action of TGF-ß and PDGF, participating in glomer- ular crescents [31].

Podocytes and crescents

Podocytes are considered in general to be terminally dif- ferentiated cells and have not been regarded as main par- ticipants in crescent formation. However, it has been dem- onstrated that new podocytes could be recruited from glo- merular parietal epithelial cells through differentiation and proliferation [32]. In murine models and in patients with anti-GBM antibody disease, podocytes already adhered to both the GBM and the parietal basement membrane can build up podocyte bridges between the glomerular tuft and Bowman’s capsule [33]. Podocyte bridging could be an important early event in the development of crescents [33] (Fig. 1). Podocytes also populate crescents and may undergo epithelial–mesenchymal transformation also at early stages of crescentic glomerular diseases [34]. Nestin, a podocyte marker, has been encountered in cres- cents [35]. Finally, CD133- and CD24-positive renal pro- genitor cells localized in Bowman’s capsule at the vascular and urinary poles of the glomerulus are capable of regenerating podocytes [36]. The formation of tight junc- tions between podocytes is an early ultrastructural abnor- mality in crescentic glomerulonephritis, preceding foot process effacement and podocyte bridging after inflamma- tory injury. Podocyte-to-podocyte tight junction formation may be a compensatory mechanism to maintain the integ- rity of the glomerular filtration barrier following severe endocapillary injury and to avoid podocyte depletion and podocyturia [37].

Fibroblasts and crescents

Interstitial fibroblasts are the second most frequent cell type after macrophages [5] (Fig. 1). They enter Bowman’s space from the periglomerular interstitium through gaps in Bowman’s capsule. In the crescent itself, fibroblasts are a major source of interstitial collagen, which characterizes the transition from cellular to fibrous crescents [15]. As men- tioned previously, PECs can undergo mesenchymal differen- tiation and can also contribute to the collagen content of cres- cents [30].

The fate of crescents

The presence of crescents does not necessarily predict ir- reversible glomerular damage. Whether crescents progress or resolve may depend upon the integrity of Bowman’s capsule and the cellular composition of the crescent. Interstitial collagen synthesis and progression to fibrous crescents are more frequent when Bowman’s capsule rup- tures and fibroblasts and macrophages act in Bowman’s space [15]. Albeit fibrous crescents generally correlate with glomerulosclerosis, there is no evidence that an initial event in crescents can cause injury to the glomerular cap- illaries. Thus, crescent formation appears to be a conse- quence and a response, and not a cause, of severe active glomerular injury. However, there is increasing evidence that aggressive crescents may occlude the outflow from Bowman’s capsule to the proximal tubule, producing atubular glomeruli with secondary degeneration of glomer- uli and tubules and the disappearance of the involved neph- ron [38, 39].

The clinical significance of crescents in the different glomerulopathies

The impact of crescents on the different glomerulopathies varies widely. It mainly depends on the underlying glomer- ular disease and on the number and stage of the crescents. As mentioned previously, the initial step that leads to cres- cent formation is the damage to the glomerular capillary wall. Thus, crescents are not frequently observed in prima- ry podocyte damage, as may occur in minimal change dis- ease, primary focal and segmental glomerulosclerosis, membranous nephropathy, or genetic podocytopathies. Similarly, crescents are rarely observed in primary GBM disorders, such as Alport’s syndrome or thin basement membrane disease. However, when the GBM is immuno- logically damaged by antibodies against one of its compo- nents, as occurs in Goodpasture’s disease, the situation is completely different. Thence, crescents are more frequent- ly observed in those situations in which the endothelium is immunologically damaged, which results in endothelial proliferation (Table 1). This phenomenon is observed in immune complex and complement activation on the subendothelial space, as in membrano-proliferative glo- merulopathies, and in neutrophil-involved processes, such as anti-neutrophil cytoplasm antibody-associated (ANCA) vasculitis and post-infectious glomerulonephritis. Finally, endothelial proliferation can also lead to varying degrees of crescent formation, as is reported in IgA nephropathy (IgAN, see below) (Table 1). Macrophages, lymphocytes, and dendritic cells all play critical roles in these settings.

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Pathogenesis of crescent formation in the different glomerulopathies

The pathophysiology of crescent formation could be divided into two main steps. The first one pertains to each of the different glomerular entities and the capability to damage the endothelial side of the glomerular filtration barrier, from where podocytes and parietal cells interact between them- selves and with the surrounding extraglomerular cells to re- spond to this insult; the second step is the development of the crescent itself, which appears to involve a stereotypical path- way, with certain distinguishing features [1].

In the first step, either loss of tolerance (crescentic glomer- ulonephritis such as ANCA-positive or anti-GBM); an anti- body response to autologous molecules such as galactose- deficient IgA (IgAN) or to external epitopes (post-infectious glomerulonephritis); or a combined loss of tolerance plus identification of own molecules as antigens (immune complex crescentic glomerulonephritis, immune complex membrano- proliferative glomerulonephritis, lupus nephritis) leads to the priming of B and T cell clones and the synthesis of circulating autoantibodies that will eventually damage the endothelial glomerular cells [1].

In the second step, the varying degrees of activation of one or more of the three different complement pathways depend- ing on the glomerulopathy under concern will contribute to the vascular damage. In this regard, vascular necrosis, throm- bosis, or endothelial proliferation will follow, with rupturing of the GBM, plasma leakage to Bowman’s space, and podocyte stress. Podocytes may respond in different ways: cellular hypertrophy, detachment, expression of an antigen- presenting cell phenotype [1], or rarely proliferation, as in collapsing FSGS (see below). Extraglomerular cells migrate to the scene through a weakened or ruptured Bowman’s cap- sule, while effector T cells then recognize nephritogenic

antigens presented by podocytes or parietal cells within the urinary space. Coagulation factors, and the already-mentioned mitogenic signals, induce parietal cell hypertrophy, tuft en- croachment, and eventually glomerular obliteration [1, 40].

Distinguishing features among certain glomerulopathies

As mentioned, the impact of crescents in the different glomer- ular diseases depends mainly on the number of crescents and on the crescent cellularity, but also the initial location of the glomerular insult and the immunogenic capability of the anti- gen. In general, the higher the number of crescents and the proliferation of cells, the worse the prognosis will be (Table 1). Endothelial and basement membrane immune de- posits tend to carry a bad prognosis due to the capability to evoke an inflammatory response that will lead to podocyte damage and parietal cell activation and proliferation. Mesangial deposits present dissimilar behavior. In general, a low number of crescents is not associated with a bad outcome; according to the Oxford classification, a C1 score is associated with a good response to immunosuppression, while over 25% of crescents is associated with a grim prognosis despite the employment of immunosuppressants [41]. It has also been proposed that the development of endothelial proliferation in IgAN is associated with the generation of crescents [42]. Finally, a primary podocyte damage, as occurs in minimal change disease or FSGS due to circulating permeability fac- tors or to genetic mutations, is rarely accompanied by cres- cents, probably due to a primary lack of damage to the endo- thelial side of the glomerular filtration barrier. Subepithelial immune complex deposition as shown in primary and idio- pathic membranous nephropathy is rarely complicated by a high number of crescents. Occasional crescents can be

Table 1 Primary glomerulopathies and crescents

Glomerulopathy Presence of endothelial proliferation

Presence of podocyte proliferation

Presence of crescents Clinical course Progression to CKD 5

Anti-GBM disease 100% No >75% Acute 50%a

ANCA+ 100% No >75% Acute 30% a

IC extra-capillary GN 100% No >50% Acute 30% a

IgA <20% No 36% Chronic 20–30%*

Collapsing FSGS 0% Yes 24% Acute 50% a

MN 0% No 0.25% Chronic 30%**

IC MPGN 70% No 30% Chronic 12%***

C3 glomerulopathy 60% No 30% Chronic 25%***

Abbreviations: IC immune complex; GN glomerulonephritis; FSGS focal and segmental glomerulosclerosis; MN membranous nephropathy; MPGN membrano-proliferative glomerulonephritis; CKD 5 stage 5 chronic kidney disease

Symbols: a days to weeks; *at 20 years; **at 30 years; ***at 5 years

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observed and may not play a role in the outcome of membra- nous nephropathy (Table 1).

Crescentic glomerulonephritis

Glomerular crescents are the result of the proliferation of Bowman’s capsule parietal cells that encroach into the capil- lary tuft (Fig. 1). When crescents predominate, the clinical course is that of a rapidly progressive glomerulonephritis and a nephritic profile, with acute loss of kidney function, hypertension, hematuria, and varying degrees of proteinuria [43]. Three types of crescentic or extra-capillary glomerulo- nephritis are observed: anti-GBM glomerulonephritis, im- mune complex–mediated glomerulonephritis, and ANCA vasculitis, the most common form of crescentic glomerulone- phritis, accounting for >60% of all cases [44] (Table 1).

It has been proposed that the immune-related chemo- kine stromal cell–derived factor-1 (SDF-1)/C-X-C chemo- kine receptor (CXCR) 4 axis, known to be crucial for cell migration and proliferation [45], might play a role in all three types of primary glomerulonephritis. In human and rat crescentic glomerulonephritis, marked SDF-1 and CXCR4 upregulation in podocytes and PECs, respectively, suggests that SDF-1 produced by podocytes might trigger PEC/progenitor cell activation via CXCR4, leading to the formation of crescentic lesions [45]. Angiotensin II can promote cell proliferation and migration through the angio- tensin II type-1 (AT1) receptor [46]. Macrophages, possi- bly recruited by neutrophils [44], accumulate in the inter- stitial compartment, in the periglomerular area, and in the tuft itself (Fig. 1). This infiltration progressively increases inside the glomeruli, paralleling the progression of cellular hyperplasia. Interstitial macrophages produce MMP-12 elastase, a proteolytic enzyme that contributes to focal rup- tures of Bowman’s capsule, typically encountered in pa- tients with extra-capillary glomerulonephritis [47], sug- gesting a possible relationship between blood leakage from the glomerular capillaries and crescent formation [48]. Thus, in patients with extra-capillary glomerulonephritis, macrophage-driven lysis of glomerular membranes could induce PEC activation by promoting blood spillage through the GBM (Fig. 1) and producing angiotensin II (ang II), which via AT1 receptors stimulates podocytes to express SDF-1. On the other hand, excessive production of ang II stimulates podocytes to produce SDF-1 as well, pro- moting parietal progenitor activation via CXCR4 and CXCR7 receptors [44]. Finally, in a murine anti-GBM glo- merulonephritis model, crescent formation was preceded by the already-mentioned formation of podocyte bridges with PECs, considered the initiating event for cell prolif- eration on the capsular side and the formation of cellular crescents [49].

IgA nephropathy

In IgAN, it appears that fibrinogen and fibrin-related antigens but not fibrin are persistently positive in the crescents of this nephropathy. In addition, components of the GBM, such as type IV and V collagens, laminin, fibronectin, and cytokeratin, were consistently positive at all stages of cres- cents. Vimentin, usually distributed in podocytes and parietal and interstitial cells, was also found at all stages of the cres- cents. These findings may suggest that in the early stages of crescent formation in IgAN, podocytes play an important role, and that the accumulation of intrinsic basement membrane constituents is associated with the formation and progression of the crescents (Fig. 1). It appears that monocytes/ macrophages do not play a key role in the development of crescents in IgAN [50]. This finding may explain why the appearance of some crescents in kidney biopsies may not be regarded as severe lesions when compared to extra-capillary glomerulonephritis. As mentioned above, in IgAN, endothe- lial proliferation—a histological pattern frequently overlooked in this nephropathy—is associated with the devel- opment of crescents [41, 42].

Crescent formation in IgAN is associated with activation of the lectin and alternative pathways [51]. Some studies have reported that IgAN patients with increased glomerular mannose-binding lectin (MBL)–associated serine protease type 1 (MASP-1) deposition had a higher level of proteinuria and an increased rate of extra-capillary proliferation, glomer- ular sclerosis, and kidney dysfunction [52]. Hashimoto et al. observed increased intensity of properdin and factor B stain- ing in murine IgAN with more severe glomerular injury in- cluding crescent formation, suggesting the involvement of an activated alternative pathway [53]. Complement components and factors related to complement activation are partly pro- duced by intrinsic glomerular cells including mesangial and endothelial cells [54]. Glomerular C4d-positive IgAN patients are associated with an increased rate of stage 5 chronic kidney disease. Espinosa et al. showed that kidney survival at 10 years was significantly lower in C4d-positive IgAN patients than in C4d-negative IgAN patients and reported that IgAN with crescents was associated with higher levels of tissular C5b-9, MASP 1/3, MASP2, properdin, and factor B than those without crescents [51]. Therefore, crescent formation in IgAN may be associated with activation of both alternative and lectin pathways.

Collapsing FSGS

A recent study in experimental crescentic glomerulonephritis found that CD44, a cell surface glycoprotein that plays a key role in various cellular processes, is expressed in activated PECs and that its deficiency was associated with a reduced number of PECs in Bowman’s space [1]. In addition, CD44

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deficiency reduced glomerular cell proliferation and reduced albuminuria, indicating a link among CD44-expresing activat- ed PECs, the formation of crescents, and the development of albuminuria. In association with CD44 expression, CD9, a tetraspanin involved in cell proliferation, migration, adhesion, and survival, was found in PECs of a collapsing glomerulosclerosis rodent model [55]. Blocking CD9 attenu- ated the ability of PECs to proliferate and migrate, and atten- uated glomerulosclerosis. One possible mechanism of PEC activation via CD9 relates to the activation of epidermal growth factor receptor, a key driver of kidney damage in early stages of glomerulonephritis [54]. Thus, the local expression of CD9 is related to crescent formation in collapsing glomer- ulonephritis [1].

One interesting observation is that the anti-Thy1.1 model for collapsing FSGS is induced by targeting podocytes with anti-Thy1.1 antibody, which results in podocyte injury and activation of PECs. The subsequent formation of adhesions between the glomerular capillary tuft and Bowman’s capsule provides an entry site for activated PECs [2]. Activated PECs contribute to deposition of extracellular matrix material, which ultimately results in segmental and global glomerulosclerosis. The number of glomerular proliferating cells is reduced in the absence of CD44, which most likely can be explained by a reduction in proliferating PECs, because the anti-Thy1.1 model is not characterized bymassive glomer- ular infiltration of immune cells. Despite a decreased number of proliferating glomerular cells and reduced proteinuria, the absence of CD44 did not result in fewer histologically affected glomeruli. In summary, it appears that acquired glomerular CD44 expression by activated PECs is required for the path- ogenesis of experimental crescent formation in collapsing FSGS [2]. Finally, it is worthy of mention that in collapsing FSGS, the expression “pseudocrescents” is frequently employed to describe the occupation of Bowman’s space by hypertrophic and stressed podocytes. This term must not be confused with the cellular proliferation and intense inflamma- tion encountered in crescents as described in this manuscript.

The dynamics of crescents: from proliferation to fibrosis

As already mentioned, an initial microvascular injury leads to rupture of the GBM, which leads to the leakage of plasma proteins into Bowman’s space, driving hyperplasia of PECs as the key cellular component of the crescent and encroaching upon the involved glomerulus. Single-nephronGFR decreases because tuft collapse, rupture of the Bowman’s capsule, and influx of inflammatory cells and fibroblasts are secondary events (Fig. 1). Periglomerular immune cell infiltrates or fi- brosis due to local intense inflammation are subsequent events that may affect the dynamics and prognosis of the disease [1].

In this regard, the development of fibrosis in a proliferative crescent will eventually lead the clinical picture from an acute to a chronic setting. However, although the time it takes for such transformation in clinical practice would be of utmost relevance, there are no clinical studies addressing this topic, except in the case of rapidly progressive glomerulonephritis.

In a rat model of crescentic glomerulonephritis reproducing anti-GBM disease, the time elapsed from the development of cellular crescents to scarring and fibrosis was 4 to 6 weeks with almost 100% of glomeruli affected by global and diffuse glomerulosclerosis and severe interstitial medullary damage [39].

In clinical practice, when the number of cellular crescents exceeds 50% of the kidney sample, the prompt immunosup- pressive intervention may delay, decrease, or even be unable to stop the evolution of cellular to fibrotic crescents. Without intervention, in ANCA glomerulopathies, the time it takes to develop fibrosis may progress from an acute to a chronic phase within 1 to 2 weeks [56]. A similar extrapolation can be made with anti-GBM disease and immune complex extra- capillary glomerulonephritis: all entities with a rapidly pro- gressive course. In general, the number of crescents evolving to fibrosis parallels the degree of GFR decline. In the case of IgAN, if the number of crescents exceeds 25% in a kidney biopsy, the evolution to fibrosis and a bad prognosis appears to be the rule despite immunosuppression [41, 42].

Novel and potential therapeutic approaches

Based on the molecular mechanisms of crescent formation detailed in the manuscript, some of the intervening molecules may be plausible for pharmacologic assessment. Most of the already-tested antagonists have been explored in other fields outside nephrology. The blockade of specific molecules at different stages of crescent formation may be useful to im- prove the prognosis of glomerulopathies in which extra- capillary lesions are usually associated with poor outcomes. At the initial steps, Th17 CD4 effector lymphocytes express chemokine receptor CCR6 [8, 9]. Besides oncology, chemo- kine receptor CCR6 antagonist CO339589 has been success- fully assessed in autoimmune diseases such as psoriasis, rheu- matoid arthritis, and multiple sclerosis [57]. Already-available inhibitors of TLR-4 and/or TLR-9, whose activity has been proven in the development of crescents, could be useful to ascertain in the glomerular setting. Ibudilast, a TLR4 antago- nist, has been tested in autoimmune asthma and NI-101 in rheumatoid arthritis [58, 59]. In the same line, TLR 9 ODN 2088 has been shown to modulate macrophage chemotaxis in spinal cord injury [60]. A natural antagonist of CCR2, 747, s e l e c t i v e l y a t t enua t e s mac rophage ac t i v i t y i n hepatocarcinoma [61]. In a clinical trial, a novel selective MCP-1 receptor antagonist CCX140-B was given in addition to standard care in a randomized, double-blind study [62].

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Patients were randomized to placebo, 5 mg, or 10 mg of CCX140-B daily. Reduction in albuminuria was greatest in patients receiving low-dose CCX140-B, indicating MCP-1 inhibition on top of ACE inhibitors or ARBs conferred further renoprotection in diabetic nephropathy [62]. Finally, PAR-2, the already-mentioned receptor that intervenes in crescent de- velopment [16], has been antagonized by AZ3451 in patients with osteoarthritis [63]. As previously commented, the ex- pression of the tetraspanin CD9 increases markedly in PECs in mouse models of crescentic glomerulonephritis and FSGS. CD9 gene targeting in PECs prevents glomerular damage, and CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and β1 integrin expression, offering another potential therapeutic pathway to target formation of crescents [55].

Conclusion

Crescents constitute the hallmark of inflammation in active glomerulonephritis and are a marker of glomerular injury. However, the presence of cellular crescents may not lead to the same bad outcome in the different types of primary glo- merulopathies. The pathophysiology of parietal cell prolifera- tion varies among these entities. Crescents are a non-specific morphological pattern of glomerular injury with different im- plications in the clinical outcome of the different glomerular diseases. Unraveling the diverse actors that play in each of the glomerulopathies may lead to a better understanding of the pathophysiology of these entities, as well as to tailored and more specific therapies.

Author contribution The author entirely contributed to the development of the manuscript.

Declarations

Conflict of interest The author declares no competing interests.

References

1. Anguiano L, Kain R, Anders HJ (2020) The glomerular crescent: triggers, evolution, resolution, and implications for therapy. Curr Opin Nephrol Hypertens 29:302–309

2. Eymael J, Sharma S, Loeven MA, Wetzels JF, Mooren F, Florquin S, Deegens JK, Willemsen BK, Sharma V, van Kuppevelt TH, Bakker MA, Ostendorf T, Moeller MJ, Dijkman HB, Smeets B, van der Vlag J (2018) CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal and segmental glomerulosclerosis. Kidney Int 93:626–642

3. Fogo AB, LuscoMA,Najafian B, Alpers CE (2016) AJKDAtlas of renal pathology: pauci-immune necrotizing crescentic glomerulo- nephritis. Am J Kidney Dis 68:e31–e32

4. Puelles VG, Fleck D, Ortz L, Papadouri S et al (2019) Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis. Kidney Int 96: 505–516

5. Lan HY, Nikolic-Paterson DJ, Atkins RC (1992) Involvement of activated periglomerular leukocytes in the rupture of Bowman’s capsule and glomerular crescent progression in experimental glo- merulonephritis. Lab Investig 67:743–751

6. Jennette JC (2003) Rapidly progressive crescentic glomerulone- phritis. Kidney Int 63:1164–1177

7. Kitching AR, Holdsworth SR, Tipping PG (1999) IFN-gammame- diates crescent formation and cell-mediated immune injury in mu- rine glomerulonephritis. J Am Soc Nephrol 10:752–759

8. Hopfer H, Holzer J, Hünemörder S, Paust HJ, Sachs M, Meyer- Schwesinger C, Turner JE, Panzer U, Mittrücker HW (2012) Characterization of the renal CD4+ T-cell response in experimental autoimmune glomerulonephritis. Kidney Int 82:60–71

9. Kluger MA, Luig M, Wegscheid C, Goerke B et al (2014) Stat3 programs Th17-specific regulatory T cells to control GN. J Am Soc Nephrol 25:1291–1302

10. Giorgini A, Brown HJ, Sacks SH, Robson MG (2010) Toll-like receptor 4 stimulation triggers crescentic glomerulonephritis by multiple mechanisms including a direct effect on renal cells. Am J Pathol 177:644–653

11. Naish P, Penn GB, Evans DJ, Peters DK (1972) The effect of defibrination on nephrotoxic serum nephritis in rabbits. Clin Sci 42:643–646

12. Tipping PG, Erlich JH, Apostolopoulos J, Mackman N, Loskutoff D, Holdsworth SR (1995) Glomerular tissue factor expression in crescentic glomerulonephritis. Correlations between antigen, activ- ity, and mRNA. Am J Pathol 147:1736–1748

13. Cunningham MA, Kitching AR, Tipping PG, Holdsworth SR (2004) Fibrin independent proinflammatory effects of tissue factor in experimental crescentic glomerulonephritis. Kidney Int 66:647– 654

14. Kitching AR, Holdsworth SR, Ploplis VA, Plow EF, Collen D, Carmeliet P, Tipping PG (1997) Plasminogen and plasminogen activators protect against renal injury in crescentic glomerulone- phritis. J Exp Med 185:963–968

15. Atkins RC, Nikolic-Paterson DJ, Song Q, Lan HY (1996) Modulators of crescentic glomerulonephritis. J Am Soc Nephrol 7:2271–2278

16. Moussa L, Apostolopoulos J, Davenport P, Tchongue J, Tipping PG (2007) Protease-activated receptor-2 augments experimental crescentic glomerulonephritis. Am J Pathol 171:800–808

17. Tipping PG, Holdsworth SR (1986) The participation of macro- phages, glomerular procoagulant activity, and factor VIII in glo- merular fibrin deposition. Studies on anti-GBM antibody-induced glomerulonephritis in rabbits. Am J Pathol 124:10–17

18. Lloyd CM, Dorf ME, Proudfoot A, Salant DJ, Gutierrez-Ramos JC (1997) Role of MCP-1 and RANTES in inflammation and progres- sion to fibrosis during murine crescentic nephritis. J Leukoc Biol 62:676–680

19. Segerer S, Cui Y, Hudkins KL, Goodpaster T, Eitner F, Mack M, Schlöndorff D, Alpers CE (2000) Expression of the chemokine monocyte chemoattractant protein-1 and its receptor chemokine receptor 2 in human crescentic glomerulonephritis. J Am Soc Nephrol 11:2231–2242

20. Nishikawa K, Guo YJ, Miyasaka M, Tamatani T, Collins AB, Sy MS, McCluskey RT, Andres G (1993) Antibodies to intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 pre- vent crescent formation in rat autoimmune glomerulonephritis. J Exp Med 177:667–677

21. Timoshanko JR, KitchingAR, Semple TJ, Holdsworth SR, Tipping PG (2005) Granulocyte macrophage colony-stimulating factor ex- pression by both renal parenchymal and immune cells mediates

1212 Pediatr Nephrol (2022) 37:1205–1214

murine crescentic glomerulonephritis. J Am Soc Nephrol 16:2646– 2656

22. Song CY, Kim BC, Hong HK, Lee HS (2007) TGF-beta type II receptor deficiency prevents renal injury via decrease in ERK ac- tivity in crescentic glomerulonephritis. Kidney Int 71:882–888

23. Han Y, Ma FY, Tesch GH, Manthey CL, Nikolic-Paterson DJ (2013) Role of macrophages in the fibrotic phase of rat crescentic glomerulonephritis. Am J Physiol Ren Physiol 304:F1043–F1053

24. Sanders JS, van Goor H, Hanemaaijer R, Kallenberg CG, Stegeman CA (2004) Renal expression of matrix metalloproteinases in human ANCA-associated glomerulonephritis. Nephrol Dial Transplant 19: 1412–1419

25. Hochheiser K, Engel DR, Hammerich L, Heymann F, Knolle PA, Panzer U, Kurts C (2011) Kidney dendritic cells become pathogen- ic during crescentic glomerulonephritis with proteinuria. J Am Soc Nephrol 22:306–316

26. Evers BD, Engel DR, Böhner AM, Tittel AP, Krause TA, Heuser C, Garbi N, Kastenmüller W, Mack M, Tiegs G, Panzer U, Boor P, Ludwig-Portugall I, Kurts C (2016) CD103+ kidney dendritic cells protect against crescentic GN by maintaining IL-10-producing reg- ulatory T cells. J Am Soc Nephrol 27:3368–3382

27. Li HL, Hancock WW, Dowling JP, Atkins RC (1991) Activated (IL-2R+) intraglomerular mononuclear cells in crescentic glomeru- lonephritis. Kidney Int 39:793–798

28. Kitching AR, Turner AL, Wilson GR, Semple T, Odobasic D, Timoshanko JR, O'Sullivan KM, Tipping PG, Takeda K, Akira S, Holdsworth SR (2005) IL-12p40 and IL-18 in crescentic glomeru- lonephritis: IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation and leukocyte recruit- ment. J Am Soc Nephrol 16:2023–2033

29. Smeets B, Uhlig S, Fuss A, Mooren F, Wetzels JF, Floege J, Moeller MJ (2009) Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells. J Am Soc Nephrol 20:2604– 2615

30. Shirato I, Asanuma K, Takeda Y, Hayashi K, Tomino Y (2000) Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman’s capsule in the rat kidney. J Am Soc Nephrol 11: 2381–2386

31. Fujigaki Y, Sun DF, Fujimoto T, Suzuki T, Goto T, Yonemura K, Morioka T, Yaoita E, HishidaA (2002)Mechanisms and kinetics of Bowman’s epithelial-myofibroblast transdifferentiation in the for- mation of glomerular crescents. Nephron 92:201–213

32. Appel D, Kershaw DB, Smeets B, Yuan G, Fuss A, Frye B, Elger M, Kriz W, Floege J, Moeller MJ (2009) Recruitment of podocytes from glomerular parietal epithelial cells. J AmSocNephrol 20:333– 343

33. Le Hir M, Keller C, Eschmann V, Hähnel B, Hosser H, Kriz W (2001) Podocyte bridges between the tuft and Bowman’s capsule: an early event in experimental crescentic glomerulonephritis. J Am Soc Nephrol 12:2060–2071

34. Bariéty J, Bruneval P, Meyrier A, Mandet C, Hill G, Jacquot C (2005) Podocyte involvement in human immune crescentic glomer- ulonephritis. Kidney Int 68:1109–1119

35. Thorner PS, Ho M, Eremina V, Sado Y, Quaggin S (2008) Podocytes contribute to the formation of glomerular crescents. J Am Soc Nephrol 19:495–502

36. Ronconi E, Sagrinati C, Angelotti ML, Lazzeri E, Mazzinghi B, Ballerini L, Parente E, Becherucci F, Gacci M, Carini M, Maggi E, Serio M, Vannelli GB, Lasagni L, Romagnani S, Romagnani P (2009) Regeneration of glomerular podocytes by human renal pro- genitors. J Am Soc Nephrol 20:322–332

37. Succar L, Boadle RA, Harris DC, Rangan GK (2016) Formation of tight junctions between neighboring podocytes is an early ultra- structural feature in experimental crescentic glomerulonephritis. Int J Nephrol Renovasc Dis 9:297–312

38. Kriz W, Hähnel B, Hosser H, Ostendorf T, Gaertner S, Kränzlin B, Gretz N, Shimizu F, Floege J (2003) Pathways to recovery and loss of nephrons in anti-Thy-1 nephritis. J Am Soc Nephrol 14:1904– 1926

39. Tam FWK, Smith J, Morel D, Karkar AM, Thompson EM, Cook HT, Pusey CD (1999) Development of scarring and renal failure in a rat model of crescentic glomerulonephritis. Nephrol Dial Transplant 14:1658–1666

40. Kitching AR, Aikhan MA (2018) CD8+ cells and glomerular cres- cent formation: outside-in as well as inside-out. J Clin Invest 128: 3231–3233

41. Haas M, Verhave JC, Liu ZH, Alpers CE, Barratt J, Becker JU, Cattran D, H. Cook T, Coppo R, Feehally J, Pani A, Perkowska- Ptasinska A, Roberts ISD, Soares MF, Trimarchi H, Wang S, Yuzawa Y, Zhang H, Troyanov S, Katafuchi R (2017) The predic- tive value of crescents in IgA nephropathy: a large retrospective multicenter study. J Am Soc Nephrol 28:691–701

42. Trimarchi H, Barratt J, Cattran DC, Cook HT, Coppo R, Haas M, Liu ZH, Roberts ISD, Yuzawa Y, Zhang H, Feehally J (2017) Oxford classification for IgA nephropathy 2016. An update from the IgANephropathy ClassificationWorkingGroup. Kidney Int 91: 1014–1021

43. Couser WG (1988) Rapidly progressive glomerulonephritis: classi- fication, pathogenetic mechanisms, and therapy. Am J Kidney Dis 11:449–464

44. Rizzo P, Novelli R, Rota C, Gagliardini E, Ruggiero B, Rottoli D, Benigni A, Remuzzi G (2017) The role of angiotensin II in parietal epithelial cell proliferation and crescent formation in glomerular diseases. Am J Pathol 187:1441–1450

45. Rizzo P, Perico N, Gagliardini E, Novelli R, Alison MR, Remuzzi G, Benigni A (2013) Nature and mediators of parietal epithelial cell activation in glomerulonephritides of human and rat. Am J Pathol 183:1769–1778

46. Kim S, Iwao H (2000) Molecular and cellular mechanisms of an- giotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev 52:11–34

47. Kaneko Y, Sakatsume M, Xie Y, Kuroda T, Igashima M, Narita I, Gejyo F (2003) Macrophage metalloelastase as a major factor for glomerular injury in anti-glomerular basement membrane nephritis. J Immunol 170:3377–3385

48. Ryu M, Migliorini A, Miosge N, Gross O, Shankland S, Brinkkoetter PT, Hagmann H, Romagnani P, Liapis H, Anders HJ (2012) Plasma leakage through glomerular basement membrane ruptures triggers the proliferation of parietal epithelial cells and crescent formation in non-inflammatory glomerular injury. J Pathol 228:482–494

49. Moeller MJ, Soofi A, Hartmann I, Le Hir M, Wiggins R, Kriz W, Holzman LB (2004) Podocytes populate cellular crescents in a mu- rine model of inflammatory glomerulonephritis. J Am Soc Nephrol 15:61–67

50. Yoshioka K, TakemuraT AN,Miyamoto H, Iseki T, Maki S (1987) Cellular and non-cellular compositions of crescents in human glo- merulonephritis. Kidney Int 32:284–291

51. Itami H, Hara S, Samejima K, Tsushima H, Morimoto K, Okamoto K, Kosugi T, Kawano T, Fujiki K, Kitada H, Hatakeyama K, Tsuruya K, Ohbayashi C (2020) Complement activation is associ- ated with crescent formation in IgA nephropathy. Virchows Arch 477:565–572

52. Roos A, Rastaldi MP, Calvaresi N, Oortwijn BD, Schlagwein N, van Gijlswijk-Janssen DJ, Stahl GL, Matsushita M, Fujita T, van Kooten C, Daha MR (2006) Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease. J Am Soc Nephrol 17:1724–1734

53. Hashimoto A, Suzuki Y, Suzuki H, Ohsawa I, Brown R, Hall S, Tanaka Y, Novak J, Ohi H, Tomino Y (2012) Determination of severity of murine IgA nephropathy by glomerular complement

1213Pediatr Nephrol (2022) 37:1205–1214

activation by aberrantly glycosylated IgA and immune complexes. Am J Pathol 181:1338–1347

54. Daha MR, van Kooten C (2000) Is there a role for locally produced complement in renal disease? Nephrol Dial Transplant 15:1506– 1509

55. Lazareth H, Henique C, Lenoir O, Puelles VG et al (2019) The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression. Nat Commun 10:3303

56. Jennette JC, Nachman PH (2017) ANCA glomerulonephritis and vasculitis. Clin J Am Soc Nephrol 12:1680–1691

57. Ranasinghe R, Eri R (2018) Modulation of the CCR6-CCL20 axis: a potential therapeutic target in inflammation and cancer. Medicina 54:88

58. Jia ZJ, Wu FX, Huang QH, Liu JM (2012) Toll-like receptor 4: the potential therapeutic target for neuropathic pain. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 34:168–173

59. Monnet E, Lapeyre G, Poelgeest EV, Jacqmin P, Graaf K, Reijers J, Moerland M, Burggraaf J, Min C (2017) Evidence of NI-0101 pharmacological activity, an anti-TLR4 antibody, in a randomized phase I dose escalation study in healthy volunteers receiving LPS. Clin Pharmacol Ther 101:200–208

60. Li L, Ni L, Heary RF, Elkabes S (2020) Astroglial TLR9 antago- nism promotes chemotaxis and alternative activation of macro- phages via modulation of astrocyte derived signals: implications for spinal cord injury. J Neuroinflammation 17:73

61. YaoW, BaQ, Li X, Li H, Zhang S, Yuan Y,Wang F, Duan X, Li J, Zhang W, Wang H (2017) A natural CCR2 antagonist relieves tumor-associated macrophage-mediated immunosuppression to produce a therapeutic effect for liver cancer. EBioMedicine 22: 58–67

62. de Zeeuw D, Bekker P, Henkel E, Hasslacher C, Gouni-Berthold I et al (2015) The effect of CCR2 inhibitor CCX140-B on residual albuminuria in patients with type 2 diabetes and nephropathy: a randomised trial. Lancet Diabetes Endocrinol 3:687–696

63. Huang X, Ni B, Xi Y (2019) Protease-activated receptor 2 (PAR-2) antagonist AZ3451 as a novel therapeutic agent for osteoarthritis. Aging 11:12533–12545

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