Crescentic Glomerulonephritis: A Single Center Observational Cohort Study

Background The spectrum and outcomes of crescentic glomerulonephritis in South Asia is vastly different from that reported worldwide and there is a paucity of information. Methods It was an observational cohort study of renal biopsies done in the largest tertiary center in South India over a period of 10 years with [≥]50percentage crescents on histology. Results A total of 8645 kidney biopsies were done at our center from January 2006 to December 2015, and 200 were crescentic glomerulonephritis. Patients were categorized into three etiological groups- anti-GBM (type I), immune complex (type II) and pauci-immune (type III). The most common was type II (96, 46.5percentage), followed by type III (73, 38percentage) and then type I (31, 15.5percentage). Female preponderance was seen across all three types. About half of all the three types presented with recent onset hypertension. Type II had the highest median proteinuria (4.2 (2.1-6) g/day, p=0.06) and the median eGFR was lowest in type I (5 (4-8) ml/min/1.73m2, p<0.001). Among type III, ANCA associated vasculitis (AAV) was seen only in about half of the patients. Nearly one third patients with type I was also positive for ANCA making them double positive. Severe glomerular insults like tuft necrosis and chronicity as evidenced by moderate to severe interstitial fibrosis was a predominant feature of type I. Conclusions ANCA negative vasculitis as well as double positive types are reported for the first time from South-Asia. Prevalence of ANCA negative vasculitis (type III subgroup) was much higher in our population. Renal survival was significantly worse in type I and III compared to type II. Types I or III, moderate to severe IFTA, presence of oliguria or anuria and increasing percentage of crescents in renal biopsy were significant predictors of dialysis dependence at index visit or of end stage kidney disease at follow-up in our cohort.


INTRODUCTION
Crescentic glomerulonephritis (Cr.GN) is characterized by the presence of extensive glomerular crescents (usually greater than 50%) as the principal histologic finding. Because it often clinically presents with a rapid decline in kidney function, it is also known as rapidly progressive glomerulonephritis (RPGN). It can complicate any glomerular disease 1 and is present in approximately 4% to 10% of native kidney biopsies. 2 The natural course of the disease renders it a "medical emergency", as the decline in renal function is relentless and leads to end stage renal failure in a few weeks or months. 3 Understanding the clinical presentation, natural history and outcomes of crescentic glomerulonephritis is of major concern for nephrologists worldwide. Various studies have been conducted worldwide and epidemiologic data are available from large national kidney biopsy registries including those from the United States 4 , China 2 , Japan 5 , Spain 6 and Saudi Arabia. 7 An important thing to realise here is that pathogenesis of glomerular diseases involves a complex and as yet incompletely understood interplay between epigenetic, immunoregulatory, hormonal, and environmental factors on a background of genetic predisposition. 8 This translates into a broad spectrum of disease presentation, a variable tempo of progression and heterogenous outcomes which are evident from these studies. To this end, exploring the features of crescentic glomerulonephritis in our population, which is yet another genetically and demographically diverse group of individuals may help to provide useful insights into the causality, and predictors of severe outcomes.

METHODS
This was an observational retrospective cohort study done in outpatient and inpatient services of the Department of Nephrology in our hospital. We included all patients (≥18 years) who underwent native renal biopsy at our centre between January 2006 to December 2015 and had ≥50% crescents on histology. Data on their demographic profile, clinical features, biochemical parameters, histopathology, treatments, morbidity and mortality were retrieved from the electronic patient records (Clinical WorkStation) maintained in the hospital. Followup clinical and outcome data with regards to their serum creatinine, dialysis requirement, and complications were collected for each review visit till August 2016. During the index and follow up visits, patients were classified into chronic kidney disease (CKD) stages as per estimated glomerular filtration rate (eGFR) calculated by CKD-EPI equation. 9 Cr.GN was defined as the presence of ≥50% glomerular crescents as the principal histologic finding. Patients were categorized into three groups on the basis of etiology of crescentic GN-anti-GBM crescentic GN (type I Cr.GN), immune complex . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
Microhematuria was defined as >5 red blood cells (RBC's) per high power field. Proteinuria was assessed from 24-hour timed collection as is the standard practice in our center.
Interstitial fibrosis and tubular atrophy (IFTA) was classified into: focal-<25%, moderate-25% to 50% and severe->50%. Qualitative and semiquantitative determination of antinuclear antibody (ANA) in serum was done manually by EUROIMMUN Mosaic Hep-20-10 indirect immunofluorescence test (IIFT). The test was done with a sample dilution starting point of 1:100. It is graded on a scale of 1+ to 5+. The sensitivity of the test was 100% with a specificity of 96%. Quantitative determination of anti-double stranded DNA (anti-dsDNA) in serum was done by Anti-dsDNA-NcX ELISA (IgG). The upper limit of the normal range (cut-off) was 100 IU/ml. Anti-neutrophil cytoplasmic antibodies (ANCA) were determined by measuring anti-myeloperoxidase (anti-MPO) and anti-proteinase3 (anti-PR3). Quantitative determination of anti-MPO was done by Anti-Myeloperoxidase ELISA (IgG) test kit. The upper limit of the normal range (cut-off) is 20 RU/ml. The ELISA had a sensitivity of 93.3% and a specificity of 99.8% with regard to IIFT. Quantitative determination of anti-PR3 was done by Anti-PR3-hn-hr ELISA (IgG). The upper limit of the normal range (cut-off) was 20 RU/ml. The ELISA had a sensitivity of 94% and a specificity of 99%. The tests kits for antibodies were from EUROIMMUN, Luebeck, Germany. Quantitative determination of complement factors (C3 and C4) was done by means of endpoint nephelometry on the BN ProSpec System by Siemens Health Care Diagnostics Products, Marburg, Germany. Antisera used were liquid animal sera produced by immunization of rabbits with highly purified human complement factors (C3c or C4). The following reference intervals applied for serum samples from healthy adults: C3/C3c: 0.9-1.8 g/L, C4/C4c: 0.1-0.4 g/L.

Statistical Analysis
Data are presented as mean ± standard deviation or medians (interquartile range) or frequency & percent according to the types and distribution of variables. Differences among groups of normally distributed variables were analyzed by t test or one-way analysis of variance (ANOVA). Post-hoc comparisons were performed using t Test with Bonferroni correction. Differences among groups of non-parametric variables were analyzed by Mann-Whitney U-test or the Kruskal-Wallis Test. Categorical variables were compared using chisquared or Fisher's exact test. Multivariable logistic regression was used to identify predictors of end stage kidney disease (ESKD). Statistical calculations were performed using SPSS software for Windows, version 21.0 (SPSS Inc., Chicago, IL) and graphs were made . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint using Graph Pad Prism 7.0e (Graph Pad Software Inc., San Diego, CA). A P value of <0.05 was taken as significant.

Demography
A total of 8645 kidney biopsies were done at our center from January 2006 to December 2015, out of which 200 were found to have Cr.GN (2.31%). The most common cause of Cr.GN was type II (96, 46.5%), followed by type III (73, 38%) and least common was type I (31, 15.5%). The various etiologies of Cr.GN are depicted in Figure 1. Females constituted 60% of the patients with a female: male ratio of 1.5:1. Female preponderance was seen across all three types of Cr.GN. The mean age of presentation was 40.6±14.6 years with the highest mean age seen in type III Cr.GN. Demographic and baseline clinical and laboratory parameters of the study cohort are summarized in table 1.

Clinical and laboratory features
Non-visible hematuria was near universal (95%). More than half of the patients (56%) were oliguric at presentation. Anuria at presentation was seen in 10% patients. Oliguria and anuria were more common in type I Cr.GN patients (oliguria in 74%; p=0.08 and anuria in 16%; p=0.04) who also had significantly more uremic symptoms (64%, p=0.006). About half of all the three types presented with recent onset hypertension. Among extra-renal symptoms, skin lesions and arthritis were rare in type I but hemoptysis was seen only in type I & III Cr.GN.
Serum complement levels were low in nearly 50% patients with Cr.GN (low C3 in 43% and low C4 in 11%), predominantly in those with type II Cr.GN (p <0.001). Interestingly, low C3 levels were also found in about 25% patients with either type I or type III Cr.GN. Low C4 levels, on the other hand was seen only in type II Cr.GN. Though ANA was positive to various extents in all types of Cr.GN (23% in type I, 9% in type II & 47% in type III), dsDNA positivity was seen only type II Cr.GN (33%). Among type III Cr.GN, ANCA associated vasculitis (AAV) was seen only in 51% patients (anti-MPO 26%; anti-PR3 29%). . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint

Histopathological features
Characteristic histopathological features noted in kidney biopsies have been summarized in table 2. The highest percentage of glomeruli with crescents was seen in type I (83±17%, p=0.001) followed by type III (74±19%) and type II Cr.GN (69±18%). The most common type of crescents in all three groups was fibrocellular. Glomerular proliferative lesions as evidenced by mesangial hypercellularity (68%, p=0.02), endocapillary proliferation (100%) and neutrophilic exudation (50%, p=0.01), were predominantly seen in type II Cr.GN. Severe glomerular insults like tuft necrosis was a predominant feature of type I (32%, p=0.002) and type III Cr.GN (25%). Chronicity as evidenced by moderate to severe IFTA was most severe in type I Cr.GN (68%; p=0.02).

Treatment differences
Standard treatment protocols as per KDIGO 2012 10 guidelines were followed for all patients.
Nearly half of the patients required dialysis at presentation, with significantly more in type I and type III Cr.GN (81% & 62% respectively, p<0.001). About 1/5 th of patients received therapeutic plasma exchange (PLEX) and this was significantly more in type I and type III Cr.GN (42% & 26% respectively, p<0.001). The frequency and indications of PLEX are summarized in table 3. Immunosuppression with/without PLEX was given in 90% of patients with significantly more in type II & III Cr.GN (92% & 91% respectively, p<0.001).

Patient and renal outcomes
The mean follow-up period was 9.4±15 months. Table 4 summarizes the outcomes observed in three types of Cr.GN. In the entire cohort, nearly half (45%) developed ESKD requiring renal replacement therapy. This was significantly more in type I (77%, p<0.001). Overall mortality rate was 4% and did not vary significantly between the groups. Sepsis was found to be the most common cause of death.
. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint Rates of ESKD and renal survival did not differ between the two groups (Table 5 & Figure   2).

Predictors of severe outcomes
We analyzed the risk factors associated with dialysis dependence at discharge from index visit in the entire cohort (Table 6). In an adjusted regression analysis, type I/type III Cr.GN, moderate to severe IFTA, presence of oliguria/anuria and increasing percentage of crescents in renal biopsy were significant predictors of dialysis dependence at discharge.
We also analyzed various risk factors which predicted the development of ESKD at followup in this cohort. (Table 7). Similarly, in an adjusted regression analysis, type I/type III Cr.GN, moderate to severe IFTA, presence of oliguria/anuria and increasing percentage of crescents in renal biopsy were significant predictors of ESKD at follow-up.
Kaplan-Meier estimates of renal survival differed significantly between the three groups ( Figure 2) but did not differ in any subgroups ( Figure 2). Patient survival was similar in all three groups ( Figure 3).

DISCUSSION
Crescentic GN (Cr.GN) is one of the leading causes of rapidly progressive renal failure.
There are a few studies of crescentic GN from South Asia but there is dearth of data on the different types of Cr.GN and their outcomes.
In this study Cr.GN accounted for 2.3% of all biopsies conducted over a period of 10 years, which is comparable to previously reported rates of Cr.GN from India. 11,12 Type II Cr.GN was found to be the most common type which is unique to Asian continent. Various studies conducted in different parts of the world have shown type III Cr.GN to be the most common. 5,13 However studies from China, Saudi Arabia and a smaller Indian study have reported type II Cr.GN to be the most common. 2,11,14 Reasons cited for increased incidence of type II Cr.GN in these parts have been an increased incidence of infections and a higher prevalence of IgA nephropathy. Lupus nephritis (45%) is the most common cause of type II Cr.GN, followed by infection related GN (24%) and IgA nephropathy (23%). This is similar to one of the largest reviews of Cr.GN from China in which lupus nephritis was listed as most common cause of type II Cr.GN (34%) followed by IgA nephropathy (17%). 2 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint 8 Gender differences in Cr.GN have mostly been observed in type II Cr.GN with female preponderance. The gender distribution has been variable in type I and type III Cr.GN in different studies. 2,11,[13][14][15] As is already known and established in other studies, patients with type I Cr.GN had the most severe renal failure at presentation. However, in this study, more than half of the patients with type II & type III Cr.GN presented with severe renal failure highlighting the severity of presentation in this patient population. These rates are much higher than previously reported. 2,15 Serum complement C3 levels have been shown to be low in type I and type III Cr.GN in addition to type II Cr.GN. The same was confirmed in our study. However, we found no cases of low C4 levels in type I and III Cr.GN, undersigning the statement that alternate complement pathway has a role in the pathogenesis of these GNs. Our observed rates of ANCA seropositivity in type III Cr.GN are much lower than those reported in other studies. 2,15 An earlier study from India also reports similar rates of ANCA seropositivity. 16 ANCA negative vasculitis could be attributed to other antibodies such as anti-endothelial cell antibodies, or to cell mediated immune mechanisms which lead to neutrophilic activation. 17 The high prevalence of ANCA negative vasculitis in this cohort highlights the need for research into its pathogenesis to elucidate factors specific to our population.

Data availability
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Competing interests
No competing interests were disclosed. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint  P value is significant at <0.05 between @ Type 1 and Type III, # Type II and Type III, $ Type 1 and Type II analyzed by One-way ANOVA with Bonferroni correction.
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(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint   is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.12.20129122 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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