Proteinuria Indicates Decreased Normal Glomeruli in ANCA-Associated Glomerulonephritis Independent of Systemic Disease Activity

Background: Renal involvement is a common and severe complication of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), potentially resulting in a pauci-immune necrotizing and crescentic ANCA glomerulonephritis (GN) with acute kidney injury (AKI), end-stage renal disease (ESRD) or death. There is recent evidence that the degree of proteinuria at diagnosis is associated with long-term renal outcome in ANCA GN. Therefore, we here aimed to systematically describe the association between proteinuria and clinicopathological characteristics in 53 renal biopsies with ANCA GN and corresponding urinary samples at admission. Methods: A total number of 53 urinary samples at admission and corresponding renal biopsies with confirmed renal involvement of AAV were retrospectively included from 2015 to 2021 in a single-center study. Results: Proteinuria correlated with myeloperoxidase (MPO) subtype, diagnosis of microscopic polyangiitis (MPA) and severe deterioration of kidney function. Proteinuria was most prominent in sclerotic class ANCA GN and ANCA renal risk score (ARRS) high risk attributed to nonselective proteinuria, including both glomerular and tubular proteinuria. Finally, there was no association between proteinuria and systemic disease activity, suggesting that proteinuria reflected specific renal involvement in AAV rather that systemic disease activity. Conclusions: In conclusion, proteinuria correlated with distinct clinicopathological characteristics in ANCA GN, mostly attributed to a reduced fraction of normal glomeruli. Furthermore, proteinuria in ANCA GN reflected specific renal involvement in AAV rather than systemic disease activity. Therefore, urinary findings could further improve our understanding of mechanisms promoting kidney injury and progression of ANCA GN.

The aforementioned previous studies mainly focused on deterioration of kidney function in combination with histopathological findings in ANCA GN. However, there is recent evidence that the degree of proteinuria at diagnosis is associated with long-term renal outcome in ANCA GN [25,30,31]. At disease manifestation, the majority of patients with ANCA GN have proteinuria with variable amounts [18]. Previously, we also reported that proteinuria is observed in a considerable subset of ANCA GN and associated with acute deterioration of kidney function at disease onset, implicating an association with active lesions and ANCA manifestation in the kidney [32]. This is confirmed by recent findings that proteinuria associates with established ANCA scoring systems showed the lowest proteinuria in biopsies categorized as focal class, followed by mixed and crescentic class ANCA GN [33]. However, correlation of proteinuria and its subclassification with regard to histopathological findings in ANCA GN has not been systematically described yet. Therefore, we here aimed to systematically describe the association between proteinuria and clinicopathological characteristics in 53 renal biopsies with ANCA GN and corresponding urinary samples at admission.

Study Population
A total number of 53 renal biopsies with confirmed renal involvement of AAV and 53 corresponding urinary samples primarily admitted to the Department of Nephrology and Rheumatology, University Medical Center Göttingen, Germany were retrospectively included from 2015 to 2021; the patient cohort was in part previously described [32]. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of University Medical Center Göttingen, Germany. Medical records were used to obtain data on age, sex, diagnosis (MPA or GPA) and laboratory results. The estimated glomerular filtration rate (GFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [34]. The worst measurement during the initial course of the disease was used to determine the severity of kidney injury. At admission, the Birmingham Vasculitis Activity Score (BVAS) version 3 was calculated as described previously [35]. The BVAS is assessed on a scale of 0 to 63, with a score of 0 indicating the absence of disease activity and higher scores indicating active disease.

Renal Histopathology
A pathologist (SH) evaluated all biopsies, independently validated by a second renal pathologist (PS) blinded to clinical data collection and analysis. Within a renal biopsy specimen, each glomerulus was scored separately for the presence of necrosis, crescents and global sclerosis. Consequently, the percentage of glomeruli with any of these features was calculated as a fraction of the total number of glomeruli in each renal biopsy. Based on these scorings, histopathological subgroupings according to Berden et al. (focal, crescentic, mixed or sclerotic class) and ARRS according to Brix et al. (low, medium or high risk) were performed [8,29]. Apart from these categories, the degree of interstitial fibrosis and tubular atrophy (IF/TA) was quantified. Renal pathologists were blinded to clinical data collection and analysis.

Statistical Methods
Variables were tested for normal distribution using the Shapiro-Wilk test. Nonnormally distributed continuous variables are expressed as median and interquartile range (IQR), categorical variables are presented as frequency and percentage. Statistical comparisons were not formally powered or prespecified. For group comparisons, the Mann-Whitney U-test was used to determine differences in medians. Nonparametric between-group comparisons were performed with Pearson's Chi-square test. Data analyses were performed with GraphPad Prism (version 8.4.3 for MacOS, GraphPad Software, San Diego, California, U.S.). Multiple regression analyses were performed using IBM SPSS Statistics (version 27 for MacOS, IBM Corporation, Armonk, NY, U.S.). A probability (p) value of <0.05 was considered statistically significant.

Parameter Value
Clinical data Female sex-no.  Association with clinical and laboratory findings revealed that uPCR correlated with MPO subtype, diagnosis of MPA and severe deterioration of kidney function assessed by eGFR loss (Figure 2), in line with our previous observations [32]. Proteinuria subclassification established that higher levels of proteinuria in MPO subtype was attributed to increased uACR and urinary IgG levels (Figure 2), reflecting nonselective glomerular proteinuria. Association between severe deterioration of kidney function was observed for uACR, IgG, α 1 -microglobulin and α 1 -macroglobulin ( Figure 2). Interestingly, increased levels of C-reactive protein (CRP) were specifically associated with α 1 -microglobulin ( Figure 2), confirming previous observations that urinary α 1 -microglobulin reflects systemic inflammation [38]. In addition, leukocyturia was more frequently observed in patients with relapsing ANCA GN ( Figure 2). In summary, ANCA GN presents with subnephrotic proteinuria in most cases, including glomerular and/or tubular proteinuria. Furthermore, proteinuria correlated with more severe deterioration of kidney function in ANCA GN. Therefore, we next aimed to correlate proteinuric findings in association with histopathological findings in ANCA GN.

Proteinuric Findings in Correlation with Histopathological Findings in ANCA GN
By applying scoring of ANCA GN according to Berden et al., highest levels of proteinuria (uPCR) were observed in sclerotic and lowest in focal class ANCA GN (Table 2), in line with previous observations [8,33]. Comparable results were also observed for proteinuria subclassification; uACR, IgG, α 1 -microglobulin and α 2 -macroglobulin were most prominent in sclerotic and lowest in focal class ANCA GN ( Table 2). Categorization of ANCA GN in ARRS revealed that increased risk class was associated with higher levels of uPCR, equally reflected by proteinuria subclassification in uACR, IgG, α 1 -microglobulin and α 2 -macroglobulin (Table 3).   Based on these observations that categorization of ANCA GN into established histopathological scoring systems into sclerotic class ANCA GN and ARRS high risk were associated with highest levels of proteinuria attributed to nonselective proteinuria, we next correlated proteinuric findings in association with distinct histopathological lesions in ANCA GN. Nonselective proteinuria, including uPCR, uACR, urinary IgG, α 1microglobulin and α 2 -macroglobulin was associated with a decreased fraction of normal glomeruli attributed to crescentic ANCA GN ( Figure 3A,B). Furthermore, IF/TA also correlated with uPCR, uACR, urinary IgG and α 2 -macroglobulin, but not α 1 -microglobulin ( Figure 3B).
Multiple regression analyses revealed a stronger correlation between glomerular proteinuria and a decreased fraction of normal glomeruli as compared to other glomerular lesions or IF/TA in ANCA GN (Table 4), consistent with the concept that each glomerular lesion contributing to a decreased fraction of normal glomeruli needs to be considered [29]. In summary, proteinuria is an independent indicator of decreased normal glomeruli in ANCA GN. Because proteinuria and decreased fraction of normal glomeruli could reflect both specific renal involvement and systemic severity of AAV, we next determined extrarenal manifestation of AAV in association with proteinuria. Abbreviations: ANCA, antineutrophil cytoplasmic antibodies; GN, glomerulonephritis; IF/TA, interstitial fibrosis/tubular atrophy; IgG, immunoglobulin G; uACR, urinary albumin-to-creatinine ratio; uPCR, urinary protein-to-creatinine ratio. Abbreviations: ANCA, antineutrophil cytoplasmic antibodies; GN, glomerulonephritis; IgG, immunoglobulin G; SE, standard error; uACR, urinary albumin-to-creatinine ratio; uPCR, urinary protein-to-creatinine ratio.

Proteinuric Findings in Correlation with Extrarenal Manifestations of AAV
We next examined the correlation between proteinuria and extrarenal manifestation of AAV. We observed no association between proteinuria and systemic disease activity assessed by the BVAS despite previously observed correlation between proteinuria with more severe deterioration of kidney function in ANCA GN (Figure 4), implicating that proteinuria associates with decreased extrarenal manifestations of AAV. By systematic analysis of lung, sinus, joint, ear, eye, peripheral nerve, skin involvement or alveolar hemorrhage, we confirmed less involvement of sinus and joint in association with proteinuria ( Figure 4). In summary, these observations suggested that proteinuria reflected specific renal involvement in AAV rather that systemic disease activity.

Discussion
We here aimed to systematically describe the correlation between urinary findings and clinicopathological characteristics in ANCA GN. Proteinuria correlated with MPO subtype, diagnosis of MPA and severe deterioration of kidney function, in line with our previous observations [32]. Proteinuria subclassification established that higher levels of proteinuria in MPO subtype were attributed to nonselective glomerular proteinuria. At disease manifestation, renal involvement of AAV can either present with active lesions including glomerular crescents and necrosis, or with chronic lesions including global glomerular sclerosis. The pathologic activity and chronicity of ANCA GN can be classified by histopathological subgrouping (crescentic, mixed, focal and sclerotic) or ARRS (high, intermediate and low risk) [8,29]. Aforementioned previous studies have mainly focused on deterioration of kidney function in combination with histopathological findings in ANCA GN. However, there is recent evidence that the degree of proteinuria at diagnosis is associated with long-term renal outcome in ANCA GN [25,30,31]. Proteinuria is the hallmark of GN and the most important predictor of outcome, including diabetes-related and idiopathic glomerular kidney diseases [39][40][41][42][43][44][45][46][47]. We here show that categorization of ANCA GN into established histopathological scoring systems revealed that sclerotic class ANCA GN and ARRS high risk were associated with highest levels of proteinuria attributed to nonselective proteinuria, including both glomerular and tubular proteinuria. In contrast, lowest proteinuria was observed in focal class ANCA GN, in line with recent findings that proteinuria associates with established ANCA scoring systems showing lowest proteinuria in biopsies categorized as focal class, followed by mixed and crescentic class ANCA GN [33]. Multivariate regression analyses revealed that only decreased fraction of normal glomeruli independently associated with proteinuria. It has previously been proposed that damaged glomeruli have the capability to recover and active lesions may be reversible in principle, at least to a certain extent [5,30]. In addition, the fraction of normal glomeruli (without scarring, crescents or necrosis within the tuft) was the strongest independent predictor of death-censored ESRD in ANCA GN [29]. Therefore, each glomerular lesion needs to be considered, emphasizing the importance of normal glomeruli and an early diagnosis. Studies of repeat biopsies strengthen this hypothesis since previous studies have demonstrated the progression of active into chronic lesions in ANCA GN over time [30,48]. This could in part explain the variable outcomes of patients that are classified as crescentic and mixed in the validation of the classification [9,14,22,24].
Finally, there was no association between proteinuria and systemic disease activity assessed by the BVAS despite previously observed correlation between proteinuria with more severe deterioration of kidney function in ANCA GN. Since the BVAS also includes deterioration of kidney function, these results implicate that proteinuria associates with decreased extrarenal manifestations of AAV. We here provide evidence that proteinuria correlated with less involvement of sinus and joint in AAV. These observations suggested that proteinuria reflected specific renal involvement in AAV rather that systemic disease activity. This interesting phenomenon needs further investigation and has independently been observed [49].
The main limitations of our study are its retrospective design in a single center, a selection bias towards more severe cases of ANCA GN with limited information of kidney function before disease manifestation as a tertiary referral center and lack of long-term renal survival rates. Nevertheless, the number of patients with ANCA GN and severe deterioration of kidney function at our center is considerable.

Conclusions
In conclusion, proteinuria correlated with distinct clinicopathological characteristics in ANCA GN, mostly attributed to a reduced fraction of normal glomeruli. Furthermore, proteinuria in ANCA GN reflected specific renal involvement in AAV rather than systemic disease activity. Therefore, urinary findings could further improve our understanding of mechanisms promoting kidney injury and progression of ANCA GN.
Author Contributions: B.T. conceived the study, collected and analyzed data and wrote the first draft. D.T. and P.K. collected and analyzed data. P.S. and S.H. evaluated histopathological findings. D.T., P.K., P.S. and S.H. edited the manuscript. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Deidentified data are available on reasonable request from the corresponding author.