Sclerotic Glomeruli Research Articles

Quantifying measurement uncertainty in renal transplant biopsy assessment.

Renal transplant biopsies provide insights into graft health and support decision making. The current evidence on links between biopsy scores and transplant outcomes suggests there may be numerous factors affecting biopsy scores. Here we adopt measurement science approach to investigate the sources of uncertainty in biopsy assessment and suggest techniques to improve its robustness. Histological assessments, Remuzzi scores, biopsy processing and clinical variables are obtained from 144 repeat biopsies originating from 16 deceased-donor kidneys. We conducted sensitivity analysis to find the morphometric features with highest discriminating power and studied the dependencies of these features on biopsy and stain type. The analysis results formed a basis for recommendations on reducing the assessment variability. Most morphometric variables are influenced by the biopsy and stain types. The variables with the highest discriminatory power are sclerotic glomeruli counts, healthy glomeruli counts per unit area, percentages of interstitial fibrosis and tubular atrophy as well as diameter and lumen of the worst artery. A revised glomeruli adequacy score is proposed to improve the robustness of the glomeruli statistics, whereby a minimum of 104 µm2 of cortex tissue is recommended to keep type 1 and type 2 error probabilities below 0.15 and 0.2. The findings are transferable to several biopsy scoring systems. We hope that this work will help practitioners to understand the sources of statistical uncertainty and improve the utility of renal biopsy.

Super-resolved highly multiplexed immunofluorescence imaging for precise protein localization and podocyte ultrastructure.

Deep insights into the complex cellular and molecular changes occurring during (patho-)physiological conditions are essential for understanding the interactions and regulation of proteins. This understanding is crucial for research and diagnostics. However, the effectiveness of conventional immunofluorescence and light microscope, tools for visualizing the spatial distribution of cells or proteins, are limited both in resolution and multiplexity in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, the inherent variability of samples and the optical resolution limit. The herein demonstrated combination of multiplex immunofluorescence imaging and super resolution microscopy offers a solution to these limitations by enabling the identification of different cell types and precise subcellular localization of proteins in tissue sections. In this study, we demonstrate the cyclic staining and de-staining of paraffin kidney sections, making it suitable for routine use and compatible with super-resolution microscopy for podocyte ultrastructural studies. We have further developed a computerized workflow for data processing which is accessible through available reagents and open-access code. As a proof of principle, we identified CDH2 as a marker for cellular lesions of sclerotic glomeruli in the nephrotoxic serum nephritis mouse model and cross-validated this finding with a human Nephroseq dataset indicating its translatability. In summary, our work represents an advance in multiplex imaging, which is crucial for understanding the localization of numerous proteins in a single FFPE kidney section and the compatibility with super-resolution microscopy to study ultrastructural changes of podocytes.

#2615 Altered peroxidasin expression pattern in IgA nephropathy

Abstract Background and Aims PXDN is a multifunctional heme peroxidase responsible for the crosslinking of type IV collagen protomers at basement membranes, playing a crucial role in the extracellular matrix dynamics. In renal pathophysiology, increased PXDN expression has been associated with pro-fibrotic and pro-carcinogenic mechanisms and we have shown that PXDN is expressed throughout all kidney compartments since the early stages of nephrogenesis. However, the role of PXDN in kidney health and disease remains largely unknown. In this work we performed a comparative analysis of PXDN expression between renal biopsy samples from IgA nephropathy (IgAN) patients and healthy kidney biopsy samples. IgAN was the selected CKD model due to its straightforward diagnosis by standard protocols for histopathological evaluation of kidney biopsies, and to the fact that the renal outcomes are consistently associated with several key pathologic features, including tubular atrophy and interstitial fibrosis. Correlations between PXDN expression and IgAN disease and kidney function parameters as well as with ECM proteins were also explored. Method Fluorescence immunohistochemistry for PXDN was performed in biopsy samples from IgAN patients (n = 20) and in biopsies from healthy kidney donors (n = 7). Proximal tubules were assessed by anti-CD15 fluorescent staining. Collagen III (COLIII) deposition was investigated in IgAN samples (n = 7) by immunofluorescence. Brightfield images were manually segmented into glomeruli, proximal and distal tubules, and interstitium, using QuPath software, and the fluorescence intensity measurements were obtained with ImageJ/Fiji. Results Results showed that, in comparison with healthy kidney samples, PXDN is significantly decreased in proximal and distal tubules of IgAN patients (P < 0.001 and P < 0.05, respectively) whereas it is increased in IgAN interstitium (P < 0.001). When IgAN samples were stratified by the presence or absence of chronic lesions (glomerular sclerosis and interstitial fibrosis) we observed that in the proximal tubules, PXDN is decreased even in areas without lesions while in the distal tubules the expression is compromised only in the lesioned areas. In the interstitium, PXDN shows a tendency of increase from healthy tissue to IgAN in areas without and with tubulointerstitial fibrosis, presenting a significant increase in the lesioned areas (P < 0.001). Concerning ECM deposition, results showed a significant correlation between the expression of PXDN and COLIII in sclerotic glomeruli (R = 0.8214; P = 0.0341) and in the tubulointerstitial fibrotic areas (R = 0.93; P = 0.0067). Moreover, although no differences were observed in the glomerular expression of PXDN between healthy and IgAN samples, when considering eGFR, a significant increase of PXDN was observed in both non-lesioned and sclerotic glomeruli from IgAN patients with eGFR bellow 60 ml/min/1,73 m2. In the interstitium, the expression of PXDN was also increased in tubulointerstitial fibrotic areas of IgAN patients with eGFR bellow 60 ml/min/1,73 m2, in comparison with the same areas in patients with higher eGFR values. Conclusions Overall, our study shows a differential expression of PXDN in IgAN, with loss in the tubular region that relates with the state of interstitial fibrosis, and gain in the glomerular and interstitial region where it correlates with COLIII compartment-specific deposition and with kidney function as assessed by eGFR. These data support, for the first time, the enrollment of PXDN in the topographic mechanism of IgA-mediated kidney lesion that deserves further investigations. Given that sclerosis and fibrosis are a common pathogenic mechanism of CKD, irrespective of the underlying etiology, we postulate that PXDN may serve as a novel therapeutic target for this disease.

Protein N-glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease.

Diabetes is expected to directly impact renal glycosylation, yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). We used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in FFPE sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single nuclei transcriptomics (snRNAseq) data was used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them identified twelve of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine (LacNAc) epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that are common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and LacNAc synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

Clinicopathological characteristics and predictors of outcome of rapidly progressive glomerulonephritis: a retrospective study.

Globally, there are regional and time-based variations in the prevalence, etiology, and prognosis of rapidly progressive glomerulonephritis (RPGN). Prognosis of RPGN is poor, with a higher risk of death and end stage renal disease (ESRD) even with immunosuppressive medications. In the Middle East and North Africa, the studies on this disease are very limited. Therefore, we determined the predictors of outcome of RPGN. We retrospectively assessed 101 adult patients over age of 18, diagnosed with RPGN based on renal biopsy illustrating crescents in ≥ 50% of the glomeruli. Patients who had crescents in their renal biopsies that were < 50% and those who refused to consent to a renal biopsy were excluded. We categorized the patients into 3 groups based on immunohistochemistry; type I, type II and type III. Then, depending on renal loss, we divided them into ESRD and non-ESRD groups. The clinical history and physical examination were retrieved. Additionally, 24-hour urine protein, urine analysis, renal function tests, serum albumin, complete blood count, antinuclear antibodies, anti-double stranded DNA antibodies, ANCA antibodies and serum complement levels were checked. Each patient underwent a kidney biopsy for immunohistochemistry and light microscopy. The percentage of crescentic glomeruli, number of sclerosed glomeruli, tertiary lymphoid organ (TLO), neutrophil infiltration, endocapillary or mesangial hypercellularity, interstitial fibrosis with tubular atrophy (IFTA) were analyzed. Primary outcomes (remission, ESRD and mortality) and secondary outcomes were assessed. Type II was the most frequent cause of RPGN (47.5%), followed by type III (32.7%) and type I (19.8%). 32 patients (31.7%) died during follow up, whereas 60 patients (59.4%) developed ESRD. In 41 patients (40.6%), remission occurred. Oliguria, serum creatinine, and need for HD at presentation were significantly increased in ESRD group compared to non-ESRD group (P < 0.001 for each). Mesangial proliferation, IFTA, TLO formation, sclerotic glomeruli and fibrous crescents were also significantly increased in ESRD group in comparison to non-ESRD group (P < 0.001 for each). Glomerulosclerosis (P = 0.036), and IFTA (P = 0.008) were predictors of ESRD. Infections (P = 0.02), respiratory failure (P < 0.001), and heart failure (P = 0.004) were mortality risk factors. Type II RPGN was the most common. Infection was the most frequent secondary outcome. Oliguria, glomerulosclerosis, the requirement for hemodialysis at presentation, IFTA and TLO formation were predictors of ESRD. Respiratory failure, heart failure and infections were significant predictors of mortality.

Clinical and pathological findings of IgA nephropathy following SARS-CoV-2 infection

The features of IgA nephropathy (IgAN) after SARS-CoV-2 infection have not been well characterized. In this study, we compared the clinical and pathological characteristics of patients with IgAN who had experienced SARS-CoV-2 infection to those who had not. We conducted a retrospective study that enrolled 38 patients with biopsy-proven IgAN following SARS-CoV-2 infection with 4 months (post-SARS-CoV-2 infection group) and 1154 patients with IgAN prior to the pandemic (pre-SARS-CoV-2 infection group). Among the SARS-CoV-2 group cases, 61% were females. The average duration from SARS-CoV-2 infection to renal biopsy was 78.6 days. Prior to SARS-CoV-2 infection, the patients had different presentations of nephropathy. One patient had isolated hematuria, two had isolated proteinuria, twenty presented with both hematuria and proteinuria, and one patient had elevated serum creatinine. Additionally, there were eight cases with uncertain nephropathy history, and six cases did not have a history of nephropathy. Following SARS-CoV-2 infection, five patients experienced gross hematuria, one case exhibited creatinine elevation, and five cases showed an increase in proteinuria. The group of patients infected with SARS-CoV-2 after the COVID-19 pandemic exhibited older age, higher hypertension ratio and lower eGFR values compared to the pre-SARS-CoV-2 infection group. As for pathological parameters, a higher proportion of patients in the post-SARS-CoV-2 infection group exhibited a higher percentage of sclerotic glomeruli and glomerular ischemic sclerosis. There were no significant differences observed between the two groups in terms of therapy involving steroids, immunosuppressants, or RAS inhibitors. IgA nephropathy patients who were infected with SARS-CoV-2 were generally older and experienced more severe kidney damage compared to those without SARS-CoV-2 infection.

A Machine Learning-Driven Virtual Biopsy System For Kidney Transplant Patients

In kidney transplantation, day-zero biopsies are used to assess organ quality and discriminate between donor-inherited lesions and those acquired post-transplantation. However, many centers do not perform such biopsies since they are invasive, costly and may delay the transplant procedure. We aim to generate a non-invasive virtual biopsy system using routinely collected donor parameters. Using 14,032 day-zero kidney biopsies from 17 international centers, we develop a virtual biopsy system. 11 basic donor parameters are used to predict four Banff kidney lesions: arteriosclerosis, arteriolar hyalinosis, interstitial fibrosis and tubular atrophy, and the percentage of renal sclerotic glomeruli. Six machine learning models are aggregated into an ensemble model. The virtual biopsy system shows good performance in the internal and external validation sets. We confirm the generalizability of the system in various scenarios. This system could assist physicians in assessing organ quality, optimizing allograft allocation together with discriminating between donor derived and acquired lesions post-transplantation.

Development of a Kidney Prognostic Score in a Japanese Cohort of Patients With Antineutrophil Cytoplasmic Autoantibody Vasculitis

Glomerulonephritis is frequent in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and crucial to disease outcomes. We conducted a detailed assessment of renal pathology in Japanese patients with AAV, and developed a new score that would predict renal outcome. Two hundred twenty-one patients who were diagnosed with AAV and underwent a kidney biopsy were enrolled. Data on glomerular, tubular, interstitial, and vascular lesions from kidney biopsies were analyzed; the 3 established classification and prognostic scoring systems (Berden Classification, Mayo Clinic/RPS Chronicity Score [MCCS], and ANCA Renal Risk Score [ARRS]) were validated. Further, we developed a new prognostic score by including variables relevant for Japanese patients with ANCA-glomerulonephritis. Median follow-up was 60 months (interquartile range: 6-60). End-stage kidney disease (ESKD) risk prediction by the MCCS and the ARRS was confirmed. Moreover, our analysis identified 4 items with significant ESKD risk prediction capacity, namely percentage of cellular, fibrocellular, and fibrous crescents; and sclerotic glomeruli. Based on our findings, we created a score evaluating the percentage of these lesions to total glomeruli, the Percentage of ANCA Crescentic Score (PACS). The area under the receiver operating characteristic (ROC) curve evaluating PACS was 0.783. The PACS had a comparable performance as the ARRS in predicting ESKD. The optimal PACS cut-off for ESKD risk over 60 months was 43%. In addition, the percentage of cellular crescents and presence of interstitial inflammation were independent predictors of kidney function recovery. We developed a new score predicting renal prognosis using histopathological data of Japanese patients with ANCA-glomerulonephritis. Studies are needed to validate our results in international cohorts.

Application of cloud server-based machine learning for assisting pathological structure recognition in IgA nephropathy

BackgroundMachine learning (ML) models can help assisting diagnosis by rapidly localising and classifying regions of interest (ROIs) within whole slide images (WSIs). Effective ML models for clinical decision support require...

Pre-Treatment Serum Complement 3 Levels And Rituximab Response In Idiopathic Membranous Nephropathy

Objective: The efficacy of rituximab has been reported in patients with idiopathic membranous nephropathy (MN). We aimed to evaluate the relationship between biochemical tests at diagnosis, immunohistochemical profile, and rituximab response in patients with idiopathic MN unresponsive to other therapies in our center. Material and Methods: In this study, nine patients with idiopathic MN who received other immunosuppressive therapies between 2017-2022 and who underwent renal biopsy in our center were evaluated. Pre-treatment phospholipase A2 antibody levels were positive in 6 patients, and antibodies could not be analyzed in 3 patients. All patients received rituximab ≥2 grams after renin-angiotensin-aldosterone system (RAAS) blocker, cyclophosphamide, steroid, and calcineurin inhibitors. Results: Of the nine patients included in the study, 7 (78.2%) were male, and the mean age was 39.713.2 years. Four patients had complete remission (CR) with rituximab treatment, and five had partial remission (PR). Sclerotic glomeruli count, IgG, A, M, C1q, C3, C4d, fibrinogen, kappa and lambda staining, tubular atrophy, and interstitial fibrosis findings on renal biopsy were similar. However, the serum complement 3 (C3) level was significantly lower within normal limits (1.22  0.26 vs 1.560  0.56 p=0.016). The mean arterial pressure was significantly higher (96.22.5 mmHg vs 84.754.27 mmHg, p=0.018) in patients with partial remission compared to those with complete remission. Conclusion: A low baseline serum C3 level within normal limits before treatment in patients with idiopathic membranous nephropathy may help predicting unresponsiveness to other immunosuppressive therapies and partial response to rituximab treatment.

Correlating Deep Learning-Based Automated Reference Kidney Histomorphometry with Patient Demographics and Creatinine.

Reference histomorphometric data of healthy human kidneys are largely lacking due to laborious quantitation requirements. Correlating histomorphometric features with clinical parameters through machine learning approaches can provide valuable information about natural population variance. To this end, we leveraged deep learning, computational image analysis, and feature analysis to associate the relationship of histomorphometry with patient age, sex, serum creatinine (SCr), and estimated glomerular filtration rate (eGFR) in a multinational set of reference kidney tissue sections. A panoptic segmentation neural network was developed and used to segment viable and sclerotic glomeruli, cortical and medullary interstitia, tubules, and arteries/arterioles in the digitized images of 79 periodic acid-Schiff-stained human nephrectomy sections showing minimal pathologic changes. Simple morphometrics (e.g., area, radius, density) were quantified from the segmented classes. Regression analysis aided in determining the association of histomorphometric parameters with age, sex, SCr, and eGFR. Our deep-learning model achieved high segmentation performance for all test compartments. The size and density of glomeruli, tubules, and arteries/arterioles varied significantly among healthy humans, with potentially large differences between geographically diverse patients. Glomerular size was significantly correlated with SCr and eGFR. Slight, albeit significant, differences in renal vasculature were observed between sexes. Glomerulosclerosis percentage increased and cortical density of arteries/arterioles decreased, as a function of increasing age. Using deep learning, we automated precise measurements of kidney histomorphometric features. In the reference kidney tissue, several histomorphometric features demonstrated significant correlation to patient demographics, SCr, and eGFR. Deep learning tools can increase the efficiency and rigor of histomorphometric analysis.

A large-scale retrospective study enabled deep-learning based pathological assessment of frozen procurement kidney biopsies to predict graft loss and guide organ utilization

Lesion scores on procurement donor biopsies are commonly used to guide organ utilization for deceased-donor kidneys. However, frozen sections present challenges for histological scoring, leading to inter- and intra-observer variability and inappropriate discard. Therefore, we constructed deep-learning based models to recognize kidney tissue compartments in hematoxylin &eosin-stained sections from procurement needle biopsies performed nationwide in years 2011-2020. To do this, we extracted whole-slide abnormality features from 2431 kidneys, and correlated with pathologists’ scores and transplant outcomes. A Kidney Donor Quality Score (KDQS) was derived and used in combination with recipient demographic and peri-transplant characteristics to predict graft loss or assist organ utilization. The performance on wedge biopsies was additionally evaluated. Our model identified 96% and 91% of normal/sclerotic glomeruli respectively; 94% of arteries/arterial intimal fibrosis; 90% of tubules. Whole-slide features of Sclerotic Glomeruli (GS)%, Arterial Intimal Fibrosis (AIF)%, and Interstitial Space Abnormality (ISA)% demonstrated strong correlations with corresponding pathologists’ scores of all 2431 kidneys, but had superior associations with post-transplant estimated glomerular filtration rates in 2033 and graft loss in 1560 kidneys. The combination of KDQS and other factors predicted one- and four-year graft loss in a discovery set of 520 kidneys and a validation set of 1040 kidneys. By using the composite KDQS of 398 discarded kidneys due to “biopsy findings”, we suggest that if transplanted, 110 discarded kidneys could have had similar survival to that of other transplanted kidneys. Thus, our composite KDQS and survival prediction models may facilitate risk stratification and organ utilization while potentially reducing unnecessary organ discard.

Fibroblast Growth Factor 23 Neutralizing Antibody Ameliorates Abnormal Renal Phosphate Handling in Sickle Cell Disease Mice.

We assessed the involvement of fibroblast growth factor 23 (FGF23) in phosphaturia in sickle cell disease (SCD) mice. Control and SCD mice were treated with FGF23 neutralizing antibody (FGF23Ab) for 24 hours. Serum ferritin was significantly increased in SCD mice and was significantly reduced in female but not male SCD mice by FGF23Ab. FGF23Ab significantly reduced increased erythropoietin in SCD kidneys. Serum intact FGF23 was significantly increased in SCD female mice and was markedly increased in SCD male mice; however, FGF23Ab significantly reduced serum intact FGF23 in both genotypes and sexes. Serum carboxy-terminal-fragment FGF23 (cFGF23) was significantly reduced in SCD IgG male mice and was markedly but not significantly reduced in SCD IgG female mice. FGF23Ab significantly increased cFGF23 in both sexes and genotypes. Serum 1,25-dihydroxyvitamin D3 was significantly increased in SCD IgG and was further significantly increased by FGF23Ab in both sexes and genotypes. Significantly increased blood urea nitrogen in SCD was not reduced by FGF23Ab. The urine phosphate (Pi)/creatinine ratio was significantly increased in SCD in both sexes and was significantly reduced by FGF23Ab. Increased SCD kidney damage marker kidney injury molecule 1 was rescued, but sclerotic glomeruli, increased macrophages, and lymphocytes were not rescued by short-term FGF23Ab. FGF23Ab significantly reduced increased phospho-fibroblast growth factor receptor 1, αKlotho, phosphorylated extracellular signal-regulated kinase, phosphorylated serum/glucocorticoid-regulated kinase 1, phosphorylated sodium-hydrogen exchanger regulatory factor-1, phosphorylated janus kinase 3, and phosphorylated transducer and activator of transcription-3 in SCD kidneys. The type II sodium Pi cotransporter (NPT2a) and sodium-dependent Pi transporter PiT-2 proteins were significantly reduced in SCD kidneys and were increased by FGF23Ab. We conclude that increased FGF23/FGF receptor 1/αKlotho signaling promotes Pi wasting in SCD by downregulating NPT2a and PIT2 via modulation of multiple signaling pathways that could be rescued by FGF23Ab.

Molecular architecture of proliferative lupus nephritis as elucidated using 50-plex imaging mass cytometry proteomics

Due to unique advantages that allow high-dimensional tissue profiling, we postulated imaging mass cytometry (IMC) may shed novel insights on the molecular makeup of proliferative lupus nephritis (LN). This study interrogates the spatial expression profiles of 50 target proteins in LN and control kidneys. Proliferative LN glomeruli are marked by podocyte loss with immune infiltration dominated by CD45RO+, HLA-DR+ memory CD4 and CD8 T-cells, and CD163+ macrophages, with similar changes in tubulointerstitial regions. Macrophages are the predominant HLA-DR expressing antigen presenting cells with little expression elsewhere, while macrophages and T-cells predominate cellular crescents. End-stage sclerotic glomeruli are encircled by an acellular fibro-epithelial Bowman's space surrounded by immune infiltrates, all enmeshed in fibronectin. Proliferative LN also shows signs indicative of epithelial to mesenchymal plasticity of tubular cells and parietal epithelial cells. IMC enabled proteomics is a powerful tool to delineate the spatial architecture of LN at the protein level.

Long-term follow-up of IgA nephropathy: clinicopathological features and predictors of outcomes.

The establishment of the Oxford classification and newly developed prediction models have improved the prognostic information for immunoglobulin A nephropathy (IgAN). Considering new treatment options, optimizing prognostic information and improving existing prediction models are favorable. We used random forest survival analysis to select possible predictors of end-stage kidney disease among 37 candidate variables in a cohort of 232 patients with biopsy-proven IgAN retrieved from the Norwegian Kidney Biopsy Registry. The predictive value of variables with relative importance >5% was assessed using concordance statistics and the Akaike information criterion. Pearson's correlation coefficient was used to identify correlations between the selected variables. The median follow-up period was 13.7 years. An isolated analysis of histological variables identified six variables with relative importance >5%: T %, segmental glomerular sclerosis without characteristics associated with other subtypes (not otherwise specified, NOS), normal glomeruli, global sclerotic glomeruli, segmental adherence and perihilar glomerular sclerosis. When histopathological and clinical variables were combined, estimated glomerular filtration rate (eGFR), proteinuria and serum albumin were added to the list. T % showed a better prognostic value than tubular atrophy/interstitial fibrosis (T) lesions with C-indices at 0.74 and 0.67 and was highly correlated with eGFR. Analysis of the subtypes of segmental glomerulosclerosis (S) lesions revealed that NOS and perihilar glomerular sclerosis were associated with adverse outcomes. Reporting T lesions as a continuous variable, normal glomeruli and subtypes of S lesions could provide clinicians with additional prognostic information and contribute to the improved performance of the Oxford classification and prognostic tools.

#4834 THE PROPORTION OF UNAFFECTED GLOMERULI IS A ROBUST PROGNOSTIC FACTOR OF KIDNEY OUTCOME IN PATIENTS WITH ANCA-ASSOCIATED GLOMERULONEPHRITIS

Abstract Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA) associated glomerulonephritis (GN), the most common form of secondary GN in elderly (&amp;gt;60 years), requires immunosuppressive treatment that may increase risk of opportunistic infections. This study evaluated the prognostic value of clinical factors and histopathologic findings affecting kidney outcome in ANCA-associated GN patients. Method From 2000 to 2018, we identified 106 adults (≥18 years old) who were pathologically confirmed as ANCA-associated GN. The number of normal, crescent, and sclerotic glomeruli was recorded for each biopsy by slide review. The primary outcome was incident end stage kidney disease (ESKD). Results The age was 67 (57-73) years, the estimated glomerular filtration rate (eGFR) was 19 (11-36) mL/min/1.73 m2, and % of normal glomeruli in kidney specimen was 25 (11-47) %. Overall kidney survival was 85% and 76% at 1 and 5 years, respectively. Among clinical variables, lower eGFR (adjusted hazard ratio [aHR], 0.339; 95% confidence interval [CI], 0.180-0.638; p&amp;lt;0.001) was independently associated with increased risk of incident of ESKD. Multivariate Cox proportional hazard model including both clinical and histological variables demonstrated that % of normal glomeruli (aHR, 0.960; 95% CI, 0.935-0.986; p&amp;lt;0.001) was a sole risk factor of incident ESKD, independently of eGFR and other pathological findings. When the % of normal glomeruli was divided by quartiles, the risk of incident ESKD in the lowest quartile was significantly increased compared to the highest quartile (&amp;lt;11%; HR, 5.986; 95% CI, 2.114-16.950 vs. &amp;gt;46%; p&amp;lt;0.001). Conclusion The proportion of unaffected glomeruli was independent predictor of kidney outcome in patients with ANCA-associated GN.

#6881 ELEVATED LEVELS OF THE C5B9 TERMINAL COMPLEMENT COMPLEX IN PATIENTS RECEIVING RENAL REPLACEMENT THERAPY

Abstract Background and Aims The complement cascade and its inhibitors play an important role in health and disease. Complement activation represents a key pathogenic mechanism underlying inflammatory diseases affecting the kidney. The study aimed to investigate the C5b9 system in renal biopsies, where we stained all obtained kidney biopsies from the year 2017 (n = 256). Method Antibody staining against terminal C5b9 was performed, and a C5b9 score for each glomerulus (n = 1942) was generated based on automated image analysis. Mean scores for the interstitium, tubules and blood vessels, and whole biopsy sections were also calculated. Results Globally and segmentally, sclerosed areas in glomeruli stained much stronger than their non-sclerosed glomerular counterparts. The results were linked with Renal Replacement Therapy (RRT) status and clinical data from the Norwegian Kidney Biopsy Registry. The study's main findings were that elevated levels of C5b9 were present in patients on RRT in glomeruli, blood vessels and whole section staining. Fibrillar glomerulopathy and Membranous Glomerulonephritis had the highest C5b9 scores, while glomeruli from Hypertensive Nephrosclerosis were represented with a low score. C5b9 scores correlated weakly or non-significantly with clinical variables like age, blood pressure and eGFR. The degree of fibrosis showed an associated positive correlation to C5b-9 staining intensities. Additionally, patients with an elevated C5b9 score (from the 50th percentile and above) showed a significantly elevated risk of receiving RRT within 60 months of biopsy. Conclusion C5b9 levels were significantly elevated in sclerotic glomeruli as well as in patients receiving RRT. A weak correlation between C5b9 scores and clinical variables for renal function suggest that the C5b9 complement system may be an independent effector in CKD.

#3084 EXPLORATION OF PROGNOSTIC FACTORS IN DIABETIC NEPHROPATHY WITH NODULAR LESION USING TRANSCRIPTOME ANALYSIS OF HUMAN KIDNEY TISSUES

Abstract Background and Aims Although the speed of eGFR decline is usually rapid in patients with diabetic nephropathy (DMN) and a nodular lesion (NL), some patients with NL experience a slow eGFR decline. The factors associated with the difference in prognosis remain unknown. In this study, we aimed to explore prognostic factors by performing transcriptome analysis on preserved residual renal biopsy specimens from patients with pathologically diagnosed and prognostically known DMN with nodules. Method Patients who had an annual eGFR drop rate &amp;gt; 5 mL/min/1.73 m2 were defined as rapid decliners (RD) and the others as slow decliners (SD). Twelve patients (RD = 6 and SD = 6) who underwent kidney biopsy, had no other renal comorbidities, and had been followed up for at least one year were included. Total RNA was isolated from renal biopsy tissues in formalin-fixed, paraffin-embedded blocks, and microarray analysis was done. Results At the time of renal biopsy, the eGFR, urine protein, and rate of sclerotic glomeruli were not statistically different between the RD and SD groups, at 49.7±10.1 mL/min/1.73 m2 and 57.5±12.4 mL/min/1.73 m2 (p = 0.258), 7.5±4.2 g/gCre and 4.0±2.7 g/gCre (p = 0.123), and 19.2±13.4% and 25.5±14.4% (p = 0.450), respectively. In microarray analyses (total 58,341 gene probes), genes with little expression (raw signals &amp;lt;50) and with little differences between RD and SD (processed signals &amp;lt;0.3) were excluded, leaving 14,227 genes for further analysis. A total of 1,496 differentially expressed genes (DEGs) were selected with p-values &amp;lt;0.05 and fold change ≥2 between the RD and SD group for further bioinformatic analysis. Using the Ingenuity Pathway Analysis system, 15 canonical pathways and 9 diseases or functions annotations were found to be enriched. In addition, upstream regulator analysis identified 73 upstream regulators. Among them, only two regulators, CBX5 and CCN5, showed p &amp;lt;0.05 and absolute z-score &amp;gt; = 2 as activated and inhibited regulators, respectively, in the RD group. We next performed regulator effects analysis, indicating one regulator network which identified the key upstream regulator CCN5. The regulator network also showed a small number of genes involved in the viral infection, which included CDH1, CD24, and ESR1. Additionally, IPA showed 25 interaction networks ranging from 11 to 38. The fifth-ranked network was associated with Cancer, Organismal Injury, and Abnormalities (score = 32), in which CD24 and ESR1 were included. We, therefore, performed qRT-PCR to compare the expression levels of CDH1, CD24, and ESR1 in the RD group with those in the SD group. The expression levels of CD24 and ESR1 in the RD group were downregulated compared with those in the SD group (p = 0.004 and p = 0.022, respectively) although there was little difference in the level of CDH1 between them (p = 0.638). Conclusion Transcriptomic analysis using renal biopsied tissue revealed that ESR1 and CD24 might be candidates for renoprotective factors in DMN with NL.

#6722 A RARE CASE OF ACUTE KIDNEY INJURY IN A PATIENT GRANULOMATOSIS WITH POLYANGIITIS, MONOCLONAL GAMMOPATHY WITH RENAL SIGNIFICANCE AND AMYLOIDOSIS

Abstract Introduction Anti - neutrophil cytoplasmic antibody /ANCA/- associated vasculitis is a small vessel vasculitis. It is a rare, autoimmune, inflammatory disease, with undetermined etiology, including granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis. It could lead to pauci - immune necrotizing and crescentic glomerulonephritis and rapidly progressive glomerulonephritis. Monoclonal gammopathy of renal significance /MGRS/ is a plasma cell or B-cell disorder that could lead to renal impairment due to production of monoclonal protein, which do not meet the criteria for myeloma multiplex. There is a significant higher risk in progression to end stage renal disease/ESRD/ in groups with renal vasculitis and concomitant disease due to nonrenal factors. Clinical case We present a rare clinical case of a patient with combination of ANCA -vasculitis, MGRS and amyloidosis. A 60 year-old male patient with deteriorated renal function was presented to the clinic. Chronic congestive heart failure, arterial hypertension, situs inversus, anemic syndrome, hepatosplenomegaly were reported as concomitant diseases. Due to progressive worsening of renal function emergency hemodialysis treatment was initiated through temporary vascular access. Against the background of several chronic diseases, which could lead to chronic kidney failure, the echographic image of the kidneys stands out. Both kidneys are enlarged, with increased echogenicity of the parenchyma as in a diffuse renal process. The performed immunological tests confirm the diagnosis of granulomatosis with polyangiitis /formerly called Wegener's/, PR3-105,7. The results for the screened viral infections was negative. An episode of severe pulmonary edema was registered in the context of obstruction of the hemodialysis catheter due to coagulation disorders. The renal biopsy supports the diagnosis of vasculitis, crescentic glomerulonephritis - in advanced stage of development, probably a morphological manifestation of secondary nephropathy. An interesting finding is the presence of amyloidosis /AL/. The immunofixation blood test identify monoclonal IgG kappa type component. A sternal puncture, SPECT/CT was performed and diagnosis of multiple myeloma was excluded. Pathogenic treatment with corticosteroids and cyclophosphamide was initiated. In addition a short-term change in vision is observed in the context of the underlying disease. A consultation with ophthalmologist and neurologist was held, confirming cerebral arteritis. At this stage the recovery of renal function is incomplete. High flux membrane were used during dialysis treatment to reduce large and middle sized molecules. In addition, for the treatment of light chain amyloidisis, bortezomib was considered. Conclusion This clinical case emphasizes on the need to precisely clarify the cause that led to acute kidney injury. A thorough search for the underlying disease causing the deterioration of kidney function is critical for the renal outcome. The reasons for incomplete recovery of renal function during active treatment of vasculitis should be sought in underlying diseases-arterial hypertension, chronic heart failure, amyloidosis. Discussion Younger patients with lower eGFR at the onset of ANCA-associated vasculitis and no concomitant disease show significant improvement in renal function by the end of the first year regardless the need of dialysis treatment. Patients with severe accompanying disease have slow and insufficient improvement in renal function due to the prevalence of higher percentage of globally sclerotic glomeruli. Considering the severe prognosis of ANCA-associated vasculitis it is crucial to start early treatment. The state of the renal function at the first year of pathogenic treatment along with the pathological findings of the renal biopsy could be a predictors for the renal function course.

#2775 USING A DEEP LEARNING MODEL TO EVALUATE PATHOLOGICAL INJURY OF DIABETIC KIDNEY DISEASE

Abstract Background and Aims Early diagnosis and evaluation play an important role in preventing the progression of diabetic kidney disease (DKD). Renal biopsy is the gold standard of DKD diagnosis. In 2010, the Renal Pathology Society (RPS) developed a consensus classification for DKD, which classifies DKD glomerular lesions and scores for tubulointerstitial lesion. However, as the pathologic heterogeneity of DKD patients and unparallel relationship between pathology features and clinical symptoms, it remains controversial whether is reliable to use RPS classification for renal outcomes prediction. Besides, the inconsistence between pathologists might magnify the gap between prediction and outcome. More reliable methods for DKD pathology assessment are needed. The development of machine learning (ML) algorithms especially convolutional neural networks (CNNs) enables it possible, which can provide automatic and precise analysis for complicated images. Thus, this study aimed to construct a ML-based model to assist and provide automatic decision supports to clinical doctors. Method We examined 204 DKD renal biopsied whole-slide images (WSIs) of Periodic Acid Schiff staining from Southeast University Institute of Nephrology whose database came from more than 30 hospital centers. All the WSIs were randomly annotated by three pathologists. We established a consecutive CNNs framework: DKD pathology evaluation system (DPES). It consists of three parts: 1. Based on classic UNet architecture, we developed a segmentation model whose backbone is replaced by ResNet 50 architecture for detecting glomeruli. 2. According to RPS classification, a CNN added with channel attention mechanism is built to discriminate sclerotic glomeruli, glomeruli with K-W node, and glomeruli with mesangial expansion. Through this method, the entire WSIs were divided into RPS grades I-IV. 3. For the analysis of tubulointerstitial lesion, we constructed a segmentation model as previous description to calculate the area of the atrophic tubule and its percentage of the total tubules. Results For glomerular assessment, compared with other ML networks, DPES achieved better performance in segmentation for glomeruli (Intersection over Union (IOU): 0.82, Precision: 0.89, Accuracy: 0.91) and tubulointerstitial (atrophy (TA): IOU: 0.9;Precision: 0.94; Accuracy: 0.95; non-tubular atrophy (NT): IOU: 0.84; Precision: 0.90; Accuracy: 0.93). Interstitial fibrosis and tubular atrophy (IFTA) from network compared with three pathologists didn’t show statistically difference. Conclusion Our findings demonstrated that the DPES achieved similar performance with three pathologists. It suggests that machine learning algorithms can generate reliable results and provide more supports for clinical decision.