Non-sclerotic Glomeruli Research Articles

Podocyte density as a predictor of long-term kidney outcome in obesity-related glomerulopathy

Glomerulomegaly and focal segmental glomerulosclerosis are histopathological hallmarks of obesity-related glomerulopathy (ORG). Podocyte injury and subsequent depletion are regarded as key processes in the development of these glomerular lesions in patients with ORG, but their impact on long-term kidney outcome is undetermined. Here, we correlated clinicopathological findings and podocyte depletion retrospectively in patients with ORG. Relative (podocyte density) and absolute (podocyte number per glomerulus) measures of podocyte depletion were estimated using model-based stereology in 46 patients with ORG. The combined endpoint of kidney outcomes was defined as a 30% decline in estimated glomerular filtration rate (eGFR) or kidney failure. Patients with lower podocyte density were predominantly male and had larger body surface area, greater proteinuria, fewer non-sclerotic glomeruli, larger glomeruli and higher single-nephron eGFR. During a median follow-up of 4.1 years, 18 (39%) patients reached endpoint. Kidney survival in patients with lower podocyte density was significantly worse than in patients with higher podocyte density. However, there was no difference in kidney survival between patient groups based on podocyte number per glomerulus. Cox hazard analysis showed that podocyte density, but not podocyte number per glomerulus, was associated with the kidney outcomes after adjustment for clinicopathological confounders. Thus, our study demonstrates that a relative depletion of podocytes better predicts long-term kidney outcomes than does absolute depletion of podocytes. Hence, the findings implicate mismatch between glomerular enlargement and podocyte number as a crucial determinant of disease progression in ORG.

#1324 The antiproteinuric effect of Gliflozins is associated with the mitochondrial density of endothelial cells in diabetic patients

Abstract Background and Aims The sodium-glucose co-transporter type 2 inhibitors (SGLT2i) such as dapagliflozin and empagliflozin, are a new class of nephro- and cardio-protective drugs in diabetic and non-diabetic patients. SGLT2 is greatly expressed in the proximal tubule cells, and therefore the protective effects of gliflozins are thought to be mediated by a hemodynamic mechanism on glomerular hyperfiltration. However, they could be acting also in other cell types, particularly in diabetic patients, because hyperglycemia induces the expression of SGLT2 in endothelial cells, mesangial cells, cardiomyocytes, etc. Because SGLT2 changes the intracellular concentration of sodium and glucose, gliflozins may indirectly change mitochondrial swelling and activity. In this study, we have tested the hypothesis that gliflozins act on mitochondria in patients suffering from diabetes. Method We have analyzed kidney biopsies from diabetic patients in treatment with a gliflozin (n = 10) or without gliflozin treatment (n = 10) stained with fuchsin red, which gives a color intensity proportional to mitochondrial content. Clinical variables (albumin-creatinine ratio (ACR), blood pressure, eGFR, glycated albumin) have been measured before and after 4 months of treatment. Images have been acquired using a Zeiss Axioscope with 100x objective and analyzed using ImageJ software to quantify staining intensity (expressed as optical density) after background subtraction. Two different cell types have been considered: proximal tubule cells and endothelial cells in glomeruli. Only non-sclerotic glomeruli have been considered. Results The systolic blood pressure was linearly correlated to the intensity of staining of mitochondria in the proximal tubule cells in all patients irrespective of gliflozin treatment. In non-treated diabetic patients, the mitochondrial content of endothelial cells was not related to the extent of ACR reduction over time. In diabetic patients treated with gliflozins, the extent of ACR reduction after treatment was correlated with the intensity of mitochondrial staining in glomerular endothelial cells. This correlation was not present in proximal tubule cells. Gliflozin treatment may induce a change in the mitochondrial content of endothelial cells, particularly in patients responding with a reduction in proteinuria. Conclusion Proteinuria in diabetic patients depends on damage in both the endothelial and podocyte compartments. Gliflozins may improve proteinuria by a hemodynamic mechanism; however, our data suggest an effect of gliflozins specific on mitochondria of the endothelial cells in the glomeruli, which is proportional to their anti-proteinuric effect. Though we do not analyze the mechanism of gliflozins on mitochondria, it is possible to speculate that the changes of intracellular sodium-induced by gliflozins may induce an altered mitochondrial activity.

#2555 C4d—a novel predictor of the disease progression and treatment failure in primary focal segmental glomerulosclerosis?

Abstract Background and Aims Complement system seems to play a role in the pathogenesis of primary focal segmental glomerulosclerosis (pFSGS) where, due to unpredictable clinical course and outcome, novel prognostic factors are sought. The split product of C4 and tissue marker of complement activation, C4d, is a potential candidate whose prognostic significance was determined in this study. Method Patients >18 years with features consistent with pFSGS (full blown nephrotic syndrome, diffuse podocyte foot processes effacement and exclusion of known causes of FSGS), were recruited from our Hospital registry of kidney biopsies, from 2003 to 2021. Patients with end-stage renal disease (ESRD) defined as eGFR <15 ml/min/1.73 m2 were not included. Every renal biopsy specimen was analyzed by light, immunofluorescence and electron microscopy with additional immunochemistry for C4d performed on paraffin-embedded tissue using monoclonal rabbit antibody (detection system Ventana BenchMark Ultra). Samples with at least one non-globally sclerosed glomerulus (GSG) were examined by two renal pathologists who classified every non-GSG into C4d positive and negative. Based on ratio of C4d+ non-GGS/total N of non-GSG and C4d+ nonsclerotic glomeruli (NSG)/total N of NSG, patients were stratified into two groups: <50% and ≥50%. Primary outcome was defined as composite of ESRD and initial eGFR declining >50%. Failure to meet criteria for at least partial remission at the last follow-up visit according to KDIGO 2021 Guidelines was defined as treatment failure. Numeric variables are shown as median with interquartile range (IQR) and P value <0.05 was considered as significant. Results Relevant data of 58 included patients with renal outcomes are shown in Table. Median follow up was 90.7 (IQR 48-167) months. Patients with ≥50% of C4d+ non-GSG had higher proportion of treatment failure (Table) and worse renal survival (log-rank test, p = 0.006). In multivariate Cox regression analysis, after inclusion of age, sex, initial eGFR and 24-h proteinuria, IFTA, serum albumin and C4d category, only C4d ≥50% (HR = 4.077, 95% CI 1.101-15.09, p = 0.035) and IFTA (HR = 1.052, 95% CI 1.019-1.085, p = 0.002) remained independent predictors of progression to defined endpoint. Likewise, patients with ≥50% of C4d+ NSG/total N of NSG had higher proportion of treatment failure (chi-square, p = 0.03) and worse renal survival (log-rank, p = 0.037). Conclusion C4d deposition is an independent predictor of disease progression, treatment failure and renal survival in pFSGS. Significant influence of NSG-C4d deposition on disease progression may indicate pathophysiological role of the complement system activation in pFSGS independently of the presence of glomerular sclerosis.

#5390 INTENSIFIED B CELL DEPLETION THERAPY IN ANCA-VASCULITIS WITH SEVERE RENAL IMPAIRMENT AS COMPARED TO CONVENTIONAL IMMUNOSUPPRESSION

Abstract Background and Aims Rituximab (RTX) has shown to be an effective induction treatment for small-vessel vasculitides associated with antineutrophil cytoplasm antibodies (AAV) in both newly diagnosed and relapsing patients. However, the role of RTX in the management of the most severe cases of AAV remains to be fully elucidated. The aim of this study was to assess both safety and efficacy of an intensified B-cell depletion therapy (IBCDT) protocol, including RTX, cyclophosphamide (CYC), and methylprednisolone pulses without additional maintenance immunosuppressive therapy in a cohort of 15 AAV patients with the most severe features of AVV renal involvement (as <15 ml/min GFR and histological findings of paucimmune necrotizing glomerulonephritis with more than 50% crescents of non-sclerotic glomeruli at the renal biopsy). Method Results of the IBCDT regimen have been compared to those obtained in a control cohort of 10 patients with AAV treated with a conventional therapy regimen based on oral CYC and steroids followed by a prolonged maintenance therapy with azathioprine (AZA). Plasma exchange was equally employed in the study and the control group. Results Complete clinical remission (BVAS 0) was observed at 6 months in 14 of 15 patients treated with IBCDT (93%). All cases who achieved a complete clinical remission experienced a depletion of peripheral blood B cells at the end of therapy. Of the 10 dialysis dependent patients at onset, 6 subjects (60%) experienced a functional recovery allowing the suspension of dialysis treatment. When compared to the control group, no statistically significant difference was observed in patients treated with IBCDT in terms of overall survival, 6-month therapeutic response rate, and 6-, and 12-month functional renal recovery. The cumulative total dose of CYC in the case group was on average 1 g/patient while in the control group on average 8.5 g/patient (p = 0.00008). Conclusion Despite the retrospective design and relative limited sample size, IBCDT appeared to be safe and had the same efficacy profile when compared to the conventional therapy with CYC plus AZA in the management of the most severe patients with AAV. Additionally, this avoided the need of prolonged maintenance therapy for long, and limited the exposure to CYC with consequent reduced toxicity and drug-related side effect rates.

Single-Nephron GFR in Different Glomerular Basement Membrane Stages of Membranous Nephropathy.

Alterations in single-nephron dynamics have been demonstrated in animal models of membranous nephropathy (MN). This study applied a recently developed technique to estimate single-nephron parameters in human MN. Single-nephron GFR (SNGFR) and single-nephron urinary protein excretion (SNUPE) were calculated by dividing total GFR and UPE by the total estimated number of non-sclerotic glomeruli (NSG). The NSG number per kidney was estimated using cortical volume assessment and biopsy-based stereology. MN staging by electron microscopy was performed using Ehrenreich-Churg (EC) criteria. Single-nephron parameters were analyzed in relation to clinicopathological factors known to associate with disease outcomes. The study included 109 MN patients (mean age 65 years; 73% male; eGFR 62 mL/min, 36% on renin-angiotensin aldosterone system [RAAS] inhibitors pre-biopsy). EC stages were I, 19%;II, 49%; III, 26%; and IV, 6%. There was no difference in glomerular volume among EC stage groups. With advancing EC stage, SNGFR and SNUPE decreased from mean 56 to 42 nL/min and 5.1 to 3.8 μg/day, respectively. In multivariable models, EC stage was associated with SNGFR even after adjustment for key clinicopathological factors, such as reduced GFR, serum albumin, UPE, segmental glomerulosclerosis, chronic tubulointerstitial damage and pre-biopsy use of RAAS inhibitors. In contrast, EC stage was not associated with glomerular volume and SNUPE after multivariable adjustment. These results provide the first clinical evidence of alterations in single-nephron dynamics with advancing EC stage of human MN and support a role for disease-specific GBM structural lesions as determinants of SNGFR.

De novo posttransplant membranous nephropathy following BNT162b2 mRNA COVID-19 vaccine in a kidney transplant recipient

To the Editor: Mass COVID-19 vaccination programs in kidney transplant patients (KTRs) have demonstrated an excellent safety profile, without increased rates of ensuing alloimmune events. However, autoimmune diseases may seldom occur after vaccination. We report the case of a KTR who presented de novo posttransplant membranous nephropathy (MN) following administration of COVID-19 vaccine. A 66-year-old patient underwent first kidney transplantation for autosomal-dominant polycystic-kidney-disease-related end-stage kidney disease in June 2020. His medical history included hypertension and left nephrectomy in 2016. After basiliximab induction, the maintenance regimen consisted of tacrolimus, mycophenolic acid, and steroids. His baseline creatinine level was 100 μmol/L, and he remained free of proteinuria during the posttransplant course. A 3-month protocol graft biopsy was unremarkable. Immunohistochemistry (IHC) C4d staining was negative (Figure 1A,B). The patient received the second injection of the BNT162b2 mRNA COVID-19 vaccine (Pfizer-BioNTech) in March 2021. Subsequent serology tests showed negative results, consistent with a blunted antibody response to the vaccine, as widely reported in KTRs. Eight weeks after the second vaccine dose, the patient underwent a 1-year protocol graft biopsy. Concurrent creatinine level and proteinuria were 120 μmol/L and negative, respectively. Light microscopy showed normal glomeruli and ruled out subclinical rejection(Figure 1D). However, C4d staining revealed the presence of granular staining along the glomerular basement membrane consistent with stage 1 MN (Figure 1E). Anti-phospholipase-A2-receptor (PLA2R) immunochemistry analysis showed strong positive staining on the glomerular capillary walls (Figure 1F). Circulating PLA2R antibodies were negative. Notably, IHC staining for PLA2R and C4d was retrospectively negative in the 3-month protocol graft biopsy (Figure 1E) and in the native kidney specimen (nephrectomy). The residual renal parenchyma on the native kidney contained non-sclerotic glomeruli. We retrospectively performed Jones staining which did not show irregularities of the glomerular basement membrane suggestive of membranous nephropathy. There was no available frozen sample on the renal parenchyma. Hence, the diagnosis of de novo posttransplant MN was firmly established. Screening for malignancy and infections remained negative, excluding secondary causes of MN. COVID-19 tests remained also negative (SARS-CoV-2 PCR was negative at MN diagnosis and 3 months after the diagnosis. Anti-Spike IgG were negative 1 and 2 months after the diagnosis). This is the first case of de novo posttransplant MN following mRNA COVID-19 vaccine. Interestingly, cases of de novo or relapsing MN after COVID-19 vaccine have recently been reported.1Aydin MF Yildiz A Oruc A et al.Relapse of primary membranous nephropathy after inactivated SARS-CoV-2 virus vaccination.Kidney Int. 2021; 100 (doi:10.1016/j.kint.2021.05.001): 464-465Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 2Da Y Goh GH Khatri P A case of membranous nephropathy following Pfizer-BioNTech mRNA vaccination against COVID-19.Kidney Int. 2021; 100 (doi:10.1016/j.kint.2021.07.016): 938-939Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 3Gueguen L Loheac C Saidani N Khatchatourian L Membranous nephropathy following anti-COVID-19 mRNA vaccination.Kidney Int. 2021; S0085-2538 (doi:10.1016/j.kint.2021.08.006): 00752-00757Google Scholar Additionally, rare cases of MN (including one PLA2R+ case) have been described after COVID-19 infection.4Nasr SH Alexander MP Cornell LD et al.Kidney biopsy findings in patients with COVID-19, kidney injury, and proteinuria.Am J Kidney Dis. 2021; 77 (doi:10.1053/j.ajkd.2020.11.002): 465-468Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar The temporality between COVID-19 vaccination and PLA2R+ MN in a patient without history of MN and in whom the etiological workup has remained otherwise negative, supports a causal link. However, we must acknowledge that a coincidental association could not be ruled out. Given the lack of proteinuria, no specific treatment was introduced. The subclinical presentation of MN may be related to the mitigating effects of maintenance immunosuppressive therapy. In conclusion, auto-immune reactivity and MN may occur after mRNA COVID-19 vaccine. This report will raise awareness of nephrologists for the need to closely monitor patients with primary MN, following COVID-19 vaccination, including KTRs. If proteinuria occurs after vaccination, a graft biopsy should be promptly performed to firmly establish the diagnosis. We would like to thank Ms. Roberta Vellve for the English language review. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. Additional supporting information can be found online in the Supporting Information section at the end of this article. 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Associations between nephron number and podometrics in human kidneys

Podocyte loss and resultant nephron loss are common processes in the development of glomerulosclerosis and chronic kidney disease. While the cortical distribution of glomerulosclerosis is known to be non-uniform, the relationship between the numbers of non-sclerotic glomeruli (NSG), podometrics and zonal differences in podometrics remain incompletely understood. To help define this, we studied autopsy kidneys from 50 adults with median age 68 years and median eGFR 73.5 mL/min/1.73m2 without apparent glomerular disease in a cross-sectional analysis. The number of NSG per kidney was estimated using the physical dissector/fractionator combination, while podometrics were estimated using model-based stereology. The number of NSG per kidney was directly correlated with podocyte number per tuft and podocyte density. Each additional 100,000 NSG per kidney was associated with 26 more podocytes per glomerulus and 16 podocytes per 106 μm3 increase in podocyte density. These associations were independent of clinical factors and cortical zone. While podocyte number per glomerulus was similar in the three zones, superficial glomeruli were the smallest and had the highest podocyte density but smallest podocytes. Increasing age and hypertension were associated with lower podocyte number, with age mostly affecting superficial glomeruli, and hypertension mostly affecting juxtamedullary glomeruli. Thus, in this first study to report a direct correlation between the number of NSG and podometrics, we suggest that podocyte number is decreasing in NSG of individuals losing nephrons. However, another possible interpretation may be that more nephrons might protect against further podocyte loss.

Deep Learning Approaches for the Segmentation of Glomeruli in Kidney Histopathological Images

Deep learning is widely applied in bioinformatics and biomedical imaging, due to its ability to perform various clinical tasks automatically and accurately. In particular, the application of deep learning techniques for the automatic identification of glomeruli in histopathological kidney images can play a fundamental role, offering a valid decision support system tool for the automatic evaluation of the Karpinski metric. This will help clinicians in detecting the presence of sclerotic glomeruli in order to decide whether the kidney is transplantable or not. In this work, we implemented a deep learning framework to identify and segment sclerotic and non-sclerotic glomeruli from scanned Whole Slide Images (WSIs) of human kidney biopsies. The experiments were conducted on a new dataset collected by both the Siena and Trieste hospitals. The images were segmented using the DeepLab V2 model, with a pre-trained ResNet101 encoder, applied to 512 × 512 patches extracted from the original WSIs. The results obtained are promising and show a good performance in the segmentation task and a good generalization capacity, despite the different coloring and typology of the histopathological images. Moreover, we present a novel use of the CD10 staining procedure, which gives promising results when applied to the segmentation of sclerotic glomeruli in kidney tissues.

FC046: Automated Mest-C Classification in IGA Nephropathy using Deep-Learning based Segmentation

Abstract BACKGROUND AND AIMS IgA nephropathy prognosis depends on histological factors. The MEST-C score is used to grade those factors and assess the renal prognosis of this disease. Nevertheless, this manual evaluation is time-consuming, tedious and has a poor reproducibility. An automated analysis would be faster and more objective. This work aimed to use deep-learning techniques on whole kidney biopsies from patients suffering from IgA nephropathy to obtain an automated MEST-C score. METHOD We used a previously developed convolutional neural network (CNN) to isolate the cortical area. Then, two additional CNNs were independently trained. The first one was used to evaluate the Interstitial Fibrosis/Tubular Atrophy (IF/TA) and segment the glomeruli. The second one was used to segment the relevant glomerular lesions (mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis and crescents). A total of 95 kidney biopsies from patients suffering from IgA nephropathy were randomly sorted into either the training set or the validation set. From those samples, regions of interest (ROI) were selected and annotated to develop the two CNNs (358 ROI and 458 ROI, respectively). The main annotation categories for the first CNN were `non-sclerotic glomeruli’, `sclerotic glomeruli’, `normal tubules’, `atrophic tubules’, `veins’ and `arteries’. For the second CNN, the main categories were `mesangial hypercellularity’, `endocapillary hypercellularity’, `segmental glomerulosclerosis’ and `crescents. Then, the three CNNs were sequentially applied to the biopsies of 109 patients suffering from IgA nephropathy. From the data provided by the CNNs, an automated MEST-C was calculated. We assessed its performances to predict the criteria of the gold standard MEST-C (scored by nephropathologists) using Receiver Operator Characteristic (ROC). RESULTS The CNNs detected the renal structures of interest with good precision and recall up to 0.98 and 0.96 respectively for the non-sclerotic glomeruli. Normal and atrophic were also reliably recognized with 0.88 and 0.71 f-scores. Glomerular lesions were harder to recognize with a 0.67 f-score for mesangial hypercellularity, 0.76 f-score for endocapillary hypercellularity, 0.47 f-score for segmental glomerulosclerosis and 0.50 f-score for the crescents. Manual and automated evaluation of IF/TA were well correlated with a 0.76 Spearman coefficient (P < 0.001). In the application cohort, the criteria of the automated adapted MEST-C were compared with the criteria of the visual MEST-C using Receiver Operator Characteristic (ROC), we assessed the performances of the criteria of the automated MEST-C to predict the criteria of the manual MEST-C. Areas under the curve (AUC) were 0.82 for mesangial hypercellularity, 0.79 for endothelial hypercellularity and 0.82 for segmental glomerulosclerosis. Regarding IF/TA (the T criteria), the AUC were 0.86 for T1 and 0.84 for T2. Regarding the evaluation of crescents, the AUC were 0.76 for the C1 criteria and 0.88 for the C2 criteria. Those ROC were used to determine adapted thresholds for each criterion. CONCLUSION This deep-learning based tool can be used to segment relevant renal cortical structures and provide a reliable assessment of IF/TA. An automated MEST-C score can be provided by our tool. Nevertheless, improvements are needed to consider using this tool in clinical practice and obtain an objective evaluation on large and numerous samples. FIGURE 1:Kidney samples stained with Masson's trichrome before and after the two first consecutive convolutional neural networks predictions (B, C). B: Cortical area (red), capsule (deep blue), C: normal and atrophic tubules (red and orange), non-sclerotic glomeruli (yellow), sclerotic glomeruli (light blue), arteries (purple), vein (deep blue).FIGURE 2:Kidney samples stained with Masson's trichrome before (A) and after the last convolutional neural networks predictions (B). B: hilum (yellow), mesangial hypercellularity (red), endothelial hypercellularity (purple), segmental glomerulosclerosis (green).

Treating Patients With ANCA-Associated Vasculitis and Very Severe Renal Injury With an Intensified B Cell Depletion Therapy: Comparison With a Control Cohort Receiving a Conventional Therapy.

Rituximab (RTX), an anti-CD20 monoclonal antibody, has shown to be an effective induction treatment for small-vessel vasculitides associated with antineutrophil cytoplasm antibodies (AAV) in both newly diagnosed and relapsing patients. However, the role of RTX in the management of the most severe cases of AAV remains to be fully elucidated. The aim of this study was to assess both safety and efficacy of an intensified B-cell depletion therapy (IBCDT) protocol, including RTX, cyclophosphamide (CYC), and methylprednisolone pulses without additional maintenance immunosuppressive therapy in a cohort of 15 AAV patients with the most severe features of AVV renal involvement (as <15 ml/min GFR and histological findings of paucimmune necrotizing glomerulonephritis with more than 50% crescents of non-sclerotic glomeruli at the renal biopsy). Results of the IBCDT regimen have been compared to those obtained in a control cohort of 10 patients with AAV treated with a conventional therapy regimen based on oral CYC and steroids followed by a prolonged maintenance therapy with azathioprine (AZA). Plasma exchange was equally employed in the study and the control group. Complete clinical remission (BVAS 0) was observed at 6 months in 14 of 15 patients treated with IBCDT (93%). All cases who achieved a complete clinical remission experienced a depletion of peripheral blood B cells at the end of therapy. Of the 10 dialysis dependent patients at onset, 6 subjects (60%) experienced a functional recovery allowing the suspension of dialysis treatment. When compared to the control group, no statistically significant difference was observed in patients treated with IBCDT in terms of overall survival, 6-month therapeutic response rate, and 6-, and 12-month functional renal recovery. The cumulative total dose of CYC in the case group was on average 1 g/patient while in the control group on average 8.5 g/patient (p = 0.00008). Despite the retrospective design and relative limited sample size, IBCDT appeared to be safe and had the same efficacy profile when compared to the conventional therapy with CYC plus AZA in the management of the most severe patients with AAV. Additionally, this avoided the need of prolonged maintenance therapy for long, and limited the exposure to CYC with consequent reduced toxicity and drug-related side effect rates.

Identification of glomerulosclerosis using IBM Watson and shallow neural networks.

BackgroundAdvanced stages of different renal diseases feature glomerular sclerosis at a histological level which is observed by light microscopy on tissue samples obtained by performing a kidney biopsy. Computer-aided diagnosis (CAD) systems leverage the potential of artificial intelligence (AI) in healthcare to support physicians in the diagnostic process.MethodsWe propose a novel CAD system that processes histological images and discriminates between sclerotic and non-sclerotic glomeruli. To this goal, we designed, tested, and compared two artificial neural network (ANN) classifiers. The former implements a shallow ANN classifying hand-crafted features extracted from Regions of Interest (ROIs) by means of image-processing procedures. The latter, instead, employs the IBM Watson Visual Recognition System, which uses a deep artificial neural network making decisions taking the images as input, without the need to design any procedure for describing images with features. The input dataset consisted of 428 sclerotic glomeruli and 2344 non-sclerotic glomeruli derived from images of kidney biopsies scanned by the Aperio ScanScope System.ResultsBoth AI approaches allowed to very accurately distinguish (mean MCC 0.95 and mean Accuracy 0.99) between sclerotic and non-sclerotic glomeruli. Although the systems may seem interchangeable, the approach based on feature extraction and classification would allow clinicians to gain information on the most discriminating features. In fact, further procedures could explain the classifier’s decision by analysing which subset of features impacted the most on the final decision.ConclusionsWe developed a customizable support system that can facilitate the work of renal pathologists both in clinical and research settings.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s40620-021-01200-0.

Podometrics in Japanese Living Donor Kidneys: Associations with Nephron Number, Age, and Hypertension.

Podocyte depletion, low nephron number, aging, and hypertension are associated with glomerulosclerosis and CKD. However, the relationship between podometrics and nephron number has not previously been examined. To investigate podometrics and nephron number in healthy Japanese individuals, a population characterized by a relatively low nephron number, we immunostained single paraffin sections from 30 Japanese living-kidney donors (median age, 57 years) with podocyte-specific markers and analyzed images obtained with confocal microscopy. We used model-based stereology to estimate podometrics, and a combined enhanced-computed tomography/biopsy-specimen stereology method to estimate nephron number. The median number of nonsclerotic nephrons per kidney was 659,000 (interquartile range [IQR], 564,000-825,000). The median podocyte number and podocyte density were 518 (IQR, 428-601) per tuft and 219 (IQR, 180-253) per 106μm3, respectively; these values are similar to those previously reported for other races. Total podocyte number per kidney (obtained by multiplying the individual number of nonsclerotic glomeruli by podocyte number per glomerulus) was 376 million (IQR, 259-449 million) and ranged 7.4-fold between donors. On average, these healthy kidneys lost 5.63 million podocytes per kidney per year, with most of this loss associated with glomerular loss resulting from global glomerulosclerosis, rather than podocyte loss from healthy glomeruli. Hypertension was associated with lower podocyte density and larger podocyte volume, independent of age. Estimation of the number of nephrons, podocytes, and other podometric parameters in individual kidneys provides new insights into the relationships between these parameters, age, and hypertension in the kidney. This approach might be of considerable value in evaluating the kidney in health and disease.

Clinicopathological Correlation with Mesangial C4d Deposition in Primary Immunoglobulin A Nephropathy - A Descriptive study.

In immunoglobulin A (IgA) nephropathy, activation of lectin pathway leads to severe renal damage and more pronounced histological damage. As C4d is a marker of lectin pathway activation, the presence of mesangial C4d positivity will help in identifying those patients at risk. The study was conducted to study the prevalence of mesangial C4d positivity in patients with primary IgA nephropathy and to compare the clinical and histopathological features with C4d-positive and C4d-negative cases. It is a retrospective study conducted for four years. The inclusion criterion was IgA nephropathy with a minimum of four viable glomeruli. Biopsies with >25% of nonsclerotic glomeruli with mesangial positivity will be considered as positive for C4d. Seventy-six patients of IgA nephropathy were included of which mesangial C4d positivity was noted in 33 patients (43%). The mean age was 35 years. The male:female ratio was 2.3:1. The most common presentation was edema (56%) followed by microscopic hematuria (28%). Fifty-four patients were hypertensive. Among the clinical and laboratory parameters, absence of hematuria (P = 0.04) and presence of proteinuria (P = 0.02) showed a significant association with C4d positivity. The histological parameters in Oxford classification which had significant association with C4d positivity were segmental sclerosis (P = 0.01) and tubular atrophy (P = 0.001). Among 45 patients on follow-up with a maximum duration of 51 months, 10 developed end-stage renal disease of which four had C4d expression (0.05%) in the biopsy. Nearly half of IgA nephropathy patients have mesangial C4d positivity. Elevated creatinine with chronicity changes is more common in C4d-positive patients. Hence, C4d can be used as a marker for poor prognosis.

Nephron Number and Time to Remission in Steroid-Sensitive Minimal Change Disease

Rationale & ObjectiveThe response to corticosteroid therapy may differ among patients with minimal change disease (MCD). Previous studies have suggested that glomerular hypertrophy or low areal glomerular density in biopsy specimens, which may be related to fewer nephrons, is associated with such a difference. We examined the associations between nephron number and the therapeutic response to corticosteroids in patients with MCD.Study DesignRetrospective cohort study.Setting & Participants75 adult patients with a histologic diagnosis of MCD.ExposureNephron number per kidney estimated based on the combination of unenhanced computed tomography and nonsclerotic volumetric glomerular density in kidney biopsy specimens.OutcomesComplete remission and relapse following corticosteroid therapy.Analytical ApproachMultivariable Cox proportional hazard analyses of associations between factors, including nephron number, and outcomes.ResultsMean age of patients was 45.9 years and 60.0% were men. Patients had an estimated glomerular filtration rate of 64.6 mL/min/1.73 m2 and proteinuria of 8.7 g/d. The estimated total number of nonsclerotic glomeruli ranged from 1.07 to 18.77 ×105 per kidney among all patients. There were no significant differences in total amounts or selectivity of urinary protein excretion at biopsy among the tertile groups categorized by nephron number. All patients responded to corticosteroid therapy, but those with fewer nephrons had a delayed achievement of complete remission. Multivariable Cox proportional hazard analyses identified nephron number as a significant independent explanatory variable for the achievement of complete remission, with a hazard ratio of 1.10 (95% CI, 1.02-1.19)/100,000 nephrons per kidney. Nephron number in these patients was not associated with achievement of partial remission or relapse following complete remission.LimitationRetrospective design and sampling bias of needle biopsy.ConclusionsA small nephron number in patients with MCD is associated with longer time to complete remission.

P0119ARTIFICIAL INTELLIGENCE IN RENAL PATHOLOGY: IBM WATSON FOR THE IDENTIFICATION OF GLOMERULOSCLEROSIS

Abstract Background and Aims Artificial Intelligence (AI) is the branch of computer technology aimed at creating hardware and software systems solving problems similarly to human intelligence, whereas specifically machine learning (ML) is the “field of study that gives computers the ability to learn without being explicitly programmed”. Our study was carried out taking advantage of the Watson Visual Recognition System by IBM, an advanced AI tool based on ML able to classify complex visual content. The aim of the study was to train and test the ability of the system in recognizing sclerotic and non-sclerotic glomeruli. Method A dataset of 26 renal biopsies performed at the Nephrology unit of the Department of Emergency and Organ Transplantation (DETO), University of Bari, Italy, was used for the analysis. All biopsies were stained with Periodic Acid-Schiff (PAS) staining. Each bioptic section was acquired using Aperio ScanScope; glomeruli were identified and sclerotic glomeruli were marked in yellow, non-sclerotic ones in green. Annotations were validated by two renal pathologists. The final dataset consisted of 2772 glomeruli: 428 with sclerosis, 2344 with no sclerosis (ratio 1/5.5). The dataset was divided in three parts: training set (about 70% of the entire dataset), validation set (about 10%) and test set (about 20%). Watson Visual Recognition Service is customizable and the system can be trained on recognizing any visual content. Classifiers are created and trained uploading both positive (in our study, sclerotic glomeruli) and negative (non-sclerotic glomeruli) classes. The IBM Watson learning algorithm is not open, therefore in order to improve the performance of the system, it is necessary to train it with different models and choose the best one. Results We created all the possible models derived from the arrangement of the following 4 variables: color of the image (PAS staining or grey scales), dimension (original or resized), number of images in order to balance the positive and negative classes, binary (one class containing sclerotic glomeruli) or multi-class (two classes: sclerotic and non-sclerotic glomeruli). Every test had a cut-off of 0.5: if &amp;gt;0.5, the system considered the glomerulus belonging to the tested class. After validating all the models with all the variables considered, the best performing model was the following: grey scaled, resized and multi-class. This model was then tested with a different number of input images (300, 600, 900, 1200, and 1600). The models with the most numerous dataset have been created using data augmentation, a technique that virtually increases the number of available samples. Results show that the use of larger input datasets does not yield a better linear performance. In fact, models 900 and 1200 had worst performances than other models, the best performances of the system were reached with model 1600, both in recognizing sclerotic (positive class) and non-sclerotic (negative class) glomeruli (Table 1, Figure 1 and 2). Conclusion In our study, renal biopsy images were analyzed and classified using the IBM Watson Visual Recognition tool which was able to distinguish automatically and with very high accuracy between sclerotic and non-sclerotic glomeruli. This study focuses only on the glomerular compartment and the next step will be the recognition of intermediate lesions and other portion of renal tissue. Our results prove the potential of AI and ML techniques in supporting the activity of renal pathologists.

Single-Nephron GFR in Patients With Obesity-Related Glomerulopathy

IntroductionObesity-related glomerulopathy (ORG) is a slowly progressive kidney disease occurring in association with obesity. It is characterized histopathologically by glomerulomegaly, likely caused by single-nephron hyperfiltration that has not been demonstrated in humans because of technical difficulty in measuring single-nephron glomerular filtration rate (SNGFR) in the clinical setting.MethodsTotal glomerular number per kidney, with or without global glomerulosclerosis, was estimated by the combination of cortical volume assessment via unenhanced computed tomography and biopsy-based stereology. Mean glomerular volume was calculated from the measured area of glomerular tufts. Both SNGFR and single-nephron urinary protein excretion (SNUPE) were estimated by dividing values for estimated glomerular filtration rate and urinary protein excretion by the number of nonsclerotic glomeruli. Living kidney donors were used as healthy controls.ResultsA total of 48 ORG patients with average nonsclerotic glomerular numbers of 456,000 ± 235,000 per kidney were included. The values for SNGFR in ORG patients with chronic kidney disease (CKD) stages 1 and 2 were higher than for nonobese and obese controls (97 ± 43 vs. 59 ± 21 vs. 64 ± 21 nl/min, respectively, P = 0.001). Nonsclerotic glomerular number decreased with advancing stages of renal functional impairment. The presence of ORG with more advanced CKD stages was associated with lower SNGFR and marked elevation in SNUPE levels, with no difference in the mean glomerular volume between the stages.ConclusionsThese results provide functional evidence for single-nephron hyperfiltration in patients with ORG, and identify compensatory failure to maintain effective SNGFR as a feature of advanced-stage ORG.

Semantic Segmentation Framework for Glomeruli Detection and Classification in Kidney Histological Sections

The evaluation of kidney biopsies performed by expert pathologists is a crucial process for assessing if a kidney is eligible for transplantation. In this evaluation process, an important step consists of the quantification of global glomerulosclerosis, which is the ratio between sclerotic glomeruli and the overall number of glomeruli. Since there is a shortage of organs available for transplantation, a quick and accurate assessment of global glomerulosclerosis is essential for retaining the largest number of eligible kidneys. In the present paper, the authors introduce a Computer-Aided Diagnosis (CAD) system to assess global glomerulosclerosis. The proposed tool is based on Convolutional Neural Networks (CNNs). In particular, the authors considered approaches based on Semantic Segmentation networks, such as SegNet and DeepLab v3+. The dataset has been provided by the Department of Emergency and Organ Transplantations (DETO) of Bari University Hospital, and it is composed of 26 kidney biopsies coming from 19 donors. The dataset contains 2344 non-sclerotic glomeruli and 428 sclerotic glomeruli. The proposed model consents to achieve promising results in the task of automatically detecting and classifying glomeruli, thus easing the burden of pathologists. We get high performance both at pixel-level, achieving mean F-score higher than 0.81, and Weighted Intersection over Union (IoU) higher than 0.97 for both SegNet and Deeplab v3+ approaches, and at object detection level, achieving 0.924 as best F-score for non-sclerotic glomeruli and 0.730 as best F-score for sclerotic glomeruli.

The number of nephrons in different glomerular diseases.

BackgroundThe total number of nephrons has been measured mainly from post-mortem studies and only in selected populations. Data from living subjects are scanty, and direct comparisons among different glomerular diseases are lacking. The present work exploits modern methodology to estimate the total nephron number in glomerulopathies with prevalent proteinuria/nephrotic syndrome versus glomerulopathies with nephritic syndrome (IgA nephropathy (IgAN), lupus nephritis), thus extending previous observations about the number and function of glomeruli in different physiological and pathological states.MethodsThis is a retrospective study based on one hundred and seven patients who have undergone renal biopsy. The glomerular density has been estimated from the biopsy specimens and the total cortical volume has been obtained from ultrasound recordings. Stereological methods have been applied to calculate the total number of nephrons and their volume. The correlation between clinical parameters and quantitative morphological data have studied using the Pearson correlation coefficient (r).ResultsThe total number of nephrons inversely correlated with the systolic blood pressure (r = −0.4, p < 0.05). In proteinuric diseases, such as focal segmental glomerulo-sclerosis (FSGS), membranous nephropathy (MN) and diabetes, the change in estimated GFR (eGFR) directly correlated with the total number of non-sclerotic glomeruli (NSG) (r = 0.62, p < 0.01), whereas in nephritic syndrome no significant correlation was observed. The alterations in eGFR occurring in nephritic syndromes such as IgAN cannot be explained on the basis of the number of NSG.DiscussionThe fusion of the podocyte foot-processes that typically occurs in purely proteinuric diseases does not modify the glomerular filtration rate: therefore in these situations, the change in eGFR depends mainly on the number of available glomeruli. On the other side, the eGFR decrease occurring in nephritic syndromes, such as IgAN, cannot be explained simply on the basis of the number of NSG and likely depends on the substantial involvement of the mesangial axis. Future studies should verify whether these changes are reversible with appropriate therapy, thus reversing eGFR decrease.

Clinical and Pathology Findings Associate Consistently with Larger Glomerular Volume.

Background Glomerular volume increases when demand exceeds nephron supply, which may lead to glomerulosclerosis. It is unclear if determinants of glomerular volume are consistent between populations that differ by severity of comorbidities.Methods We studied kidney biopsy specimens from living kidney donors (n=2453) and patients who underwent radical nephrectomy for a renal tumor (n=780). We scanned specimen sections into high-resolution digital images, manually traced glomerular profiles, and calculated mean glomerular volumes using the Weibel-Gomez stereologic formula (separately for nonsclerosed glomeruli and globally sclerosed glomeruli). We then assessed the relationship of glomerular volume with age, clinical characteristics, and nephrosclerosis on biopsy specimen.Results Compared with kidney donors, patients with tumors were older and more frequently men, obese, diabetic, or hypertensive, had more glomerulosclerosis and interstitial fibrosis on biopsy specimen, and had 12% larger nonsclerosed glomeruli (P<0.001). In both populations, male sex, taller height, obesity, hypertension, and proteinuria associated with larger nonsclerosed glomeruli to a similar extent. In patients with tumors, diabetes, glomerulosclerosis >25%, and interstitial fibrosis >25% also associated with larger nonsclerosed glomeruli. Independent clinical predictors of larger nonsclerotic glomeruli were family history of ESRD, male sex, taller height, obesity, diabetes, and proteinuria. After adjustment for these characteristics, nonsclerotic glomerular volume did not differ between populations and was stable up to age 75 years, after which it decreased with age. Many of these findings were also evident with globally sclerotic glomerular volume.Conclusions Characteristics associated with glomerular volume are consistent between patient populations with low and high levels of comorbidity.

The Substantial Loss of Nephrons in Healthy Human Kidneys with Aging.

Nephron number may be an important determinant of kidney health but has been difficult to study in living humans. We evaluated 1638 living kidney donors at Mayo Clinic (MN and AZ sites) and Cleveland Clinic. We obtained cortical volumes of both kidneys from predonation computed tomography scans. At the time of kidney transplant, we obtained and analyzed the sections of a biopsy specimen of the cortex to determine the density of both nonsclerotic and globally sclerotic glomeruli; the total number of glomeruli was estimated from cortical volume×glomerular density. Donors 18-29 years old had a mean 990,661 nonsclerotic glomeruli and 16,614 globally sclerotic glomeruli per kidney, which progressively decreased to 520,410 nonsclerotic glomeruli per kidney and increased to 141,714 globally sclerotic glomeruli per kidney in donors 70-75 years old. Between the youngest and oldest age groups, the number of nonsclerotic glomeruli decreased by 48%, whereas cortical volume decreased by only 16% and the proportion of globally sclerotic glomeruli on biopsy increased by only 15%. Clinical characteristics that independently associated with fewer nonsclerotic glomeruli were older age, shorter height, family history of ESRD, higher serum uric acid level, and lower measured GFR. The incomplete representation of nephron loss with aging by either increased glomerulosclerosis or by cortical volume decline is consistent with atrophy and reabsorption of globally sclerotic glomeruli and hypertrophy of remaining nephrons. In conclusion, lower nephron number in healthy adults associates with characteristics reflective of both lower nephron endowment at birth and subsequent loss of nephrons.