Renal Cell Carcinoma in End-Stage Renal Disease: A Retrospective Study in Patients from Hungary.

INTRODUCTION: End-stage renal disease (ESRD) and acquired cystic kidney disease (ACKD) are known risk factors for renal cell carcinoma (RCC). Hereby, the clinicopathological features of RCCs developed in ESRD were investigated. METHODS: A database consisting of 34 tumors from 31 patients with ESRD among 2,566 nephrectomy samples of RCC was built. The demographic, clinical, and follow-up data along with pathological parameters were analyzed. The RCCs were diagnosed according to the current WHO Classification of Urinary and Male Genital Tumors. RESULTS: Twenty-two tumors developed in men and 12 in women, with a median age of 56 years (range: 27-75 years). The causes of ESRD were glomerulonephritis (n = 7), hypertensive kidney disease (n = 6), autosomal dominant polycystic kidney disease (n = 6), chronic pyelonephritis (n = 4), diabetic nephropathy (n = 3), chemotherapy-induced nephropathy (n = 1), and undetermined (n = 4). ACKD complicated ESRD in 12 patients. The following histological subtypes were identified: clear cell RCC (n = 19), papillary RCC (n = 5), clear cell papillary tumor (n = 5), ACKD RCC (n = 3), and eosinophilic solid and cystic RCC (n = 2). The median tumor size was 31 mm (range: 10-80 mm), and 32 tumors were confined to the kidney (pT1-pT2). There was no tumor-specific death during the period of this study. Progression was registered in 1 patient. CONCLUSION: In our cohort, the most common RCC subtype was clear cell RCC (55%), with a frequency that exceeded international data appreciably (14-25%). The incidence of clear cell papillary tumor and ACKD RCC (14.7% and 8.5%) was lower than data reported in the literature (30% and 40%). Our results indicate a favorable prognosis of RCC in ESRD.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies.

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

A unique case of anti-GBM disease with concomitant anti-PLA2R positivity.

BACKGROUND: Concomitant occurrence of anti-GBM disease and anti-PLA2R positive membranous nephropathy have been previously described. However, to the best of our knowledge, this is the first case report that documents the co-occurrence of the diseases proven by both serologic and histologic methods. CASE PRESENTATION: A 51-year-old woman presented to hospital with nausea, bilateral lower extremity edema, dyspnea, dark urine, and then anuria. Symptoms developed one month after an upper respiratory tract infection. Laboratory results showed acute kidney injury, and hypoalbuminemia. Immunologic examination revealed both anti-GBM and anti-PLA2R positivity. Kidney biopsy demonstrated the histological features of Goodpasture's disease and anti-PLA2R positive membranous nephropathy. Steroid, cyclophosphamide, and plasmapheresis were commenced. Despite the combined immunosuppressive, the patient remained on renal replacement therapy. CONCLUSIONS: Microbial kidney injury can trigger multiple autoimmune diseases. The simultaneous occurrence of anti-glomerular basement (anti-GBM) disease and membranous nephropathy is extremely rare. Delayed recognition leads to delayed treatment, causing worse renal and patient outcomes, as well as increased financial costs.

A reference tissue atlas for the human kidney.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Kidney biopsy-based epidemiologic analysis shows growing biopsy rate among the elderly.

Systematic registration and examination of biopsy-related data in Central and Eastern Europe are scarce, while the health condition of the population is worse compared to other more developed countries. We aim to create a database and analyze the distribution and temporal variation of the renal biopsy diagnoses in Hungary, including the effect of the recent coronavirus pandemic. The diagnoses were standardized according to the recommendation of the European Renal Association. Native biopsy samples processed between January 1, 2006, and December 31, 2020, were analyzed. During the 15 years, 2140 native kidney biopsies were performed. The number of samples increased from 24.5 to 57.9 per million person-years and the median age from 37 to 51 years (p < 0.0001). The predominance of glomerular diseases was stable. The most frequent glomerulopathy was IgA nephropathy (21.5%), followed by focal segmental glomerulosclerosis (17.7%), and membranous nephropathy (15.7%). Trends showed the rise of ANCA-associated vasculitis. During the coronavirus pandemic, there was a decrease in the number of kidney biopsies and the proportion of membranous nephropathies. The diagnostic trends in our database showed increasing biopsy rates among the elderly and the growing frequencies of age-related diseases, which emphasizes the importance of altering medical focus according to demographic changes in this area.

A COVID-19-association-dependent categorization of death causes in 100 autopsy cases.

From March through December 2020, 100 autopsies were performed (Semmelweis University, Budapest, Hungary), with chart review, of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection demonstrated by real-time reverse-transcription polymerase chain reaction testing (mean age, 74.73 years, range 40-102 years; 50 males, mean age 71.96 years, and 50 females, mean age 77.5 years). Classified by the date of death, 21 cases were from the pandemic's "first wave" (March through July) and 79 from the "second wave" (August through December). Three mortality categories were defined by relevance of SARS-CoV-2 infection: (1) "strong" association (n=57), in which COVID-19 was primary responsible for death; (2) "contributive" association (n=27), in which a pre-existing condition independent of COVID-19 was primary responsible for death, albeit with substantial COVID-19 co-morbidity; (3) "weak" association (n=16), in which COVID-19 was minimally or not at all responsible for death. Distributions among categories differed between the first wave, in which the "contributive" association cases dominated (strong: 24%, contributive: 48%, weak: 28%), and the second wave, in which the "strong" association cases dominated (strong: 66%, contributive: 21%, weak: 13%). Charted co-morbidities included hypertension (85 %), cardiovascular diseases (71 %), diabetes (40 %), cerebrovascular diseases (31 %), chronic respiratory diseases (30 %), malignant tumors (20 %), renal diseases (19 %), diseases of the central nervous system (15 %), and liver diseases (6 %). Autopsy evaluation analyzed alterations on macroscopy as well as findings on microscopy of scanned and scored sections of formalin-fixed, paraffin-embedded tissue samples (50-80 blocks/case). Severity of histological abnormalities in the lung differed significantly between "strong" and "contributive" (p<0.0001) and between "strong" and "weak" categories (p<0.0001). Abnormalities included diffuse alveolar damage, macrophage infiltration, and vascular and alveolar fibrin aggregates (lung), with macro- and microvascular thrombi and thromboemboli (lung, kidney, liver). In conclusion, autopsies clarified in what extent COVID-19 was responsible for death, demonstrated the pathological background of clinical signs and symptoms, and identified organ alterations that led to the death. Clinicopathologic correlation, with conference discussions of severity of co-morbidities and of direct pathological signs of disease, permitted accurate categorization of cause of death and COVID-19 association as "strong," "contributive," or "weak." Lung involvement, with reduced ventilatory capacity, was the primary cause of death in the "strong" and "contributive" categories. Shifts in distribution among categories, with "strong" association between COVID-19 and death dominating in the second wave, may reflect improved clinical management of COVID-19 as expertise grew.

A multimodal and integrated approach to interrogate human kidney biopsies with rigor and reproducibility: guidelines from the Kidney Precision Medicine Project.

Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and personalized therapies for diseases. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate three-dimensional (3-D) molecular atlases of healthy and diseased kidney biopsies by using multiple state-of-the-art omics and imaging technologies across several institutions. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single-cell level or in 3-D space is a significant challenge that can be a futile exercise if not well controlled. We describe a "follow the tissue" pipeline for generating a reliable and authentic single-cell/region 3-D molecular atlas of human adult kidney. Our approach emphasizes quality assurance, quality control, validation, and harmonization across different omics and imaging technologies from sample procurement, processing, storage, shipping to data generation, analysis, and sharing. We established benchmarks for quality control, rigor, reproducibility, and feasibility across multiple technologies through a pilot experiment using common source tissue that was processed and analyzed at different institutions and different technologies. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before their being approved to interrogate clinical biopsy specimens. The process established economizes the use of valuable biopsy tissue for multiomics and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and serves as a model for precision medicine projects across laboratories, institutions and consortia.

The impact of belatacept on the phenotypic heterogeneity of renal T cell-mediated alloimmune response: The critical role of maintenance treatment and inflammatory load.

Belatacept offers superior long-term outcome relative to calcineurin inhibitor (CNI)-based immunosuppression. However, the higher frequency of early T cell-mediated rejection (TCMR) in belatacept-treated patients hampered the widespread adoption of costimulation blockade. Here, we applied gene expression analysis and whole-slide inflammatory cell quantification to assess the impact of belatacept on intragraft immune signature. We studied formalin-fixed, paraffin-embedded renal biopsies from 92 patients stratified by histopathologic diagnosis (TCMR, borderline changes, or normal) and immunosuppression regimen (belatacept, CNI). An interaction model was built to explore maintenance treatment-dependent expression level changes of immune response-related genes across diagnostic categories of normal, borderline changes, and TCMR. Ninety-one percent of genes overexpressed in TCMR showed significant correlation with whole section inflammatory load. There were 27 genes that had a positive association with belatacept treatment. These were mostly related to myeloid cells and innate immunity. Genes negatively associated with costimulation blockade (n = 14) could be linked to B-cell differentiation and proliferation. We concluded that expression levels of genes characteristic of TCMR are strongly interconnected with quantitative changes of the biopsy inflammatory load. Our results might suggest differential involvement of the innate immune system, and an altered B-cell engagement during TCMR in belatacept-treated patients relative to CNI-treated referents.

Novel In Situ Hybridization and Multiplex Immunofluorescence Technology Combined With Whole-slide Digital Image Analysis in Kidney Transplantation.

The elusive nature of assessing immunological processes in situ in organ transplantation is one of the major impediments to improve diagnostics and treatment. Here, we present a proof-of-concept study using multiplexed in situ hybridization (ISH) (RNAscope) to detect low-abundance cytokines in formalin-fixed paraffin-embedded (FFPE) human transplant kidney biopsies in combination with immunofluorescence (IF) for cell phenotyping. We show that a multiplex IF and ISH (mIFISH) assay is feasible to identify the cellular source of cytokines and chemokines (tumor necrosis factor-α, interferon-γ, and CXCL9) in FFPE transplant kidney biopsies and that quantification of the mRNA and protein signal is also possible at single-cell resolution in the context of tissue complexity. Furthermore, the mIFISH assay allows precise quantitative assessment of tubulitis, one of the key morphological correlates of alloimmune injury. Simultaneous in situ identification and quantification of multiple cellular phenotypes and mRNA expression of proinflammatory cytokines in FFPE tissues offer a novel insight into the biology of alloimmune injury in kidney transplantation and may contribute to improved diagnostic accuracy and patient care.

Further Evidence That the Soluble Urokinase Plasminogen Activator Receptor Does Not Directly Injure Mice or Human Podocytes.

BACKGROUND: The role of the soluble urokinase plasminogen activator receptor (suPAR) in focal segmental glomerulosclerosis (FSGS) as the circulating factor or as a predictor of recurrence after transplantation remains controversial. Previously published studies in mice and isolated podocytes produced conflicting results on the effect of suPAR on podocyte injury, effacement of foot processes, and proteinuria. These discordant results were in part due to diverse experimental designs and different strains of mice. The aim of our study was to determine the reasons for the inconsistencies of the previous studies results with suPAR by using uniform methods and studies in different strains of mice. METHODS: We utilized a primary culture of human podocytes and 2 mouse models, the wild type (WT) and the urokinase plasminogen activator receptor (uPAR) KO (uPAR), in an attempt to resolve the reported conflicting results. RESULTS: In both WT and uPAR mouse models, injection of recombinant uPAR, even at a high dose (100 µg), did not induce proteinuria, effacement of podocytes, or disruption of the cytoskeleton. Injection of suPAR resulted in its deposition exclusively in the glomerular endothelial cells and not in the podocytes of WT mice and was not detected at the uPAR KO mice. Kidneys from patients with recurrent FSGS had negative immunostaining for uPAR. We also evaluated the effect of recombinant uPAR on primary culture of human podocytes. uPAR did not result in podocytes damage. CONCLUSIONS: suPAR by itself is not the cause for direct podocyte injury, in vitro or in vivo. These findings suggest a more complex and still poorly understood role of suPAR in FSGS.

Assessment of 19 Genes and Validation of CRM Gene Panel for Quantitative Transcriptional Analysis of Molecular Rejection and Inflammation in Archival Kidney Transplant Biopsies.

Background: There is an urgent need to develop and implement low cost, high-throughput standardized methods for routine molecular assessment of transplant biopsies. Given the vast archive of formalin-fixed and paraffin-embedded (FFPE) tissue blocks in transplant centers, a reliable protocol for utilizing this tissue bank for clinical validation of target molecules as predictors of graft outcome over time, would be of great value. Methods: We designed and optimized assays to quantify 19 target genes, including previously reported set of tissue common rejection module (tCRM) genes. We interrogated their performance for their clinical utility for detection of graft rejection and inflammation by analyzing gene expression microarrays analysis of 163 renal allograft biopsies, and subsequently validated in 40 independent FFPE archived kidney transplant biopsies at a single center. Results: A QPCR (Fluidigm) and a barcoded oligo-based (NanoString) gene expression platform were compared for evaluation of amplification of gene expression signal for 19 genes from degraded RNA extracted from FFPE biopsy sections by a set protocol. Increased expression of the selected 19 genes, that reflect a combination of specific cellular infiltrates (8/19 genes) and a graft inflammation score (11/19 genes which computes the tCRM score allowed for segregation of kidney transplant biopsies with stable allograft function and normal histology from those with histologically confirmed acute rejection (AR; p = 0.0022, QPCR; p = 0.0036, barcoded assay) and many cases of histological borderline inflammation (BL). Serial biopsy shaves used for gene expression were also processed for in-situ hybridization (ISH) for a subset of genes. ISH confirmed a high degree of correlation of signal amplification and tissue localization. Conclusions: Target gene expression amplification across a custom set of genes can identify AR independent of histology, and quantify inflammation from archival kidney transplant biopsy tissue, providing a new tool for clinical correlation and outcome analysis of kidney allografts, without the need for prospective kidney biopsy biobanking efforts.

Targeted Transcriptional Profiling of Kidney Transplant Biopsies.

INTRODUCTION: Studies are needed to assess the quality of transcriptome analysis in paired human tissue samples preserved by different methods and different gene amplification platforms to enable data comparisons across experimenters. METHODS: RNA was extracted from kidney biopsies, either submerged in RNA-stabilizing solution (RSS) or stored in formalin-fixed, paraffin-embedded (FFPE) blocks. RNA quality and integrity were compared. Gene expression of the common rejection module and other immune cell genes were quantified for both tissue preservation methods in the same sample using conventional quantitative polymerase chain reaction (QPCR) by 2 different commercial platforms, (fluidigm [FD]) or barcoded-oligos (nanostring [NS]). RESULTS: RNA quality was inferior in FFPE tissues. Despite this, gene expression for 19 measured genes on the same sample, stored in FFPE or RSS, were strongly correlated on the FD (r = 0.81) or NS platforms (r = 0.82). For the same samples, interplatform gene expression correlations were excellent (r = 0.80) for RSS and moderate (r = 0.66) for FFPE. Significant differences in gene expression were confirmed on both platforms (FD: P = 1.1E-03; NS: P = 2.5E-04) for biopsy-confirmed acute rejection. CONCLUSION: Our study provided supportive evidence that despite a low RNA quality of archival FFPE kidney transplantation tissue, small quantities of this tissue can be obtained from existing paraffin blocks to provide a viable and rich biospecimen source for focused gene expression assays. In addition, reliable and reproducible gene expression evaluation can be performed on these FFPE tissues using either a QPCR-based or a barcoded-oligo approach, which provides opportunities for collaborative analytics.

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

In Situ Hybridization and Double Immunohistochemistry for the Detection of VEGF-A mRNA and CD34/Collagen IV Proteins in Renal Transplant Biopsies.

Quantitative metrics on the tissue distribution of different cell phenotypes, extracellular matrix components, and signaling/cell cycle markers hold the promise for the advent of new-generation tissue-based predictive/prognostic biomarkers in clinical diagnostics. The workflow of this approach is composed of three major phases: (1) detection of multiple molecular targets on a single histologic section, (2) image acquisition, and (3) digital image processing and analysis. Here, we present the most prevalent current alternatives for step (1) and describe a three-plex staining and image acquisition platform that captures the spatial distribution of macromolecules from two different species.

Peritubular capillary basement membrane multilayering in early and advanced transplant glomerulopathy: quantitative parameters and diagnostic aspects.

The ultrastructural quantitative aspects of peritubular capillary basement membrane multilayering (PTCBML) were examined in 57 kidney transplant biopsies with transplant glomerulopathy (TG). The measurements included three cutoffs [permissive: 1 PTC with 5 basement membrane (BM) layers, intermediate: 3 PTCs with 5 layers or 1 PTC with 7 layers, strict: 1 PTC with 7 layers and 2 PTCs with 5 layers] and the mean number of BM layers (PTCcirc). Two groups were assigned, namely patients with mild TG (Banff cg1a and cg1b) and those with moderate-to-severe TG (cg2 and cg3). Their respective clinical, serological, and morphological characteristics were then compared. The clinical data revealed that mild TG corresponded to early chronic antibody-mediated rejection (cABMR), while moderate-to-severe TG corresponded to the advanced stage of the disease. The permissive threshold displayed the lowest specificity (73 %) and the highest sensitivity (83 %) for moderate-to-severe TG, and its corresponding PTCcirc value was 3 layers. In contrast, the strict threshold-adopted by the Banff 2013 classification-displayed a specificity and sensitivity of 93 and 52 %, respectively, and the corresponding PTCcirc was 4 layers. In mild TG, 26 % of the cases met the permissive cutoff and 6 % the strict cutoff. Mild TG was associated with a lower PTCcirc (2.6 layers vs 4.5 layers in moderate-to-severe TG; p < 0.0001). Amongst the various criteria, the permissive criterion was associated most frequently with mild TG, and had prognostic relevance. Because of this, we propose its usage as a marker of early cABMR-induced PTCBML if non-alloimmune causes of PTCBML can be ruled out.

Morphologic Features and Clinical Impact of Arteritis Concurrent with Transplant Glomerulopathy.

Little is known about the morphology and clinical relevance of arteritis in renal allograft biopsies with transplant glomerulopathy. We retrospectively reviewed the morphologic findings and clinical course of 59 patients with cg, 16 of which featured concurrent arteritis (fibrosing intimal arteritis with luminal narrowing in 15, and acute intimal arteritis in 1 case). Fifteen out of the 16 cases with arteritis fulfilled the morphological diagnostic criteria for chronic active antibody-mediated rejection, and 11 cases with arteritis showed morphological evidence of concurrent, ongoing T-cell-mediated alloimmune response (acute T-cell-mediated rejection in 5, borderline changes in 6 cases). Further, the Banff grades of interstitial inflammation in scarred and nonscarred cortex, total cortical inflammation, and arterial luminal narrowing were significantly higher in biopsies with arteritis. By immunohistochemistry, T-lymphocyte predominance over macrophages was found in the intimal infiltrates in 14 out of 16 cases, and cytotoxic T-lymphocytes were identified among intimal mononuclears in 10 cases. Patients with arteritis demonstrated a significantly shorter renal survival (7.5 vs. 29 months). In conclusion, T-cell-mediated mechanisms could play a role in the development of arteritis concurrent with cg. However, this finding does not exclude the possibility that antibody-mediated rejection can also contribute to the evolution of the lesion. Importantly, the lesion carries negative prognostic value likely via severe arterial luminal narrowing.