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Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

The major goals of the Kidney Precision Medicine Project (KPMPP) are to establish a molecular atlas of the kidney in health and disease and improve our understanding of the molecular drivers of CKD and AKI. In this clinical-pathologic-molecular correlation, we describe the case of a 38-year-old woman without any history of CKD who underwent a research kidney biopsy in the setting of AKI suspected to be due to nonsteroidal anti-inflammatory use after cesarean section delivery. The participant's histopathology was consistent with mild acute tubular injury, without significant interstitial fibrosis or tubular atrophy. This diagnosis was supported by analysis of the glomerular and tubulointerstitial proteomes. The proteomic interrogation revealed a molecular landscape that demonstrated differences in kidney prostaglandin synthesis that may be in response to nonsteroidal anti-inflammatory drugs and signs of intrarenal inflammation and fibrosis that were not evident by histopathology alone., (Copyright © 2022 by the American Society of Nephrology.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with increased levels of autoantibodies targeting immunological proteins such as cytokines and chemokines. Reports further indicate that COVID-19 patients may develop a broad spectrum of autoimmune diseases due to reasons not fully understood. Even so, the landscape of autoantibodies induced by SARS-CoV-2 infection remains uncharted territory. To gain more insight, we carried out a comprehensive assessment of autoantibodies known to be linked to diverse autoimmune diseases observed in COVID-19 patients in a cohort of 231 individuals, of which 161 were COVID-19 patients (72 with mild, 61 moderate, and 28 with severe disease) and 70 were healthy controls. Dysregulated IgG and IgA autoantibody signatures, characterized mainly by elevated concentrations, occurred predominantly in patients with moderate or severe COVID-19 infection. Autoantibody levels often accompanied anti-SARS-CoV-2 antibody concentrations while stratifying COVID-19 severity as indicated by random forest and principal component analyses. Furthermore, while young versus elderly COVID-19 patients showed only slight differences in autoantibody levels, elderly patients with severe disease presented higher IgG autoantibody concentrations than young individuals with severe COVID-19. This work maps the intersection of COVID-19 and autoimmunity by demonstrating the dysregulation of multiple autoantibodies triggered during SARS-CoV-2 infection. Thus, this cross-sectional study suggests that SARS-CoV-2 infection induces autoantibody signatures associated with COVID-19 severity and several autoantibodies that can be used as biomarkers of COVID-19 severity, indicating autoantibodies as potential therapeutical targets for these patients., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

In patients with severe #COVID19, increased levels of autoantibodies against PAR1 were found. These might serve as allosteric agonists of PAR1 on endothelial cells and platelets, and thus might contribute to the pathogenesis of microthrombosis in COVID-19. https://bit.ly/3pqM9Vv., Competing Interests: Conflict of Interest: T. Bahmer reports grants from BMBF (unrestricted research grant for the German Center for Lung Research (DZL) and National Pandemic Cohort Network (NAPKON) – Coordinating Study Site for population-based cohort platform); lecture fees from Novartis, AstraZeneca, and Chiesi; support for attending the American Thoracic Society Conference from Chiesi; and advisory board participation with GlaxoSmithKline, Boehringer Ingelheim, Roche and AstraZeneca, outside the submitted work. D. Frank reports grants from DFG (467267736), BMBF and DZHK; consulting fees and support for attending meetings from Edwards Lifesciences and Medtronic; lecture honoraria from Edwards Lifesciences, Medtronic, Astra Zeneca, Pfizer, BMS, Novartis, Bayer and Abbott; and participation on advisory boards with BMS, Boehringer Ingelheim, Daiichi Sankyo, outside the submitted work. A.Z. Rosenberg reports grants from the NIH (NIDDK and NHLBI) outside the submitted work. CellTrend is owned by K. Schulze-Forster; CellTrend produces ELISA kits for the determination of antibodies against GPCR. G. Riemekasten reports consulting fees from CellTrend GmbH outside the submitted work. S. Schreiber reports consulting fees from Abbvie, Arena, BMS, Biogen, Celltrion, Celgene, IMAB, Gilead, MSD, Mylan, Pfizer, Fresenius, Janssen, Takeda, Theravance, Prevention Bio, Protagonist and Falk, outside the submitted work. All other authors have nothing to disclose., (Copyright ©The authors 2022.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

Introduction: The mechanisms in podocytes that mediate the pathologic effects of the APOL1 high-risk (HR) variants remain incompletely understood, although various molecular and cellular mechanisms have been proposed. We previously established conditionally immortalized human urine-derived podocyte-like epithelial cell (HUPEC) lines to investigate APOL1 HR variant-induced podocytopathy., Methods: We conducted comprehensive transcriptomic analysis, including mRNA, microRNA (miRNA), and transfer RNA fragments (tRFs), to characterize the transcriptional profiles in undifferentiated and differentiated HUPEC with APOL1 HR (G1/G2, 2 cell lines) and APOL1 low-risk (LR) (G0/G0, 2 cell lines) genotypes. We reanalyzed single-cell RNA-seq data from urinary podocytes from focal segmental glomerulosclerosis (FSGS) subjects to characterize the effect of APOL1 genotypes on podocyte transcriptomes., Results: Differential expression analysis showed that the ribosomal pathway was one of the most enriched pathways, suggesting that altered function of the translation initiation machinery may contribute to APOL1 variant-induced podocyte injury. Expression of genes related to the elongation initiation factor 2 pathway was also enriched in the APOL1 HR urinary podocytes from single-cell RNA-seq, supporting a prior report on the role of this pathway in APOL1 -associated cell injury. Expression of microRNA and tRFs were analyzed, and the profile of small RNAs differed by both differentiation status and APOL1 genotype., Conclusion: We have profiled the transcriptomic landscape of human podocytes, including mRNA, miRNA, and tRF, to characterize the effects of differentiation and of different APOL1 genotypes. The candidate pathways, miRNAs, and tRFs described here expand understanding of APOL1 -associated podocytopathies.

Cross-reactive immunity between SARS-CoV-2 and other related coronaviruses has been well-documented, and it may play a role in preventing severe COVID-19. Epidemiological studies early in the pandemic showed a geographical association between high influenza vaccination rates and lower incidence of SARS-CoV-2 infection. We, therefore, analyzed whether exposure to influenza A virus (IAV) antigens could influence the T cell repertoire in response to SARS-CoV-2, indicating a heterologous immune response between these 2 unrelated viruses. Using artificial antigen-presenting cells (aAPCs) combined with real-time reverse-transcription PCR (RT-qPCR), we developed a sensitive assay to quickly screen for antigen-specific T cell responses and detected a significant correlation between responses to SARS-CoV-2 epitopes and IAV dominant epitope (M158-66). Further analysis showed that some COVID-19 convalescent donors exhibited both T cell receptor (TCR) specificity and functional cytokine responses to multiple SARS-CoV-2 epitopes and M158-66. Utilizing an aAPC-based stimulation/expansion assay, we detected cross-reactive T cells with specificity to SARS-CoV-2 and IAV. In addition, TCR sequencing of the cross-reactive and IAV-specific T cells revealed similarities between the TCR repertoires of the two populations. These results indicate that heterologous immunity shaped by our exposure to other unrelated endemic viruses may affect our immune response to novel viruses such as SARS-CoV-2.

Introduction: Proliferative lupus nephritis (LN) progresses to end-stage kidney disease (ESKD) in roughly 10% of the cases despite treatment. Other than achieving <0.8 g/24h proteinuria at 12 months after treatment, early biomarkers predicting ESKD or death are lacking. Recent studies encompassing not only LN have highlighted the central role of the alternative complement pathway (ACP), with or without histological evidence of thrombotic microangiopathy (TMA), as a key promotor of renal death., Methods: We assessed whether persistent isolated C3 hypocomplementemia (PI-LowC3), that is not accompanied by C4 hypocomplementemia, 6 months after kidney biopsy, is associated with an increased risk of death or ESKD in proliferative LN., Results: We retrospectively followed-up 197 patients with proliferative LN (51 with PI-LowC3) for a median of 4.5 years (interquartile-range: 1.9-9.0), 11 of whom died and 22 reached ESKD. After adjusting for age, gender, ethnicity, hypertension, mycophenolate, or cyclophosphamide use, PI-LowC3 was associated with a hazard ratio [HR] of the composite outcome ESKD or death of 2.46 (95% confidence interval [CI]: 1.22-4.99, P  = 0.012). These results were confirmed even after controlling for time-varying estimated glomerular filtration rate (eGFR) measurements in joint longitudinal-survival multiple regression models. After accounting for the competing risk of death, PI-LowC3 patients showed a strikingly increased risk of ESKD (adjusted HR 3.41, 95% CI: 1.31-8.88, P  = 0.012)., Conclusion: Our findings support the use of PI-LowC3 as a low-cost readily available biomarker, allowing clinicians to modify treatment strategies early in the course of disease and offering a rationale for complement blockade trials in this particularly at-risk subgroup of LN patients., (© 2022 International Society of Nephrology. Published by Elsevier Inc.)

Background: Image-based machine learning tools hold great promise for clinical applications in pathology research. However, the ideal end-users of these computational tools (e.g., pathologists and biological scientists) often lack the programming experience required for the setup and use of these tools which often rely on the use of command line interfaces., Methods: We have developed Histo-Cloud , a tool for segmentation of whole slide images (WSIs) that has an easy-to-use graphical user interface. This tool runs a state-of-the-art convolutional neural network (CNN) for segmentation of WSIs in the cloud and allows the extraction of features from segmented regions for further analysis., Results: By segmenting glomeruli, interstitial fibrosis and tubular atrophy, and vascular structures from renal and non-renal WSIs, we demonstrate the scalability, best practices for transfer learning, and effects of dataset variability. Finally, we demonstrate an application for animal model research, analyzing glomerular features in three murine models., Conclusions: Histo-Cloud is open source, accessible over the internet, and adaptable for segmentation of any histological structure regardless of stain., Competing Interests: Competing interestsJ.E.Z. is a paid consultant for Leica Biosystems. The remaining authors declare no competing interests., (© The Author(s) 2022.)

Pathogenic autoreactive antibodies that may be associated with life-threatening coronavirus disease 2019 (COVID-19) remain to be identified. Here, we show that self-assembled genome-scale libraries of full-length proteins covalently coupled to unique DNA barcodes for analysis by sequencing can be used for the unbiased identification of autoreactive antibodies in plasma samples. By screening 11,076 DNA-barcoded proteins expressed from a sequence-verified human ORFeome library, the method, which we named MIPSA (for Molecular Indexing of Proteins by Self-Assembly), allowed us to detect circulating neutralizing type-I and type-III interferon (IFN) autoantibodies in five plasma samples from 55 patients with life-threatening COVID-19. In addition to identifying neutralizing type-I IFN-α and IFN-ω autoantibodies and other previously known autoreactive antibodies in patient plasma, MIPSA enabled the detection of as yet unidentified neutralizing type-III anti-IFN-λ3 autoantibodies that were not seen in healthy plasma samples or in convalescent plasma from ten non-hospitalized individuals with COVID-19. The low cost and simple workflow of MIPSA will facilitate unbiased high-throughput analyses of protein-antibody, protein-protein and protein-small-molecule interactions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Podocyte depletion is a histomorphologic indicator of glomerular injury and predicts clinical outcomes. Podocyte estimation methods or podometrics are semiquantitative, technically involved, and laborious. Implementation of high-throughput podometrics in experimental and clinical workflows necessitates an automated podometrics pipeline. Recognizing that computational image analysis offers a robust approach to study cell and tissue structure, we developed and validated PodoCount (a computational tool for automated podocyte quantification in immunohistochemically labeled tissues) using a diverse data set., Methods: Whole-slide images (WSIs) of tissues immunostained with a podocyte nuclear marker and periodic acid-Schiff counterstain were acquired. The data set consisted of murine whole kidney sections ( n  = 135) from 6 disease models and human kidney biopsy specimens from patients with diabetic nephropathy (DN) ( n  = 45). Within segmented glomeruli, podocytes were extracted and image analysis was applied to compute measures of podocyte depletion and nuclear morphometry. Computational performance evaluation and statistical testing were performed to validate podometric and associated image features. PodoCount was disbursed as an open-source, cloud-based computational tool., Results: PodoCount produced highly accurate podocyte quantification when benchmarked against existing methods. Podocyte nuclear profiles were identified with 0.98 accuracy and segmented with 0.85 sensitivity and 0.99 specificity. Errors in podocyte count were bounded by 1 podocyte per glomerulus. Podocyte-specific image features were found to be significant predictors of disease state, proteinuria, and clinical outcome., Conclusion: PodoCount offers high-performance podocyte quantitation in diverse murine disease models and in human kidney biopsy specimens. Resultant features offer significant correlation with associated metadata and outcome. Our cloud-based tool will provide end users with a standardized approach for automated podometrics from gigapixel-sized WSIs., (© 2022 International Society of Nephrology. Published by Elsevier Inc.)

COVID-19 shares the feature of autoantibody production with systemic autoimmune diseases. In order to understand the role of these immune globulins in the pathogenesis of the disease, it is important to explore the autoantibody spectra. Here we show, by a cross-sectional study of 246 individuals, that autoantibodies targeting G protein-coupled receptors (GPCR) and RAS-related molecules associate with the clinical severity of COVID-19. Patients with moderate and severe disease are characterized by higher autoantibody levels than healthy controls and those with mild COVID-19 disease. Among the anti-GPCR autoantibodies, machine learning classification identifies the chemokine receptor CXCR3 and the RAS-related molecule AGTR1 as targets for antibodies with the strongest association to disease severity. Besides antibody levels, autoantibody network signatures are also changing in patients with intermediate or high disease severity. Although our current and previous studies identify anti-GPCR antibodies as natural components of human biology, their production is deregulated in COVID-19 and their level and pattern alterations might predict COVID-19 disease severity., (© 2022. The Author(s).)

Non-alcoholic steatohepatitis (NASH) is a most common chronic liver disease that is manifested by steatosis, inflammation, fibrosis, and tissue damage. Hepatocytes produce giant mitochondria termed megamitochondria in patients with NASH. It has been shown that gene knockout of OPA1, a mitochondrial dynamin-related GTPase that mediates mitochondrial fusion, prevents megamitochondria formation and liver damage in a NASH mouse model induced by a methionine-choline-deficient (MCD) diet. However, it is unknown whether blocking mitochondrial fusion mitigates NASH pathologies. Here, we acutely depleted OPA1 using antisense oligonucleotides in the NASH mouse model before or after megamitochondria formation. When OPA1 ASOs were applied at the disease onset, they effectively prevented megamitochondria formation and liver pathologies in the MCD model. Notably, even when applied after mice robustly developed NASH pathologies, OPA1 targeting effectively regressed megamitochondria and the disease phenotypes. Thus, our data show the efficacy of mitochondrial dynamics as a unique therapy for megamitochondria-associated liver disease., Competing Interests: T. Yamada, DM, DEK, RA, AZR, JK, JPH, ML, NYW, TMD, T. Yanagawa, MI, and HS declare that they have no competing interests. AFP and MA are employees and shareholders of Ionis Pharmaceuticals., (© 2022 The Authors.)

Tyrosine kinase inhibitors play an important role in the armamentarium against cancer. Lenvatinib is a multiple kinase inhibitor approved by the Food and Drugs Administration (FDA) for the treatment of advanced and radioresistant thyroid carcinomas and, in combination with everolimus, for renal cell carcinoma and unresectable hepatocellular carcinoma. The anti-tumoral activity is largely dependent on inhibition of neo-angiogenesis, and established side effects of anti-angiogenetic therapeutics include renal thrombotic microangiopathy (TMA). Here, we describe three cases of biopsy-proven renal TMA clinically presenting with proteinuria and stable serum creatinine in patients receiving lenvatinib for thyroid cancer. Microangiopathic lesions included glomerular basement membrane reduplication with segmental cellular interposition, mesangiolysis, and focal intracapillary and arteriolar thrombi. Drug-dose reduction or withdrawal was effective in renal function preservation, but cancer progressed in all patients. The management of lenvatinib-induced renal TMA remains a challenge. The best therapy in these patients is still uncertain. Earlier and more precise measurement of urine protein levels, allowing for early dose adjustment, could be effective in preventing further damage and drug discontinuation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Training convolutional neural networks using pathology whole slide images (WSIs) is traditionally prefaced by the extraction of a training dataset of image patches. While effective, for large datasets of WSIs, this dataset preparation is inefficient., Methods: We created a custom pipeline (histo-fetch) to efficiently extract random patches and labels from pathology WSIs for input to a neural network on-the-fly. We prefetch these patches as needed during network training, avoiding the need for WSI preparation such as chopping/tiling., Results & Conclusions: We demonstrate the utility of this pipeline to perform artificial stain transfer and image generation using the popular networks CycleGAN and ProGAN, respectively. For a large WSI dataset, histo-fetch is 98.6% faster to start training and used 7535x less disk space., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Pathology Informatics.)

Background: Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19., Methods: We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy., Findings: The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury., Interpretation: Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2., Funding: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health., Competing Interests: Declaration of interests None exist., (Copyright © 2022. Published by Elsevier B.V.)

Introduction: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time., Methods: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership)., Results: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies., Conclusion: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes., (© 2021 Published by Elsevier, Inc., on behalf of the International Society of Nephrology.)

The extracellular matrix (ECM) has historically been explored through proteomic methods. Whether or not global transcriptomics can yield meaningful information on the human matrisome is unknown. Gene expression data from 17,382 samples across 52 tissues, were obtained from the Genotype-Tissue Expression (GTEx) project. Additional datasets were obtained from The Cancer Genome Atlas (TCGA) program and the Gene Expression Omnibus for comparisons. Gene expression levels generally matched proteome-derived matrisome expression patterns. Further, matrisome gene expression properly clustered tissue types, with some matrisome genes including SERPIN family members having tissue-restricted expression patterns. Deeper analyses revealed 382 gene transcripts varied by age and 315 varied by sex in at least one tissue, with expression correlating with digitally imaged histologic tissue features. A comparison of TCGA tumor, TCGA adjacent normal and GTEx normal tissues demonstrated robustness of the GTEx samples as a generalized matrix control, while also determining a common primary tumor matrisome. Additionally, GTEx tissues served as a useful non-diseased control in a separate study of idiopathic pulmonary fibrosis (IPF) matrix changes, while identifying 22 matrix genes upregulated in IPF. Altogether, these findings indicate that the transcriptome, in general, and GTEx in particular, has value in understanding the state of organ ECM., (© 2021. The Author(s).)

Background: Podocyte depletion precedes progressive glomerular damage in several kidney diseases. However, the current standard of visual detection and quantification of podocyte nuclei from brightfield microscopy images is laborious and imprecise., Methods: We have developed PodoSighter, an online cloud-based tool, to automatically identify and quantify podocyte nuclei from giga-pixel brightfield whole-slide images (WSIs) using deep learning. Ground-truth to train the tool used immunohistochemically or immunofluorescence-labeled images from a multi-institutional cohort of 122 histologic sections from mouse, rat, and human kidneys. To demonstrate the generalizability of our tool in investigating podocyte loss in clinically relevant samples, we tested it in rodent models of glomerular diseases, including diabetic kidney disease, crescentic GN, and dose-dependent direct podocyte toxicity and depletion, and in human biopsies from steroid-resistant nephrotic syndrome and from human autopsy tissues., Results: The optimal model yielded high sensitivity/specificity of 0.80/0.80, 0.81/0.86, and 0.80/0.91, in mouse, rat, and human images, respectively, from periodic acid-Schiff-stained WSIs. Furthermore, the podocyte nuclear morphometrics extracted using PodoSighter were informative in identifying diseased glomeruli. We have made PodoSighter freely available to the general public as turnkey plugins in a cloud-based web application for end users., Conclusions: Our study demonstrates an automated computational approach to detect and quantify podocyte nuclei in standard histologically stained WSIs, facilitating podocyte research, and enabling possible future clinical applications., (Copyright © 2021 by the American Society of Nephrology.)

Renal injury resulting from obesity is a growing concern caused by the global obesity epidemic. We discuss the glomerular structure, obesity-related glomerular changes, and diagnostic pathologic criteria for obesity-related glomerulopathy. The three main hypothesized mechanisms of podocyte injury are mechanical stress on the podocytes, metabolic derangement, and genetic/molecular factors. Weight loss, renin-angiotensin-aldosterone system inhibitors, and improved insulin resistance may slow the progression. A more comprehensive understanding of obesity-related glomerulopathy will help in developing more effective therapies., (Copyright © 2021. Published by Elsevier Inc.)

[Figure: see text].

Background: Deceased-donor kidneys experience extensive injury, activating adaptive and maladaptive pathways therefore impacting graft function. We evaluated urinary donor uromodulin (UMOD) and osteopontin (OPN) in recipient graft outcomes., Methods: Primary outcomes: all-cause graft failure (GF) and death-censored GF (dcGF). Secondary outcomes: delayed graft function (DGF) and 6-month estimated glomerular filtration rate (eGFR). We randomly divided our cohort of deceased donors and recipients into training and test datasets. We internally validated associations between donor urine UMOD and OPN at time of procurement, with our primary outcomes. The direction of association between biomarkers and GF contrasted. Subsequently, we evaluated UMOD:OPN ratio with all outcomes. To understand these mechanisms, we examined the effect of UMOD on expression of major histocompatibility complex II in mouse macrophages., Results: Doubling of UMOD increased dcGF risk (adjusted hazard ratio [aHR], 1.1; 95% confidence interval [CI], 1.02-1.2), whereas OPN decreased dcGF risk (aHR, 0.94; 95% CI, 0.88-1). UMOD:OPN ratio ≤3 strengthened the association, with reduced dcGF risk (aHR, 0.57; 0.41-0.80) with similar associations for GF, and in the test dataset. A ratio ≤3 was also associated with lower DGF (aOR, 0.73; 95% CI, 0.60-0.89) and higher 6-month eGFR (adjusted β coefficient, 3.19; 95% CI, 1.28-5.11). UMOD increased major histocompatibility complex II expression elucidating a possible mechanism behind UMOD's association with GF., Conclusions: UMOD:OPN ratio ≤3 was protective, with lower risk of DGF, higher 6-month eGFR, and improved graft survival. This ratio may supplement existing strategies for evaluating kidney quality and allocation decisions regarding deceased-donor kidney transplantation., Competing Interests: S.G.M. supported by the American Heart Association (18CDA34110151) and Patterson Trust Fund. P.P.R. supported by epidemiology consulting from COHRDATA related to dialysis outcomes. This study received grant/research support from Merck, CVS and initiated grants from Merck to the University of Pennsylvania to support the THINKER (kidney), USHER (heart), and SHELTER (lung) trials of transplanting organs from donors with Hepatitis C into Hepatitis C negative recipients. It initiated grants from CVS to the University of Pennsylvania to support studies of medication adherence. I.E.H. received NIH grant support from NCATS (UL1TR002538 and KL2TR002539). T.M.E.-A. received grant support from NIDDK and US Department of Veterans Affairs. M.D.D. obtained the salary support from NIDDK. S.M. received grant/research support from NIH (NIDDK/NIAID/NIHMD/NIBIB) and consulting fees from Angion Pharmaceuticals. A.Z.R. received NDAs with Pliant Therapeutics and xMD Diagnostics, advisory board of Escala Therapeutics. C.R.P. received consulting fee from Renalytix and grant/research support from National Institute of Diabetes and Digestion and Kidney Diseases, National Heart, Lung, and Blood Institute and Data Safety and Monitoring Board of Genfit, Abbott. M.N.H. received grant support from NIH/NIDDK. The other authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Histologic examination of interstitial fibrosis and tubular atrophy (IFTA) is critical to determine the extent of irreversible kidney injury in renal disease. The current clinical standard involves pathologist's visual assessment of IFTA, which is prone to inter-observer variability. To address this diagnostic variability, we designed two case studies (CSs), including seven pathologists, using HistomicsTK- a distributed system developed by Kitware Inc. (Clifton Park, NY). Twenty-five whole slide images (WSIs) were classified into a training set of 21 and a validation set of four. The training set was composed of seven unique subsets, each provided to an individual pathologist along with four common WSIs from the validation set. In CS 1, all pathologists individually annotated IFTA in their respective slides. These annotations were then used to train a deep learning algorithm to computationally segment IFTA. In CS 2, manual and computational annotations from CS 1 were first reviewed by the annotators to improve concordance of IFTA annotation. Both the manual and computational annotation processes were then repeated as in CS1. The inter-observer concordance in the validation set was measured by Krippendorff's alpha (KA). The KA for the seven pathologists in CS1 was 0.62 with CI [0.57, 0.67], and after reviewing each other's annotations in CS2, 0.66 with CI [0.60, 0.72]. The respective CS1 and CS2 KA were 0.58 with CI [0.52, 0.64] and 0.63 with CI [0.56, 0.69] when including the deep learner as an eighth annotator. These results suggest that our designed annotation framework refines agreement of spatial annotation of IFTA and demonstrates a human-AI approach to significantly improve the development of computational models.

In diabetic kidney disease (DKD), podocyte depletion, and the subsequent migration of parietal epithelial cells (PECs) to the tuft, is a precursor to progressive glomerular damage, but the limitations of brightfield microscopy currently preclude direct pathological quantitation of these cells. Here we present an automated approach to podocyte and PEC detection developed using kidney sections from mouse model emulating DKD, stained first for Wilms' Tumor 1 (WT1) (podocyte and PEC marker) by immunofluorescence, then post-stained with periodic acid-Schiff (PAS). A generative adversarial network (GAN)-based pipeline was used to translate these PAS-stained sections into WT1-labeled IF images, enabling in silico label-free podocyte and PEC identification in brightfield images. Our method detected WT1-positive cells with high sensitivity/specificity (0.87/0.92). Additionally, our algorithm performed with a higher Cohen's kappa (0.85) than the average manual identification by three renal pathologists (0.78). We propose that this pipeline will enable accurate detection of WT1-positive cells in research applications.

The Human Phenotype Ontology (HPO, https://hpo.jax.org) was launched in 2008 to provide a comprehensive logical standard to describe and computationally analyze phenotypic abnormalities found in human disease. The HPO is now a worldwide standard for phenotype exchange. The HPO has grown steadily since its inception due to considerable contributions from clinical experts and researchers from a diverse range of disciplines. Here, we present recent major extensions of the HPO for neurology, nephrology, immunology, pulmonology, newborn screening, and other areas. For example, the seizure subontology now reflects the International League Against Epilepsy (ILAE) guidelines and these enhancements have already shown clinical validity. We present new efforts to harmonize computational definitions of phenotypic abnormalities across the HPO and multiple phenotype ontologies used for animal models of disease. These efforts will benefit software such as Exomiser by improving the accuracy and scope of cross-species phenotype matching. The computational modeling strategy used by the HPO to define disease entities and phenotypic features and distinguish between them is explained in detail.We also report on recent efforts to translate the HPO into indigenous languages. Finally, we summarize recent advances in the use of HPO in electronic health record systems., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: In patients with systemic lupus erythematosus (SLE) without concurrent active urinary sediment or unexplained acute kidney injury (AKI), current guidelines recommend performing a kidney biopsy in those with at least 500 mg/24-hour (European League Against Rheumatism/European Renal Association-European Dialysis and Transplant Association [EULAR/ERA-EDTA]) or 1000 mg/24-hour (American College of Rheumatology [ACR]) proteinuria. To evaluate the relevance of these indications, we studied histopathologic findings in patients with SLE with proteinuria below these cutoffs., Methods: We retrospectively reviewed the clinical, laboratory and histological characteristics of patients with SLE with <1000 mg/24-hour proteinuria (or mg/g urinary protein-to-creatinine ratio [UPCR]) who underwent their first kidney biopsy between 2003 and 2018., Results: We identified 87 patients with SLE with proteinuria less than 1000 mg/24-hour (or mg/g UPCR); 52 of 87 (60%) with isolated proteinuria, that is, without AKI or active urinary sediment (hematuria). Histologic evidence of lupus nephritis (LN) was present in 40 of 52 (76%). Of the 40 patients with LN, 12 had class I or II, 14 had class III or IV, 8 had class V, 6 had a combined proliferative and membranous LN. Non-lupus diagnoses included focal segmental glomerulosclerosis, acute interstitial nephritis, and others. Patient's age, low C3, low C4, and positivity for anti-double-stranded DNA (anti-dsDNA) antibodies predicted the histological diagnosis of LN on univariate logistic regression; however, a multivariate model including these parameters as independent covariates failed to predict LN., Conclusions: Patients with SLE with low-level proteinuria may have significant lupus- or non-lupus-related kidney disease with management implications. There was a significant burden of severe forms of LN. The presence of LN was not predicted by laboratory abnormalities. Based on our findings, we suggest current guidelines be revised to expand kidney biopsy indications to include isolated proteinuria of any grade., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Objectives: This study prospectively evaluated endomyocardial biopsies in patients with heart failure with preserved ejection fraction (HFpEF) to identify histopathologic phenotypes and their association with clinical characteristics., Background: Myocardial tissue analysis from a prospectively defined HFpEF cohort reflecting contemporary comorbidities is lacking., Methods: Patients with HFpEF (EF ≥50%) referred to the Johns Hopkins HFpEF Clinic between August 2014 and September 2018 were enrolled for right heart catheterization and endomyocardial biopsy. Clinical features, echocardiography, hemodynamics, and tissue histology were determined and compared with controls (unused donor hearts) and HF with reduced EF (HFrEF)., Results: Of the 108 patients enrolled, median age was 66 years (25th to 75th percentile: 57 to 74 years), 61% were women, 57% were African American, 62% had a previous HF hospitalization, median systolic blood pressure was 141 mm Hg (25th to 75th percentile: 125 to 162 mm Hg), body mass index (BMI) was 37 kg/m 2 (25th to 75th percentile: 32 to 45 kg/m 2 ), and 97% were on a loop diuretic. Myocardial fibrosis and myocyte hypertrophy were often present (93% and 88%, respectively); however, mild in 71% with fibrosis and in 52% with hypertrophy. Monocyte infiltration (CD68+ cells/mm 2 ) was greater in patients with HFpEF versus controls (60.4 cells/mm 2 [25th to 75th percentile: 36.8 to 97.8] vs. 32.1 cells/mm 2 [25th to 75th percentile: 22.3 to 59.2]; p = 0.02) and correlated with age and renal disease. Cardiac amyloidosis (CA) was diagnosed in 15 (14%) patients (HFpEF-CA: 7 patients with wild-type transthyretin amyloidosis [ATTR], 4 patients with hereditary ATTR, 3 patients with light-chain amyloidosis, and 1 patient with AA (secondary) amyloidosis), of which 7 cases were unsuspected. Patients with HFpEF-CA were older, with lower BMI, higher left ventricular mass index, and higher N-terminal pro-B-type natriuretic peptide and troponin I levels., Conclusions: In this large, prospective myocardial tissue analysis of HFpEF, myocardial fibrosis and hypertrophy were common, CD68+ inflammation was increased, and CA prevalence was 14%. Tissue analysis in HFpEF might improve precision therapies by identifying relevant myocardial mechanisms., (Copyright © 2020. Published by Elsevier Inc.)

Introduction: The term "acute tubular injury" (ATI) represents histopathologic renal tubular injury and often manifests clinically as acute kidney injury (AKI). Studies systematically summarizing the clinical presentation and histological changes in human ATI are limited., Methods: We used a comprehensive search strategy to search human studies of ATI from 1936 to July 2019. We extracted study characteristics, clinical characteristics, and histologic descriptions of ATI by bright field, immunofluorescence, electron microscopy, and immunohistochemistry. We compared ATI histology as a function of tissue procurement type, timing, and etiologies., Results: We included 292 studies comprising a total of 1987 patients. The majority of studies (222 of 292, 76%) were single-center case reports. The mean age of included patients was 47 years. In native kidney biopsy cases, baseline, peak, and latest creatinine were 1.3 mg/dl, 7.19 mg/dl, and 1.85 mg/dl respectively, and biopsy was performed mostly after peak creatinine (86.7%, 391 of 451). We identified 16 histologic descriptions of tubular injury, including tubular cell sloughing (115 of 292, 39.4%), tubular epithelial flattening/simplification (110 of 292, 37.7%), tubular dilatation (109 of 292, 37.3%), and tubular cell necrosis (93 of 292, 31.8%). There was no difference in tubular injury histology among different tissue procurement types (native kidney biopsy, transplant kidney biopsy, and autopsy), among different etiologies, or between different tissue procurement timing (before or after creatinine peaks in native kidneys). Electron microscopy and immunohistochemistry were used in a minority of studies., Conclusion: ATI manifests with diverse histologic changes. Efforts to establish protocols to harmonize biopsy practices, to handle kidney biopsy for tissue interrogation, and to report results across clinical practice are needed to improve our understanding of this complex disease., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Mitochondria regulate ATP production, metabolism, and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Because most measurements of mtDNA use homogenates of complex tissues, little is known about cell-type-specific mtDNA copy number heterogeneity in normal physiology, aging, and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, an in situ hybridization approach to generate a single-cell-resolution atlas of mtDNA content in mammalian tissues was validated. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared with differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared with cells within glomeruli and collecting duct epithelial cells. In mice, decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. This study provides a novel approach for quantifying species- and cell-type-specific mtDNA copy number and dynamics in any normal or diseased tissue that can be used for monitoring the effects of interventions in animal and human studies., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Successful integration of artificial intelligence into extant clinical workflows is contingent upon a number of factors including clinician comprehension and interpretation of computer vision. This article discusses how image analysis and machine learning have enabled comprehensive characterization of kidney morphology for development of automated diagnostic and prognostic renal pathology applications., Recent Findings: The primordial digital pathology informatics work employed classical image analysis and machine learning to prognosticate renal disease. Although this classical approach demonstrated tremendous potential, subsequent advancements in hardware technology rendered artificial neural networks '(ANNs) the method of choice for machine vision in computational pathology'. Offering rapid and reproducible detection, characterization and classification of kidney morphology, ANNs have facilitated the development of diagnostic and prognostic applications. In addition, modern machine learning with ANNs has revealed novel biomarkers in kidney disease, demonstrating the potential for machine vision to elucidate novel pathologic mechanisms beyond extant clinical knowledge., Summary: Despite the revolutionary developments potentiated by modern machine learning, several challenges remain, including data quality control and curation, image annotation and ontology, integration of multimodal data and interpretation of machine vision or 'opening the black box'. Resolution of these challenges will not only revolutionize diagnostic pathology but also pave the way for precision medicine and integration of artificial intelligence in the process of care.

A challenge of next generation sequencing is read contamination. We use Genotype-Tissue Expression (GTEx) datasets and technical metadata along with RNA-seq datasets from other studies to understand factors that contribute to contamination. Here we report, of 48 analyzed tissues in GTEx, 26 have variant co-expression clusters of four highly expressed and pancreas-enriched genes (PRSS1, PNLIP, CLPS, and/or CELA3A). Fourteen additional highly expressed genes from other tissues also indicate contamination. Sample contamination is strongly associated with a sample being sequenced on the same day as a tissue that natively expresses those genes. Discrepant SNPs across four contaminating genes validate the contamination. Low-level contamination affects ~40% of samples and leads to numerous eQTL assignments in inappropriate tissues among these 18 genes. This type of contamination occurs widely, impacting bulk and single cell (scRNA-seq) data set analysis. In conclusion, highly expressed, tissue-enriched genes basally contaminate GTEx and other datasets impacting analyses.

Sex disparities modulate cardiac function, although the proteins and mechanisms remain to be elucidated. We recently demonstrated a mosaic pattern of protein expression in the heart for over 100 proteins. Here we investigate one of these proteins, myosin light chain 4 (MYL4), which is important for contractile functions by increasing force production. We assayed the expression pattern of MYL4 across 756 ventricular myocardial samples from 668 individuals utilizing a semi-automated Cell Profiler method on five tissue microarrays (TMAs) of cardiac tissues across a diverse set of diseases. The percentage of MYL4 positive cells was significantly higher in male subjects independently across all five TMAs, regardless of disease state (p = 8.66e-15). Higher MYL4 expression was also modestly associated with hypertrophic cardiomyopathy (p = 6.3e-04). MYL4 expression did not associate with sudden cardiac death or other cardiomyopathies. This study demonstrates a new mosaic pattern of protein expression that underlies sex disparities in the human heart.

Genetic variants in APOL1, encoding apolipoprotein L1, are major drivers of glomerular disease in peoples of sub-Saharan African descent. APOL1-associated primary glomerular diseases include focal segmental glomerulosclerosis, human immunodeficiency virus-associated nephropathies, and arterionephrosclerosis. Other conditions where APOL1 variants affect outcomes include membranous nephropathy, lupus nephritis, diabetic nephropathy, preeclampsia, and kidney transplant. In focal segmental glomerulosclerosis, APOL1 variants are associated with upregulation of RNA encoding chemokine C-X-C motif receptor 3 ligands and ubiquitin D; the significance of these findings remains unclear but may provide valuable insight into disease mechanisms.

Lewy body diseases involve neurogenic orthostatic hypotension (nOH), cardiac noradrenergic deficiency, and deposition of the protein AS (alpha-synuclein) in sympathetic ganglion tissue. Mechanisms linking these abnormalities are poorly understood. One link may be AS deposition within sympathetic neurons. We validated methodology to quantify AS colocalization with TH (tyrosine hydroxylase), a marker of sympathetic noradrenergic innervation, and assessed associations of AS/TH colocalization with myocardial norepinephrine content and cardiac sympathetic neuroimaging data in nOH. Postmortem sympathetic ganglionic AS/TH colocalization indices and myocardial norepinephrine contents were measured in 4 Lewy body and 3 rare non-Lewy body nOH patients. Sixteen Lewy body and 11 non-Lewy body nOH patients underwent in vivo skin biopsies and thoracic 18 F-dopamine positron emission tomographic scanning, with cutaneous colocalization indices expressed versus cardiac 18 F-dopamine-derived radioactivity. Ganglionic AS/TH colocalization indices were higher and myocardial norepinephrine lower in Lewy body than non-Lewy body nOH ( P=0.0020, P=0.014). The Lewy body nOH group had higher AS/TH colocalization indices in skin biopsies and lower myocardial 18 F-dopamine-derived radioactivity than did the non-Lewy body nOH group ( P<0.0001 each). All Lewy body nOH patients had colocalization indices >1.5 in skin biopsies and 18 F-dopamine-derived radioactivity <6000 nCi-kg/cc-mCi, a combination not seen in non-Lewy body nOH patients ( P<0.0001). In Lewy body nOH, AS deposition in sympathetic noradrenergic nerves is related to postmortem neurochemical and in vivo neuroimaging evidence of myocardial noradrenergic deficiency. These associations raise the possibility that intraneuronal AS deposition plays a pathophysiological role in the myocardial sympathetic neurodegeneration attending Lewy body nOH.

Accurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple pathways, including stimulation of a signaling cascade involving AMP-activated protein kinase, sirtuin 1, PGC-1 α , sirtuin 3, estrogen-related receptor- α , and Nrf-1; inhibition of endoplasmic reticulum stress; and inhibition of enhanced renal fatty acid and cholesterol metabolism. Additionally, in mice with diet-induced obesity, INT-767 prevented mitochondrial dysfunction and oxidative stress determined by fluorescence lifetime imaging of NADH and kidney fibrosis determined by second harmonic imaging microscopy. These results identify the renal signaling pathways regulated by FXR and TGR5, which may be promising targets for the treatment of nephropathy in diabetes and obesity., (Copyright © 2018 by the American Society of Nephrology.)

Background and Objectives: This analysis from the Nephrotic Syndrome Study Network (NEPTUNE) assessed the phenotypic and pathology characteristics of proteinuric patients undergoing kidney biopsy and defined the frequency and factors associated with complete proteinuria remission (CRever)., Design, Setting, Participants, & Measurements: We enrolled adults and children with proteinuria ≥0.5 g/d at the time of first clinically indicated renal biopsy at 21 sites in North America from April 2010 to June 2014 into a prospective cohort study. NEPTUNE central pathologists assigned participants to minimal-change disease (MCD), FSGS, membranous nephropathy, or other glomerulopathy cohorts. Outcome measures for this analysis were (1) CRever with urine protein-to-creatinine ratio (UPC) <0.3 g/g with preserved native kidney function and (2) ESRD. Continuous variables are reported as median and interquartile range (IQR; 25th, 75th percentile). Cox proportional hazards modeling was used to assess factors associated with CRever., Results: We enrolled 441 patients: 116 (27%) had MCD, 142 (32%) had FSGS, 66 (15%) had membranous nephropathy, and 117 (27%) had other glomerulopathy. The baseline UPC was 4.1 g/g (IQR, 1.9, 7.7) and the eGFR was 81 ml/min per 1.73 m(2) (IQR, 50, 105). Median duration of observation was 19 months (IQR, 11, 30). CRever occurred in 46% of patients, and 4.6% progressed to ESRD. Multivariate analysis demonstrated that higher prebiopsy proteinuria (hazard ratio, 0.3; 95% confidence interval, 0.2 to 0.5) and pathology diagnosis (FSGS versus MCD; hazard ratio, 0.2; 95% confidence interval, 0.1 to 0.5) were inversely associated with CRever. The effect of immunosuppressive therapy on remission varied by pathology diagnosis., Conclusions: In NEPTUNE, the high frequency of other pathology in proteinuric patients affirms the value of the diagnostic kidney biopsy. Clinical factors, including level of proteinuria before biopsy, pathology diagnosis, and immunosuppression, are associated with complete remission., (Copyright © 2016 by the American Society of Nephrology.)