Plasma Proteins associated with Chronic Histopathologic Lesions on Kidney Biopsy.

BACKGROUND: The severity of chronic histopathologic lesions on kidney biopsy is independently associated with higher risk of progressive chronic kidney disease (CKD). Because kidney biopsies are invasive, identification of blood markers that report on underlying kidney histopathology has the potential to enhance CKD care. METHODS: We examined the association between 6592 plasma protein levels measured by aptamers and the severity of interstitial fibrosis and tubular atrophy (IFTA), glomerulosclerosis, arteriolar sclerosis, and arterial sclerosis among 434 participants of the Boston Kidney Biopsy Cohort. For proteins significantly associated with at least one histologic lesion, we assessed renal arteriovenous protein gradients among 21 individuals who had undergone invasive catheterization and assessed the expression of the cognate gene among 47 individuals with single cell RNA sequencing data in the Kidney Precision Medicine Project. RESULTS: In models adjusted for estimated glomerular filtration rate (eGFR), proteinuria, and demographic factors, we identified 35 proteins associated with one or more chronic histologic lesions, including 20 specific for IFTA, 8 specific for glomerulosclerosis, and 1 specific for arteriolar sclerosis. In general, higher levels of these proteins were associated with more severe histologic score and lower eGFR. Exceptions included testican-2 and NELL1, which were associated with less glomerulosclerosis and IFTA, respectively, and higher eGFR; notably, both of these proteins demonstrated significantly higher levels from artery to renal vein, demonstrating net kidney release. In the Kidney Precision Medicine Project, 13 of the 35 protein hits had cognate gene expression enriched in one or more cell types in the kidney, including podocyte expression of select glomerulosclerosis markers (including testican-2) and tubular expression of several IFTA markers (including NELL1). CONCLUSIONS: Proteomic analysis identified circulating proteins associated with chronic histopathologic lesions, some of which have concordant site-specific expression within the kidney.

Integrated multiomics implicates dysregulation of ECM and cell adhesion pathways as drivers of severe COVID-associated kidney injury.

COVID-19 has been a significant public health concern for the last four years; however, little is known about the mechanisms that lead to severe COVID-associated kidney injury. In this multicenter study, we combined quantitative deep urinary proteomics and machine learning to predict severe acute outcomes in hospitalized COVID-19 patients. Using a 10-fold cross-validated random forest algorithm, we identified a set of urinary proteins that demonstrated predictive power for both discovery and validation set with 87% and 79% accuracy, respectively. These predictive urinary biomarkers were recapitulated in non-COVID acute kidney injury revealing overlapping injury mechanisms. We further combined orthogonal multiomics datasets to understand the mechanisms that drive severe COVID-associated kidney injury. Functional overlap and network analysis of urinary proteomics, plasma proteomics and urine sediment single-cell RNA sequencing showed that extracellular matrix and autophagy-associated pathways were uniquely impacted in severe COVID-19. Differentially abundant proteins associated with these pathways exhibited high expression in cells in the juxtamedullary nephron, endothelial cells, and podocytes, indicating that these kidney cell types could be potential targets. Further, single-cell transcriptomic analysis of kidney organoids infected with SARS-CoV-2 revealed dysregulation of extracellular matrix organization in multiple nephron segments, recapitulating the clinically observed fibrotic response across multiomics datasets. Ligand-receptor interaction analysis of the podocyte and tubule organoid clusters showed significant reduction and loss of interaction between integrins and basement membrane receptors in the infected kidney organoids. Collectively, these data suggest that extracellular matrix degradation and adhesion-associated mechanisms could be a main driver of COVID-associated kidney injury and severe outcomes.

Pax protein depletion in proximal tubules triggers conserved mechanisms of resistance to acute ischemic kidney injury preventing transition to chronic kidney disease.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Identifying the molecular and genetic regulators unique to nephron segments that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. Here, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI and found them upregulated after severe AKI and correlated with chronic injury. Surprisingly, proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to pre-conditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of proximal tubule cells in the S3 segment that displayed features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic pre-conditioning, and female sex. Thus, our results identified a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both the injury response and protection from ischemic AKI.

Pax Protein Depletion in Proximal Tubules Triggers Conserved Mechanisms of Resistance to Acute Ischemic Kidney Injury and Prevents Transition to Chronic Kidney Disease.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. In this report, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI. We found that Pax2 and Pax8 are upregulated after severe AKI and correlate with chronic injury. Surprisingly, we then discovered that proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to preconditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of S3 proximal tubule cells that display features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic preconditioning, and female sex. Taken together, our results identify a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both injury response and protection from ischemic AKI. TRANSLATIONAL STATEMENT: Identifying the molecular and genetic regulators unique to the nephron that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are two homologous nephron-specific transcription factors that are critical for kidney development and physiology. Here we report that proximal-tubule-selective depletion of Pax2 and Pax8 protects against both acute and chronic injury and induces an expression profile in the S3 proximal tubule with common features shared among diverse conditions that protect against ischemia. These findings highlight a new role for Pax proteins as potential therapeutic targets to treat AKI.

Endogenous adenine mediates kidney injury in diabetic models and predicts diabetic kidney disease in patients.

Diabetic kidney disease (DKD) can lead to end-stage kidney disease (ESKD) and mortality; however, few mechanistic biomarkers are available for high-risk patients, especially those without macroalbuminuria. Urine from participants with diabetes from the Chronic Renal Insufficiency Cohort (CRIC) study, the Singapore Study of Macro-angiopathy and Micro-vascular Reactivity in Type 2 Diabetes (SMART2D), and the American Indian Study determined whether urine adenine/creatinine ratio (UAdCR) could be a mechanistic biomarker for ESKD. ESKD and mortality were associated with the highest UAdCR tertile in the CRIC study and SMART2D. ESKD was associated with the highest UAdCR tertile in patients without macroalbuminuria in the CRIC study, SMART2D, and the American Indian study. Empagliflozin lowered UAdCR in nonmacroalbuminuric participants. Spatial metabolomics localized adenine to kidney pathology, and single-cell transcriptomics identified ribonucleoprotein biogenesis as a top pathway in proximal tubules of patients without macroalbuminuria, implicating mTOR. Adenine stimulated matrix in tubular cells via mTOR and stimulated mTOR in mouse kidneys. A specific inhibitor of adenine production was found to reduce kidney hypertrophy and kidney injury in diabetic mice. We propose that endogenous adenine may be a causative factor in DKD.

Sodium glucose co-transporter 2 inhibition increases epidermal growth factor expression and improves outcomes in patients with type 2 diabetes.

Underlying molecular mechanisms of the kidney protective effects of sodium glucose co-transporter 2 (SGLT2) inhibitors are not fully elucidated. Therefore, we studied the association between urinary epidermal growth factor (uEGF), a mitogenic factor involved in kidney repair, and kidney outcomes in patients with type 2 diabetes (T2D). The underlying molecular mechanisms of the SGLT2 inhibitor canagliflozin on EGF using single-cell RNA sequencing from kidney tissue were examined. Urinary EGF-to-creatinine ratio (uEGF/Cr) was measured in 3521 CANagliflozin cardioVascular Assessment Study (CANVAS) participants at baseline and week 52. Associations of uEGF/Cr with kidney outcome were assessed using multivariable-adjusted Cox regression models. Single-cell RNA sequencing was performed using protocol kidney biopsy tissue from ten young patients with T2D on SGLT2i, six patients with T2D on standard care only, and six healthy controls (HCs). In CANVAS, each doubling in baseline uEGF/Cr was associated with a 12% (95% confidence interval 1-22) decreased risk of kidney outcome. uEGF/Cr decreased after 52 weeks with placebo and remained stable with canagliflozin (between-group difference +7.3% (2.0-12.8). In young persons with T2D, EGF mRNA was primarily expressed in the thick ascending loop of Henle. Expression in biopsies from T2D without SGLT2i was significantly lower compared to HCs, whereas treatment with SGLT2i increased EGF levels closer to the healthy state. In young persons with T2D without SGLT2i, endothelin-1 emerged as a key regulator of the EGF co-expression network. SGLT2i treatment was associated with a shift towards normal EGF expression. Thus, decreased uEGF represents increased risk of kidney disease progression in patients with T2D. Canagliflozin increased kidney tissue expression of EGF and was associated with a downstream signaling cascade linked to tubular repair and reversal of tubular injury.

Role of endogenous adenine in kidney failure and mortality with diabetes.

Diabetic kidney disease (DKD) can lead to end-stage kidney disease (ESKD) and mortality, however, few mechanistic biomarkers are available for high risk patients, especially those without macroalbuminuria. Urine from participants with diabetes from Chronic Renal Insufficiency Cohort (CRIC), Singapore Study of Macro-Angiopathy and Reactivity in Type 2 Diabetes (SMART2D), and the Pima Indian Study determined if urine adenine/creatinine ratio (UAdCR) could be a mechanistic biomarker for ESKD. ESKD and mortality were associated with the highest UAdCR tertile in CRIC (HR 1.57, 1.18, 2.10) and SMART2D (HR 1.77, 1.00, 3.12). ESKD was associated with the highest UAdCR tertile in patients without macroalbuminuria in CRIC (HR 2.36, 1.26, 4.39), SMART2D (HR 2.39, 1.08, 5.29), and Pima Indian study (HR 4.57, CI 1.37-13.34). Empagliflozin lowered UAdCR in non-macroalbuminuric participants. Spatial metabolomics localized adenine to kidney pathology and transcriptomics identified ribonucleoprotein biogenesis as a top pathway in proximal tubules of patients without macroalbuminuria, implicating mammalian target of rapamycin (mTOR). Adenine stimulated matrix in tubular cells via mTOR and stimulated mTOR in mouse kidneys. A specific inhibitor of adenine production was found to reduce kidney hypertrophy and kidney injury in diabetic mice. We propose that endogenous adenine may be a causative factor in DKD.

Defining the molecular correlate of arteriolar hyalinosis in kidney disease progression by integration of single cell transcriptomic analysis and pathology scoring.

Arteriolar hyalinosis in kidneys is an independent predictor of cardiovascular disease, the main cause of mortality in chronic kidney disease (CKD). The underlying molecular mechanisms of protein accumulation in the subendothelial space are not well understood. Using single cell transcriptomic data and whole slide images from kidney biopsies of patients with CKD and acute kidney injury in the Kidney Precision Medicine Project, the molecular signals associated with arteriolar hyalinosis were evaluated. Co-expression network analysis of the endothelial genes yielded three gene set modules as significantly associated with arteriolar hyalinosis. Pathway analysis of these modules showed enrichment of transforming growth factor beta / bone morphogenetic protein (TGFß / BMP) and vascular endothelial growth factor (VEGF) signaling pathways in the endothelial cell signatures. Ligand-receptor analysis identified multiple integrins and cell adhesion receptors as over-expressed in arteriolar hyalinosis, suggesting a potential role of integrin-mediated TGFß signaling. Further analysis of arteriolar hyalinosis associated endothelial module genes identified focal segmental glomerular sclerosis as an enriched term. On validation in gene expression profiles from the Nephrotic Syndrome Study Network cohort, one of the three modules was significantly associated with the composite endpoint (> 40% reduction in estimated glomerular filtration rate (eGFR) or kidney failure) independent of age, sex, race, and baseline eGFR, suggesting poor prognosis with elevated expression of genes in this module. Thus, integration of structural and single cell molecular features yielded biologically relevant gene sets, signaling pathways and ligand-receptor interactions, underlying arteriolar hyalinosis and putative targets for therapeutic intervention.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

SGLT2 inhibitors mitigate kidney tubular metabolic and mTORC1 perturbations in youth-onset type 2 diabetes.

The molecular mechanisms of sodium-glucose cotransporter-2 (SGLT2) inhibitors (SGLT2i) remain incompletely understood. Single-cell RNA sequencing and morphometric data were collected from research kidney biopsies donated by young persons with type 2 diabetes (T2D), aged 12 to 21 years, and healthy controls (HCs). Participants with T2D were obese and had higher estimated glomerular filtration rates and mesangial and glomerular volumes than HCs. Ten T2D participants had been prescribed SGLT2i (T2Di[+]) and 6 not (T2Di[-]). Transcriptional profiles showed SGLT2 expression exclusively in the proximal tubular (PT) cluster with highest expression in T2Di(-) patients. However, transcriptional alterations with SGLT2i treatment were seen across nephron segments, particularly in the distal nephron. SGLT2i treatment was associated with suppression of transcripts in the glycolysis, gluconeogenesis, and tricarboxylic acid cycle pathways in PT, but had the opposite effect in thick ascending limb. Transcripts in the energy-sensitive mTORC1-signaling pathway returned toward HC levels in all tubular segments in T2Di(+), consistent with a diabetes mouse model treated with SGLT2i. Decreased levels of phosphorylated S6 protein in proximal and distal tubules in T2Di(+) patients confirmed changes in mTORC1 pathway activity. We propose that SGLT2i treatment benefits the kidneys by mitigating diabetes-induced metabolic perturbations via suppression of mTORC1 signaling in kidney tubules.

PKD2 founder mutation is the most common mutation of polycystic kidney disease in Taiwan.

Autosomal Dominant polycystic kidney disease (ADPKD) is the most common inherited adult kidney disease. Although ADPKD is primarily caused by PKD1 and PKD2, the identification of several novel causative genes in recent years has revealed more complex genetic heterogeneity than previously thought. To study the disease-causing mutations of ADPKD, a total of 920 families were collected and their diagnoses were established via clinical and image studies by Taiwan PKD Consortium investigators. Amplicon-based library preparation with next-generation sequencing, variant calling, and bioinformatic analysis was used to identify disease-causing mutations in the cohort. Microsatellite analysis along with genotyping and haplotype analysis was performed in the PKD2 p.Arg803* family members. The age of mutation was calculated to estimate the time at which the mutation occurred or the founder arrived in Taiwan. Disease-causing mutations were identified in 634 families (68.9%) by detection of 364 PKD1, 239 PKD2, 18 PKHD1, 7 GANAB, and 6 ALG8 pathogenic variants. 162 families (17.6%) had likely causative but non-diagnostic variants of unknown significance (VUS). A single PKD2 p.Arg803* mutation was found in 17.8% (164/920) of the cohort in Taiwan. Microsatellite and array analysis showed that 80% of the PKD2 p.Arg803* families shared the same haplotype in a 250 kb region, indicating those families may originate from a common ancestor 300 years ago. Our findings provide a mutation landscape as well as evidence that a founder effect exists and has contributed to a major percentage of the ADPKD population in Taiwan.

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.

Glomerular endothelial cell-podocyte stresses and crosstalk in structurally normal kidney transplants.

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

Endoplasmic reticulum-associated degradation is required for nephrin maturation and kidney glomerular filtration function.

Podocytes are key to the glomerular filtration barrier by forming a slit diaphragm between interdigitating foot processes; however, the molecular details and functional importance of protein folding and degradation in the ER remain unknown. Here, we show that the SEL1L-HRD1 protein complex of ER-associated degradation (ERAD) is required for slit diaphragm formation and glomerular filtration function. SEL1L-HRD1 ERAD is highly expressed in podocytes of both mouse and human kidneys. Mice with podocyte-specific Sel1L deficiency develop podocytopathy and severe congenital nephrotic syndrome with an impaired slit diaphragm shortly after weaning and die prematurely, with a median lifespan of approximately 3 months. We show mechanistically that nephrin, a type 1 membrane protein causally linked to congenital nephrotic syndrome, is an endogenous ERAD substrate. ERAD deficiency attenuated the maturation of nascent nephrin, leading to its retention in the ER. We also show that various autosomal-recessive nephrin disease mutants were highly unstable and broken down by SEL1L-HRD1 ERAD, which attenuated the pathogenicity of the mutants toward the WT allele. This study uncovers a critical role of SEL1L-HRD1 ERAD in glomerular filtration barrier function and provides insights into the pathogenesis associated with autosomal-recessive disease mutants.

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.

SARS-CoV-2 receptor networks in diabetic and COVID-19-associated kidney disease.

COVID-19 morbidity and mortality are increased via unknown mechanisms in patients with diabetes and kidney disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Because ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of kidney biopsies from healthy living donors and patients with diabetic kidney disease revealed ACE2 expression primarily in proximal tubular epithelial cells. This cell-specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin-angiotensin-aldosterone system inhibitors in diabetic kidney disease. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing proximal tubular epithelial cells in diabetic kidney disease (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The diabetic kidney disease ACE2-positive proximal tubular epithelial cell module overlapped with expression patterns seen in SARS-CoV-2-infected cells. Similar cellular programs were seen in ACE2-positive proximal tubular epithelial cells obtained from urine samples of 13 hospitalized patients with COVID-19, suggesting a consistent ACE2-coregulated proximal tubular epithelial cell expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19-related kidney damage.

SARS-CoV-2 receptor networks in diabetic and COVID-19 associated kidney disease.

COVID-19 morbidity and mortality is increased in patients with diabetes and kidney disease via unknown mechanisms. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Since ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease (DKD) and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of healthy living donor (LD) and DKD kidney biopsies revealed ACE2 expression primarily in proximal tubular epithelial cells (PTEC). This cell specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin angiotensin aldosterone system inhibitors in DKD. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing PTEC in DKD (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The DKD ACE2-positive PTEC module overlapped with expression patterns seen in SARS-CoV-2 infected cells. Similar cellular programs were seen in ACE2-positive PTEC obtained from urine samples of 13 COVID-19 patients who were hospitalized, suggesting a consistent ACE2-coregulated PTEC expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19 related kidney damage.

Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

Single cell transcriptomics identifies focal segmental glomerulosclerosis remission endothelial biomarker.

To define cellular mechanisms underlying kidney function and failure, the KPMP analyzes biopsy tissue in a multicenter research network to build cell-level process maps of the kidney. This study aimed to establish a single cell RNA sequencing strategy to use cell-level transcriptional profiles from kidney biopsies in KPMP to define molecular subtypes in glomerular diseases. Using multiple sources of adult human kidney reference tissue samples, 22,268 single cell profiles passed KPMP quality control parameters. Unbiased clustering resulted in 31 distinct cell clusters that were linked to kidney and immune cell types using specific cell markers. Focusing on endothelial cell phenotypes, in silico and in situ hybridization methods assigned 3 discrete endothelial cell clusters to distinct renal vascular beds. Transcripts defining glomerular endothelial cells (GEC) were evaluated in biopsies from patients with 10 different glomerular diseases in the NEPTUNE and European Renal cDNA Bank (ERCB) cohort studies. Highest GEC scores were observed in patients with focal segmental glomerulosclerosis (FSGS). Molecular endothelial signatures suggested 2 distinct FSGS patient subgroups with α-2 macroglobulin (A2M) as a key downstream mediator of the endothelial cell phenotype. Finally, glomerular A2M transcript levels associated with lower proteinuria remission rates, linking endothelial function with long-term outcome in FSGS.

Organoid single cell profiling identifies a transcriptional signature of glomerular disease.

Podocyte injury is central to many forms of kidney disease, but transcriptional signatures reflecting podocyte injury and compensation mechanisms are challenging to analyze in vivo. Human kidney organoids derived from pluripotent stem cells (PSCs), a potentially new model for disease and regeneration, present an opportunity to explore the transcriptional plasticity of podocytes. Here, transcriptional profiling of more than 12,000 single cells from human PSC-derived kidney organoid cultures was used to identify robust and reproducible cell lineage gene expression signatures shared with developing human kidneys based on trajectory analysis. Surprisingly, the gene expression signature characteristic of developing glomerular epithelial cells was also observed in glomerular tissue from a kidney disease cohort. This signature correlated with proteinuria and inverse eGFR, and it was confirmed in an independent podocytopathy cohort. Three genes in particular were further characterized as potentially novel components of the glomerular disease signature. We conclude that cells in human PSC-derived kidney organoids reliably recapitulate the developmental transcriptional program of podocytes and other cell lineages in the human kidney and that transcriptional profiles seen in developing podocytes are reactivated in glomerular disease. Our findings demonstrate an approach to identifying potentially novel molecular programs involved in the pathogenesis of glomerulopathies.