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Kidney fibrosis constitutes the shared final pathway of nearly all chronic nephropathies, but biomarkers for the non-invasive assessment of kidney fibrosis are currently not available. To address this, we characterize five candidate biomarkers of kidney fibrosis: Cadherin-11 (CDH11), Sparc-related modular calcium binding protein-2 (SMOC2), Pigment epithelium-derived factor (PEDF), Matrix-Gla protein, and Thrombospondin-2. Gene expression profiles in single-cell and single-nucleus RNA-sequencing (sc/snRNA-seq) datasets from rodent models of fibrosis and human chronic kidney disease (CKD) were explored, and Luminex-based assays for each biomarker were developed. Plasma and urine biomarker levels were measured using independent prospective cohorts of CKD: the Boston Kidney Biopsy Cohort, a cohort of individuals with biopsy-confirmed semiquantitative assessment of kidney fibrosis, and the Seattle Kidney Study, a cohort of patients with common forms of CKD. Ordinal logistic regression and Cox proportional hazards regression models were used to test associations of biomarkers with interstitial fibrosis and tubular atrophy and progression to end-stage kidney disease and death, respectively. Sc/snRNA-seq data confirmed cell-specific expression of biomarker genes in fibroblasts. After multivariable adjustment, higher levels of plasma CDH11, SMOC2, and PEDF and urinary CDH11 and PEDF were significantly associated with increasing severity of interstitial fibrosis and tubular atrophy in the Boston Kidney Biopsy Cohort. In both cohorts, higher levels of plasma and urinary SMOC2 and urinary CDH11 were independently associated with progression to end-stage kidney disease. Higher levels of urinary PEDF associated with end-stage kidney disease in the Seattle Kidney Study, with a similar signal in the Boston Kidney Biopsy Cohort, although the latter narrowly missed statistical significance. Thus, we identified CDH11, SMOC2, and PEDF as promising non-invasive biomarkers of kidney fibrosis.

Autosomal dominant tubulointerstitial kidney diseases (ADTKDs) are a group of rare genetic diseases that lead to kidney failure. Mutations in the MUC1 gene cause ADTKD-MUC1 (MUC1 kidney disease, MKD), a disorder with no available therapies. Recent studies have identified the molecular and cellular mechanisms that drive MKD disease pathogenesis. Armed with patient-derived cell lines and pluripotent stem cell (iPSC)-derived kidney organoids, it was found that MKD is a toxic proteinopathy caused by the intracellular accumulation of misfolded MUC1 protein in the early secretory pathway. We discuss the advantages of studying rare monogenic kidney diseases, describe effective patient-derived model systems, and highlight recent mechanistic insights into protein quality control that have implications for additional proteinopathies beyond rare kidney diseases.

Purpose To evaluate self-referral from the Internet for genetic diagnosis of several rare inherited kidney diseases. Methods Retrospective study from 1996–2017 analyzing data from an academic referral center specializing in autosomal dominant tubulo-interstitial kidney disease (ADTKD). Individuals were referred by academic health care providers (HCPs) non-academic HCPs, or directly by patients/families. Results Over 21 years, there were 665 referrals, with 176(27%) directly from families, 269(40%) from academic HCPs, and 220(33%) from non-academic HCPs. 42(24%) of direct family referrals had positive genetic testing vs 73(27%) of families from academic HCPs and 55(25%) from non-academic HCPs (P=.72). 99% of direct family contacts were white and resided in zip code locations with a mean median income of $77,316±34,014 vs. US median income $49,445. Conclusions Undiagnosed families with Internet access bypassed their physicians and established direct contact with an academic center specializing in inherited kidney disease to achieve a diagnosis. Twenty-five per cent of all families diagnosed with ADTKD were the result of direct family referral and would otherwise have been un-diagnosed. If patients suspect a rare disorder that is un-diagnosed by their physicians, actively pursuing self-diagnosis using the Internet can be successful. Centers interested in rare disorders should consider improving direct access to families.

BACKGROUND: Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell–targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. METHODS: We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. RESULTS: In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. CONCLUSIONS: Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.

High resolution spatial transcriptomics is a transformative technology that enables mapping of RNA expression directly from intact tissue sections; however, its utility for the elucidation of disease processes and therapeutically actionable pathways remain largely unexplored. Here we applied Slide-seqV2 to mouse and human kidneys, in healthy and in distinct disease paradigms. First, we established the feasibility of Slide-seqV2 in human kidney by analyzing tissue from 9 distinct donors, which revealed a cell neighborhood centered around a population ofLYVE1+macrophages. Second, in a mouse model of diabetic kidney disease, we detected changes in the cellular organization of the spatially-restricted kidney filter and blood flow regulating apparatus. Third, in a mouse model of a toxic proteinopathy, we identified previously unknown, disease-specific cell neighborhoods centered around macrophages. In a spatially-restricted subpopulation of epithelial cells, we also found perturbations in 77 genes associated with the unfolded protein response (UPR). Our studies illustrate and experimentally validate the utility of Slide-seqV2 for the discovery of disease-specific cell neighborhoods.One-Sentence SummaryHigh resolution Slide-seqV2 spatial transcriptomics in human and mouse kidneys.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is an increasingly recognized. cause of end-stage kidney disease, primarily due to mutations in UMOD and MUC1. The lack of clinical recognition and the small size of cohorts have slowed the understanding of disease ontology and development of diagnostic algorithms. To expand on this, we analyzed two registries from Europe and the United States to define genetic and clinical characteristics of ADTKD-UMOD and ADTKD-MUC1 and develop a practical score to guide genetic testing. Our study encompassed 726 patients from 585 families with a presumptive diagnosis of ADTKD along with clinical, biochemical, genetic and radiologic data. Collectively, 106 different UMOD mutations were detected in 216/562 (38.4%) of families with ADTKD (303 patients), and 4 different MUC1 mutations in 72/205 (35.1%) of the families that are UMOD-negative (83 patients). The median kidney survival was significantly shorter in patients with ADTKD-MUC1 compared to ADTKD-UMOD (46 vs. 54 years respectively), whereas the median gout-free survival was dramatically reduced in patients with ADTKD-UMOD compared to ADTKD-MUC1 (30 vs. 67 years respectively). In contrast to patients with ADTKD-UMOD, patients with ADTKD-MUC1 had normal urinary excretion of uromodulin and distribution of uromodulin in tubular cells. A diagnostic algorithm based on a simple score coupled with urinary uromodulin measurements separated patients with ADTKD-UMOD from those with ADTKD-MUC1 with a sensitivity of 94.1%, a specificity of 74.3% and a positive predictive value of 84.2% for a UMOD mutation. Thus, ADTKD-UMOD is more frequently diagnosed than ADTKD-MUC1, ADTKD subtypes present with distinct clinical features, and a simple score coupled with urine uromodulin measurements may help prioritizing genetic testing.

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.

The health of the kidney filtration barrier requires communication among podocytes, endothelial cells, and mesangial cells. Disruption of these cell-cell interactions is thought to contribute to disease progression in chronic kidney diseases (CKDs). Podocyte ablation via doxycycline-inducible deletion of an essential endogenous molecule, CTCF [inducible podocyte-specific CTCF deletion (iCTCF(pod−/−))], is sufficient to drive progressive CKD. However, the earliest events connecting podocyte injury to disrupted intercellular communication within the kidney filter remain unclear. Single-cell RNA sequencing of kidney tissue from iCTCF(pod−/−) mice after 1 week of doxycycline induction was performed to generate a map of the earliest transcriptional effects of podocyte injury on cell-cell interactions at single-cell resolution. A subset of podocytes had the earliest signs of injury due to disrupted gene programs for cytoskeletal regulation and mitochondrial function. Surviving podocytes up-regulated collagen type IV ɑ5, causing reactive changes in integrin expression in endothelial populations and mesangial cells. Intercellular interaction analysis revealed several receptor-ligand-target gene programs as drivers of endothelial cell injury and abnormal matrix deposition. This analysis reveals the earliest disruptive changes within the kidney filter, pointing to new, actionable targets within a therapeutic window that may allow us to maximize the success of much needed therapeutic interventions for CKDs.

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.

Mouse models are a tool for studying the mechanisms underlying complex diseases; however, differences between species pose a significant challenge for translating findings to patients. Here, we used single-cell transcriptomics and orthogonal validation approaches to provide cross-species taxonomies, identifying shared broad cell classes and unique granular cellular states, between mouse and human kidney. We generated cell atlases of the diabetic and obese kidney using two different mouse models, a high-fat diet (HFD) model and a genetic model (BTBR ob/ob), at multiple time points along disease progression. Importantly, we identified a previously unrecognized, expanding Trem2high macrophage population in kidneys of HFD mice that matched human TREM2high macrophages in obese patients. Taken together, our cross-species comparison highlights shared immune and metabolic cell-state changes.

Dysfunction of podocytes, cells critical for glomerular filtration, underlies proteinuria and kidney failure. Genetic forms of proteinuric kidney disease can be caused by mutations in several podocyte genes, including nephrin, a critical component of the kidney filter. In contrast, the etiology of acquired acute-onset nephrotic syndrome has remained elusive. Here we identify autoantibodies against nephrin in serum and glomeruli of a subset of adults and children with non-congenital acute nephrotic syndrome. Our findings align with published experimental animal studies and elucidate a novel autoimmune phenomenon in proteinuric kidney disease interfering with glomerular filter integrity.

Progressive kidney diseases are often associated with scarring of the kidney’s filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases., National Institutes of Health (U.S.) (Grant DK095045), National Institutes of Health (U.S.) (Grant DK099465), National Institutes of Health (U.S.) (Grant DK103658), National Institutes of Health (U.S.) (Grant DK083511), National Institutes of Health (U.S.) (Grant DK093746)

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

The health of the kidney filtration barrier requires communication between podocytes, endothelial cells and mesangial cells. Disruption of these cell-cell interactions is thought to contribute to disease progression in chronic kidney diseases (CKD). We recently demonstrated that podocyte ablation via doxycycline-inducible deletion of an essential endogenous molecule, CTCF (iCTCFpod-/-), is sufficient to drive progressive CKD. However, the earliest events connecting podocyte injury to disrupted intercellular communication within the kidney filter remain unclear. Here we performed single-cell RNA sequencing of kidney tissue from iCTCFpod-/- mice after one week of doxycycline induction to generate a map of the earliest transcriptional effects of podocyte injury on cell-cell interactions at single cell resolution. A subset of podocytes showed the earliest signs of injury due to disrupted gene programs for cytoskeletal regulation and mitochondrial function. Surviving podocytes upregulated Col4a5, causing reactive changes in integrin expression in endothelial populations and mesangial cells. Intercellular interaction analysis revealed several receptor-ligand-target gene programs as drivers of endothelial cell injury and abnormal matrix deposition. This analysis reveals the earliest disruptive changes within the kidney filter, pointing to new, actionable targets within a therapeutic window that may allow us to maximize the success of much needed therapeutic interventions for CKD.

Summary Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI., Graphical Abstract, Highlights Elevated MUC1 levels predict the development of acute lung injury (ALI) A high-content screen of 3,713 compounds identifies repurposing candidates R406 removes MUC1 from the apical surface of epithelial cells Fostamatinib treatment reduces MUC1 in a mouse model of lung injury, In a high-content screen, Kost-Alimova et al. identify R406, the active metabolite of fostamatinib, as an FDA-approved candidate repurposing compound for the reduction of MUC1 protein levels in lung epithelium in the setting of acute lung injury.

SARS-CoV-2, the coronavirus that causes COVID-19, binds to angiotensin-converting enzyme 2 (ACE2) on human cells. Beyond the lung, COVID-19 impacts diverse tissues including the kidney. ACE2 is a key member of the Renin-Angiotensin-Aldosterone System (RAAS) which regulates blood pressure, largely through its effects on the kidney. RAAS blockers such as ACE inhibitors (ACEi) and Angiotensin Receptor Blockers (ARBs) are widely used therapies for hypertension, cardiovascular and chronic kidney diseases, and therefore, there is intense interest in their effect on ACE2 expression and its implications for SARS-CoV-2 pathogenicity. Here, we analyzed single-cell and single-nucleus RNA-seq of human kidney to interrogate the association of ACEi/ARB use with ACE2 expression in specific cell types. First, we performed an integrated analysis aggregating 176,421 cells across 49 donors, 8 studies and 8 centers, and adjusting for sex, age, donor and center effects, to assess the relationship of ACE2 with age and sex at baseline. We observed a statistically significant increase in ACE2 expression in tubular epithelial cells of the thin loop of Henle (tLoH) in males relative to females at younger ages, the trend reversing, and losing significance with older ages. ACE2 expression in tLoH increases with age in females, with an opposite, weak effect in males. In an independent cohort, we detected a statistically significant increase in ACE2 expression with ACEi/ARB use in epithelial cells of the proximal tubule and thick ascending limb, and endothelial cells, but the association was confounded in this small cohort by the underlying disease. Our study illuminates the dynamics of ACE2 expression in specific kidney cells, with implications for SARS-CoV-2 entry and pathogenicity.

Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2(kd/kd) mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2(kd/kd) mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.

Introduction Treatment-resistant nephrotic syndrome is a rare form of glomerular disease that occurs in children and adults. No Food and Drug Administration−approved treatments consistently achieve remission of proteinuria and preservation of kidney function. CD80 (B7-1) can be expressed on injured podocytes, and administration of abatacept (modified CTLA4-Ig based on a natural ligand to CD80) has been associated with sustained normalization of urinary protein excretion and maintenance of glomerular filtration rate in experimental and clinical settings. Methods In this report, we describe the rationale for and design of a randomized, placebo-controlled, clinical trial of abatacept in patients with treatment-resistant nephrotic syndrome caused by focal segmental glomerulosclerosis or minimal change disease. The design is a hybrid of a parallel-group and crossover design (switchover) with the primary objectives assessed in the first period of the study and the secondary objectives assessed using data from both periods. All participants will receive the active agent in 1 of the periods. The duration of treatment will be 4 months per period. Results The primary outcome will be improvement in nephrotic-range proteinuria to subnephrotic range, that is, reduction from baseline to 4 months in urine protein:creatinine ratio ≥ 50% and to a level < 3. The projected sample size is 90 patients, which has 80% power to detect a treatment difference of 28%. Conclusion This study advances efforts to validate CD80 as a therapeutic target for treatment-resistant nephrotic syndrome, and implements a precision medicine-based approach to this serious kidney condition in which the selection of a therapeutic agent is guided by the underlying disease mechanism operating in individual patients.

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells., How reproducible human kidney organoids derived from different iPSC lines are, and how faithful they are to human kidney tissue remain unclear. Here, the authors use four human iPSC lines to derive kidney organoids and show how organoid composition is reproducible, comparable to human tissue and of improved quality after transplantation.

Ion channels are critical to kidney function, and their dysregulation leads to several distinct kidney diseases. Of the diversity of ion channels in kidney cells, the transient receptor potential (TRP) superfamily of proteins plays important and varied roles in both maintaining homeostasis as well as in causing disease. Recent work showed that TRPC5 blockers could successfully protect critical components of the kidney filter both in vitro and in vivo, thus revealing TRPC5 as a tractable therapeutic target for focal and segmental glomerulosclerosis (FSGS), a common cause of kidney failure. Human genetics point to three additional TRP channels as plausible therapeutic targets: TRPC6 in FSGS, PKD2 in polycystic kidney disease, and TRPM6 in familial hypomagnesemia with secondary hypocalcemia (HSH). We conclude that targeting TRP channels could pave the way for much needed therapies for kidney diseases.

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolongedin vitroculture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

Calcium ions (Ca2+) are crucial for a variety of cellular functions. The extracellular and intracellular Ca2+concentrations are thus tightly regulated to maintain Ca2+homeostasis. The kidney, one of the major organs of the excretory system, regulates Ca2+homeostasis by filtration and reabsorption. Approximately 60% of the Ca2+in plasma is filtered, and 99% of that is reabsorbed by the kidney tubules. Ca2+is also a critical signaling molecule in kidney development, in all kidney cellular functions, and in the emergence of kidney diseases. Recently, studies using genetic and molecular biological approaches have identified several Ca2+-permeable ion channel families as important regulators of Ca2+homeostasis in kidney. These ion channel families include transient receptor potential channels (TRP), voltage-gated calcium channels, and others. In this review, we provide a brief and systematic summary of the expression, function, and pathological contribution for each of these Ca2+-permeable ion channels. Moreover, we discuss their potential as future therapeutic targets.

PURPOSE OF REVIEW In this review, we take a combined membrane biologist's and geneticist's view of the podocyte, to examine how genetics have informed our understanding of membrane receptors, channels, and other signaling molecules affecting podocyte health and disease. RECENT FINDINGS An integral part of the kidney, the glomerulus, is responsible for the kidney's filter function. Within the glomerulus, the podocyte is a unique cell serving a critically important role: it is exposed to signals from the urinary space in Bowman's capsule, it receives and transmits signals to/from the basement membrane upon which it elaborates, and it receives signals from the vascular space with which it also communicates, thus exposed to toxins, viruses, chemicals, proteins, and cellular components or debris that flow in the blood stream. Our understanding of how podocytes perform their important role has been largely informed by human genetics, and the recent revolution afforded by exome sequencing has brought a tremendous wealth of new genetic data to light. SUMMARY Genetically defined, rare/orphan podocytopathies, as reviewed here, are critically important to study as they may reveal the next generation targets for precision medicine in nephrology.

Progressive chronic kidney diseases (CKDs) are on the rise worldwide. However, the sequence of events resulting in CKD progression remain poorly understood. Animal models of CKD exploring these issues are confounded by systemic toxicities or surgical interventions to acutely induce kidney injury. Here we report the generation of a CKD mouse model through the inducible podocyte-specific ablation of an essential endogenous molecule, the chromatin structure regulator CCCTC-binding factor (CTCF), which leads to rapid podocyte loss (iCTCFpod–/–). As a consequence, iCTCFpod–/– mice develop severe progressive albuminuria, hyperlipidemia, hypoalbuminemia, and impairment of renal function, and die within 8–10 weeks. CKD progression in iCTCFpod–/– mice leads to high serum phosphate and elevations in fibroblast growth factor 23 (FGF23) and parathyroid hormone that rapidly cause bone mineralization defects, increased bone resorption, and bone loss. Dissection of the timeline leading to glomerular pathology in this CKD model led to the surprising observation that podocyte ablation and the resulting glomerular filter destruction is sufficient to drive progressive CKD and osteodystrophy in the absence of interstitial fibrosis. This work introduces an animal model with significant advantages for the study of CKD progression, and it highlights the need for podocyte-protective strategies for future kidney therapeutics.

Single-cell genomics provide a powerful approach to investigate the intrinsic complexity of the kidney and understand the diverse cell types and states that exist during kidney development, homeostasis and disease. Several advances were made in 2019 that enhance our understanding of kidney immune cell states in health and disease and the quality of current kidney organoid model systems for studying human diseases.

Visual Overview Download figureOpen in new tabDownload powerpoint Background Autosomal dominant tubulointerstitial kidney disease caused by mucin-1 gene (MUC1) mutations (ADTKD-MUC1) is characterized by progressive kidney failure. Genetic evaluation for ADTKD-MUC1 specifically tests for a cytosine duplication that creates a unique frameshift protein (MUC1fs). Our goal was to develop immunohistochemical methods to detect the MUC1fs created by the cytosine duplication and, possibly, by other similar frameshift mutations and to identify novel MUC1 mutations in individuals with positive immunohistochemical staining for the MUC1fs protein. Methods We performed MUC1fs immunostaining on urinary cell smears and various tissues from ADTKD-MUC1–positive and –negative controls as well as in individuals from 37 ADTKD families that were negative for mutations in known ADTKD genes. We used novel analytic methods to identify MUC1 frameshift mutations. Results After technique refinement, the sensitivity and specificity for MUC1fs immunostaining of urinary cell smears were 94.2% and 88.6%, respectively. Further genetic testing on 17 families with positive MUC1fs immunostaining revealed six families with five novel MUC1 frameshift mutations that all predict production of the identical MUC1fs protein. Conclusions We developed a noninvasive immunohistochemical method to detect MUC1fs that, after further validation, may be useful in the future for diagnostic testing. Production of the MUC1fs protein may be central to the pathogenesis of ADTKD-MUC1. 29 9 2418 2431

With more than 6,000 new pediatric patients with treatment-resistant nephrotic syndrome in the US each year alone, the unmet need for novel, podocyte-specific therapies is substantial. Recently, the established therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) was used as a starting point to gain insight into the pathomechanism of primary podocytopathies. A calcium (Ca(2+))-mediated pathway has been identified that connects the angiotensin type 1 receptor (AT1R) to podocyte cytoskeletal dynamics, essential for a functioning glomerular filtration barrier. This discovery provided an important missing piece in our understanding of the pathomechanism of filter barrier damage, revealing Ca(2+) signaling as critical for podocyte health and disease. The identification of the two Ca(2+) permeant channels TRPC5 and TRPC6 as mediators of this pathway not only bolstered the importance of podocyte cytoskeleton dynamics but also revealed promising drug targets for treatment-resistant nephrotic syndrome. This review will focus on this novel signaling pathway in primary podocytopathies and its implications for next-generation therapies for glomerular disease.

Abatacept (cytotoxic T-lymphocyte–associated antigen 4–immunoglobulin fusion protein [CTLA-4–Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize β1-integrin activation in podocytes and reduce proteinuria in patients with B7-1–positive glomerular disease.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome. 178 3 521 535.e23

Abatacept (cytotoxic T-lymphocyte–associated antigen 4–immunoglobulin fusion protein [CTLA-4–Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize β1-integrin activation in podocytes and reduce proteinuria in patients with B7-1–positive glomerular disease.

From the Departments of Medicine (A.G., R.S.B.), Radiology (S.H.S.), and Pathology (J.P.D.), Massachusetts General Hospital; and the Departments of Medicine (A.G., R.S.B.), Radiology (S.H.S.), and Pathology (J.P.D.), Harvard Medical School — both in Boston.

Mitochondria play a crucial role in tubular injury in diabetic kidney disease (DKD). MitoQ is a mitochondria-targeted antioxidant that exerts protective effects in diabetic mice, but the mechanism underlying these effects is not clear. We demonstrated that mitochondrial abnormalities, such as defective mitophagy, mitochondrial reactive oxygen species (ROS) overexpression and mitochondrial fragmentation, occurred in the tubular cells of db/db mice, accompanied by reduced PINK and Parkin expression and increased apoptosis. These changes were partially reversed following an intraperitoneal injection of mitoQ. High glucose (HG) also induces deficient mitophagy, mitochondrial dysfunction and apoptosis in HK-2 cells, changes that were reversed by mitoQ. Moreover, mitoQ restored the expression, activity and translocation of HG-induced NF-E2-related factor 2 (Nrf2) and inhibited the expression of Kelch-like ECH-associated protein (Keap1), as well as the interaction between Nrf2 and Keap1. The reduced PINK and Parkin expression noted in HK-2 cells subjected to HG exposure was partially restored by mitoQ. This effect was abolished by Nrf2 siRNA and augmented by Keap1 siRNA. Transfection with Nrf2 siRNA or PINK siRNA in HK-2 cells exposed to HG conditions partially blocked the effects of mitoQ on mitophagy and tubular damage. These results suggest that mitoQ exerts beneficial effects on tubular injury in DKD via mitophagy and that mitochondrial quality control is mediated by Nrf2/PINK. Keywords: Mitoq, Diabetic kidney disease, Mitophagy, Tubular

Age-associated cerebrovascular disease impacts brain tissue integrity, but other factors, including normal variation in blood markers of systemic health, may also influence the structural integrity of the brain. This cross-sectional study included 139 individuals between 40 to 86 years old who were physically healthy and cognitively intact. Eleven markers (total-cholesterol, high-density lipoprotein, low-density lipoprotein, triglyceride, insulin, fasting glucose, glycated hemoglobin, creatinine, blood urea nitrogen, albumin, total protein) and five derived indicators (estimated glomerular filtration rate, creatinine clearance rate, insulin-resistance, average glucose, and cholesterol/high-density lipoprotein ratio) were obtained from blood sampling. Diffusion tensor imaging was used to evaluate white matter tissue health. Blood markers were clustered into five factors. The first factor (defined as insulin/high-density lipoprotein factor) was associated with markers of integrity in the deep white matter and projection fiber systems, while the third factor (defined as kidney function factor) was associated with different markers of integrity in the periventricular and watershed white matter regions. Differential segregated associations for insulin and high-density lipoprotein levels and serum markers of kidney function may provide information about distinct mechanisms of brain changes across the lifespan. These results emphasize the need to determine whether therapeutic modulation of systemic health and organ function may prevent decline in brain structural integrity.

Ca(2+)-mediated remodeling of the actin cytoskeleton is a dynamic process that regulates cell motility through the modulation of rho guanosine triphosphatase (GTPase) signaling. Kidney podocytes are unique, pericyte-like cells with a complex cellular organization consisting of a cell body, major processes, and foot processes (FPs). The FPs form a characteristic interdigitating pattern with FPs of neighboring podocytes, leaving in between filtration slits that are covered by the slit diaphragm (SD). The actin-based FP and the SD form the final barrier to proteinuria. Mutations affecting several podocyte proteins cause disruption of the filtration barrier and rearrangement of the highly dynamic podocyte actin cytoskeleton. Proteins regulating the plasticity of the podocyte actin cytoskeleton are therefore of critical importance for sustained kidney barrier function. Dynamic regulation of the actin-based contractile apparatus in podocyte FPs is essential for sustained kidney filter function. Thus, the podocyte represents an excellent model system to study calcium signaling and actin dynamics in a physiologic context. Here, we discuss the regulation of podocyte actin dynamics by angiotensin or bradykinin-mediated calcium influx and downstream Rho GTPase signaling pathways and how these pathways are operative in other cells including fibroblasts and cancer cells.

Calcium (Ca(2+)) ions are important mediators of cellular homeostasis owing to their ability to elicit a dynamic, transient, and tightly regulated range of biochemical responses. More than a decade ago, a nonselective, Ca(2+)-permeable, cationic conductance was identified in podocytes downstream of angiotensin II (Ang II) signaling, but its molecular structure remained elusive. Six years ago, transient receptor potential canonical 6 (TRPC6) mutations were found in families with hereditary FSGS, and TRPC5 and TRPC6 channels are now known as the Ca(2+) influx pathways for this previously described, nonselective, cationic current in podocytes. Ang II activation engages this Ca(2+) influx to modulate the actin cytoskeleton in podocytes. These discoveries dovetail with previously described regulation of actin dynamics by the Ca(2+)-activated phosphatase, calcineurin, and the emergence of Rho GTPases as critical regulators of podocyte function in health and disease. Understanding the interconnected signaling regulated by Ca(2+) currents offers potential new therapeutic targets and highlights the notion that synergistic therapies targeting multiple levels of biochemistry may be useful in treating proteinuric kidney disease.

Tumor suppressor p53 linked to immune function We thought we knew all we needed to about the tumor suppressor p53. However, Yoon et al. now describe a previously unrecognized function of p53 (see the Perspective by Zitvogel and Kroemer). p53 induces expression of the gene encoding DD1α, a receptor-like transmembrane protein of the immunoglobulin superfamily. In conditions of stress, p53 activation can lead to cell death. p53-induced expression of DD1α also promotes the clearance of dead cells by promoting engulfment by macrophages. Furthermore, expression of DD1α on T cells inhibits T cell function. Thus, p53 offers protection from inflammatory disease caused by the accumulation of apoptotic cells, and its suppression of T cells might help cancer cells to escape immune detection. Science , this issue 10.1126/science.1261669 ; see also p. 476

PURPOSE OF REVIEW A core mission for modern medicine is the development of precision therapeutics. Cancer therapies have been at the leading edge of this effort, while nephrology has lagged on the path to precision medicine. Breaking the stalemate, recent work revealed CD80 (B7-1) as a candidate for targeted therapy in the treatment of resistant nephrotic syndrome. This review aims to summarize the current state of our understanding of podocyte CD80 biology, its therapeutic implications and the challenges that lie ahead in essential future validation studies. RECENT FINDINGS The CD80 targeting agent abatacept (CTLA4-Ig), approved to treat rheumatoid arthritis, was shown to induce remission of nephrotic range proteinuria in four patients with recurrence of disease posttransplant and one patient with primary, treatment resistant nephrotic syndrome. The concept of 'CD80-positive' proteinuric kidney disease due to podocyte CD80 staining in patient kidney biopsies was introduced as a molecular biomarker to define disease and guide treatment. The mechanism of action of CTLA4-Ig in podocytes was shown to centre on β1 integrin activation in a T-cell independent fashion. Subsequent work revealed a putative role for podocyte CD80 in diabetic kidney disease. SUMMARY These studies have direct implications for patient care, and intense interest has focused on validating these findings in upcoming clinical trials.

In this prospective, non-comparative case series, four patients with severe and highly active adult-onset Still's disease (AOSD), refractory to high doses of corticosteroids (which had been combined with methotrexate in three of them) and methotrexate were treated with infliximab (initial dose 3-5 mg/kg, continuing at intervals depending on the patient's individual disease activity). Resolution of their symptoms, which was evident within few days after the first infusion, and a parallel rapid improvement of the acute inflammatory response indices were observed in all. Concomitant corticosteroid treatment was reduced after the first courses of treatment with infliximab, which was well tolerated, and complete disease remission was sustained during a 5-18-month follow-up period. Although further studies to confirm long-term efficacy and safety in larger numbers of patients are needed, we suggest that administration of infliximab with observation for objective improvement is the treatment of choice in cases of AOSD refractory to conventional treatment.

An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

In the face of ever-increasing incidence and prevalence of kidney disease worldwide, the unmet need for new treatments is unprecedented. Precision medicine is defined as the use of modern technologies to identify mechanisms of diseases in individual patients, and thus deploy treatment using tailored, targeted approaches, in the hopes of avoiding unnecessary toxicities and complications. Is there a place for kidney disease therapeutics in this space? If so, what is required to make significant progress toward precision nephrology? To answer these critical questions, we present a series of personalized comments corresponding to the responses offered to these very questions during the Inaugural Glom-NExT Symposium held at Harvard Medical School on October 23, 2014, a national meeting focused exclusively on kidney disease therapeutics.

As an integral member of the filtration barrier in the kidney glomerulus, the podocyte is in a unique geographical position: It is exposed to chemical signals from the urinary space (Bowman's capsule), it receives and transmits chemical and mechanical signals to/from the glomerular basement membrane upon which it elaborates, and it receives chemical and mechanical signals from the vascular space with which it also communicates. As with every cell, the ability of the podocyte to receive signals from the surrounding environment and to translate them to the intracellular milieu is dependent largely on molecules residing on the cell membrane. These molecules are the first-line soldiers in the ongoing battle to sense the environment, to respond to friendly signals, and to defend against injurious foes. In this review, we take a membrane biologist's view of the podocyte, examining the many membrane receptors, channels, and other signaling molecules that have been implicated in podocyte biology. Although we attempt to be comprehensive, our goal is not to capture every membrane-mediated pathway but rather to emphasize that this approach may be fruitful in understanding the podocyte and its unique properties.

ESRD is a state of small-molecule disarray. We applied liquid chromatography/tandem mass spectrometry-based metabolite profiling to survey350 small molecules in 44 fasting subjects with ESRD, before and after hemodialysis, and in 10 age-matched, at-risk fasting control subjects. At baseline, increased levels of polar analytes and decreased levels of lipid analytes characterized uremic plasma. In addition to confirming the elevation of numerous previously identified uremic toxins, we identified several additional markers of ESRD, including dicarboxylic acids (adipate, malonate, methylmalonate, and maleate), biogenic amines, nucleotide derivatives, phenols, and sphingomyelins. The pattern of lipids was notable for a universal decrease in lower-molecular-weight triacylglycerols, and an increase in several intermediate-molecular-weight triacylglycerols in ESRD compared with controls; standard measurement of total triglycerides obscured this heterogeneity. These observations suggest disturbed triglyceride catabolism and/or beta-oxidation in ESRD. As expected, the hemodialysis procedure was associated with significant decreases in most polar analytes. Unexpected increases in several metabolites, however, indicated activation of a broad catabolic program, including glycolysis, lipolysis, ketosis, and nucleotide breakdown. In summary, this study demonstrates the application of metabolite profiling to identify markers of ESRD, provide perspective on uremic dyslipidemia, and broaden our understanding of the biochemical effects of hemodialysis.

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time- and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.

The incidence of FLT3 mutations (internal tandem duplication and Asp835) was investigated in bone marrow samples from 97 patients with myelodysplastic syndrome [(MDS); excluding cases with refractory anaemia with excess blasts in transformation] at the time of diagnosis and several time points thereafter. Three patients had FLT3 mutations at presentation. Forty-two patients progressed to acute myeloid leukaemia (AML), including the three patients with FLT3 mutations at MDS diagnosis. Three additional patients acquired FLT3 mutations and progressed to AML in 1 month. FLT3 mutations seem to be a critical additional genetic event that transforms a minority of MDS patients to AML.