Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition.

Insulin signaling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near-complete knockout of the closely related insulin-like growth factor 1 receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria in vivo and podocyte cell death in vitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near-complete loss of podocyte IGF1R results in the downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that the partial suppression of podocyte IGF1R could have therapeutic benefits in treating albuminuric kidney disease.

Genetic imputation of kidney transcriptome, proteome and multi-omics illuminates new blood pressure and hypertension targets.

Genetic mechanisms of blood pressure (BP) regulation remain poorly defined. Using kidney-specific epigenomic annotations and 3D genome information we generated and validated gene expression prediction models for the purpose of transcriptome-wide association studies in 700 human kidneys. We identified 889 kidney genes associated with BP of which 399 were prioritised as contributors to BP regulation. Imputation of kidney proteome and microRNAome uncovered 97 renal proteins and 11 miRNAs associated with BP. Integration with plasma proteomics and metabolomics illuminated circulating levels of myo-inositol, 4-guanidinobutanoate and angiotensinogen as downstream effectors of several kidney BP genes (SLC5A11, AGMAT, AGT, respectively). We showed that genetically determined reduction in renal expression may mimic the effects of rare loss-of-function variants on kidney mRNA/protein and lead to an increase in BP (e.g., ENPEP). We demonstrated a strong correlation (r = 0.81) in expression of protein-coding genes between cells harvested from urine and the kidney highlighting a diagnostic potential of urinary cell transcriptomics. We uncovered adenylyl cyclase activators as a repurposing opportunity for hypertension and illustrated examples of BP-elevating effects of anticancer drugs (e.g. tubulin polymerisation inhibitors). Collectively, our studies provide new biological insights into genetic regulation of BP with potential to drive clinical translation in hypertension.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles.

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.

The EWAS Catalog: a database of epigenome-wide association studies.

Epigenome-wide association studies (EWAS) seek to quantify associations between traits/exposures and DNA methylation measured at thousands or millions of CpG sites across the genome. In recent years, the increase in availability of DNA methylation measures in population-based cohorts and case-control studies has resulted in a dramatic expansion of the number of EWAS being performed and published. To make this rich source of results more accessible, we have manually curated a database of CpG-trait associations (with p<1x10 -4) from published EWAS, each assaying over 100,000 CpGs in at least 100 individuals. From January 7, 2022, The EWAS Catalog contained 1,737,746 associations from 2,686 EWAS. This includes 1,345,398 associations from 342 peer-reviewed publications. In addition, it also contains summary statistics for 392,348 associations from 427 EWAS, performed on data from the Avon Longitudinal Study of Parents and Children (ALSPAC) and the Gene Expression Omnibus (GEO). The database is accompanied by a web-based tool and R package, giving researchers the opportunity to query EWAS associations quickly and easily, and gain insight into the molecular underpinnings of disease as well as the impact of traits and exposures on the DNA methylome. The EWAS Catalog data extraction team continue to update the database monthly and we encourage any EWAS authors to upload their summary statistics to our website. Details of how to upload data can be found here: http://www.ewascatalog.org/upload. The EWAS Catalog is available at http://www.ewascatalog.org.

Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

BACKGROUND: Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. METHODS: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. RESULTS: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. CONCLUSIONS: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

Podocyte Bioenergetics in the Development of Diabetic Nephropathy: The Role of Mitochondria.

Diabetic nephropathy (DN) is the leading cause of kidney failure, with an increasing incidence worldwide. Mitochondrial dysfunction is known to occur in DN and has been implicated in the underlying pathogenesis of disease. These complex organelles have an array of important cellular functions and involvement in signaling pathways, and understanding the intricacies of these responses in health, as well as how they are damaged in disease, is likely to highlight novel therapeutic avenues. A key cell type damaged early in DN is the podocyte, and increasing studies have focused on investigating the role of mitochondria in podocyte injury. This review will summarize what is known about podocyte mitochondrial dynamics in DN, with a particular focus on bioenergetic pathways, highlighting key studies in this field and potential opportunities to target, enhance or protect podocyte mitochondrial function in the treatment of DN.

IGFBP-1 expression is reduced in human type 2 diabetic glomeruli and modulates ß1-integrin/FAK signalling in human podocytes.

AIMS/HYPOTHESIS: Podocyte loss or injury is one of the earliest features observed in the pathogenesis of diabetic kidney disease (DKD), which is the leading cause of end-stage renal failure worldwide. Dysfunction in the IGF axis, including in IGF binding proteins (IGFBPs), is associated with DKD, particularly in the early stages of disease progression. The aim of this study was to investigate the potential roles of IGFBPs in the development of type 2 DKD, focusing on podocytes. METHODS: IGFBP expression was analysed in the Pima DKD cohort, alongside data from the Nephroseq database, and in ex vivo human glomeruli. Conditionally immortalised human podocytes and glomerular endothelial cells were studied in vitro, where IGFBP-1 expression was analysed using quantitative PCR and ELISAs. Cell responses to IGFBPs were investigated using migration, cell survival and adhesion assays; electrical cell-substrate impedance sensing; western blotting; and high-content automated imaging. RESULTS: Data from the Pima DKD cohort and from the Nephroseq database demonstrated a significant reduction in glomerular IGFBP-1 in the early stages of human type 2 DKD. In the glomerulus, IGFBP-1 was predominantly expressed in podocytes and controlled by phosphoinositide 3-kinase (PI3K)-forkhead box O1 (FoxO1) activity. In vitro, IGFBP-1 signalled to podocytes via ß1-integrins, resulting in increased phosphorylation of focal-adhesion kinase (FAK), increasing podocyte motility, adhesion, electrical resistance across the adhesive cell layer and cell viability. CONCLUSIONS/INTERPRETATION: This work identifies a novel role for IGFBP-1 in the regulation of podocyte function and that the glomerular expression of IGFBP-1 is reduced in the early stages of type 2 DKD, via reduced FoxO1 activity. Thus, we hypothesise that strategies to maintain glomerular IGFBP-1 levels may be beneficial in maintaining podocyte function early in DKD.

An information theoretic approach to insulin sensing by human kidney podocytes.

Podocytes are key components of the glomerular filtration barrier (GFB). They are insulin-responsive but can become insulin-resistant, causing features of the leading global cause of kidney failure, diabetic nephropathy. Insulin acts via insulin receptors to control activities fundamental to GFB integrity, but the amount of information transferred is unknown. Here we measure this in human podocytes, using information theory-derived statistics that take into account cell-cell variability. High content imaging was used to measure insulin effects on Akt, FOXO and ERK. Mutual Information (MI) and Channel Capacity (CC) were calculated as measures of information transfer. We find that insulin acts via noisy communication channels with more information flow to Akt than to ERK. Information flow estimates were increased by consideration of joint sensing (ERK and Akt) and response trajectory (live cell imaging of FOXO1-clover translocation). Nevertheless, MI values were always <1Bit as most information was lost through signaling. Constitutive PI3K activity is a predominant feature of the system that restricts the proportion of CC engaged by insulin. Negative feedback from Akt supressed this activity and thereby improved insulin sensing, whereas sensing was robust to manipulation of feedforward signaling by inhibiting PI3K, PTEN or PTP1B. The decisions made by individual podocytes dictate GFB integrity, so we suggest that understanding the information on which the decisions are based will improve understanding of diabetic kidney disease and its treatment.

A role for NPY-NPY2R signaling in albuminuric kidney disease.

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.

Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease.

The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease.

An In Vitro Method to Analyze Glucose Uptake in Podocytes.

Podocytes are terminally differentiated, insulin-sensitive cells of the glomerular filtration barrier, with a central role in filtration barrier maintenance. Podocyte injury is one of the earliest features observed in diabetic nephropathy (DN) and plays a key role in the development of albuminuria. Several factors are associated with diabetes-mediated podocyte injury, including dysregulated metabolic pathways and insulin sensitivity (Lay & Coward, Nephrol Dial Transplant 29:1127-1133, 2014); thus, reliable assays to study these responses are key in understanding podocyte alterations in DN. Here, we detail an in vitro method to analyze glucose uptake in conditionally immortalized human podocytes (Saleem, J Am Soc Nephrol 13:630-638, 2002; Coward, Diabetes 54:3095-3102, 2005); this assay is useful for detecting changes in podocyte metabolism, nutrient sensing, and insulin sensitivity.

TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

BACKGROUND: Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. METHODS: We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. RESULTS: Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6-/- podocytes. CONCLUSIONS: Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.

The Evolving Importance of Insulin Signaling in Podocyte Health and Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide, occuring in approximately one-third of diabetic patients. One of the earliest hallmarks of DKD is albuminuria, often occurring following disruptions to the glomerular filtration barrier. Podocytes are highly specialized cells with a central role in filtration barrier maintenance; hence, podocyte dysfunction is a major cause of albuminuria in many settings, including DKD. Numerous studies over the last decade have highlighted the importance of intact podocyte insulin responses in the maintenance of podocyte function. This review summarizes our current perspectives on podocyte insulin signaling, highlighting evidence to support the notion that dysregulated podocyte insulin responses contribute toward podocyte damage, particularly during the pathogenesis of DKD.

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor.

AIMS/HYPOTHESIS: Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. METHODS: Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. RESULTS: Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. CONCLUSIONS/INTERPRETATION: Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.