Adiponectin Reduces Glomerular Endothelial Glycocalyx Disruption and Restores Glomerular Barrier Function in a Mouse Model of Type 2 Diabetes.

Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.

A small molecule chaperone rescues keratin-8 mediated trafficking of misfolded podocin to correct genetic Nephrotic Syndrome.

Podocin is a key membrane scaffolding protein of the kidney podocyte essential for intact glomerular filtration. Mutations in NPHS2, the podocin-encoding gene, represent the commonest form of inherited nephrotic syndrome (NS), with early, intractable kidney failure. The most frequent podocin gene mutation in European children is R138Q, causing retention of the misfolded protein in the endoplasmic reticulum. Here, we provide evidence that podocin R138Q (but not wild-type podocin) complexes with the intermediate filament protein keratin 8 (K8) thereby preventing its correct trafficking to the plasma membrane. We have also identified a small molecule (c407), a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator protein defect, that interrupts this complex and rescues mutant protein mistrafficking. This results in both the correct localization of podocin at the plasma membrane and functional rescue in both human patient R138Q mutant podocyte cell lines, and in a mouse inducible knock-in model of the R138Q mutation. Importantly, complete rescue of proteinuria and histological changes was seen when c407 was administered both via osmotic minipumps or delivered orally prior to induction of disease or crucially via osmotic minipump two weeks after disease induction. Thus, our data constitute a therapeutic option for patients with NS bearing a podocin mutation, with implications for other misfolding protein disorders. Further studies are necessary to confirm our findings.

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).

Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome.

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03-mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9-mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches.

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.

Ethnic differences in kidney function in childhood: the Born in Bradford Cohort Renal Study.

Background: Endstage kidney failure rates are higher in South Asians than in White Europeans. Low birth weight is associated with adult chronic kidney disease and is more common in South Asians. Foetal kidney size was smaller in South Asians in the Born in Bradford (BiB) birth cohort. As part of BiB follow up, we aimed to investigate if there were ethnic differences in kidney function and blood pressure in early childhood and whether this was different by foetal kidney size. Methods: Serum creatinine, cystatin C, urea, and urinary albumin to creatinine ratio (ACR), protein to creatinine ratio (PCR) and retinol binding protein (RBP) were analysed in blood and urine samples from those who participated in the BiB follow-up at 7-11 years. Ethnicity was categorised by parental self-report as White European and South Asian. Estimated glomerular filtration rate (eGFR) was calculated using Schwartz, and cystatin C Zappitelli and Filler equations. Linear regression was used to examine the association between ethnicity and eGFR, PCR and blood pressure. Results: 1591 children provided blood (n=1403) or urine (n=625) samples. Mean eGFR was 92 ml/min/1.73m 2 (standard deviation (SD) 9) using Schwartz (n=1156) and 94 (SD 11) using Zappitelli (n=1257). CKD prevalence was rare (1 with eGFR <60 ml/min/1.73m 2, 14 (2.4%) had raised ACR (>2.5 mg/mmol in boys/3.5 mg/mmol in girls). Diastolic blood pressure was higher in South Asian children (difference 2.04 mmHg, 95% CI 0.99 to 3.10) but was not significant in adjusted analysis. There was no evidence of association in adjusted models between ethnicity and any eGFR or urinary measure at this age. Conclusions: There was no evidence of significant ethnic differences in kidney function at pre-pubertal age despite differences in kidney volume at birth. Longitudinal follow-up is required to track ethnic patterns in kidney function and blood pressure as children develop through puberty.

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.

Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress.

Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.

The molecular biology of pelvi-ureteric junction obstruction.

Over recent years routine ultrasound scanning has identified increasing numbers of neonates as having hydronephrosis and pelvi-ureteric junction obstruction (PUJO). This patient group presents a diagnostic and management challenge for paediatric nephrologists and urologists. In this review we consider the known molecular mechanisms underpinning PUJO and review the potential of utilising this information to develop novel therapeutics and diagnostic biomarkers to improve the care of children with this disorder.

VEGF regulates local inhibitory complement proteins in the eye and kidney.

Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists.

IRS2 and PTEN are key molecules in controlling insulin sensitivity in podocytes.

Insulin signaling to the glomerular podocyte is important for normal kidney function and is implicated in the pathogenesis of diabetic nephropathy (DN). This study determined the role of the insulin receptor substrate 2 (IRS2) in this system. Conditionally immortalized murine podocytes were generated from wild-type (WT) and insulin receptor substrate 2-deficient mice (Irs2(-/-)). Insulin signaling, glucose transport, cellular motility and cytoskeleton rearrangement were then analyzed. Within the glomerulus IRS2 is enriched in the podocyte and is preferentially phosphorylated by insulin in comparison to IRS1. Irs2(-/-) podocytes are significantly insulin resistant in respect to AKT signaling, insulin-stimulated GLUT4-mediated glucose uptake, filamentous actin (F-actin) cytoskeleton remodeling and cell motility. Mechanistically, we discovered that Irs2 deficiency causes insulin resistance through up-regulation of the phosphatase and tensin homolog (PTEN). Importantly, suppressing PTEN in Irs2(-/-) podocytes rescued insulin sensitivity. In conclusion, this study has identified for the first time IRS2 as a critical molecule for sensitizing the podocyte to insulin actions through its ability to modulate PTEN expression. This finding reveals two potential molecular targets in the podocyte for modulating insulin sensitivity and treating DN.

Clinical and genetic analysis of patients with cystinuria in the United Kingdom.

BACKGROUND AND OBJECTIVES: Cystinuria is a rare inherited renal stone disease. Mutations in the amino acid exchanger System b(0,+), the two subunits of which are encoded by SLC3A1 and SLC7A9, predominantly underlie this disease. The work analyzed the epidemiology of cystinuria and the influence of mutations in these two genes on disease severity in a United Kingdom cohort. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Prevalent patients were studied from 2012 to 2014 in the northeast and southwest of the United Kingdom. Clinical phenotypes were defined, and genetic analysis of SLC3A1 and SLC7A9 combining Sanger sequencing and multiplex ligation probe-dependent amplification was performed. RESULTS: In total, 76 patients (42 men and 34 women) were studied. All subjects had proven cystine stones. Median age of presentation (first stone episode) was 24 years old, but 21% of patients presented after 40 years old. Patients had varied clinical courses, with 37% of patients having ≥10 stone episodes; 70% had evidence of CKD, and 9% had reached ESRD as a result of cystinuria and its complications. Patients with cystinuria received a variety of different therapies, with no obvious treatment consensus. Notably, 20% of patients had staghorn calculi, with associated impaired renal function in 80% of these patients. Genetic analysis revealed that biallelic mutations were present in either SLC3A1 (n=27) or SLC7A9 (n=20); 22 patients had only one mutated allele detected (SLC3A1 in five patients and SLC7A9 in 17 patients). In total, 37 different mutant variant alleles were identified, including 12 novel mutations; 22% of mutations were caused by large gene rearrangements. No genotype-phenotype association was detected in this cohort. CONCLUSIONS: Patients with cystinuria in the United Kingdom often present atypically with staghorn calculi at ≥40 years old and commonly develop significant renal impairment. There is no association of clinical course with genotype. Treatments directed toward reducing stone burden need to be rationalized and developed to optimize patient care.

Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction.

The endothelial surface glycocalyx is a hydrated mesh in which proteoglycans are prominent. It is damaged in diseases associated with elevated levels of tumor necrosis factor α (TNF-α). We investigated the mechanism of TNF-α-induced disruption of the glomerular endothelial glycocalyx. We used conditionally immortalized human glomerular endothelial cells (GEnCs), quantitative PCR arrays, Western blotting, immunoprecipitation, immunofluorescence, and dot blots to examine the effects of TNF-α. TNF-α induced syndecan 4 (SDC4) mRNA up-regulation by 2.5-fold, whereas cell surface SDC4 and heparan sulfate (HS) were reduced by 36 and 30%, respectively, and SDC4 and sulfated glycosaminoglycan in the culture medium were increased by 52 and 65%, respectively, indicating TNF-α-induced shedding. Small interfering (siRNA) knockdown of SDC4 (by 52%) caused a corresponding loss of cell surface HS of similar magnitude (38%), and immunoprecipitation demonstrated that SDC4 and HS are shed as intact proteoglycan ectodomains. All of the effects of TNF-α on SDC4 and HS were abrogated by the metalloproteinase (MMP) inhibitor batimastat. Also abrogated was the associated 37% increase in albumin passage across GEnC monolayers. Specific MMP9 knockdown by siRNA similarly blocked TNF-α effects. SDC4 is the predominant HS proteoglycan in the GEnC glycocalyx. TNF-α-induced MMP9-mediated shedding of SDC4 is likely to contribute to the endothelial glycocalyx disruption observed in diabetes and inflammatory states.

Podocytes, signaling pathways, and vascular factors in diabetic kidney disease.

Alterations and injury to glomerular podocytes play a key role in the initiation and progression of diabetic kidney disease (DKD). Multiple factors in diabetes cause abnormalities in podocyte signaling that lead to podocyte foot process effacement, hypertrophy, detachment, loss, and death. Alterations in insulin action and mammalian target of rapamycin activation have been well documented to lead to pathology. Reduced insulin action directly leads to albuminuria, increased glomerular matrix accumulation, thickening of the glomerular basement membrane, podocyte apoptosis, and glomerulosclerosis. In addition, podocytes generate factors that alter signaling in other glomerular cells. Prominent among these is vascular endothelial growth factor-A, which maintains glomerular endothelium viability but causes endothelial cell pathology when generated at too high a level. Finally, circulating vascular factors (eg, activated protein C) have a profound effect on podocyte stability and survival. This cytoprotective factor is critical for podocyte health, and its deficiency promotes podocyte injury and apoptosis. Thus, the podocyte sits in the center of a network of paracrine and hormonal signaling systems that in health keep the podocyte adaptable and viable, but in diabetes they can lead to pathologic changes, detachment, and death.

Recent advances in our understanding of insulin signalling to the podocyte.

It is becoming increasingly clear that the insulin responses of a number of different cell types within the kidney are important in the maintenance of normal renal function. This review summarizes our current understanding of renal insulin signalling, with specific focus on the podocyte, presenting recent evidence that suggests these responses are altered in systemic insulin-resistant states and chronic kidney disease via a number of different mechanisms.

Regulation of hypoxia-inducible factor 2-a is essential for integrity of the glomerular barrier.

Deletion of the von Hippel-Lindau tumor suppressor (Vhl) gene from renal podocytes of mice (podVhl KO) leads to rapidly progressive glomerulonephritis (RPGN), a clinical syndrome characterized by rapid loss of renal function and crescents on renal biopsy. Genomic profiling of glomeruli isolated from podVhl knockout (KO) mice and from patients with RPGN identified a fingerprint of genes regulated by hypoxia-inducible factors (HIF), important substrates of the product of the VHL gene. Here, we show that stabilization of Hifs in podocytes is both required and sufficient for the glomerular phenotype observed in podVhl KO mice. Genetic deletion of the obligate dimerization partner Arnt/Hif1b that is essential for Hif transcriptional function rescues the phenotype. Conversely, stabilization of HIF2A alone in podocytes results in crescentic glomerular disease. Together, our results show that the Hif pathway and Hif2a in particular are key players in maintenance of the glomerular barrier.

Advances in our understanding of the pathogenesis of glomerular thrombotic microangiopathy.

Glomerular thrombotic microangiopathy is a hallmark feature of haemolytic uraemic syndrome, the leading cause of acute renal failure in childhood. This paper is a review of the different mechanistic pathways that lead to this histological picture in the kidney. It will focus on atypical HUS and complement dysregulation, but will also highlight some other recent advances in our understanding of this condition, including the potential role of the molecule vascular endothelial growth factor-A (VEGF-A).

An initial evaluation of post-cardiopulmonary bypass acute kidney injury in swine.

OBJECTIVE: Acute kidney injury (AKI) post-cardiac surgery is associated with mortality rates approaching 20%. The development of effective treatments is hindered by the poor homology between rodent models, the mainstay of research into AKI, and that which occurs in humans. This pilot study aims to characterise post-cardiopulmonary bypass (CPB) AKI in an animal model with potentially greater homology to cardiac surgery patients. METHODS AND RESULTS: Adult pigs, weighing 50-75 kg, underwent 2.5 h of CPB. Pigs undergoing saphenous vein grafting procedures served as controls. Pre-CPB measures of porcine renal function were within normal ranges for adult humans. The effect of CPB on renal function; a 25% reduction in (51)Cr-EDTA clearance (p=0.068), and a 33% reduction in creatinine clearance (p=0.043), was similar to those reported in clinical studies. CPB resulted in tubular epithelial injury (median NAG/creatinine ratio 2.6 u mmol(-1) (interquartile range (IQR): 0.81-5.43) post-CPB vs 0.48 u mmol(-1) (IQR: 0.37-0.97) pre-CPB, p=0.043) as well as glomerular and/or proximal tubular injury (median albumin/creatinine ratio 6.8 mg mmol(-1) (IQR: 5.45-13.06) post-CPB vs 1.10 mg mmol(-1) (IQR: 0.05-2.00) pre-CPB, p=0.080). Tubular injury scores were significantly higher in kidneys post-CPB (median score 2.0 (IQR: 1.0-2.0) relative to vein graft controls (median score 1.0 (IQR 1.0-1.0), p=0.019). AKI was associated with endothelial injury and activation, as demonstrated by reduced DBA (dolichos biflorus agglutinin) lectin and increased endothelin-1 and vascular cell adhesion molecule (VCAM) staining. CONCLUSIONS: The porcine model of post-CPB AKI shows significant homology to AKI in cardiac surgical patients. It links functional, urinary and histological measures of kidney injury and may offer novel insights into the mechanisms underlying post-CPB AKI.

Saturated fatty acids induce insulin resistance in human podocytes: implications for diabetic nephropathy.

BACKGROUND: Cellular insulin resistance is the hallmark of type 2 diabetes and predominantly affects adipose and muscle cells. The saturated free fatty acid palmitate is elevated in insulin-resistant states and may directly contribute to cellular insulin resistance. A spectrum of renal disease is associated with increased markers of insulin resistance, although direct causal mechanisms are not known. In the kidney, glomerular podocytes are novel insulin-sensitive cells that have the ability to rapidly transport glucose. In this study, we tested the hypothesis that palmitate would induce insulin resistance in podocytes. METHODS: Conditionally immortalized human podocytes were cultured for up to 24 h with 375-750 muM palmitate. Functional effects on glucose uptake and ceramide production were measured. Gene expression was investigated using a focused gene array, and protein signalling and trafficking were studied with Western blotting and immunofluorescence. RESULTS: We found that palmitate blocked insulin-stimulated glucose uptake in human podocytes. This was associated with increased ceramide production, and use of the ceramide inhibitors myriocin and fumonisin B1 partially recovered the insulin sensitivity. At the level of transcription, palmitate downregulated genes associated with several pathways involved in insulin signalling. At the protein level, phosphorylation of the insulin receptor, IRS1 and PKB was reduced and there was impaired translocation of GLUT4 to the cell surface. CONCLUSION: This is the first study to demonstrate a direct effect of saturated fatty acids on podocyte function. These findings may represent a novel link between systemic insulin resistance and the development of nephropathy.

Hemopexin induces nephrin-dependent reorganization of the actin cytoskeleton in podocytes.

Hemopexin is an abundant plasma protein that effectively scavenges heme. When infused into rats, hemopexin induces reversible proteinuria, and activated hemopexin is increased in children with minimal change nephrotic syndrome. These observations suggest a role for hemopexin in glomerular disease; in this study, the effects of active hemopexin on human podocytes and glomerular endothelial cells, the two cell types that compose the glomerular filtration barrier, were investigated. Within 30 min of treatment with hemopexin, actin reorganized from stress fibers to cytoplasmic aggregates and membrane ruffles in wild-type podocytes. This did not occur in nephrin-deficient podocytes unless they were transfected with nephrin-expressing plasmids. Furthermore, hemopexin did not affect actin organization in cells that do not express nephrin, specifically human glomerular endothelial cells, fibroblasts, and HEK293 cells. The effects of hemopexin on wild-type podocytes reversed within 4 h and were inhibited by preincubation with human plasma. Treatment with hemopexin activated protein kinase B in both wild-type and nephrin-deficient podocytes but activated RhoA only in wild-type cells. In addition, hemopexin led to a selective increase in the passage of albumin across monolayers of glomerular endothelial cells and to a reduction in glycocalyx. In summary, active hemopexin causes nephrin-dependent remodeling of podocytes and affects permeability of the glomerular filtration barrier by degrading the glycocalyx.