Richard J M Coward

Nephrin is critical for the action of insulin on human glomerular podocytes

The leading causes of albuminuria and end-stage renal failure are secondary to abnormalities in the production or cellular action of insulin, including diabetes and hyperinsulinemic metabolic syndrome. The human glomerular podocyte is a critical cell for maintaining the filtration barrier of the kidney and preventing albuminuria. We have recently shown this cell to be insulin sensitive with respect to glucose uptake, with kinetics similar to muscle cells. We now show that the podocyte protein nephrin is essential for this process. Conditionally immortalized podocytes from two different patients with nephrin mutations (natural human nephrin mutant models) were unresponsive to insulin. Knocking nephrin down with siRNA in wild-type podocytes abrogated the insulin response, and stable nephrin transfection of nephrin-deficient podocytes rescued their insulin response. Mechanistically, we show that nephrin allows the GLUT1- and GLUT4-rich vesicles to fuse with the membrane of this cell. Furthermore, we show that the COOH of nephrin interacts with the vesicular SNARE protein VAMP2 in vitro and ex vivo (using yeast-2 hybrid and coimmunoprecipitation studies). This work demonstrates a previously unsuspected role of nephrin in vesicular docking and insulin responsiveness of podocytes.

Nephrotic Plasma Alters Slit Diaphragm–Dependent Signaling and Translocates Nephrin, Podocin, and CD2 Associated Protein in Cultured Human Podocytes

Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm (SD) proteins nephrin, podocin, and CD2-associated protein (CD2AP) to function optimally. The effects of normal human plasma and nephrotic plasma on podocytes were tested, focusing particularly on the SD complex. With the use of a conditionally immortalized human podocyte cell line, it first was shown that exposure to normal and non-nephrotic human plasma leads to a concentration of nephrin, podocin, CD2AP, and actin at the cell surface. Next, the effects of plasma from patients with nephrotic conditions to non-nephrotic conditions were compared. When exposed to all nephrotic plasma samples (and a non-human serum control), nephrin podocin and CD2AP assumed a cytoplasmic distribution; nephrin and synaptopodin were selectively downregulated, and the relocation of nephrin induced by nephrotic plasma could be rescued back to the plasma membrane by co-incubation with non-nephrotic plasma. Furthermore, intracellular calcium signaling was altered by nephrotic plasma, which was mediated by tyrosine kinase phosphorylation. With the use of nephrin mutant human cell lines, it was shown that this signaling and translocation response to normal plasma is nephrin dependent. This work demonstrates that nephrotic plasma seems to be deficient in factors that act via the podocyte SD complex, which are essential in maintaining its physiologic function.

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.

Diabetic Nephropathy: Perspective on Novel Molecular Mechanisms

Diabetes mellitus (DM) is the major cause of end-stage renal disease (ESRD) globally, and novel treatments are urgently needed. Current therapeutic approaches for diabetic nephropathy (DN) are focussing on blood pressure control with inhibitors of the renin-angiotensin-aldosterone system, on glycaemic and lipid control, and life-style changes. In this review, we highlight new molecular insights aiding our understanding of the initiation and progression of DN, including glomerular insulin resistance, dysregulation of cellular substrate utilisation, podocyte-endothelial communication, and inhibition of tubular sodium coupled glucose reabsorption. We believe that these mechanisms offer new therapeutic targets that can be exploited to develop important renoprotective treatments for DN over the next decade.

Insulin directly stimulates VEGF-A production in the glomerular podocyte.

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

Initial Steroid Sensitivity in Children with Steroid-Resistant Nephrotic Syndrome Predicts Post-Transplant Recurrence

Of children with idiopathic nephrotic syndrome, 10%-20% fail to respond to steroids or develop secondary steroid resistance (termed initial steroid sensitivity) and the majority progress to transplantation. Although 30%-50% of these patients suffer disease recurrence after transplantation, with poor long-term outcome, no reliable indicator of recurrence has yet been identified. Notably, the incidence of recurrence after transplantation appears reduced in patients with steroid-resistant nephrotic syndrome (SRNS) due to monogenic disorders. We reviewed 150 transplanted patients with SRNS to identify biomarkers that consistently predict outcome of SRNS after transplantation. In all, 25 children had genetic or familial SRNS and did not experience post-transplant recurrence. We reviewed phenotypic factors, including initial steroid sensitivity, donor type, age, ethnicity, time to ESRD, and time on dialysis, in the remaining 125 children. Of these patients, 57 (45.6%) developed post-transplant recurrence; 26 of 28 (92.9%) patients with initial steroid sensitivity recurred after transplantation, whereas only 26 of 86 (30.2%) patients resistant from the outset recurred (odds ratio, 30; 95% confidence interval, 6.62 to 135.86; P<0.001). We were unable to determine recurrence in two patients (one with initial steroid sensitivity), and nine patients did not receive initial steroids. Our data show that initial steroid sensitivity is highly predictive of post-transplant disease recurrence in this pediatric patient population. Because a pathogenic circulating permeability factor in nephrotic syndrome remains to be confirmed, we propose initial steroid sensitivity as a surrogate marker for post-transplant recurrence.

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1

Aims/hypothesisPeroxisome proliferator-activated receptor (PPAR) γ agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARγ agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARγ agonist rosiglitazone on human podocytes.MethodsConditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling.ResultsRosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate.Conclusions/interpretationIn conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARγ agonists may improve podocyte function in diabetic nephropathy.

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.—Ramnath, R., Foster, R. R., Qiu, Y., Cope, G., Butler, M. J., Salmon, A. H., Mathieson, P. W., Coward, R. J., Welsh, G. I., Satchell, S. C., Matrix metalloproteinase 9‐mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction. FASEB J. 28, 4686–4699 (2014). www.fasebj.org

Podocytes, glucose and insulin

Purpose of reviewIn the last decade the importance of the podocyte in maintaining the integrity of the glomerular filtration barrier and prevention of albuminuria has become obvious. The leading cause of renal failure in the developed world is diabetic nephropathy. This has a defined natural history of progressive albuminuria. The two fundamental cellular changes that occur in diabetes mellitus are a failure of insulin to signal to cells and an environment of hyperglycaemia. The emerging role of these factors on the biological function of the podocyte will be reviewed. Recent findingsHyperglycaemia causes both deleterious and protective cellular pathways to be initiated in the podocyte, which communicate with other cell types in the glomerulus. Furthermore, the podocyte is an insulin-sensitive cell which can be directly modulated by factors that increase and decrease its sensitivity to insulin. SummaryOur understanding of the cellular processes that affect the podocyte in diabetes and insulin resistance has progressed greatly in recent years and hopefully will result in new treatment strategies against the leading cause of renal failure in the developed world.