Podocyte-specific VEGF-a gain of function induces nodular glomerulosclerosis in eNOS null mice.

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF(164) gain of function in eNOS(-/-) mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF(164) gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS(-/-) mice with podocyte-specific VEGF(164) gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF(164) gain of function decreased glomerular S-nitrosylation of laminin in eNOS(-/-) mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson-like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin.

Excess podocyte semaphorin-3A leads to glomerular disease involving plexinA1-nephrin interaction.

Semaphorin-3A (Sema3a), a guidance protein secreted by podocytes, is essential for normal kidney patterning and glomerular filtration barrier development. Here, we report that podocyte-specific Sema3a gain-of-function in adult mice leads to proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Reversibility of the glomerular phenotype upon removal of the transgene induction provided proof-of-principle of the cause-and-effect relationship between podocyte Sema3a excess and glomerular disease. Mechanistically, excess Sema3a induces dysregulation of nephrin, matrix metalloproteinase 9, and αvß3 integrin in vivo. Sema3a cell-autonomously disrupts podocyte shape. We identified a novel direct interaction between the Sema3a signaling receptor plexinA1 and nephrin, linking extracellular Sema3a signals to the slit-diaphragm signaling complex. We conclude that Sema3a functions as an extracellular negative regulator of the structure and function of the glomerular filtration barrier in the adult kidney. Our findings demonstrate a crosstalk between Sema3a and nephrin signaling pathways that is functionally relevant both in vivo and in vitro.

Vascular endothelial growth factor receptor 2 direct interaction with nephrin links VEGF-A signals to actin in kidney podocytes.

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

Podocyte VEGF-A Knockdown Induces Diffuse Glomerulosclerosis in Diabetic and in eNOS Knockout Mice.

Vascular endothelial growth factor-a (VEGF-A) and nitric oxide (NO) are essential for glomerular filtration barrier homeostasis, and are dysregulated in diabetic kidney disease (DKD). While NO availability is consistently low in diabetes, both high and low VEGF-A have been reported in patients with DKD. Here we examined the effect of inducible podocyte VEGF-A knockdown (VEGFKD ) in diabetic mice and in endothelial nitric oxide synthase knockout mice (eNOS-/- ). Diabetes was induced with streptozotocin using the Animal Models of Diabetic Complications Consortium (AMDCC) protocol. Induction of podocyte VEGFKD led to diffuse glomerulosclerosis, foot process effacement, and GBM thickening in both diabetic mice with intact eNOS and in non-diabetic eNOS-/-:VEGFKD mice. VEGFKD diabetic mice developed mild proteinuria and maintained normal glomerular filtration rate (GFR), associated with extremely high NO and thiol urinary excretion. In eNOS-/-:VEGFKD (+dox) mice severe diffuse glomerulosclerosis was associated with microaneurisms, arteriolar hyalinosis, massive proteinuria, and renal failure. Collectively, data indicate that combined podocyte VEGF-A and eNOS deficiency result in diffuse glomerulosclerosis in mice; compensatory NO and thiol generation prevents severe proteinuria and GFR loss in VEGFKD diabetic mice with intact eNOS, whereas VEGFKD induction in eNOS-/-:VEGFKD mice causes massive proteinuria and renal failure mimicking DKD in the absence of diabetes. Mechanistically, we identify VEGFKD -induced abnormal S-nitrosylation of specific proteins, including ß3-integrin, laminin, and S-nitrosoglutathione reductase (GSNOR), as targetable molecular mechanisms involved in the development of advanced diffuse glomerulosclerosis and renal failure.

High prevalence of vitamin D deficiency in north Indian adults is exacerbated in those with chronic kidney disease.

AIM: Vitamin D is being increasingly recognized as an important player in disease. Hypovitaminosis D is widespread in chronic kidney disease (CKD) populations around the world. The vitamin D status of Indian CKD patients is not known. METHODS: Levels of 25(OH) vitamin D and parathyroid hormone (PTH) were measured in adult north Indian male patients with newly diagnosed stage IV-V CKD and matched control subjects drawn from the same population. A total of 100 (34 stage IV and 66 stage V) patients with CKD and 72 controls were studied. RESULTS: Only 4% control and 1% of CKD subjects had normal (>30 ng/mL) vitamin D levels. Approximately 68% of control and 77% of the CKD population had vitamin D deficiency (<15 ng/ml) whereas the remaining 38% control and 22% CKD patients had insufficient (15-30 ng/mL) vitamin D levels. Levels were lower in CKD subjects compared to their family members, and the CKD patients were significantly more likely to have severe vitamin D deficiency (<5 ng/mL). A strong negative correlation was noted between vitamin D and PTH. No significant correlation was found between vitamin D levels and body mass index, bodyfat percentage, serum albumin or calcium levels. CONCLUSION: Vitamin D deficiency is highly prevalent in north Indians, and this is more pronounced in CKD subjects. There is a significant inverse correlation between the vitamin D and PTH levels. The clinical significance of this deficiency and the potential benefits to be derived from vitamin D supplementation in this population merits further studies.

FAT1 mutations cause a glomerulotubular nephropathy.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.

Semaphorin3a promotes advanced diabetic nephropathy.

The onset of diabetic nephropathy (DN) is highlighted by glomerular filtration barrier abnormalities. Identifying pathogenic factors and targetable pathways driving DN is crucial to developing novel therapies and improving the disease outcome. Semaphorin3a (sema3a) is a guidance protein secreted by podocytes. Excess sema3a disrupts the glomerular filtration barrier. Here, using immunohistochemistry, we show increased podocyte SEMA3A in renal biopsies from patients with advanced DN. Using inducible, podocyte-specific Sema3a gain-of-function (Sema3a(+)) mice made diabetic with streptozotocin, we demonstrate that sema3a is pathogenic in DN. Diabetic Sema3a(+) mice develop massive proteinuria, renal insufficiency, and extensive nodular glomerulosclerosis, mimicking advanced DN in humans. In diabetic mice, Sema3a(+) exacerbates laminin and collagen IV accumulation in Kimmelstiel-Wilson-like glomerular nodules and causes diffuse podocyte foot process effacement and F-actin collapse via nephrin, αvß3 integrin, and MICAL1 interactions with plexinA1. MICAL1 knockdown and sema3a inhibition render podocytes not susceptible to sema3a-induced shape changes, indicating that MICAL1 mediates sema3a-induced podocyte F-actin collapse. Moreover, sema3a binding inhibition or podocyte-specific plexinA1 deletion markedly ameliorates albuminuria and abrogates renal insufficiency and the diabetic nodular glomerulosclerosis phenotype of diabetic Sema3a(+) mice. Collectively, these findings indicate that excess sema3a promotes severe diabetic nephropathy and identifies novel potential therapeutic targets for DN.

CYP3A5*1/*3 genotype influences the blood concentration of tacrolimus in response to metabolic inhibition by ketoconazole.

OBJECTIVES: Ketoconazole retards metabolic degradation of tacrolimus through its effect on the cytochrome P-450 enzyme system and allows reduction in treatment costs. Enzyme activity is determined by a single nucleotide polymorphism (*1/*3) in the CYP3A5 gene. METHODS: We prospectively investigated the impact of this polymorphism on tacrolimus concentration in a cohort of 79 renal transplant recipients on ketoconazole. Genotyping was carried out by using polymerase chain reaction-restriction fragment length polymorphism technique. Dose-adjusted trough level (C0) was calculated at baseline and at 3, 7, 15, 30, and 60 days. RESULTS: The baseline C0 was significantly lower in those with at least one *1 allele [44.95+/-14.12 vs. 63.43+/-14.72 (ng/ml)/(mg/kg/day), P<0.0001]. After starting ketoconazole in all genotypes, dose-normalized C0 increased and the cost of therapy decreased. Compared with baseline, the magnitude of increase was 112% and 79% in those without and with *1 allele, respectively (P<0.001). The cost savings were 32% and 39% in mycophenolate mofetil-treated and 47% and 61% in azathioprine-treated patients who were with and without one *1 allele, respectively. CONCLUSION: We show that the CYP3A5*1/*3 polymorphism is an important determinant of the response to inhibition of tacrolimus metabolism by ketoconazole, with a 30% greater inhibition in those lacking *1 allele. This finding will allow better dose adjustment and minimize exposure to subtherapeutic or toxic concentrations.