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Insulin resistance and diabetic nephropathy - development of novel in vivo models for drug discovery

Final Report Summary - DIADRUG (Insulin resistance and diabetic nephropathy - development of novel in vivo models for drug discovery)

Diabetic nephropathy, the renal complication of diabetes, develops in up to one third of diabetic patients. It is characterized by early development of proteinuria followed by a progressing decline in glomerular filtration and glomerulosclerosis. The molecular mechanisms of the disease have remained mostly unknown, but data indicate that insulin resistance of podocytes may be a causative factor initiating the development of certain pathological processes observed in diabetic nephropathy.

We identified several new molecules which play key roles in the regulation of insulin signalling and glucose transport in podocytes, which may thus be involved in the processes leading to the development or progression of diabetic nephropathy. Our novel findings show that adaptor protein CD2AP, a molecule essential for kidney function, associates with regulators of insulin signaling and glucose transport including lipid phosphatase SHIP2 (SH2-domain-containing inositol polyphosphate 5-phosphatase 2). SHIP2 was found to negatively regulate insulin signalling in podocytes leading to development of insulin resistance similarly as in adipocytes and muscle cells. Upregulation of SHIP2 in Zucker rat glomeruli prior to the age of onset of albuminuria suggested a possible role for SHIP2 in the development of podocyte injury. We also studied the genetic association of SHIP2 (INPPL1) and CD2AP gene polymorphisms with diabetic nephropathy and the metabolic syndrome that is a frequent phenomenon in patients with type 1 diabetes and associates with diabetic nephropathy. We found that two INPPL1 SNPs associated with the metabolic syndrome in males but no association between the genotyped SNPs and nephropathy was observed. This suggests that INPPL1 gene variants may contribute to susceptibility to the metabolic syndrome but not to diabetic nephropathy in male patients with type 1 diabetes. Three CD2AP SNPs were found to associate with end-stage renal disease indicating that CD2AP gene variants may contribute to susceptibility to end-stage renal disease in patients with type 1 diabetes.

The small filamentous GTPase septin 7 was found to form a complex with CD2AP and nephrin and to negatively regulate glucose transporter trafficking by forming a filamentous barrier between the GSVs and the plasma membrane. Interestingly, knockdown of sept7b in zebrafish caused severe dysfunction of cilia and disturbed the glomerular and tubular function of the zebrafish pronephric kidney.

In a project utilizing a quantitative proteomics approach we aimed to characterize the pathophysiological mechansms of early podocyte damage during progression of diabetic nephropathy. This led to identification of an actin binding protein ezrin and its interaction partner NHERF2 as proteins downregulated in streptozotocin-induced diabetic glomeruli. We found that ezrin regulates actin reorganization and glucose uptake via GLUT 1 in podocytes suggesting that ezrin may play a role in the development of diabetic nephropathy.

Collectively, we found several regulators of insulin signaling and glucose transport in podocytes suggesting that a previously uncharacterized molecular pathway(s) may modulate insulin sensitivity of podocytes and thereby affect glomerular ultrafiltration function. The data suggest a link between insulin resistance and development of proteinuria. These results advance the knowledge of the maintenance and regulation of kidney ultrafiltration under normal conditions and during development of diabetic nephropathy.