PKCα Mediates β-Arrestin2-dependent Nephrin Endocytosis in Hyperglycemia

Abstract

Nephrin, the key molecule of the glomerular slit diaphragm, is expressed on the surface of podocytes and is critical in preventing albuminuria. In diabetes, hyperglycemia leads to the loss of surface expression of nephrin and causes albuminuria. Here, we report a mechanism that can explain this phenomenon: hyperglycemia directly enhances the rate of nephrin endocytosis via regulation of the ␤-arrestin2-nephrin interaction by PKC␣. We identified PKC␣ and protein interacting with c kinase-1 (PICK1) as nephrin-binding proteins. Hyperglycemia induced up-regulation of PKC␣ and led to the formation of a complex of nephrin, PKC␣, PICK1, and ␤-arrestin2 in vitro and in vivo. Binding of ␤-arrestin2 to the nephrin intracellular domain depended on phosphorylation of nephrin threonine residues 1120 and 1125 by PKC␣. Further, cellular knockdown of PKC␣ and/or PICK1 attenuated the nephrin-␤-arrestin2 interaction and abrogated the amplifying effect of high blood glucose on nephrin endocytosis. In C57BL/6 mice, hyperglycemia over 24 h caused a significant increase in urinary albumin excretion, supporting the concept of the rapid impact of hyperglycemia on glomerular permselectivity. In summary, we have provided a molecular model of hyperglycemia-induced nephrin endocytosis and subsequent proteinuria and highlighted PKC␣ and PICK1 as promising therapeutic targets for diabetic nephropathy.Diabetes mellitus is a major health problem in Western countries. Despite therapeutic advances, diabetic nephropathy remains the leading cause of end stage renal disease (1). Chronic hyperglycemia causes glomerular damages such as endothelial dysfunction, loss of negative charges in the basement membrane, and podocyte damage (2). Microalbuminuria is an early symptom of a "leaky" glomerular barrier and is associated with a markedly increased cardiovascular risk (3).Podocytes are visceral epithelial cells wrapped around glomerular capillaries and are connected by the slit diaphragm. This specialized cell junction is a dynamic multiprotein complex that functions as a size-selective sieve to prevent the loss of plasma proteins through urine (4, 5). Nephrin serves as the backbone of this diaphragm by functioning as a regulator of podocyte signaling and mediator of actin dynamics (6 -8). Several podocyte signaling abnormalities have been noted in diabetes. For example, mice and humans with diabetes show altered distribution and expression of nephrin (9 -11), and hyperglycemia increases diacylglycerol generation, causing PKC activation (2). However, it is not fully understood how "hyperglycemic" signaling in diabetes affects the expression and distribution of nephrin.Menne et al. (12) reported a major step elucidating podocyte signaling in diabetes: they showed that albuminuria does not develop in diabetic PKC␣-deficient mice and concluded that decreased nephrin expression in diabetes is a result of altered transcriptional regulation. All of the studies until now were conducted in chronic models of diabetes (13). Therefore, their conclusions were based...

Podocyte-specific Nox4 deletion affords renoprotection in a mouse model of diabetic nephropathy

“…Indeed, we found that the diabetes-induced increase in glomerular PKC-α expression was attenuated in diabetic podNox4KO mice. Quack et al have demonstrated that acute hyperglycaemia increases nephrin endocytosis in a PKC-α-dependent manner [45] and this effect is considered to promote albuminuria. In this study, we found similar changes in VEGF and nephrin expression as well as in albuminuria, in diabetic podNox4KO mice to those seen in diabetic PkcαKO mice, and also noted a reduction in glomerular PKC-α expression in podNox4KO mice.…”

Vascular Endothelial Growth Factor Receptor 2 Direct Interaction with Nephrin Links VEGF-A Signals to Actin in Kidney Podocytes

“…Upon VEGF-A binding, VEGFR2 autophosphorylation at Tyr-1175 mediates PLC␥ binding, leading to PKC activation (19,21,22). Interestingly, PKC␣ mediates the effect of hyperglycemia on nephrin-␤-arrestin2 interaction, increasing nephrin endocytosis in podocytes (45,46). PKC␣ negatively regulates VEGF signaling and endothelial nitric oxide synthase phosphorylation in endothelial cells (53).…”

Bone marrow-derived mesenchymal stem cells ameliorate nephrosis through repair of impaired podocytes

“…Nephrin was the first globulin transmembrane protein found specifically on the SD of podocytes and is important in maintaining slit integrity [18][19]; therefore, nephrin serves as an early marker of podocyte damage [20]. In nephrin knockout mice, podocyte foot processes disappear, SD are deformed and excess proteinuria occurs.…”