Effect of Genistein in Reducing Albuminuria Due to Albumin Endocytosis Through Caveolae into Glomerular Endothelial and Epithelial Cells by Decreasing Src Kinase Activity

Background: Previously, we have been reported about the new etiology of albuminuria by caveolar mediated intracellular trafficking pathway of albumin in glomerular endothelial cells (GEnCs) and podocytes (Pod), and also researched novel therapeutic approach to reduce albuminuria by decreasing caveolae. Src kinase plays an important role of internalization of caveolae coated pits into cytoplasm as caveolae endocytosis. In this study, we analyzed whether Genistein (Gen), which was the non-steroidal phytoestrogen belonged to soy isoflavone, inhibited Src activity in GEnCs and pod, and resulted in decreasing albuminuria of puromycin aminonucleoside (PAN) induced minimal change nephrotic syndrome (MCNS) modeled mice. Methods: In vitro study, GEnCs or Pod were pre-treated with 50, 100, or 200 µM of Gen for 60min, then each cell was co-incubated with albumin for 15, 30, and 60 min. After that, the expression of albumin, caveolin-1 (Cav-1), and p-Src were evaluated by immunofluorescence (IF) and western blots (WB) analysis. In vivo study, PAN mice (n=8) were treated with 7 days intraperitoneal injection of Gen or DMSO (as control), after that , the amount of albuminuria between both groups and between day 0 and day7 in each group were compared, and the expressions of p-Src and Cav-1 in glomeruli by IF and immunohistochemistry (IHC) were also compared between both groups. Results: In each cell, albumin, Cav-1, and p-Src expression levels were significantly decreased with dose dependent manner of Gen in IF and WB analysis. In vivo study, albuminuria in control was significantly increased from day 0 to 7 (0.011 to 0.065, p=0.021), but it was not increased by Gen (0.003 to 0.019, p=0.083). Moreover, albuminuria on day 7 was significantly lower by Gen than control (p=0.021). The relative expression levels of Cav-1 (p<0.001) and p-Src (p=0.001) in glomeruli were significantly decreased by Gen in IF and IHC Conclusion: Gen interfered albumin endocytosis through caveolae by suppressing caveolae expression according to inhibit Src kinase activity and resulted in reduced albuminuria in PAN mice. These results indicated the possibility of Src kinase inhibitor, Gen as the novel therapeutic approach to reduce albuminuria in nephrotic syndrome. Funding: Veterans Affairs Support