Effect Of Angiotensin â…¡ On Expression And Phosphorylation Of Adaptor Protein P66Shc In Human Proximal Tubular Cells

Background: Diabetic nephropathy (DN) is the most common and serious micro vascular complication of diabetes mellitus, and it is the main cause of death of patients with diabetes. Overproduction of mitochondrial ROS induced by oxidative stress plays an important role in the development of DN. Recent studies have showed that tubular oxidative injury is the early and primary damage of DN. The renin-angiotensin system (RAS) plays a central role in regulating physiologic and pathophysiologic processes in the kidneys , and angiotensin II (Ang II ) is the primary effector of this system. Angiotensin II induces overproduction of intracellular ROS, causes oxidative damages of organs and cells in rodents which contribute to the progression of renal disease. Recent studies have demonstrated that over-expression of adaptor protein p66Shc up-regulates the production of mitochondrial ROS, aggravates cell oxidative damages. Furthermore, the phosphorylation of the unique Ser36 site of p66Shc appears to be a critical regulatory site which influences the intracellular oxidative stress. However, how does adaptor protein p66Shc express in renal tissues of diabetic nephropathy and the relationship between angiotensin II and p66Shc is still unclear.Objective: To observe the expression of adaptor protein p66Shc in the renal tissues of db/db mouse; To study the effect of Ang II on expression and phosphorylation of p66Shc in human proximal tubular cells (HK-2).Methods:1,The mice were divided into two groups, the db/db group and the db/m group as control, each group has 4 mice. Kidneys of the mice were harvested at the age of 14 weeks. The pathological changes were observed by HE and Masson staining, and the expression of adaptor protein was detected by immunohistochemistry and Western Blot.2,Human proximal tubular cells (HK-2) were treated with different concentration of angiotensin II (Ang II) to observe the effect of Ang II on the expression of p66Shc by Real Time PCR and Western Blot. Then HK-2 cells were treated with high concentration of Ang II (1μM) to detect the phosphorylation of the Ser36 site of p66Shc at different time by Western Blot.Results:1,In vivo: In db/db mice, HE and Masson staining showed the renal tissues of the db/db mice suffered glomerular hypertrophy, cell proliferation, mesangial expansion, parts of the tubular cells had vacuolar degeneration and detachment, no visible atrophy of the tubules. Immunohistochemistry showed that the expression of p66Shc increased especially in the tubules of the db/db mice (P<0.05), and Western Blot showed the similar results(P<0.01).2,In vitro: Both expression of p66Shc mRNA and protein increased remarkably in HK-2 cells induced by angiotensin II in a dose-dependent manner (P<0.01); Compare with the control, the phosphorylation of the Ser36 site of p66Shc began to increase at 10min, and increased significantly at 30min, then reached the peak at 60min then decreased gradually, but still kept at a high level (P<0.01).Conclusion:1,Expression of adaptor protein p66Shc increases in the tubular cells of the db/db mouse.2,Angiotensin II up-regulates the expression of p66Shc in human proximal tubular cells in a dose-dependent manner.3,High concentration of angiotensin II increases phosphorylation of the Ser36 site of p66Shc in human proximal tubular cells.