Role of PKCδ on the phosphorylation and mitochondrial translocation of p66Shc in HK-2 cells induced by high glucose

Abstract

To observe the effect of PKCδ on the phosphorylation of p66Shc and its mitochondrial translocation in human proximal tubular cells (HK-2) under high glucose (HG). HK-2 cells were incubated with different concentrations of D-glucose (5-45 mmol/L) for indicated time (0-48 h). Then the mRNA expressions of PKCδ and p66Shc and the phosphorylation levels of PKCδ (p-PKCδ) and p66Shc (p-p66Shc) were determined by real-time polymerase chain reaction (PCR) and Western blot analysis respectively. In addition, the effect of PKCδ inhibitor on the phosphorylation and mitochondrial translocation of p66Shc in HK-2 cells exposed to HG was also observed. HK-2 cells were divided into 3 groups of 5 mmol/L glucose, 30 mmol/L glucose and 30 mmol/L glucose + 1.0 µmol/L Rottlerin. Cell immunofluorescence and Western blotting were used to observe the phosphorylation and mitochondrial translocation of p66Shc. Both mRNA expression and phosphorylation level of p66shc and PKCδ significantly increased in HK-2 cells after exposure to HG (15, 30, 45 mmol/L). And it was in a concentration- and time-dependent manner as compared with control group (up-regulated 0.9, 1.3 and 1.6-fold in mRNA of PKCδ, 0.4, 1.5 and 2.0-fold in protein of p-PKCδ respectively (all P < 0.05). PKCδ inhibitor Rottlerin dramatically inhibited the phosphorylation and mitochondrial translocation of p66Shc induced by HG in HK-2 cells (down-regulated 3.1 folds in protein of p-p66Shc in mitochondria, P < 0.01). HG increases the transcription and phosphorylation of PKCδ and p66Shc in HK-2 cells. And PKCδ may modulate the phosphorylation and mitochondrial translocation of p66Shc under HG.