Effect Of Adaptor Protein P66Shc On Oxidative Injury And Apoptosis In Renal Tubular Epithelial Cells And Its Possible Mechanism In Diabetic Nephropathy

Background Diabetic nephropathy (DN) is one of the leading causes to end stage renal disease(ESRD). Approximately 30-40% of patients with type I and 15% with type II DM develop renal dysfunction. In both instances, poor glycemic control contributes to the development of diabetic nephropathy, but the mechanisms underlying hyperglycemia induced injury are not fully understood. Intracellular ROS is increased in kidney in diabetes and the overproduction of ROS is a direct consequence of hyperglycemia. ROS mediates hyperglycemia-induced activation of signal transduction cascades (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (NF-κB, activated protein-1, and specificity protein-1) leading to upregulation of TGF-(31 and ECM accumulation, and finally result in cellular injury in kidney. However, the mechanism of ROS generation in DN is still unknown.The ShcA gene encodes three isoforms:p46Shc, p52Shc and p66Shc and the p66Shc controls cellular oxidative stress responses and life span. Recently, studies showed that the mice lacking p66Shc reduced intracell-ular ROS and live 30% longer than control animals, suggesting that dele-tion of p66Shc increased oxidative resistance and prolonged a life span. It has been demonstrated that p66Shc is localized partly in the cytoplasm, while almost 10% to 40% within the mitochondrial intermembrane space. In the mitochondria, p66Shc binds to an inhibitory complex which inclu-des members of the Hsp70 and TIM-TOM import. system. Furthermore, it has been shown that the p66Shc protein might be a mediator traducing oxidative stress to injury and apoptosis in MEFs. It is inactive in normal condition, and activated by oxidative stress mediated by the phosphoryl-ation of Ser36. As recently reported, oxidative stress activates PKC(3, induces phosphorylation of p66Shc in the cytoplasm and triggers mitoc-hondria accumulation of the protein after it is recognized by Pinl; On the other hand, oxidative stress also induces the dissociation of the inhibitory complex and the release of monomeric p66Shc. Once it activated, p66Shc generates ROS through oxidation of cytochrome c, which then induces opening of the mitochondrial permeability transition pore (mPTP), with subsequent increase of mitochondrial membrane permeability to ions, solutes and water, swelling and disruption of the organelle, release of cytochrome c into the cytosol, and consequent apoptosis.Recently, p66Shc has been involved in diabetes mellitus and its complications. Pagnin et al found that p66Shc mRNA expression was increased in human peripheral blood mononuclear cells in type II diabetic patients compared with healthy subjects, and it is positively correlated with total plasma 8-isoprostanes, a validated marker of oxidative stress. It is also reported by Menini that Streptozotocin-induced diabetic p66Shc knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas.Serum and renal tissue advanced glycation end products and activa-tion of NF-κB were also lower in diabetic KO than in wild-type mice. These data suggest that p66Shc might be involved in the pathogenesis of diabetic nephropathy. However, the mechanism is unknown. Therefore, we think that p66Shc may play a key role of ROS generation and cell apoptosis in diabetic nephropathy. To this end, experimental research is carried out as follow. Objective To investigate the expression of both adaptor protein p66Shc and reactive oxygen species (ROS) in the kidneys in STZ-induced diabetic nephropathy.Methods Twenty SD male rats, weight ranged from 190g to 220g, were random divided into 2 groups(n=10 each group):Normal control group(Con group) and diabetes model group(STZ group). Diabetes was induced by the injection of STZ (The control group only with injection of PBS). Rats with blood glucose levels> 250mg/dl were selected for studies. Rats were killed 8 weeks after STZ injection. Kidney tissues were harvested for HE and MASSON test and DHE staining. The expression of p66Shc was examined by immunohistochemically staining and Western blot, respectively.Results Compared with control, the decrease of body weight, increase of kidney weight and urine protein were seen in STZ group; Pathologic changes of renal tubule were observed by HE and MASSON staining. Severe oxidative stress was seen by DHE staining in kidneys of STZ rats. The expression of p66Shc significantly increased in STZ rats in comparison to that of control group.Conclusion The expression of p66Shc was significantly up-regulated in rat kidneys with diabetes which associated with severe oxidative stress. Objective To detect the expression of p66Shc and the phosphorylation of p66shc-Ser36 induced by high glucose in the human renal proximal epithilial cell line(HK-2).Methods The cells were exposed respectively in different concentrations of D-glucose(5.5mM D-glucose as normal control and D-mannitol as the isoosmotic control). The expression of p66Shc mRNA was examined by Real-time PCR. Expressions of p66Shc and phospho-Ser36 protein was detected by Western blot.Results A single band with a predicted molecular weight (66kD) was seen in HK-2 cell by Western blot, which represents p66shc expression. Stimulation of HK-2 with 30mM D-glucose resulted in a significant increase in the expression of p66Shc protein, compared with medium control group. However, there was no significant difference between the control and D-mannitol group. HK-2 treated with 30mM D-glucose resulted in a time-dependent increase in the expression of p66shc mRNA, with the peak at 24h. In additon, an increase of p66Shc protein in HK-2 treated by 30mM D-glucose was also seen, compared to that of control group. Interestingly, after treatment with 30mM D-glucose, the level of phospho p66Shc-Ser36 protein began to increase at 15min, and reached the peak at 90min in HK-2.Conclusion It was demonstrated by first time that expression of p66Shc was seen and high glucose up-regulated p66shc expression in a time and dosage dependent manner in HK-2. Moreover, high glucose induces phosphorylation of p66shc-Ser36 in HK-2. Objective To study the role of adaptor protein p66Shc in high glucose induced oxidative injury and apoptosis in the human renal proximal epithilial cell line(HK-2).Methods Wild-Type p66Shc plasmid (p66Shc WT) and the p66Shc mutant plasmid(Ser36 was mutated to Ala, called p66Shc S36A) were transfected into HK-2 cells using lipofectamine 2000, respectively. After selection of stable transfectants, cells were maintained in the defined medium.30mM D-glucose was added (5.5mM D-glucose as control). Cell apoptosis was measured by AnnexinⅤstaining using flow cytometry and Hoechst 33258 staining using fluorescence microscopy. The mitochondrial ROS was detected by Mitosox staining using confocal microscopy. Mitochondrial membrane potential was detected by TMRE, then measured by confocal microscopy. The mtDNA damage was evaluated by amplifying of mitochondrial long and short DNA. The expression of cytochrome c and p66Shc in mitochondria and caspase9 were by Western blot. Cell lactate dehydrogenase (LDH) activity was examined by Colorimetric assay and Malondialdehyde (MDA) was examined by TBA assay. Results1. p66Shc protein is overexpressed with p66Shc constructs(p66Shc WT/S36A) transfection in HK-2 cells2. Overexpression of p66Shc in HK-2, which then treated with 30mM D-glucose induced an increase of mitochondrial ROS, LDH activity, MDA level and apoptotic rates, in comparison to HG group. A decrease of mitochondrial membrane potential, release of cytochrome c, accumulation of p66Shc in mitochondrial, activation of caspase9 and damage of mitochondrial DNA was also observed in p66Shc overexpression group, compared with HG group. While the effect was significantly abolished in p66Shc mutant plasmid (p66Shc S36A) transfected HK-2 cells, compared with p66Shc transfected HK-2 cells.Conclusion Overexpression of p66Shc intensified the effects induced by 30mM D-glucose in HK-2, p66Shc mediates oxidative injury and cell apoptosis induced by high glucose via mitochondria dependent pathway in HK-2. p66Shc may play a key role in tubular atrophy and cell apoptosis during early stage of diabetic nephropathy.