The Effect And Mechanism Of Probucol On Diabetic Nephropathy Tubulointerstitial Injury Via Down-regulating P66Shc

Background:Diabetic nephropathy (DN) is a known micro-vascular complication in patients with diabetes mellitus, a metabolic disorder in which chronic hyperglycemia induces dysfunction in various cell types of the kidney, ultimately leading to progressive renal failure. At present it accounts for almost50%of all end-stage renal diseases. It is an important cause of chronic renal failure both in the Western world and China. However, the pathogenesis underlying DN is complicated and yet not well clarified and current treatments remain suboptimal. Many factors, such as the increased accumulation of advanced glycation end products (AGEs), dyslipidemia, activation of protein kinase C (PKC), local activation of the renin-angiotensin system (RAS), increased expression of transforming growth factor (3(TGF-β) and GTP-binding proteins, influence the progression of this disease. It is important to determine the molecular mechanisms and treatment of this disease.Although DN was traditionally considered a primarily glomerular disease, it is now widely accepted that the rate of deterioration of function correlates best with the degree of renal tubulointerstitial fibrosis. In addition, urinary biomarker data in human beings support the view that proximal tubule injury contributes in a primary way, rather than in a secondary manner, to the development of early DN. Recent observations indicate that hyperglycemia triggers the generation of free radicals and oxidant stress in renal tubular epithelial cells, which might be one of the important mechanisms in the development of renal dysfunction in DN. 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 factor (NF-Kb, et al) leading to upregulation of TGF-β and ECM accumulation, and finally result in cellular injury in kidney.Adaptor protein P66Shc is known as a master regulator of cellular oxidative stress and apoptosis, as well as the signalling pathways of cell cycle. Under hyperglycemia stimulation, P66Shc is activated and translocated to mitochondria, which induce mitochondrial ROS overproduction and lead to tubular cell oxidative stress and apoptosis. Our previous research has demonstrated that the expression of P66Shc is significantly up-regulated in peripheral blood mononuclear cell of DN patients. In addition, P66Shc could interact with Drpl, then induce mitochondrial ROS overproduction and result in renal tubular cells (HK-2) oxidative injury under hyperglycemia conditions. These findings suggest that pharmacologic modulation of P66Shc expression and activity may serve as a novel and effective target for the treatment of DN. Probucol is a lipid-lowering drug which has the renal protective effect. However, the mechanism remains poorly understood. We have preliminary data in the lab demonstrating that probucol treatment could decrease the level of P66Shc in HK-2cells induced by high glucose. Here we hypothesized that probucol via inhibition of P66Shc in HK-2cells attenuates DN. To this end, experimental research is carried out as follows.Chapter I The expression of adaptor protein P66Shc and acetylation of histones in the kidney of diabetic nephropathy patientsObjective:To observe the expression of P66Shc and acetylation of histones in order to investigate the relationship between P66Shc and acetylation of histones&renal tubular interstitial injury in diabetic nephropathy patients.Methods:16patients were enrolled into this study, they were diagnosed as DN (n=8) or primary minimal changes disease (control group, n=8) based on clinical manifestations and renal pathology. Renal pathological changes were observed by HE, Masson, PAS and PASM staining. Interstitial damage score were evaluated. The expression of P66Shc, Ac-H3, P300, SIRT1were tested by immunohistochemistry and the correlationship between P66Shc and acetylation of histones&tubular interstitial damage in DN was analyzed.Results:HE, Masson, PAS and PASM staining showed that there were many lesions in the kidney of DN patients such as proliferation of mesangial matrix, nodular sclerosis, diffuse or segmental thickening of the basement membrane, tubular atrophy, local interstitial fibrosis. While the lesions in the kidneys of minimal change disease patients were less severe. Interstitial damage score and collagen deposition score in the kidney of DN patients were higher than that in the control group. Immunohistochemistry showed that the expression of P66Shc was significantly up-regulated, while the expression of SIRT1were decreased in DN patients compared with that in the control group.Conclusion:The expression of P66Shc was significantly up-regulated, while the expression of SIRT1was decreased in the kidney of DN patients, the level of P66Shc is positively correlated with renal interstitial fibrosis and P300, while negative correlated with the expression of SIRT1. Chapter Ⅱ Probucol attenuates tubulointerstitial injury of diabetic nephropathy mice via down-regulating P66ShcObjective:To investigate whether the pretreatment of probucol could reduce tubulointerstitial injury of diabetic nephropathy by inhibiting P66Shc.Methods:ICR mices were randomly divided into control (n=10), diabetic nephropathy (n=10), probucol (10mg/kg/d) group (n=10), DMSO group (n=10). The diabetic nephropathy model was induced by injection intra-peritoneally with40mg/kg body weight STZ in0.lmol/L sodium citrate solution(pH4.5) for four times. The rats in the control group were injected with0.lmol/L sodium citrate solution. Probucol was intraperitoneally injected to the mice every other day for12weeks after the model was built. Mices were sacrificed at day90and the kidneys were harvested and subjected to the studies. Renal lesions and the expression of SIRT1, P300, AcH3, P66Shc, FN were detected by HE, Masson staining, TUNEL, DHE, immunohistochemistry and western-blot respectively. On the other hand, HK-2cells were incubated with different concentrations of D-glucose (5,30mM) or high glucose (30mM) combimed with different dose of probucol for indicated time (0-72h). The cells were collected to extracte total RNA and protein at different time after incubatiom. The expression of P66Shc, SIRT1, P300, AcH3were detected by realtime-PCR,Western-blot and immunoflurescence assays. Supernatants were harvested to measure MDA by commercialized kit.Results:Extracellular matrix deposition, proliferation of mesangial matrix and vacuolar degeneration of tubular epithelial cells were observed in diabetic nephropathy mices, while the pathological changes were reduced in the probucol-treated group. The expressions of P300, P66Shc, FN were increased in the kidney of diabetic nephropathy mices compared with the control. Probucol treatment could attenuate renal ROS levels and apoptosis. Compared with diabetic nephropathy group, the level of serum creatinine were significantly decreased in probucol group. In addition, The expressions of P66Shc and FN were decreased in the kidney of probucol group.Stimulation of HK-2cells with30mM D-glucose resulted in a significant increase of ROS, MDA level and apoptotic rates. The expressions of AcH3, P300, P66Shc were increased in the HK-2cells exposed to high glucose concentration compared with the control. While pretreated with probucol could decrease ROS, apoptotic rates and the expressions of P66Shc; while boost the expression of SIRT1in HK-2cells induced by high glucose.Conclusion:Pretreatment with probucol effectively protects diabetic nephropathy renal tubulointerstitial injury, reduces ROS and apoptotic rates in HK-2cells induced by high glucose in a manner dependent on suppression of P66Shc. Chapter III The mechanism of probucol on tubulointerstitial injury of diabetic nephropathyObjective:To explore the possible mechanisms involved of probucol on oxidative damage and apoptosis in HK-2cells induced by high glucoseMethods:HK-2cells were starved for24hours in serum-free medium before starting the experiment. Thereafter,1mM AICAR or20uM Dorsomorphin or luM EX-527were added into medium for2hours. After treatment, HK-2cells were incubated with30mmol/L D-glucose combined with or without40uM probucol for indicated time (24,48,72h). After treatment, cell total proteins and RNA were extracted for western blot and RT-PCR analysis of the expression of P66Shc、 SIRT1、AMPK、p-AMPK. In addition, chromatin immunoprecipitation (ChIP) assay was used for determining the effect of probucol on acetylation of histone in P66Shc gene promoter regions of HK-2cells in high glucose ambience.Results:Probucol treatment could boost the expressions of p-AMPK and SIRT1; while decrease the expression of P66Shc in HK-2cells induced by high glucose. While pretreatment with the selective AMPK inhibitor Dorsomorphin or SIRT1inhibitor EX-527could block the inhibitory efficiencies of probucol on the expression of P66Shc. The ChIP analysis showed that probucol treatment could decrease the acetylation of histone H3in p66Shc gene promoter regions (-535bp to-276bp) of HK-2cells in high glucose ambience, and then decrease the expression of p66Shc.Conclusion:Probucol could epigenetically suppress the expression of P66Shc through AMPK-SIRT1-AcH3pathway, then ameliorate the apoptosis and oxidative injury in HK-2cells induced by high glucose.