Effect Of P102 On High Glucose - Activated Renal Mesangial Cells And Glomerular RAS

Along with the changes in the life style and diet habits, the prevalence rate of diabetes mellitus (DM) increases continually recent years. Diabetic nephropathy (DN) is one of the common complications of DM and considered as a microvascular complication of DM, in which the glomerulus is a key site of lesion. In the early stage of DN, the kidneys are enlarged and the glomerular blood flow and filtration rate are increased. These changes are followed by extracellular matrix (ECM) accumulation, thickening in glomerular basement membrane, nodular hypertrophy of glomeruli and glomerular scleroses in succession. With the prolongation of the course, the renal dysfunction progresses into the end stage of renal failure. The renal failure is one of the leading causes of death in the patients of DM.DN has been reported to be caused by different etiological factors including hyperglycemia, hypertension, renal hemodynamic abnormalities and genetic background. However the exact mechanism is still not clear. Renin-angiotensin system (RAS) plays an important role in regulating body blood pressure and water, electrolyte balance. Besides the circulatory RAS, local RAS is distributed in the kidneys. The local RAS takes a vital role in the regulation of renal function and tissue remodeling. The excessive activation of renal RAS has been identified as a key factor in the pathogenesis of DN. The inhibition of RAS helps in preventing degeneration of renal function. So currently, two types of RAS inhibitors, angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor inhibitor (ARBs), have been used as the first line medications in the clinical DN treatments.Some researches have confirmed that reactive oxygen species (ROS) over generation induced by high glucose (HG) is a major cause of renal damage in diabetes. In the early stage of DM, HG stimulates NADPH oxidase and leads to ROS elevation. Meanwhile, ROS has been reported to have complex interactions with RAS. However, the exact mechanism between ROS and RAS interaction has not been revealed,p100(SND1) was originally identified as a transcriptional co-activator by Tong X in1995. In the past few years, pi00has been identified as a multifunctional protein. It acts as a transcriptional co-activator that interacts with EBNA-2, STAT5, STAT6, and c-Myb to enhance dependent-them transcription. It is a component of RNA-induced gene silencing complexes (RISC), and participates in the mRNA clipping, degradation and maturation. Papers have also been reported that p100was involved in cancer-related genes over-expression and some researchers believe that p100can be used as a cancer indicator, p100also has been demonstrated to bound to AT1R3’-UTR to reduce AT1R mRNA degradation and increase its translation efficiency. In rat, p102is the homologous of human p100, which shows the similar function to p100. So it is speculated that the homolog p102may also be involved in regulating the expression of RAS components. The kidney is an important target organ of RAS in vivo, and RAS abnormalities are involved in the occurrence and development of diabetic-induced renal damage, so that whether RAS dysfunction is associated with p102in diabetes; whether p102is involved in the occurrence and development of DN are the questions desired to be answered for us.The aim of this study was to identify the expression of p102in rat kidney and observe the changes of p102expression in hyperglycemic condition, and to explore whether RAS is under the regulation of p102. By these researches, we attempted to elucidate the role and mechanism of p102in DN.Results:Part I. The effects and mechanisms of p102on RAS activation induced by high glucose in the cultured renal mesangial cells (MCs)1. HG elevated ROS generation in the cultured renal MCsWe firstly observed the effect of high glucose (HG,25mmol/L D-glucose) on ROS generation in the cultured rat renal MCs in vitro. ROS production was elevated significantly1h after hyperglycemic stimulation and kept at relatively high level until the end of the experiment (48h). The ROS generation reached the peak around24h of HG incubation. Diphenylene-chloride iodonium (DPI,10-6mol/L), a NADPH oxidase inhibitor, inhibited the ROS elevation induced by HG. These results demonstrated that HG stimulated ROS generation in MCs mainly by NADPH oxidase.2. Identification of p102expression in the cultured rat renal MCs and the changes of p102under hyperglycemic condition.Both Western blot and RT-PCR results demonstrated that the rat renal MCs expressed p102. HG caused a significantly elevation in p102expression. DPI (10-6mol/L) not only inhibited the elevation of p102, but also remarkably decreased the p102expression in the normal cultured MCs. Because DPI reduces ROS generation by inhibiting NAPDH oxidase, this result proved that p102expression was influenced by ROS. At the same time, we observed that DPI (10-6mol/L) inhibited HG-induced TGF-β1and fibronectin increases, which suggested that TGF-β1and fibronectin expressions were also regulated by ROS.3. Effects of p102knock-down on HG-induced RAS activation in the cultured rat renal MCsHG induced the elevation of p102expression in renal MCs. After p102siRNA (50nM) transfection for24h or48h, the elevations of p102protein and mRNA induced by HG were inhibited remarkably in the cultured MCs. HG treatment for24h or48h, the angiotensinigen (AGT), angiotensin converting enzyme (ACE), angiotensin II type1receptor (AT1R) mRNA levels and transforming growth factor-β1(TGF-β1), fibronectin protein levels in the cultured MCs were significantly increased, p102siRNA transfection (50nM) completely prohibited those elevations. These results suggested that p102participated in the upregulation of multiple RAS members as well as its downstream proteins.4. Effects of AT1R receptor blockade on HG-induced p102expression in the cultured rat renal MCsIn order to determine the causal relationship between p102and AT1R, Candesartan (10-6mol/L), an AT1R antagonist, was added into the medium30min ahead of HG. Pretreatment of Candesartan inhibited the TGF-β1and fibronectin protein increases induced by HG, but had no effect on p102expression. Those results suggested that HG caused TGF-(31and fibronectin expression increases were mediated by AT1R activation, however p102was located at the upstream of AT1R, and AT1R expression was regulated by p102.The experiments in the rat renal MCs verified that HG caused ROS elevation through NADPH oxidase. The ROS elevation subsequently stimulated p102expression, which activated local RAS expression and finally increased TGF-β1and fibronectin expressions.Part II. The changes in local RAS in the isolated rat renal glomeruli under hyperglycemic condition and its related mechanisms1. HG stimulated ROS generation in the isolated rat renal glomeruliThe rat glomeruli were separated from SD rat kidneys and incubated in HG (25mmol/L D-glucose). ROS generation was significantly elevated from0.5h to the end of experiment (24h). The ROS generation reached its peak about3h after HG incubation. The pretreatment of DPI (10-6mol/L30min ahead) significantly inhibitedthe elevation of ROS induced by HG. This result was consistent to the observations from renal MCs which revealed that HG stimulated ROS generation through NADPH oxidase.2. The elevation in ROS under hyperglycemic condition was related to the increase in p102expressionBoth Western blot and RT-PCR results demonstrated that p102was expressed in the isolated rat renal glomeruli, and HG caused p102levels significantly increased. DPI (10-6mol/L) not only inhibited completely the elevation of p102induced by HG, but also reduced the p102expression in the normal isolated rat glomeruli. This result was also consistent with the changes in MCs and suggested that HG increased p102expression through ROS.3. ROS mediated HG-induced increases in ACE and AT1R expression in the cultured rat renal glomeruliAfter incubating in HG culture medium for48h, the ACE and AT1R mRNA levels in the isolated rat glomeruli were significantly increased. The pretreatment of DPI (10-6mol/L) completely inhibited the elevations in ACE and AT1R mRNA levels. These results suggested that the increased ROS mediated the elevations in local RAS expression in the isolated rat glomeruli under HG condition.These results suggested that p102was expressed in the glomerular of the rat kidneys and the expression of p102was regulated by the local ROS. ROS increased rapidly after HG stimulation. The elimination of ROS elevation dismissed the elevation in p102and its downstream targets including ACE and AT1R.In summary, our results demonstrated that p102was expressed in the kidneys. The MCs in glomerular is a source of p102. The expression of p102was elevated under hyperglycemic condition. Over production of ROS induced by hyperglycemia stimulated the expression of p102. In subsequent, the elevated p102up regulated multiple components in RAS, including AGT, ACE and AT1R. Over activation in RAS stimulated the synthesis and secretion of ECM. These functional changes of renal MCs might lead to over accumulation of ECM in glomeruli of kidney and thickness in glomerular basement membrane.