The Role Of HIF-1α Regulating ET-1Expression In High Glucose-induced Tubular Epithelial-mesenchymal Transition

Background and aimsTubular epithelial-mesenchymal transition (TEMT) is one of the important mechanisms of renal interstitial fibrosis.36%of the renal interstitial myofibroblasts derive from proximal tubular epithelial cells. The cells which undergo transition lose their epithelial phenotype, obtain the characteristic of mesenchymal cells. Studies have shown that TEMT exists in diabetic nephropathy (DN). High glucose can induce TEMT by a variety of ways, such as advanced glycation end products(AGEs), angiotensin Ⅱ(Ang Ⅱ), as well as some cytokines.Hypoxia inducible factor-1(HIF-1) is a nucleoprotein with transcriptional activity, it is produced by cells under hypoxic conditions and can mediate the response to hypoxic-ischemic state. HIF-1mainly expresses in renal tubular epithelial cells in kidney, and is composed of functional a subunit and structural β subunit. Sun S and his colleagues found that HIF-la can induce TEMT, our previous study confirmed that the expression of HIF-1α increases and participates in RIF in DN. In recent years, more and more studies have demonstrated that a strong association exists between ET-1and a variety of acute and chronic kidney disease. In the kidney, vascular endothelial cells, podocytes, mesangial cells and tubular epithelial cells can secrete ET-1, and play a wide range of biological effects in autocrine and paracrine manner via ETA and ETB. In physiological state, ET-1is involved in the regulation of glomerular filtration rate, renal blood flow, tubular reabsorption and a variety of other important functions in renal. But under pathological condition, ET-1mediates kidney injury not only through its strong contraction of renal vessels, promoting mesangial cell contraction and chemotaxis of monocytes, but also through its effect in RIF. ET-1can lead to myocardial fibrosis in diabetes through the induction of myocardial cell transdifferentiation. Whether it can lead to RIF by promoting tubular epithelial-mesenchymal transition in diabetic nephropathy is unclear. The purpose of our study is to investigate the specific role of HIF-1α and ET-1in TEMT in DN.MethodsNormal rat proximal renal tubular epithelial cells(NRK52E) were cultured in DMEM medium containing10%fetal bovine serum. Cells were divided into5group: low glucose control group, high osmotic control group, high glucose group, high glucose+HIF-la siRNA group and high glucose+BQ123group. Morphologic changes of cells were observed by phase-contrast microscope. The supernatant level of ET-1was dectected by ELISA, and mRNA and protein expression level of HIF-1α, a-SMA, and E-cadherin was detected by RT-PCR and western blot.Results1. Morphologic changes of cells were observed by phase-contrast microscope:cells of low glucose control group packed tightly, with typical cobblestone morphology. After induced by30mmol/L glucose for48h, the cells showed obvious morphologicak changes with elongated and fibroblast-like morphology. Compared with low glucose control group, cells in high glucose+HIF-1α siRNA group and high glucose+BQ123group remained elliptical or polygonal epithelial cell morphology, but obtain increased cell space.2. In NRK52E induced by high glucose, the expression of HIF-1α, ET-1and α-SMA was up-regulated while the expression of E-cadherin was down-regulated.3. Compared with high glucose group, the expression of a-SMA was up-regulated, while the expression of E-cadherin was down-regulated in high glucose+HIF-la siRNA transfection group and high glucose+BQ123group.4. Compared with high glucose group, the expression of ET-1was lower in high glucose+HIF-la siRNA transfection group, while the expression of HIF-1α had no obvious chang in high glucose+BQ123group.Conclusion1. High glucose induces HIF-1α and ET-1production of tubular epithelial cells.2. ETA receptor antagonist can inhibite high glucose-induced tubular epithelial-mesenchymal transition.3. The pathway that activation of HIF-la induced by high glucose could stimulate the ET-1might be involved in the pathogenesis of renal interstitial fibrosis in DN.