Study On The Role Of Urotensin â…¡ In Diabetic Nephropathy And Its Mechanism

UrotensinⅡ(UⅡ) is the most potent vasoconstrictive peptide identified to date. UⅡwidely exists in the cardiovascular system, kidney, liver, brain and so on from mollusks to mammals. Accumulating evidences indicate that UⅡcan upregulate the expression of collagenⅠ,Ⅲ, FN and PAI-1, decrease MMP-1 expression and activity, promote ET synthesis and release, as well as increase transforming growth factorβ1 (TGF-β1) expression in both gene and protein levels. All of the above show its effects on regulation of bioactive compound beyond its effects on vascular tension. In addition, UⅡacts as a mitogen and promotes the proliferation and DNA synthesis of vascular smooth muscle cells, cardiac fibroblasts, mesangial cells and other cells.UⅡcombined with its specific receptor GPR14 can trigger various biological events that play an important pathophysiological role in the development and progression of primary hypertension, congestive heart failure, renal diseases, cancer and other diseases. Although the mechanism of UⅡin these diseases has not been clarified, it is likely that UⅡmay become a new target for clinical treatment of certain diseases after the specific UⅡreceptor antagonist is discovered.Diabetic nephropathy is one of important microvascular complications of diabetes, a common cause of end-stage renal disease. In diabetic nephropathy, renal tubulopathy can succeed glomerulopathy and also is a primarily change independent of glomerular pathological and hemodynamic changes, it is to take place before glomerular morphological changes, after glomerular pathological changes and synchronization. The main pathological changes in diabetic tubule lession are renal tubular basement membrane thickening, tubule number increasing and tubule hypertrophy in the initial stage, gradually renal atrophy and interstitial fibrosis in later stage. These can lead to renal function damage. In recent years, the action of renal tubular interstitial changes in diabetic nephropathy has been paid attention, especially proximal tubules.Both UⅡand its receptor are abundantly expressed in kidney. In healthy kidney, UⅡand its receptor primarily exist in the epithelial cells of the distal convoluted tubule, proximal convoluted tubule and collecting tubules. UⅡis secreted and excreted by kidney and it acts as a growth factor for the renal epithelial cells by autocrine and (or) paracrine ways. In addition, UⅡacts as a mitogen and promotes the proliferation and DNA synthesis. The Langham,s studies showed that expression of UⅡand GPR14 mRNA in kidney was significantly increased in patients with diabetic nephropathy, mainly in the tubular epithelial cells. All of the above results suggest that UⅡis associated with physiopathology and pathogenesis of the kidney diseases. However, the role of UⅡin diabetic nephropathy and the interaction with other causative agent have not been reported.In the present study, the model of diabetic nephropathy was prepared in rats using streptozotocin by intraperitoneal injection and cultured rat renal tubular epithelial cells NRK-52E were utilized, in the present experiments, the biochemical, morphological, immunohistochemical staining, ELISA, RT-PCR and RNA interference technique were employed to invesigetet the following issues: the contents of both UⅡand its receptor in renal medulla of different periods in rats with diabetic nephropathy; effects of urotensinⅡon proliferation and cell cycle in NRK-52E cells; effects of advanced glycation end products(AGEs) on UⅡexpression in NRK-52E cells; the relationship between angiotensinⅡ(AngⅡ) and TGF-β1 with UⅡ. The aim of these investigations was to reveal the roles of UⅡin the development and progression of diabetic nephropathy and possible action mechanism, and to offer new clue for prevention and cure of diabetic nephropathy.The main findings of this study are as follows: The urinary NAG and urinary RBP contents were significantly increased in different periods in rats with diabetic nephropathy; the urinary NAG and the urinary RBP contents were obviously increased at 4-, 8- and 12-wk DM compared with 2-wk DM (P<0.05); Immunohistochemical detection showed that the area and intensity of immunological staining for UⅡ, GPR14, TGF-β1, FN and ColⅣwere increased in diabetic rat kidney compare with control groups (P<0.05); The expression levels of UⅡand GPR14 mRNA in diabetic renal medulla were enhanced, respectively, compared with control (in all cases, P<0.05); The expressions of UⅡmRNA in diabetic renal medulla were positively correlated with NAG contents (r=0.837, P<0.01) and with 24-hour urinary RBP excretion rates (r=0.910, P<0.01) that reflected the tubular lesion; UⅡexerted the stimulative effect on NRK-52E cell proliferation and DNA synthesis in a concentration-dependent manner when UⅡat concentrations from 10-10 to 10-8mol/L, Nimodipine and ethylene diamine tetraacetic acid (EDTA) treatment weakend this effect; AGEs significantly upregulated UⅡmRNA expression and increased FN and ColⅣsecretion in cultured NRK-52E cells; UⅡpromoted TGF-β1 mRNA expression in NRK-52E cells, the addition of nimodipine and EDTA can attenuate this effect; RNA interference of GPR14 suppressed an increase in TGF-β1 mRNA expression caused by UⅡ; TGF-β1 and AngⅡpromoted the expression of UⅡand GPR14 mRNA in NRK-52E cells.The main conclusions are as follows: The expression levels of UⅡand GPR14 mRNA and protein in diabetic nephropathy model are increased compared with control, UⅡmRNA expression in diabetic renal medulla is positively correlated with NAG contents (r=0.837, P<0.01) and with 24-hour urinary RBP excretion rates (r=0.910, P<0.01); UⅡis able to cause proliferation and DNA synthesis of NRK-52E cells in a concentration-dependent manner when UⅡat concentrations from 10-10 to 10-8mol/L. Calcium ion inflow may partly mediate UⅡ-induced cell multiplication in NRK-52E cells; UⅡcoordinated with AGE-BSA enhance the expression of FN and ColⅣprotein in NRK-52E cells thereby promotes the renal fibrosis; GPR14 mediates UⅡ-induced TGF-β1 mRNA expression increase in NRK-52E cells. Nimodipine and EDTA can partly block this effect.