| UrotensinⅡ(urotensinⅡ,UⅡ) is a cycic neuropeptide initially isolated from the caudal neurosecretory system of teleost fish, its human homolog (hUⅡ) has subsequently been cloned in 1998. UⅡis the most potent vasoconstrictive peptide ever identified, it widely exists in the brain, cardiovascular system, liver, kidney, and so on from mollusks to mammals. G-protein-couple receptor is its specific receptor, originally termed GPR14. UⅡexerts biological effects via interaction with its receptor GPR14. Accumulating evidences indicate that UⅡshows both hemodynamic effects and non-hemodynamic effects, such as stimulating cell proliferation and regulating endocrine and metabolic, etc. So UⅡplays an important pathophysiological role in the development and progression of some diseases.Especially, its non-hemodynamic effects have become the focus of attention. Diabetic nephropathy (DN) is one of important microvascular complications of diabetes, its pathogenesis is complex and the consequences could be serious. Because of the lack of effective prevention and treatment means, it has become the common cause of end-stage renal disease. DN can also increase the incidence of cardiovascular events and mortality, it is one of the important disabled and death reasons of patients with diabetes.The main pathological changes of diabetic nephropathy is glomerular hypertrophy, hyperfiltration and the accumulation of extracellular matrix in the initial stage and glomerular sclerosis in later stage. Mesangial cells (MC) is the central factor. Usually glomerular sclerosis is divided into nodular, exudative and diffuse three kinds of types, the nodular type is characteristic for DN. The pathogenesis of DN is complex and still not clear, it is generally considered that it has relations with genetic factors, high blood sugar-related metabolic disorders, etc. AGEs, TGF-β1 and AngⅡare the important factors in the development and progression of DN.The recent studies showed that expression of UⅡand GPR14 mRNA in kidney was exceptionally upregulated in patients with diabetic nephropathy. In addition, UⅡcan be secreted and excreted by kidney. Acts as a growth factor, UⅡcan promote the proliferation of mesangial cells. These suggest that UⅡmay be involved in the occurrence and development of DN. However, the role of UⅡin pathogenesis of diabetic nephropathy and its molecular mechanism have not been elucidated.In the present study, diabetic renal lesion was prepared in rats using streptozotocin by intraperitoneal injection, and cultured rat mesangial cells were utilized, and the following issuses were investigated:①the contents of both UⅡand its receptor in renal cortex of rat at different periods of diabetes (212 weeks);②effects of UⅡon proliferation in MC;③the relationship between AGEs, TGF-β1 and AngⅡwith UⅡ;④the effects of RNA interference of GPR14 on the expression of TGF-β1 mRNA caused by UⅡ.The main findings are as follows:1. It was detected that the body weight reduced, the kidney weight, the kidney volume and the kidney index significantly increased in rats at different periods of diabetes compared with the control group (P<0.05).2. The GSP, Glu, Scr and 24 hour urinary protein contents were obviously increased in diabetic rats compared with the control group.3. Immunohistochemical detection showed that the area and intensity of immunological staining for UⅡ, GPR14, TGF-β1, FN and ColⅣwere increased in kidney of diabetic rats compared with the control group (P<0.05).4. Compared with the control group, the expression of UⅡand GPR14 mRNA in renal cortex of rats with DM2W and DM4W had no obviously change (P>0.05), but these were significantly increased in renal cortex of rats with DM8W and DM12W (P<0.05).5. UⅡat concentration 10-9 and 10-8 mol/L promoted the proliferation and DNA synthesis of mesangial cells, Treatment wih nicardipine, ethylene diaine tetraacetic acid (EDTA) and anti-UⅡantibody weakend the effect of UⅡ.6. The expression of UⅡand GPR14 mRNA in AGEs-, TGF-β1- or AngⅡ-treated MC was significantly increased, anti-TGF-β1 antibody reduced the effect caused by TGF-β1.7. UⅡat concentration 10-9 and 10-8 mol/L markedly increased the level of FN, ColⅣ, TGF-β1 and AngⅡin MC culture medium.8. UⅡpromoted the expressions of FN, ColⅣ, TGF-β1 and AngⅡ(AT1) (P<0.05). The increased expressions were significantly reduced by addition of nicardipine, ethylene diaine tetraacetic acid (EDTA) and anti-UⅡantibody.9. RNA interference of GPR14 suppressed the increased expression of TGF-β1 mRNA caused by UⅡ.The main conclusions are as follows:1. The expression of UⅡand GPR14 in renal cortex is significantly increased in diabetic rats prepared using streptozotocin by intraperitoneal injection.2. UⅡat certain concentrations can promote the proliferation of mesangial cells, the action may partly be mediated by calcium ion inflow.3. AGEs can significantly increase the expression of UⅡand GPR14 mRNA in MC.4. UⅡat certain concentrations can promote the secretion and mRNA expressions of FN, ColⅣ, TGF-β1 and AngⅡ. TGF-β1 and AngⅡcan also increase the expression of UⅡand GPR14 mRNA in MC. UⅡ,TGF-β1 and AngⅡmay have a synergistic effect in the occurrence and development of diabetic nephropathy.5. The effect of UⅡincreased TGF-β1 mRNA expression in MC is mediated by UⅡreceptor GPR14.This originality of dissertation lies in the following:It is demonstrated that the expressions of UⅡand GPR14 protein and mRNA in renal cortex in streptozotocin-induced diabetic rats are significantly increased; UⅡat certain concentrations can promote the proliferation and DNA synthesis of mesangial cells, and increase the secretion and mRNA expression of FN, ColⅣ, TGF-β1 and AngⅡ(AT1). The findings suggest that UⅡmay directly or indirectly participate in the development and progression of diabetic nephropathy.
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