Study On The Roles And Mechanism Of Urotensin â…¡ In Rat Renal Interstitial Fibrosis

UrotensinⅡ(UⅡ) is a cyclic neuropeptide that was first isolated from the caudalneurosecretory system of teleost fish. UⅡcombined with its specific receptor GPR14 cantrigger various biological events that play an important pathophysiological role. UⅡandGPR14 widely exist in the cardiovascular system, kidney, liver, brain and so on. UⅡis themost potent vasoconstrictor peptide to date, it has identified to have cardiovascular actions andcontribute to vasoconstriction. UⅡis not only a potent vasoconstrictor peptide, but also havethe effect of non-hemodynamics. With the study an increasing amount of evidence hasaccumulated for the presence of both UⅡand its receptor in kidney, UⅡis secreted andexcreted 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 andDNA synthesis of renal intercapillary and tubular cells. All of the above results suggest thatUⅡis associated with physiopathology and pathogenesis of the kidney diseases. However, therole and the mechanism of UⅡin renal interstitial fibrosis have not been addressed.Renal interstitial fibrosis, which is the common pathway and main pathology foundationfor almost all kidney disease, is the common turnover of various kinds of chronic renopathy. Itis characterized by renal interstitial fibroblasts proliferation and excess extracellular matrix(ECM) accumulation in the renal interstitium. It is reported that UⅡinvolved in the adjustmentof the cardiomyocyte hypertrophy and cardiac fibrosis. Well, the study on effects of UⅡonrenal interstitial fibrosis and its mechanism in rats have not been reported. So it has importantsignificant to discover the effects of urotensinⅡon renal interstitial fibrosis and its mechanism,and find a new target for clinical treatment of renal disease by antagonism of UⅡ.In the present study, the renal interstitial fibroblast model induced by unilateral ureteralobstruction (UUO) in rats was established and the rat renal interstitium fibroblasts NRK-49Fwere cultured in vitro. The light microscope, immunohistochemistry, flow cytometry, ELISA, RT-PCR, Western blot and RNA interference techniques were employed, and the followingissues were investigated: the contents of both UⅡand its receptor in renal medulla of renalinterstitial fibrosis model induced by UUO; effects of UⅡon proliferation and cell cycle,transdifferentiation and ECM accumulate in NRK-49F cells; the relationship of UⅡand somebioactive compound like TGF-β1, CTGF; the effects of Nimodipine, EDTA and UⅡantibodyon NRK-49F cells. The aim of these investigations was to reveal the roles of UⅡin thedevelopment and progression of renal interstitial fibrosis and possible action mechanism, andto offer new clue for prevention and cure of interstitial fibroblast.The main results of the present study are as follows:1. The left kidney which be ligated with ureter was bigger than the right, the renalcortex was thinner than normal in section, renal pelvis and renal calices were stretched. Allthese changes were obvious in the UUO rat (obstruction for 7 days, UUO 7d).2. The pathological features with HE staining included renal interstitium edema andinflammatory cell infiltration in the UUO 3d. When in the UUO 7d, prominent interstitialexpansion, inflammatory cell infiltration, renal tubules show varying degrees of vacuolardegeneration, necrosis and cast formation, renal tubule expansion, interstitial collagen wereaccumulation.3. The pathological features with Masson's trichrome-staining included collagengirdle-shaped in the interstitial of kidney which be ligated.4. The urine creatinine, serum creatinine and blood urea nitrogen contents weresignificantly increased in the UUO 7d compared with the Sham group (P<0.05).5. Immunohistochemical detection showed that the area and intensity of immunologicalstaining for UⅡ, GPR14, and tTG were increased in the UUO group(3d and 7d) comparedwith the Sham group (P<0.01).6. Compared with the Sham group, the expression levels of UⅡand GPR14 mRNA andprotein in UUO group(3d and 7d) were significantly increased ( P<0.01). The expressions ofUⅡmRNA in UUO group were positively correlated with Scr contents (r＝0.601, P=0.039)and Ucr contents (r＝0.599, P=0.04) that reflected the renal function injury.7. UⅡexerted the stimulative effect on NRK-49F cell proliferation and DNA synthesiswhen UⅡat concentrations of 10-9 and 10-8mol/L, compared with the Sham group (P<0.01).8. UⅡpromoted the transdifferentiation of NRK-49 F cells by stimulate the expressionof theα-SMA and increased the accumulation of ECM, enhanced the TGF-β1, CTGF and tTG mRNA expression in NRK-49F cells. Especially in 10-9mol/L and 10-8mol/L of UⅡgroupcompared with the control group.9. Nimodipine, EDTA and UⅡantibody treatment weakend the effect includingNRK-49F cell proliferation, ECM accumulate, the expression of TGF-β1,CTGF and tTG..10. RNA interference of GPR14 suppressed an increase in TGF-β1 and CTGF mRNAexpression caused by UⅡ.The conclusions of this study are as follows:1. Renal interstitial fibrosis animal model is established by UUO in rats.2. UUO can induce the rats with renal function injury, kidney index increasing, renalfeature changed and the accumulation of the collagen.3. UⅡand GPR14 mRNA and protein are significantly enhanced in UUO groupcompared with the Sham group, it may suggest UⅡand GPR14 have important roles in thedevelopment of the renal fibrosis.4. UⅡis able to cause proliferation and DNA synthesis of NRK-49F cells in aconcentration-dependent manner, Calcium ion inflow may partly mediate UⅡ-induced cellmultiplication in NRK-49F cells.5. UⅡincreases the expression of ColI,Col III and FN in NRK-49F cells, promote theexpression ofα-SMA, thereby promote the transdifferentiatie of the NRK-49F cells.6. The effects of UⅡcan be blocked partly by Nimodipine, EDTA and UⅡantibody.especially the effect of the UⅡantibody compared with the other group.The creative points of the present study are: it is studied that UⅡhad intimaterelationships with renal diease, well, the mechanism of which UⅡplayed in the renalinterstitial fibrosis has not been reported. and its receptor GPR14 increase significantly in themodel of renal interstitial fibrosis induced by UUO; UⅡcan promote the proliferation andDNA synthesize of NRK-49F; UⅡcan increase the production of ECM andtransdifferentiation of of NRK-49F; UⅡis involved in renal interstitial fibrosis; conbined withRNAi, it is demonstrated that UⅡcan increased the expression of TGF-β1 in NRK-49F cell byinduced of GPR14.