| Objective: Kinin is a vasoactive peptide, regulating bloodstream,blood pressue, capillary permeability, and inflammation. Byaminopeptidase, Kinin will turn to Bradykinin. Recently there is a noveldiscovery bradykinin effects cells mitogenesis related withMAPK(Mitogen-activated protein kinases). The most important signalingtransduction pathway, reacting to extracellular stimulation, MAPK,comprise ERK, JNK, P38MAPK, BMK1/ERK5. Through changing geneexpression, MAPK alters phenotype and overproduces ECM. In renalfibrosis, MAPK can assume all necessary cellular reaction. The pharmiccontrol to it may give a new theraputic way. (1) To investigate the effects ofBradykinin(BK) on proliferation, collagen secretion together with theplatelet derived growth factor (PDGF) in cultured rat mesangial cells andrenal interstitial fibroblasts. (2) To study the signaling pathway by whichBK upregulates mitogenesis or down-regulates the mitogenesis ofPDGF-BB in mesangial cells. Method: (1) Cultured immortalized rat mesangial cells line andprimary cultured human renal interstitial fibroblasts were stimulated by BKwith different concentratin(0,10,100,1000ng/ml) respectively as well aswith PDGF-BB(20ng/ml). MTT colorrnetric method and ELISA assay wereperformed to detect the levels of proliferation and type â…£ collagensecretion after treatment for 48 hours. Cells without stimulation and wereused for control and with PDGF-BB only for treated, others for stimulated.(2) Western blotting was performed to observe the instant expression ofERK1/2 phosphorylation in mesangial cells stimulated by different doses ofBK for 2 minutes. (3) Cultured mesangial cells were incubated withdifferent doses of BK for 15 minutes, and with 20ng/ml of PDGF-BB for 2minutes. ERK1/2 activation were tested by Western Blotting. (4) Culturedmesangial cells were divided into four groups: the control group,PDGF-BB with 100ng/ml of BK stimulated group, pretreated by B2antagonist HOE-140 for 15 minutes stimulated group, and pretreated bytyrosine phosphatase inhibitor orthovanadate(OV) for 15 minutesstimulated group. The activation of ERK1/2 were evaluated after the lastobstance acted for 2 minutes by Western blotting. Result: (1) Through the stimulation on the phophorylation ofERK1/2,BK induce the proliferation and the secretion of type â…£ collagenon rMC. (2) On the contrary, in rMC pretreated with BK, the mitogenesisand ERK1/2 activation induced by PDGF-BB was dose-dependentlyreduced(p<0.01). The inhibitory effects of BK on PDGF-BB-inducedphosphorylation of ERK1/2 was abolished completely by addition ofHOE-140 and OV. (3) In renal interstitial fibroblasts,when added separately,BK promote the secretion of type â…£ collagen,but had no effects on thecombined with PDGF-BB and proliferation whether added separately ornot. Conclusion:(1)BK has the double-faced effects in mesangial cellsmitogenesis including proliferation and collagen secretion, dependentlyacting separately or combinedly, and the pathway was found related with orwithout expression of phophorylated-ERK1/2. Since these effects wereabolished completely by HOE-140 and OV, so BK has thenegative-cross-talking effects with some mitogenetic factors throughattenuating the phosphorylation of signal transduction pathway moleculeactivated by such factors. (2)In renal intersitial fibroblasts, different effectsof BK from mesangial cells was detected consisting with stimulation ofcollagen when added separately but has noncorrelation when combinedlyand with proliferation. (3)The different effects of BK on MC mitogenesisand fibroblasts proliferation were related with the presence of BK receptorin MC and the absence on fibroblasts. And for the promotion of BK oncollagen secretion in fibroblasts, the mechanism is still waiting forresolvement. |
