| Renal interstitial fibrosis is a marked characteristic in the end-stage renal disease,and is a mainly manifested during progressive renal damage.Some studies have shown that,cytokines such as transforming growth factorβ1 (TGFβ1) plays a key role in the development of renal tubulointerstitial fibrosis from human and animals.Most of those cytokinse were produced by the renal tubulointerstitial cells,so antagonistic adverse effects of cytokines has become a important part.TGFβ1can stimulate the expression by the gene encoding the extracellular matrix(ECM),such as type I collagen fibers.But the TGFβ1 of signal transduction mechanism still not clear understanding completely.So strengthen the studies of tubulointerstitial fibrosis-related cytokine pathway are important way to understand the mechanism of tubulointerstitial fibrosis and explore new treatment means.TGF-activated kinase 1 (TAK1) is the first discovered member of the mitogen-activated protein kinase kinase kinase (MAP3K) family.It can be activated by different stimuli to participate in different signaling pathways,including TGFβ1.In the process of renal interstitial fibrosis of the different stages,the release of inflammatory factors and tubulointerstitial fibrosis-related cytokine,extracellular matrix generation,and the activation of renal cells-myofiblast in phenotyph conversion,there may be signaling pathway involved in the different TGFβ1/TAK1. Because of the pathogenesis of renal tubulointerstitial fibrosis is more complex.At present the results of their research is still in the initial stage.Therefore,in this study we well adopt a unilateral ureteral obstruction (UUO) rat model and in vitro experiments.further study of TAK1 in renal interstitial fibrosis,in order to further reveal the tubulointerstitial fibrosis of the biological mechanisms and provide a theoretical basis.Fosinopril (FOS) is an angiotensin-converting enzyme inhibitor (ACEI),it enables reduce the generation of angiotensinⅡ(Ang II) and inhibit kininase to reduce the degradation of bradykinin.It has been confirmed that FOS can delay the deterioration of renal function and protect the kidneys by a large number of animal models of kidney disease and clinical studies.However,their renal interstitial fibrosis associated signal pathway and the role of the mechanism is not yet clear.To further explore the possible mechanism of fosinopril on TGF-B/TAK1 signaling pathway in UUO-induced renal tubulointerstitial fibrosis,we were studied the modern pharmacology on animal experiments and cell test for clinical treatment of RIF to provide theoretical and experimental basis.Part One Effect of Fosinopril on expression of TAK1 in rats with renal interstitial fibrosisObjective:To investigate the mechanism of renal interstitial fibrosis after unilateral ureteral obstruction (UUO) and renal protective effect of fosinopril on the rat kidney,for further provide experimental evidence for studies on renal interstitial fibrosis.Methods:UUO model was induced by ligating the left ureter in rats.A total of 72 Male Sprague-Wistar rats were randomly divided into three groups:sham-operated group(n=24),UUO model group(n=24) and UUO+fosinopril group (n=24).the rats were treated with 10 mg/(kg-d) by gastric gavage in the fosinopril treated group from 2 d after the operation, and the rats were treated with the identical dose of normal saline in the other 2 groups.eight rats of each group were sacrificed at 7,14 and 21 days after UUO.Pathological changes of the renal tissue were observed by HE and Masson staining.The expression of TAK1 was detected by In situ hybridization.Western bloting was used to examine the protein expression of TAKl.Finally,the mRNA expression of TAK1 was measured by real-time quantitative PCR.Results:1) Histological staining,in compared with the sham-operated group,model group obstructed renal tubular epithelial cells appeared vacuolar degeneration,necrosis,some tubular atrophy,luminal expansion,renal interstitial edema,widened,increased collagen fibers,and have inflammatory cell infiltration and fibrosis.It show that renal interstitial fibrosis model was successfully prepared.The renal interstitial damage index of UUO group was increased more than the sham-operation group(P<0.05),but decreased in fosinopril treatment group(P<0.05).2) The results of in stiu hybridization showed that,TAKl mRNA expression were only in the nuclei of renal tubular epithelial cells within the brown-yellow granular positive staining in sham-operation group.In model group,the TAKl hign expression in renal tubular epithelial cell and fibroblasts within the diffuse brown cord-like distribution (P<0.05).In compared with model group during the same period, the expression of TAKl in fosinopril treatment group were decreased significantly,but still higher than sham-operation group, were scattered in the yellow-brown thin-like distribution (P<0.05).3) The results of western-bloting showed that,TAK1 protein has a small amount of basal expression in sham-operated group.In compared with sham-operated group,The protein expression of TAKl were upregulated at 7 d after UUO in model group (P<0.05),but decreased in fosinopril treatment group at the same time points(P <0.05).4) The results of RT-PCR showed that,TAKl mRNA has a small amount of basal expression in sham-operated group.In compared with sham-operated group,The mRNA expression of TAKl were upregulated at 7 d after UUO in model group (P <0.05),but decreased in fosinopril treatment group at the same time points(P<0.05).Conclusion:TGF-β/TAK1 signal transduction pathway in the UUO-induced renal interstitial fibrosis in rat model of early,middle and late stage are not the same,showing a decreasing trend after the first increased.It indicate that activating the signal transduction pathway of cytokines and its regulation factors may play an important role in the pathological process of renal interstitial fibrosis.Fosinopril may play a protective role in the kidney by downregulating the expression of TAK1 and alleviating renal interstitial fibrosis after UUO. Part Two Effects of Fosinopril on LPS Induced TAK1 expression in Human Renal Tubular Epithelial CellsObjective:To investigate the effects of fosinopril(FOS) on expression of TAKl and proliferation of the muman renal tubular epithelial cells induced by LPS.Methods:Human renal tubular epithelial (HK-2) cells were divided into four groups:blank control group(Ctol),LPS group (10 ug/L),FOS group (1 X 106 mol/L),LPS+FOS group (LPS 10 ug/L+FOS 1×106 mol/L).At 12,24,48 hours,HK-2 proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay.The change of fibronectin (FN) in the supernatants of the cultured HK-2 was detected by enzymelinked immunosorbent assay (ELISA).The protein expressions of TAK1 and FN were measured by Western blot.The mRNA expressions of TAK1 was measured by real-time quantitative PCR.Results:The cell proliferation and the expression of FN were increased, And the expressions of protein and mRNA of TAK1 in LPS group were upregulating significantly compared with control group from 12 h (P<0.01, P<0.05),but they were downregulating in FOS and LPS+FOS group compared with LPS group(P<0.01).Conclusion:LPS could induce increased proliferation of HK-2 and secretion of FN.Fos could inhibit proliferation of HK-2, downregulate the expression of FN,and inhibit TAK1 expression induced by LPS in the proliferation of HK-2.Its mechanism may be partly related to inhibiting TAK1 signaling pathway.
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