| Acute kidney injury(AKI) represents a very important and potentially ischemic acute renal failure devastating disorder in clinical medicine. Ischemic renal injury is the leading cause of acute renal failure. For its reversibility, early detection and treatment of ischemic acute kidney injury will be helpful to prevent its development to kidney failure. Because the early clinical symptom is untypical, ischemia and hypoxia of organism for a long time will lead to renal failure if low perfusion in kidney can't be retrieved.In current clinical practice, there has no certain index for early diagnosis and therapeutic effect in ischemic renal injury. Serum creatinine is an unreliable indicator during acute changes in kidney function. Some urine proteins reflecting renal damage can't be early detected also. Early diagnosis to ischemic renal injury is an imminent work.Recently study has shown that Cyr61 protein was markedly up-regulated in the kidney following renal ischemia. It was detected in urine at three to six hours after renal injury, which is earlier not only than Scr and urokinase protein,but also than kidney injury molecule-1. But the detection of Cyr61 protein was measured followed capture with heparin beads, which limit the clinical application of Cyr61. Therefore, a convenient and precise method for Cyr61 protein detection has more practical importance.The study was consist of three parts:1. Distribution and expression of Cyr61 in kidney tissues,body fluid in ischaemia acute renal injuryThe analog of ischemic acute renal injury was made. Distribution and expression of Cyr61 in kidney was investigated by immunohistochemistry and fluorescence quantu PCR with the Cyr61 polyclonal antibody and fluorescent probe designed by ourself. The content of Cyr61 in tissue of the ischemic renal and in body fluid were detected by Western blotting. Our experiments showed that Cyr61 was up-regulated in the proximal tubule epithelial cells of the ischemic renal tissue, while positive result was not found in distal tubule and collect tubule. The experiment of fluorescence quantu PCR show: mRNA for Cyr61 was obviously up-regulated at 1 hour in the ischemia kidney and peaked at four hours.Cyr61 protein in serum in ischemic group was markedly raised at one to four hours. While in urine, it was up-regulated at two hours, peaked at four to eight hours.2.The quantitative determination of the Cyr61 in body fluid following ischemic acute kidney injuryTotal RNA was extracted in breast cancer tissue. A gene sequences which code domain of Cyr61 protein (according to GenBank Z98053)were amplified with RT-PCR, which product is segment of Cyr61 protein (133-433 AA) . The fragment was inserted into pQE80L.After enzyme digest and DNA sequencing verified, the expression vector were transformed into competent E.coli Rosetta-gamiTM 2 cells. The fusion protein expression was induced by IPTG and purified by Ni-NTA affinity chromatography. The products were identified by SDS-PAGE analysis and Western blotting. Fusion protein of Cyr61 was obtained, with a molecular weight of 32kDa.In order to have the quantitative information about Cyr61 in body fluid in ischemic acute kidney injury following operation on heart. We established a sandwich ELISA method by using two polyclonal antibodies which aim at different sites in carboxyl terminus and middle piece respectively. Cyr61 in body fluid following operation on heart were detected. The quantitative analysis revealed that:Cyr61 in serum in acute kidney injury following operation on heart was markly raised up, peaked at four to eight hours, decreased at 12 hours, while the amplification of Cyr61 in serum in control group was lower than it in AKI group at four to eight hours. Cyr61 in urine in acute kidney injury group raised up at two hours after operation on heart, peaked at six to eight hours, decreased at 12 hours until 60hours(p<0.01).3. Effect of mitogen-activated protein kinase on the trans-activation of Cyr61 induced by hypoxia in HKC cellsThe expression levels of Cyr61, p38, ERK1/2, JNK, and HIF-1αin HKC cells induced by hypoxia. We analyzed by Northern and Western blot. Full length of Cyr61 promoter region was amplified by PCR and recombinant plasmid of Cyr-luc were constructed. After transfection alone or coexpression with vector overexpressing constitutively active forms of either MEK1 (Ca-MEK1),or MKK6 (Ca-MKK6),the trans-activation of Cyr61 in HKC cells induced by hypoxia, MAPK pathway inhibitors or MAPK kinase were assayed by means of luciferase reporter gene assay system. The experiments showed that Cyr61 and HIF-1αwere expressed at an obviously high level in HKC cells cultured under hypoxia. The phosphorylation content of ERK, JNK, p38 in the HKC cells increased along with the time of hypoxia, while there was no significently difference in total ERK, JNK and p38 expression. The luciferase activity was inhibited separately by PD98059 or SB203580 in HKC cells transfected with Cyr-luc plasmid, and it was obviously enhanced by the cotreated with PD98059 and SB203580. The trans-activation of Cyr61 in HKC cells transfected with Cyr-luc was not affected after cotransfected with Ca-MEK1,but was greatly increased with Ca-MKK6 .The protein expression of Cyr61 and HIF-1αwas inhibited by PD98059 in HKC cells under hypoxia, the same effect of SB203580 on Cyr61 was seen also. The inhibition of Cyr61 protein expression was great improved when co-treated by PD98059 and SB203580.Our study suggest that Cyr61 protein express in the proximal tubule epithelial cells of the ischemic renal tissue and could be detected in urine after renal ischemia. Cyr61 in urine raised up at two hours after ischemia, peaked at six to eight hours; A sandwich ELISA method was established and used to quantitative detect Cyr61 protein in body fluid after operation on heart. Cyr61 in serum in acute kidney injury group raised up obviously at four to eight hours than the group without kidney injury, which in both group raised up after two to twelve hours of operation.Cyr61 in urine in acute kidney injury group raised up at two hours after operation on heart, peaked at six to eight hours, decreased at 12 hours until 60 hours. In HKC cells, hypoxia stimulates the trans-activation of Cyr61 through p38 MAPK pathway directly, and it also adjusted HIF-1αexpression through ERK1/2 pathway, which could stimulates the trans-activation of Cyr61 indirectly. |
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