An Experimental Study On HSP70 In Renal Ischemia Preconditiong Protect Renal Tubular Cells Against Oxdation And Ischemia/reperfusion Injury

Background: Acute renal failure is a frequent clinical entity with an increasing incidence,yet there is no specific treatment.Now there are more studies about ischemia preconditioning (IP)against ischemia injury.Repeated transient ischemia in advance could stimulate planted organs to tolerate longer subsequent ischemia injury,This kind of special phenomenon is called ischemia preconditioning.Ischemic preconditioning, was first described in the heart,in fact there is almost a century of literature on the kidney that supports the concept that prior injury protects against a second insult.The mechanisms of IP are very complex. Overpression of Heat Shock Protein70(HSP70)has been shown in kidney after ischemia preconditioning,but there are still no reports about that up-regulation of HSP70 maybe one of the protective mechanisms brought to kidney by IP.We employed adeno-associated virus as the vector to transfect HSP70 gene into cells and rats.we intended to find the role of HSP70 gene in the protection of renal tubular cells against oxdative injury and ischemia /reperfusion injury in rats and prove that renal ischemia preconditioning is concerned with the overexpression of HSP70. Objective: To research on the effectiveness of HSP70 gene transfection in renal tubular cells against against oxdative injury and ischemia/reperfusion injury in rats and to prove that renal ischemia preconditioning is concerned with the overexpression of HSP70. Enhancement of the protective mechanisms about preconditioning has potential benefit in preventing ischemia-reperfusion damage and improving long-term graft survival in kidney transplantation. Methods: 1. Full-length HSP70 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) method from cell line HepG2 of liver cancer and cloned into eukaryotic expressing vector pAAV-IRES-hrGFP, using the T/A vector as intermediary. The correct cloning of HSP70 gene in pAAV-IRES-hrGFP was confirmed by restrictive enzyme digestion and sequencing. Eukaryotic expressing vector PAAV-IRES-hrGFP transferred AAV-293 cells with pAAV-Helper,pAAV-RC,then the recombinant AAV-HSP70 was constructed and high titer of the AAV-HSP70 was gained. 2. The renal tubular cells were transfected by AAV-HSP70, The expressions of HSP70 were measured by immunocytochemistry and western blotting.The viability of the cells were measured by the MTT ,also the cytotoxic activity of H2O2 were observed by 51Cr release assay. The rats were transfected by AAV-HSP70 by mainline,The expressions of HSP70 were measured by immunocytochemistry。The rat model of ischemia/reperfusion and ischemia preconditioning were established, renal function was determined by measuring serum creatinine after 45 min of ischemia followed by 24 hours reperfusion,histopathological changes were analyzed for tubular necrosis. Results: 1. Full-length HSP70 cDNA was cloned successfully,and the sequence of HSP70 was the same as which was reported in Genbank. 2. The HSP70 gene was cloned into the adeno-associated virus vector pAAV-IRES-hrGFP, eukaryotic expressing vector PAAV-IRES-hrGFP transferred AAV-293 cells with pAAV-Helper,pAAV-RC and gain high titer of the AAV-HSP70,the titer of the prepared AAV was 2.2×106(pfu/ml) which was enough to transfect in vitro and vivo. 3. The transfection of AAV-HSP70 into renal tubular cells could successfully induce the up-regulation of HSP70. 4. Up-regulation of HSP70 could protect the renal tubular cells against oxdative injury, the probable mechanisms maybe concered with the inhibition of cell apoptosis. 5. The transfection of AAV-HSP70 in rats could successfully induce the up-regulation of HSP70,which could protect the rats against ischemic /reperfusion injury. 6. Up-regulation of HSP70 induced by renal ischemia preconditioning could protect the rats against ischemia /reperfusion injury. Conclusion: The transfection of AAV-HSP70 into renal tubular cells could successfully induce the up-regulation of HSP70 and protect the cells against oxdative injury, its probablemechanism maybe concered with the inhibition of cell apoptosis. Up-regulation of HSP70 induced by renal ischemia preconditioning could protect the rats against ischemia /reperfusion injury.The mechanism of renal ischemia preconditioning maybe concered with the up-regulation of HSP70.