Protective Effect And Mechanism Of Taurine On Renal Ischemia Reperfusion Injury In Rats

Renal ischemia reperfusion injury (RIRI) is the main reason that cause ischemic acute renal failure (ARF). The apoptosis of renal tubular epithelial cell is one of the most important mechanisms. Excessive endoplasmic reticulum stress (ERS) can induce apoptosis, involved in RIRI. Taurine(Tau) has function in adjusting calcium homeostasis in cells, anti-inflammatory, anti-oxygen and so on. In this study, our objective was to investigate the protective effect and mechanism of taurine on renal ischemia reperfusion injury in rats from the perspective of endoplasmic reticulum stress through establishing renal tubular epithelial cell line of rat NRK-52E cells hypoxia/reoxygenation (H/R) injury model and the animal model of acute renal ischemia reperfusion injury in rats. It could provide experimental evidence for clinical prevention and treatment of renal ischemia reperfusion injury .This research project consisted of three parts:Part I: The effect of different hypoxia/reoxygenation time on renal tubular epithelial cells.Objective: To establish an ideal H/R model of renal tubular epithelial cells and select a suitable H/R time.Methods: Chose rat's renal tubular epithelial cell line NRK-52E cells as the research object and established the H/R model .The cells were randomly divided into control groups and H/R groups. Cells of the H/R groups were exposed to 4,8,12 h of hypoxia followed by 1,6,12 and 24 h of reoxygenation.①c ell count and calculated cell survival (trypan blue exclusion test);②Determined released lactate dehydrogenase (LDH) content;③Determined the mRNA expression of Caspase-12 gene by RT-PCR.Results:①T he live cell count and cell survival decreased gradually with the prolongation of hypoxia time and reached the lowest in H12. They further reduced after reoxygenation and reached the minimum in H12/R12.②L DH content of H/R groups was significantly higher than control groups, and it reached a peak in H12. After reoxygenation, LDH increased slowly, reached a maximum in H12/R12.③The mRNA expression of Caspase-12 gene began to increase significantly in H4/R6 and reached a peak in H8/R12. It began to decrease in H12, but was still significantly higher than control group.Conclusion: Through 12 hours of reoxygenation after 8 hours of hypoxia, we got an ideal H/R model of renal tubular epithelial cells.Part II: Protective effect and mechanism of taurine on hypoxia/reoxygenation damage in renal tubular epithelial cells.Objective: To study on the protective effect and mechanism of taurine on hypoxia/reoxygenation damage in renal tubular epithelial cells.Methods: Chose NRK-52E cells as the research object. The cells were randomly divided into control group, H/R group and H/R+Tau group. Chose H8/R12 as hypoxia/reoxygenation time because the expression of Caspase-12 mRNA is highest in this time. H/R+Tau group: supplemented with taurine solution(final concentration:10,20,40,80 mmol/L) in medium and the operations for the rest followed as the H/R group.①c ell count and calculated cell survival (trypan blue exclusion test);②Detected fluorescence intensity of intracellular freeing Ca2+ and apoptosis rate of renal tubular epithelial cells by flow cytometry;③Determined the mRNA expression of GRP78, Caspase-12, Caspase-3 gene by RT-PCR;④Determined the protein expression of GRP78, Caspase-12, Caspase-3 gene by Western-blotting.Results:①T he live cell count and the cell survival rate of H/R +Tau group were significantly higher than H/R group.②The average fluorescence intensity of intracellular freeing Ca2+ (reflecting the concentration of intracellular freeing Ca2+) increased significantly after hypoxia/reoxygenation. Compared with H/R group, the average fluorescence intensity of intracellular freeing Ca2+ significantly decreased after the taurine treatment. The apoptosis rate of H/R group significantly increased. It significantly decreased after the taurine treatment and showed a dose-dependent.③Compared with the control group, the mRNA and protein expression of GRP78, Caspase-12, Caspase-3 gene in H/R group was significantly higher. While the genes'expression of the group accepting the taurine treatment dramatically fell and showed a dose-dependent. Conclusion: Taurine could improve hypoxia/ reoxygenation damage of renal tubular epithelial cells. The mechanism was mainly to relieve the intracellular calcium overload and down regulate the expression of GRP78 and apoptosis related gene Caspase-12, Caspase-3.Part III: Protective effect and mechanism of taurine on renal ischemia reperfusion injury in ratsObjective: To study on the protective effect and mechanism of taurine on renal ischemia reperfusion injury in rats.Methods: Chose SD rats as the research object.The rats were randomly divided into sham operation group (Sham group), I/R group and I/R+Tau group. Prepared the animal model of acute RIRI through clamping bilateral renal pedicle 45min and then reperfusion 24h. I/R+Tau group: intraperitoneal injection of 400mg/kg taurine solution 3h before surgery and the operations for the rest followed as the I/R group.①Determined blood urea nitrogen (BUN) and creatinine (Cr);②O bserved the changes of renal tubular epithelial cells with electron microscope;③O bserved the histological changes of renal by HE staining;④D etermined the mRNA expression of GRP78, Caspase-12, Caspase-3 gene by RT-PCR;⑤Determined the protein expression of GRP78, Caspase-12, Caspase-3 gene by immunohistochemistry.Results:①I schemia reperfusion injury could significantly reduce kidney function.The BUN, Cr of I/R group were significantly increased. They significantly decreased after the taurine treatment.②We discovered that the damage of renal tissue resulting from Ischemia reperfusion was alleviated after the taurine treatment through HE staining and electron microscope.③Compared with the Sham group, the mRNA and protein expression of GRP78, Caspase-12, Caspase-3 gene in I/R group was significantly increased. While the genes'expression of the group accepting the taurine treatment dramatically fell.Conclusion: Taurine was a good protector of the renal ischemia reperfusion injury mediated by ERS in rats.The molecule mechanism was mainly to down regulate the expression and activity of GRP78 and Caspase-12 which is role in ER-associated death.Synthetized all of the results, we got the conclusion: endoplasmic reticulum stress response existed in the renal ischemia reperfusion injury. Taurine could improve the renal ischemia reperfusion injury. The mechanism was mainly to relieve the intracellular calcium overload and down regulate the expression and activity of GRP78 and apoptosis related gene Caspase-12, Caspase-3.