The Effects Of Response Gene To Complement 32 In Renal Tubular Repair In Acute Kidney Injury And Their Probable Mechanisms

Part 1: The Expression and Significance of Response Gene to Complement 32 in Rat with Acute Kidney InjuryObjective: To investigate the expression of Response Gene to Complement 32(RGC-32) in rat with acute kidney injury(AKI), and to explore the role of RGC-32 in renal injury induced by ischemia reperfusion.Methods: Sprague Dawley rats were randomly divided into 2 groups, including sham operation group(n=64) and acute ischemia reperfusion injury(IRI) group(n=64). Rats were sacrificed following reperfusion 2h, 6h, 24 h, 48 h, 72 h, 1week(w), 2w and 4w. Serum samples were taken from aorta abdominalis for Serum creatinine(Scr) measurement at different reperfusion time points. The distribution and expression of RGC-32 in renal tissue were observed by means of immunohistochemistry. The mean density of the images detected by Image-Pro Plus 6 was designated as the representative RGC-32 expression levels. Meanwhile, RGC-32 m RNA expression was measured by q PCR.Results:(1) The location of RGC-32 in normal renal tissues: RGC-32 majorly expressed in cytoplasm of epithelial cells of proximal tubular, distal renal tubular and collecting ducts, and expressed slightly in glomerular, while RGC-32 did not expressed in renal interstitium and the vessels of kidney.(2) The expression levels of RGC-32 measured by immunohistochemistry at different reperfusion time were 0.0168±0.0029, 0.0156±0.0021, 0.0065±0.0013, 0.0075±0.0013, 0.0096±0.0014, 0.0132±0.0016, 0.0169±0.0014, 0.0179±0.0022, respectively. Compared with the sham operation group, the level of RGC-32 expression in IRI group was significant lower at 24 h,48h,72 h after IRI(P<0.05). The expression levels of RGC-32 m RNA at different reperfusion time also measured by q PCR were corroborated the immunohistochemistry finding. And the expression of RGC-32 was correlated with renal tubule-interstitial injury score(r= -0.514,P<0.01).Conclusions:(1) We firstly found that RGC-32 protein located in renal tubular in renal tissues of rat, and expressed slightly in glomerular, while RGC-32 did not express in renal interstitium and the vessels of kidney.(2) Our data showed that the RGC-32 expression in AKI rat decreased significantly reduces with different reperfusion time and performs a time-dependent manner.RGC-32 may play an important role in the pathogenesis of AKI following ischemia-reperfusion injury in rat.Part 2: Response Gene to Complement 32 acts as a novel cell cycle factor on renal tubular epithelial cells repairObjective.: This study aimed to evaluate the cell cycle influence of response gene to complement-32(RGC-32) in processing renal tubular epithelial cells injury and repair. Methods.:We cultured NRK-52E cells in vitro and treated with TNF-α, and then determinate the RGC-32 expression levels of cell injury. Then we made the NRK-52E cells expressed in either high or low expression by transient transfection, and determinate the cell cycle distribution, and the expression and significance of fibrosis factors.Results : First, when the NRK-52E cells treated with TNF-α, the cells were injured by TNF-α. NGAL and RGC-32 expression was significantly increased. Second, RGC-32 regulates the cell cycle distribution of the NRK-52E cells by controlling G2/M checkpoint in cell cycle. Our data showed that the cell number of G2/M phases increased dramatically, indicating that the low RGC-32 expression group induced G2/M arrest. Third, the low RGC-32 expression group NRK-52E cells whose G2/M phases were prevented, had significantly increased the expression of fibrosis factors.Conclusion: In this study, we indicated that in vitro RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells by regulating G2/M phase checkpoint. However, the exact mechanism needs to be further elucidated.