The Effects Of G2/M Phase In Renal Tubular Remodeling After Acute Kidney Injury

Acute kidney injury(AKI)is a major clinical critical disease,and it refers to the various causes of acute renal function decline,with or without oliguria/anuria.The incidence and mortality of AKI is higher.The most common cause of AKI is acute ischemia-reperfusion kidney injury(AIKI).The pathological basis of AKI is acute tubular necrosis.Renal tubular epithelial cells are mainly damaged cells in AKI.Renal tubular epithelial cell remodeling plays a vital role in AKI.The regulatory mechanism of renal tubule remodeling is complex,and it is involved in cell cycle regulation,cell signal transduction,cell microenvironment and various cytokines.However,the regulatory mechanism of renal tubule remodeling is not yet clear,so we conducted the following research:Part Ⅰ: The cell cycle of renal tubular epithelial cells in AIKI rats Objective: To study that the cell cycle of renal tubular epithelial in AIKI rats model.Methods: SD rats were randomly divided into AIKI group(n=64)and Sham group(n=64).The rats of AIKI group were clip bilateral renal pedicle for 45 min,and the rats of Sham group were not clip bilateral renal pedicle.The rats were sacrificed following reperfusion 2 h,6 h,24 h,48 h,72 h,1 week,2 weeks and 4 weeks.Serum samples were taken for serum creatinine measurement,and histomorophology method and fluorescence quantitative PCR of renal tissue was measured.The cell cycle distribution of renal tubular epithelial cells was measured by K67(cell proliferation marker),p-H H3(M phase marker)and BrdU(S phase marker)immunohistochemical method.Results:(1)The renal damage degree changes of AIKI group rats after AIKI: The serum creatinine levels of AIKI group increased significantly than Sham group at 2 h to 72 h after AIKI(P<0.05).The kidney tissues NGAL mRNA expression of AIKI group was significantly higher than Sham group at 2 h to 4 weeks after AIKI(P<0.05).The tubulointerstitial injury scores of AIKI group were significantly higher than Sham group at 2 h to 4 weeks after AIKI(P<0.05).(2)The E-Cadherin,α-SMA and FN expression of AIKI group rats after AIKI: The E-Cadherin expression of AIKI group increased significantly than Sham group at 2 h and 6 h after AIKI,and decreased significantly than Sham group at 24 h to 72 h after AIKI(P<0.05).The α-SMA expression of AIKI group increased significantly than Sham group in renal tissue(P<0.05).The FN expression of AIKI group decreased significantly than Sham group at 2 h and 6 h after AIKI(P<0.05).(3)The cell cycle distribution of renal tubular epithelial cells of AIKI group rats after AIKI: The renal tissues Ki-67 staining positive cells percent of AIKI group increased significantly than Sham group at 2 h to 2 weeks after AIKI(P<0.05);The p-H H3 staining positive cells percent of AIKI group increased significantly than Sham group at 2 h to 72 h after AIKI(P<0.05).The BrdU staining positive cells percent of AIKI group increased significantly than Sham group at 24 h to 2 weeks after AIKI(P<0.05).Conclusion: The cell cycle distribution of renal tubular epithelial cells in AIKI rats were obvious changes.The percent of G2/M phase cells was increased at 2 h to 72 h after AIKI,and the percent of G1/S phase cells was increased at 24 h to 72 h after AIKI.Part Ⅱ: The effects of G2/M phase in AIKI rats Objective: To study the effects of G2/M phase in renal tubular epithelial cells of AIKI rats.Methods: SD rats were randomly divided into TAX+AIKI group(n=64),PIF+AIKI group(n=64),TAX+Sham group(n=24)and PIF+Sham group(n=24).The rats of TAX+AIKI group and TAX+Sham group were treated with TAX for G2/M phase arrest,and the rats of PIF+AIKI group and PIF+Sham group were treated with PIF for promote G2/M phase.The rats of TAX+AIKI group and PIF+AIKI group were clip bilateral renal pedicle for 45 min,and the rats of TAX-S group and PIF-S group were not clip bilateral renal pedicle.Serum samples were taken for serum creatinine measurement,and histomorophology method,western blot and fluorescence quantitative PCR of renal tissue was measured.The cell cycle distribution of renal tubular epithelial cells was measured by immunohistochemical method.Results:(1)The cell cycle distribution of renal tubular epithelial cells: The p-H H3 staining positive cells percent of TAX+Sham group were significantly higher than Sham group,and the p-H H3 staining positive cells percent of TAX+AIKI group were significantly higher than AIKI group(P<0.05).The p-H H3 staining positive cells percent of PIF+AIKI group were significantly lower than AIKI group at 2 h after AIKI(P<0.05).(2)The renal damage degree changes: The serum creatinine levels of TAX+AIKI group similar to AIKI group and the serum creatinine levels of PIF+AIKI group significantly decreased than AIKI group at 6 h to 72 h after AIKI(P<0.05).The tubulointerstitial injury scores of TAX+AIKI group significantly increased than AIKI group at 2 h to 6 h after AIKI,and the scores of PIF+AIKI group significantly decreased than AIKI group at 24 h to 4 weeks after AIKI(P<0.05).The renal tissues NGAL mRNA expression of TAX+AIKI group was higher than AIKI group at 24 h after AIKI,and the NGAL mRNA expression of PIF+AIKI group was lower than AIKI group at 6 h to 1 week after AIKI(P<0.05).The renal tissue NGAL protein expression of TAX+AIKI group significantly increased than AIKI group,and the NGAL protein expression of PIF+AIKI group significantly decreased than AIKI group at 72 h to 4 weeks after AIKI(P<0.05).(3)The E-Cadherin,α-SMA and FN expression: The renal tissue E-Cadherin mRNA expression of TAX+AIKI group was higher than AIKI group at 24 h,48 h and 2 weeks after AIKI,and the E-Cadherin mRNA expression of PIF+AIKI group was lower than AIKI group at 6 h after AIKI,and was higher than AIKI group at 24 h and 72 h after AIKI(P<0.05).The renal tissue α-SMA mRNA expression of TAX+AIKI group was higher than AIKI group at 6 h to 24 h after AIKI,and the α-SMA mRNA expression of PIF+AIKI group was higher than AIKI group at 24 h after AIKI(P<0.05).The α-SMA protein expression of TAX+AIKI group was higher than AIKI group,and the α-SMA protein expression of PIF+AIKI group was lower than AIKI group at 2 weeks and 4 weeks after AIKI(P<0.05).The renal tissue FN mRNA expression of TAX+AIKI group was higher than AIKI group at 6 h after AIKI,and the FN mRNA expression of PIF+AIKI group was lower than AIKI group at 24 h after AIKI,and was higher than AIKI group at 6 h after AIKI(P<0.05).Conclusion: The G2/M phase of renal tubular epithelial cells was changed by TAX or PIF.TAX may make renal tubular epithelial cells arrest in G2/M phase,and make the rat renal damage degree aggravating.PIF can improve the injury of the renal tubular epithelial cells,and may be related to fibrosis factor expression.Part Ⅲ: The effects of G2/M phase in NRK-52 E cells Objective: To study the effects of G2/M phase in NRK-52 E cells by with TNF-α treated and the cell cycle changes.Methods: NRK-52 E cells were cultured in vitro and induced cell damage by TNF-α,and treated with TAX or Pifithrin-α for change cell cycle distribution.The cell cycle distribution was measured by flow cytometry,and fluorescence quantitative PCR,western blot and immunofluorescence was measured.Results:(1)The cell cycle distribution: NRK-52 E cells treated with TAX for 2 h to 24 h,then the G2/M phase cells increased gradually,and NRK-52 E cells treated with PIF for 2 h to 24 h,then the G2/M phase cells decreased gradually(P<0.05).(2)The NGAL expression: NRK-52 E cells induced by TNF-α for 2 h to 24 h,the NGAL mRNA expression gradually increased(P<0.05).NRK-52 E cells treated with TNF-α and TAX for 12 h,the NGAL mRNA expression was significantly higher than the cells without treated(P<0.05).NRK-52 E cells treated with TNF-α and PIF for 6 h,the NGAL mRNA expression was significantly lower than the cells without treated(P<0.05).NRK-52 E cells treated with TNF-α and TAX for 24 h,the NGAL protein expression had higher trend than the cells without treated.NRK-52 E cells treated with TNF-α and PIF for 24 h,the NGAL protein expression had lower trend than the cells without treated.(3)The α-SMA expression: NRK-52 E cells induced by TNF-α for 6 h or 24 h,the α-SMA mRNA expression increased(P<0.05).NRK-52 E cells treated with TNF-α and TAX for 24 h,the α-SMA mRNA expression was significantly higher than the cells without treated.NRK-52 E cells treated with TNF-α and PIF for 24 h,the α-SMA mRNA expression were significantly lower than the cells without treated(P<0.05).NRK-52 E cells treated with TNF-α and TAX for 24 h,the α-SMA protein expression had higher trend than the cells without treated.NRK-52 E cells treated with TNF-α and PIF for 24 h,theα-SMA protein expression was significantly lower than the cells without treated(P<0.05).(3)The FN expression: NRK-52 E cells treated with TNF-α and TAX,the FN mRNA expression had higher trend than the cells without treated(P<0.05).NRK-52 E cells treated with TNF-α and PIF for 24 h,the FN mRNA expression were significantly lower than the cells without treated(P<0.05).Conclusion: NRK-52 E cells can be induced damage by TNF-α.TAX or Pifithrin-α can affect the cell injury degree and the formation of fibrosis factor through intervention the cell cycle of NRK-52 E cells.