Study Of The Effect Of P53 And It's Mechanism On The Changes Of Renal Tubular Epithelial Cells After Ischemia/reperfusion Injury

Background: The high incidence rate of acute renal failure(ARF) remains an unsolved problem in clinical medicine. In america adult, the year incidence rate of ARF is 0.02%, and in general ward, it is 5%, but in intensive care unit, which is up to 30-50%. Several studies have reported older individuals to be at a higher risk for ARF , which is not surprising because kidney function declines in the elderly. ARF not only endangers human life, but also increases the economic burden to our society. Renal ischemia/reperfusion injury(IRI) is the majority reason for the occurrence of ARF in native kidneys, and none graft kidney can avoid ARF. Tubular injury is the primary pathology changes during the initial stage after renal IRI, which present renal tubule necrosis and tubular cell apoptosis. These changes result in not only the acute pathology bases of kidney dysfunction , but also the"Long-term effects"of renal structure and function. In the IRI kidney, oxidative stress and DNA damage persistently exist, even in the late phase after IRI, which may results in the activation of senescence signaling pathway, and eventually, triggers and maintains the tubular cell senescence in late phase of IRI. So we proposed that IRI can induce and accelerate renal tubular cell senescence, which is presumed to be one of the mechanisms of"Long-term effect"of IRI, on the other hand, sesescent tubular epithelial cells in aging kidney are easily subjected to necrosis and apoptosis after IRI , which may play a vital role for old organisms to subject ARF more easily than young organisms. p53 is one of the important cell cycle regulatory proteins, either the cell death or the cell divided retention depends on the expression level of p53, and the environmental stress of cells or the activation of p53 target gene.The aim of this study was to approach the acute and chronic changes of renal tubular cells after renal IRI, and investigate the effects of p53 and it's mechanism on these changes. The results of our study would provide some theories for the prevention and cure of ARF and age-related kidney disease.The study consisted of four parts as following:Part I Senescence variation and its significance of renal tubular epithelial cells after kidney ischemia/reperfusion injury1. Objective To observe the outcome of normal and senescent renal tubular epithelial cells after kidney ischemia/reperfusion injury(IRI), and investigate the role of cell senescence in the aging-related pathological changes in kidney.2. Methods Wild-type male mice at ages of 2 (young group) and 12 months (aged group) were used to make ischemic models by clamping left renal hila for 45 minutes. At 0,1,3 and 7d,1,3 and 6m after reflow, renal tissues were examined for histomorphology, cell proliferation(PCNA),apoptosis and senescence (SA-β-gal).3. Results In both young and aged mice, renal tubule necrosis was the primary changes at day 0 after kidney IRI, but in aged mice,that was much more severely. Apoptotic tubular cells were detected at day 1 after IRI in young and aged mice,and was much more widely distributed spread in aged mice,the most severe apoptosis occurred in tubular epithelial cells of both young and aged mice at day 7 after reflow(p<0.05). In young animals , faint staining for SA-β-gal activity occasionally appeared in IRI kidney begin at month 1 , and increased at month 3 and 6 after IRI(p<0.05), but in contralateral kidney,there was no positive staining for SA-β-gal at any time mentioned above.contra-lateral Another pattern of the expression of SA-β-gal was detected in aged mice, both kindeys had widely positive staining for SA-β-gal at day 0 after IRI ,but decreased notably at day 1 in the IRI kidney(p<0.05), then increased again at month 3,but still less than the contralateral kidney,and more than the young mice at the same time point(p<0.05). 6 months after IRI, in both the IRI kidney and the contralateral kidney, positive staining for SA-β-gal almost reached the same level.Positive staining for nuclear PCNA in young and aged mice has no statistical significance(p>0.05),although the number of positively stained nuclear PCNA are bigger in young mice than that in aged mice. Correlation analysis between senescent and apoptotic cells in aged mice was made at day 1 after IRI, striking negative correlation was found between them (r=-0.82, p<0.001).4.Conclusions IRI can promote the senescence process of normal tubular cells,and can accelerate the death(necrosis and apoptosis)process of senescent tubular cells. These variations may play an important role in the development and progression of aging-related pathological changes in kidney.PartⅡEffect of p21 on the changes of renal tubular epithelial cells after kidney ischemia/reperfusion injury1.Objective To observe the changes of renal tubular epithelial cells in p21(+/+) and p21(-/-) mice with young and old ages at different times after kidney ischemia/reperfusion injury(IRI), and investigate the contribution of p21 gene in these variations.2.Mehtods p21(+/+)and p21(-/-) male mice at the ages of 2 and 12 months were made ischemic by clamping left renal hila for 45 minutes, at 0,1,3 and 7d,1,3 and 6m after reflow, renal tissues were processed for morphometric,proliferating cell nuclear antigen(PCNA),apoptosis and senescence-associatedβ-galactosidase(SA-β-gal) analysis, using hematoxylin and eosin stain,immunofluorescence,terminal deoxynucleotidyl trans-ferase-mediated dUTP-biotin in situ nick-end labeling ( TUNEL) and histochemical stain, respectively.3.Results Renal tubule necrosis and cell apoptosis were more severely in p21(-/-) mice and aged mice as compared with p21(+/+)mice and young mice (P<0.05),respectively . In young p21(+/+) mice,occasionally faint staining for SA-β-gal activity appeared begin at 1 m, and obviously significantly increased at 3 and 6 m after IRI(P<0.05), but in contralateral kidney,and in both kidneys in p21(-/-) mice, there was no positive staining for SA-β-gal at any time as mentioned above;another manner of the expression of SA-β-gal was detected in aged p21(+/+) mice, both kindey had widely positive staining for SA-β-gal at 0d after IRI ,then decreased notably at 1d in the IRI kidney(P<0.05), and increased again at 3 months,but still less than the contralateral kidney,and more than the young mice at the same time (P<0.05);3 months after IRI,in both the IRI kidney and the contralateral kidney, positive staining for SA-β-gal almost reached the same level;On the contral,only occasionally faint staining for SA-β-gal activity was observed in aged p21(-/-) mice at any time as mentioned above .Positive stain of nuclear PCNA in young and aged p21(+/+)mice has no statistical significance(P>0.05),although the numbers of positive stain nuclear PCNA are more in young mice than in aged mice.But in p21(-/-)mice ,significant positive staining for PCNA was tested, especially in young mice and in IRI kidneys(P<0.05). Correlation analysis between senescent and apoptotic cells in aged mice was made at 1d after IRI, then striking negative correlation was found between both of them (r=-0.82, P<0.001, p21(+/+)mice;r=-0.76, P<0.001, p21(-/-)mice).4.Conclusions IRI can promote the senescence process of normal tubular cells,and can accelerate the death(necrosis and apoptosis)proceeding of senescent tubular cells.p21 gene may play an important role in the senescence variation of tubular epithelial cells after kidney ischemia/reperfusion injury.PartⅢEffect of p53 on the variation of renal tubular epithelial cells after kidney ischemia/reperfusion injury1.Objective To observe the variation of renal tubular epithelial cells in p53(+/+) and p53(-/-)mice with young or old age at different time after kidney ischemia/reperfusion injury (IRI), and to investigate the contribution of p53 gene in the variation.2.Mehtods p53(+/+) and p53(-/-) male mice at age of 2 and 12 months were made ischemic by clamping left renal hila for 45 min. At 0, 1, 3 and 7d,1, 3 and 6 month after reflow, renal tissues were processed for morphometric observation and proliferating cell nuclear antigen(PCNA), apoptosis and senescence-associatedβ-galactosidase (SA-β-gal) analysis, using hematoxylin and eosin stain,immunofluorescence,terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick-end labeling ( TUNEL) and histochemical staining, respectively.3.Results Renal tubule necrosis was more severely in p53(-/-)mice and aged mice compared to p53(+/+) mice and young mice (P<0.05),respectively. Apoptotic cells in p53(+/+)mice increased obviously compared to p53(-/-) mice (P<0.05) at 7 d after IRI. In young wild-type mice, occasionally faint staining for SA-β-gal activity begain to appear at 1 month, and obviously significantly increased at 3 and 6 month after IRI (P<0.05), but in contralateral kidney at any time as mentioned above, and in the IRI kidneys in p53(-/-)mice at 1 and 3 month ,there was almost no positive staining for SA-β-gal; occasionally positive staining for SA-β-gal was observed in the IRI kidney in p53(-/-) mice at 6 month after IRI. In p53 (-/-) and p53 (+/+) aged mice, both kindeys had positive staining for SA-β-gal activity at 0d after IRI, but the level of the activity in p53(-/-) mice was much more lower than that in p53(+/+) mice (P<0.05), then the level of the activity decreased notably at 1d in the IRI kidney (P<0.05). Positive stain of nuclear PCNA in p53(+/+) young mice had no statistical significance compared to p53(+/+) aged mice (P>0.05); But in p53(-/-) mice ,significant positive staining for PCNA was tested, especially in young mice and in IRI kidneys(P<0.05). Correlation analysis between senescent and apoptotic cells in aged mice was made at 1d after IRI, then striking negative correlation was found between both of them in p53(+/+) mice (r=-0.82, P<0.05), but no statistical correlation in p53(-/-) mice(r=0.26,P>0.05).4.Conclusions IRI can accelerate renal tubular cell senescence and cellular death(both necrosis and apoptosis)after that. p53 gene may play an important role in the variation of tubular epithelial cells after kidney IRI.PartⅣp53-p21-Rb Signaling Pathways is Involved in Tubular Cell Senescence in Renal Ischemia/Reperfusion Injury1.Objective To investigate the course of tubular cell senescence and expression of p53, p21 and Rb during the late phase of IRI in kidney, and assess the effects of p53-Rb pathway on tubular cell senescence.2.Methods Experimental models of unilateral renal IRI were used in p53(+/+) and p53(-/-) mice ; histological changes at the tubular level, progress of cell senescence and expression of Rb, p21 and/or p53 proteins in tubular cells were studied at different time points after IRI .3.Results Chronic tubulointerstitial fibrosis was much more severe and widely distributed in IRI kidneys of p53(+/+) mice in later stages; senescent tubular cells were significantly increased at 3 and 6 months after IRI. In contrast, in contralateral p53(+/+) kidneys and in both kidneys from p53(-/-) mice, almost no senescent cells were observed at 1 and 3 month after IRI; a few senescent cells was detected in IRI kidneys of p53(-/-) mice at 6 month . Changes of cell senescence were correlated with the expression level of p53, p21 and Rb proteins in mice of either genotype.4.Conclusion These results suggested that IRI can accelerate tubular cell senescence, which is presumed to be one of the mechanisms of"Long-term effect"of IRI. The activation of p53-Rb signaling pathway may play a vital role in tubular cell senescence induced by IRI.