[Effect of p21 on the changes in renal tubular epithelial cells after ischemia/reperfusion injury of kidney].

OBJECTIVE: To investigate the contribution of p21 gene in renal tubular epithelial cells in p21 (+/+) and p21 (-/-) mice of young and old ages at different times after kidney ischemia/reperfusion injury (IRI). METHODS: In p21 (+/+) and p21 (-/-) male mice at the ages of 2 and 12 months the kidneys were made ischemic by clamping the left renal artery for 45 minutes followed by declamping. On 0, 1, 3 and 7 days, 1, 3 and 6 months after reflow, renal tissue was processed for pathological study, determination of proliferating cell nuclear antigen (PCNA), apoptosis and senescence-associated beta-galactosidase (SA-beta-gal) analysis, using hematoxylin and eosin staining, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick-end labeling (TUNEL), and histochemical staining, respectively. RESULTS: Renal tubule necrosis and cell apoptosis were more severe in p21 (-/-) mice and old mice as compared with p21 (+/+) mice and young mice (both P<0.05), respectively. In young p21 (+/+) mice, occasionally faint staining for SA-beta-gal activity began to appear after 1 month, and significantly increased 3 and 6 months after IRI (P<0.05), but there was no positive staining for SA-beta-gal in the contralateral kidney or both kidneys in p21 (-/-) mice at any time. Another manner of the expression of SA-beta-gal was detected in aged p21 (+/+) mice, as both kidneys showed intensely positive staining for SA-beta-gal at 0 day after IRI, it then subsided notably on 1 day in the IRI kidney (P<0.05), but increased again at 3 months, though still less intense than the contralateral kidney, albeit more intense than the young mice at the same time (P<0.05). Three months after IRI, in both the IRI kidney and the contralateral kidney, positive staining for SA-beta-gal almost reached the same level. On the contrary, only occasional faint staining for SA-beta-gal activity was observed in aged p21 (-/-) mice at any time. No significant difference in positive staining of nuclear PCNA was found between in young and aged p21 (+/+) mice (P>0.05), although the numbers of positively stained nuclear PCNA were more in number in young mice than in aged mice. But in p21 (-/-) mice, significantly more cells were positively stained for PCNA, especially in young mice and in IRI kidneys (P<0.05). Correlation analysis between senescent and apoptotic cells in aged mice made at 1 day after IRI showed striking negative correlation between both of them [p21 (+/+) mice: r=-0.82; P<0.001; p21 (-/-) mice: r=-0.76, P<0.001]. CONCLUSION: IRI can promote the senescence process of normal tubular cells, and can accelerate death (necrosis and apoptosis) process of senescent tubular cells. p21 gene may play an important role in the senescence changes in tubular epithelial cells after kidney ischemia/reperfusion injury.

[Gly374Arg mutation in Fgfr3 causes achondroplasia in mice].

OBJECTIVE: To establish the mouse model of Gly374Arg mutation in fibroblast growth factor receptor 3(Fgfr3) and to analyze the phenotype of the mutant mice. METHODS: The double PCR was used to introduce Gly374Arg point mutation into mouse Fgfr3. The electroporation of embryonic stem(ES) cells was carried out with targeting vector. The targeted ES cells were screened by Positive-Negative Selection of G418 and Ganciclovir, and Southern blot. The correct targeted ES cells were microinjected into blastula. Finally, mutant mice were obtained by crossing between EIIa-Cre transgenic mice and mice carrying recombined mutant Fgfr3 allele. The mice were genotyped by PCR, and phenotype was observed by skeleton staining, histology, etc. RESULTS: Fgfr3-Gly374Arg mutant mice exhibited small size, short tail, macrocephaly and had dome-shaped heads, the epiphyseal growth plates of mutant mice were narrower, and the hypertrophic chondrocyte zone was also obviously decreased. Meanwhile, the majority of female mice were infertile, and the uterus, ovary and mammal gland in mutant female mice were also smaller and underdeveloped. CONCLUSION: The model of Fgfr3-Gly374Arg mutation causing achondroplasia in mice has been established successfully.

[Effect of p53 on the variation of renal tubular epithelial cells after kidney ischemia/reperfusion injury].

AIM: 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. METHODS: 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 7 d, 1, 3 and 6 month after reflow, renal tissues were processed for morphometric observation and proliferating cell nuclear antigen(PCNA), apoptosis and senescence-associated beta-galactosidase (SA-beta-gal) analysis, using hematoxylin and eosin stain, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick-end labeling (TUNEL) and histochemical staining, respectively. 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-beta-gal activity began to appear at 1 month, and obviously significantly increased at 3 and 6 months 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 months, there was almost no positive staining for SA-beta-gal; occasionally positive staining for SA-beta-gal was observed in the IRI kidney in p53(-/-) mice at 6 months after IRI. In p53 (-/-) and p53(+/+) aged mice, both kindeys had positive staining for SA-beta-gal activity at 0 d 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 1 d 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 1 d 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). CONCLUSION: 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.