p21对缺血-再灌注损伤后肾小管上皮细胞演变的影响

目的 探讨p21对缺血-再灌注损伤（IRI）后肾小管上皮细胞演变的影响。方法 选择低龄（2个月龄）和高龄（12个月龄）p21（＋／＋）和p21（-／-）鼠，建立左肾IRI模型。于IRI后0、1、3、7d及1、3、6个月光镜下观察肾小管组织学变化，采用免疫组化法检测肾小管上皮细胞增殖细胞核抗原（PCNA）表达，组织化学染色观察肾小管上皮细胞衰老相关β-半乳糖苷酶（SA-β-gal）活力，末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺刻标记技术（TUNEL）检测肾小管上皮细胞凋亡。结果 IRI后0d，肾小管以坏死为主，高龄鼠比低龄鼠严重、p21（-／-）鼠比p21（＋／＋）鼠严重（P均〈0．05）。肾小管上皮细胞凋亡在IRI 1d后出现，7d达高峰，且高龄鼠比低龄鼠明显、p21（-／-）鼠比p21（＋／＋）鼠明显（P均d0．05）。低龄鼠IRI后1个月出现SA—β-gal染色阳性的肾小管上皮细胞，而对侧肾此时未见衰老细胞，3和6个月时衰老的肾小管上皮细胞显著增多，且p21（＋／＋）鼠比p21（-／-）鼠明显（P〈0．05）；p21（＋／＋）高龄鼠IRI后0d双肾即可见大量的SA-β-gal染色阳性肾小管上皮细胞，且较p21（-／-）鼠显著增多（P〈O．05），但1d后，p21（＋／＋）和p21（-／-）鼠IRI肾衰老细胞均明显减少（P均〈0．05），1个月后又呈进行性增加，且p21（＋／＋）鼠始终比p21（-／-）鼠严重。高龄和低龄p21（＋／＋）鼠PCNA阳性染色细胞出现的几率差异无显著性（P〉0．05），但低龄鼠细胞增殖能力要强于高龄鼠；而p21（-／-）鼠的细胞增殖能力明显强于p21（＋／＋）鼠，低龄鼠更为显著（P均〈0．05）。对高龄鼠IRI后1d细胞衰老和凋亡进行相关分析显示，二者呈显著负相关Cp21（＋／＋）鼠：r=-0．82，P〈0．001,p21（-／-）鼠：r=-0．76，P〈0．0013。结论 ①IRI可促进正常肾小管上皮细胞衰老的进程；②已经进入衰老状态的肾小管上皮细胞在遭受IRI刺激后，更易走向死亡[坏死和（或）凋亡]；③p21在IRI所致肾小管上皮细胞演变过程中发挥重要的调控作用。

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.