Chang In Youb, Lee Jung-Hee, Kim Jin Nam, Lee Kun Ho, Park Kyung Soo, Yoon Sang Pil
Department of Anatomy, College of Medicine, Chosun University , Gwangju , Republic of Korea.
Free Radic Res. 2015 Jan;49(1):95-101. doi: 10.3109/10715762.2014.981261. Epub 2014 Nov 22.
We have reported a possible involvement of apurinic/apyrimidinic endonuclease 1 (APE1), one of the DNA repair pathways, in various nephropathy models and found that there is a close connection between APE1 and p53-dependent apoptosis. Therefore, we investigated the changes of APE in aging rat kidney since aging is the consequence of increased susceptibility to apoptosis and impaired repair. Characteristics of chronological aging were compared among 6-, 24- and 28-month-old male Sprague-Dawley rats. Serum blood urea nitrogen and creatinine were measured for renal function. Western blot assay was compared for p53, bax, cleaved caspase 3, rH2AX, and APE1. Immunohistochemical staining of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and APE1 was performed. Cellular senescence was checked by beta-galactosidase staining. Compared with young rats, aged rats showed significant increase in creatinine level with cellular senescence in the proximal convoluted tubules confirmed by beta-galactosidase staining. All the checked variables were significantly increased with aging: 1) increased p53, bax, and caspase 3 may activate the apoptotic pathway, 2) increased rH2AX and 8-OHdG immunolocalization in the proximal convoluted tubules might mean augmented DNA damage, and 3) increased APE1 might be caused by the immunoreactivity in the distal convoluted tubules while decreased in the proximal convoluted tubules. These results suggested that APE1 might have little protective effects on p53-dependent apoptosis irrespective of DNA repair activities in aged renal proximal tubules. Therefore, researchers should use older animals than 24-month-old rats in future studies for investigating the relationship between the apoptosis and DNA repair in the aging kidneys.
我们已经报道了脱嘌呤/脱嘧啶内切酶1（APE1）（一种DNA修复途径）可能参与多种肾病模型，并发现APE1与p53依赖性凋亡之间存在密切联系。因此，由于衰老会导致细胞凋亡敏感性增加和修复受损，我们研究了衰老大鼠肾脏中APE的变化。比较了6、24和28月龄雄性Sprague-Dawley大鼠的自然衰老特征。测量血清尿素氮和肌酐以评估肾功能。比较了p53、bax、裂解的半胱天冬酶3、rH2AX和APE1的蛋白质免疫印迹分析结果。进行了8-羟基-2'-脱氧鸟苷（8-OHdG）和APE1的免疫组织化学染色。通过β-半乳糖苷酶染色检查细胞衰老情况。与年轻大鼠相比，衰老大鼠的肌酐水平显著升高，β-半乳糖苷酶染色证实近端曲管存在细胞衰老。所有检测变量均随衰老显著增加：1）p53、bax和半胱天冬酶3增加可能激活凋亡途径；2）近端曲管中rH2AX和8-OHdG免疫定位增加可能意味着DNA损伤加剧；3）远端曲管中APE1免疫反应性增加而近端曲管中减少可能导致APE1增加。这些结果表明，无论老年肾近端小管中的DNA修复活性如何，APE1对p53依赖性凋亡可能几乎没有保护作用。因此，在未来研究衰老肾脏中细胞凋亡与DNA修复之间的关系时，研究人员应使用超过24月龄的大鼠。
