Department of Nephrology, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
Department of Internal Medicine, Jackson Memorial Hospital, 1611 Northwest 12 Avenue, Miami, FL, 33139. USA.
Biomed Pharmacother. 2019 Jul;115:108917. doi: 10.1016/j.biopha.2019.108917. Epub 2019 May 3.
Aristolochic acid nephropathy (AAN) is a chronic, progressive interstitial nephritis. To date, treatment strategies remain limited. Mounting evidence shows that relaxin (RLX) possesses powerful anti-fibrotic and anti-apoptotic characteristics, therefore, the present study aimed to investigate the possible protective role of RLX in aristolochic acid (AA) induced nephrotoxicity.
The in vitro cell tests were performed: the embryonic kidney cells 293 were treated with AA-I (40 μg/mL) or with AA-I (40 μg/mL) plus RLX (100 ng/mL) and the cell groups were then tested and the normal 293 cells were set as blank control. In addition, the in vivo animal tests were performed: mice were randomly divided into three groups: a control group injected intraperitoneally with an equal volume of saline every other day for 6 weeks, an AA group injected intraperitoneally with AA-I (5 mg/kg) every other day for 6 weeks, and an AA + RLX group treated with the AA-I for 6 weeks and subsequently received RLX (0.25 mg/kg/day) using an implanted osmotic mini-pump for an additional 2 weeks. 8 weeks post-AA-I, mice were sacrificed for analysis.
In the in vivo animal tests, RLX administration prevented increased plasma creatinine and nitrogen levels caused by aristolochic acid as well as alleviated the severity of renal ultrastructural lesions induced by aristolochic acid. Both in the in vitro cell tests and in the in vivo animal tests, Western blotting of the AA-I group showed increased expression of the pro-apoptotic protein genes Bax and the cleaved form of caspase-3, both of which were reversed by RLX. In contrast, the expression of the anti-apoptotic gene Bcl-2 correlated inversely to Bax in RLX treated mice. RLX restored the decreased phosphorylated Akt induced by AA-I. The protein expression of eNOS was also reduced in AA-I treated group compared with control, which was reversed in the presence of RLX. Immunohistochemical staining of the animal tissue revealed that RLX markedly reduced the overexpression of type IV collagen, fibronectin, and alpha-smooth muscle actin in AA-I treated mice.
Our results suggest that RLX alleviates AA-I induced kidney fibrosis by reducing apoptosis and up-regulation the expression of p-Akt.
马兜铃酸肾病（AAN）是一种慢性进行性间质肾炎。迄今为止，治疗策略仍然有限。越来越多的证据表明松弛素（RLX）具有强大的抗纤维化和抗细胞凋亡特性，因此，本研究旨在探讨 RLX 在马兜铃酸（AA）诱导的肾毒性中的可能保护作用。
进行体外细胞试验：用 AA-I（40μg/mL）或 AA-I（40μg/mL）加 RLX（100ng/mL）处理胚胎肾细胞 293，然后对各组细胞进行检测，将正常 293 细胞设为空白对照。此外，还进行了体内动物试验：将小鼠随机分为三组：对照组每隔一天腹腔注射等体积生理盐水 6 周，AA 组每隔一天腹腔注射 AA-I（5mg/kg）6 周，AA+RLX 组用植入的渗透微型泵给予 RLX（0.25mg/kg/天）治疗 2 周。AA-I 注射 8 周后处死小鼠进行分析。
在体内动物试验中，RLX 给药可防止马兜铃酸引起的血浆肌酐和氮水平升高，并减轻马兜铃酸引起的肾脏超微结构损伤的严重程度。无论是在体外细胞试验还是在体内动物试验中，AA-I 组的 Western blot 均显示促凋亡蛋白基因 Bax 和 caspase-3 的裂解形式表达增加，RLX 可逆转这两种表达。相反，在 RLX 处理的小鼠中，抗凋亡基因 Bcl-2 的表达与 Bax 呈负相关。RLX 恢复了 AA-I 诱导的磷酸化 Akt 的减少。与对照组相比，AA-I 处理组的 eNOS 蛋白表达降低，而 RLX 存在时则恢复。动物组织的免疫组织化学染色显示，RLX 可显著减少 AA-I 处理小鼠中 IV 型胶原、纤维连接蛋白和α-平滑肌肌动蛋白的过度表达。
我们的结果表明，RLX 通过减少细胞凋亡和上调 p-Akt 的表达来减轻 AA-I 诱导的肾脏纤维化。
