Protective role of relaxin in a mouse model of aristolochic acid nephropathy.


Department of Nephrology, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China. Electronic address: [Email]


BACKGROUND : 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.
METHODS : 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.
RESULTS : 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.
CONCLUSIONS : Our results suggest that RLX alleviates AA-I induced kidney fibrosis by reducing apoptosis and up-regulation the expression of p-Akt.


Apoptosis,Aristolochic acid nephropathy,Relaxin,Renal fibrosis,