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. 2021 Jul;24(1):495.
doi: 10.3892/mmr.2021.12134. Epub 2021 May 6.

Rapamycin protects against aristolochic acid nephropathy in mice by potentiating mammalian target of rapamycin‑mediated autophagy

Fan Lin #  1 , Yunqi Liu #  1 , Lili Tang  2 , Xiaohui Xu  3 , Xueli Zhang  3 , Yifan Song  3 , Bicheng Chen  4 , Yeping Ren  3 , Xiangdong Yang  1
Affiliations

Rapamycin protects against aristolochic acid nephropathy in mice by potentiating mammalian target of rapamycin‑mediated autophagy

Fan Lin et al. Mol Med Rep. 2021 Jul.

Abstract

Autophagy serves a crucial role in the etiology of kidney diseases, including drug‑induced renal impairment, inherited kidney disease, diabetic nephropathy and aristolochic acid nephropathy (AAN) and is, therefore, a potential target for treatment. We previously demonstrated that rapamycin could attenuate AAN in mice; however, the underlying mechanism remains to be elucidated. Therefore, whether the renal protective effect of rapamycin (an autophagy activator) is related to autophagy in aristolochic acid (AA)‑treated mice was of particular interest. The pathophysiological roles of rapamycin were investigated in AA‑induced nephrotoxicity in mice and the mechanisms of rapamycin action were explored by evaluating the modulation of autophagy in rapamycin‑treated mice and cultured renal tubular epithelial cells. Supplementation with rapamycin reversed AA‑induced kidney injury in mice and improved AA‑induced autophagy damage in vivo and in vitro. Mechanistically, rapamycin inhibited the renal expression of phosphorylated (p‑)mammalian target of rapamycin (mTOR) and p‑ribosomal S6 protein kinase 1, which in turn activated renal autophagy and decreased apoptosis, probably by removing AA‑elicited damaged mitochondria and misfolded proteins. The findings of the present study demonstrated that rapamycin protects against AA‑induced nephropathy by activating the mTOR‑autophagy axis and suggested that rapamycin may be a promising pharmacological target for the treatment of AAN.

Keywords: apoptosis; aristolochic acid nephropathy; autophagy; mTOR; rapamycin.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Rapamycin improves AA-induced renal dysfunction and histopathological abnormalities in mice. (A) Levels of blood urea nitrogen and (B) serum creatinine in each group detected using ELISA (n=6/group). (C) Representative microscopic images showing H&E and PAS staining in the kidney tissues of mice in all groups (scale bar=50 µm; n=6/group). Results are presented as the mean ± standard deviation. *P<0.05; **P<0.01. All experiments were performed three times. AA, aristolochic acid; H&E, hematoxylin and eosin staining; PAS, Periodic acid-Schiff staining; Ctrl, control group; AAI, AA-induced group; RAP, rapamycin treatment group.
Figure 2.
Figure 2.
Rapamycin promotes AA-induced renal autophagy and suppresses apoptosis in the kidney tissues of mice by inhibiting mTOR activity. (A) Representative western blots showing the protein expression levels of p-mTOR, mTOR p62, Beclin1, LC3 and Bcl-2 in kidney tissues; GAPDH was used as a loading and normalization control. (B) Representative western blots showing the protein expression levels of p-S6K1 and S6K1 in kidney tissues; GAPDH was used as a loading and normalization control. Renal expression of (C) p-mTOR/mTOR, (D) p62, (E) Beclin1, (F) LC3, (G) Bcl-2, (H) Bax and (I) p-S6K1/S6K1 were quantified using ImageJ. Results are presented as the mean ± standard deviation. **P<0.01. All experiments were performed three times; n=10/group. AA, aristolochic acid; p-, phosphorylated; LC3, light chain 3; S6K1, ribosomal S6 protein kinase 1; Ctrl, control group; AAI, AA-induced group; RAP, rapamycin treatment group.
Figure 3.
Figure 3.
Rapamycin inhibits p-mTOR expression and promotes AA-induced renal autophagy in HK-2 cells. (A) Representative images showing the protein expression of p-mTOR, mTOR, p62, Beclin1 and LC3 in HK-2 cells determined using western blotting. GAPDH was used as an internal control. Relative protein expression of (B) p-mTOR/mTOR, (C) p62, (D) Beclin1 and (E) LC3 were quantified using ImageJ. (F) Representative images showing the protein expression of p-S6K1 and S6K1 in HK-2 cells determined using western blotting. GAPDH was used as an internal control. (G) Relative protein expression of p-S6K1/S6K1 was quantified using ImageJ. (H) Detection of autophagosomes by LC3B immunofluorescence staining in HK-2 cells; small green dots indicate autophagosome formation (scale bar=25 µm). (I) Mean area fraction of LC3B in each group was quantified using Image-Pro plus 6.0. Results are presented as the mean ± standard deviation. *P<0.05; **P<0.01. All experiments were performed three times. p-, phosphorylated; LC3, light chain 3; AA, aristolochic acid; S6K1, ribosomal S6 protein kinase 1; Ctrl, control group; AAI, AA-induced group; RAP, rapamycin treatment group.
Figure 4.
Figure 4.
Rapamycin reduces AA-induced apoptosis in HK-2 cells. (A) Flow cytometry results showing apoptosis in HK-2 cells; Q2 for early apoptotic cell death and Q3 for late apoptotic cell death were used to calculate the rates of apoptosis. (B) Cell apoptosis rate (%) was analyzed using FlowJo software. (C) Representative western blots showing the protein expression of Bcl-2 and Bax in HK-2 cells in each group; GAPDH was used as a loading and normalization control. Protein expression of (D) Bax and (E) Bcl-2 in HK-2 cells was quantified using ImageJ. Results are presented as the mean ± standard deviation. *P<0.05; **P<0.01. All experiments were performed three times. AA, aristolochic acid; Ctrl, control group; AAI, AA-induced group; RAP, rapamycin treatment group.
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Grants and funding

The present study was supported by grants from the National Natural Science Foundation of China of Xiangdong Yang (grant no. 81670660); the Shandong Important Research Plans Fund of Xiangdong Yang (grant no. GG201809250293); the Natural Science Foundation of Shenzhen University General Hospital of Fan Lin (grant no. SUGH2018QD071); and the Natural Science Foundation of Shenzhen University General Hospital of Yeping Ren (grant no. SUGH2020QD011).