Anlotinib Alleviates Renal Fibrosis via Inhibition of the ERK and AKT Signaling Pathways

Figures

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  Figure 1. Anlotinib improved albuminuria and serum creatinine in an FA-induced mouse model. FA-induced mouse models of nephropathy were treated with or without anlotinib in this study. (a) Intervention dosing regimen of folic acid in an experimental mouse model of kidney fibrosis. (b) 24-hour urinary albumin excretion and serum creatinine were measured at 14 days after folic acid injection, and both were significantly increased and later reduced after the mice were treated with anlotinib in FA-induced mice compared to the control group. (c) Established UUO-induced mouse models of kidney fibrosis. (d) UUO-induced mouse models of nephropathy showed no significance when treated with or without anlotinib in 24-hour urinary albumin excretion and serum creatinine. Results are presented as mean ± SEM. ∗∗P < 0.01, n.s indicates not significant (P > 0.05), n = 3.

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  Figure 2. Anlotinib reduced the accumulation of extracellular matrix components and blocked EMT in UUO mice. (a) Quantitative RT-PCR was performed to determine the RNA expression of α-SMA, TGF-β1, collagen І, and collagen ІV in the kidney tissue of UUO mice. (b) Western blotting was performed to determine the protein expression of α-SMA, collagen І, and collagen ІV in the kidney tissue of UUO mice. (c) The representative photographs of the effects of anlotinib against OUU-induced pathological changes (HE and Masson stain) at day 14; interstitial fibrosis was evident in mouse kidney from the UUO group, compared with the sham group. The pathological changes were obviously relieved in the kidney of the anlotinib-treated group. This was immunohistochemical staining of α-SMA in the renal cortex from the experimental mice. (Bars, 100 μm. All data are represented as mean ± SD. ∗P < 0.05 and ∗∗P < 0.01, n = 3. Scale bars, 100 μm.).

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  Figure 3. Anlotinib attenuated inflammatory responses in a UUO mouse model. (a) The mRNA expression of CCL2, CCL5, and IL-6 in the mouse kidneys was examined by real-time PCR. Quantitative RT-PCR showed increased mRNA expression of CCL2, CCL5, and IL-6 in UUO mice compared to the sham group. These markers of inflammation were reduced in the mice treated with anlotinib. (b, c) Immunochemical staining of CD163 and F4/80 in the kidney tissue of OUU mice. Results are presented as mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01, n = 3.Scale bars: 100 μm.

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  Figure 4. Anlotinib inhibited TGF-β1-stimulated collagen and α-SMA expression in human proximal tubular cells. (a) Human proximal tubular cells pretreated with/without anlotinib for 4 hours were incubated with TGF-β1 for 48 hours. Real-time RT-PCR results showed that anlotinib inhibited TGF-β1-induced α-SMA, collagen I, and collagen IV mRNA expression. (b, c) Western blot results showed that anlotinib inhibited the induction of α-SMA, collagen I, and collagen IV in TGF-β1-treated human proximal tubular cells. Results are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, n = 3.

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  Figure 5. Anlotinib attenuated kidney fibrosis via the mediation of TGF-β1 signaling via ERK and AKT pathways. (a) Venn diagram showed the overlaps between anlotinib targets and renal fibrosis-related targets. (b) Protein-protein interaction (PPI) network of common targets between anlotinib and renal fibrosis. (c) The KEGG enrichment analysis of 70 targets of common targets. (d) The GO enrichment for each section is listed. (e, f) Western blot results showed that anlotinib inhibited TGF-β1-induced increases in p-AKT and p-ERK in human proximal tubular cells. Results are presented as mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01, n = 3.