MK-2206 Alleviates Renal Fibrosis by Suppressing the Akt/mTOR Signaling Pathway In Vivo and In Vitro

Abstract

Renal fibrosis is a common pathological feature of various kidney diseases, leading to irreversible renal failure and end-stage renal disease. However, there are still no effective treatments to reverse renal fibrosis. This study aimed to explore the potential mechanism of a targeted drug for fibrosis. Here, unilateral ureteral obstruction (UUO)-treated mice and a TGF-β1-treated human renal tubular epithelial cell line (HK-2 cells) were used as models of renal fibrosis. Based on the changes of mRNA in UUO kidneys detected by transcriptome sequencing, MK-2206, an Akt inhibitor, was predicted as a potential drug to alleviate renal fibrosis through bioinformatics. We dissolved UUO mice with MK-2206 by gastric gavage and cultured TGF-β-induced HK-2 cells with MK-2206. Histopathological examinations were performed after MK-2206 intervention, and the degree of renal fibrosis, as well as the expression of Akt/mTOR pathway-related proteins, were evaluated by immunohistochemical staining, immunofluorescence staining, and Western blot. The results showed that MK-2206 significantly improved the pathological structure of the kidney. Furthermore, MK-2206 intervention effectively inhibited UUO- and TGF-β1-induced epithelial-mesenchymal transition, fibroblast activation, and extracellular matrix deposition. Mechanistically, MK-2206 treatment attenuated the activation of the Akt/mTOR signaling pathway. Taken together, our study revealed for the first time that MK-2206 is a promising drug for the improvement of renal fibrosis.

Keywords: Akt/mTOR signaling pathway; MK-2206; chronic kidney diseases; renal fibrosis.

Figure 1

Renal fibrosis models established by UUO. (A) Histomorphology (including H&E and Masson staining) of the kidneys in both groups (scale bar: 100 μm). (B–G) Analysis of E-cadherin, Vimentin, α-SMA, Collagen I, and Fibronectin relative protein levels by Western blot in both groups. (H,I) Levels of biochemical parameters (Scr and BUN) in both groups. Protein levels were normalized with GADPH. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Transcriptome sequencing results analyzed by bioinformatics. (A,B) Visualization of differentially expressed genes between sham and UUO groups using volcano and heat maps. (C,D) Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes. (E) Construction of protein-protein interaction (PPI) networks to identify and visualize key differentially expressed genes. (F) Top 150 selected key differential genes. (G–L) Exploration of potential pathways using Gene Set Enrichment Analysis (GSEA). (M) Prediction of potential anti-fibrotic drugs by the connectivity map (CMap). (N) Chemical structure of MK-2206.

Figure 3

MK-2206 attenuated renal injury and reduced inflammatory factors. (A) H&E staining and Masson staining were performed to examine the extent of renal injury and collagen fiber deposition in each group of mice, respectively (scale bar: 100 μm). (B–D) mRNA levels of inflammatory factors TGF-β1, IL-1β, and IL-6 were detected by real-time PCR in each group of mice. (E,F) mRNA levels of Collagen I and Fibronectin in HK-2 cells treated by different concentrations of MK-2206 were detected by real-time PCR. The levels of mRNA expression were normalized with GADPH. ** p < 0.01, *** p < 0.001. MU: UUO + MK-2206.

Figure 4

MK-2206 inhibited the EMT process in UUO mice and TGF-β1-induced HK-2 cells. (A,B) Immunofluorescence staining of E-cadherin in renal tissue and HK-2 cells (scale bar: 50 μm). (C,D) Western blot analysis of E-cadherin and transcription factors (Snail and Twist). Protein levels were normalized with GADPH. * p < 0.05, ** p < 0.01, *** p < 0.001. MU: UUO + MK-2206, MT: TGF-β1 + MK-2206.

Figure 5

MK-2206 reduced myofibroblast formation and the deposition of extracellular matrix in UUO mice and TGF-β1-induced HK-2 cells. (A–C) Immunofluorescence staining and Western blot analysis of Vimentin, α-SMA in kidney tissues (scale bar: 100 μm). (D–F) Immunofluorescence staining and Western blot analysis of Vimentin, α-SMA in HK-2 cells (scale bar: 100 μm). (G) Western blot was used to detect the protein expression levels of Collagen I and Fibronectin in the kidney of each group of mice. (H) Protein expression levels of Collagen I and Fibronectin in HK-2 cells were detected by Western blot. Protein levels were normalized with GADPH. * p < 0.05, ** p < 0.01, *** p < 0.001. MU: UUO+ MK-2206, MT: TGF-β1 + MK-2206.

Figure 6

MK-2206 inhibited the activation of the Akt/mTOR signaling pathway in vivo and in vitro. (A,B) Expression levels of p-Akt and p-mTOR in the kidneys of each group of mice were detected by immunofluorescence staining (scale bar: 50 μm). (C,H) Protein expression levels of p-Akt and p-mTOR in vivo were detected by Western blot. (D,E) Expression levels of p-Akt and p-mTOR in HK-2 cells were detected by immunofluorescence staining (scale bar: 50 μm). (F,I) Western blot was used to detect the protein expression levels of p-Akt and p-mTOR in vitro. (G) Immunohistochemical staining of the kidneys in each group of mice using p-Akt and p-mTOR (scale bar: 30 μm). Protein levels were normalized with GADPH. * p < 0.05, ** p < 0.01. MU: UUO + MK-2206, MT: TGF-β1 + MK-2206.