Targeting CC chemokine ligand (CCL) 20 by miR-143-5p alleviate lead poisoning-induced renal fibrosis by regulating interstitial fibroblasts excessive proliferation and dysfunction

Abstract

Environmental lead contamination can cause chronic renal disease with a common clinical manifestation of renal fibrosis and constitutes a major global public health threat. Aberrant proliferation and extracellular matrix (ECM) accumulation in renal interstitial fibroblasts are key pathological causes of renal fibrosis. However, the mechanism underlying lead-induced kidney fibrosis remains unclear. The present study analyzed gene expression prolifes in lead acetate-treated primary mice renal interstitial fibroblasts and confirmed the aberrant expression of CC chemokine ligand (CCL) 20, one of the most obvious up-regulated genes. Analogously, lead acetate exposure dose-dependently increased CCL20 transcription, protein expression and release. Knockdown of CCL20 suppressed lead acetate-induced fibroblast proliferation, hydroxyproline contents, transforming growth factor-beta production and ECM-related protein (Collagen I and fibronectin) expression. Bioinformatics analysis predicted five top miRNAs targeting CCL20. Among them, miR-143-5p expression was dose-dependently decreased in lead acetate-treated fibroblasts. Mechanistically, miR-143-5p directly targeted CCL20. Elevation of miR-143-5p antagonized lead acetate-induced fibroblast proliferation, hydroxyproline and ECM-related protein expression, which were reversed by CCL20 overexpression. Additionally, CCL20 knockdown suppressed lead acetate-mediated Smad2/3 and AKT pathway activation. Notably, miR-143-5p overexpression attenuated the activation of the Smad2/3 and AKT pathway in lead acetate-exposed fibroblasts, which was counteracted by CCL20 elevation. miR-143-5p injection ameliorated renal fibrosis progression in mice in vivo. Thus, targeting CCL20 by miR-143-5p could alleviate renal fibrosis progression by regulating fibroblast proliferation and ECM deposition via the Smad2/3 and AKT signaling, providing a potential therapeutic target for environmental lead contamination-evoked fibrotic kidney disease.

Keywords: CCL20; Renal fibrosis; lead contamination; miR-143; renal interstitial fibroblast.

Graphical abstract

Figure 1.

Aberrant expression of CCL20 is observed in lead acetate-stimulated mice primary renal interstitial fibroblasts. (a) Staining of renal interstitial fibroblasts with CK19 and Vimentin. (b) Heat map of gene expression in lead acetate-treated fibroblasts. The red shades represent high expression, and blue shades indicate low expression. (c) Volcano plots exhibited gene expression profiles. (d, e) Isolated renal fibroblasts were stimulated with the indicated doses of lead acetate for 24 h. Then, the mRNA (d) and protein expression (e) levels were analyzed by qRT-PCR and western blotting. (f) ELISA assay was performed to quantify the contents of CCL20 in supernatants from lead acetate-treated renal fibroblasts. *P < 0.05 vs. control groups.

Figure 2.

CCL20 knockdown inhibits lead acetate-evoked renal interstitial fibroblast proliferation and dysfunction. (a, b) Renal interstitial fibroblasts were transfected with si-CCL20-1, si-CCL20-2 and si-CCL20-3 for 48 h. Then, the mRNA levels of CCL20 (a) and protein expression (b) were detected by qRT-PCR and western. (c) Cells were transfected with si-CCL20-1, si-CCL20-2, and prior to lead acetate exposure. The contents of CCL20 in supernatants were analyzed by ELISA kits. (d) Cell proliferation was evaluated in lead acetate- and/or si-CCL20 transfected fibroblasts. (e-h) After si-CCL20 transfection, cells were exposed to lead acetate. Then, the effects on hydroxyproline contents (e), fibrosis-associated gene transcripts (f) and protein expression (g, h) were subsequently determined. *P < 0.05 vs. control group. ^#P < 0.05 vs. LA-treated group.

Figure 3.

Identification of miR-143-5p that directly targets CCL20. (a) Prediction of miRNAs targeting CCL20 by three bioinformatics tools (TargetScan, miRWalk and miRanda). The five top potential miRNAs were shown in red box. (b) qRT-PCR was used to determine the expression of five miRNAs in fibroblasts under the indicated doses of lead acetates (LA). (c) The predicted binding site of miR-143-5p in the 3’-UTR of CCL20. (d) Dual luciferase reporter assay system was used to evaluate the targeted correlation between CCL20 and miR-143-5p. (e) Cells were transfected with miR-143-5p mimics or miR-NC. Then, the expression of miR-143-5p was analyzed by qRT-PCR. (f) The mRNA levels of CCL20 were determined in fibroblasts treated with lead acetate and miR-143-5p transfections. *P < 0.05 vs. control group. ^#P < 0.05 vs. LA-treated group.

Figure 4.

Elevation of miR-143-5p reverses lead acetate-induced proliferation and dysfunction of renal fibroblasts by targeting CCL20. Fibroblasts were transfected with miR-143-5p mimics or recombinant CCL20 plasmids, prior to lead acetate exposure. Then, the transcripts (a) and production of CCL20 (b) were analyzed by qRT-PCR and ELISA assay. The subsequent effects on cell proliferation (c), hydroxyproline levels (d), ECM-related gene expression (e-g) and TGF-β1 (h) were detected. *P < 0.05 vs. control group. ^#P < 0.05 vs. LA-treated group. ^&P < 0.05 vs. LA+miR-143-5p-treated group.

Figure 5.

miR-143-5p regulates lead acetate-activated the pathways of the Smad2/3 and AKT by targeting CCL20. (a, b) Cells were treated with si-CCL20 transfection and lead acetate exposure. Then, the expression of p-Smad2/3, Smad2/3, p-AKT and AKT were determined by western blotting. The corresponding quantify of protein bands was performed by Image J software. (c, d) The activation of the Smad2/3 and AKT signaling was analyzed in lead acetate-stimulated cells that were pre-transfected with miR-143-5p mimics or CCL20 plasmids. *P < 0.05 vs. control group. ^#P < 0.05 vs. LA-treated group. ^&P < 0.05 vs. LA+miR-143-5p-treated group.

Figure 6.

Injection with miR-143-5p alleviates the development of renal fibrosis in lead acetate-treated mice. (a) Mice under lead acetate injected were underwent miR-143-5p overexpression. Then, the expression of miR-143-5p was analyzed. (b, c) Kidney fibrosis was evaluated by H&E (b) and Masson detection (c) Scale bar = 50 μm. (d) The contents of hydroxyproline were determined in plasma from mice. (e, f) Western assay was then performed to detect the protein expression of CCL20, TGF-β1, CCR6, Collagen I and FN. (g) qRT-PCR was performed to determine the mRNA levels of fibrosis-related gene expression. (h) ELISA assay was used to measure plasma levels of CCL20 and TGF-β1. (i, j) Effects on the activation of the Smad2/3 and AKT pathways were analyzed. *P < 0.05 vs. control group. ^#P < 0.05 vs. LA-treated group.