Relaxin abrogates renal interstitial fibrosis by regulating macrophage polarization via inhibition of Toll-like receptor 4 signaling

Abstract

Renal fibrosis is a common feature of chronic kidney disease (CKD). To inhibit the CKD process, it is important to prevent renal fibrosis, though CKD remains incurable. Renal fibrosis can be inhibited by relaxin in several experimental models, but the mechanism of relaxin for antifibrotic potential is still not clear. And here we have studied the role of relaxin in macrophage polarization and renal inflammation after unilateral ureteral obstruction (UUO). Our results show that relaxin can downregulate the Toll-like receptor (TLR) 4 signaling, shift macrophage polarization toward the M2 phenotype and ameliorat renal fibrosis in the early stages of UUO. In vitro experiments, it has been confirmed that relaxin can downregulate the TLR4 signaling and induce the M2 macrophage transition. Furthermore, the transitional actions of macrophage phenotype induced by relaxin are significantly blocked by TAK-242, a TLR4 antagonist, in vitro experiments. Thus, there is a novel mechanism of relaxin for antifibrosis that shifts macrophage polarization toward the M2 phenotype via inhibition of TLR4 signaling.

Keywords: Toll-like receptor 4; macrophage polarization; relaxin; renal fibrosis.

Figure 1. Relaxin alleviates fibrosis following UUO

(A–B) Representative images (eight visual fields for each tissue analyzed) of H&E (A), Sirus red (B) staining of kindrys frong the indicated experimental groups. Scale bars, 50 μm. (C) Interstitial fibrosis on basis of Sirius red staining. n = 8 per group;*p < 0.05. (D, E) Whole kidney lysates from kidneys of the mice pretreated with deionized water or relaxin following UUO (sham, UUO, sham + relaxin and UUO + relaxin) were analyzed for changes in fibrosis (fibronectin) by western blot analysis. Expression of the indicated proteins in the kidneys was analyzed by densitometry normalized to glyceraldehyde 3-phosphate dehydrogenase (GADPH) and expressed as mean ± sd. n = 8 per group; *p < 0.05.

Figure 2. Relaxin shifts macrophage polarization toward the M2 phenotype in vivo following UUO

(A–J) Gene expression analysis of M1 markers, including tumor necrosis factor-α (TNF-α), interleukin (IL)-23, chemokine (C-C motif) ligand 3 (CCL3), inducible nitric oxide synthase (iNOS), and M2 markers, including arginase, CX3CR1, IL-4, mannose receptor (MRC), IL-10, Ym1 on macrophages isolated from kidneys of the mice pretreated with deionized water or relaxin following UUO. Experiments were performed at least three times and gene expression data were normalized to GADPH, analyzed, and represented as mean ± sd; n = 8 per group; *p < 0.05.

Figure 3. Relaxin shifts macrophage polarization toward the M2 phenotype in vitro

Raw 264.7 cells were treated with deionized water (M0), interferonγ(IFNγ) (M1) or IL-4(M2) and (A–C) analyzed for the expression of M1 marker iNOS and M2 marker arginase (Arg) after treated with vehicle (deionized water) or relaxin by western blot analysis. Protein expression data were normalized to GADPH, analyzed, and represented as mean ± sd; *p < 0.05 vs. vehicle treated groups(control groups). (D–F) M0, M1 and M2 macrophages were analyzed for the expression of M1 (TNF-α, IL-23, CCL3, iNOS) and M2 (arginase, CX3CR1, IL-4, MRC, IL-10, Ym-1) genes after treated with vehicle (deionized water) or relaxin by real-time PCR. Experiments were performed at least three independent times and gene expression data were normalized to GAPDH, analyzed, and represented as mean ± sd; *p < 0.05 vs. vehicle treated groups (control groups).

Figure 4. Relaxin downregulates the TLR4-NF-κB signaling pathway in vivo following UUO

(A, B) Whole kidney lysates from kidneys of the mice pretreated with deionized water or relaxin following UUO (sham, UUO, sham + relaxin and UUO + relaxin) were analyzed for changes in TLR4, Myd88, p65, p-p65 by western blot analysis. Expression of the indicated proteins in the kidneys was analyzed by densitometry normalized to GADPH and expressed as mean ± sd; n = 8 per group; *p < 0.05. (C–E) Macrophages isolated from kidneys of the mice pretreated with deionized water or relaxin following UUO were analyzed for the expression of TLR4, Myd88 and p65 genes. Experiments were performed at least three independent times and gene expression data were normalized to GAPDH, analyzed, and represented as mean ± sd; n = 8 per group;*p < 0.05.

Figure 5. Relaxin downregulates the TLR4-NF-κB signaling pathway in vitro

Raw 264.7 cells were treated with deionized water (M0), interferonγ (IFNγ) (M1) or IL-4(M2) and (A–L) analyzed for the expression of TLR4, Myd88, p65 after treated with vehicle (deionized water) or relaxin by western blot analysis. Protein expression data were normalized to GADPH, analyzed, and represented as mean ± sd; *p < 0.05 vs. vehicle treated groups (control groups). (M, N, O) M0, M1 and M2 macrophages were analyzed for the expression of TLR4, Myd88, p65, p-p65 genes after treated with vehicle (deionized water) or relaxin by real-time PCR. Experiments were performed at least three independent times and gene expression data were normalized to GAPDH, analyzed, and represented as mean ± sd; *p < 0.05 vs. vehicle treated groups (control groups).

Figure 6. The effects of relaxin on macrophages polarization regulation are blocked by the TLR4 antagonist TAK-242 in vitro

(A, B) Raw 264.7 cells were treated with IFNγ (M1) to induce the macrophage to M1 macrophages, the cells were pre-treated with TAK-242 or vehicle (deionized water) for 1 h and were continuously treated with vehicle (deionized water) or relaxin for over 72 h and analyzed for the expression of M1 marker iNOS (A) and M2 marker arginase (Arg) (B) by western blot analysis. Protein expression data were normalized to GADPH, analyzed, and represented as mean ± sd; *p < 0.05. (C–E) M0, M1 and M2 macrophages were analyzed for the expression of M1 (TNF-α, IL-23, CCL3, iNOS) and M2 (arginase, CX3CR1, IL-4, MRC, IL-10, Ym-1) genes after treated with relaxin and TAK-242 or TAK-242 only by real-time PCR. Experiments were performed at least three independent times and gene expression data were normalized to GAPDH, analyzed, and represented as mean ± sd; *p < 0.05 vs. treated with TAK-242 only.