The long noncoding RNA Meg3 mediates TLR4-induced inflammation in experimental obstructive nephropathy

Abstract

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked down-regulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared with wild-type control. Meg3 was also induced by lipopolysaccharide in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

Keywords: Meg3; TLR4 signaling; kidney fibrosis; large intervening non-coding RNA; tubular injury.

Figure 1. Generation of tubule-specific deletion of TLR4 in mice

(A) Schematic illustration of breeding strategy for generating Ksp-TLR4^f/f mice. (B) TLR4 mRNA expression determined by real-time qPCR in TLR4^f/f sham mice, TLR4^f/f UUO mice, and Ksp-TLR4^f/f UUO mice. (C) TLR4 protein expression determined by Western blot analysis. ***P<0.001 and **P<0.01 vs. TLR4^f/f sham mice; ^##P<0.01 vs. TLR4^f/f UUO mice (n=7 in each group).

Figure 2. Deletion of tubular TLR4 ameliorates kidney fibrosis

(A) Representative PAS staining of renal tissues in TLR4^f/f sham mice, TLR4^f/f UUO mice and Ksp-TLR4^f/f UUO mice and quantitative analysis. Bar = 100 µm. (B) Representative immunohistochemical staining of Col-3, α-SMA and Masson’s Trichrome staining in different groups and the corresponding quantitative analysis; bar = 100 µm. (C) Western blotting analyses shows renal expression of Fn and KIM-1 in different groups and the corresponding quantitative analysis. ***P<0.001 vs. TLR4^f/f sham mice; ^###P<0.001, ^##P<0.01 and ^#P<0.05 vs. TLR4^f/f UUO mice (n=7 in each group).

Figure 3. Deletion of tubular TLR4 reduces renal inflammation

(A) Quantitative real-time PCR shows relative mRNA and protein expression levels of CCL-2, CXCL-2, and TNF-α in renal tissues from TLR4^f/f sham mice, TLR4^f/f UUO mice and Ksp-TLR4^f/f UUO mice. (B) Representative immunohistochemical staining of F4/80 in different groups and the corresponding quantitative analysis. Bar = 100 µm (C) Western blotting analyses shows phosphorylation level of IκB and total IκB in different groups and the corresponding quantitative analysis. (D) Serum creatinine (Cr) and blood urea nitrogen (BUN) levels in different groups. ***P<0.001, **P<0.01, and *P<0.05 vs. TLR4^f/f sham mice; ^###P<0.001, ^##P<0.01, and ^#P<0.05 vs. TLR4^f/f UUO mice (n=7 in each group).

Figure 4. RNA sequencing reveals differentially expressed genes (DEGs) between TLR4^f/f UUO mice and Ksp-TLR4^f/f UUO mice

(A) The volcano plot of transcriptomic changes between renal tissues from TLR4^f/f UUO mice and Ksp-TLR4^f/f UUO mice. (B) Enrichment analysis of DEGs that were down-regulated and up-regulated in Ksp-TLR4^f/f UUO mice vs. TLR4^f/f UUO mice. (C) The volcano plot of lncRNA changes between renal tissues from TLR4^f/f UUO mice and Ksp-TLR4^f/f UUO mice. (D) Quantitative real-time PCR shows relative expression levels of H-19, Rian, and Meg3 in renal tissues in different groups and the corresponding quantitative analysis. (E) In situ hybridization of Meg3 and TLR4 expression in kidney tissue from different groups. Red color indicated positive signal for TLR4 and cyan color indicated positive signal for Meg3. ***P<0.001 and *P<0.05 vs. TLR4^f/f sham mice; ^##P<0.01 and ^#P<0.05 vs. TLR4^f/f UUO mice.

Figure 5. Meg3 mediates TLR4-driven inflammatory responses in tubular cells

Quantitative real-time PCR shows (A) the time- and dose-dependent expression of Meg3 in response to LPS in cultured tubular cells, and the effect of TLR4 inhibitor (CLI-095) on LPS-induced Meg3 expression. (B) The effect of p38 MAPK inhibitor SB203580 (SB), ERK MAPK inhibitor PD98059 (PD), and NF-κB inhibitor Bay11-7085 (Bay) on LPS-induced Meg3 expression. (C) The effect of p53 inhibitor (PFT- α) and p53 activator (RITA) on LPS-induced Meg3 expression. (D) Knockdown efficiency of Meg3 in LPS-treated cells. (E) ELISA measurement of CCL-2 and CXCL-2 from culture supernatant of LPS-treated groups. (F) Western blotting analyses show phosphorylation level of p38 MAPK and total p38 in different groups and the corresponding quantitative analysis. ***P<0.001, **P<0.01 and *P<0.05 vs. 0 h, ctl and siRNA (NC); ^###P<0.001, ^##P<0.01 and ^#P<0.05 vs. ctl/LPS and siRNA (NC)/LPS.

Figure 6. Kidney-specific knockdown of Meg3 in UUO mice

(A) Quantitative real-time PCR shows relative expression levels of Meg3 at day 3 and day 7 after UUO. (B) The experimental protocol of ultrasound microbubble-mediated shRNA-Meg3 gene knockdown in UUO mice. (C) The knockdown efficiency of Meg3 in renal tissues of UUO mice. ***P<0.001 and **P<0.01 vs. sham and TLR4^f/f sham mice; ^##P<0.01 vs. NC-shRNA UUO mice.

Figure 7. Knockdown of Meg3 by shRNA attenuates kidney fibrosis in UUO mice

(A) Representative PAS staining of renal tissues in NC-shRNA sham mice, NC-shRNA UUO mice and Meg3-shRNA UUO mice and quantitative analysis; bar = 100 µm. (B) Representative immunohistochemical staining of Col-3, α-SMA and Masson’s Trichrome staining in different groups and the corresponding quantitative analysis. (C) Representative immunohistochemical staining of F4/80 staining and the corresponding quantitative analysis; bar = 100 µm. ***P<0.001 and **P<0.01 vs. NC-shRNA sham; ^##P<0.01 and ^#P<0.05 vs. NC-shRNA UUO mice (n=7 in each group).