LncRNA IRAR regulates chemokines production in tubular epithelial cells thus promoting kidney ischemia-reperfusion injury

Abstract

Increasing evidence demonstrates that long noncoding RNAs (lncRNAs) play an important role in several pathogenic processes of the kidney. However, functions of lncRNAs in ischemic acute kidney injury (AKI) remain undefined. In this study, global lncRNA profiling indicated that many lncRNA transcripts were deregulated in kidney after ischemia reperfusion (IR). Among them, we identified IRAR (ischemia-reperfusion injury associated RNA) as a potential lncRNA candidate, which was mostly expressed by the tubular epithelial cells (TECs) after IR, involved in the development of AKI. GapmeR-mediated silencing and viral-based overexpression of IRAR were carried out to assess its function and contribution to IR-induced AKI. The results revealed that in vivo silencing of IRAR significantly reduced IR-induced proinflammatory cells infiltration and AKI. IRAR overexpression induced chemokine CCL2, CXCL1 and CXCL2 expression both in mRNA and protein levels in TECs, while, silencing of IRAR resulted in downregulation of these chemokines. RNA immunoprecipitation and RNA pulldown assay validated the association between IRAR and CCL2, CXCL1/2. Further examination revealed that specific ablation of CCL2 in TECs reduced macrophages infiltration and proinflammatory cytokine production, attenuated renal dysfunction in IR mice. Inhibition of CXC chemokine receptor 2 (receptor of CXCL1/2) reduced neutrofils infiltration, but had no overt effect on kidney function. To explore the mechanism of IRAR upregulation in kidney during IR, we analyzed promoter region of IRAR and predicted a potential binding site for transcription factor C/EBP β on IRAR promoter. Silencing of C/EBP β reduced IRAR expression in TECs. A dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) confirmed that IRAR was a transcriptional target of the C/EBP β. Altogether, our findings identify IRAR as a new player in the development of ischemic AKI through regulating chemokine production and immune cells infiltration, suggesting that IRAR is a potential target for prevention and/or attenuation of AKI.

Fig. 1. IRAR is induced by ischemia reperfusion (IR) in mice.

A Screening strategy of IR-induced murine lncRNAs derived from microarray profiling. B A volcano plot showed the differentially expressed lncRNAs in IR group compared to Sham group. Red and green dots represent upregulated and downregulated lncRNAs in kidneys 24 h after IR, respectively (fold-change ≥ 2.0 and p-value ≤ 0.05). Black dots represent the genes that were not differentially expressed. IRAR is highlighted in red. C Time course of IRAR expression. Data represent mean ± SEM. n = 6-8, **p < 0.01 versus Sham group. D RNA fluorescence in situ hybridization (FISH) assay of IRAR in the mouse kidney samples at 24 h after IR. Scale bar, 100 μm. E FISH assay of IRAR in the hypoxia-treated mTECs. Scale bar, 25 μm. F The expression level of IRAR in cytoplasm and nuclei of hypoxia-treated mTECs. GAPDH (cytoplasm retained) and U1 (nuclear retained) were used as controls. G 3′ and 5′ rapid amplification of cDNA ends (RACE). Agarose gel electrophoresis of PCR products from the 5′-RACE and 3′-RACE procedure.

Fig. 2. Ischemia-reperfusion (IR) induces production of chemokines and infiltration of immune cells.

A Hierarchical clustering analysis of the differentially expressed mRNAs in IR group compared to Sham group (green, low; red, high). Three significantly upregulated mRNAs: CXCL1, CXCL2, and CCL2 (fold-change > 10) were highlighted in red box. B KEGG pathway enrichment analysis for upregulated mRNAs. C–E Time course of chemokine CXCL1 (C), CXCL2 (D), and CCL2 (E) expression. Data represent mean ± SEM. n = 6–8. **p < 0.01 versus Sham group. F Immunofluorescence staining of Gr-1 and F4/80 in the kidney 6 h and 24 h after renal IR. Gr-1 was used as a marker of neutrophils, and F4/80 was used as a marker of macrophages. Scale bar, 100 μm. G Quantification of Gr-1-positive neutrophils and F4/80-positive macrophages in the kidneys. Data represent mean ± SEM. n = 6. **p < 0.01.

Fig. 3. Inhibition of IRAR in vivo attenuates inflammation and acute kidney injury.

The GapmeR IRAR was injected into mice 1 h before IR operaton. GapmeR-control was a scrambled sequence. A Quantitative RT-PCR analysis of IRAR. B Serum creatinine 24 h after renal IR. C, D ELISA of IL-6 in circulation and in the kidney 6 h after renal IR. E Hematoxylin–eosin staining for kidneys. Original magnification, ×200. Arrow indicates renal tubular epithelial cell necrosis. Scale bar, 100 μm. F Tubular damage score in renal cortical tissues. G Serum creatinine 72 h after cisplatin injection. GapmeR IRAR or Negative control was injected into mice via tail vein 1 h before cisplatin injection. H qRT-PCR analysis of IL-6 in kidney. Data represent mean ± SEM. n = 6–8. **p < 0.01.

Fig. 4. Inhibition of IRAR reduces ischemia-reperfusion (IR)-induced production of chemokines and infiltration of immune cells.

A–C ELISA of CXCL1 (A), CXCL2 (B), CCL2 (C) in the kidney 24 h after renal IR (n = 6–8). D Immunofluorescence staining of Gr-1 and F4/80 in the kidney 6 h after renal IR. Gr-1 was used as a marker of neutrophils and F4/80 was used as a marker of macrophages. Scale bar, 100 μm (n = 6–8). E Quantification of Gr-1-positive neutrophils and F4/80-positive macrophages in the kidneys. Data represent mean ± SEM. n = 4. *p < 0.05, **p < 0.01.

Fig. 5. IRAR regulates chemokine expression.

A Co -expression network of IRAR and differentially expressed mRNAs. The solid line indicates positive regulation and the dotted line indicates negative regulation. B qRT-PCR analysis of IRAR in mTECs transfected with IRAR overexpressing lentivirus (LncRNA-OE) or Negative lentivirus (LncRNA-NC). C–E qRT-PCR analysis of CXCL1 (C), CXCL2 (D), CCL2 (E) in mTECs transfected with IRAR overexpressing lentivirus or Negative lentivirus. F Western blot analysis of CXCL1, CXCL2 and CCL2 in mTECs transfected with IRAR overexpressing lentivirus or Negative lentivirus.. Data are means from three independent experiments. G–J qRT-PCR analysis of IRAR (G), CXCL1 (H), CXCL2 (I) and CCL2 (J) in mTECs. mTECs were transfected with GapmeR IRAR (GapmeR-Hypoxia) or Negative control (Control-Hypoxia) for 48 h, then treated with hypoxia. K RNA fluorescence in situ hybridization (FISH) assays indicated co-expression between IRAR and CXCL1, CXCL2, CCL2 in the kidney at 24 h after ischemia reperfusion. Scale bar, 100 μm. L RNA immunoprecipitation (RIP) experiments were performed using antibodies against CXCL1, CXCL2 and CCL2. RIP enrichment was determined relative to the input controls. M RNA pulldown assays were performed in tubular epithelia cells to examine the association of IRAR with CXCL1, CXCL2 and CCL2. Data represent mean ± SEM. n = 4. **p < 0.01.

Fig. 6. Inhibition of CXC chemokine receptor 2 (CXCR2) attenuates ischemia-reperfusion (IR)-induced inflammation.

The mice received intraperitoneal administrations of antagonist of CXCR2 (CXCR2(-)-IR) or vehicle (NC-IR) 1 h prior and 1 h after renal IR. A Immunofluorescence staining of Gr-1 in the kidney 6 h after renal IR. Scale bar, 100 μm. B Quantification of Gr-1-positive neutrophils. C Concentration of serum creatinine 24 h after renal IR. D ELISA of IL-6 in the kidney 6 h after renal IR. Data represent mean ± SEM. n = 6–8. **p < 0.01.

Fig. 7. Tubule-specific ablation of CCL2 reduces macrophages infiltration and kidndy injury.

A Breeding protocol for generating conditional tubular epithelial cell knockout mice (TE-CCL2-KO). B Representative gel images of genotyping. DNA was extracted from mouse tail and amplified, wild-type and floxed alleles of CCL2 and Cdh16-cre allele were detected. Lane 1 showed the genotyping of the wild-type mice used in this study (CCL2^fl/fl), lane 2 showed the genotyping of the tubule-specific CCL2 knockout mice (CCL2^fl/flCre, designated as TE-CCL2-KO). C Western blot analysis indicated a substantial reduction of renal CCL2 expression in TE-CCL2-KO mice 24 h after renal IR. D Representative images showed CCL2 staining in renal cortical and medullar regions of the WT and TE-CCL2-KO mice at 24 h after IR. Scale bar, 50 or 100 μm. E Immunofluorescence staining of F4/80 in the kidney 6 h after renal IR. Scale bar, 100 μm. F Quantification of F4/80-positive cells. G Concentration of serum creatinine 24 h after renal IR. H ELISA of IL-6 in the kidney 6 h after renal IR. Data represent mean ± SEM. n = 6–8. *p < 0.05, **p < 0.01.

Fig. 8. IRAR is regulated by the transcription factor C/EBP β during renal ischemia reperfusion.

A Western blot analysis of C/EBP β during renal ischemia reperfusion. B qRT-PCR analysis of C/EBP β. mTECs were transfected with C/EBP β siRNA (Hypoxia-siRNA) or negative control oligonucleotides (Hypoxia-Neg), and then subjected to hypoxia. C qRT-PCR analysis of IRAR in hypoxia-treated mTECs transfected with C/EBP β siRNA or negative control. D Luciferase reporter assays were performed to determine the C/EBP β binding on the IRAR promoter region. Prediction and mutation of C/EBP β-binding site in the IRAR promoter region (left). Luciferase activity was shown as relative luciferase activity normalized to Renilla activity (right). E ChIP assays were performed to detect C/EBP β occupancy in the IRAR promoter region. IgG was as negative control. F Schematic representation of lncRNA-IRAR-mediated renal ischemia-reperfusion injury. Data represent mean ± SEM. n = 4. *p < 0.05, **p < 0.01.