Identification of a microRNA signature in renal fibrosis: role of miR-21

Abstract

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-β1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-β1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.

Fig. 1.

microRNA (miRNA) expression is altered in kidneys with unilateral ureteral obstruction (UUO)-induced fibrosis. At 0, 3, and 7 days after UUO surgery, mice were killed and the obstructive kidneys (left kidneys) were harvested. Kidney RNA was isolated and miRNA array analysis was performed. Unsupervised hierarchical clustering is presented (n = 3/group).

Fig. 2.

Expression of specific miRNAs is altered in kidneys with UUO-induced fibrosis. A and B: mice experiments were performed as in Fig. 1. The levels of specific miRNAs were determined by real-time PCR assays. *P < 0.05, **P < 0.01, ***P < 0.001 vs. day 0.

Fig. 3.

miR-21 expression is upregulated in fibrotic kidneys. A: mice underwent sham or UUO surgery. At 0 and 6 h and 1, 2, 3, and 7 days after UUO, the mice were killed and both kidneys [left: obstructive kidneys (UUO); right: contralateral kidneys (CL)] were harvested. Kidney RNA was isolated and Northern blotting was performed. The PAGE gels were stained with ethidium bromide before transfer to manifest 5S rRNA serving as loading controls. B: at 7 days after UUO, the mice were killed and both kidneys were harvested. Frozen sections were prepared and in situ hybridization (ISH) assays were performed as described in materials and methods using specific miR-21 probes and probes with scrambled sequence serving as negative controls. The original digital microphotographs were ×4. C: experiments were performed as in B. The original digital microphotographs were ×40. The images in the squares were enlarged for detailed manifestation (UUO ×120).

Fig. 4.

miR-21 expression is upregulated in TNF-α-, transforming growth factor (TGF)-β1-, or aristolochic acid (AA)-treated human renal epithelial cells. A: human renal tubular epithelial cells were treated without or with 10 ng/ml TNF-α or 10 ng/ml TGF-β1 for 48 h. Cells were harvested and RNA was isolated. The levels of miR-21 were determined by Northern blotting. B: at 0 and 7 days after UUO, mice were killed and both kidneys were harvested. Kidney RNA was isolated and TGF-β1 expression was determined by real-time PCR (n = 7). C: renal tubular epithelial cells were treated without or with 5 μg/ml AA for 48 h. Cells were harvested and RNA was isolated. The levels of miR-21, Col1A1, and smooth muscle actin (SMA)-α were determined by real-time PCR assays; n = 3/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. time 0.

Fig. 5.

Blocking miR-21 in vivo suppresses UUO-induced kidney fibrosis. A: mice were injected intraperitoneally with control probes or anti-miR-21 probes (10 mg/kg in 200 μl saline) 3 times separated by 1 day between injections. On the second day after the last injection, UUO surgery was performed. At 12 days after UUO, mice were killed and both kidneys were harvested. Kidney RNA was isolated and the levels of miR-21 and U6 were determined by Northern blotting. B: experiments were performed as in A. Kidney sections were prepared and Picrosirius red staining was performed to manifest collagen deposition. C: ten random fields were selected from each section for digital quantification (n = 7/group). *P < 0.05 vs. UUO kidneys in the control group. D: experiments were performed as in A. Kidney RNA was isolated and the expression of Col1A1, Col1A2, Fn, SMA-α, PAI-1, and TGF-β1 was determined by real-time PCR (n = 7/group). *P < 0.05, **P < 0.01 vs. UUO kidneys in the control group.

Fig. 6.

Blocking miR-21 reduces macrophage infiltration in UUO kidneys. A: mice were injected intraperitoneally with control probes or anti-miR-21 probes (10 mg/kg in 200 μl saline) 3 times separated by 1 day between injections. On the second day after the last injection, UUO surgery was performed. At 4 days after UUO, mice were killed and both kidneys were harvested. Kidney sections were prepared and immunofluorescence assays were performed using anti-CD11b antibodies to determine macrophage infiltration in the kidneys. DAPI was used to stain nuclei. B: quantification of macrophage infiltration was performed as described in materials and methods (n = 7/group). *P < 0.05 vs. UUO kidneys in the control group.