MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy

Abstract

Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3'-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3'-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation.

FIGURE 1.

miRNA expression profiling under hyperglycemic conditions in vitro and in vivo. Comparative microarray analysis indicated that nine miRNAs were differentially up-regulated within all samples. Specifically, 58 miRNAs were up-regulated in db/db glomeruli, 149 miRNAs were up-regulated in high glucose (HG)-treated kidney microvascular endothelial cells, and 42 miRNAs were up-regulated in podocytes treated with HG (25 mm) for 24 h as compared with normal glucose conditions.

FIGURE 2.

Up-regulation of miR-29c expression in vivo and in vitro. A, expression of miR-29c in different tissues of a control db/m mouse as detected by Northern blot analysis. U6 snRNA served as a loading control. B, Northern blot analysis shows representative results from glomerular miR-29c expression in control db/m and diabetic db/db mice. C, real-time qPCR analysis shows glomerular miR-29c expression in control db/m and diabetic db/db mice. Measured transcript levels were normalized to U6 snRNA expression. Data are shown as mean ± S.E. (n = 5). D, Northern blot analysis shows representative results of miR-29c expression in cultured podocytes treated with normal glucose (NG, 5 mm) or HG (25 mm) for 24 h. E, real-time qPCR analysis shows miR-29c expression in podocytes treated with high glucose (25 mm) for 24 h as compared with podocytes treated with either normal glucose (5 mm) or mannitol (25 mm) for 24 h. Measured transcript levels were normalized to U6 snRNA expression. Data are shown as mean ± S.E. (n = 3).

FIGURE 3.

miR-29c is predicted to target Spry1 gene. A (upper panel), a predicted miR-29c target site resides at nucleotides 773–779 (shown in the gray box) of the mouse Spry1 3′-UTR and is highly conserved in several species. Lower panel, sequence alignment of miR-29c with the mouse Spry1 3′-UTR. Mmu, Mus musculus; Hsa, Homo sapiens; Rno, Rattus norvegicus; and Cfa, Canis lupus familiaris. B, miR-29c can potentially form a strong secondary structure with the target sequence of 3′-UTR of Spry1 (predicted by the RNA Hybrid program). C, differentiated podocytes were co-transfected with plasmid 3.1-luc-Spry1 wild type 3′-UTR (luc-Spry1-wt) or miR-29c mutant 3′-UTR (luc-Spry1-mut) and the indicated miRNA mimics. Luciferase activities were normalized to β-gal activities. Results were obtained from three independent experiments. Data are shown as mean ± S.E.

FIGURE 4.

miR-29c targets Spry1. A, schematic diagram of lentiviral miR-29c construct. CAGGS, CMV enhancer-chicken β-actin-globin intron promoter; WPRE, woodchuck hepatitis post-transcriptional regulatory element. B, real-time qPCR analysis of miR-29c expression in stable podocytes infected with empty vector lentivirus (Lenti-CAG) or miR-29c lentivirus (Lenti-miR-29c). Measured transcript levels were normalized to U6 snRNA expression. Data are shown as mean ± S.E. (n = 3). C, expression of Spry1 (red) in control podocytes or stable podocytes infected with miR-29c lentivirus (Lenti-miR-29c) harboring GFP marker (green) was assessed by deconvolution microscopy. Original magnification, ×400. D, quantitative analysis based on fluorescence intensity of Spry1 (n = 3). E, podocytes were transfected with miR-29c mimics or inhibitor as indicated and treated with HG (25 mm) for 24 h as compared with normal glucose. Spry1 expression in total cell lysates was analyzed by immunoblot. GAPDH served as a loading control. F, densitometric analysis of Spry1 expression in E (n = 3).

FIGURE 5.

miR-29c promotes cell apoptosis, promotes accumulation of fibronectin, and activates Rho kinase by targeting Spry1. Lenti-CAG and A and B, Lenti-miR-29c podocytes were transfected with miR-29c inhibitor as indicated under NG (5 mm) conditions. Cell apoptosis was analyzed by FACS (A) and caspase-3 activity assay (B). Results were obtained from three independent experiments. Data are shown as mean ± S.E. C, control podocytes were transfected with Spry1 siRNA (siSpry1, 30 nm) and/or miR-29c inhibitor as indicated and treated with HG (25 mm) for 36 h. Apoptosis in the cells was analyzed by FACS. Results were obtained from three independent experiments. Data are shown as mean ± S.E. D, mesangial cells stably infected with empty vector lentivirus (Lenti-CAG) or miR-29c lentivirus (Lenti-miR-29c) were transfected with miR-29c inhibitor or FLAG-Spry1 as indicated and treated with HG (25 mm) for 48 h as compared with NG. Expression of fibronectin (Fn1) in the cells was analyzed by real-time qPCR normalized to GAPDH. Data are shown as mean ± S.E. (n = 3). E, podocytes were transfected with Spry1 siRNA (si Spry1, 30 nm) and/or miR-29c inhibitor as indicated and treated with HG (25 mm) for 36 h as compared with NG. Rho kinase activity in cell lysates was measured as described under “Experimental Procedures.” β-Actin served as a loading control. F, densitometric analysis of Rho kinase activity in E (n = 3).

FIGURE 6.

In vivo inhibition of miR-29c ameliorates progression of DN. A, miR-29c expression level was significantly down-regulated in the kidney cortices of miR-29c ASO-treated db/db mice as compared with control db/db mice. Representative RT-qPCR resulting from three different experiments is shown (n = 3). Expression levels were normalized to U6 snRNA expression. Data are shown as mean ± S.E. B, injection of miR-29c ASO led to improved proteinuria in db/db mice. Urine albumin and creatinine from miR-29c ASO-treated and control db/db mice (n = 5/group) were measured at the indicated weeks of age, and the relative albumin/creatinine ratio (ACR) was calculated. C, representative TUNEL assay to detect the apoptosis in miR-29c ASO-treated db/db mice as compared with control db/db mice. Apoptotic cells in the glomeruli are indicated by arrows. D, quantitative analysis of apoptosis in miR-29c ASO-treated mice as compared with control as shown in C (n = 3). E, caspase-3 activity in the kidney cortex lysates of miR-29c ASO-treated db/db mice as compared with control mice. Data are shown as mean ± S.E. (n = 5). F, representative fibronectin immunostaining from kidney cortex of miR-29c ASO-treated db/db mice as compared with control mice. G, the mesangial matrix index of miR-29c ASO-treated db/db mice was significantly decreased as compared with control mice.

FIGURE 7.

In vivo inhibition of miR-29c reduces Rho kinase activation through Spry1. A, Spry1 expression in the kidney cortex extracts of miR-29c ASO-treated db/db mice as compared with control mice was analyzed by Western blot. GAPDH served as a loading control. B, densitometric analysis of Spry1 expression in A (n = 3). C, representative Spry1 staining from kidney cortex of miR-29c ASO-treated db/db mice as compared with control mice. D, Rho kinase activity in the kidney cortex extracts of miR-29c ASO-treated db/db mice as compared with control mice. Representative results from three different experiments are shown. β-Actin served as a loading control. E, densitometric analysis of Rho kinase activity in D (n = 3). F, proposed mechanism for the putative effects of high glucose on the miR-29c-mediated signaling pathway leading to diabetic nephropathy. GBM, glomerular basement membrane.