MicroRNA-27a promotes podocyte injury via PPARγ-mediated β-catenin activation in diabetic nephropathy

Abstract

Podocyte injury has a pivotal role in the pathogenesis of diabetic nephropathy (DN). MicroRNA-27a (miR-27a), peroxisome proliferator-activated receptor γ (PPARγ) and β-catenin pathways have been involved in the pathogenesis of DN. Herein, we asked whether miR-27a mediates podocyte injury through PPARγ/β-catenin signaling in DN. The functional relevance of miR-27a, PPARγ and β-catenin were investigated in cultured podocytes and glomeruli of diabetic rats and patients using in vitro and in vivo approaches. Podocyte injury was assessed by migration, invasion and apoptosis assay. Biological parameters were analyzed using enzyme-linked immunosorbent assay. We found that high glucose stimulated miR-27a expression, which, by negatively targeting PPARγ, activated β-catenin signaling as evidenced by upregulation of β-catenin target genes, snail1 and α-smooth muscle actin (α-SMA) and downregulation of podocyte-specific markers podocin and synaptopodin. These changes caused podocyte injury as demonstrated by increased podocyte mesenchymal transition, disrupted podocyte architectural integrity and increased podocyte apoptosis. Furthermore, we provide evidence that miR-27a contributed to unfavorable renal function and increased podocyte injury in diabetic rats. Notably, miR-27a exhibited clinical and biological relevance as it was linked to elevated serum creatinine, proteinuria and reduced creatinine clearance rate. In addition, miR-27a upregulation and activation of PPARγ/β-catenin signaling were verified in renal biopsy samples from DN patients. We propose a novel role of the miR-27a/PPARγ/β-catenin axis in fostering the progression toward more deteriorated podocyte injury in DN. Targeting miR-27a could be a potential therapeutic approach for DN.

Figure 1

High glucose induces miR-27a expression in cultured podocytes. (a) qRT-PCR analysis shows the level of miR-27a in various conditions as indicated. (b) Representative western blotting shows the expression of PPARγ and β-catenin target genes in various conditions as indicated. Cell lysates were immunoblotted with specific antibodies against PPARγ, active β-catenin, snail1, α-SMA, podocin and β-actin. qRT-PCR shows that miR-27a was increased in a (c) time and (d) dose-dependent manner. (e-f) qRT-PCR and (g-h) western blot analyses show the expression level of PPARγ and β-catenin target genes in a time- and dose-dependent manner. (i) PPARγ gene transcription was amplified by miR-27ai and diminished by miR-27am. Mouse podocytes were co-transfected with miR-27ai, miR-27am, and control or wild-type or mutant 3′-UTR of PPARγ and transfection efficiency was evaluated by luciferase reporter assay. *P<0.05; ^#P<0.001. Active β-cat, active β-catenin; CTNNB1, catenin beta-1; HG, high glucose; miR-iNC: miRNA inhibitor negative control; mt: mutant type; NG, normal glucose; wt: wild type

Figure 2

MiR-27ai attenuates podocyte injury via PPARγ-mediated β-catenin inactivation in high glucose. (a) qRT-PCR analysis shows miR-27ai reduced miR-27a expression in HG cultured podocytes. (b) Representative western blotting shows the expression of phosphorylated and total PPARγ and β-catenin target genes in various conditions as indicated. (c) qRT-PCR analysis shows miR-27ai upregulated the level of PPARγ and podocin but downregulated β-catenin target genes. (d) HG enhanced the interaction of phosphorylated PPARγ and active β-catenin by co-immunoprecipitation. (e) Transwell migration assay and quantitative data show decreased migration of HG cultured podocytes. Scale bar, 100 μm. (f) Wound-healing assay and quantitative data show decreased invasion of HG cultured podocytes. (g) Flow cytometric analysis shows decreased podocyte apoptosis. *P<0.05; ^#P<0.001. Active β-cat, active β-catenin; 7-AAD, 7-aminoactinomycin D; CTNNB1, catenin beta-1; HG, high glucose; IB, immunoblotting; IP, immunoprecipitation; miR-iNC: miRNA inhibitor negative control; miR-NC: miRNA negative control; NG, normal glucose; PE, phycoerythrin; pPPARγ, phosphorylated peroxisome proliferator-activated receptor γ

Figure 3

PPARγ-mediated β-catenin activation induces podocyte injury in high glucose. (a) Representative western blotting shows the expression of phosphorylated and total PPARγ and β-catenin target genes in various conditions as indicated. (b) qRT-PCR analysis shows PPARγ siRNA decreased PPARγ and podocin but increased β-catenin target genes. (c) Transwell migration assay and quantitative data show increased migration. Scale bar, 100 μm. (d) Wound-healing assay and quantitative data show increased invasion. (e) Summarized data showing increased podocyte apoptosis by flow cytometric analysis. (f) Immunofluorescence microscopy and quantitative data show PPARγ siRNA-induced β-catenin activation was attenuated by co-transfection with miR-27ai. Scale bar, 20 μm. Representative (g) qRT-PCR and (h) western blotting show the expression of PPARγ and β-catenin target genes in various conditions as indicated. Representative (i) transwell migration assay and (j) wound-healing assay show the increased ability of migration and invasion caused by PPARγ abolishment were mitigated by co-transfection with miR-27ai. *P<0.05; ^#P<0.001. Active β-cat, active β-catenin; CTNNB1, catenin beta-1; NT, non-targeting; pPPARγ, phosphorylated peroxisome proliferator-activated receptor γ

Figure 4

MiR-27a promotes PPARγ-mediated β-catenin activation in diabetic rats. Representative (a) micrographs of ISH and (b) quantitative data illustrate the expression of miR-27a in podocytes in various groups as indicated. Representative (c) immunohistochemical staining and (d) quantitative data show the expression of PPARγ and β-catenin target genes in various groups as indicated. The level of miR-27a in (e) glomeruli and (f) plasma samples of diabetic rats in various groups as indicated. Frozen rat kidney sections were hybridized with digoxygenin (DIG)-labeled miRCURY Locked Nucleic Acid (LNA) microRNA Detection Probes. Paraffin-embedded sections were immunostained for PPARγ (total and phosphorylated), active β-catenin, snail1, α-SMA and synaptopodin. Scale bar, 50 μm. Glomeruli were dissected using laser microdissection and detected with qRT-PCR. *P<0.05; ^#P<0.001. Active β-cat, active β-catenin; DM, diabetes mellitus; DM_miR-iNC, diabetic rats treated with miRNA inhibitor negative control; DM_miR-27ai, diabetic rats treated with miR-27ai; NC, normal control; pPPARγ, phosphorylated peroxisome proliferator-activated receptor γ

Figure 5

miR-27a contributes to podocyte depletion and disrupts podocyte architectural integrity in diabetic rats. (a) Immunofluorescence staining shows glomerular synaptopodin and WT1 expression in different groups. Paraffin-embedded rat kidney sections were co-immunostained for synaptopodin (red) and WT1 (green). Nuclei were visualized by DAPI. Scale bar, 20 μm. (b) Transmission electron microscopic analysis shows morphological changes in the podocyte foot process in different groups as indicated. Scale bar, 1.0 μm. Red asterisks specify podocyte foot processes. (c) Level of synaptopodin, (d) podocyte number, (e) level of WT1 and (f) percentage of podocyte foot process effacement in different groups were shown. (g) Representative immunofluorescence micrographs show glomerular pPPARγ and active β-catenin expression in diabetic rats with magnified white insets on the lower right quadrant. Paraffin-embedded rat kidney sections were co-immunostained for pPPARγ (green) and active β-catenin (red). Nuclei were visualized by DAPI. Arrows indicate the colocalization (yellow). Scale bar, 20 μm. (h) Representative co-immunoprecipitation analysis shows the interaction of pPPARγ and active β-catenin in laser capture microdissected glomeruli in different groups as indicated. *P<0.05; ^#P<0.001. Active β-cat, active β-catenin; DM, diabetes mellitus; DM_miR-iNC, diabetic rats treated with miRNA inhibitor negative control; DM_miR-27ai, diabetic rats treated with miR-27ai; IB, immunoblotting; IP, immunoprecipitation; NC, normal control; pPPARγ, phosphorylated peroxisome proliferator-activated receptor γ; WT1, Wilm's tumor 1

Figure 6

Expression patterns of miR-27a and PPARγ/β-catenin signaling molecules in human renal biopsy samples. Representative (a) ISH staining and (b) quantitative data illustrate the expression of miR-27a in human renal biopsy tissues from DN patients (n=62) and control healthy donor transplant kidney tissues (n=13). Representative (c) immunohistochemical staining and (d) quantitative data illustrate the expression of pPPARγ and β-catenin target genes in different groups as indicated. Fresh snap-frozen human renal biopsies were sectioned and hybridized with digoxygenin (DIG)-labeled miRCURY Locked Nucleic Acid (LNA) microRNA Detection Probes. Paraffin-embedded human renal biopsy sections were immunostained for pPPARγ, active β-catenin, snail1, α-SMA and synaptopodin. Red insets in the left side images of each panel were magnified and shown on the right side. Black arrows indicate podocytes. Representative (e) immunofluorescence images and quantitative data show (f) glomerular synaptopodin (green) and WT1 (red) expression and (g) podocyte number in human renal biopsy samples. (h) Representative immunofluorescence micrographs show glomerular pPPARγ (green) and active β-catenin (red) expression in human renal biopsy samples. Scale bar, 50 μm. (i) Magnified white insets show the colocalization of pPPARγ and active β-catenin (yellow). Scale bar, 20 μm. (j) Quantitative data show the level of pPPARγ (green) and active β-catenin (red). Nuclei were visualized by DAPI. White arrow indicates colocalization of pPPARγ and active β-catenin. Glomeruli were demarcated with white dashed lines. (k) A hypothetical model illustrating that miR-27a, via PPARγ-mediated β-catenin activation, promotes podocyte injury in DN. MiR-27a inhibits PPARγ gene transcription whereas indirectly stimulates PPARγ phosphorylation, which activates β-catenin signaling and triggers β-catenin-dependent reprogramming and target gene expression levels. These events promote podocyte injuries as demonstrated by increased migration, invasion, and apoptosis, and decreased adhesion abilities. #P<0.001. Active β-cat, active β-catenin; LEF1, lymphoid-enhancer factor 1; P, phosphorylation; pPPARγ, phosphorylated peroxisome proliferator-activated receptor γ; WT1, Wilm's tumor 1