Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1

Abstract

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a^-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFβ signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a^-/- animals. Treatment of podocytes in vitro with TGF-β1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-β1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

Keywords: Type 1 diabetes; diabetic glomerulopathy; diabetic nephropathy; kidney; microRNA (miRNA); mir-146a; podocyte.

FIGURE 1.

Down-regulation of miR-146a correlates with increased albuminuria in diabetic patients. A and B, scatter plots showing correlation between relative expression of miR-146a versus urinary ACR in the isolated glomeruli of kidney biopsies from diabetic patients (n = 45). Each dot represents an individual patient. Data are from two time points; one at the time of biopsy (A) and one at the end of observation period (5 ± 1 years post-biopsy, B). Relative miR-146a level of 0.4 is shown to divide the plot into two groups with different ACR values.

FIGURE 2.

miR-146a levels are reduced in the diabetic human and mouse kidney glomeruli. A and B, representative ISH images of human (A) and mouse (B) kidney sections to detect the expression pattern of miR-146a (indicated with an arrow). Each kidney section was stained with the indicated probe (against miR-146a, a scrambled control or against U6 RNA). Confocal images of immunofluorescently labeled glomeruli (A), stained with the podocyte marker synaptopodin (Synpo, green), show relative podocyte density in the representative healthy and diabetic human kidney sections. Representative ISH images of kidney sections from C57BL/6 WT and miR-146a^−/− animals stained with a specific probe against miR-146a are also shown (B). Scale bar, 50 μm (A and B). C, a bar graph showing urinary ACR in 12-week-old BTBR WT and BTBR ob/ob animals. Data shown are mean ± S.E. (n = 5/group). ***, p < 0.001. D, a bar graph showing relative expression level of miR-146a in kidney sections from 12-week-old BTBR WT and BTBR ob/ob mice, as measured by qRT-PCR. Data shown are mean ± S.E. (n = 3). *, p < 0.05.

FIGURE 3.

miR-146a targets Notch-1 and ErbB4 are up-regulated in the diseased glomeruli. A, primary sequence and predicted secondary structure of pre-miR-146a. miRNA is transcribed in the nucleus as primary miRNA, processed by endonuclease Drosha into pre-miRNA, exported into the cytoplasm, where it is cleaved by RNase Dicer to yield a mature 22-nt duplex miRNA. The sequence of 22-nucleotide (22 nt) mature miR-146a duplex is highlighted in red and blue. Typically, only one strand of the 22-nt duplex gets loaded into an RNA-induced silencing complex (RISC) to suppress target gene expression. In miR-146a, this is typically the 5′-strand (shown in red), also known as miR-146a-5p. B, alignment of miR-146a target sites in the 3′ UTRs of Notch-1 and ErbB4 mRNA (26, 40). C, graph showing relative expression levels of ErbB4 mRNA in various human kidney biopsies, data were derived from Schmid et al. (46). The published microarray data were generated using renal biopsies from patients with histological evidence of DN (n = 13), where the biopsies from cadaveric donors (n = 4), related living donors (n = 3), and from patients with minimal change disease (MCD) (n = 4) without histological or clinical evidence of impaired renal function served as controls. Data shown are mean ± S.E. ****, p < 0.0001. D, representative images showing histochemical analyses of kidney tissue samples from 12-week-old BTBR WT (healthy) and BTBR ob/ob (diabetic) mice using periodic acid-Schiff (PAS) and Masson's trichrome (trichrome) staining showing increased mesangial sclerosis and fibrosis in the diabetic kidneys. Scale bar, 50 μm. E, expression of EGFR, Notch-1, and ErbB4 is up-regulated in the glomeruli of BTBR ob/ob mice. Representative confocal microscopy images of immunofluorescently labeled glomeruli from 12-week-old BTBR WT (healthy) and BTBR ob/ob (diabetic) mice. Kidney sections were imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4, and Synaptopodin (Synpo). Merged DAPI, EGFR and Synpo, DAPI, Notch-1 and Synpo, and DAPI, ErbB4 and Synpo channels are also presented that show podocyte colocalization for these proteins. Scale bar, 50 μm. F, expression of EGFR, Notch-1, and ErbB4 is up-regulated in the glomeruli of diabetic patients. Representative images showing histochemical analyses of kidney tissue samples (left panels) after PAS and trichrome staining showing extensive glomerular expansion, mesangial sclerosis, and fibrosis in the diabetic kidneys. Scale bar, 50 μm. Representative confocal microscopy images of immunofluorescently labeled kidney sections that imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4 and Synpo (right panels). Merged EGFR and Synpo, Notch-1 and Synpo, and ErbB4 and Synpo channels are shown that show podocyte colocalization for these proteins, respectively. Scale bar, 50 μm. Bottom panels present higher magnification views of the boxed regions in the middle panel. G, miR-146a reduces the activity of luciferase linked with 3′ UTRs of ErbB4 and Notch1. Bar graph reporting results from luciferase activity assay from HEK293T cells co-transfected with pre-miR-146a or pre-miR-control and a luciferase reporter plasmid containing either the WT or mutated (mut) sequence of 3′ UTRs of ErbB4 or Notch-1. Luciferase activity was normalized with the activity of β-galactosidase from a co-transfected plasmid. Data shown are mean ± S.E. (n = 4). *, p < 0.05. ***, p < 0.0005.

FIGURE 4.

Erlotinib attenuates accelerated development of STZ-induced albuminuria in miR-146a^−/− animals. A, both WT and miR-146a^−/− animals developed similar and sustained levels of hyperglycemia within 2 weeks of STZ treatment. Treatment with erlotinib starting at 4 weeks after STZ induction did not result in any change in level of hyperglycemia in either strain. Blood glucose levels remained unchanged in the untreated WT and miR-146^−/− mice. Data presented on these graphs are mean ± S.E. (n = 3–10/group). B, WT and miR-146a^−/− animals displayed equal levels of weight loss upon STZ-induced hyperglycemia that was unaffected by treatment with erlotinib. Data presented on these graphs are mean body weight at the end of the experiment (16 weeks post-STZ) ± S.E. (n = 3–10/group). C, miR-146a^−/− animals showed increased albuminuria after ∼8 weeks post-STZ and a significantly increased albuminuria 14 weeks post-STZ, as compared with the WT animals. Treatment with erlotinib significantly reduced the development of albuminuria and both the WT and the miR-146a^−/− animals. Data shown are mean ± S.E. (n = 4–10/group). * and #, p < 0.05.

FIGURE 5.

STZ-induced hyperglycemia increases glomerulopathy and foot process effacement in the mouse glomeruli that is suppressed by erlotinib. A, representative images showing PAS-stained kidney tissue samples from WT and miR-146a^−/− animals treated with vehicle alone (Control), STZ and vehicle (STZ), or STZ and erlotinib (STZ + Erl) and analyzed 16 weeks post-STZ showing increased mesangial sclerosis in STZ-treated animals that is reduced with erlotinib treatment. Scale bar, 50 μm. B, graphs showing quantification of mesangial matrix expansion from the PAS-stained sections in A. Data shown are normalized to the level of staining in control tissue and are mean ± S.E. (n = 3–5/group). *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, erlotinib protects against STZ-induced podocyte foot process (FP) effacement. Representative electron microscopy images of WT (top panels) and miR-146a^−/− (bottom panels) mouse glomeruli treated with vehicle alone (Control), with STZ and vehicle (STZ), or with STZ and erlotinib (STZ + Erl). Scale bar, 2 μm. FP, foot processes; BM, basement membrane. A higher magnification view is presented below every image. Scale bar, 500 nm. A graph on the right shows quantification of the number of FPs per unit glomerular length in each of the samples. Data shown are mean ± S.E. (n = 3/group) and significant difference comparison was performed as compared with the respective controls. *, p < 0.05; **, p < 0.01; ns, not significant.

FIGURE 6.

STZ treatment of WT and miR-146a^−/− mice results in increased glomerular injury and induction of miR-146a targets in the mouse glomeruli that is suppressed by erlotinib. A, STZ-induced up-regulation of EGFR, Notch-1, and ErbB4 expression in the mouse glomeruli is reduced by erlotinib. Representative confocal microscopy images of immunofluorescently labeled glomeruli from WT (top panels) and miR-146a^−/− (bottom panels) mice treated with vehicle alone (Control), with STZ and vehicle (STZ) or with STZ and erlotinib (STZ + Erl). Kidney sections were imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4, and Synaptopodin (Synpo). Merged DAPI, EGFR and Synpo, DAPI, Notch-1 and Synpo, and DAPI, ErbB4 and Synpo channels are also presented that show podocyte colocalization for these proteins. Scale bar, 50 μm. B, graphs showing the quantification of relative glomerular signal intensity of EGFR, Notch-1, and ErbB4 in samples from A. Data shown are mean ± S.E. (n = 5/group). ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 7.

Reduction in glomerular miR-146a levels and podocyte numbers by STZ treatment is suppressed by erlotinib. A, representative ISH images of kidney sections from WT mice treated with vehicle alone (Control), with STZ and vehicle (STZ), or with STZ and erlotinib (STZ + Erl) and stained to detect expression pattern of miR-146a show protection of glomerular miR-146a levels by erlotinib. Each kidney section was stained with the indicated probe (against miR-146a, a scrambled control, or against U6 RNA). Representative ISH image of kidney sections from miR-146a^−/− animals stained with a specific probe against miR-146a is also shown. Scale bar, 50 μm. B, erlotinib protects against STZ-induced podocyte loss. Representative immunohistochemical images of glomeruli stained with an antibody against WT1 from WT mice treated with vehicle alone (Control), with STZ and vehicle (STZ), or with STZ and erlotinib (STZ + Erl). Scale bar, 50 μm. Graph on the right shows quantification of WT1 positive cells per glomeruli in these samples. Data shown are mean ± S.E. (n = 3). *, p < 0.05; ****, p < 0.0001.

FIGURE 8.

Pan-ErbB inhibitor suppresses inflammation by rescuing podocyte expression of miR-146a and suppressing its targets Notch-1 and ErbB4. A, TGF-β1 treatment reduces F-actin fibers in cultured podocytes and erlotinib protects against this loss. Representative confocal microscopy images of cultured mouse podocytes treated with vehicle alone (Control), TGF-β1 (5 ng/ml), or with TGF-β1 (5 ng/ml) and erlotinib (10 μm) for 24 h and stained with CellMask blue (nuclear stain) and Alexa Fluor 568-labeled phalloidin (to stain F-actin fibers). Scale bar, 20 μm. B and C, TGF-β1 treatment induces expression of Notch-1 and ErbB4 in cultured WT and miR-146a^−/− podocytes and erlotinib suppresses it. Immunoblot analysis of various phosphorylated (p-) and total proteins in the lysates from WT (B) and miR-146a^−/− (C) podocytes stimulated without or with 5 ng/ml of TGF-β1 (TGF-β1) in the absence or presence of erlotinib (10 μm). GAPDH was used as the loading control. Relative position of the molecular weight markers is shown on the left.

FIGURE 9.

TGF-β1 induced up-regulation of MCP-1 expression in podocytes is suppressed by erlotinib. A bar graph showing the relative expression level of MCP-1 in WT podocytes treated with vehicle DMSO (Control), 10 μm erlotinib (Erl), 5 ng/ml of TGF-β1 (TGF-β1), or 5 ng/ml of TGF-β1 and 10 μm erlotinib (TGF-β1 + Erl) for 24 h, as measured by qRT-PCR. Data were normalized using GAPDH mRNA controls and are mean ± S.E. (n = 3). **, p < 0.005.

FIGURE 10.

Working model. A diagram showing our working model. Under homeostatic conditions of normoglycemia, podocyte expressed miR-146a suppresses expression of Notch-1 and ErbB4 to maintain healthy cells. Diabetic milieu, including TGF-β1 (and MCP-1), induces MCP1 and MCPIP1 in podocytes, which decreases miR-146a levels. This results in de-repression of Notch-1 and ErbB4, which together with EGFR, induce podocyte injury. Blocking this signaling pathway with ErbB4/EGFR inhibitors suppresses the harmful signaling and decreases podocyte injury in vitro and glomerulopathy in vivo.