Genetic and pharmacological inhibition of microRNA-92a maintains podocyte cell cycle quiescence and limits crescentic glomerulonephritis

Abstract

Crescentic rapidly progressive glomerulonephritis (RPGN) represents the most aggressive form of acquired glomerular disease. While most therapeutic approaches involve potentially toxic immunosuppressive strategies, the pathophysiology remains incompletely understood. Podocytes are glomerular epithelial cells that are normally growth-arrested because of the expression of cyclin-dependent kinase (CDK) inhibitors. An exception is in RPGN where podocytes undergo a deregulation of their differentiated phenotype and proliferate. Here we demonstrate that microRNA-92a (miR-92a) is enriched in podocytes of patients and mice with RPGN. The CDK inhibitor p57^Kip2 is a major target of miR-92a that constitutively safeguards podocyte cell cycle quiescence. Podocyte-specific deletion of miR-92a in mice de-repressed the expression of p57^Kip2 and prevented glomerular injury in RPGN. Administration of an anti-miR-92a after disease initiation prevented albuminuria and kidney failure, indicating miR-92a inhibition as a potential therapeutic strategy for RPGN. We demonstrate that miRNA induction in epithelial cells can break glomerular tolerance to immune injury.

Fig. 1

Increased miR-92a expression in crescents and podocytes during nephrotoxic nephritis and human crescentic glomerulonephritis. a Representative microRNA profiling on dynabeads-isolated glomeruli from control or NTS-challenged mice (NTN). Values are means ± s.e.m., *p < 0.05 vs. control condition (n = 3 samples per condition). b Relative abundance of miR-92a assessed by RT-qPCR in sorted podocytes from normal healthy mice (control) and NTS-challenged mice (NTN). Values are means ± s.e.m., *p < 0.05 vs. control condition (n = 5 mice per condition). c Double miR-92a in situ hybridization (blue staining) and WT1 staining (brown staining) of kidney sections from normal mice (control) and NTS-challenged mice (NTN). Pictures in the bottom show a higher magnification of the top panel. Scale bars, 10 μm. d miR-92a in situ hybridization of kidney sections from random biopsies from individuals diagnosed with non-crescentic glomerulopathies, including minimal change disease (MCD) and membranous nephropathy (MN), and from patients with RPGN of various etiologies including stage III and IV lupus nephritis (LN), microscopic polyangiitis (MPA), and granulomatosis with polyangiitis (GPA). Scale bars, 50 μm. The lower panel shows higher magnification of middle panels (black box). Black stars (*) show miR-92a-positive cells. e RT-qPCR analysis of the relative abundance of miR-92a in microdissected glomeruli from four patients with non-crescentic glomerulopathies (black bars) and six patients with crescentic RPGN (white bars). Values are means ± s.e.m., *p < 0.05 vs. non-crescentic glomerular diseases. f Fluorescent in situ hybridization of miR-92a (red) and WT1 (green) on patients biopsies described in d. Bottom panel shows higher magnification of top panel (white box). White arrows show colocalization of WT1-positive cells and miR-92a expression. Scale bars, 50 μm. Statistical analysis: Mann–Whitney test to compare two groups

Fig. 2

Inhibition of miR-92a in podocytes upregulates its target p57 and impairs proliferation. a RT-qPCR analysis of the relative abundance of miR-92a in podocytes transfected with an anti-miR-control (anti-miR-ctrl) or an anti-miR-92a. Values are normalized to U6snRNA and are relative to control (non-transfected cells). b, c Representative pictures (b) and quantification (c) of podocyte proliferation assay involving decapsulated mouse glomeruli. Podocyte proliferation was assessed after 4 days. Scale bars, 50 μm. d RT-PCR analysis of Ki67 mRNA abundance in intact and anti-miR-ctrl primary podocytes or in anti-miR-92a primary podocytes. e Dual luciferase assay wild-type or mutated p57 3′UTR in HEK293 cells transfected with pre-miR-ctrl, pre-miR-92a, or pre-miR-126. n = 2 independent experiments (each experiment assayed each condition in triplicate). p57 3′UTR miR-92a-binding sequence binding site is indicated in bold and mutated sequence is labeled in red. ***p < 0.001 vs. 3′UTR +Pre-miR- ctrl. f, g Western blot analysis (f) and quantification of p57 protein abundance (g) in untreated or anti-miR-ctrl primary podocytes vs. in anti-miR-92a primary podocytes. Tubulin is shown as a loading control. h Staining of p57 protein (green) in podocyte outgrowths. DAPI-stained nuclei (blue). Adherent glomeruli are indicated by arrows. Scale bars, 100 μm. Statistical analysis: Kruskal–Wallis one-way analysis of variance followed by Dunn’s multiple comparaison test. Values are means ± s.e.m. (n = 4 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control podocytes (control)

Fig. 3

p57^Kip2 expression is lost during RPGN and p57^Kip2 silencing decreases podocyte proliferation. a, b Western blot analysis (a) and quantification (b) of p57 on magnetic beads isolated glomeruli from unchallenged (control) or NTS-challenged (NTN) mice. Values are means ± s.e.m. (n = 5 per group). *p < 0.05 vs. control. c Immunostaining of p57 (strong brown staining) and immunofluorescent p57 (red)/WT1 (green) staining in kidney sections from normal mice (control) and NTS-challenged mice (NTN). Scale bars, 10 µm. d Quantification of p57-positive cells per glomerular section in mice described in a. e Representative photomicrographs of p57 immunostaining (strong brown staining) in kidney sections from random biopsies from individuals diagnosed with non-crescentic glomerulopathies, including minimal change disease (MCD) and membranous nephropathy (MN), and from patients with RPGN of various etiologies including stage III and IV lupus nephritis (LN) and microscopic polyangiitis (MPA). Scale bars, 50 µm. f Quantification of p57-positive cells per glomerular section in biopsies described in e. Values are means ± s.e.m. *p < 0.05 vs. non-crescentic glomerular diseases. g Representative pictures of p57 staining (red) and podocyte proliferation assay in control cells (non-transfected) or transfected with a control siRNA (siRNA-control) or a siRNA for p57 (siRNA-p57). DAPI-stained nuclei (blue). Top panel (scale bars, 50 μm); bottom panel (scale bars, 100 µm). h Quantification of podocyte proliferation assay involving decapsulated mouse glomeruli. Podocyte proliferation was assessed after 4 days. Values are means ± s.e.m. (n = 6 per group). ***p < 0.001 vs. non-transfected cells. Statistical analysis: Kruskal–Wallis one-way analysis of variance followed by Dunn’s multiple comparaison test or Mann–Whitney test to compare groups. Values are means ± s.e.m

Fig. 4

miR-92a-specific deletion in podocytes reduces nephrotoxic nephritis. a Fluorescent in situ hybridization of miR-92a (red) and WT1 (green) on kidney sections from NTS-challenged iPod-miR92a WT mice and iPod-miR92a lox mice. DAPI-stained nuclei (blue). Right panel shows higher magnification of the left panel (white box). Scale bars, 50 μm. b RT-qPCR analysis of the relative abundance of miR-92a in freshly isolated glomeruli from NTS-challenged iPod-miR92a WT mice and NTS-challenged iPod-miR92a lox mice. c Urinary albumin excretion rates at baseline and 10 days after NTS injection. d Masson trichrome- and silver-stained kidney sections of glomeruli from NTS-challenged iPod-miR92a WT mice and iPod-miR92a lox mice at day 10 after NTS injection. Scale bars, 10 μm. e Proportion of crescentic glomeruli in NTS-challenged iPod-miR92a WT and iPod-miR92a lox mice at day 10 after NTS injection. f Blood urea nitrogen concentration at day 10 after NTS injection in iPod-miR92a WT and iPod-miR92a lox mice. g Immunostaining of p57 (strong brown staining, *) in kidney sections from mice described in a. Scale bars 10 µm. h Quantification of p57-positive cells per glomerular section in mice described in a. i Representative photomicrophotographs of dual immunofluorescent satining of p57 (red) and WT1 (green) in kidney sections from mice described in a. Statistical analysis: Mann–Whitney test to compare two groups. Values are means ± s.e.m. (n = 7 per group). *p < 0.05, **p < 0.01 vs. iPod-miR-92a WT mice

Fig. 5

Silencing miR-92a prevents kidney injury in a mouse model of nephrotoxic nephritis. a Study design of the in vivo preventative antagomir experiment. b RT-qPCR analysis of the relative abundance of miR-92ain freshly isolated glomeruli from normal mice (control), NTS-challenged mice (NTN), NTS-challenged mice treated with anti-miR-control (NTN + anti-miR-ctrl) and NTS-challenged mice treated with anti-miR-92a (NTN + anti-miR-92a) after 10 days. All values are normalized to U6snRNA and are relative to control. Values are means ± s.e.m. (n = 4 per group). *p < 0.05 vs. control, ^# p < 0.05 vs. NTN alone. c Representative photomicrographs of p57 staining (scale bars, 10 µm), Masson trichrome- (scale bars, 20 µm), silver-stained kidney sections (scale bars, 10 µm) and transmission electron microscopy (scale bars, 0.5 µm) from groups of mice described in a. d Quantification of p57^Kip2-positive cells per glomerular section in mice described in a. e Proportion of glomerular crescents in kidney sections, f albuminuria, and g blood urea nitrogen concentrations in mice as described in a. Values are means ± s.e.m. (n = 4 per group). *p < 0.05 vs. control, ^# p < 0.05 vs. NTN alone. Statistical analysis: Kruskal–Wallis one-way analysis of variance followed by Dunn’s multiple comparaison test

Fig. 6

miR-92a in vivo silencing abolishes nephrotoxic nephritis development. a Study design of the in vivo antagomir experiment. b Relative miR-92a expression in dynabeads-isolated glomeruli from normal mice (control), NTS-challenged mice (NTN), NTS-challenged mice treated with anti-miR-control (NTN + anti-miR-ctrl), and NTS-challenged mice treated with anti-miR-92a (NTN + anti-miR-92a) after 10 days. All values are normalized to U6 and are relative to control. Values are means ± s.e.m. (n = 5 per group). *p < 0.05 vs. control, ^# p < 0.05 vs. NTS alone. c Urinary albumin excretion rates at day 4 (before antagomir injection) and at day 10 after NTS injection. d Blood urea nitrogen concentration at day 10 after NTS injection in control or NTS-challenged mice. e Silver-stained kidney sections of mice described in b. Scale bars, 10 µm. f Proportion of crescentic glomeruli in kidney from mice described in b. Values are means ± s.e.m. (n = 10 mice per group). g Representative immunostaining of p57 (strong brown staining) in kidney sections from mice described in a. Scale bars, 10 µm. h Quantification of p57-positive cells per glomerular section in mice described in a. Values are means ± s.e.m. (n = 10 per group). *p < 0.05, ^# p < 0.05 vs. NTS alone (NTN). Statistical analysis: Kruskal–Wallis one-way analysis of variance followed by Dunn’s multiple comparaison test