MicroRNA-23b Targets Ras GTPase-Activating Protein SH3 Domain-Binding Protein 2 to Alleviate Fibrosis and Albuminuria in Diabetic Nephropathy

Abstract

Diabetic nephropathy (DN) is a frequent and severe complication of diabetes that is structurally characterized by glomerular basement membrane thickening, extracellular matrix accumulation, and destabilization of podocyte foot processes. MicroRNAs (miRNAs) are dysregulated in DN, but identification of the specific miRs involved remains incomplete. Here, we confirm that the peripheral blood from patients with diabetes and the kidneys of animals with type 1 or 2 diabetes have low levels of miR-23b compared with those of their nondiabetic counterparts. Furthermore, exposure to high glucose downregulated miR-23b in cultured kidney cells. In contrast, renal expression of Ras GTPase-activating protein SH3 domain-binding protein 2 (G3BP2), a putative miR-23b target, increased in DN. In vitro, overexpression of miR-23b decreased, and inhibition of miR-23b increased, G3BP2 expression levels. Bioinformatics analysis also revealed p53 binding sites in the miR-23b promoter; in vitro inhibition of p53 or the upstream p38 mitogen-activated protein kinase (p38MAPK) upregulated miR-23b expression in high-glucose conditions. In turn, inhibition of G3BP2 or overexpression of miR-23b downregulated p53 and p38MAPK expression in high-glucose conditions. In vivo, overexpression of miR-23b or inhibition of p53 in db/db mice reversed hyperalbuminuria and kidney fibrosis, whereas miR-23b antagomir treatment promoted renal fibrosis and increased albuminuria in wild-type mice. These data suggest that hyperglycemia regulates pathogenic processes in DN through an miR-23b/G3BP2 feedback circuit involving p38MAPK and p53. In conclusion, these results reveal a role for miR-23b in DN and indicate a novel potential therapeutic target.

Keywords: chronic kidney disease; diabetes; diabetic nephropathy.

Figure 1.

miR-23b levels and G3BP2 expression. (A) Quantification of miR-23b levels in the serum of patients with type 1 (T1DM, n=8) or type 2 (T2DM, n=21) diabetes, DN (n=19), and healthy controls (Con). (B, C) miR-23b expression levels in the kidneys of type 1/2 diabetic mice (Dia, db/db, n=5) and controls (Con, n=5). (D–F) Quantification of miR-23b levels in HEK 2 cells, HRGE cells, and podocytes exposed to high glucose (HG; 25 mmol/L D-glucose) compared with normal glucose (NG; 5 mmol/L D-glucose) and osmotic control (OSM; 25 mmol/L L-glucose) levels. (G) miR-23b expression level correlated with the urine protein levels of patients with type 2 diabetes. (H) miR-23b expression levels in various tissues of normal C57BL/6J mice (n=4). (I, J) Quantification of G3BP2 gene expression in the HEK 2 cells exposed to HG and in the kidneys of type 2 diabetic db/db mice (n=5). Data are expressed as the mean±SEM. *P<0.05; **P<0.01; ***P<0.001. HEK, human embryonic kidney.

Figure 2.

G3BP2 is a target of miR-23b. (A) G3bp2 mRNA expression levels in various tissues of normal C57BL/6J mice (n=4). (B) Effects of miR-23b agomir [miR-23b(+)] and miRNA negative controls (miRNEG) on G3BP2 protein levels, compared with HG, NG, and OSM (lower panel shows quantification). (C) Immunofluorescence staining for G3BP2 from HG-, NG-, miR-23b antagomir [miR-23b(-)]-, p38 inhibitor (SB203580)-, miRNEG-, and OSM-treated HK-2 cells. (D) Luciferase activity in HEK 293A cells that were transfected with the indicated 3′-UTR reporter constructs showing binding of miR-23b with the 3′-UTR of G3BP2. (E) Immunofluorescence staining for FN from HEK 2 cells treated with HG, miR-23b agomir [miR-23b(+)], G3BP2 siRNA, SB203580, miR-23b antagomir [miR-23b(-)], siRNEG, miRNEG, and miRNA antagomir negative control (anti-miRNEG). Data are expressed as the mean±SEM. *P<0.05; ***P<0.001. DAPI, 4′,6-Diamidino-2-phenylindole dihydrochloride; HEK, human embryonic kidney; Mut1, mutant 1; Mut2, mutant 2; NC, negative control; wt1, wild-type 1; wt2, wild-type 2.

Figure 3.

miR-23b/G3BP2 feedback circuitry. (A–C) Quantification of P53 mRNA expression in HK-2 cells treated with HG, p38MAPK inhibitor SB203580, miR-23b(+), and G3BP2 siRNA (G3BP2 siR1, 2, 3). (D–F) Quantification of miR-23b expression levels in HK-2 cells exposed to HG, p53 siRNA, SB203580, and G3BP2 siRNA. (G) Quantification of G3BP2 mRNA expression in HK-2 cells incubated with SB203580 exposed to HG. (H) Western blot analyses for p38, phosphorylated p38 (p-p38), p53, and phosphorylated p53 (p-p53) from HG, NG, miR-23b agomir, miRNEG, and OSM (right panels showing quantification). (I) A schematic model of potential regulation and function of miR-23b in diabetic nephropathy. Data are expressed as the mean±SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

Intravenous injection of miR-23b and G3BP2 siRNA alleviate albuminuria in diabetes. (A–C) Quantification of body weight (BW), abdominal fat, and food consumption of db/db mice with or without miR-23b agomir [miR-23b(+)] or G3bp2 siRNA (G3BP2siR) injection weekly for 1 month (for P values [see below], * indicates miR-23b versus db/db, and ^# indicates G3bp2 siRNA versus db/db, n=5). (D–G) Quantification of serum glucose and insulin levels, insulin resistance index, and insulin sensitivity index from db/db mice and miR-23b agomir- or G3bp2 siRNA-treated mice. (H) Quantification of p53 gene expression from the kidney of db/db, miR-23b agomir-, G3bp2 siRNA-, and miRNA- or siRNA-negative-control-injected mice. (I) Western blot analysis for p38, phosphorylated p38 (p-p38), p53, and phosphorylated p53 (p-p53) protein expression from db/db, miR-23b agomir-, G3bp2 siRNA-, miRNA-, or siRNA-negative-control-treated mice; right panel shows the quantification of p-p38/p38 and p-p53/p53. (J–K) Quantification of MAU (microalbuminuria) and albumin-to-creatinine (ACR) ratio levels from db/db, miR-23b agomir-, and G3bp2 siRNA-treated mice. (L) Representative electron microscopic image of the glomeruli staining from kidney sections of miR-23b agomir- and G3bp2 siRNA-treated mice (dotted red line in left-hand boxes shows area expanded in right-hand boxes; scale bar =10 μm (left-hand boxes), 2 μm (right-hand boxes); arrowhead = GBM; white asterisk = mesangial expansion). (M) Quantification of GBM thickening after miR-23b or G3bp2 siRNA injection into db/db mice. Data are shown as the mean±SEM. *P<0.05; **P<0.01; ***P<0.001. Con, nondiabetic control.

Figure 5.

Intravenous injection of miR-23b and G3bp2 siRNA alleviate fibrosis in diabetic kidneys. (A) Representative images of kidney sections for PAS-stained sections (top panel), FN-stained sections (second panel), collagen I–stained sections (third panel), and collagen IV–stained sections (fourth panel). (B) Quantitation of mRNA expression levels of various markers of fibrosis in db/db mouse kidneys with or without miR-23b agomir or G3bp2 siRNA injection. Data are shown as the mean±SEM. *P<0.05; **P<0.01; ***P<0.001. Acta2, alpha-actin-2; Colla1, collagen I; Coll4a1, collagen IV; Con, nondiabetic control; miR-23b(+), miR-23b agomir; Pai1, plasminogen activator inhibitor 1; Tgfβ1, transforming growth factor beta 1; Timp1, TIMP metallopeptidase inhibitor 1.

Figure 6.

Inhibition of miR-23b in normal mice promoted kidney fibrosis and increased urine protein excretion. (A–C) Representative images of PAS and FN staining of glomeruli (first panel) and renal tubules (second panel, inset image is augmentative tubules) in normal C57BL/6J mice after miR-23b antagomir (Anti–miR-23b) injection for 1 month (n=4). (D) Quantification of mRNA expression levels of various markers of fibrosis in normal mouse kidneys after miR-23b antagomir injection. (E) Electron microscopic analyses of the podocyte foot-processes showed miR-23b antagomir [miR-23b(-)] caused kidney dysfunction (dotted red line in left-hand boxes shows area expanded in right-hand boxes; scale bar =10 μm (left-hand boxes), 2 μm (right-hand boxes); arrowhead = GBM; white asterisk = mesangial expansion). (F–H) miR-23b antagomir injection in the normal mice increased microalbuminuria (MAU), albumin-to-creatinine ratio (ACR), and N-acetyl-β-d-glucosaminidase (NAG) in parallel. (I) Quantification of increased GBM thickening in miR-23b antagomir-injected mice compared with controls. (J–K) miR-23b antagomir injection increased p53 expression and phosphorylated p38MAPK (p-p38MAPK)/p38 ratio. Data are shown as the mean±SEM. *P<0.05; **P<0.01; ***P<0.001. Acta2, alpha-actin-2; Colla1, collagen I; Coll4a1, collagen IV; Con, control; Pai1, plasminogen activator inhibitor 1; p-p38, phosphorylated p38; p-p53, phosphorylated p53; Tgfβ1, transforming growth factor beta 1; Timp1, TIMP metallopeptidase inhibitor 1.