Targeting of Rho kinase ameliorates impairment of diabetic endothelial function in intrarenal artery

Abstract

Endothelial dysfunction in kidney vasculature is the initial and key element for nephropathy in diabetes mellitus. Accumulating evidence suggests the protective role of Rho kinase inhibitors in endothelial dysfunction via modulating eNOS activity and NO production. However, the role of Rho kinase in diabetes-related endothelial dysfunction in kidney vasculature and the relevant mechanisms remain unknown. We assessed whether pharmacological inhibition of Rho kinase attenuates endothelial dysfunction in intrarenal arteries from type 1 diabetic rats. Fasudil, a Rho kinase inhibitor effectively decreased the phosphorylated level of MYPT1 without affecting the expression of ROCKs in the kidney. Fasudil treatment showed no improvement in diabetes-related abnormality in metabolic indices, but it significantly ameliorated endothelial dysfunction in intrarenal arteries and lessened the mesangial matrix expansion in the kidney cortex. Mechanistically, superoxide production in the intrarenal artery and NOX4 member of NADPH oxidase in the renal cortex that contribute to diabetic nephropathy were also prevented by the Rho kinase inhibitor. In conclusion, the present results indicate that Rho kinase is involved in endothelial dysfunction in type 1 diabetes via enhancement of oxidative stress and provides new evidence for Rho kinase inhibitors as potential therapeutic agents for the treatment of diabetic nephropathy.

Figure 1

Effects of Rho kinase inhibition on metabolic parameters in diabetic rats. (A) Body weight of rats after 4-week treatment with fasudil; (B) Blood glucose of rats after 4-week treatment with fasudil; (C–E) Total cholesterol level (C), triglyceride level (D) and creatinine level (E) in plasma of rats after 4-week treatment with fasudil; (F) Blood pressure of rats during 4-week treatment with fasudil; (G,H) kidney weight, kidney weight/body weight ratio (G) and liver weight, liver weight/body weight ratio (H) of rats after 4-week treatment with fasudil. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group. Data are mean ± S.E.M. from five to six different rats.

Figure 1

Effects of Rho kinase inhibition on metabolic parameters in diabetic rats. (A) Body weight of rats after 4-week treatment with fasudil; (B) Blood glucose of rats after 4-week treatment with fasudil; (C–E) Total cholesterol level (C), triglyceride level (D) and creatinine level (E) in plasma of rats after 4-week treatment with fasudil; (F) Blood pressure of rats during 4-week treatment with fasudil; (G,H) kidney weight, kidney weight/body weight ratio (G) and liver weight, liver weight/body weight ratio (H) of rats after 4-week treatment with fasudil. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group. Data are mean ± S.E.M. from five to six different rats.

Figure 2

Effect of Rho kinase inhibition on vascular dysfunction in intrarenal artery from diabetic rats. (A) Representative traces showing acetylcholine (ACh)-induced relaxations in phenylephrine (Phe)-contracted artery rings from control rats (a); diabetic rats (b); diabetic rats treated with fasudil (c) and diabetic rats but with rings acutely incubated in 1 μM Y27632 for 30 min (d); (B) Concentration-response curves for ACh in artery rings from rats treated with or without fasudil; (C) Concentration-response curves for ACh in artery rings with or without acute incubation of Y27632; (D) Representative traces showing acetylcholine (ACh)-induced contractions of artery rings in presence of L-NAME from control rats (a); diabetic rats (b) and diabetic rats treated with fasudil (c); (E) Concentration-response curves in artery rings for ACh with L-NAME pretreatment; (F–H) Concentration-response curves in artery rings in response to U46619 (F), phenylephrine (G) and KCl (H). *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group at respective concentration. Data are mean ± S.E.M. from four–five different animals.

Figure 3

Effects of Rho kinase inhibition on oxidative stress in intrarenal artery and EDCFs in plasma from diabetic rats. (A) The concentrations of TxB₂ and PGF_2α in plasma of rats after 4-week treatment with fasudil. (B) Fluorescent photomicrographs at identical settings of sections of intrarenal artery labeled with dihydroethidium and quantification of the fluorescent ethidium signal in the artery by average intensity. Scale bar is 50 μm shown in the picture. *p < 0.05, ***p < 0.001 compared with control group; ^##p < 0.01 when compared diabetic rats with and without treatment of fasudil. Data are mean ± S.E.M. from four–six different animals.

Figure 3

Effects of Rho kinase inhibition on oxidative stress in intrarenal artery and EDCFs in plasma from diabetic rats. (A) The concentrations of TxB₂ and PGF_2α in plasma of rats after 4-week treatment with fasudil. (B) Fluorescent photomicrographs at identical settings of sections of intrarenal artery labeled with dihydroethidium and quantification of the fluorescent ethidium signal in the artery by average intensity. Scale bar is 50 μm shown in the picture. *p < 0.05, ***p < 0.001 compared with control group; ^##p < 0.01 when compared diabetic rats with and without treatment of fasudil. Data are mean ± S.E.M. from four–six different animals.

Figure 4

Effects of Rho kinase inhibition on mesangial matrix expansion and expression of NADPH oxidase subunits in renal cortex from diabetic rats. (A) Representative photomicrographs of PAS–stained kidney. a, control rats; b, diabetic rats; c, diabetic rats treated with fasudil. Scale bar is 50 μm shown in the picture. B–D. Quantitative analysis of mesangial area (B), glomerular area (C), and matrix fraction (D); (E) Effect of Rho kinase inhibition on gene expression of NADPH oxidase subunits/members in the renal cortex of diabetic rats. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group; ^#p < 0.05, ^##p < 0.01 when compared diabetic rats with and without treatment of fasudil. Data are mean ± S.E.M. from five different animals.

Figure 5

Effects of Rho kinase inhibition on Rho kinase expression and activity in kidney of diabetic rats. (A) Representative bands and analysis of protein levels of ROCK I and ROCK II in renal cortex of rats; (B) Representative bands and analysis of protein levels of phosphorylated form of MYPT1 (P-MYPT1) from the renal cortex and medulla; (C) Quantitative analysis and representative images of immunohistochemistry for P-MYPT1 from the renal cortex. a, control rats; b, diabetic rats; c, diabetic rats treated with fasudil; d, statistical analysis of immunohistochemistry for P-MYPT1 in renal cortex. **p < 0.01, ***p < 0.001 compared with control group; ^###p < 0.001 when compared diabetic rats with and without treatment of fasudil. Data are mean ± S.E.M. from five different rats.