Benidipine protects kidney through inhibiting ROCK1 activity and reducing the epithelium-mesenchymal transdifferentiation in type 1 diabetic rats

Abstract

We investigated the protective effect of benidipine, by testing the changes of the activity of Rho kinase and transdifferentiation of renal tubular epithelium cells in vivo. Wistar rats were randomly divided into two groups: normal (N) and diabetes. STZ were used to make the rats type 1 diabetic and were randomly assigned as diabetes without treatment (D), diabetes treated with benidipine (B), and diabetes treated with fasudil (F) and treated for 3 months. Immunohistochemistry and western blotting were for protein expressions of ROCK1, α-SMA, and E-cadherin and real-time PCR for the mRNA quantification of ROCK1. Compared with N group, D group had significant proliferation of glomerular mesangial matrix, increased cell number, thickened basement membrane, widely infiltrated by inflammatory cells and fibrosis in the renal interstitial, and dilated tubular. Those presentations in F and B groups were milder. Compared with N group, D group showed elevated MYPT1 phosphorylation, increased expression of ROCK1, α-SMA protein, and ROCK1 mRNA and decreased expression of E-cadherin protein. B group showed attenuated MYPT1 phosphorylation, decreased ROCK1, α-SMA protein, and ROCK1 mRNA expression and increased expression of E-cadherin protein. In conclusion, benidipine reduces the epithelium-mesenchymal transdifferentiation and renal interstitial fibrosis in diabetic kidney by inhibiting ROCK1 activity.

Figure 1

The pathological changes of renal glomerular and interstitial space (×400). (a), (b), (c), and (d) represent N group, D group, F group, and B group, respectively, with HE staining. (a) showed normal tubular; (b) showed tubular dilated and distorted with inflammatory infiltration and fibrosis in renal interstitial space; (c) and (d) showed that the distorted tubular with inflammatory infiltration and fibrosis in renal interstitial were significantly reduced. Blue arrow: tubular dilation; red arrow: inflammatory cell infiltration.

Figure 2

Immunohistochemistry expression (envision ×400). (a), (e), and (i) are for N group; (b), (f), and (j) are for D group; (c), (g), and (k) are for F group; and (d), (h), and (l) are for B group. (a), (b), (c), and (d) are the expression of ROCK1; (e), (f), (g), and (h) are the expression of α-SMA; (i), (j), (k), and (l) are the expression of E-cadherin. The brown color in plasma, membrane represented positive expression. (a) showed little expression of ROCK1 in renal tubular epithelium cells; (b) showed widely expressed ROCK1 in renal tubular epithelium cells; (c) and (d) showed the range and degree of staining slighter than those of B group. (e) showed that α-SMA is only expressed in the smooth muscle cells of renal small artery; (f) showed that α-SMA is expressed in renal tubular epithelium cells; (g) and (h) showed no expression in renal epithelium cells; (i) showed the expression of E-cadherin in renal tubular epithelium membrane; (j) showed no expression; (k) and (l) showed partial expression of E-cadherin on the surface of the cell membrane.

Figure 3

The protein expression changes of p-MYPT1, ROCK1, E-cadherin, and α-SMA.

Figure 4

The mRNA expression of ROCK1. Compared with N group, P < 0.01; compared with D group, P < 0.01.