A Rho-kinase inhibitor, fasudil, attenuates progressive glomerulosclerosis induced by daunorubicin in rats

Abstract

Accumulating evidence suggests that the small G protein Rho and its downstream effector Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor, fasudil, on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group (sham group, n=12), unilateral nephrectomy (UNX)+daunorubicin (DRB) group (model group, n=12), UNX+DRB+Fasudil group (treatment group, n=12). Two to four weeks after the establishment of the animal model, 6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were examined by HE and PAS staining, immunohistochemistry and transmission electric microscopy (TEM). The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group (P<0.01). But this increase was significantly suppressed by fasudil (P<0.05). At 4 week, the foot process effacement in podocytes, mesangial proliferation and ECM accumulation were observed in model group, presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen (PCNA)-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover, fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.

Keywords

Rho-kinase / glomerulosclerosis / P27 / fasudil

References

[1] WettschureckN., OffermannsS.. Rho/Rho-kinase mediated signaling in physiology and pathophysiology. J Mol Med, 2002, 80(10): 629-638

[2] NishikimiT., MatsuokaH.. Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effect of Rho-kinase inhibitor in hypertensive glomerulosclerosis. J Pharmacol Sci, 2006, 100(1): 22-28

[3] UehataM., IshizakiT., StaohH., et al.. Calcium sensitization of smooth muscle mediated by Rho-associated protein kinase in hypertension. Nature, 1997, 389(6654): 990-994

[4] TakaharaA., SugiyamaA., SatohY., et al.. Cardiovascular effects of Y-27632, a selective Rho-associated kinase inhibitor, assessed in the halothane-anesthetized canine model. Eur J Pharmacol, 2003, 460(1): 51-57

[5] YasushiM., HiroakiS., TetsuyaM., et al.. Involvment of Rho kinase in hypertensive vascular disease: a novel therapeutic target in hypertention. FASEB, 2001, 15(6): 1062-1064

[6] MoriyamaT., NagatoyaK.. The Rho-ROCK system as a new therapeutic target for preventing interstitial fibrosis. Drug News Perspect, 2004, 17(1): 29-34

[7] KobayashiN., HaraK., TojoA., et al.. Eplerenone shows renoprotective effect by reducing LOX1-mediated adhesion molecule, PKCepsilon-MAPK-p90RSK, and Rho-kinase pathway. Hypertension, 2005, 45(4): 538-544

[8] NagatoyaK., MoriyamaT., KawadaN., et al.. Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int, 2002, 61(5): 1684-1695

[9] Heusinger-RibeiroJ., EberleinM., WahabN.A., et al.. Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP. J Am Soc Nephrol, 2001, 12(9): 1853-1861

[10] EndlichN., KressK.R., ReiserJ., et al.. Podocytes respond to mechanical stress in vitro. J Am Soc Nephrol, 2001, 12(3): 413-422

[11] SunG.P., KohnoM., GuoP., et al.. Involvements of Rho-kinase and TGF-beta pathways in aldosteroneinduced renal injury. J Am Soc Nephrol, 2006, 17(8): 2193-2201

[12] DaneshF.R., SadeghiM.M., AmroN., et al.. 3-hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/p21 signaling pathways: implications for diabetic nephropathy. Proc Natl Acad Sci, 2002, 99(12): 8301-8305

[13] SharpeC.C., HendryB.M.. Signaling: focus on Rho in renal disease. J Am Soc Nephrol, 2003, 14(1): 261-264

[14] SatohS., YamguchiT., HitomiA., et al.. Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction. Eur J Pharmacol, 2002, 455(2–3): 169-174

[15] TeraishiK., KurataH., NakajimaA., et al.. Preventive effect of Y-27632, a selective Rho-kinase inhibitor, on ischemia/reperfusion-induced acute renal failure in rats. Eur J Pharmacol, 2004, 505(1–3): 205-211

[16] MundelP., KrizW.. Structure and function of podocytes: An update. Anat Embryol, 1995, 192(5): 385-397

[17] MorigiM., BuelliS., AngiolettiS., et al.. In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies. Am J Pathol, 2005, 166(5): 1309-1320

[18] ShibataS., NagaseM., FujitaT., et al.. Fluvastatin ameliorates podocyte injury in proteinuric rats via modulation of excessive Rho signaling. J Am Soc Nephrol, 2006, 17(3): 754-764

[19] SeasholtzT.M., ZhangT., MorissetteM.R., et al.. Increased expression and activity of RhoA are associated with increased DNA synthesis and reduced p27Kip1 expression in the vasculature of hypertensive rats. Circ Res, 2001, 89(6): 488-495

[20] BishopA.L., HallA.. Rho GTPases and their effector proteins. Biochem J, 2000, 348(2): 241-255

[21] IshizakiT., NaitoM., FujisawaK., et al.. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett, 1997, 404(2–3): 118-124

[22] AmanoM., FukataY., KaibuchiK.. Regulation and functions of Rho-associated kinase. Exp Cell Res, 2000, 261(1): 44-51

[23] IwamotoH., NakamutaM., TadaS., et al.. A p160ROCKspecific inhibitor, Y-27632, attenuates rat hepatic stellate cell growth. J Hepatol, 2000, 32(5): 762-770

[24] ChiaraM., MenegattiE., Di SimoneD., et al.. Mycophenolate mofetil and roscovitine decrease cyclin expression and increase p27(kip1) expression in anti Thy1 mesangial proliferative nephritis. Clin Exp Immunol, 2005, 139(2): 225-235

[25] VidalA., MillardS.S., MillerJ.P., et al.. Rho activity can alter the translation of p27 mRNA and is important for Rasv12-induced transformation in a manner dependent on p27 status. J Biol Chem, 2002, 277(19): 16433-16440

[26] CroftD.R., OlsonM.F.. The Rho GTPase effector ROCK regulates cyclinA, cyclinD1 and p27kip levels by distinct mechanisms. Mol Cell Biol, 2006, 26(12): 4612-4627