Inhibitory effects of fasudil on renal interstitial fibrosis induced by unilateral ureteral obstruction

Baba,Itsuko; Egi,Yasuhiro; Utsumi,Hiroyuki; Kakimoto,Tetsuhiro; Suzuki,Kazuo

doi:10.3892/mmr.2015.4467

Inhibitory effects of fasudil on renal interstitial fibrosis induced by unilateral ureteral obstruction

Authors:
- Itsuko Baba
- Yasuhiro Egi
- Hiroyuki Utsumi
- Tetsuhiro Kakimoto
- Kazuo Suzuki
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Affiliations: Pharmacology Research Laboratories II, Research Division, Mitsubishi Tanabe Pharma Corporation, Toda‑shi, Saitama 335‑8505, Japan, Safety Research Laboratory, Research Division, Mitsubishi Tanabe Pharma Corporation, Toda‑shi, Saitama 335‑8505, Japan
Published online on: October 21, 2015 https://doi.org/10.3892/mmr.2015.4467
Pages: 8010-8020
Copyright: © Baba et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Renal fibrosis is the major cause of chronic kidney disease, and the Rho/Rho-associated coiled-coil kinase (ROCK) signaling cascade is involved in the renal fibrotic processes. Several studies have reported that ROCK inhibitors attenuate renal fibrosis. However, the mechanism of this process remains to be fully elucidated. The present study assessed the inhibitory effect of fasudil, a ROCK inhibitor using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blot analyses, in vivo and in vitro, to elucidate the mechanisms underlying renal interstitial fibrosis. In mice induced with unilateral ureteral obstruction (UUO), collagen accumulation, the expression of fibrosis‑associated genes and the content of hydroxyproline in the kidney increased 3, 7, and 14 days following UUO. Fasudil attenuated the histological changes, and the production of collagen and extracellular matrix in the UUO kidney. The expression of α‑smooth muscle actin (α‑SMA) and the transforming growth factor‑β (TGFβ)‑Smad signaling pathway, and macrophage infiltration were suppressed by fasudil in the kidneys of the UUO mice. The present study also evaluated the role of intrinsic renal cells and infiltrated macrophages using NRK‑52E, NRK‑49F and RAW264.7 cells. The mRNA and protein expression levels of collagen I and α‑SMA increased in the NRK‑52E and NRK‑49F cells stimulated by TGF‑β1. Hydroxyfasudil, a bioactive metabolite of fasudil, attenuated the increase in the mRNA and protein expression levles of α‑SMA in the two cell types. However, the reduction in the mRNA expression of collagen I was observed in the NRK‑49F cells only. Hydroxyfasudil decreased the mRNA expression of monocyte chemoattractant protein‑1 (MCP‑1) induced by TGF‑β1 in the NRK‑52E cells, but not in the NRK‑49F cells. In the RAW264.7 cells, the mRNA expression levels of MCP‑1, interleukin (IL)‑1β, IL‑6 and tumor necrosis factor α were increased significantly following lipopolysaccharide stimulation, and were not suppressed by hydroxyfasudil. These data suggested that the inhibition of ROCK activity by fasudil suppressed the transformation of renal intrinsic cells into the myofibroblast cells, and attenuated the infiltration of macrophages, without inhibiting the expression or the activation of cytokine/chemokines, in the progression of renal interstitial fibrosis.

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1	Satoh K, Fukumoto Y and Shimokawa H: Rho-kinase: Important new therapeutic target in cardiovascular diseases. Am J Physiol Heart Circ Physiol. 301:H287–H296. 2011. View Article : Google Scholar : PubMed/NCBI
2	Hahmann C and Schroeter T: Rho-kinase inhibitors as therapeutics: From pan inhibition to isoform selectivity. Cell Mol Life Sci. 67:171–177. 2010. View Article : Google Scholar
3	Olson MF: Applications for ROCK kinase inhibition. Curr Opin Cell Biol. 20:242–248. 2008. View Article : Google Scholar : PubMed/NCBI
4	Ming D, Yan BP, Liao JK, Lam YY, Yip GW and Yu CM: Rho-kinase inhibition: A novel therapeutic target for the treatment of cardiovascular diseases. Drug Discov Today. 15:622–629. 2010. View Article : Google Scholar
5	Budzyn K, Marley PD and Sobey CG: Targeting Rho and Rho-kinase in the treatment of cardiovascular disease. Trends Pharmacol Sci. 27:97–104. 2006. View Article : Google Scholar
6	Shimokawa H and Rashid M: Development of Rho-kinase inhibitors for cardiovascular medicine. Trends Pharmacol Sci. 28:296–302. 2007. View Article : Google Scholar : PubMed/NCBI
7	Kushiyama T, Oda T, Yamamoto K, Higashi K, Watanabe A, Takechi H, Uchida T, Oshima N, Sakurai Y, Miura S and Kumagai H: Protective effects of Rho kinase inhibitor fasudil on rats with chronic kidney disease. Am J Physiol Renal Physiol. 304:F1325–F1334. 2013. View Article : Google Scholar : PubMed/NCBI
8	Nishikimi T, Koshikawa S, Ishikawa Y, Akimoto K, Inaba C, Ishimura K, Ono H and Matsuoka H: Inhibition of Rho-kinase attenuates nephrosclerosis and improves survival in salt-loaded spontaneously hypertensive stroke-prone rats. J Hypertens. 25:1053–1063. 2007. View Article : Google Scholar : PubMed/NCBI
9	Kanda T, Wakino S, Hayashi K, Homma K, Ozawa Y and Saruta T: Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats. Kidney Int. 64:2009–2019. 2003. View Article : Google Scholar : PubMed/NCBI
10	Xie X, Peng J, Chang X, Huang K, Huang J, Wang S, Shen X, Liu P and Huang H: Activation of RhoA/ROCK regulates NF-κB signaling pathway in experimental diabetic nephropathy. Mol Cell Endocrinol. 369:86–97. 2013. View Article : Google Scholar : PubMed/NCBI
11	Zhou H, Li YJ, Wang M, Zhang LH, Guo BY, Zhao ZS, Meng FL, Deng YG and Wang RY: Involvement of RhoA/ROCK in myocardial fibrosis in a rat model of type 2 diabetes. Acta Pharmacol Sin. 32:999–1008. 2011. View Article : Google Scholar : PubMed/NCBI
12	Li Y, Zhu W, Tao J, Xin P, Liu M, Li J and Wei M: Fasudil protect the heart against ischemia-reperfusion injury by attenuating endoplasmic reticulum stress and modulating SERCA activity: The differential role for PI3K/Akt and JAK2/STAT3 signaling pathways. PLoS One. 7:e481152012. View Article : Google Scholar
13	Kentrup D, Reuter S, Schnöckel U, Grabner A, Edemir B, Pavenstädt H, Schober O, Schäfers M, Schlatter E and Büssemaker E: Hydroxyfasudil-mediated inhibition of ROCK1 and ROCK2 improves kidney function in rat renal acute ischemia-reperfusion injury. PLoS One. 6:e264192011. View Article : Google Scholar : PubMed/NCBI
14	Matoba K, Kawanami D, Okada R, Tsukamoto M, Kinoshita J, Ito T, Ishizawa S, Kanazawa Y, Yokota T, Murai N, et al: Rho-kinase inhibition privents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1α. Kidney Int. 84:545–554. 2013. View Article : Google Scholar : PubMed/NCBI
15	Diah S, Zhang GX, Nagai Y, Zhang W, Gang L, Kimura S, Hamid MR, Tamiya T, Nishiyama A and Hitomi H: Aldosterone induced mypfibroblastic transdifferentiation and collagen gene expression through the Rho-kinase dependent signaling pathway in rat mesangial cells. Exp Cell Res. 314:3654–3662. 2008. View Article : Google Scholar : PubMed/NCBI
16	Wei J, Li Z, Ma C, Zhan F, Wu W, Han H, Huang Y, Li W, Chen D and Peng Y: Rho kinase pathway is likely responsible for the profibrotic actions of aldosterone in renal epithelial cells via inducing epithelial-mesenchymal transition and extracellular matrix excretion. Cell Biol Int. 37:725–730. 2013. View Article : Google Scholar : PubMed/NCBI
17	Manickam N, Patel M, Griendling KK, Gorin Y and Barnes JL: RhoA/Rho kinase mediates TGF-β1-induced kidney myofi-broblast activation through Poldip2/Nox4-derived reactive oxygen species. Am J Physiol Renal Physiol. 307:F159–F171. 2014. View Article : Google Scholar : PubMed/NCBI
18	Nagatoya K, Moriyama T, Kawada N, Takeji M, Oseto S, Murozono T, Ando A, Imai E and Hori M: Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int. 61:1684–1695. 2002. View Article : Google Scholar : PubMed/NCBI
19	Satoh S, Yamaguchi T, Hitomi A, Sato N, Shiraiwa K, Ikegaki I, Asano T and Shimokawa H: Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction. Eur J Pharmacol. 455:169–174. 2002. View Article : Google Scholar : PubMed/NCBI
20	Takeda Y, Nishikimi T, Akimono K, Matsuoka H and Ishimitsu T: Benefical effects of a combination of Rho-kinase inhibitor and ACE inhibitor on tubulointerstitial fibrosis induced by unilateral ureteral obstruction. Hypertens Res. 33:965–973. 2010. View Article : Google Scholar : PubMed/NCBI
21	Kakimoto T, Kimata H, Iwasaki S, Fukunari A and Utsumi H: Automated recognition and quantification of pancreatic islets in Zucker diabetic fatty rats treated with exendin-4. J Endocrinol. 216:13–20. 2013. View Article : Google Scholar
22	Woessner JF Jr: The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys. 93:440–447. 1961. View Article : Google Scholar : PubMed/NCBI
23	Kivirikko KI, Laitinen O and Prockop DJ: Modifications of a specific assay for hydroxyproline in urine. Anal Biochem. 19:249–255. 1967. View Article : Google Scholar : PubMed/NCBI
24	Chevalier RL, Forbes MS and Thornhill BA: Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int. 75:1145–1152. 2009. View Article : Google Scholar : PubMed/NCBI
25	Chevalier RL: Obstructive nephropathy: Towards biomarker discovery and gene therapy. Nat Clin Pract Nephrol. 2:157–168. 2006. View Article : Google Scholar : PubMed/NCBI
26	Shen B, Liu X, Fan Y and Qiu J: Macrophages regulate renal fibrosis through modulating TGFβ superfamily signaling. Inflammation. 37:2076–2084. 2014. View Article : Google Scholar : PubMed/NCBI
27	Yang HC, Zuo Y and Fogo AB: Models of chronic kidney disease. Drug Discov Today Dis Models. 7:13–19. 2010. View Article : Google Scholar
28	Löpez-Novoa JM, Martinez-Salgado C, Rodriguez-Peña AB and López-Hernández FJ: Common pathophysiological mechanisms of chronic kidney disease. Therapeutic perspectives Pharmacol Ther. 128:61–81. 2010. View Article : Google Scholar
29	Truong LD, Gaber L and Eknoyan G: Obstructive uropathy. Contrib Nephrol. 169:311–326. 2011. View Article : Google Scholar : PubMed/NCBI
30	Eddy AA, López-Guisa JM, Okamura DM and Yamaguchi I: Investigating mechanizes of chronic kidney disease in mouse models. Pediatr Nephrol. 27:1233–1247. 2012. View Article : Google Scholar
31	Fu P, Liu F, Su S, Wang W, Huang XR, Entman ML, Schwartz RJ, Wei L and Lan HY: Signaling mechanism of renal fibrosis in unilateral ureteral obstructive kidney disease in ROCK1 knockout mice. J Am Soc Nephrol. 17:3105–3114. 2006. View Article : Google Scholar : PubMed/NCBI
32	Ballhause TM, Soldati R and Mertens PR: Sources of myofibroblats in kidney fibrosis: All answers are correct, however to different extent! Int Urol Nephrol. 46:659–664. 2014. View Article : Google Scholar
33	Duffield JS: Cellular and molecular mechanisms in kidney fibrosis. J Clin Invest. 124:2299–2306. 2014. View Article : Google Scholar : PubMed/NCBI
34	Pan SY, Chang YT and Lin SL: Microvascular pericytes in healthy and diseased kidneys. Int J Nephrol Renovascul Disease. 7:39–48. 2014.
35	LeBleu VS, Taduri G, O'Connell J, Teng Y, Cooke VG, Wada C, Sugimoto H and Kalluri R: Origin and function of myofibroblasts in kidney fibrosis. Nat Med. 19:1047–1053. 2013. View Article : Google Scholar : PubMed/NCBI
36	Jang HS, Kim JI, Jung KJ, Kim J, Han KH and Park KM: Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site. Biochim Biophys Acta. 1832:817–825. 2013. View Article : Google Scholar : PubMed/NCBI
37	Qin J, Xie YY, Huang L, Yuan QJ, Mei WJ, Yuan XN, Hu GY, Cheng GJ, Tao LJ and Peng ZZ: Fluorofenidone inhibits nicotinamide adeninedinucleotide phosphate oxidase via PI3K/Akt pathway in the pathogenesis of renal interstitial fibrosis. Nephrology (Carlton). 18:690–699. 2013.
38	Gu L, Gao Q, Ni L, Wang M and Shen F: Fasudil inhibits epithelialmyofibroblast transdifferentiation of human renal tubular epithelial HK-2 cells induced by high glucose. Chem Pharm Bull (Tokyo). 61:688–694. 2013. View Article : Google Scholar
39	Gande MT, Pèrez-Barriocanal F and López-Novoa JM: Role of inflammation in tubule-interstitial damage associated to obstructive nephropathy. J Inflamm (Lond). 7:192010. View Article : Google Scholar
40	Wang Y and Harris DC: Macrophages in renal disease. J Am Soc Nephrol. 22:21–27. 2011. View Article : Google Scholar : PubMed/NCBI
41	Chung AC and Lan HY: Chemokines in renal injury. J Am Soc Nephrol. 22:802–809. 2011. View Article : Google Scholar : PubMed/NCBI
42	Liu N, Tolbert E, Pang M, Ponnusamy MI, Yan H and Zhuang S: Suramin inhibits renal fibrosis in chronic kidney disease. J Am Soc Nephrol. 22:1064–1075. 2011. View Article : Google Scholar : PubMed/NCBI
43	Pang M, Ma L, Gong R, Tolbert E, Mao H, Ponnusamy M, Chin YE, Yan H, Dworkin LD and Zhuang S; A novel STAT3 inhibitor: S31–201. attenuates renal interstitial fibroblast activation and interstitial fibrosis in obstructive nephropathy. Kidney Int. 78:257–268. 2010. View Article : Google Scholar
44	Meng XM, Huang XR, Xiao J, Chen HY, Zhong X, Chung AC and Lan HY: Diverse roles of TGF-β receptor II in renal fibrosis and inflammation in vivo and in vitro. J Pathol. 227:175–188. 2012. View Article : Google Scholar
45	Anders HJ and Ryu M: Renal microenvironments and macrophage phenotypes determine progression or resolution of renal inflammation and fibrosis. Kidney Int. 80:915–925. 2011. View Article : Google Scholar : PubMed/NCBI
46	Ricardo SD, van Goor H and Eddy AA: Macrophage diversity in renal injury and repair. J Clin Invest. 118:3522–3530. 2008. View Article : Google Scholar : PubMed/NCBI