Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction

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Abstract

This study was designed to investigate possible effects of the Rho-kinase inhibitor, fasudil, on the progression of renal failure in rats with unilateral ureteral obstruction. The renal failure markers monitored were the extent of renal interstitial fibrosis and that of macrophage infiltration. In kidneys with unilateral ureteral obstruction, interstitial fibrosis was observed, using Sirius-Red staining, on day 16 after unilateral ureteral obstruction. Macrophage infiltration was observed by immunohistochemistry, using the antibody, ED1. Interstitial fibrosis and macrophage infiltration were significantly attenuated in fasudil-treated animals. The migration of monocytes in vitro elicited by N-formyl-methionyl-leucyl-phenylalanine was potently inhibited by fasudil and its active metabolite, hydroxyfasudil. These results suggest that inhibition of Rho-kinase produces a reduction of macrophage infiltration and represents a new therapeutic strategy for renal fibrosis, a major factor in the progression to end-stage renal failure.

Introduction

The final step in the process of chronic renal failure is renal interstitial fibrosis. To induce renal damage leading to tubulointerstitial fibrosis, chronic unilateral obstruction was used in various species to serve as a well-characterized experimental model Sommer et al., 1999, Ludewig et al., 2000, Moriyama et al., 2001. The molecular and cellular mechanisms of renal interstitial fibrosis are now beginning to be elucidated Guo et al., 1999, Hruska et al., 2000. One of the possible mechanisms has been identified as the inflammatory process, in which infiltrating macrophages play a major role as a source of inflammatory mediators. Inhibiting macrophage infiltration may reduce the production of such inflammatory mediators and may, therefore, present an option in the prevention of progressive renal fibrosis leading to end-stage renal failure (Anders et al., 2002).

Rho-kinase contributes to the reorganization of the actin cytoskeleton and to the formation of stress fibers, and is thought to be one of the critical elements involved in a variety of cytoskeleton-dependent cell functions such as cell migration (Niggli, 1999).

Fasudil is a protein kinase inhibitor that has shown clinical effectiveness in patients with subarachnoid hemorrhage (Shibuya et al., 1992), and that was launched for clinical use after subarachnoid hemorrhage in Japan. Studies in animal models show fasudil to be promising in the treatment of stroke and angina Asano et al., 1991, Satoh et al., 2001a, Satoh et al., 2001b, Utsunomiya et al., 2001. Fasudil and its active metabolite, hydroxyfasudil, inhibit Rho-kinase more effectively than they inhibit other protein kinases; e.g., protein kinase C, or myosin light chain kinase Shimokawa et al., 1999, Shimokawa, 2002, Davies et al., 2000, Nagumo et al., 2000. Using chemoattractants in vitro, we previously showed that neutrophil chemotaxis was inhibited by fasudil and hydroxyfasudil (Satoh et al., 1999a), as was in vivo neutrophil and macrophage infiltration Satoh et al., 1999a, Satoh et al., 2001a, Miyata et al., 2000, Ikegaki et al., 2001.

Nagatoya et al. (2002) reported that pretreatment with Y-27632, another Rho-kinase inhibitor, from 2 days before unilateral ureteral obstruction until the day of killing prevented tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. In the present study, we investigated if the inhibition of Rho-kinase with fasudil or hydroxyfasudil would reduce macrophage infiltration into the kidneys and renal fibrosis after unilateral ureteral obstruction. We endeavored to determine the therapeutic potential of fasudil in renal failure since it is currently the only Rho-kinase inhibitor approved for clinical use to our knowledge. We, therefore, tried to ascertain whether renal fibrosis could be attenuated by fasudil treatment in the post-obstructed kidneys.

Section snippets

Unilateral ureteral obstruction rat model

Male Sprague–Dawley rats (5 weeks old) were used. Rats were anesthetized with ether and underwent left unilateral ureteral ligation on day 1. The first intraperitoneal administration of fasudil (3 or 10 mg/kg) or saline was on day 2. Administration of fasudil or saline once daily was continued until day 15. On day 16, the rats were anesthetized with ether, and obstructed and contralateral kidneys were removed, sliced transversely, and fixed in buffered formalin. Sirius-Red staining was

Effects on renal interstitial fibrosis

On day 16 after unilateral ureteral obstruction, interstitial fibrotic lesions were found in obstructed kidneys (Fig. 1). The percent area of interstitial fibrosis in obstructed kidneys (control: 2.5±0.4%) was significantly greater than that in contralateral kidneys (0.3±0.0%) (Fig. 2). Fasudil at 3 and 10 mg/kg dose dependently inhibited the development of interstitial fibrosis. The mean area of fibrosis in kidneys of rats treated with fasudil 10 mg/kg (1.4±0.2%, P<0.05) was significantly

Discussion

Previous studies have shown that unilateral ureteral obstruction in rats resulted in renal interstitial fibrosis and macrophage infiltration into the obstructed kidneys Takeda et al., 2000, Sato et al., 2001; these morphological changes could also be observed in the present study. The mechanism of initiation and subsequent progression of fibrosis is still far from completely understood. Macrophages are thought to play a central role in the fibroproliferative response. Fibrogenic growth factors

Acknowledgements

The authors thank Mark Smith for pertinent comments.

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