Received July 20, 2007
Accepted on May 26, 2008

BASIC RESEARCH

Inhibition of Renal Rho Kinase Attenuates Ischemia/Reperfusion-Induced Injury

Jai Prakash ^*1, Martin H. de Borst , Marie Lacombe , Frank Opdam , Pieter A. Klok , Harry van Goor , Dirk K.F. Meijer ^*, Frits Moolenaar ^*, Klaas Poelstra ^*, and Robbert J. Kok ^*

*Department of Pharmacokinetics and Drug Delivery, Groningen Research Institute for Pharmacy, University of Groningen, and Department of Pathology and Experimental Medicine, University Medical Centre Groningen, Groningen, Kreatech Biotechnology, Amsterdam, and Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands

¹ To whom correspondence should be addressed. E-mail: j.prakash{at}rug.nl.

	Abstract

The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen I1, TGF-1, tissue inhibitor of metalloproteinase 1, and -smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of -smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.