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CELLULAR AND MOLECULAR

Effectiveness of Novel Imidazole-Dioxolane Heme Oxygenase Inhibitors in Renal Proximal Tubule Epithelial Cells

Departments of Pharmacology and Toxicology (R.T.K., Y.J., J.F.B., K.N.) and Chemistry (J.Z.V., W.A.S.), Queen's University, Kingston, Ontario, Canada; and Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, United Kingdom (R.M.)

To enhance our understanding of the physiological roles of heme oxygenase (HO) isozymes, HO-1 (inducible) and HO-2 (constitutive), we developed novel imidazole-based HO inhibitors. Unlike the metalloporphyrins, these imidazole-dioxolane compounds are selective for the in vitro inhibition of HO with minimal effects on other heme-dependent enzymes such as nitric oxide synthase and soluble guanylyl cyclase. In the current study, we tested the hypothesis that these novel HO inhibitors are effective in intact cells by extending their application to cultured, renal proximal tubule epithelial cells (LLC-PK₁). HO-1 and HO-2 protein expression was enhanced by pretreatment of cells with hemin, transduction with adenovirus encoding human HO-1, and transfection with cDNA for HO-2, respectively. Total HO activity was measured by determining the formation of carbon monoxide (CO), whereas cell viability and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the expression of activated caspase-3. Gliotoxin/tumor necrosis factor- (TNF-) produced cytotoxicity in wild-type LLC-PK₁ cells (P < 0.05) but not in HO-1 and HO-2 overexpressing or wild type cells pretreated with hemin (10 µM). The presence of imidazole-dioxolane HO inhibitors (2–25 µM) decreased cell viability (P < 0.05). A CO-releasing molecule reversed, in a dose-dependent manner, the cytotoxic effects and caspase-3 activation induced by the combination of gliotoxin/TNF- and the HO inhibitors, suggesting an important role for CO in protection against renal toxicity. These data demonstrate a protective role of both HO-1 and HO-2 against gliotoxin/TNF--induced cytotoxicity in LLC-PK₁ cells. The novel imidazole-dioxolane compounds can be used as effective inhibitors of HO activity in cell culture.

Address correspondence to: Dr. Kanji Nakatsu, Department of Pharmacology and Toxicology, Queen's University, Kingston, ON K7L 3N5, Canada. E-mail: nakatsuk{at}post.queensu.ca

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