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

Abstract

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-PK 1 ). 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-PK 1 cells (P Ͻ 0.05) but not in HO-1 and HO-2 overexpressing or wild type cells pretreated with hemin (10 M). The presence of imidazole-dioxolane HO inhibitors (2-25 M) decreased cell viability (P Ͻ 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-PK 1 cells. The novel imidazole-dioxolane compounds can be used as effective inhibitors of HO activity in cell culture.

The role of myoglobin degradation in nephrotoxicity after rhabdomyolysis

“…The H chain of ferritin demonstrates high ferroxidase activity inhibiting the redox cycling of iron [21] thus reducing the risk of oxidative stress related to iron oxidation and favoring the cytoprotective effect of heme oxygenase-1 [22,23]. Heme oxygenase-1 deficient mice demonstrated increased sensitivity to heme-containing proteins -mice in the rhabdomyolysis model demonstrated substantially higher levels of blood creatinine and lactate dehydrogenase and an increased death rate while inhibition of heme oxygenase-1 in situ caused heme-induced apoptosis of kidney cells [24]. Myoglobin detoxication depends not only on its proteolytic degradation but also on how active is the subsequent heme and iron degradation system.…”

Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors

“…The study focused on the systematic design and synthesis of analogs of QC-1, which were screened by an in vitro CO-formation assay using microsomal samples derived from rat spleen and brain as the physiological sources of HO-1 and HO-2, respectively. Moreover, analyses on the in vivo efficacy of some select compounds have also been performed in mice and rats (Dercho et al, unpublished results), as well as in cultured cells [30]. This program has led to a series of novel compounds, which do not affect NOS or sGC except at high millimolar concentrations, nor do they induce expression of NOS, sGC, or HO-1.…”

Pharmacological and Clinical Aspects of Heme Oxygenase

“…Zinc-protoporphyrin was developed to treat cancer (Fang et al, 2004b). Recently, imidazole-dioxolane compounds have been shown to act as inhibitors of HO activity (Vlahakis et al, 2006;Sugishima et al, 2007) and shown to be effective as inhibitors of specific HO isoenzymes as well as HO activity (Kinobe et al, 2007).…”