AP39, A Mitochondrially Targeted Hydrogen Sulfide Donor, Exerts Protective Effects in Renal Epithelial Cells Subjected to Oxidative Stress in Vitro and in Acute Renal Injury in Vivo

Abstract

This study evaluated the effects of AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl) phenoxy)decyl) triphenyl phosphonium bromide], a mitochondrially targeted donor of hydrogen sulfide (H2S) in an in vitro model of hypoxia/oxidative stress injury in NRK-49F rat kidney epithelial cells (NRK cells) and in a rat model of renal ischemia-reperfusion injury. Renal oxidative stress was induced by the addition of glucose oxidase, which generates hydrogen peroxide in the culture medium at a constant rate. Glucose oxidase (GOx)-induced oxidative stress led to mitochondrial dysfunction, decreased intracellular ATP content, and, at higher concentrations, increased intracellular oxidant formation (estimated by the fluorescent probe 2, 7-dichlorofluorescein, DCF) and promoted necrosis (estimated by the measurement of lactate dehydrogenase release into the medium) of the NRK cells in vitro. Pretreatment with AP39 (30-300 nM) exerted a concentration-dependent protective effect against all of the above effects of GOx. Most of the effects of AP39 followed a bell-shaped concentration-response curve; at the highest concentration of GOx tested, AP39 was no longer able to afford cytoprotective effects. Rats subjected to renal ischemia/reperfusion responded with a marked increase (over 4-fold over sham control baseline) blood urea nitrogen and creatinine levels in blood, indicative of significant renal damage. This was associated with increased neutrophil infiltration into the kidneys (assessed by the myeloperoxidase assay in kidney homogenates), increased oxidative stress (assessed by the malondialdehyde assay in kidney homogenates) and an increase in plasma levels of IL-12. Pretreatment with AP39 (0.1, 0.2 and 0.3 mg/kg) provided a dose-dependent protection against these pathophysiological alterations; the most pronounced protective effect was observed at the 0.3 mg/kg dose of the H2S donor; nevertheless AP39 failed to achieve a complete normalization of any of the injury markers measured. The partial protective effects of AP39 correlated with a partial improvement of kidney histological scores and reduced TUNEL staining (an indicator of DNA damage and apoptosis). In summary, the mitochondria-targeted H2S donor AP39 exerted dose-dependent protective effects against renal epithelial cell injury in vitro and renal ischemia-reperfusion injury in vivo. We hypothesize that the beneficial actions of AP39 are related to the reduction of cellular oxidative stress, and subsequent attenuation of various positive feed-forward cycles of inflammatory and oxidative processes.

The novel mitochondria-targeted hydrogen sulfide (H 2 S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro

“…We found that AP39 and AP123 were effective against hyperglycemic injury at >1000-fold lower concentrations than Na 2 S in endothelial cells. The cytoprotective concentrations of the compounds (30–300 nM) are similar to the values previously reported for AP39 [34], [35], [56], [57]. The mitochondrial potential normalising and antioxidant effects of the compounds also confirm that H 2 S-mediated cytoprotection depends on its mitochondrial effect in hyperglycemic endothelial cells.…”

Cardioprotection by H2S Donors: Nitric Oxide-Dependent and -Independent Mechanisms

“…A similar bell-shaped curve was also noted for thiovaline, the second most potent H 2 S donor used, exerting beneficial effects at 10 nM. H 2 S donors have been shown to behave in an analogous fashion (bell-shaped concentration-response curves) in many instances Hellmich et al, 2015;Ahmad et al, 2016).…”

Delayed Treatment with Sodium Hydrosulfide Improves Regional Blood Flow and Alleviates Cecal Ligation and Puncture (CLP)-Induced Septic Shock

“…Tissue malondialdehyde (MDA) levels, an index of tissue oxidative stress, were detected in organ homogenates using a fluorimetric MDA-specific lipid peroxidation assay kit (Enzo Life Sciences, Farmingdale, NY) as described (11). The assay is based on the BML-AK171 method in which two molecules of the chromogenic reagent N-methyl-2-phenylindole (NMPI) react with one molecule of MDA at 45°C to yield a stable carbocyanine dye with a maximum absorption at 586 nm.…”

“…Myeloperoxidase activity was measured in organ homogenates using a commercially available myeloperoxidase (MPO) fluorimetric detection kit (Enzo Life Sciences) as described (11). The assay utilizes a non-fluorescent detection reagent, which is oxidized in the presence of hydrogen peroxide and MPO to produce its fluorescent analog.…”