Blockade of calcium influx through L-type calcium channels attenuates mitochondrial injury and apoptosis in hypoxic renal tubular cells

In hypoxia, ATP depletion causes cellular Ca²⁺ increase, mitochondrial injury, and apoptosis in renal tubular cells. However, the molecular basis of these observations is incompletely delineated. IRPTC, a rat renal proximal tubular cell line, was treated with antimycin A, and disturbances in cytoplasmic calcium ([Ca²⁺]c) and mitochondrial calcium ion concentration ([Ca²⁺]m), dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c release, and resultant apoptosis were examined. Pharmacologic targeting of L-type Ca²⁺ channels in vitro and in vivo was used to clarify the involvement of voltage-dependent Ca ²⁺ channels during this process. In vitro studies indicated that ATP depletion-induced apoptosis was preceded by increased [Ca²⁺]c and [Ca²⁺]m before activation of mitochondrial signaling. Antagonizing L-type Ca²⁺ channels offset these findings, suggesting [Ca ²⁺]c and [Ca²⁺]m involvement. Azelnidipine administration ameliorated cellular and mitochondrial Ca²⁺ accumulation, mitochondrial permeability transition, cytochrome c release, caspase-9 activation, and resultant apoptosis (15.8 ± 0.8% versus 8.9 ± 0.7%; P < 0.01). Similar effects of azelnidipine were substantiated in an in vivo ischemia/reperfusion injury model. There were fewer terminal- deoxynucleotidyl transferase mediated dUTP nick-end labeling-positive cells in the azelnidipine-treated group (0.322 ± 0.038/tubule) as compared with the vehicle-treated group (0.450 ± 0.041; P < 0.05), although the antiapoptotic effect was smaller in vivo than in vitro, partly as a result of distinct levels of Bax expression. It is proposed that voltage-dependent Ca ²⁺ channels are involved in cellular and mitochondrial accumulation of Ca²⁺ subsequent to ATP depletion and play an important role in regulating mitochondrial permeability transition, cytochrome c release, caspase activation, and apoptosis.