Ca2+-Dependent Mitochondrial Permeability Transition Pore Opening in Kidney is Substrate Dependent

Abstract

Oxidative stress within the kidney is associated with disease states including hypertension, renal diseases. The metabolic functions of the mammalian kidneys result in consumption of nearly 20% of total oxygen utilization. There is limited information regarding the effects of oxygen free radicals upon kidney mitochondrial functions. Not only mitochondria oxidize numerous metabolic substrates but also play a key role in maintaining cellular Ca2+ homeostasis. Failure of cellular Ca2+ homeostasis and excessive mitochondrial Ca2+ (mCa2+) lead to opening of a mitochondrial permeability transition pore (mPTP), which is associated with loss of IMM potential and uncoupling of oxidative phosphorylation. We investigated whether mitochondrial metabolic substrates have a differential impact on the Ca2+-induced mPTP opening in the kidney, which plays a critical role in hypertension. For this purpose, mitochondria isolated from Sprague Dawley (SD) rat kidney outer medulla (OM) were energized using mechanistically distinct substrate combinations, Pyruvate+Malate, Glutamate+Malate(GM), Succinate+Rotenone, Succinate, α-KetoGlutarate+Malate, or PalmitoylCarnitine+Malate. Fluorescence-based analysis using Ca2+ indicator Fura-4F assessed the functional consequences of Ca2+-induced mPTP opening. Results show that in the presence of GM, mitochondria can tolerate significantly greater mCa2+ loads as compared to that in the presence of other substrates. Succinate+Rotenone addition significantly delayed mPTP opening over controls (succinate alone). A pore inhibitor CyclosporinA(CsA) and Na+/Ca2+exchanger inhibitor(CGP) were used with all substrates for comparisons. The present study reports variations in mitochondrial substrates sensitivity to Ca2+-induced mPTP opening and that the metabolic fluxes determine the mitochondrial functional responses to mCa2+ overload. The results underscore the effects of various mitochondrial substrates and their electron flux in regulating mPTP opening, which is a determinant of cell death in many diseases. These data are important in order to understand the different pathways/proteins involved in mPTP opening to identify effective interventions to prevent/limit it.