Troglitazone Induced Cytosolic Acidification via Extracellular Signal-Response Kinase Activation and Mitochondrial Depolarization: Complex I Proton Pumping Regulates Ammoniagenesis in Proximal Tubule-like LLC-PK1 Cells
Purpose: To determined the mechanism(s) through which troglitazone induces cytosolic acidification and glutamine-dependent ammoniagenesis in pig kidney derived LLC-PK1 cells. Experimental Design: Acute experiments measured acid extrusion, acid production and simultaneous Extracellular Signal-Regulated Kinase activation. TRO-enhanced acid production was correlated with mitochondrial membrane potential and rotenone and 5-(N-ethyl-N-isopropyl) amiloride, were employed to test specifically the role of Complex I proton pumping. Chronic experiments correlated inhibitors of Complex I with prevention of TRO-increased ammoniagenesis and affects on glutamine metabolism. Results: Exposure to TRO acutely activated Extracellular Signal-Regulated Kinase in a dose dependent manner associated with a fall in spontaneous cytosolic pH. Cytosolic acidosis was associated with both an increase in acid production and inhibition of sodium/hydrogen ion exchanger -mediated acid extrusion. Preventing TRO-induced Extracellular Signal-Regulated Kinase activation with Mitogen Activated Protein Kinase Kinase inhibitors blocked the increase in acid production, restored sodium/hydrogen ion exchanger-activity and prevented cytosolic acidification. Mechanistically, increased acid production was associated with a rapid mitochondrial depolarization and Complex I proton pumping. Blocking Extracellular Signal-Regulated Kinase activation prevented both the fall in Ψm and the increased acid production suggesting that the former underlies the accelerated mitochondrial “acid production”. Mitochondrial Complex I inhibitors EIPA and rotenone prevented increased acid production despite Extracellular Response Kinase activation and reduced sodium/hydrogen ion activity. Inhibition of Complex I prevented TRO’s effects on glutamine metabolism. Conclusion:TRO induces cellular acidosis through Extracellular Signal-Regulated Kinase activation-associated acid production and impaired acid extrusion. Acutely, increased acid production reflects mitochondrial Complex I proton pumping into the cytosol while chronically Complex I activity appears coupled to mitochondrial glutamate uptake and oxidation to ammonium at the expense of cytosolic transamination and alanine formation in these proximal tubule-like cells.