Protective role of protein kinase B/Akt against mitochondrial dysfunction and cell death in nephrotoxicant injured renal cells

Mitochondrial dysfunction is the most common mechanism of cell injury induced by ischemia/reperfusion, drugs and toxicants in renal cells. Signaling molecules regulate mitochondrial functions and cell death in a variety of cells. This study demonstrates the protective role of protein kinase B/Akt against mitochondrial dysfunction and cell death in renal proximal tubular cells (RPTC) following a nephrotoxic cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), induced injury. In RPTC, Akt was activated during 8 hours but not at 24 hours following DCVC exposure. Phosphorylated (active) Akt was localized in mitochondria of non-injured RPTC. Mitochondrial levels of active Akt decreased in DCVC injured RPTC and this decrease was associated with mitochondrial dysfunction. In RPTC, DCVC decreased basal, uncoupled and state 3 respirations, activities of complexes I, II and III, and F0F1-ATPase, mitochondrial membrane potential (DeltaPsim), ATP production and intracellular ATP levels resulting in both apoptosis and necrosis at 24 hr following the exposure. Expressing constitutively active Akt in DCVC-injured RPTC increased the activity of Akt in mitochondria, reduced the decreases in basal and uncoupled respirations, prevented the decreases in complex I-coupled state 3 respiration, activities of complex I, complex III, DeltaPsim and ATP production, and restored F0F1-ATPase activity resulting in increased intracellular ATP levels and diminished necrosis. In contrast, inhibiting Akt activation by expressing dominant negative (inactive) Akt or using 20 muM LY294002 in RPTC, exacerbated the DCVC-induced decreases in electron transport rate, complex I-coupled state 3 respiration, ATP production, DeltaPsi m and activities of complex I, complex III, and F0F 1-ATPase, resulting in further decrease in intracellular ATP content and increase in necrosis. Proteomic analysis of mitochondrial proteins phosphorylated by Akt in DCVC-injured RPTC identified beta-actin as the phosphorylated Akt substrate in RPTC mitochondria.; In conclusion, these studies demonstrate that Akt activation in DCVC injured RPTC: (1) improves the electron transport rate through the respiratory chain by preventing the decrease in the activity of respiratory complex I and complex III, (2) prevents decreases in DeltaPsim, (3) restores F0F1-ATPase activity (4) increases ATP production, (5) maintains intracellular ATP levels, (5) decreases necrosis and (6) phosphorylates mitochondrial beta-actin.