Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules

Abstract

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC.

γ-Tocotrienol Protects against Mitochondrial Dysfunction and Renal Cell Death

“…Reactive Oxygen/Nitrogen Species Generation. The carboxyl derivative of fluorescein, carboxy-H 2 DCFDA, was used to assess oxidant generation in RPTCs as described previously (Nowak et al, 2011). RPTCs were loaded with 5 M carboxy-H 2 DCFDA and incubated for 45 min.…”

Protein kinase C-α interaction with iHSP70 in mitochondria promotes recovery of mitochondrial function after injury in renal proximal tubular cells

“…RPTCs were homogenized and mitochondria were isolated as previously described (37,41). Mitochondria resulting from this isolation were free of lysosomal and endoplasmic reticular contaminations and contained small amounts of peroxisomes (37,41). The final mitochondrial pellet was resuspended in the lysis buffer used for immunoprecipitation or in Laemmli sample buffer if used for immunoblot analysis.…”

“…carboxy-H2DCFDA was used to assess oxidant generation in RPTCs as previously described (36,37). Autofluorescence was measured in unstained RPTCs in each experiment.…”

“…RPTC apoptosis was evaluated by measuring phosphatidylserine externalization on the plasma membrane using the annexin V/propidium iodide-binding assay as previously described (31,37). Cells positive for annexin V and negative for No transfection +wtPKC-α +dnPKC-α propidium iodide were considered apoptotic.…”

“…State 3 and uncoupled O 2 consumptions were measured polarographically using a Clark-type electrode as previously described (29,33,34). State 3 respiration in mitochondria energized by electron donors to the respiratory complexes I (5 mM glutamate and 5 mM malate) and complex II (10 mM succinate and 0.1 M rotenone) was measured in assay buffer containing digitonin (0.1 mg/ml) and respiratory substrates as previously described (30,37). State 3 respiration was initiated by the addition of 0.4 mM ADP.…”