Protein kinase Cα mediates recovery of renal and mitochondrial functions following acute injury

Abstract

Previously, we have shown that active protein kinase Ca (PKCa) promotes recovery of mitochondrial function after injury in vitro [Nowak G & Bakajsova D (2012) Am J Physiol Renal Physiol 303, F515-F526]. This study examined whether PKCa regulates recovery of mitochondrial and kidney functions after ischemia-induced acute injury (AKI) in vivo. Markers of kidney injury were increased after bilateral ischemia and returned to normal levels in wild-type (WT) mice. Maximum mitochondrial respiration and activities of respiratory complexes and F o F 1 -ATPase decreased after ischemia and recovered in WT mice. Reperfusion after ischemia was accompanied by translocation of active PKCa to mitochondria. PKCa deletion reduced mitochondrial respiration and activities of respiratory complex I and F o F 1 -ATPase in noninjured kidneys, indicating that PKCa is essential in developing fully functional renal mitochondria. These changes in PKCadeficient mice were accompanied by lower levels of complex I subunits (NDUFA9 and NDUFS3) and the c-subunit of F o F 1 -ATPase. Also, lack of PKCa exacerbated ischemia-induced decreases in respiration, complex I and F o F 1 -ATPase activities, and blocked their recovery after injury, indicating a crucial role of PKCa in promoting mitochondrial recovery after AKI. Further, PKCa deletion exacerbated acetylation and succinylation of key mitochondrial proteins of energy metabolism after ischemia due to decreases in deacetylase and desuccinylase (sirtuin3 and sirtuin5) levels in renal mitochondria. Thus, our data show a novel role for PKCa in regulating levels of mitochondrial sirtuins and acetylation and succinylation of key mitochondrial proteins. We conclude that PKCa deletion: (a) affects renal physiology by decreasing mitochondrial capacity for maximum respiration; (b) blocks recovery of mitochondrial functions, renal morphology, and functions after AKI; and (c) decreases survival after AKI. EnzymesProtein kinase C: EC 2.

γ-Tocotrienol Protects against Mitochondrial Dysfunction, Energy Deficits, Morphological Damage, and Decreases in Renal Functions after Renal Ischemia

“…Plasma levels of creatinine were used as a marker of renal function. Blood was drawn while mice were euthanized, spun down and plasma was used to determine creatinine concentration using the Creatinine Reagent Set from Biotron Diagnostics (Hemet, CA, USA) and the manufacturer’s protocol, as reported previously [ 56 , 57 , 58 ]. Plasma creatinine levels were determined in all mice used in the four experimental groups described on page 3.…”

“…; Ovation Pharmaceuticals, Deerfield, IL, USA) and all surgical procedures were performed under sterile conditions. Following a midline incision of the abdomen, kidneys were exposed and decapsulated, and both renal hila were clamped for 35 min with small vascular clamps to induce bilateral renal ischemia as described previously [55][56][57][58]. While renal arteries of both kidneys were clamped, mice were placed on a heating pad that maintained a temperature of 37 • C. During that time, approximately 0.5 mL sterile saline was applied intraperitoneally to prevent fluid loss and dehydration.…”

“…Immediately after mice were euthanized, both kidneys were harvested, submerged in an ice-cold isolation buffer (10 mM Hepes, 225 mM mannitol, 75 mM sucrose, 2 mM EGTA, and 0.1% BSA, fatty acid free, pH 7.4) and dissected on ice as described previously [56][57][58]. Cortical tissue separated from the kidneys was homogenized in ice-cold isolation buffer and the homogenate was spun down at 1000× g 5 min at 4 • C. The supernatant resulting from this centrifugation was spun down at 15,000× g 15 min at 4 • C to obtain a mitochondrial pellet.…”

“…Immunoblot analysis was used to assess protein levels of subunits of complexes I, III and IV, and subunits of F 0 F 1 -ATPase in renal cortical mitochondria using 20 µg of mitochondrial protein, as described previously [57,61]. Antibodies against NDUFA9, NDUFS3, and NDUFB7 subunits of complex I (Cat #A21344, A21343, and 21359) were purchased from Molecular Probes/ThermoFisher Scientific (Waltham, MA, USA).…”

Molecular pathophysiology of acute kidney injury: The role of sirtuins and their interactions with other macromolecular players

“…Their results demonstrated that to protect against AKI, SIRT5 modulates the peroxisomal and mitochondrial FAO balance in PTECs (Chiba et al, 2019). Nowak and Megyesi (2019) concluded that the activation and translocation of protein kinase Cα (PKCα) to mitochondria restores normal mitochondrial function and kidney physiology. They observed that PKCa modulates the expression levels of SIRT3 and SIRT5 which were involved in de/acetylation and de/ succinylation of mitochondrial proteins (Nowak & Megyesi, 2019).…”