Carnitine palmitoyl-transferase enzyme inhibition protects proximal tubules during hypoxia

摘要

Carnitine palmitoyl-transferase enzyme inhibition protects proximal tubules during hypoxia. The role of inhibition of the CPT enzymes responsible for accumulation of long chain acylcarnitines (LCAC) during hypoxia in the proximal tubule has not been previously examined. We have characterized CPT enzyme activities in mitochondrial fractions of rabbit proximal tubules. Malonyl CoA-sensitive CPT I activity (1.1 0.3 nmol/min/mg protein), and detergent-solubilized, malonyl CoA-insensitive CPT II activity (2.3 0.4 nmol/min/mg protein) were readily detected in proximal tubule mitochondrial fractions. Subjecting rabbit proximal tubules to various periods of hypoxia did not significantly change mitochondrial CPT I or CPT II activities. Thirty minutes of hypoxia resulted in an increase in lysophospholipid mass from 440 105 to 720 93 pmol/mg protein, N = 5, LCAC mass from 79 11 to 618 34 pmol/mg protein, N = 5, and lactate dehydrogenase (LDH) release from 9 1% to 46 3%, N = 8. Pretreatment of proximal tubules with two different CPT inhibitors, glybenclamide (Glyb) 400 M and oxfenicine (Oxfe) 1 mM, resulted in reduction in the magnitude of hypoxia-induced lysophospholipid formation 490 160 (Glyb), 342 150 pmol/mg protein (Oxfe), N = 4, hypoxia-induced LCAC formation 295 27 (Glyb), 128 16 pmol/mg protein (Oxfe), N = 5, and LDH release 25 1% (Glyb) and 19 2% (Oxfe), N = 8. The protective effect of CPT inhibition was also associated with increased production of lactate suggesting the modulation of a substrate-mediated metabolic switch. Immunoblots demonstrated that hypoxia caused a time dependent hydrolysis of fodrin-alpha subunit and that CPT inhibition protected against hypoxia-induced fodrin proteolysis. These data suggest a unifying hypothesis that links phospholipase A2 (PLA2) activation, and hypoxia-mediated fodrin proteolysis to the proximal tubule mitochondrial CPT system. I propose that CPT inhibition may represent a novel mechanism to ameliorate proximal tubule cell death during hypoxia.