title: Evidence for involvement of nonesterified fatty acid-induced protonophoric uncoupling during mitochondrial dysfunction caused by hypoxia and reoxygenation.
abstract: BACKGROUND
  Proximal tubules subjected to hypoxia in vitro under conditions relevant to ischaemia in vivo develop an energetic deficit that is not corrected even after full reoxygenation. We have provided evidence that accumulation of nonesterified fatty acids (NEFA) is the primary reason for this energetic deficit. In this study, we have further investigated the mechanism for the NEFA-induced energetic deficit.
  
  
  METHODS
  Mitochondrial membrane potential (Deltapsi) was measured in digitonin-permeabilized, freshly isolated proximal tubules by safranin O uptake. Addition of the potassium/proton exchanger nigericin enables the determination of the mitochondrial proton motive force (Deltap) and the proton gradient (DeltapH). ATP was measured luminometrically and NEFA colorimetrically.
  
  
  RESULTS
  Tubule ATP content was depleted after hypoxia and recovered incompletely, even after full reoxygenation. Mitochondrial safranin O uptake was decreased in proximal tubules after hypoxia and reoxygenation (H/R). This decrease was attenuated by delipidated bovine serum albumin (dBSA) or citrate. Addition of nigericin increased safranin O uptake of mitochondria in normoxic proximal tubules, but not in proximal tubules after H/R. Addition of dBSA restored the effect of nigericin to increase mitochondrial safranin O uptake. Addition of the NEFA oleate had the same impact on mitochondrial safranin O uptake as subjecting proximal tubules to H/R.
  
  
  CONCLUSIONS
  The mechanism of the NEFA-induced energetic deficit in freshly isolated rat proximal tubules induced by H/R is characterized by impaired ATP production after full reoxygenation, impaired recovery of Deltapsi and Deltap, abrogation of DeltapH and sensitivity to citrate, consistent with involvement of the tricarboxylate carrier. The data support the concept that protonophoric uncoupling by NEFA movement on anion carriers plays a critical role in proximal tubule mitochochondrial dysfunction after H/R.
authors.affiliation: Department of Nephrology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. thorsten.feldkamp@uni-due.de
authors.forename: Thorsten
authors.initials: T
authors.lastname: Feldkamp
authors.forename: Joel M
authors.initials: JM
authors.lastname: Weinberg
authors.forename: Markus
authors.initials: M
authors.lastname: Hörbelt
authors.forename: Christina
authors.initials: C
authors.lastname: Von Kropff
authors.forename: Oliver
authors.initials: O
authors.lastname: Witzke
authors.forename: Jens
authors.initials: J
authors.lastname: Nürnberger
authors.forename: Andreas
authors.initials: A
authors.lastname: Kribben
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019343:Citric Acid / Q000494:pharmacology; D005230:Fatty Acids, Nonesterified / Q000378:metabolism* / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000293:injuries / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D019301:Oleic Acid / Q000494:pharmacology; D018892:Proton-Motive Force / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism
chemical_list: D005230:Fatty Acids, Nonesterified; D019343:Citric Acid; D019301:Oleic Acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase