NDT Advance Access published online on August 1, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn436
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Evidence for involvement of nonesterified fatty acid-induced protonophoric uncoupling during mitochondrial dysfunction caused by hypoxia and reoxygenation
1 Department of Nephrology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany 2 Division of Nephrology, Department of Internal Medicine, Veterans Affairs Ann Arbor Healthcare System and University of Michigan, Ann Arbor, MI 48109, USA
Correspondence and offprint requests to: Thorsten Feldkamp, Department of Nephrology, University Hospital Essen, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel: +49-201-723-2552; Fax: +49-201-723-5633; E-mail: thorsten.feldkamp{at}uni-due.de
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Abstract |
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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 (
) 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 (p) and the proton gradient (pH). 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.
Conclusion. 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 and p, abrogation of pH 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.
Keywords: acute kidney injury; hypoxia/reoxygenation; mitochondrial damage; nonesterified fatty acids; proximal tubule
Received for publication: 2. 3.08
Accepted in revised form: 8. 7.08
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