Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition

C. Balut, M. Vandeven, S. Despa, I. Lambrichts, M. Ameloot, P. Steels, I. Smets

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.

Original languageEnglish
Pages (from-to)226-232
Number of pages7
JournalKidney International
Volume73
Issue number2
DOIs
StatePublished - Jan 10 2008

Bibliographical note

Funding Information:
We acknowledge Mr J Janssen for his excellent technical assistance with the cell culture. We also thank Mr M Jans, P Pirotte, W Leyssens, R Van Werde, and Mrs R Beenaerts for their technical help and Mrs M Ieven for art work. We gratefully acknowledge using ImageJ freeware (NIH, Bethesda, MD, USA; Dr W Rasband: http://rsb.info.nih.gov/ij/plugins/lsm-reader.html ) and plugins like LSM Reader and LSM Toolbox (University of Strasbourg, Strasbourg, France; Drs J Mutterer, Y Krempp and P Pirrotte) and Dr G Hamel, SVI BV. This work was supported by a bilateral research collaboration program between Flanders and Romania (BOF 05 B03), by the tUL impulse financing and by the Research Foundation—Flanders (FWO, GO270.07).

Keywords

  • 2-deoxyglucose
  • Confluent non-permeabilized renal cells
  • Cyanide
  • Ischemia/reperfusion
  • SNARF-1

ASJC Scopus subject areas

  • Nephrology

Fingerprint

Dive into the research topics of 'Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition'. Together they form a unique fingerprint.

Cite this