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

doi:10.1038/sj.ki.5002632

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

Producción científica: Article › revisión exhaustiva

68 Citas (Scopus)

Resumen

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.

Idioma original	English
Páginas (desde-hasta)	226-232
Número de páginas	7
Publicación	Kidney International
Volumen	73
N.º	2
DOI	https://doi.org/10.1038/sj.ki.5002632
Estado	Published - ene 10 2008

Nota bibliográfica

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).

Financiación

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).

Financiadores	Número del financiador
Flanders and Romania	BOF 05 B03
Research Foundation—Flanders
Fonds Wetenschappelijk Onderzoek	GO270.07

ASJC Scopus subject areas

Nephrology

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10.1038/sj.ki.5002632

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title = "Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition",

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.",

keywords = "2-deoxyglucose, Confluent non-permeabilized renal cells, Cyanide, Ischemia/reperfusion, SNARF-1",

author = "C. Balut and M. Vandeven and S. Despa and I. Lambrichts and M. Ameloot and P. Steels and I. Smets",

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).",

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AU - Balut, C.

AU - Vandeven, M.

AU - Despa, S.

AU - Lambrichts, I.

AU - Ameloot, M.

AU - Steels, P.

AU - Smets, I.

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N2 - 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.

AB - 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.

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KW - Confluent non-permeabilized renal cells

KW - Cyanide

KW - Ischemia/reperfusion

KW - SNARF-1

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

Resumen

Nota bibliográfica

Financiación

ASJC Scopus subject areas

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