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Nephron. Physiology
2010;115 (2): p9-p19.

Albumin uptake in OK cells exposed to rotenone: a model for studying the effects of mitochondrial dysfunction on endocytosis in the proximal tubule?

A M Hall, M Campanella, A Loesch, M R Duchen, R J Unwin
Author Information
  1. A M Hall: Centre for Nephrology, University of London, London , UK. andrew.hall@ucl.ac.uk
PMID: 20484937 DOI: 10.1159/000314540

Abstract

BACKGROUND: The renal proximal tubule (PT) is clinically vulnerable to mitochondrial dysfunction; sub-lethal injury can lead to the Fanconi syndrome, with elevated urinary excretion of low-molecular-weight proteins. As the mechanism that couples mitochondrial dysfunction to impaired PT low-molecular weight protein uptake is unknown, we investigated the effect of respiratory chain (RC) inhibitors on endocytosis of FITC-albumin in PT-derived OK cells.
METHODS: Uptake of FITC-albumin was quantified using confocal microscopy. Cytosolic ATP levels were measured in real time using both luciferin/luciferase assays and measurements of free [Mg(2+)]. Reactive oxygen species production was measured using mitosox.
RESULTS: RC blockade produced only a small decrease in cytosolic ATP levels and had minimal effect on FITC-albumin uptake. Inhibition of glycolysis caused a much bigger decrease in both cytosolic ATP levels and FITC-albumin endocytosis. Rotenone led to higher rates of reactive oxygen species production than other RC inhibitors. Rotenone also caused widespread structural damage on electron microscopy, which was mimicked by colchicine and prevented by taxol; consistent with inhibition of microtubule polymerisation as the underlying mechanism.
CONCLUSIONS: Endocytosis of FITC-albumin is ATP-dependent in OK cells, but the cells are very glycolytic and therefore represent a poor metabolic model of the PT. Rotenone has toxic extra-mitochondrial structural effects.

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Grants

  1. G0601943/Medical Research Council
  2. /Wellcome Trust

MeSH Term

Adenosine Triphosphate
Animals
Cell Line, Transformed
Cell Survival
Colchicine
Cyanides
Dextrans
Electron Transport
Endocytosis
Epithelial Cells
Fanconi Syndrome
Fluorescein-5-isothiocyanate
Glycolysis
Kidney Tubules, Proximal
Mitochondria
Opossums
Paclitaxel
Pyridines
Reactive Oxygen Species
Rotenone
Serum Albumin

Chemicals

Cyanides
Dextrans
FITC-albumin
Pyridines
Reactive Oxygen Species
Serum Albumin
fluorescein isothiocyanate dextran
Rotenone
Adenosine Triphosphate
Fluorescein-5-isothiocyanate
Paclitaxel
Colchicine
Journal Article Research Support, Non-U.S. Gov't

Word Cloud

Created with Highcharts 10.0.0FITC-albumincellsPTmitochondrialdysfunctionuptakeRCendocytosisOKusingATPlevelsRotenoneproximalmechanismeffectinhibitorsmicroscopymeasuredoxygenspeciesproductiondecreasecytosoliccausedstructuralmodeleffectsBACKGROUND:renaltubuleclinicallyvulnerablesub-lethalinjurycanleadFanconisyndromeelevatedurinaryexcretionlow-molecular-weightproteinscouplesimpairedlow-molecularweightproteinunknowninvestigatedrespiratorychainPT-derivedMETHODS:UptakequantifiedconfocalCytosolicrealtimeluciferin/luciferaseassaysmeasurementsfree[Mg2+]ReactivemitosoxRESULTS:blockadeproducedsmallminimalInhibitionglycolysismuchbiggerledhigherratesreactivealsowidespreaddamageelectronmimickedcolchicinepreventedtaxolconsistentinhibitionmicrotubulepolymerisationunderlyingCONCLUSIONS:EndocytosisATP-dependentglycolyticthereforerepresentpoormetabolictoxicextra-mitochondrialAlbuminexposedrotenone:studyingtubule?

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