Heat-shock protein 25 ameliorates calcium oxalate crystal-mediated oxidative stress in renal epithelial cells.
OBJECTIVE
To investigate whether the antioxidant protection attributed to small heat-shock proteins (sHSPs) affects calcium oxalate stone formation, a pro-oxidant disease.


METHODS
Canine distal tubular epithelial cells (Madin-Darby canine kidney, MDCK cells) were grown as confluent monolayers. Treatment regimens included control and HS-treated cells (37 degrees C and 42 degrees C for 1 h) with or without calcium oxalate monohydrate (COM) or free oxalate treatment (28 microg/cm2) 16 h later. In digitonin-permeabilized cells, O2- was measured by lucigenin-enhanced chemiluminescence over a 5-min period, to measure mitochondrial O2- production. Protein expression was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis Western blot analysis using specific antibodies.


RESULTS
COM significantly increased O2- production in MDCK cells. HS treatment, which up-regulated HSP25 expression, significantly decreased this O2- production (P < 0.05) but had no effect in control cells. In COM-treated cells (20 h) there was a marked and significant down-regulation of both HSP 25, HSP 70 and heme oxygenase-1 expression compared to cells treated with HS alone (P < 0.05). Free oxalate had no effect on HSP 25 expression.


CONCLUSIONS
The results suggest that the COM-induced increase in mitochondrial O2- production in MDCK cells is ameliorated by HSP 25 up-regulation via HS. Specific COM inhibition of HSP 25, HSP 70 and heme oxygenase-1 up-regulation suggests that COM-induced reactive oxygen species damage is unable to benefit from HSP-associated physiological resistance.
Patel
Amit B
AB
Centre for Treatment and Prevention of Kidney Stone disease, Institute of Urology, University College London, UK. a.b.patel@ucl.ac.uk
Robertson
William G
WG
Choong
Simon
S
Hothersall
John S
JS
D000818:Animals; D015153:Blotting, Western; D002129:Calcium Oxalate / Q000494:pharmacology*; D003460:Crystallization; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D050888:Heat-Shock Proteins, Small / Q000494:pharmacology*; D007669:Kidney Calculi / Q000517:prevention & control*; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D050888:Heat-Shock Proteins, Small; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D002129:Calcium Oxalate