Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack.
BACKGROUND
Cholesterol is a major component of plasma membranes, forming membrane microdomains ("rafts" or "caveolae") via hydrophobic interactions with sphingolipids. We have recently demonstrated that tubule cholesterol levels rise by 18 hours following diverse forms of injury, and this change helps to protect kidneys from further damage (so-called acquired cytoresistance). The present study was undertaken to better define the effects of membrane cholesterol/microdomains on tubule homeostasis and cell susceptibility to superimposed attack.


METHODS
Plasma membrane cholesterol was perturbed in normal mouse proximal tubular segments with either cholesterol esterase (CE) or cholesterol oxidase (CO). Alternatively, cholesterol-sphingomyelin complexes were altered by sphingomyelinase (SMase) treatment. Changes in cell energetics (ATP/ADP ratios + ouabain), viability [lactate dehydrogenase (LDH) release], phospholipid profiles, and susceptibility to injury (Fe-induced oxidant stress, PLA2, Ca2+ ionophore) were determined. The impacts of selected cytoprotectants were also assessed.


RESULTS
Within 15 minutes, CE and CO each induced approximately 90% ATP/ADP ratio suppressions. These were seen prior to lethal cell injury (LDH release), and it was ouabain resistant (suggesting decreased ATP production, not increased consumption). SMase also depressed ATP without inducing cell death. After 45 minutes, CE and CO each caused marked cytotoxicity (up to 70% LDH release). However, different injury mechanisms were operative since (1) CE, but not CO, toxicity significantly altered cell phospholipid profiles, and (2) 2 mmol/L glycine completely blocked CE- but not CO-mediated cell death. Antioxidants also failed to attenuate CO cytotoxicity. Disturbing cholesterol/microdomains with a sublytic CE dose dramatically increased tubule susceptibility to Fe-mediated oxidative stress and Ca2+ overload, but not PLA2-mediated damage.


CONCLUSIONS
Intact plasma membrane cholesterol/microdomains are critical for maintaining cell viability both under basal conditions and during superimposed attack. When perturbed, complex injury pathways can be impacted, with potential implications for both the induction of acute tubular damage and the emergence of the postinjury cytoresistance state.
Zager
R A
RA
The Fred Hutchinson Cancer Research Center and the University of Washington, Seattle 98109-1024, USA. dzager@fhcrc.org
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D002784:Cholesterol / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D007674:Kidney Diseases / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010042:Ouabain / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010741:Phospholipases A / Q000494:pharmacology; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D002787:Sterol Esterase / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007476:Ionophores; D010743:Phospholipids; D000001:Calcimycin; D010042:Ouabain; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002784:Cholesterol; D007501:Iron; D007770:L-Lactate Dehydrogenase; D002789:Cholesterol Oxidase; D002787:Sterol Esterase; D010741:Phospholipases A; D054467:Phospholipases A2; D013108:Sphingomyelin Phosphodiesterase; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium; D005998:Glycine