Proximal tubular cytochrome c efflux: determinant, and potential marker, of mitochondrial injury.
BACKGROUND
Cytochrome c (cyt c) is released from mitochondria after tissue injury, but little is known of its subsequent fate. This study was undertaken to ascertain: (1) does cyt c readily gain access to the extracellular space; (2) if so, what are some determinants of this process; and (3) might cyt c release be a potentially useful marker of in vivo tissue damage.


METHODS
Isolated mouse proximal tubules (PT) were subjected to site 1 (rotenone; Rot), site 2 (antimycin A, AA), or site 3 (hypoxic) respiratory chain blockade (+/- 2 mmol/L glycine, to prevent plasma membrane disruption/cell death). Alternatively, oxidant injury was imposed (Fe(2+) or cholesterol oxidase). Extra- and intracellular cyt c levels were quantified by Western blot. Plasma or urine cyt c levels were also determined after rhabdomyolysis or ischemic acute renal failure (ARF) (in mice), or clinical ARF.


RESULTS
AA, Rot, and hypoxia caused variable degrees of PT cyt c release (AA >> rot approximately hypoxia), but at most, <20% of total cell content was involved. In contrast, Fe(2+) evoked approximately 65% cyt c efflux, and cholesterol oxidation caused approximately 100% cyt c release. Glycine did not block cyt c efflux, dissociating this process from plasma membrane disruption/necrotic cell death. After rhabdomyolysis, plasma cyt c levels rose and correlated with the severity of ARF (r, 0.93 vs. BUNs). Cyt c was detected in urine after both experimental and clinical ARF.


CONCLUSIONS
Cell cyt c release is dependent on the site and the type of mitochondrial injury sustained. Oxidative injury, in general, and cholesterol oxidation, in particular, seem particularly relevant in this regard. After mitochondrial release, cyt c traverses plasma membranes, eventuating in the extracellular space. The data suggest that plasma and/or urine cyt c appearance might function as a clinically useful in vivo marker of mitochondrial stress and the tissue injury sustained.
Zager
Richard A
RA
Department of Medicine, University of Washington, and Fred Hutchinson Cancer Research Center, Seattle, 98109, USA. dzager@fhcrc.org
Johnson
Ali C M
AC
Hanson
Sherry Y
SY
D058186:Acute Kidney Injury / Q000378:metabolism / Q000652:urine; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000633:toxicity; D015415:Biomarkers / Q000378:metabolism / Q000652:urine; D002784:Cholesterol / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism* / Q000652:urine; D005110:Extracellular Space / Q000378:metabolism; D005990:Glycerol / Q000633:toxicity; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000293:injuries / Q000378:metabolism*; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012206:Rhabdomyolysis / Q000378:metabolism / Q000652:urine; D012402:Rotenone / Q000633:toxicity
D015415:Biomarkers; D012402:Rotenone; D000244:Adenosine Diphosphate; D000968:Antimycin A; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D002784:Cholesterol; D005990:Glycerol