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Methodological Report
Primary mouse renal tubular epithelial cells have variable injury tolerance to ischemic and chemical mediators of oxidative stress
Anne C. Breggia and Jonathan Himmelfarb
volume 1 | issue 1
July/August 2008Subscribe to this journal for $0/year
We have developed and evaluated an in vitro culture method for assessing ischemic injury in primary mouse renal tubular epithelial cells (RTEC) in which to explore the pathobiology underlying acute kidney injury. RTEC were predominately of proximal tubule origin which is most susceptible to ischemic injury as compared to other nephron segments. Oxidative stress was induced by chemically depleting ATP using Antimycin A and 2-Deoxy-D-Glucose and by exposing cells to a 1% oxygen environment. Necrotic injury was assessed by measuring LDH released into culture supernatants. Optimal dose and time of exposure to each injury agent was determined for induction of mild, moderate and severe ischemic injury defined as LDH release of ≤20%, 21-49% and ≥50% above baseline respectively. Antimycin A and 2-Deoxy-D-Glucose produced a progressive increase in LDH release which was time dependent but chemical concentration independent. A 1% oxygen environment also induced cell injury over time but only if glucose was absent from the culture media. Antimycin was most effective at inducing oxidative stress causing a mean LDH release of 61% at 48hr compared to19% and 50% LDH release induced by 2-Deoxy-D-Glucose and by exposure to1% oxygen respectively at the same 48hour time point.
Authors
Anne C. Breggia
Maine Medical Center Research Institute, Clinical and Translational Research; Portland, Maine USA
Jonathan Himmelfarb
Maine Medical Center, Department of Medicine, Division of Nephrology; Portland, Maine USA