Primary mouse renal tubular epithelial cells have variable injury tolerance to ischemic and chemical mediators of oxidative stress

Abstract

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 A was most effective at inducing oxidative stress causing a mean LDH release of 61% at 48 hr compared to 19% and 50% LDH release induced by 2-Deoxy-D-Glucose and by exposure to 1% oxygen respectively at the same 48 hour time point.The cell culture method described provides several advantages including the use of serum free media and the ability to grow primary cells without matrix support. The LDH assay for injury assessment is reproducible, cost effective, objective and minimizes background cell death. A simple method for the culture and injury of primary mouse renal tubular epithelial cells has thereby been established and provides a useful tool for future investigations of ischemic kidney injury.

Keywords: ATP; acute kidney injury; glycolysis; ischemia; lactate dehydrogenase; necrosis; renal tubular epithelial cells.

Figures

Figure 1

Culture of mouse primary renal tubule epithelial cells. (A) Kidney cortices, abundant in proximal tubular epithelial cells, were digested, resuspended in kidney culture media (KCM) and plated at a previously determined optimal density. (B) On day 2 of culture, tubular epithelial cells migrated out of the tubes and proliferated, forming patches on the plate surface. (C) Individual patches of epithelial cells merged and the cultures reached 90% confluency on day 7–10. (D) Epithelial cells were identified as originating from proximal tubules by the brush border specific marker megalin fluorescently tagged with Alexa fluor 488. (E) Under normoxic conditions, E cadherin was prominent on tubular epithelial cells. (F) Upon exposure to hypoxic (1% oxygen) conditions, E-cadherin staining was diminished.

Figure 2

Antimycin A depletion of ATP. Antimycin A was added to cell cultures and injury assessed by the LDH released into culture supernatants. (A) The 0.5 µM concentration did not significantly increase the LDH release unless incubated for 48 hr (p

Figure 3

2-Deoxy-D-Glucose depletion of ATP. 2-Deoxy-D-Glucose was added to cell cultures and injury assessed by the LDH released into culture supernatants. (A) At 24 hr, the 4 µM and 6 uM concentrations significantly increased the LDH release. All concentrations of 2-Deoxy-D-Glucose significantly increased the LDH release at 48 hr (p

Figure 4

Chamber hypoxia depletion of ATP. Confluent cell cultures were incubated in a 1% oxygen environment at 37°C and injury assessed by the LDH released into culture supernatants. (A) Significant increases in LDH release resulted from no dextrose and 1.0 mM dextrose addition to the culture media for 24 hr (p