Purpose: Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal models have been developed to investigate this condition, they are limited by differential renal cell inflammatory mediator production and heterogeneous cell sensitivity to ischemia. We therefore developed an in vitro model of renal tubular cell ischemia that simulates the cellular injury observed in animal models of renal IR injury.
Materials and methods: Using the established renal tubular cell line, LLC-PK1, simulated ischemia was induced by immersing the cellular monolayer in mineral oil. The effect of simulated ischemia on renal tubular cells was then determined by measuring the time course of TNF-alpha protein expression (ELISA), TNF-alpha mRNA induction (RT-PCR), and renal tubular cell apoptosis (TUNEL).
Results: Maximal TNF-alpha protein expression occurs following 60 min of simulated ischemia and 2 h of substrate replacement (reimmersion in media), and maximal TNF-alpha mRNA induction occurs following 60 min of simulated ischemia. Cellular apoptosis peaks following 60 min of simulated ischemia and 24 h of reperfusion.
Conclusion: The time course of TNF-alpha production and apoptosis induction in this model closely parallels the time course for these markers in vivo. This study constitutes the initial demonstration that an in vitro oil immersion model of ischemia simulates the cellular injury (TNF-alpha production and apoptosis) observed in animal models of renal ischemia-reperfusion. This model may be used to study cellular mechanisms of IR in the absence of the systemic confounding variables.
Copyright 2001 Academic Press.