As we are interested in the regulation of renal cortical organic anion transport after ischemia, we established an in vitro model of ischemia and reperfusion using a cell line from rat proximal tubule (NRK-52E). Therein, cells were exposed to pH 6.6, aglycemia and O₂ fraction below 1% for 2 h. Subsequently the cells were kept under standard cell culture conditions in serum free medium for 6h, 24h or 48h. We had evidence from own in vivo studies that inhibition of cyclooxygenases (by indomethacin) after ischemia re-establishes organic anion transport and positively influences renal outcome. Thus, we tested whether this is also the case in proximal tubule cells subjected to a model ischemia and reperfusion. Therefore we measured typical parameters of cell death (protein, LDH-release, caspase-3-activity) and of cortical ischemia reperfusion injury (ischemia reperfusion induced protein, alpha smooth muscle actin, inducible NO-synthase, COX1, COX2, PGE_2-release) in NRK-52E cells after model ischemia. Organic anion transport was measured by uptake of fluorescein. Indomethacin was given after ischemia at 1mM. After "ischemia" a time dependent decrease in protein was detected, which is reflected by an increase in both LDH-release and caspase-3-activity. Ischemia-reperfusion-induced-protein IRIP), alpha smooth muscle actin and inducible NO-synthase were all up regulated (with different time courses) after ischemia. COX1 expression and PGE₂-release both were increased, whereas COX2 expression decreased (which is in accordance to the data from whole animal studies in our lab). Organic anion uptake is substantially impaired 48h after ischemia. Most notably, indomethacin reduced ischemia induced changes of all the latter parameters, which is in accordance to the beneficial effect detected in vivo. In conclusion our study indicates that inhibition of cyclooxygenases impairs the appearance of typical signs of ischemic damage in proximal tubular cells, which is in accordance to the events in renal cortex in vivo.

This study was supported by the IZKF Würzburg. Grant IZKF 34(1).