INDUCTION OF APOPTOSIS IN ISCHEMIA-REPERFUSION MODEL OF MOUSE KIDNEY - POSSIBLE INVOLVEMENT OF FAS

Although ischemia-reperfusion of mouse kidney is known to cause severe renal failure due to tubular cell death, the exact cellular mechanism responsible for this phenomenon is not clear. To investigate the spat ial and temporal development of renal cell death and the role of Fas/A PO-1/CD95 (Fas) in this process, the left renal vessels were occluded in a group of mice for 30, 60, or 120 min followed by reperfusion for 24 h (n = 4 for each group). Analysis of the isolated DNA in agarose-g el electrophoresis revealed a typical ladder pattern of bands consisti ng of multiples of 180 to 200 bp, considered the hallmark of apoptosis . The intensity of the bands increased proportionately with the durati on of ischemia. Histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling showed the presenc e of nuclei with DNA double-strand breaks specifically in distal renal tubules of the outer medulla, The presence of apoptosis was also conf irmed by electron microscopy. Analysis of total RNA by Northern blotti ng revealed one appropriate-sized band for Fas mRNA in the normal kidn ey, which intensified in the ischemia-reperfused kidney. Moreover, non radioactive in situ hybridization revealed that distal renal tubular e pithelial cells were positive for Fas mRNA in the outer medulla. Fas a ntigen was also localized to the renal tubular epithelial cells of the outer medulla by immunohistochemistry. The number of apoptotic cells in the ischemia-reperfusion kidney of the lpr/lpr mouse was low. These findings strongly indicate that ischemia-reperfusion of the kidney in duces apoptosis of a specific area of tubular epithelial cells in the outer medulla through the Fas system.