Bcl-2 and Bax Expression on Rat Ischemic Kidney P. Eschwege, V. Paradis, M. Conti, S. Loric, F. Dumas, P. Berteau, M. Ahmed, S. Droupy, b. Charpentier, A. Legrand, P. Bedossa, and G. Benoit I T has been suggested that oxidative stress can cause injuries to the renal tubular epithelial cells. 1 Cells can die by two distinct pathways: necrosis or apoptosis. 2 Necro- sis is associated with metabolic collapse that leads to cell swelling, loss of plasma membrane integrity, and cell rup- ture. Apoptosis requires energy, and constitutes a strictly regulated device responsible for the ordered removal of superflous damaged cells. The family of Bcl-2–related pro- teins constitutes one of the biologically most relevant classes of apoptosis-regulatory gene products. 2,3 Bcl-2 as Bax belongs to a family of apoptosis-regulatory gene prod- ucts which may either be death antagonists (Bcl-2) or death agonists (Bax). The relative amount of death agonists and antagonists from Bcl-2 family constitutes a regulatory bal- ance. 3 Anti-apoptotic properties of Bcl-2 oncoprotein are associated with a marked decrease in the cellular genera- tion of reactive oxygen species and prevention to lipid peroxidation. 2 Moreover, Bcl-2 works by inhibiting the mitochondrial generation of reactive oxygen species (ROS) even under anaerobic conditions in which the generation of ROS is reduced. 2 Apoptosis seems to play a key role in acute tubular injury as it was described by Schumer et al. 4 after a brief period of ischemia and 24 to 48 hours after reperfusion. Most of the studies described apoptosis pro- cess after ischemia–reperfusion. Therefore, to our knowl- egde, we do not know the pathway of postischemic cell death: necrosis or apoptosis. To improve our knowlegde, we have studied the occurrence of apoptotic process and the expression of Bcl-2 and Bax proteins after warm renal ischemia. ANIMALS, MATERIALS, AND METHODS Animals and Surgical Procedure Thirty-six male Sprague-Dawley rats (body weight 350 to 400 g) were studied. They were housed in metabolic cages during the experiment with free access to feed and water. Anesthesia was induced by intramuscular administration of ketamine (0.2 mg/100 g). A coeliotomy was performed, and the left and right renal pedicles were gently dissected and clamped for 15, 30, 45, 60, and 120 minutes (n = 6 rats per group). One group was operated but not clamped. A right nephrectomy was performed after the end of the warm ischemic period for immunohistochemical analysis. The right kid- neys were collected, opened longitudinally and immediately im- mersed in Bouin liquid until analysis. Experimental Groups The animals were divided into 6 groups: group 1 (n = 6), unclamped control rats; group 2 (n = 6), renal pedicles clamped 15 minutes; group 3 (n = 6), renal pedicles clamped 30 minutes; group 4 (n = 6) renal pedicles clamped 45 minutes; group 5 (n = 6; renal pedicles clamped 60 minutes; group 6 (n = 6), renal pedicles clamped 120 minutes. Immunohistochemical Method A total of 36 Bouin-fixed paraffin-embedded kidney samples were studied. Serial sections were performed for routine histologic evaluation (HES) and for the immunohistochemical procedure. Immunohistochemistry was performed using an automated immu- nostainer (Techmate 500, Dako, Carpinteria, Calif) with the avi- dine– biotin–peroxidase method. Anti-Bax rabbit polyclonal anti- bodies 1/250 (Biogenex, San Ramon, Calif) and monoclonal mouse anti Bcl-2 1/120 (Dako, Glostrup, Denmark) were used. As positive controls, fixed paraffin-embedded rat spleen samples were used. Localization and intensity of staining were indepen- dently assessed by two pathologists who were not aware of the time of warm ischemic periods. In case of discordance, a consensual evaluation was performed by simultaneous reviewing of slides by the two pathologists. The localization of immunostaining was described and the intensity of staining was graded as absent (no staining = 0) or present (+ to ++). RESULTS No significant morphologic lesion was observed in the kidney tissue, whatever the duration of ischemia. Especially we did not detect any apoptotic bodies. In positive control experiments, several cells labeled with Bcl-2 and Bax anti- bodies in the spleen tissue. No Bax and Bcl-2 immunostain- From the Laboratoire de Chirurgie Expe ´ rimentale et Service d’Urologie, Ho ˆ pital de Bice ˆ tre, Faculte ´ de Me ´ decine (P.E., M.A., S.D., G.B.) the Service d’Anatomie Pathologique, Ho ˆ pital de Bice ˆ tre (V.P., P.B.) the Laboratoire de Biochimie, Ho ˆ pital de Bice ˆ tre (M.C., A.L.), Le kremlin-Bice ˆ tre, France, the Laboratoire de Biochimie A, Ho ˆ pital Necker, (S.L., F.D., P.B.), Paris, France and the Service de Ne ´ phrologie, Ho ˆ pital de Bice ˆ tre (B.C.), Le Kremlin-Bice ˆ tre, France. Address reprint requests to Pascal Eschwege MD, Depart- ment of Urology, Ho ˆ pital de Bice ˆ tre, 78 rue du Ge ´ ne ´ ral Leclerc, 94270 Le Kremlin-Bice ˆ tre, France. Supported by grants from Fondation de L’Avenir etude ET-5 118, and Etablissement Franc ¸ ais des Greffes. © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(98)00844-6 Transplantation Proceedings, 30, 2861–2862 (1998) 2861