Aprotinin improves kidney function and decreases tubular cell apoptosis and proapoptotic signaling after renal ischemia-reperfusion Ajay Kher, MD, Kirstan.

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Aprotinin improves kidney function and decreases tubular cell apoptosis and proapoptotic signaling after renal ischemia-reperfusion Ajay Kher, MD, Kirstan K. Meldrum, MD, Karen L. Hile, BS, Meijing Wang, MD, Ben M. Tsai, MD, Mark W. Turrentine, MD, John W. Brown, MD, Daniel R. Meldrum, MD The Journal of Thoracic and Cardiovascular Surgery Volume 130, Issue 3, Pages 662.e1-662.e11 (September 2005) DOI: 10.1016/j.jtcvs.2005.02.035 Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Renal function and apoptosis after 24 hours of reperfusion. A, Serum creatinine levels in 25-hour sham controls (Sham), 1 hour of ischemia and 24 hours of reperfusion with saline solution (I/R), and 1 hour of ischemia and 24 hours of reperfusion with aprotinin (I/R + AP). B, Apoptosis ELISA in 25-hour sham controls (Sham), 1 hour of ischemia and 24 hours of reperfusion with saline solution (I/R), and 1 hour of ischemia and 24 hours of reperfusion with aprotinin (I/R + AP). Results are depicted as enrichment factor, ratio of sample value to negative control (sham controls) value. Bars represent mean; error bars indicate SEM. Asterisk indicates P < .001 relative to sham controls; delta indicates P < .05 relative to ischemia-reperfusion with saline solution. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Photomicrographs (original magnification 200×) demonstrating renal apoptosis. Nuclear stain (bis-benzimide, blue) shows nonapoptotic cells; apoptosis stain (TUNEL assay, green) shows apoptotic cells. A, In 25-hour sham control, white arrow points to nonapoptotic cell. B, In ischemia-reperfusion with saline solution, white arrows point to apoptotic cells. C, In ischemia-reperfusion with aprotinin, white arrow points to nonapoptotic cell. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Active caspase protein expression in 1-hour sham controls (leftmost Sham), 1-hour ischemia with saline solution (1hr I), 1-hour ischemia with aprotinin (1hr I + AP), 2-hour sham controls (rightmost Sham), 1-hour ischemia and 1-hour reperfusion with saline solution (1hrI/1hrR), and 1-hour ischemia and 1-hour reperfusion with aprotinin (1hrI/1hrR + AP). A, Representative immunoblot of caspase 8. B, Densitometric data of caspase 8 (as percentage of GAPDH). C, Representative immunoblot of caspase 3. D, Densitometric data of caspase 3 (as percentage of GAPDH). Bars represent mean; error bars indicate SEM. Asterisk indicates P < .01 relative to sham controls; double asterisk indicates P < .05 relative to sham controls; delta indicates P < .05 relative to 1-hour ischemia and 1-hour reperfusion with saline solution. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 The expression of activated p38 MAPK in 1-hour sham controls (leftmost Sham), 1-hour ischemia with saline solution (1hr I), 1-hour ischemia with aprotinin (1hr I + AP), 2-hour sham controls (rightmost Sham), 1-hour ischemia and 1-hour reperfusion with saline solution (1hrI/1hrR), and 1-hour ischemia and 1-hour reperfusion with aprotinin (1hrI/1hrR + AP). A, Representative immunoblot. Top row shows nonphosphorylated p38 MAPK (total); bottom row shows phosphorylated p38 MAPK (activated). B, Densitometric data of phosphorylated p38 MAPK (percentage of total p38 MAPK). Bars represent mean; error bars indicate SEM. Asterisk indicates P < .001 relative to sham controls; double asterisk indicates P < .05 relative to sham controls. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 A1, B1, C1, Representative gel photographs depicting TNF-α (A1), IL-1β (B1), IL-6 (C1), and GAPDH RT-PCR bands in 1-hour ischemia with saline solution (1 hr I) and in 1-hour ischemia with aprotinin (1 hr I + AP). A2, B2, C2, Densitometric data of TNF-α (A2), IL-1β (B2), and IL-6 (C2) are presented as percentage of GAPDH. Bars represent mean; error bars indicate SEM. Asterisk indicates P < .05 relative to 1-hour ischemia without aprotinin. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 Renal TNF-α (A), IL-1β (B), and IL-6 (C) protein levels expressed as picograms of cytokine per milligram of protein in sham controls (Sham), 1-hour ischemia with saline solution (1 hr I), and 1-hour ischemia with aprotinin (1 hr I + AP). Bars represent mean; error bars indicate SEM. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure E2 A1, B1, C1, Representative gel photographs depicting TNF-α (A1), IL-1β (B1), IL-6 (C1), and GAPDH RT-PCR bands in 1-hour ischemia and 1-hour reperfusion with saline solution (1 hr I/1 hr R) and in 1-hour ischemia and 1-hour reperfusion with aprotinin (1 hr I/ 1 hr R + AP). A2, B2, C2, Densitometric data of TNF-α (A2), IL-1β (B2), and IL-6 (C2) are presented as percentage of GAPDH. Bars represent mean; error bars indicate SEM. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions

Figure E3 Renal TNF-α (A), IL-1β (B), and IL-6 (C) protein levels expressed as picograms of cytokine per milligram of protein in sham controls (Sham), 1-hour ischemia and 1-hour reperfusion with saline solution (1 hr I/1 hr R), and 1-hour ischemia and 1-hour reperfusion with aprotinin (1 hr I/ 1 hr R + AP). Bars represent mean; error bars indicate SEM. Asterisk indicates P < .05 relative to sham control; double asterisk indicates P < .01 relative to sham control. The Journal of Thoracic and Cardiovascular Surgery 2005 130, 662.e1-662.e11DOI: (10.1016/j.jtcvs.2005.02.035) Copyright © 2005 The American Association for Thoracic Surgery Terms and Conditions