TNFR1-Dependent NF-κB Activation and Pulmonary Apoptosis during Ischemic Acute Kidney Injury

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS protective effects of pharmacologic preconditioning with HS in an in vitro model of IRI. Methods: Murine myoblasts were differentiated into myotubes and then treated with either 10mM HS, 100mM allopurinol, both 10mM HS and 100mM allopurinol, or vehicle alone (non-treated control). The experiment was replicated with a higher concentration of allopurinol (1mM). Cells were exposed to 3h of anoxia followed by 3h of normoxia (21% O2). Parallel control groups were exposed to normoxia for 6 hours. A TUNEL assay was performed to determine the apoptotic index (AI; number of TUNEL-positive cells divided by the total number of cells) for each group. Results: The AI of myotubes treated with 100mM allopurinol alone was unchanged from that of non-treated controls (9.1%61.1% v. 10.2%61.4%, p¼0.194). For cells treated with 1mM allopurinol alone, the AI was similarly unchanged (9.5%6 1.2%, p¼0.317). Ten mM HS alone resulted in a significant decrease in AI when compared to non-treated controls (4.0%60.4%, p<0.001). However, treatment with both 100mM allopurinol and HS resulted in an AI of 11.3%60.8%, significantly greater than that of myotubes treated with HS alone (p<0.001) and unchanged from non-treated controls (p¼0.974). Likewise, treatment with both 1mM allopurinol and HS resulted in an AI of 10.5%60.9%, significantly greater than that of HS alone (p<0.001) and not significantly different from that of the untreated controls (p¼0.448). Conclusions: Even in the presence of increased concentrations of allopurinol, the protective effects of HS in the setting of IRI are lost. Furthermore, in contrast to others’ reports, allopurinol alone was found not to have a cytoprotective effect. These data suggest that the protective mechanism of HS is due at least in part to a xanthine oxidase-mediated pathway and the generation of low levels of ROS. These findings importantly expand our understanding of the pathophysiology of IRI and may help to delineate the clinical utility of HS.

45.4. TNFR1-Dependent NF-kB Activation and Pulmonary Apoptosis during Ischemic Acute Kidney Injury. L. E. White,1 R. J. Santora,1 C. M. Feltes Shelak,2 Y. Cui,1 F. A. Moore,1 H. T. Hassoun1; 1The Methodist Hospital and Research Institute, Houston, TX; 2Legacy Emanuel Children’s Hospital, Portland, OR Introduction: Acute kidney injury (AKI) alters the host immune response and induces distant organ phenotypic changes including TNF-dependent pulmonary apoptosis that contributes to lung microvascular injury. We investigated lung endothelial cell (EC) specific gene changes related to apoptosis, and hypothesized that NF-kB plays a key role in signal transduction of TNF receptor (TNFR)-dependent pulmonary endothelial apoptosis during AKI. Methods: C57BL/6J (WT) or TNFR1-/- mice underwent 60 minutes bilateral renal pedicle clamp (IRI) or sham laparotomy (sham) and were sacrificed at 24 hours for the following studies: 1) Serum creatinine (SCr, mg/dL) was obtained, 2) WT murine lung ECs were isolated by novel tissue digestion and antibody-coupled magnetic bead sorting techniques, and purified lung ECs were analyzed for changes in mRNA expression by real-time PCR ‘‘Superarray’’ of 84 genes related to apoptosis. Significant changes in gene expression between sham and IRI (FC1.5 or p<.05 by t-test) were identified, analyzed and functionally clustered, 3) WT or TNFR1-/- whole lung tissues were processed for NF-kB p65 nuclear protein, caspase-8, or cleaved caspase-3 expression by western blot (relative units (RU) normalized to b-actin), and 4) lung TUNEL staining by immunohistochemistry (cells/hpf) was performed. N4/group. *p<0.05, ANOVA. Results: Isolated WT pulmonary ECs produced significant changes in 16/84 (19%) of apoptosis genes identified by RT-PCR Superarray, including activation of NF-kB -promoting genes DR5/killer (FC ¼ 3.05), Bcl10 (FC ¼ 2.22), and caspase-8 inhibiting Birc2 (FC ¼ 1.86), and decreased expression of NF-kB inhibitors such as Osteoprotegerin (FC¼-6.34), Traf1 (FC¼-2.22) and caspase-12 (FC¼-2.31). While SCr was

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increased in both WT IRI (0.1460.07 vs 1.3460.29*) and TNFR1-/IRI (0.2260.04 vs 1.2060.38*) compared to sham, only WT IRI mice demonstrated lung apoptosis with increased caspase-3 (0.060.0 vs .246.02*) expression and TUNEL (0.961.03 vs. 3.1762.28*) positive cells. There was no difference in caspase-8 expression between WT (1.376.02 vs 1.436.03, p ¼ 0.21) or TNFR1-/- (0.6560.13 vs. 0.7660.19, p ¼ 0.37) sham and IRI, yet nuclear NF-kB p65 expression increased during IRI in WT (.266.04 vs .516.03*) but not TNFR1-/(.276.05 vs .286.03, p ¼ 0.88) mice. Conclusions: Ischemic AKI activates distant organ pulmonary endothelial pro-apoptotic pathways and the TNF receptor is a critical mediator of these events. Our findings imply that NF-kB, rather than caspase-8, is an important regulator of TNFR-dependent pulmonary EC apoptosis during AKI.

45.5. Delayed Administration Of Human AM/AMBP-1 Reduces Injury After Intestinal Ischemia And Reperfusion. K. G. Shah,1 R. Wu,1,2 J. Nicastro,1 G. F. Coppa,1 P. Wang1,2; 1 North Shore University and Long Island Jewish Medical Center, Manhasset, NY; 2Feinstein Institute for Medical Research, Manhasset, NY Introduction: Intestinal ischemia-reperfusion (I/R) injury can be caused by a variety of disease conditions, including abdominal aortic aneurysm surgery, cardio-pulmonary bypass, and strangulated hernias. Currently, it has a high morbidity and mortality rate because no specific treatment is available. The pathophysiology of intestinal I/R injury is thought to be caused by direct cellular damage during the ischemic phase, as well as the generation of oxygen free radicals and secondary organ injury during reperfusion. Adrenomedullin (AM) is a novel vasoactive peptide and adrenomedullin binding protein-1 (AMBP-1) is a specific AM binding protein which augments the activity of AM. We have shown that AM combined with AMBP-1 is advantageous in multiple disease conditions. However, it remains unknown whether human AM/ AMBP-1 ameliorates the inflammatory response and organ damage seen after intestinal I/R injury. Our objective is to determine if the delayed administration of human AM/AMBP-1 can minimize damage in a rat model of intestinal I/R injury. Methods: Male adult rats were subjected to superior mesenteric artery clamping with a microvascular clip for 90 min. At 60 min after the initiation of reperfusion (clip removal), administration of either vehicle (normal saline) or human AM (96 mg/kg) and AMBP-1 (320 mg/kg) was given intravenously over 30 min. At 4 h after reperfusion, animals were sacrificed for sampling. Blood was collected and measured for systemic markers of tissue injury and inflammation. Intestinal tissue was collected to assess the degree of small bowel edema. Results: Systemic markers of tissue injury were significantly increased following intestinal I/R and infusion of normal saline. As shown in the table below, plasma levels of the liver injury markers, AST and ALT, were markedly elevated as compared to sham animals. Intestinal water content was also significantly increased. Furthermore, systemic levels of creatinine, lactate, and IL-6 were elevated after intestinal I/R. Administration of human AM/AMBP-1 decreased plasma levels of AST and ALT by 57%

AST (IU/L

ALT (IU/L)

Intestinal Water %

Lactate (mg/dL)

Sham 2261.4 2262.1 75.360 9.760.6 I/R - Vehicle 11968.3* 7963.6* 79.460.4* 35.563.7* I/R - AM/ 5166.7*,# 4966.1*,# 76.860.6# 25.262.2*,# AMBP-1

Creatinine (mg/dL)

IL-6 (pg/mL)

0.960.1 1.760.2* 1.160.1#

28.367.8 1055.2649.9* 836.9655.1*,#

Mean6SEM, n¼6/group; One-way ANOVA & Student-Newman-Keuls; *P<0.05 vs. Sham; #P<0.05 vs. I/R - Vehicle.