Abstract 219: Tumor Necrosis Factor α Drives Lung Endothelial Cell Apoptosis During Ischemic Acute Kidney Injury

Abstract

Introduction: Ischemic acute kidney injury (AKI) alters the innate immune response, activating cellular and soluble mediators which perpetuate microvascular barrier dysfunction and distant organ injury. We have previously identified that lung TNFR1 activation is a critical pathway to programmed cell death in an in vivo lung injury model during ischemic AKI. Objectives: Hypothesizing that lung endothelial cells (ECs) are a key target for programmed cell death, we investigated lung EC-specific transcriptional and functional pathways to TNFR1-dependent apoptosis during ischemic AKI. Methods: Male Sprague-Dawley rats (∼250gm) underwent 60 minutes of bilateral renal pedicle occlusion (IRI) or sham laparotomy (sham) and were sacrificed at 24hrs. Rat lung microvascular ECs (RLMVECs) grown to confluence were treated with serum from rats obtained following sham or kidney IRI +/- Etanercept, a TNFα/TNFR1 signaling inhibitor, or vehicle. RLMVECs underwent 1) custom RT-PCR analysis for pro-apoptotic, pro-inflammatory, and TNF superfamily candidate genes, 2) caspase-3 activity assay, and 3) PARP activity assay during 2,4, or 24 hours of ischemic AKI. *p<0.05 vs. sham, paired t-test; n≥4/group. Results: Compared to sham, RLMVECs exposed to IRI serum displayed a pro-apoptotic transcriptome during ischemic AKI with activation of TNF superfamily (TNFR1, TNFR2, TNFα) and apoptosis genes (Fasl, Dapk1, Bcl10) over a time course. RLMVECs demonstrated phenotypic programmed cell death with increased caspase-3 activation (100±1.12 vs. 106.5±0.79*) and PARP activity (10±3.47 vs. 61.75±20.45*) at 24 hours during ischemic AKI. Treatment with Etanercept ameliorated activation of the pro-inflammatory EC transcriptome (E-Selectin, ICAM-1, IL-6 and RhoB) and also attenuated programmed cell death with no increase in caspase-3 activity (100±0.88 vs. 101.98±0.48, p=0.096) or PARP activity (10±2.33 vs. 10.91±2.15, p=0.783) compared to vehicle during ischemic AKI. Conclusions: Ischemic AKI produces distinct changes in the pulmonary EC phenotype with activation of pro-inflammatory and pro-apoptotic pathways driven by TNFα/TNFR1 signaling. Further investigation of potential mechanisms of kidney-lung crosstalk during AKI may identify potential therapeutic targets for this deadly disease.