Laura E. White1, Yan Cui1, Carolyn M. Feltes Shelak3, Heitham T. Hassoun2
1The Methodist Hospital and Research Institute, Houston, TX; 2The Methodist Hospital and Research Institute and The Methodist DeBakey Heart & Vascular Center, Houston, TX; 3The Johns Hopkins School of Medicine, Baltimore, MD.
OBJECTIVES: Despite advancements in renal replacement therapy, mortality with acute kidney injury (AKI) remains high, likely due to remote organ injury. Kidney ischemia-reperfusion injury (IRI) activates cellular and soluble mediators, driving lung inflammation, TNFR1-mediated apoptosis and microvascular barrier dysfunction. We hypothesized that lung microvascular endothelial cells (ECs), with their integral role maintaining the lung semipermeable barrier, were key cellular targets of TNFR1-mediated apoptosis during ischemic AKI.
METHODS: Male C57/BL6 mice and Sprague-Dawley rats underwent 60 min bilateral renal pedicle occlusion (IRI) or sham laparotomy(sham) and sacrifice at 4 or 24 hrs. TUNEL/DAPI/CD34 colocalization identified EC apoptosis in vivo and ECs [CD45-/CD31+] isolated by novel techniques underwent RT-PCR Apoptosis SuperArray analysis. In parallel, rat lung microvascular ECs(RLMVECs) treated with serum from rats after sham or IRI underwent custom RT-PCR analysis for apoptosis and TNF family gene transcription; capase-3 and PARP activity assays identified RLMVEC apoptosis. Separately, inflammatory genes and apoptosis were compared in RLMVECs treated with TNFα/TNFR1 signaling inhibition (Etanercept vs. vehicle) during ischemic AKI.
RESULTS: In vivo, TUNEL+cells colocalized with CD34 during ischemic AKI and isolated lung ECs demonstrated a proapoptotic transcriptome. In vitro, ischemic AKI activated apoptosis (FasL, Dapk1, Bcl10) and TNF family (TNFR1, TNFR2, TNFα) genes, caspase-3 (100±1.1 vs. 106.5±0.8, p=0.00325), and PARP (10±3.5 vs. 61.8±20.5, p=0.047) vs. sham. Etanercept inhibited RLMVEC inflammatory genes (E-Selectin,ICAM-1,IL-6,RhoB), caspase-3 activity (100±0.9 vs. 102±0.5, p=0.096) and PARP activity (10±2.3 vs. 10.9±2.2, p=0.783) vs. vehicle during ischemic AKI.
CONCLUSIONS: Ischemic AKI drives a proinflammatory and proapoptotic lung EC transcriptome with TNFR1-dependent apoptosis. Further study of EC-specific mechanisms of kidney-lung crosstalk during AKI may identify potential therapeutic targets.
AUTHOR DISCLOSURES: Y. Cui, Nothing to disclose; C. M. Feltes Shelak, Nothing to disclose; H. T. Hassoun, Nothing to disclose; L. E. White, Nothing to disclose.
Posted April 2012