Quantitation of apoptotic cells

Since ERK MAPK and Akt signaling pathways are recognized to protect against endothelial cell apoptosis and since hepatic IR caused AKI directly causes renal endothelial cell apoptosis with subsequent vascular Erlotinib dysfunction and neutrophil infiltration, we hypothesized that sphinganine 1 phosphate via S1P1 receptormediated activation of ERK MAPK and Akt signaling pathways protect against renal endothelial cell apoptosis and reduce AKI after liver IR. Additionally, we've shown previously that superior phosphorylation along with increased synthesis of heat shock protein 27 secured against endothelial cell apoptosis and vascular compromise after hepatic IR. For that reason, we postulated that sphinganine 1 phosphate may also improve HSP27 phosphorylation and upregulation. Eventually, since endothelial nitric-oxide synthase up-regulation with eventually increased release of NO shields against vascular endothelial cell injury, and since S1P receptor activation is well known to trigger eNOS to improve NO amounts in the vasculature, we postulated that sphinganine 1 phosphate activation of S1P1 receptors may defend against liver and kidney injury via stimulating Cellular differentiation the eNOS pathway. In this study, we tested the hypothesis that sphinganine 1 phosphate protects against liver IR induced hepatic and renal dysfunction via S1P1 receptor activation coupled to pertussis toxin sensitive G proteins with subsequent activation of cytoprotective kinases including ERK MAPK and Akt and induction of HSP27 and eNOS in the kidney and liver. We also established in this research the S1P receptor subtype involved with S1P mediated hepatic and renal protection using both pharmacologic in addition to gene knock-down approaches. Reagents Sphinganine 1 phosphate and 3 Amino 4 oxobutylphosphonic acid were purchased from Avanti Polar Lipids, Inc. 5 3 1,2,4 oxadiazole and 1 pyridin 6 yl] 4 semicarbazide were bought from Tocris Bioscience. Icotinib 2 undecyl thiazolidine 4 carboxylic acid was obtained from Cayman Chemical. Wortmannin and D N5 ornithine were obtained from EMD Chemicals, Inc. Unless otherwise specified, all other reagents including PD98059 were purchased from Sigma. Murine model of hepatic IR All protocols were accepted by the Institutional Animal Care and Use Committee of Columbia University. As described previously male C57BL/6 mice were put through liver IR injury. This process of partial hepatic ischemia for 60 min. in a segmental hepatic ischemia but spares the right lobe of the liver and prevents mesenteric venous congestion by allowing portal decompression through the right and caudate lobes of the liver. Scam controlled rats were put through laparotomy and similar liver manipulations minus the vascular occlusion. Plasma in addition to liver and kidney tissues were collected 24 hrs after liver IR injury.