Abstract
Acute renal injury is a major clinical problem directly related with increase in morbidity and mortality that is persistently increasing and remains without effectiveness therapy. Ischemia-reperfusion (IR) injury is the major cause of AKI, and develops from the interruption of blood flow and consequent deprivation of oxygen supply to the kidney. Studies showed that heme oxygenase-1 (HO-1) - enzyme which cleaves the heme molecule leading to production of biliverdin, carbon monoxide (CO) and iron - is cytoprotective and has anti-apoptotic effects not only in renal IR model, but others such as cisplatin induced renal injury model. Adenosine, an endogenous nucleotide, is capable of binding up to four different protein-coupled receptors G (A (1), A (2A), A (2B) and A (3)), is important in the adaptation, maintenance of cell function during hypoxia and to decrease hypoxia induced inflammation, and is also a potent inducer of HO-1 via ADORA-2A. Since studies have shown that both molecules are interrelated, and that HO-1 must be present in order to adenosine produce a protective effect, the aim of this project is to investigate the role of HO-1 on the receptors ADORA-2A and ADORA-2B in renal ischemia-reperfusion injury model. Since CD39, enzyme which contributes to ATP metabolism, is related to adenosine availability, CD39 knockout (KO) mice and macrophages derived from them will be used in order to confirm this relation. Macrophage culture cells stimulated with ATP, and subsequently with LPS, will be submitted to the treatment with CO using a bioactive gas-controlling system. We suppose that the inflammatory response in CD39 KO is going to be exacerbated, once that the lower adenosine availability would lead to lower HO-1 concentrations, and that CO treatment will be able to attenuate the inflammation. For in vivo model, we will perform ischemia and reperfusion C57BL6 and CD39 KO mice, and animals without renal clamping will be used as control (SHAM). The mice will be submited to CO treatment. We will analyze renal injury by urea and creatinine dosage in the serum, and the evaluation of cytokines expression and receptors ADORA-2A and ADORA-2B will be done by real time RT-PCR. Cytokines (such as TNF, IL-10, IL-6) will also be analysed by ELISA; immunofluorescence will be done in kidney in order to detect receptors ADORA-2A and-2B; and Western Blot will be performed to detect a-HIF-1a. Thus, this project aims to contribute to the understanding of the protective mechanism of HO-1 in renal ischemia-reperfusion injury model, which may have future clinical applications. (AU)
|