Evaluation of NF-kB intracellular signaling pathway and endostatin on human mesangial cell in endotoxic acute kidney injury during diabetes mellitus

Abstract

Acute Kidney Injury (AKI) is commonly associated with multiple organ failure and sepsis and its presence is a marker of high mortality in levels of 70 to 90%. The AKI mechanisms are complexes and is target of several studies, however their pathophysiology still unclear.The sepsis is characterized by general inflammatory response and activation of fibrinolytc and coagulation pathway, resulting on endothelial injury. Several substances are released in systemic circulation in consequence of endotoxemy. The cytokines rise nitric oxide synthesis, decreasing the vascular resistance, in compensatory response, the circulation of vasopressors hormones are increased, including angiotensin II.The Akt (PKB) is a serine/treonine kinase activated when cells are stimulated by several growth factors. The signaling pathway PI3Akt modulate normal cells functions like cell growth, proliferation and cell survive. The wrong regulation of this pathway has been associated with cancer and diabetes development. Despite the important participation on normal physiology, the NF-kB inappropriate regulation is related with pathophysiology of cardiovascular disease, diabetes tipe-2, chronic inflammation and cancer, however, their participation in AKI still unclear.The endostatin was characterized by its antiangiogenic property and its specific inhibition of cell proliferation, however, poor know its action, expression and function during sepsis. Moreover, its alterations are worse during diabetes mellitus by the pre-existents complications and modified responses of AKI pathophysiological mechanisms.Therefore, the aim of this study is evaluate the NF-kB, Akt and endostatin expression in models of endotoxic AKI and diabetes. Immortalized human mesangial cell will cultured in DMEM exposed to high-glucose medium (30mM) for diabetes induction during 30min, 1h and 24h, and sepsis will provoked by incubation with LPS (100mg/ml) in the same times. The overlap of these injuries also is tested. The mRNA expression levels of p65 member of NFkB family and endostatina will estimated by real-time RT-PCR and immunocytochemistry. To evaluate angiotensin II will performed immunoenzymatic assay (ELISA) and to analyse Akt protein, Western blot will be implemented. Also will be performed intracellular calcium (espectrofluorimetry), proliferation, apoptosis and necrosis cellular (flow cytometry).