Effects of glutamine supplementation on kidney of diabetic rat

Glutamine is the most important donor of NH(3) in kidney playing an important role in acid-base buffering system. Besides this effect, glutamine presents many other relevant functions in the whole body, such as a precursor of arginine in adult and neonates. In addition to these effects, some studies have shown that glutamine can potentiate renal disease. In the present study, the effect of short-term treatment (15 days) with glutamine on control and diabetic rats was investigated. Using biochemical, histological and molecular biology analysis from control and diabetic rats we verified that glutamine supplementation increase in pro-inflammatory interleukins (IL)-1 beta and IL-6 content in renal cortex and induce alteration in glomerular characteristics. This study showed that short-term treatment with glutamine in association with increased glucose levels could cause important alterations in glomerular morphology that may result in fast progression of kidney failure.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Pesquisa (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Molecular Mechanisms by Which Saturated Fatty Acids Modulate TNF-alpha Expression in Mouse Macrophage Lineage

Many macrophage functions are modulated by fatty acids (FAs), including cytokine release, such as tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is of great interest due to its role in the inflammation process observed in several diseases such as rheumatoid arthritis, atherosclerosis, and obesity. However, the mechanisms by which FA effects occur have not been completely elucidated yet. In this study, we used a mouse monocyte lineage (J774 cells) to evaluate the effect of 50 and 100 mu M of saturated (palmitic and stearic acids), monounsaturated (oleic acid) and polyunsaturated (linoleic acid) FAs on TNF-alpha production. Alterations in gene expression, poly(A) tail length and activation of transcription factors were evaluated. Oleic and linoleic acids, usually known as neutral or pro-inflammatory FA, inhibited LPS-induced TNF-alpha secretion by the cells. Saturated FAs were potent inducers of TNF-alpha expression and secretion under basal and inflammatory conditions (in the presence of LPS). Although the effect of the saturated FA was similar, the mechanism involved in each case seem to be distinct, as palmitic acid increased EGR-1 and CREB binding activity and stearic acid increased mRNA poly(A) tail. These results may contribute to the understanding of the molecular mechanisms by which saturated FAs modulate the inflammatory response and may lead to design of associations of dietary and pharmacological strategies to counteract the pathological effects of TNF-alpha.FAPESP[2006/02709-3]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPqConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPE

Dyslipidemia: a prospective controlled randomized trial of intensive glycemic control in sepsis

Metabolic disturbances are quite common in critically ill patients. Glycemic control appears to be an important adjuvant therapy in such patients. In addition, disorders of lipid metabolism are associated with worse prognoses. The purpose of this study was to investigate the effects that two different glycemic control protocols have on lipid profile and metabolism. We evaluated 63 patients hospitalized for severe sepsis or septic shock, over the first 72 h of intensive care. Patients were randomly allocated to receive conservative glycemic control (target range 140-180 mg/dl) or intensive glycemic control (target range 80-110 mg/dl). Serum levels of low-density lipoprotein, high-density lipoprotein, triglycerides, total cholesterol, free fatty acids, and oxidized low-density lipoprotein were determined. In both groups, serum levels of low-density lipoprotein, high-density lipoprotein, and total cholesterol were below normal, whereas those of free fatty acids, triglycerides, and oxidized low-density lipoprotein were above normal. At 4 h after admission, free fatty acid levels were higher in the conservative group than in the intensive group, progressively decreasing in both groups until hour 48 and continuing to decrease until hour 72 only in the intensive group. Oxidized low-density lipoprotein levels were elevated in both groups throughout the study period. Free fatty acids respond to intensive glycemic control and, because of their high toxicity, can be a therapeutic target in patients with sepsis.The Sao Paulo Research Foundation (FAPESP) [04/02161-2]The Sao Paulo Research Foundation (FAPESP

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

<div><p>Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.</p></div

Effect of PMA, fMLP and hyperglycemia on calcium influx and ER calcium content in neutrophil-like HL60 cells (dHL60).

<p>PMA (1 μM) (A) did not alter calcium dynamics in dHL60 cells. fMLP (1 μM) (B-D) promoted calcium influx in neutrophil-like HL60 cells; and hyperglycemia (C) did not disturb the calcium intake by fMLP stimulation. ER calcium content in dHL60 was not altered by hyperglycemia (E; F). Calcium dynamics were monitored by Indo-1-AM (1 μM) using fluorometry. [NG] normoglycemic media (5.5 mM of glucose); [MN] Mannitol enriched media (5.5 mM of glucose + 19.5 mM of mannitol); [HG] Hyperglycemic media (25 mM of glucose). Graphs show fluorescence intensity during the time of analysis (A; B; C; E). Histograms show the mean intracellular calcium concentration ± S.E.M. (D) and the mean ± S.E.M. of ER calcium content (F). Results are from 4 independent experiments. (**) Indicate p <0.01 and (*) indicate p<0,05.</p

Effect of ROS production on UPR regulated proteins in neutrophil-like HL60 cells (dHL60).

<p><b> </b> PMA (1 μM) treatment for 1 and 4h increased phosphorylated eIF2α (A; C) and GRP78 protein levels (B; D). This was blocked by DPI (10 μM), a NADPH oxidase inhibitor (A; B; C; D). GADD34 and ATF4 protein content was not altered by PMA (E; F). Histograms (C; D; E; F) show the mean ± S.E.M. of the optical density (OD) of the protein bands. γ Tubulin was used as loading control. Results are from at least 3 independent experiments. (***) Indicates p <0.001 and (**) Indicates p <0.01.</p

Effect of hyperglycemia and PMA on ROS production in neutrophil-like HL60 cells (dHL60) and non-differentiated HL60 cells.

<p>PMA (1μM) triggered the production of ROS only in dHL60 (A; C). Hyperglycemia did not affect the ROS production in neutrophil-like HL60 cells (B). DPI (10 μm) was used as NADPH oxidase inhibitor. [NG] normoglycemic media (5.5 mM of glucose); [MN] Mannitol enriched media (5.5 mM of glucose + 19.5 mM of mannitol); [HG] Hyperglycemic media (25 mM of glucose). DHR (10 μM) was used to monitor ROS production by flow cytometry. Graphs show median of fluorescence ± S.E.M. Results are from 6 independent experiments. (***) Indicate p <0.001.</p