Buller CL, Loberg RD, Fan MH, Zhu Q, Park JL, Vesely E, Inoki K, Guan KL, Brosius FC III. A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression.

Background: Excess nutrients induce adipose inflammation. Results: Excess glucose and palmitate generate ROS via NOX4 by a mechanism that involves the PPP and translocation of NOX4 into LRs, rather than by mitochondrial oxidation. Conclusion: NOX4 activates monocyte chemotactic factor expression. Significance: Understanding the source of ROS generation may lead to the development of new therapeutic targets for adipose tissue inflammation.

Eya1 is a critical gene for mammalian organogenesis. Mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by varying combinations of branchial, otic and renal anomalies, whereas deletion of mouse Eya1 results in the absence of multiple organ formation. Eya1 and other Eya gene products share a highly conserved 271 amino acid Eya domain that is required for protein-protein interaction. Recently, several point mutations that result in single amino acid substitutions in the conserved Eya domain region of EYA1 have been identified in BOR patients; however, the molecular and developmental basis of organ defects that occurred in BOR syndrome is unclear. To understand how these point mutations cause disease, we have analyzed the functional importance of these Eya domain missense mutations with respect to protein complex formation and cellular localization. We have demonstrated that these point mutations do not alter protein localization. However, four mutations are crucial for protein-protein interactions in both yeast and mammalian cells. Our results provide insights into the molecular mechanisms of organ defects detected in human syndromes.

Diabetic nephropathy (DN) is characterized by a plethora of signaling abnormalities that together ultimately result in the clinical and pathologic hallmarks of DN, namely progressive albuminuria followed by a gradual decline in glomerular filtration rate leading to kidney failure, and accompanied by podocyte loss, progressive glomerular sclerosis and, ultimately, progressive tubulointerstitial fibrosis. Over the past few years, the general understanding of the abnormalities in signaling pathways that lead to DN has expanded considerably. In this review, some of the important pathways that appear to be involved in driving this process are discussed, with special emphasis on newer findings and insights. Newer concepts regarding signaling changes in bradykinin, mTOR, JAK/STAT, MCP-1, VEGF, endothelial nitric oxide synthase, activated protein C and other pathways are discussed. Keywordsdiabetes; glomerulus; matrix proteins; signaling Diabetic nephropathy (DN) is usually manifested clinically by gradually worsening albuminuria, followed by a decline in glomerular filtration rate, which over years or decades leads to end-stage kidney disease in many patients with Type 1 or Type 2 diabetes. The pathologic correlates of this process are glomerular podocyte damage and loss [1], followed by the gradual, inexorable scarring of the renal glomerulus and then a similar fibrosing process

This study examines whether longitudinal antioxidant treatment initiated in prehypertensive spontaneously hypertensive rats (SHR) can attenuate vascular oxidant stress and prevent blood pressure elevation during development. Male SHR and age-matched Wistar-Kyoto rats (WKY) were treated from 6 to 11 weeks of age with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl) (1 mmol/l in drinking water), a membrane-permeable superoxide dismutase mimetic. Mean systolic blood pressures (SBPs) were measured by tail-cuff Agonist-induced and basal O2- production was measured in thoracic aortas of 6- and 11-week-old SHR and WKY by lucigenin-derived chemiluminescence and oxidative fluorescent microscopy, respectively. SBP of 6-week-old SHR (131 +/- 5 mmHg) and WKY (130 +/- 4 mmHg) were not different; however, 11-week-old SHR SBP (171 +/- 4 mmHg) was significantly greater (p = .0001) than 11-week-old WKY SBP (143 +/- 5 mmHg). Tempol treatment completely, but reversibly, prevented this age-related rise in SHR SBP (SHR + Tempol: 137 +/- 4 mmHg; p < .0001 versus untreated SHR). Agonist-induced vascular O2- was increased in 6- (p = .03) and 11-week-old SHR (p< .0001) and 11-week-old WKY (p = .03) but not in 6-week-old WKY. Long-term Tempol treatment significantly lowered O2- production in both strains. Basal O2- measurements in both 6- and 11-week-old SHR were qualitatively increased compared with age-matched WKY; this increase in SHR was inhibited with in vitro Tempol treatment. These data show that antioxidant treatment to reduce oxidative stress prevents the age-related development of high blood pressure in an animal model of genetic hypertension.

Buller CL, Heilig CW, Brosius FC 3rd. GLUT1 enhances mTOR activity independently of TSC2 and AMPK.

Chronic kidney disease (CKD) is a significant public health problem, and progression to end-stage renal disease leads to dramatic increases in morbidity and mortality. The mechanisms underlying progression of disease are poorly defined, and current noninvasive markers incompletely correlate with disease progression. Therefore, there is a great need for discovering novel markers for CKD. We utilized a glycoproteomic profiling approach to test the hypothesis that the urinary glycoproteome profile from subjects with CKD would be distinct from healthy controls. N-linked glycoproteins were isolated and enriched from the urine of healthy controls and subjects with CKD. This strategy identified several differentially expressed proteins in CKD, including a diverse array of proteins with endopeptidase inhibitor activity, protein binding functions, and acute-phase/immune-stress response activity supporting the proposal that inflammation may play a central role in CKD. Additionally, several of these proteins have been previously linked to kidney disease implicating a mechanistic role in disease pathogenesis. Collectively, our observations suggest that the human urinary glycoproteome may serve as a discovery source for novel mechanism-based biomarkers of CKD.

ABSTRACT:Reactive oxygen species (ROS) are hypothesized to play a key role in myocardial ischemia-reperfusion (IR) injury after cardiopulmonary bypass in children. Clinical studies in adults and several animal models suggest that myocardial IR injury involves cardiomyocyte apoptosis and necrosis. This study investigated a potential relationship between IR-induced ROS production and neonatal cardiomyocyte apoptosis using both in vitro and ex vivo techniques. For in vitro experiments, embryonic rat cardiomyocytes (H9c2 cells) exposed to hypoxia-reoxygenation (HR) showed a timedependent increase in gp91 phox (a marker for ROS production by NADPH oxidases), caspase-3 (a key mediator of apoptosis) expression, and a decrease in the glutathione redox ratio. N-acetylcysteine (NAC; 0.25-2 mM), a potent antioxidant, decreased gp91 phox and caspase-3 expression, inhibited apoptosis and restored the glutathione redox ratio. M yocardial ischemia-reperfusion (IR) injury associatedwith cardiopulmonary bypass and cardioplegic arrest contributes to adverse outcomes after cardiac surgery. The pathogenesis of IR injury is complex, but reactive oxygen species (ROS) generated during IR are thought to play a pivotal role leading to membrane lipid peroxidation, protein denaturation, and DNA modification, all of which may result in irreversible myocyte injury and cell death. Oxygen free radicals are cytotoxic molecules generated during reperfusion and/or reoxygenation of a previously hypoxic tissue bed. The cell damage induced by ROS can also initiate a local inflammatory response, which leads to further oxidant stressmediated tissue damage (1).Several studies have suggested that ROS are involved in the pathogenesis of perinatal asphyxia (2). Neonates, especially those born prematurely, are particularly vulnerable to ROSmediated tissue damage due, in part, to their immature native antioxidant system (3,4). Similarly, myocardial IR injury seems to be a more clinically significant problem in infants after cardiac surgery compared with adults (5-7). Several ROS-producing systems have been identified in many cell types. NADPH oxidase is reported to be a primary source of ROS production in cardiac tissue, and gp91 phox is responsible for the catalytic activity of NADPH oxidase (8,9). Increased ROS induce apoptosis in cardiomyocytes. Caspase-3 protein is a member of the cysteine-aspartic acid protease family that has been identified as being a key mediator of apoptosis in mammalian cells (10,11).N-acetylcysteine (NAC) is a thiol-containing molecule that acts as a free radical scavenger. In addition, NAC is a glutathione precursor that increases intracellular antioxidant capacity (12). Clinically, NAC is indicated for the treatment of acetaminophen overdose, for the prevention of radiocontrastinduced nephropathy, and as an adjuvant in respiratory conditions with excessive and/or thick mucus production. However, there are contradictory reports on the effects of NAC in clinical (13,14) and animal studies (15-17) of myocardial IR injury. Furthe...