Abstract. Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X L and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.Erythropoietin (EPO) is the major regulator of proliferation and differentiation of erythroid progenitor cells through its antiapoptotic actions (1). EPO gene expression is under the control of the oxygen-sensitive transcription factor hypoxiainducible factor (HIF-1), which consists of the regulatory subunit HIF-1␣ and the constitutively expressed subunit HIF-1␤. Low oxygen tension adverts enzymatic prolyl-residue hydroxylation by prolyl-4-hydroxylase, which, in normoxia, serves as a signal for polyubiquitination and proteosomal degradation, thereby preventing von-Hippel-Lindau (VHL)-dependent HIF degradation, leading to nuclear accumulation of HIF-1 (2). HIF-1 controls the expression of several cytokines that mediate the adaptive response to ischemia, including vascular endothelial growth factor and glucose metabolism. The prolyl-4-hydroxylase requires iron as a co-factor, and cobalt mimics the effect of hypoxia on HIF-1␣ activation (3). Cobalt administration to rats caused upregulation of HIF-dependent proteins, including EPO, and diminished the degree of renal injury caused by ischemia-reperfusion (I/R), suggesting the HIF/EPO pathway may play an important role in ischemic preconditioning (4). EPO is upregulated in the brain and spinal cord after hypoxic stimuli and protects neurones against ischemic or oxidative injury in vivo and in vitro (5,6). The neuroprotective effects of EPO are dependent on EPO receptormediated JAK2 phosphorylation and NF-B-dependent transcription of antiapoptotic genes, including endogenous inhibitors of apoptosis XIAP and cIAP-2 (7). In the retina, EPO upregulation is essential for hypoxic preconditioning via HIF-1␣ stabilization. The systemic administration of recombinant EPO also reduces the degree of retinal apoptosis induced by high-intensity light insult (8). EPO receptor-mediated intracellular signaling may involve nuclear translocation of the transcription factor NF-B and phosphorylation of Akt (protein kinase B) by phosph...

Abnormal elevation of hepatic gluconeogenesis is central to the onset of hyperglycaemia in patients with type 2 diabetes mellitus (T2DM). Metformin corrects hyperglycaemia through inhibition of gluconeogenesis, but its mechanism of action is yet to be fully described. SIRT1 and GCN5 (listed as KAT2A in the MGI Database) have recently been identified as regulators of gluconeogenic gene expression through modulation of levels and activity of the coactivators cAMP-response element binding protein-regulated transcription coactivator 2 (TORC2 or CRTC2 as listed in the MGI Database) and peroxisome proliferator-activated receptor-g coactivator-1a (PGC1a or PPARGC1A as listed in the MGI Database). We report that in db/db mice, metformin (250 mg/kg per day; 7 days) increases hepatic levels of GCN5 protein and mRNA compared with the untreated db/db mice, as well as increases levels of SIRT1 protein and activity relative to controls and untreated db/db mice. These changes were associated with reduced TORC2 protein level and decreased gene expression and activation of the PGC1a gene target phosphoenolpyruvate carboxykinase, and lower plasma glucose and insulin. Inhibition of SIRT1 partially blocked the effects of metformin on gluconeogenesis. SIRT1 was increased through an AMP-activated protein kinase-mediated increase in gene expression of nicotinamide phosphoribosyltransferase, the rate-limiting enzyme of the salvage pathway for NAD C . Moreover, levels of GCN5 were dramatically reduced in db/db mice compared with the controls. This indicates that loss of GCN5-mediated inhibition of gluconeogenesis appears to constitute a major mechanism for the onset of abnormally elevated hepatic glucose production in db/db mice. In conclusion, induction of GCN5 and SIRT1 potentially represents a critical mechanism of action of metformin. In addition, these data identify induction of hepatic GCN5 as a potential therapeutic strategy for treatment of T2DM.

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine g-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-kB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-kB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

Sex differences have been widely reported in neuroinflammatory disorders, focusing on the contributory role of estrogen. The microvascular endothelium of the brain is a critical component of the blood-brain barrier (BBB) and it is recognised as a major interface for communication between the periphery and the brain. As such, the cerebral capillary endothelium represents an important target for the peripheral estrogen neuroprotective functions, leading us to hypothesise that estrogen can limit BBB breakdown following the onset of peripheral inflammation.Comparison of male and female murine responses to peripheral LPS challenge revealed a short-term inflammation-induced deficit in BBB integrity in males that was not apparent in young females, but was notable in older, reproductively senescent females. Importantly, ovariectomy and hence estrogen loss recapitulated an aged phenotype in young females, which was reversible upon estradiol replacement. Using a well-established model of human cerebrovascular endothelial cells we investigated the effects of estradiol upon key barrier features, namely paracellular permeability, transendothelial electrical resistance, tight junction integrity and lymphocyte transmigration under basal and inflammatory conditions, modelled by treatment with TNFα and IFNγ. In all cases estradiol prevented inflammationinduced defects in barrier function, action mediated in large part through up-regulation of the central coordinator of tight junction integrity, annexin A1. The key role of this protein was then further confirmed in studies of human or murine annexin A1 genetic ablation models.