Autophagy is induced in renal tubular cells during acute kidney injury, however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7 knockout mouse model shown to be defective in both basal and cisplatin induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound LH- [1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (ICso = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.Nitric oxide (NO), S-nitrosothiols, and other NO donors (1) activate the soluble guanylyl cyclase (SGC) by binding to the heme moiety of the enzyme (2). This results in increased cGMP concentrations that are associated with relaxation in vascular tissue (for review, see ref.3). In addition, these compounds increase cGMP in platelets, and this is thought to be the mechanism by which they inhibit platelet function (4,5).Studies attempting to investigate the role of SGC in the mediation of the physiological actions of NO have been hampered by the lack of a potent and selective inhibitor of this enzyme. Methylene blue has been used as such; however, this compound also inhibits prostacyclin (PGI2) production (6, 7), generates superoxide anions (8-10), and directly inhibits NO synthase (11). Furthermore, LY 83583 (6-anilino-5,8-quinolinedione), which inhibits elevation of cGMP concentrations in a number of tissues (12, 13), probably does so by generating superoxide anions (14).1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) has been shown to inhibit N-methyl-D-aspartate-induced increases in cGMP concentrations in rat brain slices and to inhibit the activity of SGC purified from bovine lung (15). We have now used this compound to characterize the role of SGC in the actions of NO in platelets and vascular tissue.MATERIALS AND METHODS Washed Platelet Suspensions. Human blood was collected, and PGI2-washed platelet (WP) suspensions were prepared as described (16).Platelet cGMP Concentrations. WPs (2-2.5 X 108/ml) were incubated without stirring in a platelet-ionized calcium lumiaggregometer (Chronolog) for 20 min at 37°C in th...

Cigarette smoking is associated with impaired endothelium-dependent vasodilation and reduced nitric oxide (NO) in the exhaled air of smokers. To explore the mechanism for the impairment of NO-mediated vasodilation, we studied the effect of cigarette smoke extract (CSE) on NO synthase (eNOS) activity and content in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with CSE resulted in a time- and dose-dependent decrease in eNOS activity. The inhibitory effect of CSE on eNOS activity was not reversible. Both gas-phase and particulate-phase extracts of CSE contributed to the inhibition of eNOS activity. The protein kinase c (PKC) inhibitors staurosporine and chelerythrine did not affect the CSE-induced inhibition of eNOS activity. Catalase, superoxide dismutase (SOD), vitamin C, vitamin E, glutathione, and dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of eNOS activity, and incubation of PAEC with 3 mM nicotine did not change the activity of eNOS. Treatment of PAEC with CSE also caused a nonreversible, time-dependent decrease in eNOS protein content detected by Western blot analysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione contents or on lactate dehydrogenase (LDH) release. These results indicate that exposure to CSE causes an irreversible inhibition of eNOS activity in PAEC, and suggest that the decreased activity is secondary to reduced eNOS protein mass and mRNA. The decrease in eNOS activity may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.

Akt kinase, a member of AGC kinases, is important in many cellular functions including proliferation, migration, cell growth and metabolism. There are three known Akt isoforms which play critical and diverse roles in the cardiovascular system. Akt activity is regulated by its upstream regulatory pathways at transcriptional and post-translational levels. beta-catenin/Tcf-4, GLI1 and Stat-3 are some of few known transcriptional regulators of AKT gene. Threonine 308 and serine 473 are the two critical phosphorylation sites of Akt1. Translocation of Akt to the cell membrane facilitates PDK1 phosphorylation of the threonine site. The serine site is phosphorylated by mTORC2. Ack1, Src, PTK6, TBK1, IKBKE and IKKε are some of the non-canonical pathways which affect the Akt activity. Protein-protein interactions of Akt to actin and Hsp90 increase the Akt activity while Akt binding to other proteins such as CTMP and TRB3 reduces the Akt activity. The action of Akt on its downstream targets determines its function in cardiovascular processes such as cell survival, growth, proliferation, angiogenesis, vasorelaxation, and cell metabolism. Akt promotes cell survival via caspase-9, YAP, Bcl-2, and Bcl-x activities. Inhibition of FoxO proteins by Akt also increases cell survival by transcriptional mechanisms. Akt stimulates cell growth and proliferation through mTORC1. Akt also increases VEGF secretion and mediates eNOS phosphorylation, vasorelaxation and angiogenesis. Akt can increase cellular metabolism through its downstream targets GSK3 and GLUT4. The alterations of Akt signaling play an important role in many cardiovascular pathological processes such as atherosclerosis, cardiac hypertrophy, and vascular remodeling. Several Akt inhibitors have been developed and tested as anti-tumor agents. They could be potential novel therapeutics for the cardiovascular diseases.

Objective Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3), is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. Approach and Results Under hypoxic conditions or following treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. Additionally, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated AKT (pAKT) was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. Conclusions The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis.

Patients with chronic kidney diseases, including diabetic nephropathy, are more susceptible to acute kidney injury (AKI) and have a worse prognosis following AKI. However, the underlying mechanism is unclear. Here we tested whether diabetic mice were more sensitive to AKI and show that renal ischemia-reperfusion induced significantly more severe AKI and higher mortality in the streptozotocin and the Akita diabetic mouse models. The severity of AKI in the mice correlated with their blood glucose levels. In vitro, high glucose-conditioned renal proximal tubular cells showed higher apoptosis and caspase activation following ATP-depletion and hypoxic injury, accompanied by a heightened mitochondrial accumulation of Bax and release of cytochrome c. In response to injury, both glucose-conditioned renal proximal tubular cells and diabetic kidney tissues showed markedly higher p53 induction. Suppression of p53 diminished the sensitivity of high glucose-conditioned cells to acute injury in vitro. Moreover, blockade of p53 by pifithrin-α, siRNA, or proximal tubule-targeted gene ablation reduced ischemic AKI in diabetic mice. Insulin reduced blood glucose in diabetic mice and largely attenuated their AKI sensitivity. Thus, our results suggest the involvement of hyperglycemia, p53 and mitochondrial pathway of apoptosis in the susceptibility of diabetic models to AKI.

C/EBP homologous protein (Chop) has been shown to have altered expression in patients with idiopathic pulmonary fibrosis (IPF), but its exact role in IPF pathoaetiology has not been fully addressed. Studies conducted in patients with IPF and Chop(-/-) mice have dissected the role of Chop and endoplasmic reticulum (ER) stress in pulmonary fibrosis pathogenesis. The effect of Chop deficiency on macrophage polarization and related signalling pathways were investigated to identify the underlying mechanisms. Patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis were affected by the altered Chop expression and ER stress. In particular, Chop deficiency protected mice against BLM-induced lung injury and fibrosis. Loss of Chop significantly attenuated transforming growth factor β (TGF-β) production and reduced M2 macrophage infiltration in the lung following BLM induction. Mechanistic studies showed that Chop deficiency repressed the M2 program in macrophages, which then attenuated TGF-β secretion. Specifically, loss of Chop promoted the expression of suppressors of cytokine signaling 1 and suppressors of cytokine signaling 3, and through which Chop deficiency repressed signal transducer and activator of transcription 6/peroxisome proliferator-activated receptor gamma signaling, the essential pathway for the M2 program in macrophages. Together, our data support the idea that Chop and ER stress are implicated in IPF pathoaetiology, involving at least the induction and differentiation of M2 macrophages.

OBJECTIVE Pulmonary Hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PA) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PA and determine the functional relevance of Nox4 in PH. APPROACH AND RESULTS Elevated expression of Nox4 was detected in hypertensive PA from 3 rat PH models and human PH using qRT-PCR, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of ROS production. Small molecule inhibitors of Nox4 reduced adventitial ROS generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and non-invasive indices of PA stiffness in monocrotaline (MCT)-treated rats as determined by morphometric analysis and high resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PA. In fibroblasts, Nox4 over-expression stimulated migration and proliferation and was necessary for matrix gene expression. CONCLUSIONS These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling and the development of PH.