K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells.

P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the mitochondrial potassium pool was mobilized in a P2X7 signaling, and ATP dose-dependent manner, suggesting a role for mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further, intracellular calcium chelation with BAPTA-AM indicated that P2X7-induced potassium depletion was independent of calcium mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.

Lifestyle and metabolic approaches to maximizing erectile and vascular health.

Oxidative stress and inflammation, which disrupt nitric oxide (NO) production directly or by causing resistance to insulin, are central determinants of vascular diseases including ED. Decreased vascular NO has been linked to abdominal obesity, smoking and high intakes of fat and sugar, which all cause oxidative stress. Men with ED have decreased vascular NO and circulating and cellular antioxidants. Oxidative stress and inflammatory markers are increased in men with ED, and all increase with age. Exercise increases vascular NO, and more frequent erections are correlated with decreased ED, both in part due to stimulation of endothelial NO production by shear stress. Exercise and weight loss increase insulin sensitivity and endothelial NO production. Potent antioxidants or high doses of weaker antioxidants increase vascular NO and improve vascular and erectile function. Antioxidants may be particularly important in men with ED who smoke, are obese or have diabetes. Omega-3 fatty acids reduce inflammatory markers, decrease cardiac death and increase endothelial NO production, and are therefore critical for men with ED who are under age 60 years, and/or have diabetes, hypertension or coronary artery disease, who are at increased risk of serious or even fatal cardiac events. Phosphodiesterase inhibitors have recently been shown to improve antioxidant status and NO production and allow more frequent and sustained penile exercise. Some angiotensin II receptor blockers decrease oxidative stress and improve vascular and erectile function and are therefore preferred choices for lowering blood pressure in men with ED. Lifestyle modifications, including physical and penile-specific exercise, weight loss, omega-3 and folic acid supplements, reduced intakes of fat and sugar, and improved antioxidant status through diet and/or supplements should be integrated into any comprehensive approach to maximizing erectile function, resulting in greater overall success and patient satisfaction, as well as improved vascular health and longevity.

TNF-alpha neutralization ameliorates obstruction-induced renal fibrosis and dysfunction.

Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline in renal function. Although several inflammatory mediators have been implicated in the pathophysiology of renal obstruction, the contribution of TNF-alpha to obstruction-induced fibrosis and renal dysfunction has not been thoroughly evaluated. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1) every 84 h. The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were analyzed for TNF-alpha expression (ELISA), macrophage infiltration (ED-1 staining), transforming growth factor-beta(1) expression (ELISA, RT-PCR), collagen I and IV activity (Western Blot, immunohistochemistry), alpha-smooth muscle actin accumulation (immunohistochemistry, Western blot analysis), and angiotensinogen expression (Western blot). In a separate arm, the glomerular filtration rate (inulin clearance) of rats subjected to unilateral ureteral obstruction in the presence of either vehicle or PEG-sTNFR1 was determined. Renal obstruction induced increased tissue TNF-alpha and transforming growth factor-beta(1) levels, collagen I and IV activity, interstitial volume, alpha-smooth muscle actin accumulation, angiotensinogen expression, and renal dysfunction, whereas treatment with PEG-sTNFR1 significantly reduced each of these markers of renal fibrosis. These results demonstrate that TNF-alpha mediates obstruction-induced renal fibrosis and identify TNF-alpha neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.

TNF-alpha mediates obstruction-induced renal tubular cell apoptosis and proapoptotic signaling.

Obstruction of the upper urinary tract induces a progressive loss in renal mass through apoptotic renal cell death. Although TNF-alpha has been implicated in ischemia-reperfusion-induced apoptotic renal cell death, its role in obstructive renal cell apoptosis remains unknown. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Twenty-four hours before surgery and every 84 h thereafter, rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1). The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were subsequently analyzed for TNF-alpha (ELISA, RT-PCR), Fas ligand (RT-PCR), apoptosis (TUNEL, ELISA), and caspase 8 and 3 activity (Western blot). Renal obstruction induced increased tissue TNF-alpha and Fas ligand mRNA levels, TNF-alpha protein production, apoptotic renal tubular cell death, and elevated caspase 8 and 3 activity, whereas treatment with PEG-sTNFR1 significantly reduced obstruction-induced TNF-alpha production, renal tubular cell apoptosis, and caspase activity. PEG-sTNFR1 did not significantly alter Fas ligand expression. These results demonstrate that TNF-alpha mediates obstruction-induced renal tubular cell apoptosis and proapoptotic signaling and identify TNF-alpha neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.

p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis during simulated ischemia.

Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK(1), we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-alpha (TNF-alpha) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-alpha mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-alpha mRNA production and TNF-alpha bioactivity, and both p38 MAPK inhibition and TNF-alpha neutralization (anti-porcine TNF-alpha antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-alpha mRNA and biologically active TNF-alpha and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis after SI.

A novel model of ischemia in renal tubular cells which closely parallels in vivo injury.

PURPOSE: Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal models have been developed to investigate this condition, they are limited by differential renal cell inflammatory mediator production and heterogeneous cell sensitivity to ischemia. We therefore developed an in vitro model of renal tubular cell ischemia that simulates the cellular injury observed in animal models of renal IR injury. MATERIALS AND METHODS: Using the established renal tubular cell line, LLC-PK1, simulated ischemia was induced by immersing the cellular monolayer in mineral oil. The effect of simulated ischemia on renal tubular cells was then determined by measuring the time course of TNF-alpha protein expression (ELISA), TNF-alpha mRNA induction (RT-PCR), and renal tubular cell apoptosis (TUNEL). RESULTS: Maximal TNF-alpha protein expression occurs following 60 min of simulated ischemia and 2 h of substrate replacement (reimmersion in media), and maximal TNF-alpha mRNA induction occurs following 60 min of simulated ischemia. Cellular apoptosis peaks following 60 min of simulated ischemia and 24 h of reperfusion. CONCLUSION: The time course of TNF-alpha production and apoptosis induction in this model closely parallels the time course for these markers in vivo. This study constitutes the initial demonstration that an in vitro oil immersion model of ischemia simulates the cellular injury (TNF-alpha production and apoptosis) observed in animal models of renal ischemia-reperfusion. This model may be used to study cellular mechanisms of IR in the absence of the systemic confounding variables.

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism.

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.

Differential cellular immunolocalization of renal tumour necrosis factor-alpha production during ischaemia versus endotoxaemia.

Both renal ischaemia and endotoxaemia provoke renal dysfunction and cellular injury. Although the clinical manifestation of each insult is similar (global renal dysfunction), ischaemia and endotoxaemia induce different patterns of cellular injury. Tumour necrosis factor-alpha (TNF-alpha) has been implicated in both types of renal injury; however, it remains unknown whether differential cellular TNF-alpha expression accounts for these changes. We hypothesized that renal glomerular cells and tubular cells differentially express TNF-alpha in response to ischaemia compared with endotoxaemia. To investigate this hypothesis, male Sprague-Dawley rats were anaesthetized and exposed to various time-periods of renal ischaemia, with or without reperfusion (sham operation=negative control), or lipopolysaccharide (LPS) 0.5 mg/kg intraperitoneally (i.p.). The kidneys were harvested following renal injury, and rat TNF-alpha protein expression was determined (by enzyme-linked immunosorbent assay), as were TNF-alpha bioactivity (by WEHI-164 cell clone cytotoxicity assay) and TNF-alpha cellular localization (by immunohistochemistry). TNF-alpha protein expression and TNF-alpha bioactivity peaked following 1 hr of ischaemia and 2 hr of reperfusion (48 +/- 11 pg/mg of protein, P < 0.05, and 12 +/- 0.5 x 10-3 units/mg of protein, P < 0.05, respectively). The concentration of TNF-alpha increased to a similar extent following exposure to LPS; however, while TNF-alpha production following ischaemia-reperfusion injury localized predominantly to renal tubular epithelial cells, animals exposed to LPS demonstrated a primarily glomerular distribution of TNF-alpha production. Hence, the cellular localization of renal TNF-alpha production appears to be injury specific, i.e. renal tubular cells are the primary source of TNF-alpha following an ischaemic insult, whereas LPS induces glomerular TNF-alpha production. The cellular source of TNF-alpha following different insults may have therapeutic implications for targeted inhibition of TNF-alpha production.

The abdominal compartment syndrome is a morbid complication of postinjury damage control surgery.

BACKGROUND: The abdominal compartment syndrome (ACS) is a recognized complication of damage control surgery (DCS). The purposes of this study were to (1) determine the effect of ACS on outcome after DCS, (2) identify patients at high risk for the development of ACS, and (3) determine whether ACS can be prevented by preemptive intravenous bag closure during DCS. METHODS: Patients requiring postinjury DCS at our institution from January 1996 to June 2000 were divided into groups depending on whether or not they developed ACS. ACS was defined as an intra-abdominal pressure (IAP) greater than 20 mm Hg in association with increased airway pressure or impaired renal function. RESULTS: ACS developed in 36% of the 77 patients who underwent DCS with a mean IAP prior to decompression of 26 +/- 1 mm Hg. The ACS versus non-ACS groups were not significantly different in patient demographics, Injury Severity Score, emergency department vital signs, or intensive care unit admission indices (blood pressure, temperature, base deficit, cardiac index, lactate, international normalized ratio, partial thromboplastin time, and 24-hour fluid). The initial peak airway pressure after DCS was higher in those patients who went on to develop ACS. The development of ACS after DCS was associated with increased ICU stays, days of ventilation, complications, multiorgan failure, and mortality. CONCLUSIONS: ACS after postinjury DCS worsens outcome. With the exception of early elevation in peak airway pressure, we could not identify patients at higher risk for ACS; moreover, preemptive abdominal bag closure during initial DCS did not prevent this highly morbid complication.

Feasibility of damage control surgery in the management of military combat casualties.

Unique and expanded applications of staged operative management are undergoing careful evaluation in many civilian level I trauma centers under the rubric of damage control surgery. Recently there have been advocates for its broad application to the early management of critically injured combat casualties. However, the enormous logistic requirements for such strategies are contrary to the demands of the usual wartime scenario. On the basis of experience in civilian trauma centers and combat casualty management, we question the suggested extensive role of damage control surgery during wartime. Each decision point in damage control surgery should be analyzed as it is altered (sensitivity analysis) by conditions of war. It is unwise to adopt such indications unchanged from current civilian trauma policy.

Early renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF-alpha bioactivity in the kidney.

PURPOSE: Acute tubular necrosis (ATN) and the ensuing renal failure induced by ischemia and reperfusion injury (I/R) remain a major cause of morbidity and mortality among patients in the intensive care unit. Although it is well established that exogenous tumor necrosis factor-alpha (TNF) induces renal injury, it remains unknown whether ischemia and/or reperfusion activates the signaling mechanisms required for renal TNF production. We hypothesized that ischemia and/or reperfusion would activate the oxidant sensitive TNF transcription factor, nuclear factor kappa B (NFkappaB), and thereby lead to renal TNF production. MATERIALS AND METHODS: Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, after which various periods of renal ischemia, with or without reperfusion, were induced in rats. At different time intervals, kidneys were harvested and NFkappaB activation (electrophoretic mobility shift assay), TNF mRNA content (RT-PCR), and TNF bioactivity (WEHI-164 cell clone cytotoxicity assay) were determined. RESULTS: Results indicate that 15 minutes of ischemia alone activates NFkappaB, whereas peak activation occurred at 30 minutes of ischemia alone. NFkappaB remained activated through 60 minutes reperfusion. Thirty minutes of ischemia is required to induce renal TNF mRNA production; however, renal TNF protein expression and bioactivity peaked following 1 hour of ischemia and 2 hours reperfusion. CONCLUSIONS: These results are the initial demonstration that renal ischemia, with or without reperfusion, activates the TNF transcription factor NFkappaB and increases TNF bioactivity in the kidney.

Maximal human neutrophil priming for superoxide production and elastase release requires p38 mitogen-activated protein kinase activation.

HYPOTHESIS: Neutrophil priming has been implicated in the development of multiple organ failure, although the precise intracellular mechanisms that regulate neutrophil priming remain unclear. Our previous work characterized platelet-activating factor (PAF) priming of human neutrophils for concordant superoxide anion (O2-) generation and elastase degranulation. The p38 mitogen-activated protein kinase (MAPK) is activated by PAF stimulation. We hypothesized that PAF-induced human neutrophil priming for O2- and elastase release is mediated via the p38 MAPK pathway. DESIGN: Isolated neutrophils from 6 human donors were preincubated with the specific p38 MAPK inhibitor SB 203580 (1 micromol/L) or buffer (control) for 30 minutes. Cells were then primed with PAF (200 nmol/L), followed by receptor-dependent (N-formyl-methionyl-leucyl-phenylalanine, 1 micromol/L) or receptor-independent phorbol myristate acetate (PMA, 100 ng/mL) activation. SETTING: Urban trauma research laboratory. PATIENTS: Healthy volunteer donors of neutrophils. MAIN OUTCOME MEASURES: Maximal rate of O2- generation was measured by superoxide dismutase-inhibitable reduction of cytochrome c and elastase release by the cleavage of N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. RESULTS: SB 203580 significantly attenuated the generation of O2- and release of elastase from neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine but not with PMA. Independent of the activator receptor status, SB 203580 almost completely blocked the exaggerated neutrophil cytotoxic response due to PAF priming. CONCLUSIONS: The p38 MAPK pathway is required for maximal PAF-induced neutrophil priming for O2- production and elastase degranulation. Therefore, the MAPK signaling cascade may offer a potential therapeutic strategy to preempt global neutrophil hyperactivity rather than attempt to nullify the end products independently.

Interleukin-11 attenuates pulmonary inflammation and vasomotor dysfunction in endotoxin-induced lung injury.

Interleukin (IL)-11, like other members of the gp130 receptor class, possesses anti-inflammatory properties. We hypothesized that IL-11 pretreatment would attenuate endotoxin [lipopolysaccharide (LPS)]-induced lung inflammation and diminish injury to endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation that require cGMP in Sprague-Dawley rats. LPS (20 mg/kg ip) increased lung tumor necrosis factor (TNF)-alpha compared with the saline control (0.7 +/- 0.15 ng/g lung wet wt for control vs. 3.5 +/- 0.09 ng/g lung wet wt for LPS; P < 0.05). IL-11 (200 mg/kg ip) injected 10 min before LPS administration attenuated the LPS-induced lung TNF-alpha levels (1.6 +/- 0.91 ng/g lung wet wt; P < 0.05 vs. LPS). IL-11 also diminished LPS-induced lung neutrophil sequestration as assessed by myeloperoxidase units (2.1 +/- 0.25 U/g lung wet wt for saline and 15.6 +/- 2.02 U/g lung wet wt for LPS vs. 7.07 +/- 1.65 U/g lung wet wt for LPS plus IL-11; P < 0.05). Similarly, TNF-alpha binding protein (175 mg/kg) attenuated LPS-induced myeloperoxidase activity (6.04 +/- 0.14 U/g lung wet wt; P < 0.05). Both IL-11 and TNF-alpha binding protein similarly attenuated LPS-induced endothelium-dependent vasomotor dysfunction with improved relaxation responses to 10(-7) and 10(-6) M acetylcholine and A-23187 in phenylephrine-preconstricted isolated pulmonary artery rings (P < 0.05 vs. LPS). Endothelium-independent relaxation responses to sodium nitroprusside were also improved after LPS at 10(-6) M (P < 0.05 vs. LPS). Moreover, IL-11 decreased endotoxin-induced mortality in CF1 mice from 90 to 50% (P

Early kidney TNF-alpha expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion.

The purpose of this study was to determine whether isolated renal ischemia and reperfusion (I/R) induces renal tumor necrosis factor (TNF) mRNA production, TNF protein expression, or TNF bioactivity and, if so, whether local/early TNF production acts as mediator of ischemia-induced, neutrophil-mediated renal injury. After rats were anesthetized, varying periods of renal ischemia, with or without reperfusion, were induced. Kidney mRNA content (RT-PCR), TNF protein expression (ELISA), TNF bioactivity (WEHI-164 cell clone cytotoxicity assay), and neutrophil infiltration [myeloperoxidase (MPO) assay] were determined. In other animals, renal MPO and serum creatinine were assessed after TNF was neutralized [binding protein (TNF-BP)]. Thirty minutes of ischemia induced renal TNF mRNA. TNF protein expression and bioactivity peaked after 1 h ischemia and 2 h reperfusion, whereas neutrophil infiltration peaked at 4 h reperfusion. TNF-BP neutralized TNF bioactivity, reduced neutrophil infiltration, and protected postischemic function. These results constitute the initial demonstration that 1) early renal tissue TNF expression contributes to neutrophil infiltration and injury after I/R and 2) TNF-BP may offer a new adjunctive therapy in renal preservation prior to planned ischemic insults.

Liposomal delivery of heat-shock protein 72 into the heart prevents endotoxin-induced myocardial contractile dysfunction.

BACKGROUND: The purposes of this study were to (1) determine whether functional heat-shock protein 72 (HSP-72) may be delivered into the heart, (2) determine whether HSP-72 itself is protective against endotoxin (lipopolysaccharide [LPS]-induced cardiodepression, and (3) compare relative protection and time courses required for protection for thermally induced HSP-72 versus liposomally introduced HSP-72. METHODS: HSP-72 was introduced (liposomal HSP-72) or induced (heat shock, 42 degrees C x 15 minutes, 24 hours before) in rat heart before LPS administration (0.5 mg/kg intraperitoneal or ex vivo coronary infusion). Western blot analysis for HSP-72 was used to confirm its expression. Left ventricular developed pressure (Langendorff) was used as an index of cardiac function. RESULTS: Direct intracoronary perfusion of liposomal HSP-72 delivered functioning HSP-72 into the myocardium. LPS induced cardiodepression; however, heat shock pretreatment abolished LPS-induced contractile dysfunction. A direct connection was found between HSP-72 and protection derived from liposomal transfer experiments that similarly reduced LPS-induced cardiodepression. CONCLUSIONS: (1) HSP-72 prevents LPS-induced myocardial contractile dysfunction, (2) liposomal transfer of HSP-72 into the myocardium provides the first direct mechanistic connection between myocardial HSP-72 and protection against LPS, (3) HSP-72 induction requires 24 hours and liposomal transfer of HSP-72 requires 90 minutes, and (4) HSP-72 may offer a clinically acceptable means of protecting the heart.

Tumor necrosis factor-alpha and interleukin-1beta synergistically depress human myocardial function.

OBJECTIVE: Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta have been implicated in the pathogenesis of myocardial dysfunction in ischemia-reperfusion injury, sepsis, chronic heart failure, viral myocarditis, and cardiac allograft rejection. Although circulating TNF-alpha and IL-1beta are both often elevated in septic shock, it remains unknown whether TNF-alpha or IL-1beta are the factors induced during sepsis that directly depress human myocardial function, and if so, whether the combination synergistically depresses myocardial function. Furthermore, the mechanism(s) by which these cytokines induce human myocardial depression remain unknown. We hypothesized the following: a) TNF-alpha and IL-1beta directly depress human myocardial function; b) together, TNF-alpha and IL-1beta act synergistically to depress human myocardial function; and c) inhibition of ceramidase or nitric oxide synthase attenuates myocardial depression induced by TNF-alpha or IL-1beta by limiting proximal cytokine signaling or production of myocardial nitric oxide (NO). DESIGN: Prospective, randomized, controlled study. SETTING: Experimental laboratory in a university hospital. SUBJECTS: Freshly obtained human myocardial trabeculae. INTERVENTIONS: Human atrial trabeculae were obtained at the time of cardiac surgery, suspended in organ baths, and field simulated at 1 Hz, and the developed force was recorded. After a 90-min equilibration, TNF-alpha (1.25, 12.5, 125, or 250 pg/mL for 20 mins), IL-1beta (6.25, 12.5, 50, or 200 pg/mL for 20 mins), or TNF-alpha (1.25 pg/mL) plus IL-1beta (6.25 pg/mL) were added to the bath, and function was measured for the subsequent 100 mins after the 20-min exposure. To assess the roles of the sphingomyelin and NO pathways in TNF-alpha and IL-1beta cross-signaling, the ceramidase inhibitor N-oleoyl ethanolamine (1 microM) or the NO synthase inhibitor N(G)-monomethyl-L-arginine (10 microM) was added before TNF-alpha (125 pg/mL) or IL-1beta (50 pg/mL). MEASUREMENTS AND MAIN RESULTS: TNF-alpha and IL-1beta each depressed human myocardial function in a dose-dependent fashion (maximally depressing to 16.2 + 1.9% baseline developed force for TNF-alpha and 25.7 + 6.3% baseline developed force for IL-1beta), affecting systolic relatively more than diastolic performance (each p < .05). However, when combined, TNF-alpha and IL-1beta at concentrations that did not individually result in depression (p > .05 vs. control) resulted in contractile depression (p < .05 vs. control). Inhibition of myocardial sphingosine or NO release abolished the myocardial depressive effects of either TNF-alpha or IL-1beta. CONCLUSIONS: TNF-alpha and IL-1beta separately and synergistically depress human myocardial function. Sphingosine likely participates in the TNF-alpha and IL-1beta signal leading to human myocardial functional depression. Therapeutic strategies to reduce production or signaling of either TNF-alpha or IL-1beta may limit myocardial dysfunction in sepsis.

Inhibition of myocardial TNF-alpha production by heat shock. A potential mechanism of stress-induced cardioprotection against postischemic dysfunction.

Overproduction of tumor necrosis factor-alpha (TNF-alpha) contributes to cardiac dysfunction associated with systemic or myocardial stress, such as endotoxemia and myocardial ischemia/reperfusion (I/R). Heat shock has been demonstrated to enhance cardiac functional resistance to I/R. However, the protective mechanisms remain unclear. The purpose of this study was to determine: (1) whether cardiac macrophages express heat shock protein 72 (HSP72) after heat shock, (2) whether induced cardiac HSP72 suppresses myocardial TNF-alpha production during I/R, and (3) whether preservation of postischemic myocardial function by heat shock is correlated with attenuated TNF-alpha production during I/R. Rats were subjected to heat shock (42 degrees C for 15 min) and 24 h recovery. Immunoblotting confirmed the expression of cardiac HSP72. Immunofluorescent staining detected HSP72 in cardiac interstitial cells including resident macrophages rather than myocytes. Global I/R caused a significant increase in myocardial TNF-alpha. The increase in myocardial TNF-alpha was blunted by prior heat shock and the reduced myocardial TNF-alpha level was correlated with improved cardiac functional recovery. This study demonstrates for the first time that heat shock induces HSP72 in cardiac resident macrophages and inhibits myocardial TNF-alpha production during I/R. These observations suggest that inhibition of myocardial TNF-alpha production may be a mechanism by which HSP72 protects the heart against postischemic dysfunction.

Role of TNF in mediating renal insufficiency following cardiac surgery: evidence of a postbypass cardiorenal syndrome.

Recent evidence has implicated proinflammatory mediators such as TNF-alpha in the pathophysiology of ischemia-reperfusion (I/R) injury. Clinically, serum levels of TNF-alpha are increased after myocardial infarction and after cardiopulmonary bypass. Both cardiopulmonary bypass and renal ischemia-reperfusion injury induce a cascade of events leading to cellular damage and organ dysfunction. Tumor necrosis factor (TNF), a potent proinflammatory cytokine, is released from both the heart and the kidney in response to ischemia and reperfusion. TNF released during cardiopulmonary bypass induces glomerular fibrin deposition, cellular infiltration, and vasoconstriction, leading to a reduction in glomerular filtration rate (GFR). The signaling cascade through which renal ischemia-reperfusion induces TNF production is beginning to be elucidated. Oxidants released following reperfusion activate p38 mitogen-activated protein kinase (p38 MAP kinase) and the TNF transcription factor, NFkappaB, leading to subsequent TNF synthesis. In a positive feedback, proinflammatory fashion, binding of TNF to specific TNF membrane receptors can reactivate NFkappaB. This provides a mechanism by which TNF can upregulate its own expression as well as facilitate the expression of other genes pivotal to the inflammatory response. Following its production and release, TNF results in both renal and myocardial apoptosis and dysfunction. An understanding of these mechanisms may allow the adjuvant use of anti-TNF therapeutic strategies in the treatment of renal injury. The purposes of this review are: (1) to evaluate the evidence which indicates that TNF is produced by the heart following cardiopulmonary bypass; (2) to examine the effect of TNF on myocardial performance; (3) to outline the mechanisms by which the kidney produces significant TNF in response to ischemia and reperfusion; (5) to investigate the role of TNF in renal ischemia-reperfusion injury, (6) to describe the mechanisms of TNF-induced renal cell apoptosis, and (7) to suggest potential anti-TNF strategies designed to reduce renal insufficiency following cardiac surgery.

Calcium preconditioning, but not ischemic preconditioning, bypasses the adenosine triphosphate-dependent potassium (KATP) channel.

BACKGROUND: Recent evidence has implicated the KATP channel as an important mediator of ischemic preconditioning (IPC). Indeed, patients taking oral sulfonylurea hypoglycemic agents (i.e., KATP channel inhibitors) for treatment of diabetes mellitus are resistant to the otherwise profoundly protective effects of IPC. Unfortunately, many cardiopulmonary bypass patients, who may benefit from IPC, are chronically exposed to these agents. Calcium preconditioning (CPC) is a potent form of similar myocardial protection which may or may not utilize the KATP channel in its mechanism of protection. The purpose of this study was to determine whether CPC may bypass the KATP channel in its mechanism of action. If so, CPC may offer an alternative to IPC in patients chronically exposed to these agents. METHODS: Isolated rat hearts (n = 6-8/group) were perfused (Langendorff) and received KATP channel inhibition (glibenclamide) or saline vehicle 10 min prior to either a CPC or IPC preconditioning stimulus or neither (ischemia and reperfusion, I/R). Hearts were subjected to global warm I/R (20 min/40 min). Postischemic myocardial functional recovery was determined by measuring developed pressure (DP), coronary flow (CF), and compliance (end diastolic pressure, EDP) with a MacLab pressure digitizer. RESULTS: Both CPC and IPC stimuli protected myocardium against postischemic dysfunction (P < 0.05 vs I/R; ANOVA with Bonferroni/Dunn): DP increased from 52 +/- 4 (I/R) to 79 +/- 2 and 83 +/- 4 mmHg; CF increased from 11 +/- 0.7 to 17 +/- 2 and 16 +/- 1 ml/min; and EDP decreased (compliance improved) from 50 +/- 7 to 27 +/- 5 and 31 +/- 7 mmHg. However, KATP channel inhibition abolished protection in hearts preconditioned with IPC (P < 0.05 vs IPC alone), but not in those preconditioned with CPC (P > 0.05 vs CPC alone). CONCLUSIONS: (1) Both IPC and CPC provide similar myocardial protection; (2) IPC and CPC operate via different mechanisms; i.e., IPC utilizes the KATP channel whereas CPC does not; and (3) CPC may offer a means of bypassing the deleterious effects of KATP channel inhibition in diabetic patients chronically exposed to oral sulfonylurea hypoglycemic agents.

Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury induces a cascade of events leading to cellular damage and organ dysfunction. Tumor necrosis factor-alpha (TNF), a potent proinflammatory cytokine, is released from the kidney in response to, and has been implicated in the pathogenesis of, renal ischemia-reperfusion injury. TNF induces glomerular fibrin deposition, cellular infiltration and vasoconstriction, leading to a reduction in glomerular filtration rate (GFR). The signaling cascade through which renal ischemia-reperfusion induces TNF production is beginning to be elucidated. Oxidants released following reperfusion activate p38 mitogen activated protein kinase (p38 MAP kinase) and the TNF transcription factor, NFkappaB, leading to subsequent TNF synthesis. In a positive feedback, proinflammatory fashion, binding of TNF to specific TNF membrane receptors can reactivate NFkappaB. This provides a mechanism by which TNF can upregulate its own expression as well as facilitate the expression of other genes pivotal to the inflammatory response. TNF receptor binding can also induce renal cell apoptosis, the major form of cell death associated with renal ischemia-reperfusion injury. Anti-TNF strategies targeting p38 MAP kinase, NFkappaB, and TNF itself are being investigated as methods of attenuating renal ischemic injury. The control of TNF production and activity represents a realistic goal for clinical medicine.