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Zileuton is an orally active inhibitor of leukotriene synthesis for maintenance treatment of asthma, for which clinical usage has been associated with idiosyncratic liver injury. Mechanistic understanding of zileuton toxicity is hampered by the rarity of the cases and lack of an animal model. A promising model for mechanistic study of rare liver injury is the Diversity Outbred (J:DO) mouse population, with genetic variation similar to that found in humans. In this study, female DO mice were administered zileuton or vehicle daily for 7 days (i.g.). Serum liver enzymes were elevated in the zileuton group, with marked interindividual variability in response. Zileuton exposure-induced findings in susceptible DO mice included microvesicular fatty change, hepatocellular mitosis, and hepatocellular necrosis. Inducible nitric oxide synthase and nitrotyrosine abundance were increased in livers of animals with necrosis and those with fatty change, implicating nitrosative stress as a possible injury mechanism. Conversely, DO mice lacking adverse liver pathology following zileuton exposure experienced decreased hepatic concentrations of resistin and increased concentrations of insulin and leptin, providing potential clues into mechanisms of toxicity resistance. Transcriptome pathway analysis highlighted mitochondrial dysfunction and altered fatty acid oxidation as key molecular perturbations associated with zileuton exposure, and suggested that interindividual differences in cytochrome P450 metabolism, glutathione-mediated detoxification, and farnesoid X receptor signaling may contribute to zileuton-induced liver injury (ZILI). Taken together, DO mice provided a platform for investigating mechanisms of toxicity and resistance in context of ZILI which may lead to targeted therapeutic interventions.

The mitogen-activated protein kinase/extracellular signal–regulated kinase signaling pathway is an essential component of innate immunity necessary for mediating proinflammatory responses in the setting of sepsis. We previously demonstrated that the mitogen-activated protein kinase 1/2 inhibitor trametinib prevents endotoxin-induced renal injury in mice. We therefore assessed efficacy of trametinib in a more clinically relevant experimental model of sepsis. Controlled in vivo laboratory study. University animal research laboratory. Male C57BL/6 mice. Mice were subjected to cecal ligation and puncture to induce sepsis or underwent sham operation as controls. Six hours after cecal ligation and puncture, mice were randomized to four experimental groups as follows: 1) sham control; 2) sham control + trametinib (1 mg/kg, IP); 3) cecal ligation and puncture; and 4) cecal ligation and puncture + trametinib. All animals received buprenorphine (0.05 mg/kg, SC) and imipenem/cilastatin (14 mg/kg, SC) in 1.5 mL of warm saline (40 mL/kg) at the 6-hour time point. Mice were euthanized at 18 hours after induction of cecal ligation and puncture. Trametinib inhibition of mitogen-activated protein kinase/extracellular signal–regulated kinase signaling 6 hours after cecal ligation and puncture attenuated increases in circulating proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte macrophage colony-stimulating factor) and hypothermia at 18 hours. Trametinib also attenuated multiple organ injury as determined by serum creatinine, alanine aminotransferase, lactate dehydrogenase, and creatine kinase. At the organ level, trametinib completely restored peritubular capillary perfusion in the kidney. Restoration of microvascular perfusion was associated with reduced messenger RNA expression of well-characterized markers of proximal tubule injury. mitogen-activated protein kinase/extracellular signal–regulated kinase blockade attenuated cecal ligation and puncture-mediated up-regulation of cytokines (tumor necrosis factor-α, interleukin-1β) and restored interleukin-6 to control levels in the renal cortex, indicating the protective effects on the proximal tubule occur primarily through modulation of the proinflammatory response in sepsis. These data reveal that the mitogen-activated protein kinase/extracellular signal–regulated kinase inhibitor trametinib attenuates systemic inflammation and multiple organ damage in a clinically relevant model of sepsis. Because trametinib has been safely used in humans, we propose that this drug might represent a translatable approach to limit organ injury in septic patients.

Sepsis is a serious medical condition caused by a severe systemic inflammatory response to a bacterial, fungal, or viral infection that most commonly affects neonates and the elderly. Advances in understanding the pathophysiology of sepsis have resulted in guidelines for care that have helped reduce the risk of dying from sepsis for both children and older adults. Still, over the past three decades, a large number of clinical trials have been undertaken to evaluate pharmacological agents for sepsis. Unfortunately, all of these trials have failed, with the use of some agents even shown to be harmful. One key issue in these trials was the heterogeneity of the patient population that participated. What has emerged is the need to target therapeutic interventions to the specific patient's underlying pathophysiological processes, rather than looking for a universal therapy that would be effective in a "typical" septic patient, who does not exist. This review supports the concept that identification of the right biomarkers that can direct therapy and provide timely feedback on its effectiveness will enable critical care physicians to decrease mortality of patients with sepsis and improve the quality of life of survivors.

Acute kidney injury (AKI) is a complication of sepsis and leads to a high mortality rate. Human and animal studies suggest that mitochondrial dysfunction plays an important role in sepsis-induced multi-organ failure; however, the specific mitochondrial targets damaged during sepsis remain elusive. We used a clinically relevant cecal ligation and puncture (CLP) murine model of sepsis and assessed renal mitochondrial function using high-resolution respirometry, renal microcirculation using intravital microscopy, and renal function. CLP caused a time-dependent decrease in mitochondrial complex I and II/III respiration and reduced ATP. By 4 h after CLP, activity of manganese superoxide dismutase (MnSOD) was decreased by 50% and inhibition was sustained through 36 h. These events were associated with increased mitochondrial superoxide generation. We then evaluated whether the mitochondria-targeted antioxidant Mito-TEMPO could reverse renal mitochondrial dysfunction and attenuate sepsis-induced AKI. Mito-TEMPO (10 mg/kg) given at 6 h post-CLP decreased mitochondrial superoxide levels, protected complex I and II/III respiration, and restored MnSOD activity by 18 h. Mito-TEMPO also improved renal microcirculation and glomerular filtration rate. Importantly, even delayed therapy with a single dose of Mito-TEMPO significantly increased 96-h survival rate from 40% in untreated septic mice to 80%. Thus, sepsis causes sustained inactivation of three mitochondrial targets that can lead to increased mitochondrial superoxide. Importantly, even delayed therapy with Mito-TEMPO alleviated kidney injury, suggesting that it may be a promising approach to treat septic AKI.

Hyperpermeability of the endothelial barrier and resulting microvascular leakage are a hallmark of sepsis. Our studies describe the mechanism by which serotonin (5-HT) regulates the microvascular permeability during sepsis. The plasma 5-HT levels are significantly elevated in mice made septic by cecal ligation and puncture (CLP). 5-HT-induced permeability of endothelial cells was associated with the phosphorylation of p21 activating kinase (PAK1), PAK1-dependent phosphorylation of vimentin (P-vimentin) filaments, and a strong association between P-vimentin and ve-cadherin. These findings were in good agreement with the findings with the endothelial cells incubated in serum from CLP mice. In vivo, reducing the 5-HT uptake rates with the 5-HT transporter (SERT) inhibitor, paroxetine blocked renal microvascular leakage and the decline in microvascular perfusion. Importantly, mice that lack SERT showed significantly less microvascular dysfunction after CLP. Based on these data, we propose that the increased endothelial 5-HT uptake together with 5-HT signaling disrupts the endothelial barrier function in sepsis. Therefore, regulating intracellular 5-HT levels in endothelial cells represents a novel approach in improving sepsis-associated microvascular dysfunction and leakage. These new findings advance our understanding of the mechanisms underlying cellular responses to intracellular/extracellular 5-HT ratio in sepsis and refine current views of these signaling processes during sepsis.

Acute kidney injury is a frequent and serious complication of sepsis. To better understand the development of sepsis-induced acute kidney injury, we performed the first time-dependent studies to document changes in renal hemodynamics and oxidant generation in the peritubular microenvironment using the murine cecal ligation and puncture (CLP) model of sepsis. CLP caused an increase in renal capillary permeability at 2 hours, followed by decreases in mean arterial pressure, renal blood flow (RBF), and renal capillary perfusion at 4 hours, which were sustained through 18 hours. The decline in hemodynamic parameters was associated with hypoxia and oxidant generation in the peritubular microenvironment and a decrease in glomerular filtration rate. The role of oxidants was assessed using the superoxide dismutase mimetic/peroxynitrite scavenger MnTMPyP [Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin]. At 10 mg/kg administered 6 hours after CLP, MnTMPyP did not alter blood pressure, but blocked superoxide and peroxynitrite generation, reversed the decline in RBF, capillary perfusion, and glomerular filtration rate, preserved tubular architecture, and increased 48-hour survival. However, MnTMPyP administered at CLP did not prevent capillary permeability or the decrease in RBF and capillary perfusion, which suggests that these early events are not mediated by oxidants. These data demonstrate that renal hemodynamic changes occur early after sepsis and that targeting the later oxidant generation can break the cycle of injury and enable the microcirculation and renal function to recover.

Oxidative stress has been implicated to play a major role in multiorgan dysfunction during sepsis. To study the mechanism of oxidant generation in acute kidney injury (AKI) during sepsis, we developed an in vitro model of sepsis using primary cultures of mouse cortical tubular epithelial cells exposed to serum (2.5-10%) collected from mice at 4 h after induction of sepsis by cecal ligation and puncture (CLP) or Sham (no sepsis). CLP serum produced a concentration-dependent increase in nitric oxide (NO) (nitrate + nitrite) release at 6 h and cytotoxicity (lactate dehydrogenase release) at 18 h compared with Sham serum treatment. Before cytotoxicity there was a decrease in mitochondrial membrane potential, which was followed by increased superoxide and peroxynitrite levels compared with Sham serum. The role of oxidants was evaluated by using the superoxide dismutase mimetic and peroxynitrite scavenger manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin tetratosylate hydroxide (MnTmPyP). MnTmPyP (10-100 μM) produced a concentration-dependent preservation of ATP and protection against cytotoxicity. MnTmPyP blocked mitochondrial superoxide and peroxynitrite generation produced by CLP serum but had no effect on NO levels. Although MnTmPyP did not block the initial CLP serum-induced fall in mitochondrial membrane potential, it allowed mitochondrial membrane potential to recover. Data from this in vitro model suggest a time-dependent generation of mitochondrial oxidants, mitochondrial dysfunction, and renal tubular epithelial cell injury and support the therapeutic potential of manganese porphyrin compounds in preventing sepsis-induced AKI.

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. Understanding the development of pediatric sepsis and its effects on the kidney are critical in uncovering new therapies. The goal of this study was to characterize the development of sepsis-induced AKI in the clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17–18 days old. CLP produced severe sepsis demonstrated by time-dependent increase in serum cytokines, NO, markers of multiorgan injury, and renal microcirculatory hypoperfusion. Although blood pressure and heart rate remained unchanged after CLP, renal blood flow (RBF) was decreased 61% by 6 h. Renal microcirculatory analysis showed the number of continuously flowing cortical capillaries decreased significantly from 69 to 48% by 6 h with a 66% decrease in red blood cell velocity and a 57% decline in volumetric flow. The progression of renal microcirculatory hypoperfusion was associated with peritubular capillary leakage and reactive nitrogen species generation. Sham adults had higher mean arterial pressure (118 vs. 69 mmHg), RBF (4.2 vs. 1.1 ml·min−1·g−1), and peritubular capillary velocity (78% continuous flowing capillaries vs. 69%) compared with pups. CLP produced a greater decrease in renal microcirculation in pups, supporting the notion that adult models may not be the most appropriate for studying pediatric sepsis-induced AKI. Lower RBF and reduced peritubular capillary perfusion in the pup suggest the pediatric kidney may be more susceptible to AKI than would be predicted using adults models.

Sepsis-induced acute renal injury (AKI) occurs in 20%–50% of septic patients and nearly doubles the mortality rate of sepsis. Since treatment in the septic patient is usually only begun after the onset of symptoms, therapy that is effective even when delayed would have the greatest impact on patient survival. The metalloproteinase meprin A, an oligomeric complex made of α- and β- subunits, is highly expressed at the brush-border membranes of the kidney and capable of degrading numerous substrates including extracellular matrix proteins and cytokines. The goal of the present study was to compare the therapeutic potential of actinonin, an inhibitor of meprin A when administered before and after the onset of sepsis. Mice were treated with actinonin at 30 min prior to or 7 h post induction of sepsis by cecal ligation and puncture (CLP). Intravital videomicroscopy was utilized to image renal peritubular capillary perfusion and reactive nitrogen species. Actinonin treatment 30 min before CLP reduced IL1-β levels and prevented the fall in renal capillary perfusion at 7 h and 18 h. Actinonin also prevented the fall in renal capillary perfusion even when administered at 7 h post CLP. In addition, even late administration of actinonin preserved renal morphology and lowered blood urea nitrogen and serum creatinine concentrations. These data suggest that agents like actinonin should be evaluated further as possible therapeutic agents because targeting both the early systemic and later organ-damaging effects of sepsis should have the highest likelihood of success.

Sepsis-induced acute kidney injury (SAKI) is a frequent complication of infant sepsis that approximately doubles the mortality rate. The poor prognosis of these patients is a result of care that is mainly supportive, nontargeted, and usually begun only after symptoms of the systemic inflammatory response syndrome are observed. Preclinical studies from relevant rodent models of SAKI suggest that mitochondria-targeted antioxidants may be a new mode of therapy that could promote recovery.

Sepsis-induced acute kidney injury (AKI) is a complex disease characterized by generation of inducible nitric oxide synthase (iNOS)-derived reactive nitrogen species (RNS) by the renal tubular epithelium. While most in vitro models of sepsis use combinations of lipopolysaccharide and cytokines to simulate exposure to inflammatory mediators thought to play a role in sepsis, the relevance of these models is limited. To address the need for a model that more closely mimics the tubular microenvironment during sepsis, we developed an in vitro model where mIMCD-3 (murine tubular epithelial) cells are treated with media containing 5% serum collected from mice at 4 h after cecal ligation and puncture (CLP) or sham surgery (no sepsis). After exposure to CLP serum, induction of iNOS messenger RNA occurred and NO generation was significantly increased compared to sham. This increase was accompanied by increased RNS as measured by oxidation of 5-(and-6)-carboxy-2,7'-dichlorodihydrofluorescein diacetate (carboxy-H(2)DCF-DA) and 2-(3,6-diamino-9H-xanthen-9-yl)-benzoic acid, methyl ester (dihydrorhodamine 123) and moderate cytotoxicity in cells treated with CLP serum, similar to what is observed in mice subjected to CLP. Since iNOS has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine (L-NIL) was tested in this in vitro model. L-NIL completely blocked NO generation, RNS generation, and cytotoxicity, similar to its effects in vivo. Therefore, this new in vitro model exhibits many of the characteristics observed in vivo, suggesting that it is a relevant model for studying the mechanism of sepsis-induced renal epithelial RNS generation and injury.

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin alpha are capable of generating biologically active IL-1beta from its precursor pro-IL-1beta. Amino-acid sequencing analysis reveals that meprin A and meprin alpha cleave pro-IL-1beta at the His(115)-Asp(116) bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin beta site. The biological activity of the pro-IL-1beta cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1beta product produced by meprin beta or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1beta, meprin inhibitor actinonin significantly reduces levels of serum IL-1beta. Meprin A and meprin alpha may therefore play a critical role in the production of active IL-1beta during inflammation and tissue injury.

Sepsis affects practically all aspects of endothelial cell (EC) function and is thought to be the key factor in the progression from sepsis to organ failure. Endothelial functions affected by sepsis include vasoregulation, barrier function, inflammation, and hemostasis. These are among other mechanisms often mediated by glycocalyx shedding, such as abnormal nitric oxide metabolism, up-regulation of reactive oxygen species generation due to down-regulation of endothelial-associated antioxidant defenses, transcellular communication, proteases, exposure of adhesion molecules, and activation of tissue factor. This review covers current insight in EC-associated hemostatic responses to sepsis and the EC response to inflammation. The endothelial cell lining is highly heterogeneous between different organ systems and consequently also in its response to sepsis. In this context, we discuss the response of the endothelial cell lining to sepsis in the kidney, liver, and lung. Finally, we discuss evidence as to whether the EC response to sepsis is adaptive or maladaptive. This study is a result of an Acute Dialysis Quality Initiative XIV Sepsis Workgroup meeting held in Bogota, Columbia, between October 12 and 15, 2014.

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15–18%, and the distance between receptors was 5–6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent proteinVT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction. (Molecular Endocrinology 21: 2178–2188, 2007)

The mortality rate for septic patients with acute renal failure is extremely high. Since sepsis is often caused by lipopolysaccharide (LPS), a model of LPS challenge was used to study the development of kidney injury. Intravital video microscopy was utilized to investigate renal peritubular capillary blood flow in anesthetized male C57BL/6 mice at 0, 2, 6, 10, 18, 24, 36, and 48 h after LPS administration (10 mg/kg ip). As early as 2 h, capillary perfusion was dramatically compromised. Vessels with continuous flow were decreased from 89 ± 4% in saline controls to 57 ± 5% in LPS-treated mice ( P < 0.01), and vessels with intermittent flow were increased from 6 ± 2% to 31 ± 5% ( P < 0.01). At 2 h, mRNA for intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were elevated 50- and 27-fold, respectively, suggesting that vascular inflammation is an early event that may contribute to capillary dysfunction. By 10 h, vessels with no flow increased from 5 ± 2% in saline controls to 19 ± 3% in LPS-treated mice ( P < 0.05). By 48 h, capillary function was returning toward control levels. The decline in functional capillaries preceded the development of renal failure and was paralleled by induction of inducible nitric oxide synthase in the kidney. Using NAD(P)H autofluorescence as an indicator of cellular redox stress, we found that tubular cell stress was highly correlated with the percentage of dysfunctional capillaries ( r 2 = 0.8951, P < 0.0001). These data show that peritubular capillary dysfunction is an early event that contributes to tubular stress and renal injury.

Acute renal failure (ARF) is a frequent and serious complication of endotoxemia caused by lipopolysaccharide (LPS) and contributes significantly to mortality. The present studies were undertaken to examine the roles of nitric oxide (NO) and caspase activation on renal peritubular blood flow and apoptosis in a murine model of LPS-induced ARF. Male C57BL/6 mice treated with LPS ( Escherichia coli) at a dose of 10 mg/kg developed ARF at 18 h. Renal failure was associated with a significant decrease in peritubular capillary perfusion. Vessels with no flow increased from 7 ± 3% in the saline group to 30 ± 4% in the LPS group ( P < 0.01). Both the inducible NO synthase inhibitor l- N6-1-iminoethyl-lysine (l-NIL) and the nonselective caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD) prevented renal failure and reversed perfusion deficits. Renal failure was also associated with an increase in renal caspase-3 activity and an increase in renal apoptosis. Both l-NIL and Z-VAD prevented these changes. LPS caused an increase in NO production that was blocked by l-NIL but not by Z-VAD. Taken together, these data suggest NO-mediated activation of renal caspases and the resulting disruption in peritubular blood flow are an important mechanism of LPS-induced ARF.

The neurohypophysial hormone arginine vasotocin (AVT) stimulates adrenocorticotropin hormone (ACTH) secretion from the avian anterior pituitary gland resulting in increased adrenal secretion of corticosterone in response to stress. Here, we report molecular cloning and functional characterization of a gene encoding an AVT receptor subtype, designated the VT2 receptor, that may mediate the stimulatory effect of AVT on ACTH secretion in birds. The open reading frame predicts a 425 amino acid polypeptide that includes seven segments of 19 to 24 hydrophobic amino acids, typical of guanine nucleotide-protein coupled receptors. Phylogenetic analysis revealed that the VT2 receptor shares highest identity with the mammalian V1b-vasopressin receptor subtype. Expressed VT2 receptors in COS7 cells mediate AVT-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the domestic chicken, expression of VT2 receptor gene transcripts is limited to the pituitary gland. Based on similarities in sequence, site of expression and coupled signal transduction pathways, we conclude that the VT2 receptor is the avian homolog of the mammalian V1b-vasopressin receptor, and therefore may play an important role in the avian stress response.

NO reacts spontaneously with superoxide to produce the potent oxidant peroxynitrite. Studies were designed to examine the role of NO-derived oxidants and peroxynitrite on the regulation of Na(+),K(+)-ATPase activity by angiotensin II (ANG II) freshly isolated rat proximal tubules. At picomolar concentrations ANG II stimulates Na(+),K(+)-ATPase activity, but at nanomolar concentrations stimulation is lost. Superoxide dismutase (SOD) was used to examine the role of superoxide and deferoxamine (DFO) and uric acid (UA) were used to examine the role of peroxynitrite. SOD (200 U/mL, 5-min preincubation) restored the stimulatory effect of ANG II (1.31 +/- 0.08-fold; n = 4; P0.05 compared to 10(-7) M alone), suggesting a role for superoxide. DFO (100 microm, 5-min preincubation) also restored the stimulatory effect of ANG II (1.40 +/- 0.08-fold; n = 4; P0.05, compared to 10(-7) M alone), as did UA (1.22 +/- 0.07-fold; n = 5; P0.05, compared to 10(-7) M alone). The NO synthesis inhibitor, N-monomethyl-L-arginine (L-NMMA, 2 mM; 5-min preincubation), also unmasked a stimulatory effect of ANG II at 10(-7) M (1.4 +/- 0.1-fold; n = 7; P0.05, compared to 10(-7) M alone). The generation of peroxynitrite was further evidenced by the formation of 3-nitrotyrosine (3-NT). 3-NT increased 3.5-fold in tubules exposed to ANG II (10(-7) M) (0.0054 +/- 0.0019 3-NT/100 tyrosines for control and 0.019 +/- 0.0058 3-NT/100 tyrosines for ANG II, P.05; n = 4) and L-NMMA prevented the increase. These data suggest that peroxynitrite signaling participates in the regulation of renal of Na(+),K(+)-ATPase activity.

Acetaminophen-induced hepatotoxicity has been attributed to covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to cysteine groups on proteins as an acetaminophen-cysteine conjugate. We report a high-performance liquid chromatography with electrochemical detection (HPLC-ECD) assay for the conjugate with increased sensitivity compared with previous methods. Previous methods to quantitate the protein-bound conjugate have used a competitive immunoassay or radiolabeled acetaminophen. With HPLC-ECD, the protein samples are dialyzed and then digested with protease. The acetaminophen-cysteine conjugate is then quantified by HPLC-ECD using tyrosine as an internal reference. The lower limit of detection of the assay is approximately 3 pmol/mg of protein. Acetaminophen protein adducts were detected in liver and serum as early as 15 min after hepatotoxic dosing of acetaminophen to mice. Adducts were also detected in the serum of acetaminophen overdose patients. Analysis of human serum samples for the acetaminophen-cysteine conjugate revealed a positive correlation between acetaminophen-cysteine conjugate concentration and serum aspartate aminotransferase (AST) activity or time. Adducts were detected in the serum of patients even with relatively mild liver injury, as measured by AST and alanine aminotransferase. This assay may be useful in the diagnostic evaluation of patients with hepatotoxicity of an indeterminate etiology for which acetaminophen toxicity is suspect.

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.

ANG II exerts a biphasic effect on Na+transport in the kidney through its effects on Na+-K+-ATPase activity. Beginning at 10−13M, ANG II increased Na+-K+-ATPase activity in freshly isolated rat proximal tubules to a maximum stimulation at 10−11M of 1.43 ± 0.08-fold above control. Stimulation decreased progressively at concentrations >10−10M to a value of 0.96 ± 0.1-fold at 10−7M. In the presence of additional l-arginine, the substrate for NO synthesis, the stimulatory effect of ANG II (10−11M) was lost. Conversely, N-monomethyl-l-arginine (l-NMMA), the nitric oxide (NO) synthase inhibitor, unmasked the stimulatory effect of ANG II at 10−7M (1.40 ± 0.1-fold). 1 H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one, the soluble guanylyl cyclase inhibitor, like l-NMMA, unmasked the stimulatory effect of ANG II at 10−7M (1.30 ± 0.1-fold). The intracellular cGMP concentration was increased 1.58 ± 0.28-fold at 10−7M ANG II. The ANG II AT1receptor antagonist SK&F 108566 blocked the stimulatory effect of ANG II at 10−11M. These data suggest that the NO/cGMP signaling pathway serves as a negative component in the regulation of Na+-K+-ATPase activity by ANG II.

Lipopolysaccharide (LPS)-induced renal oxidant injury and the role of nitric oxide (NO) were evaluated using the inducible nitric oxide synthase (iNOS) inhibitor L-iminoethyl-lysine (L-NIL). One group of male rats received LPS (Salmonella minnesota; 2 mg/kg, i.v.). A second group received LPS plus L-NIL (3 mg/kg, i.p.). A third group received saline i.v. At 6 hr, iNOS protein was induced in the kidney cortex, and plasma nitrate/nitrite levels were increased from 4 +/- 2 nmol/mL in the Saline group to 431 +/- 23 nmol/mL in the LPS group. The value for the LPS + L-NIL group was reduced significantly to 42 +/- 9 nmol/mL. LPS increased blood urea nitrogen levels from 13 +/- 1 to 47 +/- 3 mg/dL. LPS + L-NIL reduced these levels significantly to 29 +/- 2 mg/dL. Plasma creatinine levels were unchanged in all groups. Tissue lipid peroxidation products in the kidney were increased from 0.16 +/- 0.01 nmol/mg in the Saline group to 0.30 +/- 0.03 nmol/mg in the LPS group. LPS + L-NIL reduced the values significantly to 0.22 +/- 0.02 nmol/mg. Intracellular glutathione levels were decreased in the kidneys from 1.32 +/- 0.1 nmol/mg in the Saline group to 0.66 +/- 0.08 nmol/mg in the LPS group. LPS + L-NIL increased the levels significantly to 0.99 +/- 0.13 nmol/mg. LPS increased the 3-nitrotyrosine-protein adducts in renal tubules as detected by immunohistochemistry, indicating the generation of peroxynitrite. L-NIL decreased adduct formation. These data indicated that LPS-induced NO generation resulted in peroxynitrite formation and oxidant stress in the kidney and that inhibitors of iNOS may offer protection against LPS-induced renal toxicity.

Hepatotoxic doses of acetaminophen to mice produce not only acetaminophen-protein adducts in the centrilobular cells of the liver, but nitrotyrosine-protein adducts in the same cells, the site of the necrosis. Nitration of tyrosine occurs with peroxynitrite, a species formed by reaction of nitric oxide (NO.) with superoxide (O2. -). Because NO. and O2.- may be produced by activated Kupffer cells and/or infiltrated macrophages, we pretreated mice with the macrophage inactivators/depeleters gadolinium chloride (7 mg/kg, intravenously [iv]) or dextran sulfate (10 mg/kg, iv) 24 hours before administration of acetaminophen (300 mg/kg). Mice treated with acetaminophen plus gadolinium chloride, or acetaminophen plus dextran sulfate, had significantly less evidence of hepatotoxicity as evidenced by lower serum alanine transaminase (ALT) levels (28 +/- 1 IU/L and 770 +/- 240 IU/L, respectively) at 8 hours compared with acetaminophen (6,380 +/- 408 IU/L). Analysis of hepatic homogenates for acetaminophen-protein adducts at 2 hours, a time of maximal covalent binding and before hepatocyte lysis, indicated that these pretreatments did not decrease covalent binding. Western blot analysis for the macrophage marker protein F4/80 in homogenates revealed not only the expected decrease by the macrophage inactivators/depleters, but also an apparent increase in acetaminophen-only-treated mice. At 8 hours nitrotyrosine-protein adducts were present in the acetaminophen-only-treated mice, but not in the acetaminophen plus gadolinium chloride-treated mice, or acetaminophen plus dextran sulfate-treated mice. High levels of heme-protein adducts, a measure of oxidative stress, were detected in livers of the 8 hour acetaminophen-only-treated mice. These data suggest that acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding, and subsequent activation of macrophages to form O2.-, NO., and peroxynitrite. Nitration of tyrosine correlates with toxicity.

Lipid A, the biologically active component of lipopolysaccharide, initiates a specific cytotoxic signaling cascade in the renal proximal tubule that involves a rapid release of intracellular calcium, the activation of nitric oxide synthase (NOS) and NO production. Superoxide (O2-) generation is also a component of this cascade and both NO and O2- are required for the development of oxidant stress and cytotoxicity. Here we examined whether NOS activity was responsible for O2- generation. In renal proximal tubules isolated from the rat, the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) but not D-NMMA blocked lipid A (50 microg/ml)-stimulated O2- generation as measured by the reduction of cytochrome c during a 30-min incubation period. When L-arginine (2 mM) was added to the tubule suspensions, O2- generation was significantly inhibited, while NO2- (a marker of NO generation) was significantly increased. The addition of L-arginine also reduced lipid A-stimulated malondialdehyde formation at 30 min (a marker of lipid peroxidation) and lactate dehydrogenase release at 90 min (a marker of cell death). Thus, lipid A-stimulated the generation of both NO and O2- via NOS activation. Furthermore, increasing L-arginine availability shifted NOS activity toward NO generation and reduced oxidant injury. These results offer an explanation of why scavengers of NO or oxygen radicals ameliorate endotoxin-induced acute renal failure in vivo.

Microcirculatory dysfunction is correlated with increased mortality among septic patients and is believed to be a major contributor to the development of acute kidney injury (AKI). Rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, has been shown to reduce microvascular permeability and in the kidney, increase renal blood flow (RBF). This led us to investigate its potential to improve the renal microcirculation and preserve renal function during sepsis using a murine cecal ligation and puncture (CLP) model to induce sepsis. Rolipram, tested at doses of 0.3–10 mg/kg i.p., acutely restored capillary perfusion in a bell-shaped dose-response effect with 1 mg/kg being the lowest most efficacious dose. This dose also acutely increased RBF despite transiently decreasing mean arterial pressure. Rolipram also reduced renal microvascular permeability. It is noteworthy that delayed treatment with rolipram at 6 hours after CLP restored the renal microcirculation, reduced blood urea nitrogen and serum creatinine, and increased glomerular filtration rate at 18 hours. However, delayed treatment with rolipram did not reduce serum nitrate/nitrite levels, a marker of nitric oxide production, nor reactive nitrogen species generation in renal tubules. These data show that restoring the microcirculation with rolipram, even with delayed treatment, is enough to improve renal function during sepsis despite the generation of oxidants and suggest that PDE4 inhibitors should be evaluated further for their ability to treat septic-induced AKI.

Renal proximal tubules isolated from the rat possess nitric oxide synthase (NOS) activity that is calcium/calmodulin dependent and stereoselectively inhibited by NG-monomethyl-arginine (NMMA). In the absence of added Ca2+ and calmodulin, activity was reduced 84 +/- 13% compared with the activity in the presence of 2 mM Ca2+ and 25 micrograms/mL calmodulin. Inhibition by EGTA (10 mM) was 95 +/- 5% and by calmidazolium (R24571, 250 microM) was 99 +/- 1%. Inhibition by L-NMMA (100 microM) was 78 +/- 13% and by D-NMMA (100 microM) was 7 +/- 7%. The majority of NOS activity was found in the soluble fraction. NOS activity in isolated proximal tubules was also examined 6 hr after a single i.v. injection of lipopolysaccharide. Activity was increased significantly (P < 0.05) in the soluble fraction by 2-fold [from 0.320 +/- 0.052 to 0.648 +/- 0.046 (nmol/mg protein/30 min)] and in the particulate fraction by 3-fold [from 0.081 +/- 0.030 to 0.256 +/- 0.034 (nmol/mg protein/30 min)]. All activities were inhibited by EGTA. These data demonstrate that proximal tubules express a calcium/calmodulin-dependent NOS activity that is increased in vivo by lipopolysaccharide.

One of the most frequent and serious complications to develop in septic patients is acute kidney injury (AKI), a disorder characterized by a rapid failure of the kidneys to adequately filter the blood, regulate ion and water balance, and generate urine. AKI greatly worsens the already poor prognosis of sepsis and increases cost of care. To date, therapies have been mostly supportive; consequently there has been little change in the mortality rates over the last decade. This is due, at least in part, to the delay in establishing clinical evidence of an infection and the associated presence of the systemic inflammatory response syndrome and thus, a delay in initiating therapy. A second reason is a lack of understanding regarding the mechanisms leading to renal injury, which has hindered the development of more targeted therapies. In this review, we summarize recent studies, which have examined the development of renal injury during sepsis and propose how changes in the peritubular capillary microenvironment lead to and then perpetuate microcirculatory failure and tubular epithelial cell injury. We also discuss a number of potential therapeutic targets in the renal peritubular microenvironment, which may prevent or lessen injury and/or promote recovery.

We have characterized the thromboxane (TX) A2/prostaglandin (PG) H2 receptor in glomeruli isolated from the rat using the agonist radioligand [125I]-BOP. Binding of [125I]-BOP was highly specific, stereoselective, and to a single class of high affinity binding sites (Kd = 1.16 +/- 0.22 nM and Bmax = 348 +/- 32 fmol/mg protein; n = 6). Binding of [125I]-BOP was competed for by the agonist ONO11113 (Kd = 50.8 +/- 8.0 nM; n = 4) and the antagonists SQ29548 (Kd = 15.8 +/- 1.0 nM; n = 3), L657925 (Kd = 12.1 +/- 2.2 nM; n = 3) and L657926 (Kd = 1642 +/- 135 nM; n = 3). I-BOP also produced a TXA2/PGH2 receptor-mediated rise in [Ca2+]i in isolated glomeruli In adriamycin-induced nephrotic syndrome in the rat, the development of proteinuria is reported to be dependent on increased renal TXA2 production. We therefore examined whether or not changes in glomerular TXA2/PGH2 receptors occur between control and nephrotic rats. No changes in expression or affinity of either glomerular or platelet TXA2/PGH2 receptors were observed. Kd and Bmax values for isolated glomeruli were 1.45 +/- 0.24 nM and 406 +/- 72 fmol/mg for controls and 1.22 +/- 0.25 nM and 321 +/- 62 fmol/mg for nephrotic rats (n = 6).

Oxidant damage from reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major contributor to the cellular damage seen in numerous types of renal injury. Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin found naturally in many common food sources. The anti-oxidant properties of resveratrol are of particular interest because of the fundamental role that oxidant damage plays in numerous forms of kidney injury. To examine whether resveratrol could block damage to the renal epithelial cell line, mIMCD-3, cells were exposed to the peroxynitrite donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1). Resveratrol produced a concentration-dependent inhibition of cytotoxicity induced by SIN-1. To examine the mechanism of protection, resveratrol was incubated with authentic peroxynitrite and found to block nitration of bovine serum albumin with an EC(50) value of 22.7 microM, in contrast to the known RNS scavenger, N-acetyl-l-cysteine, which inhibited nitration with an EC(50) value of 439 microM. These data suggested that resveratrol could provide functional protection by directly scavenging peroxynitrite. To examine whether resveratrol was a substrate for peroxynitrite oxidation, resveratrol was reacted with authentic peroxynitrite. Resveratrol nitration products and dimers were detected using liquid chromatograph with tandem electrospray mass spectrometry. Similar products were detected in the media of cells treated with SIN-1 and resveratrol. Taken collectively, the data suggest that resveratrol is able to provide functional protection of renal tubular cells, at least in part, by directly scavenging the RNS peroxynitrite. This property of resveratrol may contribute to the understanding of its anti-oxidant activities.

Receptor-mediated increase in cytosolic calcium in LLC-PK 1 cells by platelet activating factor and thromboxane A 2 . Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A 2 (TXA 2 ) in glomerular pathophysiology. However, the potential role of PAF or TXA 2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA 2 analogue, ONO11113, on the cytosolic free calcium concentration ([Ca 2+ ] i ) in cultured LLC-PK 1 , cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca 2+ ] i in a dose-dependent manner: both agonists (10 -7 M) increased [Ca 2+ ] i from 148 ± 16 to 288 ± 39 nM and from 130 ± 8 to 240 ± 18 nM, with the values of EC 50 for PAF and ONO11113 being 17 ± 4 and 17 ± 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA 2 receptor antagonist L657925. Similarly ONO1113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca 2+ ] i as well as the sustained elevations in [Ca 2+ ] i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca 2+ by 10 mM ethylene glycol-bis(β-aminomethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). In the absence of extracellular Ca 2+ 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (which has extensively been used to study the contribution of Ca 2+ released from intracellular storage sites in the increase in [Ca 2+ ] i ), blocked the increment in [Ca 2+ ] i induced by PAF or ONO1113. These results indicate that LLC-PK i cells express discrete receptors for PAF and TXA 2 that are coupled to an increase in [Ca 2+ ] i through mobilization of calcium from both intracellular storage sites and extracellular milieu, and suggest the possible importance of PAF and TXA 2 in tubular pathophysiology.

Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the beta2-adrenergic receptor (beta2AR). The VT2R/beta2AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/beta2AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2/beta2AR-chimera/CRHR pairs, and the calculated distance was observed to be10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

Acute kidney injury (AKI) remains a frequent and serious complication of human sepsis that contributes significantly to mortality. For better understanding of the development of AKI during sepsis, the cecal ligation and puncture (CLP) murine model of sepsis was studied using intravital video microscopy (IVVM) of the kidney. IVVM with FITC-dextran was used to determine the percentage of capillaries with continuous, intermittent or no flow at 0 (sham), 10, 16, and 22 h after CLP. There was a dramatic fall in capillary perfusion as early as 10 h after CLP that persisted through 22 h. The percentage of vessels with continuous flow at 16 h decreased from 73 +/- 2% in shams to 16 +/- 2% (P0.05), whereas the percentage of vessels with no flow increased from 4 +/- 1% in shams to 42 +/- 2% (P0.05). The capillary perfusion defect preceded the rise in serum creatinine. IVVM with dihydrorhodamine-123 was used to quantify in real time reactive nitrogen species (RNS) generation by renal tubules, and the inducible nitric oxide synthase inhibitor L-iminoethyl-lysine (mg/kg) was used to examine the role of inducible nitric oxide synthase inhibitor on capillary dysfunction and RNS generation. Tubular generation of RNS was significantly elevated at 10 h after CLP and was associated with tubules that were bordered by capillaries with reduced perfusion. L-iminoethyl-lysine significantly reversed the capillary perfusion defect, blocked RNS generation, and reduced AKI. These data show that capillary dysfunction and RNS generation contribute to tubular injury and suggest that RNS should be considered a potential therapeutic target in the treatment of sepsis-induced AKI.

The mortality rate for septic patients with acute renal failure is approximately doubled compared with patients with sepsis alone. Unfortunately, the treatment for sepsis-induced renal failure has advanced little during the last several decades. Because sepsis is often caused by lipopolysaccharide (LPS), a mouse model of LPS challenge was used to study the development of kidney injury. We hypothesized that inducible nitric-oxide synthase (iNOS)-catalyzed nitric oxide production and that generation of reactive nitrogen species (RNS) might play a role in the microcirculatory defect and resulting tubular injury associated with LPS administration. Fluorescent intravital videomicroscopy was used to assess renal peritubular capillary perfusion and document RNS generation by renal tubules in real time. As early as 6 h after LPS administration (10 mg/kg i.p.), RNS generation (rhodamine fluorescence), redox stress [NAD(P)H autofluorescence], and the percentage of capillaries without flow were each significantly increased compared with saline-treated mice (p < 0.05). The generation of RNS was supported by the detection of nitrotyrosine-protein adducts in the kidney using immunohistochemistry. The iNOS inhibitor l-N(6)-(1-iminoethyl)-lysine (l-NIL; 3 mg/kg i.p.) completely blocked the increase in rhodamine fluorescence and NAD(P)H autofluorescence and prevented the capillary defects at 6 h after LPS administration. These results suggest that iNOS-derived RNS is an important contributor to the peritubular capillary perfusion defects and RNS generation that occur during sepsis and emphasize that pharmacological inhibition of iNOS may provide beneficial effects during sepsis by improving renal capillary perfusion and reducing RNS generation in the kidney.

Despite the recognized role of bradykinin (BK)-induced calcium and chloride conductance in regulating salt transport in the kidney, the signaling pathway involved has not been well examined. Patch clamp of murine proximal tubule (TKPTS) cells revealed that BK (10 nM) produced an increase in an outwardly rectifying current from a basal level of 2.9 +/- 0.6 to 13.8 +/- 1.1 pA/pF following addition of BK (n = 8; p0.001). The shift in reversal potential seen with BK on changing the intracellular solution to 152 mM chloride and significant inhibition of the current by 100 microM 4,4'-di-isothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) suggested that BK activated a chloride current. BK-induced current was blocked by B2 receptor antagonist but not by B1 antagonist or pertussis toxin indicating that the current was mediated by B2 receptors possibly through Gq activation. TMB-8 completely blocked the BK-calcium rise in fura-2 studies but did not block the BK-chloride response indicating that BK-mediated chloride current is calcium-independent. BK-induced current was dependent on phospholipase C (PLC) since U73122, a PLC-beta blocker (10 microM) blocked it completely. Furthermore, chloride conductance was not modulated by bisindolylmaleimide, an inhibitor of protein kinase C (PKC), but was enhanced by dibutyryl cAMP. We conclude that BK-induced rise in chloride current is mediated by B2 receptors and dependent on PLC activation but not dependent on calcium rise. Furthermore, the current can be modulated by cAMP but not PKC.

Since inducible nitric oxide synthase (iNOS) and proximal tubule injury are known to be critical determinants of lipopolysaccharide (LPS)-induced renal failure, the role of nitric oxide (NO) in proximal tubule cell apoptosis was examined. An 18-h treatment with a combination of LPS (5 microg/ml) and interferon-gamma (IFN-gamma, 100 units/ml) synergistically induced iNOS and produced a 20-fold increase in NO generation in the TKPTS murine proximal tubule cell line. NO generation by LPS + IFN-gamma was blocked by a specific iNOS blocker, L-N6-(1-iminoethyl)-lysine (L-NIL, 1 mM). To assess the role of iNOS-derived NO in proximal tubule cell apoptosis, annexin V- and propidium iodide-labeled cells were analyzed by flow cytometry. Neither the induction of iNOS nor its inhibition produced significant apoptotic cell death in TKPTS cells. Two exogenous NO donors were used to examine the role of NO more directly in proximal tubule apoptosis. Although both sodium nitroprusside (SNP), an iron-containing, nitrosonium cation donor, and S-nitroso-N-acetylpenicillamine (SNAP), a noniron-containing, NO generator, produced a concentration-dependent increase in NO generation, only SNP increased apoptotic cell death in TKPTS cells (5.9 +/- 0.7% in control cells vs. 21.6 +/- 3.8% in SNP [500 microM]-treated cells; n = 4-9; p < 0.01). SNP-mediated tubule cell apoptosis was not dependent on the activation of caspases or p53 but was possibly related to the generation of reactive oxygen species by SNP. Thus, in TKPTS cells induction of iNOS and generation of NO by LPS does not lead to tubular epithelial cell death.

This study examined the ability of the endocannabinoids 2-arachidonoyl glycerol (2-AG) and noladin ether as well as the synthetic cannabinoid CP-55,940 [(-)- cis -3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans -4-(3-hydroxypropyl) cyclohexanol] to regulate three intracellular effectors via CB2 receptors in transfected Chinese hamster ovary cells. Although the three agonists regulate all effectors with equivalent efficacy, the rank order of potencies differs depending on which effector is evaluated. Noladin ether and CP-55,940 most potently inhibit adenylyl cyclase, requiring higher concentrations to stimulate the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (extracellular signal-regulated kinase-mitogen-activated protein kinase; ERK-MAPK) and Ca2+-transients. In contrast, 2-AG most potently activates ERK-MAPK, necessitating greater concentrations to inhibit adenylyl cyclase and even higher amounts to stimulate Ca2+-transients. Endocannabinoids also seem to be more “efficient” agonists at CB2 receptors relative to synthetic agonists. 2-AG and noladin ether require occupancy of less than one-half the number of receptors to produce comparable regulation of adenylyl cyclase and ERK-MAPK, relative to the synthetic cannabinoid CP-55,940. The CB2 antagonist 6-iodo-2-methyl-1-\[2-(4-morpholinyl)-ethyl]-1 H -indol-3-yl\](4-methoxyphenyl)-methanone (AM630) reverses the actions of all agonists except Ca2+-transient stimulation by 2-AG. However, the effect of 2-AG on Ca2+-transients is attenuated by a second CB2 antagonist N -[(1 S )-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1-pyrazole-3-carboxamide (SR144528). This suggests that 2-AG stimulates Ca2+-transients by binding to sites on CB2 receptors distinct from those occupied by AM630 and the other cannabinoids examined. Agonists produce no effects in pertussis toxin-treated cells. In summary, cannabinoid agonists distinctly bind to CB2 receptors and display different rank order of potencies and fractional receptor occupancies for regulation of intracellular effectors. These data provide direct evidence for agonist-directed trafficking of response by endocannabinoids acting at CB2 receptors.

Noladin ether (NE) is a putative endogenously occurring cannabinoid demonstrating agonist activity at CB1 receptors. Because of reported selective affinity for CB1 receptors, the pharmacological actions of NE at CB2 receptors have not been examined. Therefore, the purpose of this study was to characterize the binding and functional properties of NE at human CB2 receptors stably expressed in Chinese hamster ovary (CHO) cells as well as in HL-60 cells, which express CB2 receptors endogenously. Surprisingly, in transfected CHO cells, NE exhibits a relatively high nanomolar affinity for CB2 receptors ( K i = 480 nM), comparable to that observed for the endocannabinoid 2-arachidonoyl glycerol (2-AG) ( K i = 1016 nM). Furthermore, NE activates G proteins and inhibits the intracellular effector adenylyl cyclase with equivalent efficacy relative to the full cannabinoid agonists 2-AG and CP 55,940 (CP) \[(-)- cis -3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans -4-(3-hydroxypropyl) cyclohexanol]. The rank order of potency for G protein activation and effector regulation by the three agonists is similar to their apparent affinity for CB2 receptors; CP > NE ≥ 2-AG. Regulation of adenylyl cyclase activity by all agonists is inhibited by pertussis toxin pretreatment or by coincubation with AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1 H -indol-3-yl\](4-methoxyphenyl)-methanone], a CB2 antagonist. Chronic treatment with NE or CP results in CB2 receptor desensitization and down-regulation. All agonists also inhibit adenylyl cyclase activity in HL-60 cells. Together, these data indicate that NE acts as a full agonist at human CB2 receptors and thus might have important physiological functions at peripheral cannabinoid receptors.

Despite the recognized physiological role of bradykinin (BK) in the kidney in maintaining glomerular and tubule function and its role in pathological states such as endotoxemia, diabetes, and other diseases, relatively little is known about the mechanisms by which BK can impact kidney function. Furthermore, the signaling of BK receptors in the murine nephron has not been fully characterized. The present studies were undertaken to examine BK-stimulated Ca(2+) signaling using Fura-2 in the murine proximal tubule epithelial cell line TKPTS. BK produced a concentration-dependent rise in intracellular Ca(2+) ([Ca(2+)])(i) (pEC(50) = 8.39 +/- 0.04). Selective antagonists showed the rise in [Ca(2+)](i) was mediated through B2 receptors. The rise in [Ca(2+)](i) was rapid and reversible and was maximally stimulated at 1 microM (697 +/- 70 nM above basal level of 115 +/- 6 nM). Studies with thapsigargin and EGTA showed Ca(2+) mobilization was dependent on two events: release and influx. Both U73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) [a phospholipase C (PLC) inhibitor] and genistein (a tyrosine kinase inhibitor) partially inhibited BK-stimulated rise in [Ca(2+)](i). When combined, both agents produced a further decrease, suggesting multiple pathways for PLC activation may be involved. The ability of Ni(2+) to inhibit influx indicated the activation of a Ca(2+) release-activated channel (CRAC). Ca(2+) mobilization did not seem to be affected by cyclic nucleotides or protein kinase C. In summary, the TKPTS murine proximal tubule cell line expresses functional B2 receptors linked to Ca(2+) mobilization that is dependent on phospholipase C and activation of CRAC.

We recently reported that following a toxic dose of acetaminophen to mice, tyrosine nitration occurs in the protein of cells that become necrotic. Nitration of tyrosine is by peroxynitrite, a species formed from nitric oxide (NO) and superoxide. In this manuscript we studied the effects of the NO synthase inhibitors N-monomethyl-l-arginine (l-NMMA), N-nitro-l-arginine methyl ester (NAME), l-N-(1-iminoethyl)lysine (l-NIL), and aminoguanidine on acetaminophen hepatotoxicity. Acetaminophen (300 mg/kg) increased serum nitrate/nitrite and alanine aminotransferase (ALT) levels, indicating increased NO synthesis and liver necrosis, respectively. None of the NO synthase inhibitors reduced serum ALT levels. In fact, l-NMMA, l-NIL, and aminoguanidine significantly augmented acetaminophen hepatotoxicity at 4 h. A detailed time course indicated that aminoguanidine (15 mg/kg at 0 h and 15 mg/kg at 2 h) significantly increased serum ALT levels over that for acetaminophen alone at 2 and 4 h; however, at 6 and 8 h serum ALT levels in the two groups were identical. At 2 h following acetaminophen plus aminoguanidine NO synthesis was significantly increased; however, at 4, 6, and 8 h NO synthesis was significantly decreased. Aminoguanidine also decreased acetaminophen-induced nitration of tyrosine. Acetaminophen alone did not induce lipid peroxidation, but acetaminophen plus aminoguanidine significantly increased hepatic lipid peroxidation (malondialdehyde levels) at 2, 4, and 6 h. These data are consistent with NO having a critical role in controlling superoxide-mediated lipid peroxidation in acetaminophen hepatotoxicity. Thus, acetaminophen hepatotoxicity may be mediated by either lipid peroxidation or by peroxynitrite.

We examined the signal transduction cascade of angiotensin II in isolated rat proximal tubules. Angiotensin II induced a rapid (15 sec) concentration-dependent rise in intracellular free Ca2+ (EC50 = 1.7 nM). The rise in Ca2+ was blocked by the angiotensin II receptor AT1 specific antagonist SKF 108566. This indicates that the rise in Ca2+ is fully mediated by AT1 receptors. To characterize further the antagonism by SKF 108566, the Schild analysis was performed (pA2 = 10.9 +/- 0.14 and slope = 0.94 +/- 0.11; n=3). It indicated that SKF 108566 is a high affinity competitive antagonist at AT1 receptors in the proximal tubule. Angiotensin II signaling also induced a rapid (5 min) rise in cGMP formation. This response was blocked by SKF 108566, by inhibition of nitric oxide synthase, or by inhibition of soluble guanylyl cyclase. This indicates that the formation of cGMP elicited by angiotensin II is mediated by AT1 receptors and activation of the NO-cGMP pathway. Since cGMP can inhibit Na, K-ATPase activity, activation of the NO-cGMP pathway may act as a negative feedback component of angiotensin II signaling in renal proximal tubules.

Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) significantly increased hepatotoxicity at 4 h, as evidenced by histological necrosis in the centrilobular areas of the liver, and increased serum levels of alanine aminotransferase (ALT) (from 8 +/- 1 IU/L in saline-treated mice to 3226 +/- 892 IU/L in the acetaminophen-treated mice). Serum levels of nitrate plus nitrite (a marker of nitric oxide synthesis) were also increased from 62 +/- 8 microM in saline-treated mice to 110 +/- 14 microM in acetaminophen-treated mice (P0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P0.05). The y intercept value (nitrate plus nitrite level) was 63 +/- 15 microM. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyrosine antibody indicated tyrosine nitration in the proteins of the centrilobular cells. Tyrosine nitration has been shown to occur by peroxynitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an anti-acetaminophen antiserum indicated the centrilobular cells also contained acetaminophen-protein adducts, a reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine residues on proteins. These data are consistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an important intermediate in the toxicity.

Lipopolysaccharide is a component of the gram-negative bacterial cell wall that is responsible for 25,000-50,000 deaths in the United States each year. The sequelae of gram-negative infection and septicemia leading to death include fever, hypotension with inadequate tissue perfusion, and disseminated intravascular coagulation. It is clear that different cell types respond differently to lipopolysaccharide. Furthermore, various autacoids and cytokines are released that can affect cellular function even in cell types that do not recognize lipopolysaccharide. Despite advances made in the etiology of septic shock and organ failure, therapy is still for the most part supportive and largely ineffective. The aim of this review is to summarize the current understanding of the role of lipopolysaccharide in the development of septicemia by examining signal transduction and therapeutic approaches.

In previous studies, we found that lipid A, the biologically active component of lipopolysaccharide, triggers a rapid release of intracellular calcium, the activation of nitric oxide synthase (NOS), and nitric oxide (NO) production in rat proximal tubules. This pathway leads ultimately to cell death [as measured by the release of lactate dehydrogenase (LDH)], initiated by early generation of NO. In the present studies we found that lipid A produces a time- and concentration-dependent increase in lipid peroxidation [malondialdehyde (MDA) formation] prior to cell death. Furthermore, preventing lipid peroxidation protected against cell death. Lipid A (50 micro;g/ml) produced significant MDA formation in 30 min. The addition of two antioxidants 5 min prior to lipid A completely inhibited MDA formation and LDH release at 90 min. Preincubation with 5 mm GSH also significantly reduced MDA formation. The involvement of NOS activation in lipid A-induced lipid peroxidation was established when an NOS inhibitor and an inhibitor of intracellular calcium release completely blocked MDA formation. In addition, superoxide generation was significantly increased in the presence of lipid A, and the involvement of superoxide was established when superoxide dismutase protected against oxidant injury. The iron chelators deferoxamine (also a scavenger of peroxynitrite) and diethylenetriaminepentaacetic acid prevented lipid A-induced lipid peroxidation and cell death, indicating a role for iron and peroxynitrite. The addition of an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl, prior to lipid A also completely protected tubule cells from lipid peroxidation and subsequent cell death. These results indicate that lipid A-stimulated NO generation in the rat proximal tubule initiates oxidant injury.

Prostaglandin (PG) formation in 16 atherosclerotic human carotid endarterectomy specimens was compared systematically with that of normal carotid artery from seven white pigs and six rhesus monkeys. Prostacyclin (PGI2) formation (picomoles 6-keto-PGF1a/2 min/100 micrograms homogenate protein plus 2 mM glutathione [GSH]) of nonatheromatous intima adjacent proximal (276 +/- 32, mean +/- SEM) or distal (271 +/- 14) to carotid plaque was comparable to that of normal carotid artery from white pig (272 +/- 25, NS) and rhesus monkey (219 +/- 41, NS), and was greater than stenotic intima (156 +/- 17, p less than 0.01), subintimal plaque (168 +/- 14, p less than 0.01), and ulceration (65 +/- 16, p less than 0.01). GSH modulated PGI2 synthesis in all carotid specimens except areas of ulceration (p less than 0.05), but did not restore PGI2 formation in atheromatous fractions to basal level. No detectable arterial thromboxane A2 (TXA2) formation or GSH-dependent PGE2 isomerase activity was observed. The decrement in atherosclerotic carotid artery PGI2 formation was focal (confined to the plaque) and may have been related to loss of effective GSH modulation. These conditions could contribute to a localized imbalance between arterial PGI2 and platelet TXA2 with adverse vascular thromboregulatory consequences.

Garlic has been used in herbal medicine for thousands of years. While garlic oil contains many components and has been widely studied, the pharmacology of pure allicin, a constituent of garlic oil, is not well understood. We report that allicin inhibits human platelet aggregation in vitro without affecting cyclooxygenase or thromboxane synthase activity or cyclic adenosine monophosphate (AMP) levels. Allicin does not alter the activity of vascular prostacyclin synthase. However, it inhibits ionophore A23187-stimulated human neutrophil lysosomal enzyme release. In vivo allicin dilates the mesenteric circulation of the cat independent of prostaglandin release or a beta adrenergic mechanism.

Thromboxanes (TX), prostaglandins (PG), and leukotrienes (LT) (eicosa­ noids) are metabolites of the the 20-carbon fatty acid arachidonic acid. Current evidence supports potential pathophysiologic roles for these autocoids in many diseases (1-3). While the synthesis and metabolism ofthese auto­ coids have been studied extensively in normal and pathologic states, the study of their receptors has lagged far behind. With the development of drugs that will be targeted to these receptors, there is a pressing need to improve our understanding of the fundamental characteristics of these important receptors.

While platelets have been shown to be capable of supplying prostaglandin (PG) H 2 to endothelial cells in culture for PGI 2 synthesis, endothelial cells have been shown unable to supply PGH 2 to platelets for thromboxane (TX) A 2 synthesis. We incubated rings of the bovine coronary artery (BCAR) with human platelets treated with aspirin (to inhibit cyclooxygenase) or CGS 13080 (to inhibit TXA 2 synthase) in the presence of 20 μM arachidonic acid. BCAR, with damaged endothelium, produced significantly less PGI 2 than that with intact endothelium. However, co-incubation with CGS 13080-treated platelets resulted in an increase in PGI 2 independent of endothelium, demonstrating a shunt of PGH 2 from platelets to BCAR. Co-incubation of BCAR with aspirin-treated platelets resulted in a net increase in TXA 2 demonstrating a shunt of PGH 2 from BCAR to platelets. Employing [ 14 C]PGH 2 as substrate, BCAR with and without intact endothelium produced similar amounts of 6-keto[ 14 C]PGF 1α , Likewise, homogenates (50 μg protein) of intimal and subintimal regions of BCAR and BCAR converted similar amounts of PGH 2 to 6-keto-PGF 1α . These data suggest that vascular production of PGH 2 is more dependent on an intact endothelium than is the conversion of PGH 2 to PGI 2 . These data also suggest a potential for a bidirectional exchange of PGH 2 between platelets and vascular wall during platelet-vascular wall interactions.

Previous studies have suggested the possibility that the non-steroidal antiflammatory drug (NSAID), ibuprofen, may inhibit thromboxane (TX) A2 synthase activity in addition to inhibiting cyclooxygenase activity. Microsomal fractions isolated from the cat lung contain cyclooxygenase as well as prostacyclin (PGI2) synthase, TX synthase, and a GSH-dependent prostaglandin (PG) E2 isomerase activities. When [1-14C] PG endoperoxide H2 (PGH2) was used as substrate, ibuprofen, indomethacin, and meclofenamate exhibited differential effects on terminal enzyme activities. Ibuprofen, at concentrations up to 1 mM, had no effect on the activities of PGI2 synthase, TXA2 synthase of GSH-dependent PGE2 isomerase, whereas indomethacin selectively inhibited PGI2 synthase activity at 5 X 10(-4) M and 10(-3) M. Meclofenamate selectively inhibited TXA2 synthase activity at 5 X 10(-4) M and 10(-3) M. At concentrations of 5 X 10(-3) M, this selectivity was not observed, and indomethacin and meclofenamate decreased the formation of both 6-keto-PGF1 alpha and TXB2. These data indicate that the choice of NSAID and the concentration employed may specifically alter PGH2 metabolism. This action may affect the physiologic consequences of the exchange of PGH2 between cells. The data further indicate that indomethacin has the potential for use as a tool to specifically attenuate PGI2 synthase activity in vitro.