Te-I Weng

Inhibition of NADPH oxidase-related oxidative stress-triggered signaling by honokiol suppresses high glucose-induced human endothelial cell apoptosis.

Angiopathy is a major complication of diabetes. Abnormally high blood glucose is a crucial risk factor for endothelial cell damage. Nuclear factor-kappaB (NF-kappaB) has been demonstrated as a mediated signaling in hyperglycemia or oxidative stress-triggered apoptosis of endothelial cells. Here we explored the efficacy of honokiol, a small molecular weight natural product, on NADPH oxidase-related oxidative stress-mediated NF-kappaB-regulated signaling and apoptosis in human umbilical vein endothelial cells (HUVECs) under hyperglycemic conditions. The methods of morphological Hoechst staining and annexin V/propidium iodide staining were used to detect apoptosis. Submicromolar concentrations of honokiol suppressed the increases of NADPH oxidase activity, Rac-1 phosphorylation, p22(phox) protein expression, and reactive oxygen species production in high glucose (HG)-stimulated HUVECs. The degradation of IkappaBalpha and increase of NF-kappaB activity were inhibited by honokiol in HG-treated HUVECs. Moreover, honokiol (0.125-1 microM) also suppressed HG-induced cyclooxygenase (COX)-2 upregulation and prostaglandin E(2) production in HUVECs. Honokiol could reduce increased caspase-3 activity and the subsequent apoptosis and cell death triggered by HG. These results imply that inhibition of NADPH oxidase-related oxidative stress by honokiol suppresses the HG-induced NF-kappaB-regulated COX-2 upregulation, apoptosis, and cell death in HUVECs, which has the potential to be developed as a therapeutic agent to prevent hyperglycemia-induced endothelial damage.

The Role of Endoplasmic Reticulum Stress-Related Unfolded Protein Response in the Radiocontrast Medium-Induced Renal Tubular Cell Injury

Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect may have a role in the pathophysiology of CM-induced nephropathy. CM has been shown to affect the endoplasmic reticulum (ER)-related capacity. Unfolded protein response (UPR) is known as a prosurvival response to reduce the accumulation of unfolded proteins and restore normal ER function. However, the role of ER stress-related UPR in the CM-induced renal cell injury still remains unclear. In this study, we examined whether UPR participates in urografin (an ionic CM)-induced renal tubular cells apoptosis. Treatment with urografin in normal rat renal tubular cell line (NRK52E) markedly increased cell apoptosis and decreased cell viability with a dose- and time-dependent manner. The cell necrosis was not increased in urografin-treated cells. Urografin also enhance the induction of ER stress-related markers in NRK52E cells, including glucose-regulated protein (GRP)78 and GRP94 expressions, procaspase-12 cleavage, phosphorylation of PERK (PKR [double-stranded RNA-activated protein kinase]-like ER kinase), and eukaryotic initiation factor 2alpha (eIF2alpha). Salubrinal, a selective inhibitor of eIF2alpha dephosphorylation, effectively decreased urografin-induced cell apoptosis. Furthermore, transfection of GRP78-small interfering RNA in NRK52E cells significantly enhanced urografin-induced cell apoptosis. These results suggest that GRP78/eIF2alpha-related signals play a protective role during UPR, and the activation of ER stress-related UPR may play an important regulative role in urografin-induced renal tubular injury.

Screening and Confirmation of 62 Drugs of Abuse and Metabolites in Urine by Ultra-High-Performance Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

An ultra-high-performance liquid chromatography--quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) method for the screening and confirmation of 62 drugs of abuse and their metabolites in urine was developed in this study. The most commonly abused drugs, including amphetamines, opioids, cocaine, benzodiazepines (BZDs) and barbiturates, and many other new and emerging abused drugs, were selected as the analytes for this study. Urine samples were diluted 5-fold with deionized water before analysis. Using a superficially porous micro-particulate column and an acetic acid-based mobile phase, 54 basic and 8 acidic analytes could be detected within 15 and 12 min in positive and negative ionization modes, respectively. The MS collision energies for the 62 analytes were optimized, and their respective fragmentation patterns were constructed in the in-house library for confirmatory analysis. The coefficients of variation of the intra- and inter-day precision of the analyte responses all were <17.39%. All analytes, except barbital, showed matrix effects of 77-121%. The limits of detection of the 62 analytes were between 2.8 and 187.5 ng/mL, which were lower than their respective cut-off concentrations (20-500 ng/mL). Ten urine samples from patients undergoing methadone treatment were analyzed by the developed UHPLC-QTOF-MS method, and the results were compared with the immunoassay method.

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells.

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (−)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.

Treating baclofen overdose by hemodialysis

Because baclofen is eliminated mostly by the kidney, baclofen-related encephalopathy is usually found in patients with renal failure. Therefore, hemodialysis has been suggested for those patients to alleviate symptoms and shorten recovery time. We present a case of baclofen intoxication with normal renal function benefiting from hemodialysis. A 55-year-old man took 420 mg of baclofen and was intubated because of respiratory depression. Hisconsciousness returned 9 hours after hemodialysis was finished, and he was extubated smoothly thereafter. The elimination half-life of baclofen before and during hemodialysis was 15.7 and 3.1 hours, respectively. As patients with baclofen overdose could have prolonged elimination even with normal renal function, hemodialysis would be beneficial to those patients with normal renal function.

Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy.

Uropathogenic Escherichia coli-Induced Inflammation Alters Mouse Urinary Bladder Contraction via an Interleukin-6-Activated Inducible Nitric Oxide Synthase-Related Pathway

ABSTRACT Escherichia coli is the most common cause of urinary tract infection. Elevated blood and urine interleukin-6 (IL-6) levels have been shown in inflammatory urinary tract diseases. The role of IL-6 in mediating the urodynamic dysfunction in response to E. coli-induced urinary tract infection has not yet been fully elucidated. In this study, we investigated the role of IL-6 in the nitric oxide (NO)-triggered alteration of contractile responses in the urinary bladder under an E. coli-induced inflammatory condition. The electrical field stimulation (EFS)-evoked contractions of the isolated detrusor strips, and immunoblotting for detecting protein expression in the bladders was measured short term (1 h) or long term (6 or 24 h) after intraperitoneal injection of E. coli endotoxin (lipopolysaccharide [LPS]) or intravesical instillation of human pyelonephritogenic E. coli-J96 (O4:K6) strain or LPS into mice. IL-6 and NO productions were increased in the urinary bladders of mice 1 to 24 h after LPS or E. coli-J96 treatment. Inducible NO synthase (iNOS) expression and protein kinase C (PKC) activation and EFS-evoked detrusor contractions were increased in the bladders at 6 h after LPS or E. coli-J96 treatment, which could be reversed by anti-IL-6 antibody and iNOS inhibitor aminoguanidine. At 1 h after LPS administration, bladder NO generation, endothelial NOS expression, and EFS-evoked detrusor contractions were effectively increased, whereas anti-IL-6 antibody could not reverse these LPS-induced responses. These results indicate that IL-6 may play an important role in the iNOS/NO-triggered PKC-activated contractile response in urinary bladder during E. coli or LPS-induced inflammation.

Acute Acetaminophen Intoxication in Taiwan: Outcomes and Risk Factors

BACKGROUND AND PURPOSE There is a paucity of information about acetaminophen intoxication from Taiwan. This study investigated the outcome and risk factors for acetaminophen-induced hepatotoxicity and validated the Rumack- Matthew nomogram in Taiwanese patients with acute acetaminophen intoxication. METHODS A total of 75 patients with acetaminophen intoxication admitted through the emergency department were included in this retrospective analysis. Patients with a serum acetaminophen concentration above the possible risk line on the nomogram were treated with oral N-acetylcysteine. The primary outcome measure was the development of major hepatotoxicity, which was defined as a serum aminotransferase concentration greater than 1000 IU/L. Patient outcomes in the possible risk group and probable risk group were plotted on the modified Rumack-Matthew nomogram for validation. The risk factors for acetaminophen-induced hepatotoxicity were identified by multiple logistic regression analysis. RESULTS No hepatotoxicity developed in patients with an initial acetaminophen concentration below the possible risk line on the nomogram. One out of 8 patients in the possible risk group developed major hepatotoxicity; 8 out of 22 patients in the probable risk group developed major hepatotoxicity, representing an incidence of 12.5% and 36.4%, respectively. Patients in the major hepatotoxicity group were older (32.5 vs 24.2 years, p = 0.019), and had a longer time to presentation (28.1 vs 6.7 hours, p < 0.01) than those in the non/minor hepatotoxicity group. Multiple logistic regression revealed that age and time to presentation were independent risk factors for hepatotoxicity (p = 0.033 and p = 0.002, respectively). CONCLUSIONS The results of outcome analysis confirm that the modified Rumack-Matthew nomogram has a high sensitivity for identifying Taiwanese patients at risk for acetaminophen-induced hepatoxicity. Patient age and time to presentation were independent risk factors for acetaminophen-induced hepatotoxicity.

Involvement of Interleukin-6-Regulated Nitric Oxide Synthase in Hemorrhagic Cystitis and Impaired Bladder Contractions in Young Rats Induced by Acrolein, a Urinary Metabolite of Cyclophosphamide

Hemorrhagic cystitis is a common complication in children receiving cyclophosphamide, a chemotherapeutic alkylating agent. Acrolein is a urinary metabolite from cyclophosphamide and can induce hemorrhagic cystitis. Here, we investigated the effects and mechanisms of acrolein by intravesical instillation on urinary bladder muscle contractions and pathological alterations in rats. Acrolein instillation significantly increased the muscle contractions of rat bladder detrusor after 1 and 6 h but markedly decreased detrusor contractions after 24 h. Acrolein increased phosphorylated protein kinase C (pan-PKC) expressions in bladders after 1 and 6 h but inhibited it after 24 h. Inducible nitric oxide (NO) synthase (iNOS) protein expressions were markedly induced in bladders 24 h after acrolein treatment. Twenty-four-hour acrolein instillation increased the levels of nitrite/nitrate and interleukin-6 (IL-6) in the urinary bladder. The iNOS inhibitors significantly inhibited the acrolein-increased nitrite/nitrate levels, but not IL-6 levels. IL-6-neutralizing antibodies effectively inhibited the acrolein-increased NOx levels. The increased detrusor contractions by 1-h acrolein treatment were significantly reversed by the PKC inhibitor RO32-0432, and the decreased detrusor contractions by 24-h acrolein treatment were significantly reversed by the iNOS inhibitor and IL-6-neutralizing antibody. Both the iNOS inhibitor and IL-6-neutralizing antibody effectively reversed the increased iNOS expression, decreased PKC phosphorylation, increased bladder weight, and hemorrhagic cystitis in rats 24 h after acrolein treatment. Taken together, these results suggest that an IL-6-regulated iNOS/NO signaling pathway participates in the acrolein-triggered detrusor contraction inhibition and hemorrhagic cystitis. These findings may help us to find a new strategy to treat cyclophosphamide-induced hemorrhagic cystitis.

Uropathogenic Escherichia coli Alters Muscle Contractions in Rat Urinary Bladder via a Nitric Oxide Synthase–Related Signaling Pathway

BACKGROUND Uropathogenic Escherichia coli (UPEC) is a common cause of urinary-tract infection. The mechanisms by which bacteria cause the symptoms of cystitis remain unclear. METHODS The contractions of isolated rat detrusor strips evoked by electrical field stimulations (EFS) or by exogenous agonists and immunoblotting for the detection of protein expression in the bladder were measured in the short (1 h) and long (24 h) term after the intravesical instillation of J96 (O4:K6) strain or UPEC isolated from patients with acute E. coli pyelonephritis or E. coli lipopolysaccharide (LPS). RESULTS One hour after the instillation of UPEC, the level of endothelial nitric oxide synthase (eNOS) and the contractile response, but not protein kinase C (PKC) and extracellular signal-regulated kinase (ERK)-1/2 activation, were higher. Twenty-four hours after UPEC treatment, detrusor contractions were decreased, and inducible (i) NOS protein expression and ERK1/2 phosphorylation, but not PKC activation, were observed. Both aminoguanidine and PD98059 treatment markedly reversed the decrease of EFS- and acetylcholine-evoked detrusor contractions induced by UPEC. The instillation of LPS triggered PKC activation but not ERK1/2 phosphorylation. CONCLUSIONS Short-term intravesical instillation of UPEC enhances detrusor contractions through an eNOS-related pathway, but iNOS-regulated ERK1/2 signaling may be involved in long-term UPEC treatment-induced responses. There are different mechanisms involved in the responses induced by UPEC and LPS.