CREB transcription factor modulates Bcl2 transcription in response to C5a in HL-60-derived neutrophils.

BACKGROUND: Complement fragment C5a and neutrophils have been implicated in the pathogenesis of renal disease and C5a has also been shown to delay apoptosis of human neutrophils via a transcription-independent pathway. However, transcription-dependent pathways have not been well described. The present study examined whether activation of HL-60-derived neutrophils by C5a modulates the transcription of two members of the Bcl2 family, Bax (pro-apoptotic) and Bcl2 (anti-apoptotic) molecules, and whether the cAMP-response element-binding protein (CREB) transcription factor mediates these effects through the phosphatidylinositol 3-kinase (PI3K)/Akt and extra-cellular signal-regulated kinase (ERK) signalling pathways. MATERIALS AND METHODS: The human promyelocytic leukaemia HL-60 cell line was differentiated into neutrophils using 1.25% DMSO. Differentiated cells were incubated with recombinant human C5a for 30-120 min with, or without, pretreatment with wortmannin or PD98059. The cells were lysed and quantified for gene-specific Bax and Bcl2 mRNA. In separate experiments, cells were incubated with C5a for 5-30 min with, or without, pretreatment with wortmannin, PD98059, or alkaline phosphatase. Cells were then lysed and immunoblotted using antihuman phospho-CREB (Ser133) antibody. Apoptosis was assessed by measuring active caspase-3 in differentiated HL-60 cells. RESULTS: C5a inhibited caspase-3 activation in HL-60-derived neutrophils (P=0.003). C5a significantly increased the expression of Bcl2 mRNA (P=0.028), which was time-dependent, peaking at 30 min, and was abrogated in the presence of either wortmannin or PD98059 (both P=0.028). The C5a had no impact on Bax mRNA expression. The Bax : Bcl2 mRNA ratio markedly decreased at 30 min (P=0.028). Time-dependent effect of C5a on CREB phosphorylation was demonstrable and rapid, peaking at 5 min, and was abrogated by either wortmannin or PD98059 (both P=0.028). Phosphorylation of CREB, but not of Akt and ERK, was inhibited by alkaline phosphatase (P=0.028). The effect of C5a on Bcl2 mRNA expression was abrogated by alkaline phosphatase (P=0.028). The Bax : Bcl2 mRNA ratio markedly increased in the presence of alkaline phosphatase (P=0.046). CONCLUSIONS: This study demonstrates that C5a induces Bcl2 mRNA transcription in HL-60-derived neutrophils, which is mediated in part by CREB through the convergence of the PI3K/Akt and ERK-signalling pathways.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors upregulate inducible NO synthase expression and activity in vascular smooth muscle cells.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase ameliorate atherosclerosis by both cholesterol-dependent and cholesterol-independent mechanisms. We examined whether HMG-CoA reductase inhibitors affect the expression and activity of inducible NO synthase (iNOS) in cultured rat aortic vascular smooth muscle (VSM) cells. Atorvastatin (34 to 68 micromol/L) markedly increased nitrite production, an increase that was essentially abrogated by the NO synthase inhibitor N(G)-monomethyl-L-arginine (500 micromol/L). Activity of iNOS, determined by the conversion of L-arginine to L-citrulline, increased 9-fold after atorvastatin treatment. Western blot and semiquantitative reverse transcriptase-polymerase chain reaction revealed that atorvastatin (34 to 68 micromol/L) strongly upregulated iNOS protein and mRNA levels, respectively. These concentrations of atorvastatin did not cause cytotoxicity, as judged by the cell survival rate. Similarly, simvastatin and lovastatin (34 micromol/L) caused robust upregulation of the iNOS protein level. Transfection experiments demonstrated that the -1034- to 88-bp human iNOS promoter was strongly induced by atorvastatin (34 micromol/L). Electromobility and supershift assays using a nuclear factor-kappaB (NF-kappaB) consensus oligonucleotide and nuclear extracts from VSM cells as well as transfection studies using an NF-kappaB reporter plasmid suggested that the transcriptional activation of the iNOS gene by atorvastatin is not mediated via the NF-kappaB pathway. We conclude that HMG-CoA reductase inhibitors potently upregulate iNOS expression and activity in VSM cells, at least in part, by transcriptional mechanisms that do not depend on transcription factor NF-kappaB. These effects might have important implications for the impact of HMG-CoA reductase inhibitors on atherosclerosis.

Transcriptional regulation of the human iNOS gene by IL-1beta in endothelial cells.

BACKGROUND: Vascular endothelium participates in the control of vascular tone and function via the release of nitric oxide (NO) by the endothelial-type NO synthase (eNOS). Inducible NO synthase (iNOS) expression in endothelial cells occurs in many clinical conditions following induction by lipopolysaccharide or cytokines and generates large quantities of NO that result in endothelial cell activation and dysfunction. No information exists on the transcriptional regulation of the human iNOS gene (or that of other species) in endothelial cells. MATERIALS AND METHODS: We examined the transcriptional regulation of the human iNOS gene by interleukin-1beta (IL-1beta) in rat pulmonary microvascular endothelial cells (PVEC) by transient cotransfections of different iNOS-promoter constructs and cDNA of different transcription factors and regulatory proteins. RESULTS: The -1034/+88 bp iNOS promoter was strongly induced by IL-1beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, IkappaB isoforms, and IKK mutants suggested that the NF-kappaB site at -115/-106 bp is important, but not sufficient, for induction of iNOS promoter and that the role of NF-kappaB is partially independent of its binding site. C/EBP sites within the -205/+88 bp region were shown to be responsible, along with NF-kappaB site, for induction of iNOS promoter by IL-1beta. Overexpression of C/EBPalpha, C/EBPdelta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBPbeta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (approximately 2-fold) by IL-1beta, whereas C/EBPdelta was not constitutively expressed but was strongly induced by IL-1beta. Both C/EBPbeta and C/EBPdelta participated in DNA-protein complex formation. CONCLUSION: Both NF-kappaB and C/EBP pathways are important for the transcriptional regulation of the human iNOS gene by IL-1beta in PVEC.

Approach to patients with acid-base disorders.

Disorders of acid-base balance are commonly encountered in clinical practice and can have a substantial impact on the prognosis of the patient. Moreover, identification of a particular acid-base disturbance can provide a clue to an underlying disorder. Proper evaluation and treatment of acid-base disorders requires a systematic and analytic approach including: (1) assess the accuracy of the acid-base values using the Henderson equation or Henderson-Hasselbalch equation, (2) obtain a complete history and physical examination, (3) calculate the serum anion gap, (4) identify the primary acid-base disturbance and determine whether a simple or mixed disturbance is present, (5) examine serum electrolytes and additional laboratory data, and (6) measure urine pH and urine electrolytes and calculate the urine anion and osmolal gaps. Strict adherence to these principles will enable the clinician to diagnose the acid-base disturbance in the majority of cases. To illustrate these principles, 5 cases of patients with acid-base disturbances are analyzed.

Admission serum potassium in patients with acute myocardial infarction: its correlates and value as a determinant of in-hospital outcome.

STUDY OBJECTIVES: Although controversial, hypokalemia (LK) in patients with acute myocardial infarction (MI) is thought to predict increased in-hospital morbidity, particularly cardiac arrhythmias, and mortality. Also, the mechanism of low serum potassium in the setting of MI has not been delineated. We evaluated the frequency, attributes, and outcome, and speculated on the mechanism of LK in patients with MI. DESIGN: This was a prospective cross-sectional study of 517 consecutive patients with MI admitted to the coronary care unit (CCU). Serum potassium was measured in the emergency department and repeatedly thereafter throughout hospitalization, and was used in the analysis, along with a large array of clinical and laboratory variables. RESULTS: The patients were allocated to a LK and a normokalemic (NK) cohort, based on the emergency department serum potassium measurement. The 41 patients with LK (3.16+/-0.24 mEq/L; 7.9% of total) were comparable on admission in their baseline assessment to the 476 patients with normal serum potassium (4.28+/-0.56 mEq/L), except for lower emergency department magnesium (1.48+/-0.15 mg/dL vs. 1.96+/-0.26 mg/dL; p = 0.0005) and earlier presentation after onset of symptoms (3.0+/-4.1 h vs. 4.4+/- 6.2 h; p = 0.05). There was a poor correlation between serum potassium and magnesium on admission (r = 0.14). Peak creatine kinase (CK) and myocardial isomer of CK were higher in the LK patients (3,870+/-3, 840 IU/L vs. 2,359+/-2,653 IU/L [p = 0.018] and 358+/-312 IU/L vs. 228 +/- 258 IU/L [p = 0.013], respectively). Management of the two cohorts was the same, except for a higher rate of use of magnesium (14.6% vs. 4.6%; p = 0.007), serum potassium supplements (90.2% vs 43. 1%; p = 0.000005), and antiarrhythmic drugs (78.0% vs 50.4%; p = 0. 0007) in the LK patients. No difference was detected between the LK and NK patients in total mortality (24.4% vs. 18.3%; p = 0.34), cardiac mortality (17.1% vs. 15.3%; p = 0.52), atrial fibrillation (14.6% vs 13.9%; p = 0.89), and ventricular tachycardia (22.0% vs. 16.0%; p = 0.32), but ventricular fibrillation (VF) occurred more often (24.4% vs 13.0%; p = 0.04) in the LK patients. However, proportions of VF occurring in the emergency department, CCU, or wards in the two cohorts were not different, but they were higher during the time interval prior to emergency department admission in LK patients (17.1% vs 2.1%; p = 0.00001). CONCLUSIONS: LK is seen in approximately 8% of patients with MI in the emergency department; LK is associated with low emergency department magnesium, and low serum potassium levels in the CCU and throughout hospitalization. LK has no relationship to preadmission use of diuretics, it is associated with early presentation to the emergency department, and it is not a predictor of increased morbidity or mortality.

Marked dilutional acidosis complicating management of right ventricular myocardial infarction.

Dilutional acidosis is a poorly recognized cause of metabolic acidosis. Indeed, the prevailing view has been that even massive expansion of the extracellular fluid volume with non-bicarbonate-containing solutions would not lead to clinically significant hypobicarbonatemia. We describe the development of marked dilutional acidosis as a complication of management of right ventricular myocardial infarction. The pathogenesis, clinical significance, prevention, and treatment of the entity are discussed.

Aiding fluid prescription for the dysnatremias.

OBJECTIVE: The goal of the present study was to develop a novel approach that facilitates the prescription of fluid therapy in patients with abnormal serum sodium concentration. METHODOLOGY AND RESULTS: The novel approach is based on a simple equation, derived from established principles on the distribution of sodium in body fluids, that estimates the impact of a unit dose, i.e., 1 l of any infusate on the patient's serum sodium concentration. In accordance with the equation, the expected change in the patient's serum sodium concentration in response to 1 l of any infusate (delta[Na+]s) is obtained by subtracting the sodium concentration of the patient's serum from the sodium concentration of the infusate, each expressed in mEq/l, and dividing the result by the patient's estimated total body water expressed in liters (adding 1 l to account for the volume of the infusate). The amount of the particular infusate to be administered over the course of any given time period can be easily computed by dividing the desired delta[Na+]s at the end of the period by the calculated delta[Na+]s effected by 1 l of the infusate. The utility and limitations of the proposed approach are presented. CONCLUSIONS: The novel equation is not a means for formulating therapy. Rather, it provides, simply and expeditiously, quantitative projections that can assist the physician in implementing the selected treatment plan for patients with dysnatremias.

Admission serum magnesium level does not predict the hospital outcome of patients with acute myocardial infarction.

OBJECTIVE: To establish whether hypomagnesemia at admission predicts excessive morbidity, particularly cardiac arrhythmias, and mortality in patients with acute myocardial infarction. METHODS: We compared hypomagnesemic and normomagnesemic patients with acute myocardial infarction in 517 patients admitted to the coronary care unit. The serum magnesium concentration, along with a large array of other parameters, was measured on admission to the emergency department. Other baseline attributes and variables related to the patients' hospital course were used to compare the 2 groups. RESULTS: The 132 patients (25.9%) with low serum magnesium concentrations at admission (mean +/- SD, 0.61 +/- 0.06 mmol/L [1.48 +/- 0.15 mg/dL]) were comparable to the patients with normal serum magnesium concentrations (0.81 +/- 0.11 mmol/L [1.96 +/- 0.26 mg/dL]) except for a higher rate of prehospital use of diuretic agents (32.6% vs 22.5%, P = .02) and earlier presentation after onset of symptoms (mean +/- SD, 3.2 +/- 4.1 vs 4.8 +/- 6.6 hours, P = .003). There was no correlation between serum magnesium and potassium concentrations in the emergency department (r = 0.14). No difference was detected between the hypomagnesemic and normomagnesemic cohorts in rates of total mortality (18.9% vs 18.5%, P = .91), cardiac mortality (15.2% vs 15.3%, P = .99), atrial fibrillation (13.6% vs 13.8%, P = .97), ventricular tachycardia (18.2% vs 15.3%, P = .44), or ventricular fibrillation (15.2% vs 13.5%, P = .63). Management of the 2 cohorts was not different, except for higher rates of use of magnesium (17.4% vs 1.3%, P < .001) and potassium (59.8% vs 42.1%, P < .001) supplements and antiarrhythmic drugs (62.9% vs 48.7%, P = .005) in the hypomagnesemic patients. An endogenous rise in serum magnesium level was documented in a subgroup of 161 patients who had a repeated measurement (0.74 +/- 0.12 mmol/L [1.79 +/- 0.29 mg/dL] in the emergency department vs 0.77 +/- 0.09 mmol/L [1.88 +/- 0.23 mg/dL] in the coronary care unit, P < .001). CONCLUSIONS: We conclude that hypomagnesemia is seen in approximately one fourth of patients with myocardial infarction, is not linked to hypokalemia, has some relationship to preadmission use of diuretic agents, is associated with early presentation to the hospital, and is not a predictor of increased morbidity or mortality.

Transcriptional regulation of the human iNOS gene in vascular-smooth-muscle cells and macrophages: evidence for tissue specificity.

We have cloned the 5' upstream -1034 to +88 fragment of the human inducible nitric oxide synthase (hiNOS) gene and demonstrate its competence to promote luciferase gene transcription in vascular-smooth-muscle (VSM) cells and macrophages. Sequential 5' end-deletions localized positive regulatory elements of hiNOS transcription in VSM A7r5 cells downstream of nucleotide -205 and demonstrated the functional importance of the resident NF-kappaB site (nucleotides -115 to -106). The hiNOS promoter/enhancer was induced strongly by LPS and IFN-gamma, and modestly by IL-1beta in RAW 264.7 cells, but not in VSM cells. Truncation of the NF-kappaB site markedly diminished, but did not eliminate, LPS-inducibility. Sodium salicylate and ibuprofen down-regulated the basal transcriptional activity of the hiNOS promoter/enhancer in VSM but not in RAW 264.7 cells. These results indicate that the transcriptional regulation of the hiNOS gene features considerable complexity and tissue specificity.

Predominant tubulointerstitial lupus nephritis.

Predominant tubulointerstitial lupus nephritis is rare. Only eight cases have been described in the literature. We report the case of a 59-year-old man with systemic lupus erythematosus who presented with acute renal failure. On renal biopsy, he was found to have chronic tubulointerstitial nephritis with a mononuclear infiltrate. The immunofluorescence showed immune deposits in the tubular basement membranes, interstitium, and glomerular capsule. The glomeruli were minimally involved. He was initially treated with high-dose corticosteroids and supported with hemodialysis. Renal function improved and dialysis was discontinued after three treatments. The corticosteroid dosage was gradually tapered. Renal function after 72 months of follow-up has remained stable (serum creatinine, approximately 1.9 mg/dL) and except for one relapse, there has been no clinical or serologic evidence of lupus activity. Furthermore, 24-hour urinary protein excretion has remained within the normal range.

Role of C/EBP proteins in hepatic and vascular smooth muscle transcription of human NHE1 gene.

We have recently shown that regulatory element D (nucleotides -239 to -215) of the 0.25-kb promoter of the human growth factor-activatable Na+/H+ exchanger (NHE1) is important for gene transcription in cells of hepatic origin (Hep G2) and vascular smooth muscle origin (VSM A7r5). This element contains a sequence (nucleotides -230 to -222) with complete homology to the C/EBP binding site. We now demonstrate that nucleotide substitution mutations disrupting this C/EBP site suppressed transcription in Hep G2 cells, VSM A7r5 cells, and Sprague-Dawley VSM cells in primary culture. These mutations abolished the binding of rat liver nuclear activities as well as transcription factors C/EBP alpha, C/EBP beta, and C/EBP delta expressed in COS-1 cell lysates to element D. Anti-C/EBP antibodies supershifted DNA-protein complexes formed between hepatic nuclear activities or C/EBP proteins expressed in COS-1 cell lysates and regulatory element D. Finally, cotransfection experiments of NHE1 0.25-kb promoter-chloramphenicol acetyltransferase (CAT) construct and C/EBP expression vectors showed that C/EBP alpha and C/EBP delta are transactivators of the NHE1 proximal promoter in Hep G2 and VSM A7r5 cells. These results indicate that members of the C/EBP family of transcription factors are involved in the regulation of hepatic and vascular smooth muscle transcription of the human NHE1 gene.

Effect of chronic respiratory acidosis on calcium metabolism in the rat.

Chronic metabolic acidosis typically results in hypercalciuria and negative calcium balance. The impact of chronic respiratory acidosis on calcium metabolism has been less well studied. To address this issue, metabolic balance and static bone histomorphometric data were obtained during a 14-day exposure of rats to 10% CO2 (blood pH 7.33, PaCO2 83 mm Hg) and were compared with pair-fed controls. All rats were fed a 0.8% calcium diet. Urinary calcium excretion (mg/period, mean +/- SEM) was increased during both week 1 and week 2 (16 +/- 3 vs 9 +/- 1 and 16 +/- 2 vs 9 +/- 1, CO2 group vs controls, respectively [p < 0.05]). Net intestinal calcium absorption (intake minus fecal excretion) was increased throughout the period of hypercapnia (week 1, 213 +/- 19 mg vs 135 +/- 15 mg; week 2, 135 +/- 16 mg vs 43 +/- 14 mg; and cumulatively, 344 +/- 27 mg vs 178 +/- 20 mg, CO2 group vs controls [p < 0.01]). As a consequence of the marked increment in intestinal calcium absorption during hypercapnia, mean net calcium balance was more positive than that of controls throughout the study (week 1, 197 +/- 18 mg vs 126 +/- 15 mg; week 2, 120 +/- 15 mg vs 34 +/- 15 mg; and cumulatively, 317 +/- 25 mg vs 159 +/- 20 mg, CO2 group vs controls, respectively [p < 0.01]). There were no significant differences in calcium intake, plasma total calcium, immunoreactive parathyroid hormone, 25-hydroxyvitamin D, or creatinine clearance between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental renal failure in the rat modulates cardiac Na,K-ATPase alpha 2 mRNA but not protein.

The decreased abundance and enzymatic activity of myocardial Na,K-ATPase have been recognized previously to occur in chronic uremia. However, the activity of the cardiac sodium pump as defined by the uptake of 86Rb is normal. The discrepancies between these findings may have resulted from the inability to distinguish between the different Na,K-ATPase isoforms now known to exist in cardiac muscle. To investigate this question, steady-state levels of Na,K-ATPase alpha and beta mRNA isoforms, alpha 1, alpha 2, and beta 1 protein, and specific high-affinity binding of [3H]ouabain were quantitated in cardiac muscle from uremic and pair-fed, sham-operated control rats. Steady-state levels of alpha 2 and beta 2 mRNA were significantly decreased (percentage of control levels: alpha 2, 48 +/- 10; beta 2, 74 +/- 9; N = 10; P < 0.025) in chronic renal failure without any change in alpha 1, alpha 3, or beta 1 expression. The number of high-affinity [3H]ouabain-binding sites and Na,K-ATPase alpha 1, alpha 2, and beta 1 subunits was not different from control. In acute renal failure, alpha 2 and beta 2 mRNA levels also were significantly decreased (percentage of control levels: alpha 2, 24 +/- 5; beta 2, 44 +/- 8; N = 6; P < 0.001), but there was no change in the level of alpha 3 or beta 1 mRNA, the number of high-affinity [3H]ouabain-binding sites, or the level of Na,K-ATPase alpha 2 and beta 1 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Cisplatin nephrotoxicity.

Cisplatin treatment causes tubulointerstitial injury to the kidneys. Common clinical syndromes associated with its use include acute renal failure and a magnesium wasting state. Routine fluid infusion therapy has markedly reduced the incidence of acute renal failure. However, methodologic limitations of most recent studies preclude confident conclusions regarding long-term effects of cisplatin treatment on renal function. Experimental and clinical evidence suggests that sulfhydryl metabolism and oxidative stress are central to cisplatin renal injury. Both in animals and humans, a variety of agents appear to alter renal uptale of cisplatin and its hemodynamic consequences, thus modulating nephrotoxicity. Thiosulfates and related agents have received most study. Other agents examined have included calcium channel blockers, bismuth, selenium, glycine, cimetidine, and probenecid.

Proximal regulatory elements and nuclear activities required for transcription of the human Na+/H+ exchanger (NHE-1) gene.

We herein demonstrate competence of the 5' upstream region -1374 to +16 of the human growth factor-activatable Na+/H+ exchanger (NHE-1) gene to promote transcription of the chloramphenicol acetyltransferase gene in cells of hepatic origin (HepG2), vascular-smooth-muscle origin (VSM A7r5) and fibroblasts (3T3). We also describe the mapping of the regulatory elements required for such transcription. Sequential 5' end-deletions indicated that the 5' boundary of the positive regulatory elements of NHE-1 transcription is localized downstream of nucleotide -252 in both HepG2 and VSM A7r5 cells but downstream of nucleotide -654 in 3T3 cells. Footprinting analysis of the 0.25-kb promoter fragment using rat liver nuclear extracts identified 4 protected regions as follows: A, -31 to -9; B, -108 to -65; C, -124 to -111; and D, -239 to -215. Internal deletion and nucleotide substitutions within regulatory element D revealed its essential role for transcription of the human NHE-1 gene in HepG2 and VSM A7r5 cells. DNA binding and competition assays using rat liver nuclear extracts indicated that regulatory element D is recognized by 5 nuclear activities. Four of these activities (designated as NHE-1D1-4) are competed out completely by oligonucleotides containing the binding sites of transcription factors CREB, AP3, NFY, and other CCAAT box-binding proteins (C/EBP alpha or related proteins). This competition profile might be explained by the presence of homology between regulatory element D and the consensus sequence of C/EBP as well as the other competitor oligonucleotides. The actual relationship between these nuclear activities and the C/EBP family of proteins (or other transcription factors) remains to be determined.