Lack of antidiabetic effect of a Eugenia jambolana leaf decoction on rat streptozotocin diabetes

Streptozotocin-diabetic rats were treated for 17 days with a decoction of Eugenia jambolana (Myrtaceae) leaves (15%, w/v) as a substitute for water. Body weight, food and fluid intake, urine volume, glycemia, urinary glucose and urea were evaluated every 5 days. The animals were sacrificed by decapitation and blood samples collected for the determination of glycemia, serum cholesterol, HDL-cholesterol, triglycerides and angiotensin-converting enzyme. The weight of adipose and muscle tissues was also determined. There were no statistically significant differences between treated and untreated rats for any of the biochemical or physiological parameters. We conclude that, at least in this experimental model, Eugenia jambolana leaf decoction has no antidiabetic activity.

Rat liver responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia

Hepatic responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia was investigated. For this purpose, livers were perfused with a saturating concentration of 2 mM glycerol, 5 mM L-alanine or 5 mM L-glutamine as gluconeogenic substrates. All experiments were performed 1 h after an ip injection of saline (CN group) or 1 IU/kg of insulin (IN group). The IN group showed higher (P<0.05) hepatic glucose production from glycerol, L-alanine and L-glutamine and higher (P<0.05) production of L-lactate, pyruvate and urea from L-alanine and L-glutamine. In addition, ip injection of 100 mg/kg glycerol, L-alanine and L-glutamine promoted glucose recovery. The results indicate that the hepatic capacity to produce glucose from gluconeogenic precursors was increased during insulin-induced hypoglycemia.

Influence of acute renal failure on the mononuclear phagocytic system

Several studies show the ability of macrophages to remove particles injected into the bloodstream. This function seems to be increased in the presence of acute renal failure. The objective of the present study was to assess the phagocytic function of the main organs (spleen, liver and lung) of the mononuclear phagocytic system in renal and postrenal failures. Fifteen rats (250-350 g) were divided into three groups (N = 5): group I - control; group II - ligature of both ureters, and group III - bilateral nephrectomy. On the third postoperative day, all animals received an iv injection of 1 ml/kg 99mTc sulfur colloid. Blood samples were collected for the assessment of plasma urea, creatinine, sodium, and potassium concentrations and arterial gasometry. Samples of liver, spleen, lung and blood clots were obtained and radioactivity was measured. Samples of liver, spleen, lung and kidney were prepared for routine histopathological analysis. Plasma urea, creatinine and potassium concentrations in groups II and III were higher than in group I (P<0.05). Plasma sodium concentrations in groups II and III were lower than in group I (P<0.05). Compensated metabolic acidosis was observed in the presence of postrenal failure. Group II animals showed a lower level of radioactivity in the spleen (0.98) and lung (2.63), and a higher level in the liver (105.51) than control. Group III animals showed a lower level of radioactivity in the spleen (11.94) and a higher level in the liver (61.80), lung (11.30) and blood clot (5.13) than control. In groups II and III liver steatosis and bronchopneumonia were observed. Renal and postrenal failures seem to interfere with blood clearance by the mononuclear phagocytic system.

Biochemical profile of amniotic fluid for the assessment of fetal and renal development

Creatinine plays a key role in the function and maturation of fetal kidneys throughout pregnancy. It is important to identify other markers that may help in the diagnosis of renal dysfunction. Our aim was to determine the profile of and the correlation between biochemical markers to be used to assess renal function and maturation of the fetus in the amniotic fluid during pregnancy and to determine the distribution of normal values for creatinine, N-acetyl-ß-D-glucosaminidase (NAG), ß2-microglobulin, glucose, urea, sodium, potassium, phosphorus, calcium, uric acid, albumin, and osmolality in three gestational age groups. This was a cross-section study that assessed 115 samples of amniotic fluid during three different periods of pregnancy, i.e., 13 to 20, 27 to 34, and 36 to 42 weeks. Concentrations of creatinine, NAG, urea, potassium and uric acid increased during pregnancy (P<0.05). ß2-Microglobulin, glucose, sodium, phosphorus, calcium, and albumin concentration and osmolality decreased (P<0.05), whereas ß2-microglobulin, glucose and uric acid presented significant correlations with gestational age and creatinine, respectively (r>0.6, P<0.05). Urea, potassium and phosphorus showed mild correlations with both (r>0.5, P<0.05). NAG, sodium, albumin and osmolality did not show significant correlations (r<0.5, P<0.05). These tests confirmed the important role of creatinine in terms of correlation with gestational age. ß2-Microglobulin, glucose and uric acid were significant as markers of function and maturation of fetal kidneys, whereas NAG did not demonstrate a useful role for the assessment of renal maturation.

Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity

The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100% of subtype B (sensitivity = 1.0; specificity = 0.95), 100% of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95% of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50% of subtype A (sensitivity = 0.5; specificity = 0.95), 60% of subtype D (sensitivity = 0.6; specificity = 1.0), and 28% of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants.

Different biochemical strategies of two Neotropical fish to cope with the impairment of nitrogen excretion during air exposure

The exposure of fish to air is normally expected to interfere with the nitrogen excretion process. Hoplias malabaricus and Hoplerythrinus unitaeniatus, two teleost species, display distinct behaviors in response to decreases in natural reservoir water levels, although they may employ similar biochemical strategies. To investigate this point, plasma levels of ammonia, urea, uric acid, and the two urea cycle enzymes, ornithine carbamoyl transferase (OCT) and arginase (ARG), as well as glutamine synthetase (GS) were determined for both species after exposure to air. Plasma ammonia increased gradually during exposure to air, but only H. malabaricus showed increased concentrations of urea. Plasma uric acid remained very low in both fish. Enzymatic activities (mean ± SD, µmol min-1 g protein-1) of H. malabaricus showed significant increases (P<0.05, N = 6) in OCT from 0.84 ± 0.05 to 1.42 ± 0.03, in ARG from 8.07 ± 0.47 to 9.97 ± 0.53 and in GS from 1.15 ± 0.03 to 2.39 ± 0.04. The OCT and ARG enzymes remained constant in H. unitaeniatus (N = 6), but GS increased from 1.49 ± 0.02 to 2.06 ± 0.03. Although these species are very closely related and share the same environment, their biochemical strategies in response to exposure to air or to increased plasma ammonia are different.

Characterization of ß-trypsin at acid pH by differential scanning calorimetry

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel ß-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of ß-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM ß-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, ß-trypsin unfolded with Tm = 54ºC and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of ß-trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Effect of an isotonic rehydration sports drink and exercise on urolithiasis in rats

The objective of the present study was to evaluate the role of physical exercise as well as the influence of hydration with an isotonic sports drink on renal function in male Wistar rats. Four groups were studied over a period of 42 days: 1) control (N = 9); 2) physical exercise (Exe, N = 7); 3) isotonic drink (Drink, N = 8); 4) physical exercise + isotonic drink (Exe + Drink, N = 8). Physical exercise consisted of running on a motor-driven treadmill for 1 h/day, at 20 m/min, 5 days a week. The isotonic sports drink was a commercial solution used by athletes for rehydration after physical activity, 2 ml administered by gavage twice a day. Urine cultures were performed in all animals. Twenty-four-hour urine samples were collected in metabolic cages at the beginning and at the end of the protocol period. Urinary and plasma parameters (sodium, potassium, urea, creatinine, calcium) did not differ among groups. However, an amorphous material was observed in the bladders of animals in the Exe + Drink and Drink groups. Characterization of the material by Western blot revealed the presence of Tamm-Horsfall protein and angiotensin converting enzyme. Physical exercise and the isotonic drink did not change the plasma or urinary parameters measured. However, the isotonic drink induced the formation of intravesical matrix, suggesting a potential lithogenic risk.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Incidence, risk factors and prognostic factors of acute renal failure in patients admitted to an intensive care unit

The objective of the present study was to assess the incidence, risk factors and outcome of patients who develop acute renal failure (ARF) in intensive care units. In this prospective observational study, 221 patients with a 48-h minimum stay, 18-year-old minimum age and absence of overt acute or chronic renal failure were included. Exclusion criteria were organ donors and renal transplantation patients. ARF was defined as a creatinine level above 1.5 mg/dL. Statistics were performed using Pearsons' chi2 test, Student t-test, and Wilcoxon test. Multivariate analysis was run using all variables with P < 0.1 in the univariate analysis. ARF developed in 19.0% of the patients, with 76.19% resulting in death. Main risk factors (univariate analysis) were: higher intra-operative hydration and bleeding, higher death risk by APACHE II score, logist organ dysfunction system on the first day, mechanical ventilation, shock due to systemic inflammatory response syndrome (SIRS)/sepsis, noradrenaline use, and plasma creatinine and urea levels on admission. Heart rate on admission (OR = 1.023 (1.002-1.044)), male gender (OR = 4.275 (1.340-13642)), shock due to SIRS/sepsis (OR = 8.590 (2.710-27.229)), higher intra-operative hydration (OR = 1.002 (1.000-1004)), and plasma urea on admission (OR = 1.012 (0.980-1044)) remained significant (multivariate analysis). The mortality risk factors (univariate analysis) were shock due to SIRS/sepsis, mechanical ventilation, blood stream infection, potassium and bicarbonate levels. Only potassium levels remained significant (P = 0.037). In conclusion, ARF has a high incidence, morbidity and mortality when it occurs in intensive care unit. There is a very close association with hemodynamic status and multiple organ dysfunction.

TP53 codon 72 polymorphism as a risk factor for cardiovascular disease in a Brazilian population

TP53, a tumor suppressor gene, has a critical role in cell cycle, apoptosis and cell senescence and participates in many crucial physiological and pathological processes. Identification of TP53 polymorphism in older people and age-related diseases may provide an understanding of its physiology and pathophysiological role as well as risk factors for complex diseases. TP53 codon 72 (TP53:72) polymorphism was investigated in 383 individuals aged 66 to 97 years in a cohort from a Brazilian Elderly Longitudinal Study. We investigated allele frequency, genotype distribution and allele association with morbidities such as cardiovascular disease, type II diabetes, obesity, neoplasia, low cognitive level (dementia), and depression. We also determined the association of this polymorphism with serum lipid fractions and urea, creatinine, albumin, fasting glucose, and glycated hemoglobin levels. DNA was isolated from blood cells, amplified by PCR using sense 5'-TTGCCGTCCCAAGCAATGGATGA-3' and antisense 5'-TCTGGGAAGGGACAGAAGATGAC-3' primers and digested with the BstUI enzyme. This polymorphism is within exon 4 at nucleotide residue 347. Descriptive statistics, logistic regression analysis and Student t-test using the multiple comparison test were used. Allele frequencies, R (Arg) = 0.69 and P (Pro) = 0.31, were similar to other populations. Genotype distributions were within Hardy-Weinberg equilibrium. This polymorphism did not show significant association with any age-related disease or serum variables. However, R allele carriers showed lower HDL levels and a higher frequency of cardiovascular disease than P allele subjects. These findings may help to elucidate the physiopathological role of TP53:72 polymorphism in Brazilian elderly people.

Contribution of CD4+ T cells to the early mechanisms of ischemia- reperfusion injury in a mouse model of acute renal failure

Renal ischemia-reperfusion (IR) injury is the major cause of acute renal failure in native and transplanted kidneys. Mononuclear leukocytes have been reported in renal tissue as part of the innate and adaptive responses triggered by IR. We investigated the participation of CD4+ T lymphocytes in the pathogenesis of renal IR injury. Male mice (C57BL/6, 8 to 12 weeks old) were submitted to 45 min of ischemia by renal pedicle clamping followed by reperfusion. We evaluated the role of CD4+ T cells using a monoclonal depleting antibody against CD4 (GK1.5, 50 µ, ip), and class II-major histocompatibility complex molecule knockout mice. Both CD4-depleted groups showed a marked improvement in renal function compared to the ischemic group, despite the fact that GK1.5 mAb treatment promoted a profound CD4 depletion (to less than 5% compared to normal controls) only within the first 24 h after IR. CD4-depleted groups presented a significant improvement in 5-day survival (84 vs 80 vs 39%; antibody treated, knockout mice and non-depleted groups, respectively) and also a significant reduction in the tubular necrosis area with an early tubular regeneration pattern. The peak of CD4-positive cell infiltration occurred on day 2, coinciding with the high expression of ßC mRNA and increased urea levels. CD4 depletion did not alter the CD11b infiltrate or the IFN-g and granzyme-B mRNA expression in renal tissue. These data indicate that a CD4+ subset of T lymphocytes may be implicated as key mediators of very early inflammatory responses after renal IR injury and that targeting CD4+ T lymphocytes may yield novel therapies.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Hypomagnesemia is a risk factor for nonrecovery of renal function and mortality in AIDS patients with acute kidney injury

The objective of the present study was to determine the prevalence of electrolyte disturbances in AIDS patients developing acute kidney injury in the hospital setting, as well as to determine whether such disturbances constitute a risk factor for nephrotoxic and ischemic injury. A prospective, observational cohort study was carried out. Hospitalized AIDS patients were evaluated for age; gender; coinfection with hepatitis; diabetes mellitus; hypertension; time since HIV seroconversion; CD4 count; HIV viral load; proteinuria; serum levels of creatinine, urea, sodium, potassium and magnesium; antiretroviral use; nephrotoxic drug use; sepsis; intensive care unit (ICU) admission, and the need for dialysis. Each of these characteristics was correlated with the development of acute kidney injury, with recovery of renal function and with survival. Fifty-four patients developed acute kidney injury: 72% were males, 59% had been HIV-infected for >5 years, 72% had CD4 counts <200 cells/mm³, 87% developed electrolyte disturbances, 33% recovered renal function, and 56% survived. ICU admission, dialysis, sepsis and hypomagnesemia were all significantly associated with nonrecovery of renal function and with mortality. Nonrecovery of renal function was significantly associated with hypomagnesemia, as was mortality in the multivariate analysis. The risks for nonrecovery of renal function and for death were 6.94 and 6.92 times greater, respectively, for patients with hypomagnesemia. In hospitalized AIDS patients, hypomagnesemia is a risk factor for nonrecovery of renal function and for in-hospital mortality. To determine whether hypomagnesemia is a determinant or simply a marker of critical illness, further studies involving magnesium supplementation in AIDS patients are warranted.