Helena Serra Azul Monteiro

Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney

Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 μM) increased glomerular filtration rate from 0.77 ± 0.07 to 1.34 ± 0.3 ml ⋅ g-1 ⋅ min-1at the highest concentration. A maximal increase in Na+ excretion was achieved at 0.66 μM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 ± 1.7 to 58.8 ± 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested ( P< 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 μM) decreased fractional Na+ reabsorption from 73.9 ± 2.4 to 64.5 ± 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 μM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 microM) increased glomerular filtration rate from 0.77 +/- 0.07 to 1.34 +/- 0.3 ml . g-1 . min-1 at the highest concentration. A maximal increase in Na+ excretion was achieved at 0. 66 microM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 +/- 1.7 to 58.8 +/- 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested (P < 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 microM) decreased fractional Na+ reabsorption from 73. 9 +/- 2.4 to 64.5 +/- 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 microM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and l-amino acid oxidase

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A(2) and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA(2) (BmarPLA(2)) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA(2) was isolated from the venom, and N-terminal amino acid sequencing of sPLA(2) showed high amino acid identity with other lysine K49 sPLA(2)s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC=50 microg/mL and MLC=200 microg/mL. However, the BmarTV and BmarPLA(2) did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC(50)=2.55 microg/mL and 2.86 microg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC(50) of 86.56 microg/mL for L. amazonensis and 79.02 microg/mL for L. chagasi. BmarPLA(2) did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.

Antinociceptive and anti-inflammatory activities of a sulfated polysaccharide isolated from the green seaweed Caulerpa cupressoides

BACKGROUND Red and brown algae sulfated polysaccharides (SPs) have been widely investigated as antinociceptive and/or anti-inflammatory agents; however, no description of these biological properties concerning green algae SPs have been reported. Caulerpa curpressoides (Chlorophyta) presents three SPs fractions (Cc-SP1, Cc-SP2, and Cc-SP3). Anticoagulant (in vitro) and anti- and pro-thrombotic (in vivo) effects of Cc-SP2 had been recently reported. We evaluated the effects of Cc-SP2 using models of nociception and acute inflammation in vivo. METHODS Male Swiss mice received Cc-SP2 (iv) 30 min prior to receiving 0.6% acetic acid (10 ml/kg, ip), 1% formalin (20 μl, sc) or were subjected to thermal stimuli (51 ± 1 °C). Cc-SP2 was injected sc to male Wistar rats in a peritonitis model or a paw edema model using carrageenan (ip or ipl, 500 μg). To analyze the systemic effects, Cc-SP2 (27 mg/kg, sc) was administrated to both genders mice before waiting for 14 days. RESULTS Cc-SP2 (3, 9 or 27 mg/kg) reduced (p < 0.05) the number of writhes induced by acetic acid by 57, 89.9 and 90.6%, respectively, the licking time in the first (9 or 27 mg/kg with 42.47 and 52.1%, respectively) and the second (3, 9 or 27 mg/kg with 68.95, 82.34 and 84.61%, respectively) phases. In the hot-plate test, the antinociceptive effect of Cc-SP2 (9 mg/kg) was primarily observed at 60 min (26.7 ± 1.2 s), with its effect reversed by naloxone (8.6 ± 1.3 s), suggesting the involvement of the opioid system. Cc-SP2 (3, 9 or 27 mg/kg, sc, p < 0.05) showed anti-inflammatory effects by decreasing neutrophils migration by 64, 69 and 73%, respectively, and potently reduced the paw edema, especially at the second (0.16 ± 0.02, 0.16 ± 0.03 and 0.12 ± 0.05 ml) and third (0.16 ± 0.03, 0.18 ± 0.02 and 0.14 ± 0.04 ml) hours, respectively. Cc-SP2 did not cause hepatic or renal alterations or affect body mass or the macroscopy of the organs examined (p > 0.05). Histopathological analyses of the liver and kidney showed that both organs were affected by Cc-SP2 treatment, but these effects were considered reversible. CONCLUSION The results indicate that the analgesic and anti-inflammatory effects of Cc-SP2 could be of biomedical applicability as a new, natural tool in pain and acute inflammatory conditions.

Effects of microcystin-LR in isolated perfused rat kidney

Microcystin is a hepatotoxic peptide which inhibits protein phosphatase types 1 and 2A. The objective of the present study was to evaluate the physiopathologic effects of microcystin-LR in isolated perfused rat kidney. Adult Wistar rats (N = 5) of both sexes (240-280 g) were utilized. Microcystin-LR (1 microg/ml) was perfused over a period of 120 min, during which samples of urine and perfusate were collected at 10-min intervals to determine the levels of inulin, sodium, potassium and osmolality. We observed a significant increase in urinary flow with a peak effect at 90 min (control (C) = 0.20 +/- 0.01 and treated (T) = 0.32 +/- 0.01 ml g-1 min-1, P<0.05). At 90 min there was a significant increase in perfusate pressure (C = 129.7 +/- 4.81 and T = 175.0 +/- 1.15 mmHg) and glomerular filtration rate (C = 0.66 +/- 0.07 and T = 1.10 +/- 0. 04 ml g-1 min-1) and there was a significant reduction in fractional sodium tubular transport at 120 min (C = 78.6 +/- 0.98 and T = 73.9 +/- 0.95%). Histopathologic analysis of the perfused kidneys showed protein material in the urinary space, suggestive of renal toxicity. These data demonstrate renal vascular, glomerular and urinary effects of microcystin-LR, indicating that microcystin acts directly on the kidney by probable inhibition of protein phosphatases.

Epidemiologia dos acidentes por serpentes peçonhentas no Estado do Ceará - Brasil

From 1992 to 1995, 688 accidents by venomous snakes (mean of 192 cases/year) have been notified to the Health Ministry of the State of Ceara, with an incidence between 0.9 and 5.8/100.000 inhabitants. Among 473 cases, 88.3% were of the genus Bothrops, 10.7% Crotalus, 0.8% Micrurus and 0.2% Lachesis. The highest incidence occurred from April to September. Male (75.6%) predominated with ages from 10 to 49 years old (72.3%). The more frequently bitten anatomical region were the lower limbs (81.9%) and upper limbs (14.7%). The attendance at health unit which notified the accident took place within 6 hours in 66.9% of the cases. Lethality was 0.7%. The afflicted people were mainly peasants (62.7%), and most of the accidents took place in their own work place. The authors emphasize that the snake bites in the State of Ceara may be considered work accidents, concern mainly peasants and constitute a cause of death.

Wasting and intestinal barrier function in children taking alanyl-glutamine-supplemented enteral formula.

Objective: We examined the effect of a diet supplemented with alanyl-glutamine (AG) or placebo glycine (G) on intestinal barrier function and growth in children in northeastern Brazil. Patients and Methods: One hundred seven children ages 7.9 to 82.2 months with a weight-for-age (WAZ), height-for-age (HAZ), or weight-for-height (WHZ) z-score less than −1 were studied. From July 2003 to November 2004, 51 study patients received AG (24 g/d) and 56 received G (25 g/d; isonitrogenic concentration) control for 10 days. Lactulose/mannitol excretion ratio was used as a measure of intestinal permeability and was performed on days 1 and 10 of nutritional supplementation. Weight and height were measured on days 1, 10, 30, and 120 of the protocol. Results: The patients were similar on admission with regard to age, sex, birth weight, nutritional status, lactulose/mannitol ratio, and serum concentrations of glutamine and arginine. The percentage of lactulose urinary excretion significantly improved (decreased) in children receiving AG for 10 days but not in those receiving glycine controls. AG significantly increased cumulative change over 120 days in WHZ and WAZ scores but not HAZ scores after adjustment for age and season in comparison with the placebo glycine group. Conclusions: Children tolerated AG-supplemented enteral formula well, and it significantly improved cumulative WHZ and WAZ over 120 days in comparison with children in the placebo glycine group. The data also suggested a beneficial effect of AG in the barrier function paracellular pathway, albeit with reduced mannitol excretion. Thus, although the effect of AG on reduced mannitol concentration requires clarification, AG appears to improve nutrition and barrier function.

Actions of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney

Many studies have reported the occurrence of lethal acute renal failure after snakebites. The aim of the present investigation was to determine alterations in renal function produced by Crotalus durissus terrificus venom and crotoxin as well as the histological alterations induced by these venoms. Isolated kidneys from Wistar rats weighing 240 to 280 g were perfused with Krebs-Henseleit solution containing 6 g% of previously dialyzed bovine serum albumin. The effects of Crotalus durissus terrificus venom and crotoxin were studied on glomerular filtration rate (GFR), urinary flow (UF), perfusion pressure (PP) and percentage sodium tubular transport (%TNa+). The infusion of Crotalus durissus terrificus venom (10 microg/ml) and crotoxin (10 microg/ml) increased GFR (control80 = 0.78 +/- 0.07, venom80 = 1.1 +/- 0.07, crotoxin80 = 2.0 +/- 0.05 ml g(-1) min(-1), P<0.05) and UF (control80 = 0.20 +/- 0.02, venom80 = 0.32 +/- 0.03, crotoxin80 = 0.70 +/- 0.05 ml g(-1) min(-1), P<0.05), and decreased %TNa+ (control100 = 75.0 +/- 2.3, venom100 = 62.9 +/- 1.0, crotoxin80 = 69.0 +/- 1.0 ml g(-1) min(-1), P<0.05). The infusion of crude venom tended to reduce PP, although the effect was not significant, whereas with crotoxin PP remained stable during the 100 min of perfusion. The kidneys perfused with crude venom and crotoxin showed abundant protein material in the urinary space and tubules. We conclude that Crotalus durissus terrificus venom and crotoxin, its major component, cause acute nephrotoxicity in the isolated rat kidney. The current experiments demonstrate a direct effect of venom and crotoxin on the perfused isolated kidney.

Effects of Crotalus durissus cascavella venom in the isolated rat kidney

Crotalus durissus cascavella (C.d.c) is a snake usually found in scrubland of Brazilian Northeast and its bite constitutes an important public health problem. Isolated kidneys from wistar rats, weighing 240 to 280 g, were perfused with Krebs Henseleit solution containing 6 g% of previously dialysed bovine serum albumin. The effects of C.d.c venom were studied on the perfusion pressure (PP), urinary flow (UF), glomerular filtration rate (GFR), percent of sodium tubular transport (%TNa+) and percent of proximal tubule sodium transport (%pTNa+). All experiments were preceded by a 30 min internal control period and an external control group. The infusion of C.d.c (10 microg ml(-1)) increased the PP, UF at 60 and 90min of perfusion, and decreased the GFR, %TNa+ and %pTNa+ at 120 min of perfusion. The proximal renal tubule was the major site for this toxic effect. In the group treated with the venom we found hyalin cylinders inside all tubules and proteinaceous material, alternating from moderate to intense presence in urinary space. Dexamethasone (Dexa 20 microg ml(-1)) protected against the increase in PP, UF, and against the decrease in GFR, it produced the reversion of the effect also in %TNa+ and %pTNa+. Indomethacin (Indo 10 microg ml(-1)) antagonized the effect observed in PP and UF, but was not able to reverse the changes in GFR, %TNa+ and %pTNa+. Nifedipine (Nif 10 microg ml(-1)) promoted a reversion of almost all functional changes, except the %pTNa+ was not reversed. We conclude that these alterations may be caused by a direct action of the venom on the kidneys and indirectly by the release of mediators from endothelial cells. Dexa protected against renal lesions caused by the venom, perhaps by inhibiting phospholipase A2 a toxic component of the venom. The reversion partially induced by indo may be due to cyclooxygenase inhibition that will inhibit the formation of prostaglandins. Nif blocked the renal alterations that may involve cell calcium influx that resulted from the venom aggression.

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.

The role of phospholipase A2 and cyclooxygenase in renal toxicity induced by microcystin-LR

We have shown previously that exposure to microcystin-LR (MCLR) causes renal toxic effects in isolated perfused rat kidney. That study was extended further to approach the perspective of pharmacological blockade of renal toxic effects by MCLR through the use of experimental therapeutic agents. An isolated kidney perfusion system was utilized and samples of urine and perfusate were collected at 10min intervals to determine the levels of inulin, sodium, potassium and osmolality. Dexamethasone (20microg ml(-1)) and indomethacin (10microg ml(-1)) were administered in the beginning of the perfusion and MCLR was employed in a dose of 1microg ml(-1) after an internal control of 30min to evaluate the perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR) and urinary flow (UF). Dexamethasone and indomethacin antagonized the toxic effects of MCLR on PP, RVR, GFR and UF. Histologic analysis of dexamethasone and indomethacin treated groups did not show any vascular or interstitial alterations. MCLR potentially impairs the renal function, probably causing vascular and glomerular lesions and, promoting renal alterations through direct or indirect actions. These data seem to indicate that the renal alterations promoted by MCLR involves also phospholipase A(2) and arachidonic acid-derived mediators.