Manassés Claudino Fonteles

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.

Renal effects of supernatant from rat peritoneal macrophages activated by microcystin-LR: role protein mediators.

We have demonstrated previously that microcystin-LR promoted some renal alterations using the isolated perfused rat kidney preparation. However, these effects were not proved to be direct or indirect. The aim of the current work is to examine the renal effects promoted by supernatants from rat macrophages stimulated with microcystin-LR and the role of inflammatory mediators. Peritoneal macrophages were collected previously and were incubated for 1h in fresh medium (control) and in medium containing microcystin-LR. Dexamethasone, quinacrine, thalidomide and cycloheximide were administered 30 min before microcystin-LR. Supernatants of macrophages stimulated with or without pharmacological inhibitors were added on the perfused rat kidney model. The infusion of macrophages supernatants stimulated by microcystin-LR caused significant increases in renal vascular resistance (C: 4.93+/-0.33 vs T: 5.15+/-0.21), glomerular filtration rate (C: 0.559+/-0.008 vs T: 0.978+/-0.15) and urinary flow (C: 0.16+/-0.01 vs T: 0.23+/-0.03). Cycloheximide, quinacrine and dexamethasone blocked these effects and thalidomide blocked renal vascular resistance. Macrophages stimulated by microcystin-LR release mediators capable of promoting nephotoxicity in isolated perfused rat kidney. Phospholipase A(2), TNF-alpha and other protein mediators appear to be involved on its renal toxic mechanism.

1,8‐Cineole induces relaxation in rat and guinea‐pig airway smooth muscle

Objectives 1,8‐Cineole is a monoterpene with anti‐inflammatory, vascular and intestinal smooth muscle relaxant activity. We have evaluated the potential bronchodilatatory activity of this compound.

Purification and characterization of the biological effects of phospholipase A2 from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BcPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BcPLA(2)1 (20 microg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.

Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom

Crotalus durissus cascavella is a snake that is usually found in the scrublands of northeast Brazil. The components of its venom may have effects on the vascular and renal systems. Recently, a new bradykinin inhibitory peptide has been identified in the venom of the Crotalinae family. The aim of the present study was to investigate the renal and vascular effects of the natriuretic peptide isolated from the venom of Crotalus durissus cascavella (NP2_Casca). The chromatographic profile showed the fractionation of substances identified as convulxin, gyroxin, crotoxin and crotamine, as well as fractions V and VI. The electrophoretic profile of fraction V consisted of several bands ranging from approximately 6kDa to 13kDa, while fraction VI showed only two main electrophoretic bands with molecular weights of approximately 6 and 14kDa. Reverse-phase chromatography showed that NP2_Casca corresponds to about 18% of fraction VI and that this fraction is the main natriuretic peptide. NP2_Casca was compared to other natriuretic peptides from other sources of snake venom. All amino acid sequences that were compared showed a consensus region of XGCFGX, XLDRIX and XSGLGCX. The group treated with NP2_Casca showed an increase in perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. The percent of total and proximal tubular transport of sodium was reduced significantly after administration of the peptide. The mean arterial pressure showed a dose-dependent decrease after infusion of NP2_Casca, and an increase in nitrite production. In the aortic ring assay, NP2_Casca caused a relaxant effect in endothelium-intact thoracic aortic rings precontracted with phenylephrine in the presence and absence of isatin. NP2_Casca failed to relax the aortic rings precontracted with an isosmotic potassium Krebs-Henseleit solution. In conclusion, the natriuretic peptide isolated from Crotalus durissus cascavella venom produced renal and vascular effects. NP2_Casca reduced total and proximal sodium tubular transport, leading to an increase in sodium excretion, thereby demonstrating a diuretic action. A hypotensive effect was displayed in an arterial pressure assay, with an increase in nitrite production, suggesting a possible vasoactive action.

Effects of Thalassophryne nattereri fish venom in isolated perfused rat kidney.

Thalassophryne nattereri, popularly known as Niquim, is a venomous fish responsible for many accidents in fishermen in the Northeast of Brazil. The effects of T. nattereri venom on renal physiology has not been tested. Isolated kidneys from Wistar rats of 240-280 g weight were perfused with Krebs-Henseleit solution containing 6g% of previously dialyzed bovine serum albumin. The effects of Niquim venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). The venom of T. nattereri (0.3, 1.0, and 3.0 microg/ml) was always added to the system 30 minutes after the beginning of each experiment (n=6). All experiments were preceded by 30 minutes internal control period and an external control group, where kidneys were perfused with only Krebs-Henseleit solution. All three doses tested promoted increases in PP and RVR. The first two doses also increased GFR and UF. The higher dose promoted decreases in GFR, UF, %TNa(+), %TK(+), %TCl(-). In the treated groups we observed hyalin casts inside all tubules and proteinaceous material in the urinary space. We conclude that the effects resulted from niquim venom agents that promoted a direct effect in kidney cells causing the release of vasoactive factors.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze–thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY‐N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin‐4, tetrodotoxin and brevetoxin‐2, as well as several secretory phospholipase A2 were identified. PHY‐N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY‐N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 μ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY‐N evoked a relaxation response, which was blocked with either L‐NG‐Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY‐N (1, 3 and 10 μg ml−1) induced an increase in tonus of the biventer–cervicis neuromuscular preparation that was blocked with pre‐treatment of galamine (10 μ m). Administration of 6 mg kg−1 PHY‐N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY‐N induces nerve depolarization and nonspecifically increases neurotransmitter release. Copyright

Antivenom action on renal effects induced by Thalassophryne nattereri venom

Thalassophryne nattereri (niquim) is a venomous fish responsible for numerous accidents involving fishermen in northern and northeastern Brazil. The aim of the present investigation was to evaluate the action of antivenom on renal effects caused by Thalassophryne nattereri venom. Isolated kidneys of Wistar rats were perfused with a previously dialyzed Krebs-Henseleit solution containing 6 g% bovine serum albumin. The antivenom action was studied through perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). The niquim venom (1 µg/mL), the antivenom alone (1 µg/mL) or the venom incubated with antivenom were added to the system 30 minutes after the beginning of each perfusion. Previous works have shown venom induced-alterations of renal function parameters. In the isolated rat kidney, T. nattereri venom (1 µg/mL) increased the perfusion pressure and renal vascular resistance at 60, 90 and 120 minutes. UF and GFR also increased at 60, 90 and 120 minutes when compared with the control group; however, no effects were observed on the percent of sodium (%TNa+control = 81.1 ±0.86; %TNa+60 = 78.04 ±1.18; %TNa+90 = 76.16 ±3.34; %TNa+120 = 79.49 ±0.87) and potassium (%TK+control = 72.29 ±1.12; %TK+60 = 75.41 ±0.65; %TK+90 = 71.23 ±2.55; %TK+120 = 76.62 ±1.04) tubular transport. The administration of the antivenom (1 µg/mL) incubated with venom (1 µg/mL) reduced the changes in PP, RVR, UF and GFR provoked by Thalassophryne nattereri venom. The group perfused with venom alone showed a moderate deposit of a proteinaceous material in the tubules and urinary space. The group perfused with the antivenom presented similar results to the control group. In conclusion, the antivenom was able to decrease the effects induced by T. nattereri venom in isolated rat kidney.

High Salt Intake Promotes Different Responses to Urodilatin and Uroguanylin in the Isolated Rat Kidney

Urodilatin (UD) and uroguanylin (UGN) have been implicated in the regulation of salt and water homeostasis, particularly in the balance handling of salt intake. In this sense, the aim of the present work was to study the main effects of these peptides in kidneys from animals subjected to high NaCl (2%) intake, during 10 days in metabolic cages. The control group received only normal water, whereas the treated group drank 2% solution of NaCl (NaCl 2%). In addition, we studied effect of subthreshold UD (0.14 nM) and UGN (0.06 μM) doses in NaCl 2% after a 30-min control period. Kidney perfusion was performed with Krebs-Henseleit containing 6 g% bovine albumin previously dialyzed. The effects of UD (0.14 nM) promoted reduction of PP, RVR, and UF in the NaCl 2% group. We also observed an increase in %TNa+ and %TCl-. The main effects of UGN in NaCl 2% were increase in PP, UF, and GFR, followed by a reduction in %TNa+ and %TCl-. After an increased intake of salt, physiological pathways are activated and regulated in order to eliminate excess sodium. In this study, we observed that in a subthreshold dose, UD does not promotes natriuresis and diuresis, suggesting that UGN is an important hormone in inducing salt excretion in a chronic salt overload. Therefore, the effects herein described may play a contributory role in the regulation of kidney function after ingestion of salty meals.