Antônio Rafael Coelho Jorge

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa(+)) at 120 min and of chloride (%TCl(-)) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC(50) of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca(2+) in a concentration dependent manner, indicating that Ca(2+) may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism.

High-salt intake primes the rat kidney to respond to a subthreshold uroguanylin dose during ex vivo renal perfusion.

In a variety of animal models, uroguanylin causes diuresis, natriuresis and kaliuresis and is found in larger concentrations in the urine compared to controls after oral salt intake or in conditions of excess salt and fluid retention. It has been proposed that uroguanylin functions as an intestinal natriuretic hormone following intake of meals high in salt content. In the present work, we examined if 10 days of salt ingestion resulted in an enhanced response to uroguanylin in the isolated perfused rat kidney. Rats were given normal water, 1% NaCl (HS1%), or 2% NaCl (HS2%) for 10 days, at which time the right kidneys were surgically removed and perfused with a modified Krebs-Henseleit solution for 30 min. After a 30-min control period, the kidneys were perfused with a modified Krebs-Henseleit solution containing 0.06 microM uroguanylin for an additional 90 min. Compared to vehicle-matched time controls, 0.06 microM uroguanylin perfusion of kidneys from rats maintained on HS2% resulted in a significantly increased urine flow (UF; from 0.17+/-0.01 to 0.23+/-0.01, after 60 min, n=6, P<0.05), fractional Na(+) excretion (%E(Na+); from 16.6+/-0.7 to 30+/-2, after 60 min, n=6, P<0.05), fractional K(+) excretion (%E(K+); from 20.5+/-0.58 to 37.4+/-2.1, after 60 min, n=6, P<0.05), and fractional Cl(-) excretion increased from 18.16+/-0.52 to 35.2+/-2.0 at 60 min, n=6, P<0.05. With the exception of a significant increase in the %E(K)(+), no other effect was observed in the kidneys from the rats maintained on HS1%, and no significant effects were seen in those that were maintained on normal water. The effect of a higher dose (0.6 microM) of uroguanylin on urinary flow, sodium or potassium excretion was also significantly increased by 2% NaCl (HS2%) treatment (P<0.05). We also observed an expressive upregulation of the GC-C and a slight downregulation of the GC-A receptor in high-salt treated rats. These data demonstrate that prolonged salt ingestion primes the kidney to enhanced renal responses to uroguanylin.

Towards a better understanding of Ipomoea asarifolia toxicity: evidence of the involvement of a leaf lectin.

Natural intoxication of livestock by ingestion of Ipomoea asarifolia leaves has been reported to occur widely in Brazil. Previous studies carried out by our research group provided strong evidence that a lectin could be involved with the toxic properties of I. asarifolia. To reinforce this hypothesis, a lectin-enriched fraction (LEF) was isolated from I. asarifolia leaves and its toxic effects were assessed. Leaves of I. asarifolia were excised from plants growing widely in the field, mechanically wounded and maintained in a chamber at 25 ± 3 °C for 72h in the dark, under near 100% relative humidity. The leaf proteins were extracted, ammonium sulfate precipitated, chromatographed on DEAE-cellulose and Phenyl-Sepharose to produce LEF that under SDS-PAGE showed a molecular mass of 44.0 kDa and after N-terminal amino acid analysis a primary sequence composed of AGYTPVLDIGAEVLAAGEPY. The in vivo toxicity of LEF assessed by intraorbital injection in mice showed induced severe uncoordinated movements without death. LEF reduced the muscular contraction in a dose depend way and at 29.8 μg/mL (CE(50)) it produces 50% inhibition of contraction, suggesting that LEF blunts autonomic neurotransmission. Isolated rat kidneys were perfused with LEF and no effects on the perfusion pressure or renal vascular resistance were observed, but urinary flow and glomerular filtration rate increased. Moreover, the percentage of tubular transport of Na(+), K(+) and Cl(-) decreased. Histological examination of the kidneys perfused with LEF exhibited little alterations. These toxic effects observed above were concomitant with the increase of LEF hemagglutination activity, which strongly suggest that one of the toxic principles of I. asarifolia is a lectin present in its leaves.

l-amino acid oxidase from Bothrops marajoensis causes nephrotoxicity in isolated perfused kidney and cytotoxicity in MDCK renal cells.

Renal alterations caused by Bothrops venom and its compounds are studied to understand these effects and provide the best treatment. Previously, we studied the renal effect of the whole venom of Bothrops marajoensis and its phospholipase A2 (PLA2), but these effects could not to be attributed to PLA2. To continue the study, we report in this short communication the effects of l-amino acid oxidase from B. marajoensis venom (LAAOBm) on renal function parameter alterations observed in the same model of isolated perfused kidney, as well as the cytotoxic effect on renal cells. LAAOBm caused a decrease in PP, RVR, UF, GFR, %TNa(+) and %TCl(-), very similar to the effects of whole venom using the same model. We also demonstrated its cytotoxicity in MDCK cells with IC50 of 2.5 μg/mL and late apoptotic involvement demonstrated by flow cytometry assays. In conclusion, we suggested that LAAOBm is a nephrotoxic compound of B. marajoensis venom.

Bothropoides pauloensis venom effects on isolated perfused kidney and cultured renal tubular epithelial cells

Snake envenomation (Bothrops genus) is common in tropical countries and acute kidney injury is one of the complications observed in Bothrops snakebite with relevant morbidity and mortality. Here, we showed that Bothropoides pauloensis venom (BpV) decreased cell viability (IC50 of 7.5 μg/mL). Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by apoptosis and late apoptosis, through caspases 3 and 7 activation, mitochondrial membrane potential collapse and ROS overproduction. BpV reduced perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, percentage of sodium, chloride or potassium tubular transportation. These findings demonstrated that BpV cytotoxicity on renal epithelial cells might be responsible for the nephrotoxicity observed in isolated kidney.

Study of the safety of methylphenidate: Focus on nephrotoxicity aspects.

AIMS Methylphenidate (MPD) is increasingly prescribed for the treatment of Attention Deficit Hyperactivity Disorder and there are concerns about its appropriate use. Furthermore, little is known about the potential nephrotoxicity in patients using MPD. This study aimed to investigate the safety of MPD, with focus on the possible effects of this drug on renal function. MAIN METHODS We investigated the effects of MPD on renal perfusion system and renal tubular cells through in vivo and in vitro experimental models. KEY FINDINGS In the in vivo experiments, 24 h and 48 h after MPD administration, urea, creatinine, creatinine clearance, and the fractional excretion of sodium and potassium were not changed. In the isolated kidney perfusion, MPD significantly reduced urinary flow, glomerular filtration rate and the percentage of tubular sodium transport. However, the perfusion pressure, renal vascular resistance and the percentage of tubular potassium transport were unchanged in this system. In the canine renal epithelial cell line MDCK culture, MPD was not cytotoxic and, in histopathological analysis, MPD did not promote alterations. SIGNIFICANCE Our findings suggest a possible nephrotoxic effect of MPD, since it altered renal function by reducing the glomerular activity, urinary flow and sodium transport. These effects need to be further investigated in order to minimize potential harms associated with the use of MPD.

Differences between renal effects of venom from two Bothrops jararaca populations from southeastern and southern Brazil

ABSTRACT Components from animal venoms may vary according to the snakes age, gender and region of origin. Recently, we performed a proteomic analysis of Bothrops jararaca venom from southern (BjSv) and southeastern (BjSEv) Brazil, showing differences in the venom composition, as well as its biological activity. To continue the study, we report in this short communication the different effects induced by the BjSEv and BjSv on isolated kidney and MDCK renal cells. BjSEv decreased perfusion pressure (PP) and renal vascular resistance (RVR) and increased urinary flow (UF) and glomerular filtration rate (GFR), while BjSv did not alter PP and RVR and reduced UF and GFR. Both types of venom, more expressively BjSEv, reduced %TNa+, %TK+ and %Cl−. In MDCK cells, the two types of venom showed cytotoxicity with IC50 of 1.22 &mgr;g/mL for BjSv and 1.18 &mgr;g/mL for BjSEv and caused different profiles of cell death, with BjSv being more necrotic. In conclusion, we suggest that BjSv is more nephrotoxic than BjSEv. HighlightsBothrops jararaca venoms from south and southeast Brazil have different renal effects.B. jararaca venom (Bjv) from south is more nephrotoxic than from Bjv from southeast.Bjv from south showed characteristics of renal injury on isolated kidney.Bjv from southeast showed a diuretic effect on isolated kidney.Bjv from south showed a higher necrotic profile than Bjv from southeast in MDCK cells.

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.