Angiogenesis in cardiopulmonary dirofilariosis: does the Wolbachia surface protein have a pro- or anti-angiogenic effect?

Cardiopulmonary dirofilariosis caused by Dirofilaria immitis produces inflammation, blood vessel obstruction and hypoxia, which are required conditions for the beginning of the process of neovascularization. Since D. immitis harbours intracellular symbiotic Wolbachia bacterium, the global understanding of the angiogenic process requires the analysis of the effect of the parasite molecules, but also that of Wolbachia. Canine primary lung microvascular endothelial cells were treated with the recombinant Wolbachia surface protein (rWSP) and the expression of angiogenic factors like Vascular Endothelial Growth Factor-A (VEGF-A), sFlt, membrane Endoglin (mEndoglin) and soluble Endoglin (sEndoglin), as well as the in vitro formation of pseudocapillaries, were measured. The analyses showed a significant increase in the expression of pro-angiogenic VEGF-A and anti-angiogenic sEndoglin, together with a significant decrease in both pro-angiogenic mEndoglin and pseudocapillary formation, compared to untreated controls. Due to the complexity of the angiogenic process and its relationship with other physiological processes like inflammation and fibrinolysis, these results might suggest that rWSP participate in various mechanisms related to each other and its effects might depend either on the balance between them or on the moment of their occurrence.

Vascular endothelial cell activation by adult Dirofilaria immitis antigens.

Dirofilaria immitis is the causal agent of cardiopulmonary dirofilariosis (heartworm disease). Adult worms lodge in the pulmonary arteries and right ventricle, thus vascular endothelium is exposed to high concentrations of Dirofilaria antigenic products. Heartworm disease habitually develops as a chronic foreseeable pathology. Moreover, the simultaneous death of many adult worms, naturally or induced by a filaricide treatment, can cause acute thromboembolisms and endarteritis. To better understand the effects of the massive death of D. immitis adult worms on the blood vessel endothelium, we cultured vascular endothelial cells in the presence or absence of an antigenic extract of D. immitis adult worms (DiSA). The parasite products increased the expression of enzymes and the synthesis of eicosanoids related to inflammation, such as COX-2, 5-LO, PGE(2) and LTB(4). The expression of ICAM-1 and PECAM-1 adhesion molecules and endothelial and inducible Nitric Oxide Synthases (eNOS and iNOS) was also increased in cultures treated with DiSA. Nevertheless, DiSA decreased endothelial permeability and does not alter both proliferation and apoptosis. These results suggest that the somatic extract of D. immitis adult worms stimulate inflammatory mechanisms in endothelial cells, without altering their basic physiologic processes.

Dirofilaria immitis and Wolbachia-derived antigens: its effect on endothelial mammal cells.

Antigens of both Dirofilaria immitis and Wolbachia symbiont bacteria are implicated in the inflammatory pathology of heartworm infection. The aim of the present study was to compare the stimulatory capacity of in vitro cultures of vascular endothelial cells by the adult somatic antigens of D. immitis (DiSA) and the recombinant form of the Wolbachia surface protein (rWSP), during the first 24h of stimulation. Our results indicate a different stimulatory activity of the two antigens. Both the DiSA and rWSP stimulate the production of the enzymes responsible of the arachidonic acid metabolism, cyclooxygenase-2, 5-lipoxygenase (5-LO), and leukotriene B4. Only DiSA stimulates the production of prostaglandin E2. Related to the adhesion molecules, the DiSA stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas rWSP stimulates ICAM-1, PECAM-1, and vascular cell adhesion molecule-1 (VCAM-1). Expression of E-cadherin and vascular endothelial growth factor also were stimulated by rWSP. Neither of the two antigens altered the basic physiological mechanisms of endothelial cells, such as cell proliferation, cell cycle, or apoptosis. The biological and pathological significance of these finding are discussed.

Teaching integrative physiology using the quantitative circulatory physiology model and case discussion method: evaluation of the learning experience.

One of the problems that we have found when teaching human physiology in a Spanish medical school is that the degree of understanding by the students of the integration between organs and systems is rather poor. We attempted to remedy this problem by using a case discussion method together with the Quantitative Circulatory Physiology (QCP) program. QCP is a Windows-based computer simulation program that offers almost real-time simulation and allows users to examine the time-dependent interactions of over 750 parameters. We evaluated students' perceptions by an anonymous questionnaire. Teachers' perceptions of this teaching approach were highly positive, as it improved students' perceptions of the complexity of biological processes, their ability to differentiate between acute and chronic responses, and promoted an integrative understanding of human body function. Teachers also identified some problems with the approach, including student difficulties in adopting self-directed learning, a lack of precision in student questions during the discussion sessions, and the lack of a tradition of using several textbooks to explain the changes observed. The results of the student questionnaire revealed that >70% of the students reported that this type of learning gave them a better understanding of the complexity of physiological processes and the role of coordinated actions of several systems in the homeostatic response and enabled them to acquire a better understanding of human body functions. Thus, we conclude that this approach promotes an integrative understanding of cardiovascular and renal functions that is difficult to achieve with other methods.

Changes in the levels of eicosanoids in cats naturally and experimentally infected with Dirofilaria immitis.

Feline heartworm (Dirofilaria immitis) infection is a severe, life-threatening disease. The eicosanoids are lipid mediators derived from the metabolism of the arachidonic acid, involved in the regulation of the immune response and of inflammatory reactions. In this study, naturally infected cats showed significant higher levels of prostaglandin E(2) (PGE2), thromboxane B(2) (TXB(2)) and leukotriene B(4) (LTB4) than uninfected cats. Changes in the levels of eicosanoids during the infection were observed in experimentally infected cats. PGE2 increased significantly during the first 60 days post-infection, then progressively decreased until day 180 post-infection. At this time, PGE2 values are still significantly higher than those observed before the infection. TxB2 and LTB4 increased progressively from the beginning of infection and reached their maximum levels 180 days post-infection. In experimentally infected, ivermectin-treated cats, 15 days after treatment (45 days after infection) both PGE2 and LTB4 levels were similar to those observed in experimentally infected, untreated cats. No significant differences of PGE2 levels were found before the infection and at the end of the experiment (165 days post-treatment, 195 days post-infection). Increased levels of LTB4 were found 15 days post-treatment, afterward they progressively decreased. These data show that D. immitis infection influences the production of intravascular eicosanoids in cats. The high levels of PGE2 observed in the early phase of infection could be related to the survival of the worms, while those of TxB2 and LTB4 detected at the end of the study could mediate the inflammatory reactions and thrombi formation during the feline dirofilariosis.

5-HT2 receptor blockade exhibits 5-HT vasodilator effects via nitric oxide, prostacyclin and ATP-sensitive potassium channels in rat renal vasculature.

The aim of this study was to determine whether orally sarpogrelate (selective 5-HT2 antagonist) treatment (30 mg/kg/day; 14 days) could modify 5-HT renal vasoconstrictor responses, characterizing 5-HT receptors and mediator mechanisms involved in serotonergic responses in the in situ autoperfused rat kidney. Intra-arterial (i.a.) injections of 5-HT (0.00000125 to 0.1 µg/kg) decreased renal perfusion pressure (RPP) but did not affect the mean blood pressure (MBP). i.a. agonists 5-CT (5-HT1/7), CGS-12066B (5-HT1B), L-694,247 (5-HT1D) or AS-19 (5-HT7) mimicked renal 5-HT vasodilator effect. However, neither 8-OH-DPAT (5-HT1A) nor 1-phenylbiguanide (5-HT3) modified RPP. Moreover: (i) GR-55562 (5-HT1B antagonist) and L-NAME (nitric oxide synthase [NOS] inhibitor) blocked CGS-12066B-induced vasodilator response, (ii) LY310762 (5-HT1D antagonist) and indomethacin (non-selective cyclooxygenase inhibitor) blocked L-694,247-induced vasodilator response; (iii) SB-258719 (5-HT7 antagonist) and glibenclamide (ATP-sensitive K+ channel blocker) blocked AS-19-induced vasodilator response; and (iv) 5-HT- or 5-CT-elicited renal vasodilation was significantly blocked by the mixture of GR-55562 + LY310762 + SB-258719. Furthermore, eNOS and iNOS proteins and prostacyclin levels are overexpressed in sarpogrelate-treated rats. Our data suggest that 5-HT exerts renal vasodilator effect in the in situ autoperfused sarpogrelate-treated rat kidney, mediated by 5-HT1D, 5-HT1B and 5-HT7 receptors, involving cyclooxygenase-derived prostacyclin, nitric oxide synthesis/release and ATP-sensitive K+ channels, respectively.

Three-dimensional microcomputed tomography of renal vasculature in rats.

Current microscopic methods to view renal microvasculature reveal only a very limited portion of the total renal volume. Identification of connectivity for postglomerular vessels in the cortex and the medulla during functional states related to changes in sodium excretion will help better to understand the coupling of renal vasculature to tubular function. The purpose of this study was to investigate the possibility of visualizing the continuity of pre- and postglomerular vasculature using three-dimensional micro-computed tomography (micro-CT). Kidneys from normal rats were perfusion fixed in situ at physiological pressure, filled with latex microfil containing lead chromate, and embedded in plastic. The micro-CT scans of the intact kidneys were carried out on a rotating stage illuminated either by a synchrotron x-ray source or a conventional x-ray spectroscopy tube. Images were reconstructed by a filtered backprojection algorithm and volume-rendering techniques were utilized to display the vasculature. The reconstructed images clearly showed the large distribution vessels and the venous drainage of the kidneys, while pre- and postglomerular vessels and their vascular connections throughout the kidney were displayed in great detail. Efferent arterioles showed the characteristics of their peritubular capillary beds in the cortical and medullary regions. The vascular volume of the cortex was 27%, the outer stripe of the outer medulla 18%, the inner stripe of the outer medulla 30%, and the inner medulla 18%. In conclusion, micro-CT is a promising method to evaluate renal vascular architecture relative to physiological and pathological alterations.

Effect of hypothalamic-hypophysary inhibitory factor on mesangial cell activation.

We examined the effect of a sodium pump inhibitor isolated from bovine hypothalamus and pituitary tissues on contraction, proliferation, and calcium mobilization in primary cultures of rat mesangial cells. Hypothalamic-hypophysary inhibitory factor (HHIF) inhibited rubidium uptake in a concentration-dependent manner (0.2 U/mL: 56.8 +/- 6.3% inhibition). It also induced a concentration- and time-dependent decrease in planar cell surface area. Maximal contraction (25 +/- 5% reduction in cell size) was reached at 60 minutes with a concentration of 0.2 U/mL. This effect was inhibited by both verapamil and TMB-8 (10(-5) mol/L). HHIF was also observed to increase DNA synthesis (0.2 U/mL: 4361 +/- 168 versus 2129 +/- 162 cpm per well under control conditions) and cell proliferation (0.2 U/mL: 52,290 +/- 1931 versus 10,512 +/- 121 cells per well under control conditions). Both effects were also inhibited by verapamil and TMB-8. Moreover, HHIF induced the expression of immediate early genes c-fos and c-jun mRNA. HHIF-induced effects were accompanied by an increase in cytosolic free calcium (203 +/- 58 versus 101 +/- 2 nmol/L under control conditions), which was inhibited by verapamil and TMB-8. In summary, HHIF induces mesangial cell contraction and proliferation; these effects seem to be mediated by an increase in cytosolic free calcium levels.

Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts.

Transforming growth factor-beta (TGF-beta) plays a pivotal role in the extracellular matrix accumulation observed in chronic progressive tissue fibrosis, but the intracellular signaling mechanism by which TGF-beta stimulates this process remains poorly understood. We examined whether mitogen-activated protein kinase (MAPK) routes were involved in TGF-beta1-induced collagen expression in L(6)E(9) myoblasts. TGF-beta1 induced p38 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation whereas no effect on Jun N-terminal kinase phosphorylation was observed. Biochemical blockade of p38 but not of the ERK MAPK pathway abolished TGF-beta1-induced alpha(2)(I) collagen mRNA expression and accumulation. These data indicate that TGF-beta1-induced p38 activation is involved in TGF-beta1-stimulated collagen synthesis.

A role for platelet-activating factor in endothelin-1-induced rat mesangial cell proliferation.

Endothelin-1 stimulated the release of bioassayable platelet-activating factor-like material and the incorporation of acetate to PAF in rat mesangial cells, in a dose-dependent manner, with an EC50 of about 10(-9) M. Endothelin-1 also stimulated dose dependently [3H]thymidine incorporation into DNA, protein synthesis and cell growth. The platelet-activating factor antagonists BN-52021 (10(-5) M), and alprazolam (10(-5) M), reduced significantly endothelin-1-stimulated thymidine incorporation into DNA, protein synthesis and cell growth. Platelet-activating factor also stimulated significantly thymidine incorporation into DNA, cell proliferation and protein synthesis. In conclusion, the present results suggest that endothelin-1 induces mesangial cell proliferation through a mechanism involving among others, synthesis and release of platelet-activating factor by these cells.

Effect of felodipine on systemic hemodynamics of spontaneous mild-hypertensive aged rats.

The aim of the present study was to examine the effects of felodipine, a dihydropyridinic calcium ions channels blocker, on mean arterial blood pressure (MAP), cardiac output (CO), peripheral resistances (TPR) and blood flow distribution in spontaneously mildly hypertensive female Wistar 30-34 months old rats. Under pentobarbital anesthesia, CO and regional organ blood flow were measured by the radioactive microspheres method, before and 30 min after administration of felodipine 0.5 mumol/kg b. w. by gastric gavage. They were compared to the corresponding values in normotensive rats of the same strain and age. Fifteen (from twenty five) rats were hypertensive with a MAP averaging 139 +/- 2 mm Hg. CO and TPR were slightly higher in these hypertensive rats. Cerebral blood flow (CBF) was lower, though the difference did not reach significant values. MAP significantly decreased after felodipine with no significant changes in CO and TPR in hypertensive as well as in normotensive animals. Renal blood flow (RBF) was similar before and after felodipine which significantly decreased renal vascular resistance in both groups. Felodipine administration did not induce significant changes in CBF but a significant increase in portal venous inflow (PVI) in hypertensive rats only. In conclusion, in old female rats with mild spontaneously hypertension, acute felodipine oral administration reduced arterial blood pressure without diminishing CBF.

Adult Dirofilaria immitis excretory/secretory antigens upregulate the production of prostaglandin E2 and downregulate monocyte transmigration in an "in vitro" model of vascular endothelial cell cultures.

Canine and feline cardiopulmonary dirofilariosis caused by Dirofilaria immitis is a chronic and potentially fatal disease. Adult worms live in the pulmonary arteries of infected immunocompetent hosts for years. The aim of the present study is the identification of the influence of the metabolic products (excretory/secretory antigens, DiE/S) of D. immitis on the vascular endothelial cells, because the vascular endothelium interplays in a direct manner with the parasite and their products. For this purpose, HAAE-1 vascular endothelial cells were treated with DiE/S, using non-treated cultures as negative controls. Significant increases in the COX-2, 5-LO expression and PGE(2) level were detected in the treated cells compared with the control cells. Moreover, DiE/S decreases monocyte transmigrations across vascular endothelial cell monolayers. Treatment with DiE/S does not have a cytotoxic effect and do not alter apoptosis, necrosis or cell cycle of vascular endothelial cells. These results suggest that the DiE/S stimulates the production of mediators and mechanisms that favor the survival of the parasite, in vascular endothelial cells, contributing to restrict vascular and lung damages in the infected host, without altering the basic physiologic processes of endothelial cells.

High levels of serum thromboxane B2 are generated during human pulmonary dirofilariosis.

The canine parasite Dirofilaria immitis can infect humans. Patients with pulmonary dirofilariosis develop significantly higher thromboxane B2 levels than healthy individuals living in areas where dirofilariosis is endemic and in areas where dirofilariosis is not endemic. The possible role of Wolbachia bacteria in the appearance of this eicosanoid is discussed.

TGF-beta1 induces COX-2 expression and PGE2 synthesis through MAPK and PI3K pathways in human mesangial cells.

Transforming growth factor-beta1 (TGF-beta1) plays a fundamental role in the progression of renal diseases. Accumulating evidence has suggested that eicosanoids derived from cyclooxygenase-2 (COX-2) participate in a number of pathological processes in immune-mediated renal diseases. Mesangial cells (MC) play a major role in physiological and pathophysiological renal processes. MC express receptors for TGF-beta1, and COX-2 expression can be induced in MC. However, to date, there are no published data on the possible role of TGF-beta1 in COX-2 expression in human mesangial cells (HMC). We designed studies to determine (1) whether TGF-beta1 stimulates COX-2 expression in primary HMC, (2) whether mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades are involved in TGF-beta1-induced COX-2 expression, and (3) whether prostaglandin (PG)E2 synthesis is affected by TGF-beta1 and MAP kinases and PI3K activation. Studies were performed in primary cultures of HMC and in an immortalized line of HMC. TGF-beta1 induces COX-2 promoter activity and COX-2 mRNA and protein expression in HMC. COX-2 induction is accompanied by increased PGE2 synthesis. Extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and PI3K pathway inhibition blunted TGF-beta1-induced COX-2 overexpression. We demonstrate that TGF-beta1 regulates COX-2 expression in HMC through the activation of ERK1/2, p38 MAPK, and PI3K. These results can help to elucidate the molecular mechanisms underlying the regulation of COX-2 and open up specific strategies for the treatment of glomerular disease.

Involvement of platelet-activating factor in gentamicin nephrotoxicity in rats.

To assess whether platelet-activating factor (PAF) could be involved in gentamicin-induced nephrotoxicity, we studied the effect of PAF antagonist BN-52021 on renal function in rats after gentamicin treatment. Administration of gentamicin resulted in a progressive increase of plasma creatinine, a drop in creatinine clearance and an increase of urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) and alkaline phosphatase (AP). Rats treated with BN-52021 and injected with gentamicin showed fewer changes in plasma creatinine and creatinine clearance, but no differences in urinary excretion of NAG and AP were observed in gentamicin-treated rats. Histological examination revealed massive cortical tubular necrosis in rats treated with gentamicin, whereas in BN-5202 1-injected animals tubular damage was markedly attenuated. Glomeruli from gentamicin-treated rats produced larger amounts of PAF than glomeruli from control rats. In addition, in the group of BN-52021- and gentamicin-treated rats, glomerular PAF production was not significantly different from that of the control group. The present results suggest a role for PAF in gentamicin-induced nephrotoxicity.

Effect of platelet activating factor antagonist treatment on gentamicin nephrotoxicity.

To assess whether PAF could be involved in the gentamicin-induced nephrotoxicity, we have studied the effect of PAF antagonist BN-52021 on renal function in rats after gentamicin (GENTA) treatment. Experiments were completed in 21 Wistar rats divided into three groups: group GENTA was injected with gentamicin 100 mg kg(-1) body wt/day s.c. for 6 days. Group GENTA + BN received gentamicin and BN-52021 i.p. 5 mg kg(-1) body wt/day. A third group served as control. Rats were placed in meta-bolic cages and plasma creatinine and creatinine clearance were measured daily. GENTA group showed a progressive increase in plasma creatinine, a drop in creatinine clearance and an increase in urinary excretion of N-acetyl-beta-D-glucosaminidase and alkaline phosphatase. GENTA + BN group showed a lesser change in plasma creatinine and a creatinine clearance, but no difference with GENTA group in urinary excretion of NAG and AP were observed. Histological examination revealed a massive cortical tubular necrosis in rats treated with gentamicin, whereas in BN-52021 injected animals tubular damage was markedly attenuated. The present results suggest a role for PAF in the gentamicininduced nephro-toxicity.

Effect of ouabain and hypothalamic, hypophysary inhibitory factor on rat mesangial cell proliferation.

The mitogenic effect of a low-molecular-weight, Na+,K(+)-ATPase inhibitory factor isolated from bovine hypothalamus and hypophysis (hypothalamic hypophysary inhibitory factor, HHIF) and ouabain on cultured rat mesangial cells was examined. Ouabain induced a potent stimulation of the [3H]thymidine incorporation to DNA, ranging from threefold for a dose of 10(-7) M to seven times for 10(-5) M. The amount of proteins per well also increased in a dose-dependent way, which was already significant from basal values for the lower dose used. HHIF also induced a dose-dependent stimulation of [3H]thymidine incorporation into DNA, ranging from 1.7 times for a concentration of 0.02 U/ml to 4.5 times for the concentration of 2 U/ml. The amount of protein per well also increased with HHIF, but the increase was significant only for the higher dose used. These data allow us to conclude that HHIF could play a role in the vascular hypertrophy that is associated with the pathogenesis of hypertension.

Potential use of isolated glomeruli and cultured mesangial cells as in vitro models to assess nephrotoxicity.

The purpose of this short review is to present the potential of using isolated glomeruli and cultured mesangial cells as two different in vitro models to assess the glomerular effect of molecules with nephrotoxic properties. The advantage of using isolated renal glomeruli is that they conserve the architecture of this anatomical region of the kidney; moreover, they are free of any vascular, nervous or humoral influences derived from other regions of the kidney. Mesangial cells are perivascular pericytes located within the central portion of the glomerular tuft between capillary loops. Mesangial cells have a variety of functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, and control of glomerular hemodynamics, mainly the ultrafiltration coefficient Kf, via mesangial cell contraction or release of vasoactive hormones. Most authors agree that mesangial cells play a major role in glomerular contraction, filtration surface area, and Kf regulation. One of the major effects of toxicants on glomerular structures is contraction. We can assess quantitatively the degree of toxicant-induced mesangial cell contraction or glomerular contraction by measuring the changes in planar cell surface area or apparent glomerular cross-sectional area after exposition to the toxicant. These in vitro models can also reveal glomerular effects of xenobiotics that are difficult or impossible to observe in vivo. In addition, these studies permit a fundamental examination of the mechanism of action of xenobiotics on glomerular cells, including the possibility that at least a part of their effects are mediated by local mediators released by glomerular cells. We review the effects and the mechanisms of action of several toxicants such as gentamicin, cyclosporin, cisplatin, and cadmium on isolated glomeruli or cultured mesangial cells. As such in vitro results confirm in vivo renal hemodynamic changes caused by toxicants, we conclude that these models are fruitful tools for the study of renal toxicity. These in vitro systems might also serve as a predictive tool in the evaluation of drugs inducing changes in glomerular filtration rate and as a way to propose protective agents against these dramatic hemodynamic effects.

Role of calcium in gentamicin-induced mesangial cell activation.

Gentamicin-induced decreases in glomerular filtration rate have been associated to a marked decline in the glomerular capillary ultrafiltration coefficient which could be due to an active contraction of mesangial cells. In the present work we assessed a possible role of cytosolic Ca2+ as a mediator that leads to contraction and proliferation induced by gentamicin on mesangial cells. Gentamicin (10(-5)M) induced an increase in cytosolic free Ca2+, that was fully inhibited by the calcium channel blocker, verapamil, and by the endoplasmic reticulum calcium release blocker, TMB8. Gentamicin induced a planar surface area reduction in cultured mesangial cells, that was blunted by verapamil and TMB-8. Gentamicin also stimulated [3H]thymidine incorporation into DNA and increased viable cell number, effects that were reduced by both, verapamil and TMB-8. Gentamicin stimulated the expression of the AP1 protein; this expression was partially blunted by verapamil and TMB-8. Moreover, verapamil inhibited gentamicin-induced PAF synthesis from mesangial cells. In summary, gentamicin directly raised intracellular Ca2+ activating both calcium influx from external medium and calcium release from internal stores. This increase is responsible of cellular activation (contraction and proliferation) and PAF synthesis induced by gentamicin on mesangial cells.

Effect of NG-nitro-L-arginine methyl ester on nephrotoxicity induced by gentamicin in rats.

The present experiments were designed to assess the effect of inhibiting NO synthesis on the renal failure induced in rats by treatment with high doses of gentamicin. Eighteen Wistar rats were given gentamicin 100 mg/kg body weight/day for 5 days, whereas another 18 rats were used as control. Half of the gentamicin-treated rats and half of the controls also received the specific inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME), 0.05 mg/ml in the drinking water for 5 days. Arterial pressure and renal function were measured on the 5th day of the study. In the animals treated with L-NAME, arterial pressure was higher than in untreated rats, thus suggesting that the treatment was effective in inhibiting NO synthesis. Rats that received L-NAME and gentamicin, showed higher plasma creatinine levels and higher score of renal damage, as well as lower Na+ and K+ excretion and creatinine clearance than rats that received gentamicin alone. These data showing that NO inhibition aggravates gentamicin-induced renal failure, suggest that endogenously released NO plays a protective role in gentamicin nephrotoxicity.