Allopurinol Reduces the Lethality Associated with Acute Renal Failure Induced by Crotalus durissus terrificus Snake Venom: Comparison with Probenecid

In Brazil, among registered snake bites, those by the genus Crotalus originate the highest mortality rate. The rattlesnake Crotalus durissus terrificus is the most frequently implicated in these accidents. The kidney is a particularly vulnerable organ to the venom of this rattlesnake. In fact, the most serious complication of Crotalus snake bite is the renal dysfunction, and among the fatal cases of Crotalus bites in Brazil 5% are patients treated with antivenom. The hyperuricemia has been observed in human accidents with snake venoms, but this parameter has not received any special attention as a relevant factor in the etiology of renal dysfunction caused by these venoms. This study examined the effects of treatments with low-cost and low-risk uricostatic (allopurinol) and uricosuric (probenecid) drugs on the envenomation by C. d. terrificus, showing that allopurinol and probenecid mitigated certain nephrotoxic effects, as well as the survival of envenomed mice was improved through the effects of allopurinol on reduction of oxidative stress and intracellular formation of uric acid. This new knowledge provides consistent evidences linking uric acid with the renal dysfunction induced by rattlesnake bites and that the allopurinol deserves to be clinically evaluated as an approach complementary to anti-snake venom serotherapy

Schematic diagram depicting mechanisms and hypothetical actions of allopurinol and probenecid in <i>vCdt</i> nephrotoxicity.

<p>ARF induced by <i>vCdt</i> occurs through indirect and direct actions leading to lethality. Direct actions of <i>vCdt</i> generate hyperuricemia and renal oxidative stress. Uric acid has direct intracellular pro-oxidative effects. Allopurinol and probenecid restore uricemia and renal oxidative stress caused by <i>vCdt</i>. These beneficial effects must be, in part, exerted through a reduction of intracellular deposit of urate, as a consequence of the reduction of uric acid formation due to the inhibition of xanthine oxidase (allopurinol) or an inhibition of an organic anion transport exchanger that blocks the entry of uric acid into the cells (probenecid). Allopurinol, but not probenecid, protects against the lethality caused by <i>vCdt</i>. This differential protective effect of allopurinol is not related to the blocking of xanthine oxidase-associated oxidants, but it is likely related to the blocking of oxidant effects of increased production of uric acid in the intracellular environment more than the entry of uric acid into the cells.</p

Effects of treatments on renal oxidative stress.

<p>Reduced glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) were measured in renal cortex and medulla from mice treated with vehicle (control), allopurinol (NL), probenecid (PB) and <i>Crotalus durissus terrificus</i> venom (<i>vCdt</i>) followed by NL (<i>vCdt</i>+NL) or PB (<i>vCdt</i>+PB) after 2 h. Values are means ± SEM. Number of animals in parentheses. Comparison of the same parameter among groups: ANOVA (cortex: GSH, p = 0.6489; GSSG, p<0.0004; GSSG/GSH, p<0.0001; MDA, p<0.0001; medulla: GSH, p = 0.8440; GSSG, p<0.0001; GSSG/GSH, p<0.0009; MDA, p<0.0001). Post hoc Student-Newman-Keuls (different letters over the bars indicate statistical differences: p<0.05).</p

Effects of treatments on renal function parameters in the urine.

<p>Mice treated with vehicle (control), allopurinol (NL), probenecid (PB) and <i>Crotalus durissus terrificus</i> venom (<i>vCdt</i>) followed by NL (<i>vCdt</i>+NL) or PB (<i>vCdt</i>+PB) after 2 h. Values are means ± SEM of pooled animals, 16 (control), 9 (NL), 10 (PB), 12 (<i>vCdt</i>), 22 (<i>vCdt</i>+NL) and 12 (<i>vCdt</i>+PB) in triplicates. Comparison of the same parameter among groups: ANOVA (Osmolality, p<0.0001; Creatinine, p<0.0009; Uric acid, p<0.005; Urea, p = 0.06; Protein, p<0.001). Post hoc Student-Newman-Keuls (different letters over the bars indicate statistical differences: osmolality and creatinine, p<0.01; uric acid and protein, p<0.05).</p

Assessment of lethality after treatments.

<p>Mice treated intraperitoneally (ip) and/or <i>per oral</i> (po) with vehicle (control), allopurinol (NL), probenecid (PB) and <i>Crotalus durissus terrificus</i> venom (<i>vCdt</i>) followed by NL (<i>vCdt</i>+NL) or PB (<i>vCdt</i>+PB) after 2 h.</p><p>*Lethality at 24 h only differed in <i>vCdt</i>+PB (p = 0.0432) and <i>vCdt</i> (p = 0.0016) compared with all other groups. Lethality at 24 h did not differ between <i>vCdt</i>+PB and <i>vCdt</i> (p = 0.1811) (Two-sided Fisher's exact test).</p

Effects of treatments on hematocrit and renal function parameters in the blood plasma.

<p>Mice treated with vehicle (control), allopurinol (NL), probenecid (PB) and <i>Crotalus durissus terrificus</i> venom (<i>vCdt</i>) followed by NL (<i>vCdt</i>+NL) or PB (<i>vCdt</i>+PB) after 2 h. Values are means ± SEM. Number of animals in parentheses. Comparison of the same parameter among groups: ANOVA (Hematocrit, p = 0.1807; Osmolality, p = 0.3577; Creatinine, p<0.006; Uric acid, p<0.0001; Urea, p<0.003; Protein, p<0.0001). Post hoc Student-Newman-Keuls (different letters over the bars indicate statistical differences: creatinine and urea, p<0.05; uric acid and protein, p<0.01).</p

Histopathological features of renal samples.

<p>Slides of hematoxilin-eosin stained sagittal sections from representative kidneys of mice treated with: <b>I</b> and <b>II</b>: vehicle (control); see normal appearance of Bowman's capsule (<b>BC</b>), glomerulus (<b>G</b>), macula densa (<b>DM</b>) and distal tubule (<b>DT</b>); <b>III</b>, <b>IV</b> and <b>V</b>: <i>C. d. terrificus</i> venom; the most frequently changes detected were edema (<b>*</b>), fibrosis (<b>1</b>) with cell influx (<b>2</b>), and tubular necrosis with tubules markedly dilated and cellular debris in the lumen (<b>3</b>, <b>4</b>) and in the brush border (<b>BB</b>). Treatments of envenomed with allopurinol or probenecid acted indistinguishably to ameliorate the frequency and the intensity of detectable histological changes comparatively with untreated envenomed mice. Bars = 100 µm.</p

Dipeptidyl peptidase IV in the hypothalamus and hippocampus of monosodium glutamate obese and food-deprived rats

Proline-specific dipeptidyl peptidases are emerging as a protease family with important roles in the regulation of signaling by peptide hormones related to energy balance. The treatment of neonatal rats with monosodium glutamate (MSG) is known to produce a selective damage on the arcuate nucleus with development of obesity. This study investigates the relationship among dipeptidyl peptidase IV (DPPIV) hydrolyzing activity, CD26 protein, fasting, and MSG model of obesity in 2 areas of the central nervous system. Dipeptidyl peptidase IV and CD26 were, respectively, evaluated by fluorometry, and enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction in soluble (SF) and membrane-bound (MF) fractions from the hypothalamus and hippocampus of MSG-treated and normal rats, submitted or not to food deprivation (FD). Dipeptidyl peptidase IV in both areas was distinguished kinetically as insensitive (DI) and sensitive (DS) to diprotin A. Compared with the controls, MSG and/or FD decreased the activity of DPPIV-DI in the SF and MF from the hypothalamus, as well as the activity of DPPIV-DS in the SF from the hypothalamus and in the MF from the hippocampus. Monosodium glutamate and/or FD increased the activity of DPPIV-DI in the MF from the hippocampus. The monoclonal protein expression of membrane CD26 by enzyme-linked immunosorbent assay decreased in the hypothalamus and increased in the hippocampus of MSG and/or FD relative to the controls. The existence of DPPIV-like activity with different sensitivities to diprotin A and the identity of insensitive with CD26 were demonstrated for the first time in the central nervous system. Data also demonstrated the involvement of DPPIV-DI/CD26 hydrolyzing activity in the energy balance probably through the regulation of neuropeptide Y and beta-endorphin levels in the hypothalamus and hippocampus. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil)[05/04699-2]CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq