Allopurinol reduces the lethality associated with acute renal failure induced by Crotalus durissus terrificus snake venom: comparison with probenecid

Abstract

Background: Acute renal failure is one of the most serious complications of envenoming resulting from Crotalus durissus terrificus bites. This study evaluated the relevance of hyperuricemia and oxidative stress and the effects of allopurinol and probenecid in renal dysfunction caused by direct nephrotoxicity of C. d. terrificus venom.

Methodology/principal findings: Hematocrit, protein, renal function and redox status were assessed in mice. High ratio of oxidized/reduced glutathione and hyperuricemia induced by C. d. terrificus venom were ameliorated by both, allopurinol or probenecid, but only allopurinol significantly reduced the lethality caused by C. d. terrificus venom. The effectiveness of probenecid is compromised probably because it promoted hypercreatinemia and hypocreatinuria and worsed the urinary hypo-osmolality in envenomed mice. In turn, the highest effectiveness of allopurinol might be due to its ability to diminish the intracellular formation of uric acid.

Conclusions/significance: Data provide consistent evidences linking uric acid with the acute renal failure induced by C. d. terrificus venom, as well as that this envenoming in mice constitutes an attractive animal model suitable for studying the hyperuricemia and that the allopurinol deserves to be clinically evaluated as an approach complementary to anti-snake venom serotherapy.

Figure 1. Effects of treatments on hematocrit and renal function parameters in the blood plasma.

Mice treated with vehicle (control), allopurinol (NL), probenecid (PB) and Crotalus durissus terrificus venom (vCdt) followed by NL (vCdt+NL) or PB (vCdt+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).

Figure 2. Effects of treatments on renal function parameters in the urine.

Mice treated with vehicle (control), allopurinol (NL), probenecid (PB) and Crotalus durissus terrificus venom (vCdt) followed by NL (vCdt+NL) or PB (vCdt+PB) after 2 h. Values are means ± SEM of pooled animals, 16 (control), 9 (NL), 10 (PB), 12 (vCdt), 22 (vCdt+NL) and 12 (vCdt+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).

Figure 3. Effects of treatments on renal oxidative stress.

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 Crotalus durissus terrificus venom (vCdt) followed by NL (vCdt+NL) or PB (vCdt+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).

Figure 4. Histopathological features of renal samples.

Slides of hematoxilin-eosin stained sagittal sections from representative kidneys of mice treated with: I and II: vehicle (control); see normal appearance of Bowman's capsule (BC), glomerulus (G), macula densa (DM) and distal tubule (DT); III, IV and V: C. d. terrificus venom; the most frequently changes detected were edema (*), fibrosis (1) with cell influx (2), and tubular necrosis with tubules markedly dilated and cellular debris in the lumen (3, 4) and in the brush border (BB). 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.

Figure 5. Schematic diagram depicting mechanisms and hypothetical actions of allopurinol and probenecid in vCdt nephrotoxicity.

ARF induced by vCdt occurs through indirect and direct actions leading to lethality. Direct actions of vCdt 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 vCdt. 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 vCdt. 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.