Free radical scavengers improve the impaired endothelium-dependent responses in aorta and kidneys of diabetic rabbits

The effects of two endogenous antioxidants, alpha-lipoic acid and reduced gluthathione (GSH), were evaluated in the response of the renal vasculature and aortic rings ex vivo of 4-week alloxan-diabetic rabbits to the endothelium-dependent agonists bradykinin (BK) and acetylcholine (Ach) or to the endothelium-independent agonist sodium nitroprusside (SNP) and compared with age and sex-matched euglycemic rabbits. The maximal decrease in perfusion pressure (R(max)) after BK infusion in the renal vasculature from diabetic rabbits was 5.4+/-1.3% (PD(2) 8 [12.6-3.4]) compared with 34.2+/-4.2% (PD(2) 9 [11.3-6.7]) (P<0.05) attained in tissues obtained from euglycemic rabbits. The addition of 1 microM lipoic acid or GSH improved (P<0.05) the R(max) to BK to 18.3+/-2.4% (PD(2) 8.6 [12.4-4.8]) and 19.5+/-3.7% (PD(2) 9.1 [13.3-4.9]), respectively. Similarly, the maximal vasorelaxant response to Ach in kidneys from diabetic rabbits was 16+/-2.0% (PD(2) 7.3 [10.4-4.2] whilst the R(max) in kidneys from euglycemic animals was 52.7+/-4.9% (PD(2) 11.3 [16.4-6.2]). Incubation with 1 microM alpha-lipoic acid or GSH restored the R(max) to Ach to 31+/-3.9% (PD(2) 9.8 [14.3-5.3]) and to 23+/-5.4% (PD(2) 7.6 [11.4-3.8], respectively. The vasodilatory response to SNP was unaltered among tissues from diabetic and euglycemic rabbits and was also unaffected by the treatments utilized. In addition, the R(max) to Ach in aortic rings of diabetic rabbits was 28.7+/-2.4% (PD(2) 8.3 [11.7-4.9]) compared with 100% (PD(2) 7.9 [12.1-3.7]) obtained in tissues gathered from euglycemic rabbits. The pretreatment of the tissues with alpha-lipoic acid restores the R(max) to 47.4+/-4% (PD(2) 11.1 [14.3-7.9]) and the pretreatment with GSH to 52+/-3.2% (PD(2) 9.8 [12.7-6.9]). Similarly, the response to SNP was unaltered in all groups. Lipoic acid and reduced gluthatione directly improved the endothelium-dependent response of renal arterioles and aortic rings of diabetic rabbits.     

Action of anti-bothropic factor isolated from Didelphis marsupialis on renal effects of Bothrops erythromelas venom.

Acute renal failure is the most common complication in the lethal cases caused by snakebites in Brazil. Among the Brazilian venom snakes, Bothrops erythromelas is responsible for the majority of accidents in Northeastern Brazil. Didelphis marsupialis serum could inhibit myonecrotic, hemorrhagic, edematogenic hyperalgesic and lethal effects of envenomation determined by ophidian bites. In the present study, we evaluated the action of the anti-bothropic factor isolated from D. marsupialis on the renal effects promoted by B. erythromelas venom without systemic interference. Isolated kidneys from Wistar rats were perfused with Krebs-Henseleit solution containing 6% bovine serum albumin. We analyzed renal perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), and the percentages of sodium and potassium tubular transport (%TNa+, %TK+). The B. erythromelas venom (10 microg mL(-1)) decreased the PP (ct = 108.71+/-5.09 mmHg; BE = 65.21+/-5.6 mmHg*) and RVR (ct = 5.76+/-0.65 mmHg mL(-1) g(-1) min(-1); BE = 3.10+/-0.45 mmHg mL(-1) g(-1) min(-1)*). On the other hand, the GFR decreased at 60 min (ct60 = 0.76+/-0.07 mL g(-1) min(-1); BE60 = 0.42+/-0.12 mL g(-1) min(-1)*) and increased at 120 min (ct120 = 0.72+/-0.01 mL g(-1) min(-1); BE120 = 1.24+/-0.26 mL g(-1) min(-1)*). The UF increased significantly when compared with the control group (ct = 0.14+/-0.01 mL g(-1) min(-1); BE = 0.47+/-0.08 mL g(-1) min(-1)*). The venom reduced the %TNa(+) (ct90 = 79.18+/-0.88%; BE90 = 58.35+/-4.86%*) and %TK+ (ct90 = 67.20+/-4.04%; BE90 = 57.32+/-5.26%*) The anti-bothropic factor from D. marsupialis (10 microg mL(-1)) incubated with B. erythromelas venom (10 microg mL(-1)) blocked the effects on PP, RVR, %TNa+, and %TK+, but was not able to reverse the effects in UF and GFR promoted by venom alone. However, the highest concentration of D. marsupialis serum (30 microg mL(-1)) reversed all the renal effects induced by the venom. In conclusion, B. erythromelas venom altered all the renal functional parameters evaluated and the anti-bothropic factor from D. marsupialis was able to inhibit the effects induced by the venom in isolated kidney.     

Guanylin and its lysine-containing analogue in the isolated perfused rat kidney: interaction with chymotrypsin inhibitor

Guanylin and uroguanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GMP. Their natriuretic and kaliuretic activities are well documented. Since guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of guanylin. Using the isolated perfused rat kidney, guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-guanylin (0.1-2.5 microg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 microg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 microg/ml), guanylin (0.3 microg/ml) was placed into solution. A maximal decrease in fractional Na+ reabsorption (%TNa+) was achieved at 1.0 microg/ml of lys-guanylin (from 73.25+/-2.29 to 54.97+/-0.10, P<0.05). Lys-guanylin (1.0 microg/ml) also decreased fractional K+ reabsorption (%TK+) from 59.26+/-3.93 to 30.75+/-0.78 (P<0.05). Chymostatin had no detectable effects in electrolyte reabsorption in this assay. When introduced after chymostatin, guanylin lowered %TNa+ (from 81.2+/-1.86 to 72.6+/-2.45, P<0.05) and %TK+ (from 69.4+/-4.12 to 65.8+/-2.81, P<0.05). At this subthreshold concentration, guanylin alone lacks effects in %TNa+ or %TK+. Furthermore, the ability of both peptides to promote increases in intestinal fluid secretion was evaluated in the in vivo suckling mouse model. When administered per os, guanylin failed to stimulate intestinal secretion. When chymostatin was present in the test solution, guanylin induced intestinal secretion in this assay. In marked contrast, lys-guanylin alone induced diarrhoea in the suckling mouse. The present paper concludes that guanylin undergoes metabolism in target tissues such as the intestine and kidney and its lysine-containing analogue retains full biological activity.     

Role of phospholipase A2 and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Clostridium difficile-associated disease causes diarrhea to fulminant colitis and death. We investigated the role of phospholipase A2 (PLA2) inhibitors, aristolochic acid (AA), bromophenacyl bromide (BPB) and quinacrine (QUIN) on the C. difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion. Toxin A caused severe hemorrhagic and inflammatory fluid secretion at 6-8 h in rabbit ileal segments, an effect that was significantly inhibited by QUIN (71%, P < 0.01), AA (87%, P < 0.000l) or by BPB (51%, P < 0.01). The secretory effect of toxin A was also inhibited in segments adjacent to those with AA (89%, P < 0.01). Furthermore, QUIN or AA substantially reduced the histologic damage seen after 6-8 h in rabbit ileal segments. The cyclooxygenase inhibitor, indomethacin, also significantly inhibited (96%; n = 6) the secretory effects of toxin A in ligated rabbit intestinal segments. The destruction by toxin A of F-actin at the tight junctions of T-84 cell monolayers was not inhibited by AA or BPB. AA or QUIN had no effect on the T-84 cell tissue resistance reduction over 8-24 h after toxin A exposure. All the inhibitors were shown to be effective in the doses administered direct in ileal loops to inhibit PLA2 activity. The data suggest that PLA2 is involved in the major pathway of toxin A-induced histologic inflammatory damage and hemorrhagic fluid secretion.