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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.

In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 microg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 microg/ml) was not statistically different from the control values. The most intense effects were seen at 10 microg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 microg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 microM) and quinacrine (10 microM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indomethacin (10 microM) and nordiidroguaretic acid (1 microM) reversed almost all functional changes, except those of the perfusion pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2 and cyclooxygenase products are involved in these biologic effects.

Cholera toxin (MW 84 kDa) is now considered a pharmacological tool to study the adenylyl cyclase system and a stimulus to generate platelet activating factor in the intestinal tract. We used this toxin to evaluate the renal haemodynamics, glomerular filtration function, tubular sodium transport and toxicity in isolated perfused rat kidney. Kidneys from adult male Wistar rats were isolated for perfusion. The perfusion fluid was modified Krebs-Henseleit solution and the samples were analyzed for sodium, potassium, inulin and osmolality. Clearance techniques were used to calculate physiological parameters. Cholera toxin (1.0 microg/ml) caused a significant time-dependent reduction of glomerular filtration rate and urinary flow. This toxin also caused a small, but consistent reduction in fractional proximal sodium reabsortion (toxin = 67.43+/-2.42% versus control = 79.26+/-5.80%; P<0.025). WEB 2086, a platelet activating factor receptor antagonist at 100 microg/ml completely blocked the effects induced by cholera toxin on glomerular filtration rate, fractional proximal sodium reabsortion and urinary flow. In contrast to cholera toxin, dibutyryl-cyclic AMP (10(-5) M) significantly increased glomerular filtration rate (Db-cyclic AMP = 0.651+/-0.035 versus control = 0.514+/-0.043 ml x g(-1) x min(-1); P<0.025) in isolated perfused kidneys. Db-cyclic AMP caused a similar, but more severe reduction in fractional proximal sodium reabsortion (Db-cyclic AMP = 54.21+/-2.35% versus control = 70.10+/-3.24%; P<0.025). In addition Db-cyclic AMP increased significantly the urinary flow (Db-Cyclic AMP = 0.290+/-0.018 versus control = 0.179+/-0.026 ml x g(-1) x min.(-1); P<0.025). WEB 2086+ Db-cyclic AMP also caused a significant increase in the urinary flow with maximal effect at 90 min. (WEB+Db-cyclic AMP = 0.26+/-0.01 versus control = 0.15+/-0.01 ml x g(-1) x min.(-1); n = 8, P<0.025). Cholera toxin caused a decrease of urinary flow (toxin = 0.034+/-0.004 versus control = 0.145+/-0.02 ml x g(-1) x min.(-1); P<0.025), this effect was also completely abolished by WEB 2086 when it was injected previously to toxin. When only WEB 2086 was injected, the functional parameters remained stable throughout the perfusion time. Cholera toxin had no effect on renal vascular resistance, renal perfusate flow or tissue potassium, suggesting renal integrity in kidneys treated with this toxin. The results suggest that cholera toxin effects in the perfused rat kidney are primarily mediated by platelet activating factor.

To compare the function of sodium transport between intestine and renal tubule, we studied the effect of E. coli STa enterotoxin and 8-bromo cyclic GMP in perfused rat kidneys. Infusion of STa enterotoxin (0.017 and 0.1 micrograms/ml) caused a dose and time dependent decrease in total renal sodium tubular transport. The major site of STa effect was at the renal proximal tubule. Similar to Sta enterotoxin, 8-bromo cyclic GMP (10(-5) M) caused a significant decrease of fractional renal sodium proximal tubule transport. In contrast to STa enterotoxin, infusion of 8-bromo cyclic GMP resulted in a significant but short lasting (30 min.) increase in glomerular filtration rate. STa enterotoxin also decreased significantly the renal tissue potassium. This STa effect was related to a significant decrease in renal potassium tubular transport, resulting also in an increase of urinary potassium excretion. These studies demonstrate the specific functional effect of STa enterotoxin in promoting the decrease in renal proximal tubular sodium transport, similar to 8-bromo cyclic GMP. In perfused rat kidneys STa also decreased tissue potassium, mainly by a decrease in potassium transport and increase in urinary potassium excretion. These effects suggest the existence of an endogenous peptide resembling STa enterotoxin, that regulates the function of renal sodium tubular transport.