Manassés C. Fonteles

Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney

Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 μM) increased glomerular filtration rate from 0.77 ± 0.07 to 1.34 ± 0.3 ml ⋅ g-1 ⋅ min-1at the highest concentration. A maximal increase in Na+ excretion was achieved at 0.66 μM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 ± 1.7 to 58.8 ± 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested ( P< 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 μM) decreased fractional Na+ reabsorption from 73.9 ± 2.4 to 64.5 ± 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 μM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 microM) increased glomerular filtration rate from 0.77 +/- 0.07 to 1.34 +/- 0.3 ml . g-1 . min-1 at the highest concentration. A maximal increase in Na+ excretion was achieved at 0. 66 microM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 +/- 1.7 to 58.8 +/- 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested (P < 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 microM) decreased fractional Na+ reabsorption from 73. 9 +/- 2.4 to 64.5 +/- 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 microM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.

Myorelaxant and antispasmodic effects of the essential oil of Alpinia speciosa on rat ileum

The effects of the essential oil of Alpinia speciosa Schum (EOAS) on rat ileum were studied. EOAS (0.1–600 µg/mL) reversibly relaxed ileal basal tonus. Submaximal contractions induced by 60 mM KCl or acetylcholine were concentration dependently inhibited by EOAS with similar IC50 values (∼ 44 and 48 µg/mL, respectively). These results show that EOAS possesses both relaxant and antispasmodic actions in the ileum. Copyright

Effects of microcystin-LR in isolated perfused rat kidney

Microcystin is a hepatotoxic peptide which inhibits protein phosphatase types 1 and 2A. The objective of the present study was to evaluate the physiopathologic effects of microcystin-LR in isolated perfused rat kidney. Adult Wistar rats (N = 5) of both sexes (240-280 g) were utilized. Microcystin-LR (1 microg/ml) was perfused over a period of 120 min, during which samples of urine and perfusate were collected at 10-min intervals to determine the levels of inulin, sodium, potassium and osmolality. We observed a significant increase in urinary flow with a peak effect at 90 min (control (C) = 0.20 +/- 0.01 and treated (T) = 0.32 +/- 0.01 ml g-1 min-1, P<0.05). At 90 min there was a significant increase in perfusate pressure (C = 129.7 +/- 4.81 and T = 175.0 +/- 1.15 mmHg) and glomerular filtration rate (C = 0.66 +/- 0.07 and T = 1.10 +/- 0. 04 ml g-1 min-1) and there was a significant reduction in fractional sodium tubular transport at 120 min (C = 78.6 +/- 0.98 and T = 73.9 +/- 0.95%). Histopathologic analysis of the perfused kidneys showed protein material in the urinary space, suggestive of renal toxicity. These data demonstrate renal vascular, glomerular and urinary effects of microcystin-LR, indicating that microcystin acts directly on the kidney by probable inhibition of protein phosphatases.

Efficacy of a glutamine-based oral rehydration solution on the electrolyte and water absorption in a rabbit model of secretory diarrhea induced by cholera toxin.

BACKGROUND Glutamine is absorbed in the intestinal tract coupled with sodium and is the principal metabolic substrate for the enterocyte. Therefore, an oral rehydration solution containing this substance might provide an effective oral means of restoring electrolyte losses as well as speeding repair of mucosal damage. The objective of this work was to investigate the use of an oral rehydration solution based on glutamine in vivo in the perfused rabbit ileal loop model of secretory diarrhea induced by choleratoxin. METHODS Phenolsulfonphthalein (PSP, 50 mg/l) was used as a nonabsorbable marker for calculations of net water and electrolyte transport. Solutions tested included: (a) a glutamine-based oral rehydration solution with 111 mmol/l glutamine, (Gln-ORS); (b) the oral rehydration solution recommended by the World Health Organization; (c) modified Ringers solution. Choleratoxin (1 microg/ml) was injected into the lumen of the ileal rabbit segments for 30 minutes prior to the initiation of the perfusion. RESULTS Choleratoxin induced significant secretion of sodium in the control modified Ringers solution (10.8 +/- 2.95 vs -14.05 +/- 5.95 microEq/g/min, n = 10; p < 0.01) and of water (0.06 +/- 0.03 vs -0.15 +/- 0.06 ml/g/min, n = 10; p < 0.01) with a maximum effect at 60 minutes after initiation of perfusion. World Health Organization oral rehydration solution was able to significantly reduce the intestinal secretion of sodium (control with cholera = -14.34 +/- 2.18 vs oral rehydration solution with cholera = -0.50 +/- 0.48 microEq/g/min, n = 10; p < 0.01) and water (-0.15 +/- 0.02 vs -0.012 +/- 0.005 ml/g/min, n = 10; p < 0.01). For comparison, glutamine-based oral rehydration solution had an even greater effect on sodium and water absorption (glutamine-based oral rehydration solution with choleratoxin = 10.31 +/- 1.21 microEq/g/min, n = 5; p < 0.01 for sodium and 0.08 +/- 0.008 ml water/g/min; n = 5; p < 0.01). Choleratoxin did not change the effect of glutamine-based oral rehydration solution on sodium and water absorption (12.90 +/- -1.09 microEq sodium/g/min, n = 5; and 0.11 +/- 0.01 ml water/g/min; n = 5). In addition glutamine-based oral rehydration solution also induced a greater absorption of potassium and chloride in the intestinal ileal segments treated with choleratoxin compared with World Health Organization glutamine-based oral rehydration solution. CONCLUSIONS These results demonstrate the superior efficacy of glutamine-based oral rehydration solution in electrolyte and water absorption compared with modified Ringers control solution or even with World Health Organization-recommended oral rehydration solution.

The role of phospholipase A2 and cyclooxygenase in renal toxicity induced by microcystin-LR

We have shown previously that exposure to microcystin-LR (MCLR) causes renal toxic effects in isolated perfused rat kidney. That study was extended further to approach the perspective of pharmacological blockade of renal toxic effects by MCLR through the use of experimental therapeutic agents. An isolated kidney perfusion system was utilized and samples of urine and perfusate were collected at 10min intervals to determine the levels of inulin, sodium, potassium and osmolality. Dexamethasone (20microg ml(-1)) and indomethacin (10microg ml(-1)) were administered in the beginning of the perfusion and MCLR was employed in a dose of 1microg ml(-1) after an internal control of 30min to evaluate the perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR) and urinary flow (UF). Dexamethasone and indomethacin antagonized the toxic effects of MCLR on PP, RVR, GFR and UF. Histologic analysis of dexamethasone and indomethacin treated groups did not show any vascular or interstitial alterations. MCLR potentially impairs the renal function, probably causing vascular and glomerular lesions and, promoting renal alterations through direct or indirect actions. These data seem to indicate that the renal alterations promoted by MCLR involves also phospholipase A(2) and arachidonic acid-derived mediators.

Renal toxicity of Bothrops moojeni snake venom and its main myotoxins.

Acute renal failure is one the most common systemic complications after snakebite, however, its pathogenesis remains obscure. In this study we evaluated the renal effects of Bothrops moojeni venom and its myotoxins (Bmtx-I and BmtxII) in rat isolated perfused kidneys. The myotoxins were purified by ion-exchange chromatography and reverse phase HPLC. The whole venom (10 microg/ml) and myotoxins (5 microg/ml) were added to the perfusion system 30 min after the beginning of each perfusion. The renal effects were compared to a control group perfused with modified Krebs-Henseleit solution alone. B. moojeni venom decreased the perfusion pressure (PP), renal vascular resistance (RVR), and the percent sodium, potassium and chloride tubular transport (%TNa(+), %TK(+), %TCl(-)). In contrast, the venom increased the urinary flow (UF), glomerular filtration rate (GFR), and the sodium, potassium and chloride excretion (ENa(+), EK(+), ECl(-)). The renal effects of myotoxin I was very similar to those of the whole venom, but there was an increase rather than a decrease in the PP and RVR. Myotoxin II had no effect on renal physiology, except for a transient decrease in %TK(+). In conclusion, B. moojeni venom caused intense alterations in renal physiology, including a drop in vascular resistance associated with diuresis, natriuresis and kaliuresis. Bmtx-I had an opposite effect when compared to whole venom, showed in the parameters of PP and RVR. Bmtx-II had a mild effect in %TK(+). The apparent inability of Bmtx-II to induce the renal effect similarly to Bmtx-I should be explained by the absence in the Bmtx-II of the C-terminal lysine rich region.

Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys

Envenomation by Crotalus durissus terrificus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure (ARF). The most serious systemic change and primary cause of death is ARF. In this work, we used RP-HPLC to isolate crotoxin, convulxin and gyroxin from venom of the related subspecies Crotalus durissus cascavella and investigated the effects of these toxins on renal function in the isolated rat kidneys perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). Crotoxin (5 microg/ml) increased the PP, RVR, GFR, UF and decreased %TNa(+), %TK(+) and %TCl(-), with gyroxin (5 micro g/ml) the GFR remained stable during the 120 min of perfusion, whereas PP and RVR increased significantly and the %TNa(+), %TK(+) and %TCl(-) decreased significantly. Convulxin (5 micro g/ml) had no effect on renal function. Crotoxin caused alterations in all renal parameters. Gyroxin produced a minor effect compared to crotoxin. These results indicated that crotoxin is the main componenet responsible for acute nephrotoxicity caused by C. d. cascavella venom.

Extraction of glutathione by the isolated perfused rabbit kidney

Abstract The effects of adding exogenous reduced glutathione (GSH) to the perfusate were studied in the isolated perfused rabbit kidney. The addition of 500 mg/liter of GSH to the perfusate prevented the depletion of cortical and medullary GSH; perfusion without the addition of GSH consistently resulted in depletion of tissue levels of this tripeptide. In addition, GSH supplementation of the perfusate decreased renal vascular resistance and increased perfusate flow. GSH extraction studies revealed a progressive decrease in renal extraction with time, ranging from complete extraction at 10 min to a value of 38% at 60 min. The fractional clearance of GSH increased from 7.3% at 10 min to 17.9% after 60 min of perfusion. The results indicate a high affinity of the rabbit kidney for GSH and a relatively large net reabsorption of the tripeptide.

Functional preservation of the mammalian kidney. III. Ultrastructural effects of perfusion with dimethylsulfoxide (DMSO).

Abstract Isolated rabbit kidneys were perfused at 37 ° C with a cell-free solution containing 0.0, 1.4, 2.1, and 2.8 m DMSO for 60 min. Electron microscopic examination of cortical structures revealed relatively high sensitivity of proximal tubular cells to DMSO-induced alterations, which included cytoplasmic clarification and disruption of microvilli. Glomeruli exhibited proliferation of rough endoplasmic reticulum, but were otherwise well preserved after perfusion with and without DMSO. Vascular endothelial cells were also intact after perfusion with DMSO, suggesting that DMSO-induced resistance changes are not necessarily associated with degeneration of capillary cells. Perfusion without DMSO resulted in good preservation of all cortical structures. These studies suggest that ultrastructural effects of DMSO must be considered in assessment of damage done in whole kidneys during freeze-preservation studies.

Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom

Envenoming by Crotalus durissus subspecies leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure. The most serious systemic alteration and primary cause of death after snakebite is acute renal failure. In this work, we isolated crotapotin, an acid component (Crtp) of crotoxin from Crotalus durissus cascavella venom and we investigated its bactericidal and pro-inflammatory activities as well as its renal effects in rat isolated perfused kidneys. Crtp was bactericidal to the Gram-negative species Xanthomonas axonopodis pv. passiflorae, but was less effective against the Gram-positive Claribacteri ssp, probably because of differences in the cell wall composition. Crtp showed a high amino acid sequence homology with other Crtps described in the literature (around of 90%) and its A and B chains had high conserved regions corresponding to the calcium-binding loop, catalytic site and helix 3 of PLA2. The Crtp showed moderate pro-inflammatory activity and increased significantly the inflammation evoked by PLA2 when co-injected or co-incubated with PLA2. The renal parameters evaluated included the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR) and percent of sodium tubular transport (%TNa+). Crotapotin (5 microg/ml) significantly increased the PP and RVR, whereas the GFR, UF and %TNa+ were unaffected. These results suggest that crotoxin is the main venom component responsible for nephrotoxicity and crotapotin contributes little to this phenomenom. The biological and bactericidal actions of Crtp also suggest that this protein may have functions other than simply acting as a chaperone for PLA2.