Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 µg/ml) in the absence and presence of thalidomide (15 µM) or pentoxifylline (500 µM) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 ± 1.3; venom = 137.1 ± 1.5; Thalid = 121.0 ± 2.5; Ptx = 121.4 ± 4.0 mmHg), renal vascular resistance (control = 4.5 ± 0.2; venom = 7.3 ± 0.6; Thalid = 4.5 ± 0.9; Ptx = 4.8 ± 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 ± 0.001; venom = 0.44 ± 0.01; Thalid = 0.22 ± 0.007; Ptx = 0.21 ± 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 ± 0.06; venom = 1.91 ± 0.11; Thalid = 0.75 ± 0.04; Ptx = 0.77 ± 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 ± 0.9; venom = 73.9 ± 0.66; Thalid = 76.6 ± 1.1; Ptx = 81.8 ± 2.0 percent), percent tubular chloride transport (control = 77.1 ± 1.2; venom = 71.4 ± 1.1; Thalid = 77.6 ± 1.7; Ptx = 76.8 ± 1.2 percent), and percent tubular potassium transport (control = 72.7 ± 1.1; venom = 63.0 ± 1.1; Thalid = 72.6 ± 1.0; Ptx = 74.8 ± 1.0 percent), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.

Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom.

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 micro g/ml) in the absence and presence of thalidomide (15 micro M) or pentoxifylline (500 micro M) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 +/- 1.3; venom = 137.1 +/- 1.5; Thalid = 121.0 +/- 2.5; Ptx = 121.4 +/- 4.0 mmHg), renal vascular resistance (control = 4.5 +/- 0.2; venom = 7.3 +/- 0.6; Thalid = 4.5 +/- 0.9; Ptx = 4.8 +/- 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 +/- 0.001; venom = 0.44 +/- 0.01; Thalid = 0.22 +/- 0.007; Ptx = 0.21 +/- 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 +/- 0.06; venom = 1.91 +/- 0.11; Thalid = 0.75 +/- 0.04; Ptx = 0.77 +/- 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 +/- 0.9; venom = 73.9 +/- 0.66; Thalid = 76.6 +/- 1.1; Ptx = 81.8 +/- 2.0%), percent tubular chloride transport (control = 77.1 +/- 1.2; venom = 71.4 +/- 1.1; Thalid = 77.6 +/- 1.7; Ptx = 76.8 +/- 1.2%), and percent tubular potassium transport (control = 72.7 +/- 1.1; venom = 63.0 +/- 1.1; Thalid = 72.6 +/- 1.0; Ptx = 74.8 +/- 1.0%), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.

Microcystin-LR promote intestinal secretion of water and electrolytes in rats.

We showed previously that exposure to microcystin-LR causes renal toxic effects in isolated perfused rat kidney, and that inflammatory mediators from supernatants of macrophages stimulated by microcystin-LR are involved in this process. The aim of this research was to examine water and electrolytes secretion in vivo, induced by microcystin-LR and supernatant of macrophages stimulated for this toxin (SUP.MphiS + MCLR), using perfused rat ileal segment and ligated intestinal loop models. We found microcystin-LR at 1 microg/ml (0.09 +/- 0.003* vs. control 0.07 +/- 0.001 g of secretion/2 cm of loop; P < 0.05*) and the SUP.MphiS + MCLR after 18 h postinoculation (0.10 +/- 0.003 vs. control 0.03 +/- 0.002 g/cm) caused intestinal secretion. In addition, microcystin-LR caused significant sodium secretion (-2.18 +/- 0.72* vs. control 2.18 +/- 0.50 microEq g(-1) min(-1)), potassium (-0.26 +/- 0.04* vs. control 0.32 +/- 0.03 microEq g(-1) min(-1)), chloride (MCLR = -3.29 +/- 1.93* vs. control 0.88 +/- 1.25 microEq g(-1) min(-1)) and water (-0.012 +/- 0.004* vs. control 0.002 +/- 0.002 ml g(-1) min(-1)). We also demonstrated SUP.MphiS + MCLR to induce intestinal secretion of electrolytes (sodium, potassium, chloride) and water. These findings suggested that microcystin-LR and lamina propria macrophages-derived mediators are able to induce intestinal secretion in vivo, probably via inhibition of protein phosphatase.

Effects of Thalassophryne nattereri fish venom in isolated perfused rat kidney.

Thalassophryne nattereri, popularly known as Niquim, is a venomous fish responsible for many accidents in fishermen in the Northeast of Brazil. The effects of T. nattereri venom on renal physiology has not been tested. Isolated kidneys from Wistar rats of 240-280 g weight were perfused with Krebs-Henseleit solution containing 6g% of previously dialyzed bovine serum albumin. The effects of Niquim venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). The venom of T. nattereri (0.3, 1.0, and 3.0 microg/ml) was always added to the system 30 minutes after the beginning of each experiment (n=6). All experiments were preceded by 30 minutes internal control period and an external control group, where kidneys were perfused with only Krebs-Henseleit solution. All three doses tested promoted increases in PP and RVR. The first two doses also increased GFR and UF. The higher dose promoted decreases in GFR, UF, %TNa(+), %TK(+), %TCl(-). In the treated groups we observed hyalin casts inside all tubules and proteinaceous material in the urinary space. We conclude that the effects resulted from niquim venom agents that promoted a direct effect in kidney cells causing the release of vasoactive factors.

Renal effects of supernatant from rat peritoneal macrophages activated by microcystin-LR: role protein mediators.

We have demonstrated previously that microcystin-LR promoted some renal alterations using the isolated perfused rat kidney preparation. However, these effects were not proved to be direct or indirect. The aim of the current work is to examine the renal effects promoted by supernatants from rat macrophages stimulated with microcystin-LR and the role of inflammatory mediators. Peritoneal macrophages were collected previously and were incubated for 1h in fresh medium (control) and in medium containing microcystin-LR. Dexamethasone, quinacrine, thalidomide and cycloheximide were administered 30 min before microcystin-LR. Supernatants of macrophages stimulated with or without pharmacological inhibitors were added on the perfused rat kidney model. The infusion of macrophages supernatants stimulated by microcystin-LR caused significant increases in renal vascular resistance (C: 4.93+/-0.33 vs T: 5.15+/-0.21), glomerular filtration rate (C: 0.559+/-0.008 vs T: 0.978+/-0.15) and urinary flow (C: 0.16+/-0.01 vs T: 0.23+/-0.03). Cycloheximide, quinacrine and dexamethasone blocked these effects and thalidomide blocked renal vascular resistance. Macrophages stimulated by microcystin-LR release mediators capable of promoting nephotoxicity in isolated perfused rat kidney. Phospholipase A(2), TNF-alpha and other protein mediators appear to be involved on its renal toxic mechanism.

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 µg/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 + or- 0.01 and treated (T) = 0.32 + or - 0.01 ml g-1 min(-1), P<0.05). At 90 min there was a significant increase in perfusate pressure (C = 129.7 + or - 4.81 and T = 175.0 + or - 1.15 mmHg) and glomerular filtration rate (C = 0.66 + or - 0.07 and T = 1.10 + or - 0.04 ml g-1 min(-1) and there was a significant reduction in fractional sodium tubular transport at 120 min (C = 78.6 + or - 0.98 and T = 73.9 + or - 0.95 percent). 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