Regulation of the mitochondrial permeability transition in kidney proximal tubules and its alteration during hypoxia-reoxygenation.

Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg(2+). To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg(2+) greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg(2+). Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg(2+), or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions.

In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.

Purification and biological effects of L-amino acid oxidase isolated from Bothrops insularis venom.

Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure (C(120)=110.28+/-3.69; BiLAO(120)=82.2+/-5.6 mmHg*); renal vascular resistance (C(120)=5.48+/-0.53; BiLAO(120)=4.12+/-0.42 mmHg/mL/g/min*), urinary flow (C(120)=0.160+/-0.020; BiLAO(120)=0.064+/-0.012 mL/g/min*), glomerular filtration rate (C(120)=0.697+/-0.084; BiLAO(120)=0.176+/-0.017 mL/g/min*), sodium (C(120)=79.76+/-0.56; BiLAO(120)=65.39+/-6.19%*), potassium (C(120)=69.94+/-6.86; BiLAO(120)=60.26+/-2.24%*) and chloride tubular reabsortion (C(120)=78.53+/-2.33; BiLAO(120)=64.58+/-6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of glomerular filtration resulted in more time for filtration and tubular reabsorption, with elevation of the total percentage of sodium and chlorine reabsorption. The maintenance of the decrease in glomerular filtration rate would determine the subsequent decreases, which were noticed in these parameters. The necrosis observed was the result of damage cell induced by l-amino acid oxidase isolated from B. insularis venom.

Estudo da toxicidade induzida por fosfolipase A2 isolada do veneno de Crotalus durissus terrificus em rim isolado de rato e em túbulos proximais isolados de coelho [manuscrito] / Study of the toxicity induced by phospholipase A2 isolated from Crotalus durissus terrificus venom in isolated rat kidney proximal tubules and in isolated rabbit [manuscript]

Apesar de muito se discutir sobre os efeitos citotóxicos dos venenos ofídicos, pouco ainda é conhecido sobre os mecanismos de ação sobre as diversas células, e em especial, sobre as células renais. No caso particular da citotoxicidade dos venenos crotálicos, tem-se postulado a participação de diversos metabólicos da hidrólise de lipídios de membrana, e, mais recentemente, da disfunção mitocondrial. O presente trabalho teve como objetivo estudar o efeito da fosfolipase A2 (FLA2) isolada do veneno da Crotalus durissus terrificus sobre rim isolado de rato assim como estudar a toxicidade e as alterações da função mitocondrial induzidas pelas FLA2s de pâncreas de porco (PFLA2) e de veneno da C. d. terrificus (VSFLA2) em suspensões de túbulos proximais (TP). No rim isolado foi observado aumento no fluxo urinário, no ritmo de filtração glomerular (RFG) e na pressão de perfusão (PP) enquanto ocorreram decréscimos nos percentuais de transporte total de sódio (%TNA+), de potássio (%TK+) e de cloreto (%TCl-)...

Purification and biological effects of C-type lectin isolated from Bothrops insularis venom.

Bothrops insularis is a snake from Queimada Grande Island, which is an island located about 20 miles away from the southeastern coast of Brazil. Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. Its C-type lectin is involved in several biological processes including anticoagulant and platelet-modulating activities. We purified the C-type lectin (BiLec) from Bothrops insularis venom and investigated its effect in the isolated kidney. BiLec was purified after two chromatographic steps; firstly, the whole venom was submitted to an HPLC molecular exclusion chromatography followed by a second purification through affinity chromatography. B. insularis lectin (BiLec) was studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. The concentration of 10mug/mL increased perfusion pressure (PP; control(60)=108.27+/-4.9; BiLec(60)=112.9+/-5.4 mmHg; *p<0.05) and renal vascular resistance (RVR; control(60)=5.38+/-0.51; BiLec(60)=6.01+/-0.57 mmHg; *p<0.05). The urinary flow reduced significantly at 90 and 120 min of perfusion (UF; control(120)=0.160+/-0.020; BiLec(120)=0.082+/-0.008 mL g(-1) min(-1); *p<0.05). Glomerular filtration rate (GFR; control(120)=0.697+/-0.084; BiLec(120)=0.394+/-0.063 mL g(-1) min(-1); *p<0.05) diminished only at 120 min. BiLec did not change the percentage of sodium (TNa(+)), potassium (TK(+)) and chloride tubular transport (TCl(-)). The histological alterations probably reflected direct injury on glomerular and tubular renal cells, as demonstrated by the rise in permeability of glomerular endothelial cells, revealed by the presence of a proteinaceous material in the Bowman space. We postulate that the C-type lectin B. insularis promoted its effects probably through interactions with endothelial cells or through the release of other mediators by tubular, mesangial and endothelial cells.

Effects of Crotalus durissus collilineatus venom in the isolated rat kidney.

Ophidian accidents caused by the subspecies Crotalus durissus are responsible for high morbity and mortality rates. Acute renal failure is a common complication observed in these accidents. The aim of the present study was to investigate the renal effects promoted by the venom of C. d. collilineatus and its fractions, crotoxin and phospholipase A2. C. d. collilineatus (Cdc; 30 microg mL(-1)), crotoxin (CTX; 10 microg mL(-1)) and phospholipase A2 (PLA2; 10 microg mL(-1)) were tested in isolated rat kidney. The first 30 min of each experiment were used as an internal control and Cdc or its fractions, CTX and PLA2 were added to the system after this period. All experiments lasted 120 min. The venom of Cdc decreased perfusion pressure (PP; control120 = 110.3 +/- 3.69 mmHg; Cdc120 = 96.7+/-8.1 mmHg), renal vascular resistance (RVR; control120 = 6.42+/-0.78 mmHg mL g(-1) min(-1); Cdc120 = 4.8+/-0.56 mmHg/mL g(-1) min(-1)), urinary flow (UF; control120 = 0.19+/-0.03 mL g(-1) min(-1); Cdc120 = 0.12 +/- 0.01 mL g(-1) min(-1)), and glomerular filtration rate (GFR; control120 = 0.79 +/- 0.07 mL g(-1) min(-1); Cdc120 = 0.53 +/- 0.09 mL g(-1) min(-1)), but had no effect on the percent of sodium tubular transport (%TNa+), percent of chloride tubular transport (%TK+) and percent of potassium tubular transport (%TCl-). CTX and PLA2 reduced the GFR, while UF, PP and RVR remained stable during the full 120 min of perfusion. Crotoxin administration also diminished the %TK+ (control120 = 69.94 +/- 6.49; CTX120 = 33.28 +/- 4.78) and %TCl- (control120 = 79.53 +/- 2.67; CTX120 = 64.62 +/- 6.93). PLA2 reduced the %TK+, but exerted no effect on the %TNa+ or on that of TCl-. In conclusion, the C. d. collilineatus venom altered the renal functional parameters evaluated. We suggest that crotoxin and phospholipase A2 were involved in this process, since the renal effects observed would be due to the synergistic action of the components of the venom.