Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells.

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by approximately 50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G(1) and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

Cell cycle delay and apoptosis in response to osmotic stress.

As part of the urinary concentrating mechanism, renal inner medulla cells may be exposed to extremely variable NaCl and urea concentrations that can reach very high levels. A number of studies, reviewed herein, aim to understand how such osmotic stress affects the cells and what protective mechanisms might exist. The majority of these studies are done on inner medullary epithelial cells that grow continuously in tissue culture (mIMCD3). Cells grown at 300 mosmol/kg survive increase to 500 mosmol/kg by adding NaCl or urea, but only after a growth arrest of approximately 24 h. At a higher osmolality (650-700 mosmol/kg) most cells die within hours by apoptosis. The cells both in vitro and in vivo adapt to high osmolality by a number of mechanisms, including accumulation of variety of organic osmolytes and induction of heat shock proteins. The cell cycle delay results from blocks at the G1 and G2/M checkpoints and slowing during S. After adding NaCl, but not urea, the amount and transcriptional activity of p53 (the tumor suppressor protein) increases. The p53 is phosphorylated on ser-15 and is transcriptionally active at 500 mosmol/kg (associated with cell survival), but not at 700 mosmol/kg (associated with apoptosis). Reduction of p53 expression by p53 antisense oligonucleotide increases sensitivity of renal cells in culture to hyperosmotic stress caused by NaCl. The possible mechanisms of the protection action of p53 against hypertonic stress are discussed.

Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats.

Saxitoxin (STX) was the first known and most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel. Although the toxin's mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. Some of these questions are addressed in the present paper, which describes an experimental design which allowed us to follow the dynamics of STX poisoning in vivo. Adult cats were anaesthetized and permanently coupled to artificial ventilation, they were then intravenously injected with Low (2.7 microg of STX/kg) and high doses (10 microg of STX/kg) of toxin. Cardiovascular parameters such as blood pressure and electrocardiograms were recorded, urine and blood samples were collected during the four hours of experimental time. In order to quantify mass amount of STX, we used the post-column derivatization HPLC method. Urine and blood samples were cleansed using a C-18 Sep-Pack cartridge and ultrafree microcentrifuge filters. At the end of each experiment, the animals were killed and tissue samples from brain, liver, spleen and medulla oblongata were extracted to measure the amount of STX. As compared to control period, Low doses of STX made no difference in hemodynamics parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Administration of 2.5 microg/kg x min of dobutamine restored hemodynamics parameters and allowed the animal to overcome the shock. With high doses, the calculated STX renal clearance in cats is 0.81 ml/min x kg(-1). This valued corresponds to 20.25% of the reported inulin renal clearance. Nevertheless with Low doses the STX renal clearance is 3.99 ml/min x kg(-1). This data suggest that in cats with normal cardiovascular parameters and diuresis, the STX excretion mainly involves glomerular filtration. During experimental time, no PSP toxins other than STX was detected in the body fluids and tissue samples analyzed, indicating that the mammals can not metabolize this molecule. STX was found in intensely irrigated organs such as the liver and spleen but also in the central nervous system (brain and medulla oblongata), showing that STX was capable of crossing the blood brain barrier.

[Comparison of extra renal potassium management in hypertensive, diabetic and normal subjects]. / Estudio comparativo del manejo extrarrenal de potasio en sujetos controles, pacientes hipertensos y diabéticos.

BACKGROUND: Sodium and potassium ions are involved in the regulation of blood pressure and the genesis of hypertension. AIM: To assess internal potassium balance, as a measure of sodium pump activity, in subjects with essential hypertension and diabetic patients. PATIENTS AND METHODS: Eleven hypertensive subjects, 5 non-insulin-dependent diabetics and 16 age matched controls were studied. An acute oral load of 0.8 mEq/Kg body weight of KCl was administered and blood samples were drawn every 30 min thereafter, until 120 min, to measure plasma K+ levels. Urinary K+ excretion during this period was also measured. In eight hypertensive patients, the test was repeated after two week of supplementation with 60 mEq/day of KCl. The maximal increase in plasma potassium levels and the time required to achieve the maximum concentration was recorded. RESULTS: All patients had normal serum creatinine levels. Mean fasting blood glucose of diabetic patients was 133 +/- 15.1 mg/dl. No difference between patients and controls in maximal increase plasma potassium increase, was observed. In hypertensive patients the lapse to achieve the maximal potassium concentration was longer than in controls. After the period of potassium supplementation in hypertensive patients, there was a significant increase in basal plasma K+ levels and the temporal pattern of plasma potassium increase was similar to that of controls. Between 63 and 68% of retained K+ load was translocated to the intracellular space at 120 min in all study groups. CONCLUSIONS: Internal potassium balance is not significantly altered in subjects with essential hypertension or in non-insulin-dependent diabetics.

Biochemical evidence for adhesion-promoting role of major intrinsic protein isolated from both normal and cataractous human lenses.

In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22,000 Da did not blunt its adhesive capability, suggesting that the proteolytic modifications that major intrinsic protein undergoes in senile cataract were not related with the incompetence of cataractous lens junctions to induce adhesion. Cataractous lens junctional membranes showed protein aggregates. These membranes were treated with sodium hydroxide and reconstituted into liposomes. The sodium hydroxide treatment removed the protein aggregates and restored the adhesive capability. Furthermore, the supernatant obtained after the sodium hydroxide treatment of cataractous junctional membranes, inhibited the adhesive effect of vesicles reconstituted with bovine solubilized proteins. These experiments prove that the failure to induce adhesion of human senile cataractous lens junction proteins is due to the interaction with protein aggregates, which can be removed by sodium hydroxide.

Lens major intrinsic protein (MIP) promotes adhesion when reconstituted into large unilamellar liposomes.

The vertebrate lens behaves like a syncytium, and it is formed mainly by cells called lens fibers. Between the fibers are extensive networks of membrane junctions. The major intrinsic protein (MIP) constitutes about 50-60% of the intrinsic membrane proteins found in lens fiber junctions. The role of MIP is unknown. Nevertheless, it has been proposed that it is the protein responsible for the adhesion between the plasmatic membranes of the lens fibers. The aim of our studies was to test the adhesion-promoting role of MIP. We reconstituted MIP into large unilamellar vesicles (LUV) of phosphatidylcholine (PC) and studied the vesicle aggregation between MIP-reconstituted LUV (PC-MIP) and phosphatidylserine (PS) vesicles. The aggregation process was monitored using methods based on resonance energy transfer (RET) and turbidity measurements. Neither RET nor an increase in turbidity occurred in any combination except in the presence of both MIP and PS. The liposomes thus aggregate through protein-lipid interactions. These results show that MIP promotes adhesion with negatively charged membranes, indicating that the adhesion is electrostatic in nature. Aggregation was fastest at pH 6.0. The aggregation effect was abolished with pronase treatment. Preincubation of PC-MIP vesicles with anti-MIP polyclonal serum also inhibited the aggregation. These studies are the first experimental evidence supporting the hypothesis of an adhesive role for MIP.