Polyene toxicity in renal medulla: injury mediated by transport activity.

Polyene antibiotics such as amphotericin and nystatin increase membrane permeability and thus increase the amount of oxygen consumed in active electrolyte transport. In isolated perfused rat kidneys, the polyenes produced extensive injury to the medullary thick ascending limb, a segment of the nephron with limited oxygen supply. This damage was prevented if reabsorptive transport was inhibited by ouabain. Cell death under these circumstances thus appears to be mediated by increased oxygen demand for transport activity.

Disparate mechanisms for hypoxic cell injury in different nephron segments. Studies in the isolated perfused rat kidney.

Hypoxic injury was evaluated morphologically in the proximal tubule and in the medullary thick ascending limb of isolated rat kidneys perfused for 90 min without O2 or with various metabolic inhibitors. Inhibition of mitochondrial respiration (with rotenone, antimycin, oligomycin) or of intermediary metabolism (with monofluoroacetate, malonate, 2-deoxyglucose) caused reduction in renal oxygen consumption, renal function, and ATP content comparable with those elicited by oxygen deprivation. Metabolic inhibition produced hypoxiclike injury in the first portions of the proximal tubule, S1 and S2 ("clubbing" of microvilli, mitochondrial swelling), and the extent of damage was correlated with the degree of ATP depletion. In the third portion of the proximal tubule, S3, hypoxiclike damage (cytoplasmic edema or fragmentation) occurred most consistently when both aerobic and anaerobic metabolism were inhibited simultaneously. In the medullary thick ascending limb, none of the metabolic or mitochondrial inhibitors used could reproduce the injury of oxygen deprivation. Thus, the proximal tubule and the thick ascending limb have markedly different responses to cellular energy depletion, suggesting disparate mechanisms for hypoxic injury along the nephron.

Vascular endothelial growth factor induces manganese-superoxide dismutase expression in endothelial cells by a Rac1-regulated NADPH oxidase-dependent mechanism.

Vascular endothelial growth factor (VEGF) is a potent vascular endothelial cell-specific mitogen that modulates endothelial cell function. In the present study, we show that VEGF induces manganese-superoxide dismutase (MnSOD) mRNA and protein in human coronary artery endothelial cells (HCAEC) and pulmonary artery endothelial cells. VEGF-mediated induction of MnSOD mRNA was inhibited by pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI), and 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not with the nitric oxide synthase inhibitor L-NAME (N-monomethyl-L-arginine) or the xanthine oxidase inhibitor allopurinol. VEGF stimulation of MnSOD was also inhibited by adenoviral-mediated overexpression of catalase Cu, Zn-SOD and a dominant-negative form of the small GTPase component of NADPH oxidase Rac1 (Rac1N17). Treatment of HCAEC with VEGF resulted in a transient increase in ROS production at 20 min, as measured by 2,7-dichlorodihydrofluorescein oxidation. This effect was abrogated by expression of Rac1N17. Taken together, these findings suggest that VEGF induces MnSOD by an NADPH oxidase-dependent mechanism and that VEGF signaling in the endothelium is coupled to the redox state of the cell.

NADPH oxidase activity is required for endothelial cell proliferation and migration.

NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor- or serum-induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor-induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.

Effect of meglumine iothalamate on renal hemodynamics and function in the diabetic rat.

Diabetic rats were given an intravenous (external jugular vein) injection of 3 mg/kg of 60% meglumine iothalamate to test for this contrast agent's functional effects on the kidney. Rats were made diabetic by the intravenous injection of 60 mg/kg streptozotocin six months prior to the experiment and received no treatment during the interim. Glomerular filtration rate, renal blood flow, blood pressure, and sodium reabsorption were measured immediately before and at timed intervals after the administration of the contrast agent. In the diabetic rats, meglumine iothalamate caused a small and brief increase in renal blood flow followed by a sustained decrease; in the control animals an initial rapid rise was followed by a sustained elevation. Glomerular filtration rate also decreased slightly after contrast agent injection and remained depressed below baseline in the diabetic rats, whereas in the control animals it rose slightly above baseline and returned to control values by the end of the experiment. Sodium reabsorption was initially much higher than that of the controls, and it remained much higher throughout the experiment but decreased slightly just after injection of the contrast agent. Volume expansion of the diabetic animals decreased fractional sodium reabsorption to levels similar to those of the control rats but did not normalize the response of renal blood flow, glomerular filtration rate, or sodium reabsorption. Dehydration of control animals increased initial sodium reabsorption to levels similar to those of the diabetic animals but did not normalize the response to the contrast agent of the glomerular filtration rate, renal blood flow, or sodium reabsorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ioversol versus iothalamate on endothelin release and radiocontrast nephropathy.

RATIONALE AND OBJECTIVES: Certain radiocontrast agents, including iothalamate, iohexol, and ioxaglate, release the renal vasoconstrictor peptide endothelin from vascular endothelium in a way that might contribute to radiocontrast nephropathy. The effects of the nonionic, low osmolar agent, ioversol, on endothelin release and renal function are investigated. METHODS: Effects of ioversol were compared with equi-iodine doses of iothalamate when applied to cultured bovine aortic endothelial cells or injected into normal rats and rats preconditioned by uninephrectomy, salt depletion, and indomethacin (USIC) to develop radiocontrast nephropathy. RESULTS: In comparison with iothalamate, ioversol had a greatly reduced propensity to stimulate the release of endothelin, from cultured cells and when injected into anesthetized rats. Ioversol produced less renal vasoconstriction than did iothalamate, in control and in USIC rats, and the development of radiocontrast nephropathy, assessed by creatinine clearance and morphologic damage to the renal medulla, was largely avoided. CONCLUSIONS: These results strengthen the hypothesis that endothelin release induced by radiocontrast agents is correlated with their renal toxicity and therefore, may play a role in radiocontrast nephropathy.

Effect of myo-inositol on renal Na-K-ATPase in experimental diabetes.

The activity of Na-K-ATPase in the kidney is increased by experimental diabetes. Because the kidney is rich in myo-inositol and abnormal inositol metabolism has been implicated in early neural complications of diabetes, we studied the effect of myo-inositol supplementation on Na-K-ATPase activity in renal medullary and cortical homogenates of Sprague-Dawley rats made diabetic with streptozotocin. Myo-inositol (650 mg/kg) was administered by gavage daily for 1 and 2 weeks after induction of diabetes. Medullary Na-K-ATPase (mumol/mg protein/h) was increased at 1 week by approximately 60% in diabetic rats versus control (25.9 +/- 0.07 vs 16.3 +/- 0.7; P less than .01). This increase was completely prevented by myo-inositol supplementation, despite persistent hyperglycemia. At 2 weeks, similar results were seen; medullary Na-K-ATPase activity was increased by 50% in diabetic rats compared with control, and once again myo-inositol prevented this increase. Sorbinil, the aldose reductase inhibitor, was also administered by gavage (20 mg/kg) for 2 weeks and partially prevented the increase in medullary Na-K-ATPase activity (20.0 +/- 0.9; P less than .05). At both 7 and 14 days, Na-K-ATPase activity in the cortex of untreated diabetic rats was also significantly increased compared with nondiabetic control rats and the increase was prevented by myo-inositol or Sorbinil. Myo-inositol or Sorbinil did not reduce Na-K-ATPase activity of nondiabetic control rats, nor did they prevent the increase in medullary Na-K-ATPase in compensatory hypertrophy following uninephrectomy. Myo-inositol content of outer medulla was about five to six times that of cortex, but was unaltered by the diabetic state.(ABSTRACT TRUNCATED AT 250 WORDS)

HOVIS -- The Hertfordshire/Oxfordshire Violent Incident Study.

Violence in psychiatric inpatient units is a major and growing problem. Research interest has primarily focussed on patient characteristics. The role of staff factors and the antecedents of violent incidents has been neglected, despite the fact that staff factors and behaviour may be more readily amenable to change than patient characteristics. The HOVIS study sought to obtain the views of a sample of mental health nurses in current clinical practice about staff-related factors, which they perceive to contribute to, or protect against, the occurrence of violent incidents. A total of 108 nurses working in psychiatric acute admission, intensive care and low secure units, in two NHS Trusts were interviewed using a specially designed semistructured interview schedule. These nurses identified a variety of behaviours, clinical skills, personal characteristics and interpersonal skills that they believe impact on the occurrence of violent incidents. These findings are discussed in relation to their possible training and managerial implications.

Sympathetic system in potassium homeostasis.

The extrarenal disposal of potassium was studied in nephrectomized and adrenalectomized rats by measuring the rise in serum potassium produced during an infusion of 3 meq KCl/kg over 90 min. Adrenalectomy alone did not alter the volume of distribution of infused potassium in nephrectomized animals. When nephrectomy and adrenalectomy were combined with either insulin deficiency produced by streptozotocin or chemical sympathectomy induced by injection of 6-hydroxydopamine, potassium tolerance was significantly impaired. Hyperkalemia produced in chemically sympathectomized animals by potassium infusion was minimized by simultaneous infusion of epinephrine, an effect blocked by the beta-antagonist propranolol but not by the alpha-blocker phenoxybenzamine. These results suggest that extra renal uptake of potassium, in addition to being influenced by insulin and circulating catecholamines, is modulated by peripheral sympathetic activity.

Competition between sodium reabsorption and gluconeogenesis in kidneys of steroid-treated rats.

Kidneys of rats treated with methylprednisolone show altered substrate requirements for sodium reabsorption when perfused in vitro. Such kidneys synthesize glucose from lactate at twice the rate of control. Optimum sodium reabsorption is not seen with glucose, which is normally the preferred substrate. Sodium reabsorption is restored toward normal by the combination of glucose and butyrate, by pyruvate, or by 3-mercaptopicolinate. All of these results point to a metabolic adaptation in the kidney; butyrate may improve sodium reabsorption by sparing glucose, pyruvate is a gluconeogenic precursor and an effective fuel of respiration, and 3-mercaptopicolinate is an inhibitor of gluconeogenesis. In kidneys from rats treated with methylprednisolone there is an increased requirement for metabolic energy because of the increased rate of gluconeogenesis. It is suggested that the availability of energy from glucose oxidation is limited in part by the diversion of pyruvate back to glucose. Under these special circumstances, gluconeogenesis competes with sodium reabsorption in the intact kidney.

Polyethylene glycol effect on the oxygenated and hypoxic isolated perfused rat kidney.

Polyethylene glycol protects against O2 deprivation after clamping of the renal artery or norepinephrine infusion and in hypoxic primary cell culture. Isolated perfused kidneys under hypoxic conditions develop morphological alterations in all segments of the proximal tubule and medullary thick ascending limb. In an attempt to ameliorate the effect of hypoxia, rat kidneys were perfused for 90 min with regularly oxygenated (95% O2 + 5% CO2) or hypoxic perfusate (95% N2 + CO2) supplemented with 8-12% polyethylene glycol (MW approximately 8000). In oxygenated and hypoxic kidneys, polyethylene glycol produced similar changes in S1-S2 segments consisting of reduction of cell thickness and organelle compaction with internalization of brush border into the tubulo-vesicular system. In the S3 segment, the cellular volume loss was more limited; the brush border was transformed to membranous whorls and the cytoplasm contained large, irregular, clear zones. Mitochondrial swelling was pronounced in the hypoxic proximal tubules. Polyethylene glycol quantitatively increased and emphasized the damage in the medullary thick ascending limb. Inclusion of 10(-2) M ouabain preserved the medullary thick ascending limb from hypoxic injury and polyethylene glycol had no effect on this undamaged epithelium. Thus, polyethylene glycol affects renal tubules on the basis of their known water permeability and does not protect against but rather worsens hypoxic injury in the medullary thick ascending limb.

Role of alpha-adrenergic hormones in potassium homeostasis in the rat.

To determine whether alpha-adrenergic stimulation impairs extrarenal potassium disposal in the rat, we studied the effect of an alpha-agonist and antagonist on the rise in plasma potassium concentration produced by a potassium infusion of 3 mEq KCl/kg over a 90-minute period to nephrectomized rats. Compared with KCl alone, the alpha-agonist phenylephrine caused more than a 25% increment in the peak plasma potassium, and reduced the volume of distribution of the potassium load. Addition of the alpha-blocker, phentolamine, to phenylephrine completely abolished the increment in potassium caused by KCl infusion. There were no differences in plasma bicarbonate, arterial pH, or plasma glucose levels among the groups. When an identical protocol was performed in nephrectomized rats also prepared by adrenalectomy, induction of diabetes, and chemical sympathectomy, the rise in plasma potassium level after KCl infusion was again augmented by phenylephrine. Addition of phentolamine also markedly blunted the increment in potassium in this group. These results show that alpha-adrenergics impair extrarenal potassium disposal in the rat. The effect does not depend on endogenous insulin or other potassium-regulatory factors. alpha-Adrenergics appear to play an important role in extrarenal potassium homeostasis.

Effect of glycine on medullary thick ascending limb injury in perfused kidneys.

The addition of 2 mM glycine to the recirculating perfusate of isolated perfused rat kidneys almost completely prevented the severe morphological injury to tubular cells lining the medullary thick ascending limb (mTAL) that normally develops in this preparation. Glycine was similarly effective in reducing mTAL injury associated with hypoxic perfusion, indomethacin and amphotericin. Fractional reabsorption of sodium was increased with glycine, without any change in perfusate flow to the whole kidney and without consistent improvement in GFR. L-alanine demonstrated a similar though less pronounced cytoprotective action, but glutamine, cysteine, glutamate, cysteine plus glutamate, 1-serine and 4-aminoisobutyric acid all had little or no effect in preventing severe mTAL injury. The protective effect of glycine was unimpaired by the arginine analogue NG-monomethyl-l-arginine (L-NMMA), suggesting that the endothelial-derived relaxing factor, NO, was not involved. The action of glycine was not reduced by the addition of a substrate (benzoate) or a product (hippurate) of the glycine N-acyltransferase reaction. Glycine did not depress the respiration of dispersed mTALs prepared from rat kidneys. The cytoprotective effect of glycine in the mTAL of perfused kidneys, shared with l-alanine, appears to be relatively specific for these amino acids and probably unrelated to a diminution in cell work.

Relationship among gluconeogenesis, QO2, and Na+ transport in the perfused rat kidney.

The relationship among sodium transport (TNa), oxygen consumption (QO2), and gluconeogenesis was studied in isolated perfused rat kidneys in which glucose formation was enhanced by providing pyruvate as a substrate and by prior treatment with methylprednisolone. TNa was increased abruptly by increasing perfusion pressure as to increase GFR or by lowering the albumin concentration of a hyperoncotic perfusate as to allow glomerular filtration to occur. Increases in TNa of 40% were accompanied by little or no increase in QO2, whereas gluconeogenesis decreased 55-80%. Conversely, a decrease in perfusion pressure that lowered TNa produced an increase in glucose formation without a change in QO2. When gluconeogenesis was blocked with 0.15 mM 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, QO2 increased together with TNa as perfusion pressure was raised. The results suggest that the energy needed for ion transport by the kidney may under some circumstances be borrowed from nontransport functions and, therefore, that basal oxygen consumption may vary with the rate of reabsorptive transport.

Ouabain inhibition of gill Na-K-ATPase: relationship to active chloride transport.

Ouabain circulating in blood inhibits Na-K-ATPase in the gills of seawater eels at a concentration similar to that necessary for inhibition in vitro. By contrast, a much higher concentration is required when ouabain is applied to the exterior of the gill. Inhibition by external ouabain occurs only when the drug gains access to the circulation of the fish, as evidenced by simultaneous inhibition of Na-K-ATPase in the kidney. These results suggest that the Na-K-ATPase of gill chloride cells faces inward, lining intracytoplasmic tubular channels continuous with the extracellular fluid. Inhibition of gill Na-K-ATPase by ouabain in intact salt water eels results in almost complete inhibition of the efflux of both Na+ and Cl-. The efflux is tritiated water was much less reduced, to 60% of normal. Since chloride is actively transported outward across the gill of seawater teleosts, it is suggested that active chloride transport is coupled to Na-K-ATPase. A neutral sodium chloride carrier is postulated that is energized by the movement of sodium from extracellular fluid down its electrochemical gradient into the chloride cell.

Mechanism of glycine protection in hypoxic injury: analogies with glycine receptor.

Addition of glycine to the recirculating perfusate of isolated perfused rat kidneys protects against hypoxic injury to the medullary thick ascending limb and slows functional deterioration in the course of perfusion. This effect is dependent on dose; the earliest significant protection is seen at 0.25 mM, and the protective effects increase as glycine concentration is increased to 2 mM, the highest level tested. Two specific agonists of the strychnine-insensitive (NMDA) glycine receptor in neural membranes, 1-aminocyclopropane carboxylic acid (ACC) and d-serine, also exerted a cytoprotective effect at a concentration of 2 mM. On the other hand, 1-serine and taurine, ineffective agonists of the NMDA-glycine receptor but effective agonists of the strychnine-sensitive glycine receptor, had no protective effect in this system. Two antagonists to glycine at its binding site on the N-methyl-D-Aspartate (NMDA) receptor, 7-chlorokynurenic acid (2 mM) and indole-2-carboxylic acid (12.5 mM), did not reverse the cytoprotective action of 0.25 mM glycine. The data are consistent with a ligand-acceptor type of interaction to account for cytoprotection. The configuration of the glycine acceptor may resemble, but is not identical with, that of certain glycine receptors in the nervous system.

Lactate increases potassium secretion by perfused rat kidney.

The effect of exogenous metabolic substrates on K+ secretion was evaluated in the isolated perfused rat kidney in the presence of 2-deoxyglucose and 2-tetradecylglycidic acid to inhibit utilization of glucose and fatty acids from endogenous sources. L-Lactate (15 mM) added to the perfusion medium enhanced renal oxygen consumption (4.0 +/- 1.1 mumol X min-1 X g-1 vs. 2.0 +/- 1.0 without lactate) while decreasing fractional excretion of sodium (19.3 +/- 2.4% vs. 47.3 +/- 1.8). L-Lactate markedly increased the fractional excretion of K+ to 181 +/- 29% compared with 68 +/- 12% without lactate (P less than 0.001). The poorly metabolized isomer D-lactate did not alter these parameters. The addition of alpha-ketoglutarate only slightly increased K+ excretion. In the absence of metabolic inhibitors and in the presence of glucose (5 mM), L-lactate also increased K+ excretion significantly more than did D-lactate (108 +/- 19% vs. 69 +/- 11, P less than 0.02). At the end of 90 min of perfusion with L-lactate medium, K+ concentration in the perfusate dropped from 4.7 +/- 0.05 to 3.2 +/- 0.2 meq/liter (vs. 3.8 +/- 0.1 meq/liter with D-lactate, P less than 0.005) without differences in glomerular filtration rate or sodium excretion. L-Lactate appears to increase K+ secretion by preferential metabolic stimulation of the distal tubule, a process that may help in vivo to prevent hyperkalemia in lactic acidosis.

Effect of water diuresis and water restriction on expression of HSPs-27, -60 and -70 in rat kidney.

Expression of HSP-27, HSP-60 and HSP-70 was estimated in the cortex, outer medulla and inner medulla (papilla) of rats undergoing water diuresis or water restriction for two days. The mRNAs for HSP-27 and HSP-60 in renal papilla were two- to threefold greater in rats during water restriction than in those excreting a dilute urine, but levels of mRNA for HSP-70 were not reduced by water diuresis and Western analysis for HSP-70 protein showed no difference between water-loaded and water-restricted animals.

Glycine reduces early renal parenchymal uptake of cisplatin.

We evaluated the effect of glycine infusions on the early renal uptake of cisplatin, measured one hour after cisplatin was injected, as well as five days following cisplatin administration. Glycine (1.25 mmol per 100 g body wt) markedly attenuated the early uptake of platinum by the kidney, an effect not observed with control infusions of saline or of L-alanine. The kidney content of platinum at five days, on the other hand, was similar in glycine-treated animals and saline controls. Early inhibition of renal uptake of platinum may be responsible for glycine's protective action in experimental cisplatin nephrotoxicity.

Transport-dependent anoxic cell injury in the isolated perfused rat kidney.

The hypothesis that decrease in energy demand may prevent anoxic cell damage has been examined in the medullary thick ascending limb of isolated perfused rat kidneys exposed to oxygen deprivation. The effects of decreasing active reabsorptive transport in the medullary thick ascending limb were observed on the extensive damage regularly induced by hypoxic perfusion (gassed with no oxygen) or potassium cyanide. Anoxic injury was consistently attenuated or abolished if reabsorptive transport was decreased with ouabain or furosemide or by halting the glomerular filtration rate with the use of a hyperoncotic medium (nonfiltering kidney). Comparison of the injury generated by warm ischemia for identical time periods showed that complete ischemia does not reproduce the severe lesions seen during hypoxic perfusion. These results suggest that transport activity is a determining factor of anoxic cell death in the thick ascending limb of Henle's loop.