Obstruction of proximal tubules initiates cytoresistance against hypoxic damage

Following acute tubular necrosis (ATN), cytoresistance to further renal injury results. However, the initiating events and the subcellular determinants of this phenomenon have not been defined. Since tubular obstruction is a consequence of ATN, this study evaluated whether it alters tubular susceptibility to hypoxic damage. Extrarenal obstruction (ureteral ligation in rats) was used for this purpose to dissociate obstructive effects from those of ATN. Twenty-four hours following ureteral ligation or sham surgery, cortical proximal tubular segments (PTS) were isolated and subjected to hypoxic (15 or 30 min)/reoxygenation injury. Since oxidant stress, cell Ca2+ overload, and PLA2 attack are purported mediators of hypoxic/reoxygenation injury, degrees of FeS04, Ca2+ ionophore, and phospholipase A2-induced PTS damage also were assessed. The cell injury (% LDH release) which resulted from each of the above was consistently less in PTS obtained from obstructed kidneys. This cytoresistance: (a) did not require prior uremia to develop (seen with unilateral obstruction); (b) it did not appear to correlate with a tubular proliferative response (assessed by proliferating cell nuclear antigen expression); (c) it was uninfluenced by early tubular repair (unchanged by 24 hrs of obstruction release); and (d) it occurred without increased heat shock protein (HSP-70) or antioxidant enzyme (superoxide dismutase, catalase) expression. Total adenylate pools were higher in obstructed versus control PTS during injury; however, this appeared to be a correlate of the protection, rather than a mediator of it. In contrast, obstructed tubules manifested a primary increase in plasma membrane resistance to PLA2 attack (approximately 3-fold less lysophosphatidylcholine and free fatty acid generation in obstructed vs. control PTS during incubation with exogenous PLA2). In sum, these results indicate that: (1) tubular obstruction protects PTS from injury, suggesting that its development during ATN may initiate cytoresistance; and (2) this cytoresistance appears to be mediated, at least in part, by a direct increase in plasma membrane resistance to PLA2 and potentially other forms (such as, oxidant stress, cytosolic Ca2+ loading) of attack.     

Prostaglandin E1 induces c-Fos and Myc proteins and protects rat hippocampal cells against hypoxic injury

A procedure for synthesizing a nucleoside 1-thiotriphosphate in a single reaction vessel beginning with the nucleoside, PSCl3, and PPi is described. Reaction of the dried nucleoside with PSCl3 in anhydrous triethylphosphate is followed by addition of the tetrabutylammonium salt of PPi. Addition of excess triethylamine precipitates the nucleotides. The crude product is dissolved in water, and the nucleoside 1-thiotriphosphate is purified in 24% to 50% yield by chromatography. The method is applied to the synthesis of dATP alpha S, dGTP alpha S, UTP alpha S and dTTP alpha S.     

Loop diuretics reduce hypoxic damage to proximal tubules of the isolated perfused rat kidney

The straight portion (S3) of the proximal tubule lies in close proximity to the thick ascending limbs (TALs) at the cortico-medullary junction. Since a delicate balance exists between oxygen demand and the limited oxygen supply in this region, we hypothesized that reduction of thick limb metabolic activity might augment oxygen availability to S3 segments, which depend heavily upon aerobic metabolism, and prevent hypoxic damage. The degree of functional deterioration and morphological damage to S3 was assessed in isolated rat kidneys perfused with an erythrocyte-free medium. Bumetanide (10(-5) M) and furosemide (10(-4) M) reduced S3 fragmentation from 9.8 +/- 3.9% of tubules in controls to 0 and 1.4 +/- 0.9%, respectively (P < 0.0005). Tubular glucose reabsorption was better preserved in kidneys exposed to loop diuretics than in control kidneys (P < 0.01), and urinary alkaline phosphatase (P < 0.05) and the total amount of LDH released into the perfusate and urine (P < 0.01) were lower in the treatment groups. Morphological damage to S3 was closely correlated with medullary TAL necrosis (r = 0.66, P < 0.001), urinary alkaline phosphatase excretion (r = 0.89, P < 0.001) and glycosuria (r = 0.83, P < 0.001). We conclude that under hypoxic conditions TALs and S3 segments may compete with each other for a limited oxygen supply. Reduction of active transport in the mTAL might augment oxygen availability to S3 segments and improve their survival.     

Hypoxia decreases calcium influx into rat proximal tubules

Renal ischemia results in adenosine triphosphate (ATP) depletion, particularly in cells of the proximal tubule (PT), which rely heavily on oxidative phosphorylation for energy supply. Lack of ATP leads to a disturbance in intracellular homeostasis of Na+, K+ and Cl-. Also, cytosolic Ca2+ levels in renal PTs may increase during hypoxia [1], presumably by a combination of impaired extrusion and enhanced influx [2]. However, Ca2+ influx was previously measured using radiolabeled Ca2+ and at varying partial oxygen tension [2]. We have now used to Mn2(+)-induced quenching of fura-2 fluorescence to study Ca2+ influx in individual rat PTs during normoxic and hypoxic superfusion. Normoxic Ca2+ influx was indeed reflected by the Mn2+ quenching of fura-2 fluorescence and this influx could be inhibited by the calcium entry blocker methoxyverapamil (D600; inhibition 50 +/- 2% and 35 +/- 3% for 10 and 100 mumol, respectively). La3+ completely blocked normoxic Ca2+ influx. Hypoxic superfusion or rat PTs did not induce an increase in Ca2+ influx, but reduced this influx to 79 +/- 3% of the normoxic control. We hypothesize that reducing Ca2+ influx during hypoxia provides the cell with a means to prevent cellular Ca2+ overload during ATP-depletion, where Ca2+ extrusion is limited.     

Estradiol protects against ischemic injury

Clinical studies demonstrate that estrogen replacement therapy in postmenopausal women may enhance cognitive function and reduce neurodegeneration associated with Alzheimer's disease and stroke. This study assesses whether physiologic levels of estradiol prevent brain injury in an in vivo model of permanent focal ischemia. Sprague-Dawley rats were ovariectomized; they then were implanted, immediately or at the onset of ischemia, with capsules that produced physiologically low or physiologically high 17beta-estradiol levels in serum (10 or 60 pg/mL, respectively). One week after ovariectomy, ischemia was induced. Estradiol pretreatment significantly reduced overall infarct volume compared with oil-pretreated controls (mean+/-SD: oil = 241+/-88; low = 139+/-91; high = 132+/-88 mm3); this protective effect was regionally specific to the cortex, since no protection was observed in the striatum. Baseline and ischemic regional CBF did not differ between oil and estradiol pretreated rats, as measured by laser Doppler flowmetry. Acute estradiol treatment did not protect against ischemic injury. Our finding that estradiol pretreatment reduces injury demonstrates that physiologic levels of estradiol can protect against neurodegeneration.     

Fatty acid metabolism in hypoxic rat liver

Fatty acid metabolism was investigated in adult male albino rats exposed to hypobaric hypoxia at 25,000 ft simulated altitude for 6 h at 32 degrees C. Oxidation and esterification of palmitic acid-1-14C and de novo lipogenesis from acetate-1-14C were studied. Palmitic acid-1-14C oxidation in liver slices was normal while acetoacetate formation was increased. In vivo esterification of palmitic acid-1-14C to form triglycerides was increased while formation of phosphatidylcholine and phosphatidylethanolamine was observed to decrease. Decreased incorporation into plasma phosphatidylcholine with unaltered total activity in plasma triglycerides was observed. The incorporation of acetate-1-14C was observed to remain unaltered in triglycerides and phospholipids of liver with a similar pattern in the plasma indicating unaltered de novo lipogenesis. There appears to be increased esterification of fatty acids with probably impaired release of triglycerides into plasma while fatty acid biosynthesis remains unaffected.     

Ursodeoxycholate protects against ethanol-induced liver mitochondrial injury

The purpose of this work was to examine whether ursodeoxycholate (UDC), a hydrophilic bile salt, could reduce mitochondrial liver injury from chronic ethanol consumption in rats. Animals were pair-fed liquid diets containing 36% of calories as ethanol or isocaloric carbohydrates. They were randomly assigned into 4 groups of 7 rats each and received a specific treatment for 5 weeks: control diet, ethanol diet, control diet + UDC, and ethanol diet + UDC. Respiratory rates of isolated liver mitochondria were measured using a Clark oxygen electrode with sodium succinate as substrate. Mitochondria from rats chronically fed ethanol demonstrated an impaired ability to produce energy. At the fatty liver stage, the ADP-stimulated respiration (V3) was depressed by 33%, the respiratory control ratio (RC) by 25% and the P/O ratio by 15%. In ethanol-fed rats supplemented with UDC, both the rate and efficiency of ATP synthesis via the oxidative phosphorylation were improved: V3 was increased by 35%, P/O by 8%. All the respiratory parameters were similar in control group and control + UDC group. On the other hand, the number and size of mitochondria were assessed by electron microscopy and computer-assisted quantitative analysis. The number of mitochondria from ethanol-treated rats was decreased by 29%, and they were enlarged by 74%. Both parameters were normalized to control values by UDC treatment. These studies demonstrate that UDC has a protective effect against ethanol-induced mitochondrial injury by improving ATP synthesis and preserving liver mitochondrial morphology. These UDC positive effects may contribute to the observed decrease in fat accumulation and may delay the progression of alcoholic injury to more advanced stages.     

Arachidonic and palmitic acid utilization in aged rat brain areas

We have previously demonstrated that the arachidonic acid (20:4) incorporation into brain lipids differs according to the age of the animals used and the experimental conditions adopted. These differences led to a further investigation of arachidonic acid uptake in both aged and adult rat brains, its transformation into CoA derivatives, its incorporation into diacyl-glycerols and polar lipids, and finally its oxidation to CO2. These metabolic parameters were then compared with those obtained after using the saturated fatty acid palmitate (16:0). In both cases slices or mitochondria from different brain areas of 24-month-old and 4-month-old rats were examined. The results obtained indicate that the uptake of the fatty acids into cells is not modified by age. However, the successive metabolic transformations of the acids are altered to a considerable extent. In particular, in 24-month-old animals (compared with 4-month-old rats) there is a significant decrease of 20:4 in its incorporation into lipids as well as its oxidation to CO2, while arachidonoyl-CoA content increases by about 50%. This increased amount of CoA derivative, which has a potent detergent effect, may interfere with membrane structure and affect membrane physiological functions. Furthermore, because the free arachidonate pool is maintained in a dynamic equilibrium with its esterified forms, the final result may be a perturbation of this equilibrium.     

Andrographolide protects rat hepatocytes against paracetamol-induced damage

The authors' objective was to determine if, in the absence of known coronary artery disease, ST-T changes suggestive of silent ischemia on the admission electrocardiogram (ECG) identify a group of patients at high risk for cardiac event or death. A prospective cohort study was undertaken at the university hospital of a tertiary care center. All patients admitted to the hospital during the 5-month study period were screened. The authors found 54 patients with risk factors but no symptoms of coronary artery disease whose admission ECGs showed silent ischemia (ischemia group), and 71 patients with similar risk of coronary artery disease but without admission ECGs showing silent ischemia (control group). Three-week and 6-month incidences of angina, myocardial infarction, and death among patients in the silent ischemia and control groups were compared. Seven (13%) patients in the silent ischemia group had cardiac events or noncardiac death in the subsequent 3 weeks versus one (1%) noncardiac death in the control group (p < 0.02). At 6 months, eight (15%) patients in the silent ischemia group versus two (3%) in the control group had cardiac events (p = 0.02). It is concluded that among patients with risk factors but no symptoms of coronary artery disease, silent ischemia on the admission ECG is associated with an increased likelihood of short-term death or cardiac event.     

Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo

Uric acid is a well-known natural antioxidant present in fluids and tissues throughout the body. Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke. We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide. Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult. Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid. Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid. These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function. Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome. These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions.     

Acetylsalicylic acid increases tolerance against hypoxic and chemical hypoxia

BACKGROUND AND PURPOSE: Treatment with acetylsalicylic acid (ASA) is established for secondary stroke prevention. Recent studies showed neuroprotection of ASA against glutamatergic excitants. The goal of this study was to investigate the time course of neuroprotection of ASA against indirect excitotoxicity by hypoxic hypoxia and chemical hypoxia. METHODS: Population spike amplitude (PSA) and ATP content were measured in hippocampal slices from untreated control animals (c-slices) and slices prepared from animals pretreated in vivo with a single intraperitoneal injection of 20 mg/kg body wt ASA 1 to 48 hours before slice preparation (p-slices). RESULTS: Posthypoxic recovery of PSA was 30% in c-slices (15 minutes of hypoxia, 45 minutes of recovery). When c-slices were treated in vitro for 15 minutes with 20 mg/L ASA 30 minutes before hypoxia, posthypoxic recovery improved to 82 +/- 4% (mean +/- SE, P < .01). In p-slices, posthypoxic recovery of PSA improved in a time-dependent manner. With a time interval of 1 hour between in vivo pretreatment with ASA and slice preparation, posthypoxic recovery of PSA was 64 +/- 16% (P < .05). With time intervals of 6 hours, 24 hours, and 48 hours, posthypoxic recovery of PSA was 87 +/- 19% (P < .01), 59 +/- 12%, and 40 +/- 9%, respectively. Pretreatment with ASA in vitro or in vivo decreased the decline of ATP content during hypoxic hypoxia and chemical hypoxia (inhibition of succinic dehydrogenase by 3-nitropropionic acid). When extracellular glucose was reduced to 4 mmol/L, no difference was observed between c-slices and p-slices. CONCLUSIONS: We conclude that ASA is neuroprotective against hypoxic hypoxia and chemical hypoxia and delays the decline of intracellular ATP content.     

Progesterone protects against lipid peroxidation following traumatic brain injury in rats

Large scale use of lysozyme for periplasmic release has been impeded by the cost of the pure enzyme and its subsequent presence as a contaminant in later downstream processing steps. In this paper, we discuss the use of lysozyme for pilot scale recovery of a periplasmic enzyme from E. coli. The effects of concentration of sucrose, lysozyme and cells on periplasmic enzyme release were examined. Lysozyme concentration can be reduced 5-fold from previous reports and a reduction in sucrose concentration from 20 to 15% (w/v) allows an improvement in centrifugal harvesting by reducing viscosity. High levels of release were still achieved using this technique and further improvements in yield were obtained by optimising other components of the releasing mixture. Results show that some release is still achieved in circumstances where no lysozyme use is possible. Results also indicate that a substantial proportion (up to 70%) of lysozyme remains bound to the cellular debris after its action and is removed with this material.     

Postexposure treatment with aminophylline protects against phosgene-induced acute lung injury

Our model of the human m1 muscarinic receptor has been refined on the basis of the recently published projection map of bovine rhodopsin. The refined model has a slightly different helix arrangement, which reveals the presence of an extra hydrophobic pocket located between helices 3, 4 and 5. The interaction of series of agonists and antagonists with the m1 muscarinic receptor has been studied experimentally by site-directed mutagenesis. In order to account for the observed results, three-dimensional models of m1 ligands docked in the target receptor are proposed. Qualitatively, the obtained models are in good agreement with the experimental observations. Agonists and partial agonists have a relatively small size. They can bind to the same region of the receptor using, however, different anchoring receptor residues. Antagonists are usually larger molecules, filling almost completely the same pocket as agonists. They can usually produce much stronger interactions with aromatic residues. Experimental data combined with molecular modelling studies highlight how subtle and diverse receptor-ligand interactions could be.     

Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug-induced damage

The multidrug resistance protein 1 (MRP1) gene encodes a transporter protein that helps to protect cells against xenobiotics. Elevated levels of MRP1 in tumor cells can result in active extrusion of a wide range of (anticancer) drugs with different cellular targets, a phenomenon called multidrug resistance (MDR). To explore the protective function of the mouse mrp1 protein during drug treatment, we investigated the toxicity caused by the anticancer drug etoposide-phosphate (ETOPOPHOS) in mice lacking the mrp1 gene (mrp1(-/-) mice). We show here that the lack of mrp1 protein results in increased etoposide-induced damage to the mucosa of the oropharyngeal cavity and to the seminiferous tubules of the testis. The high concentrations of mrp1 that we find in the basal layers of the oropharyngeal mucosa and in the basal membrane of the Sertoli cells in the testis apparently protect wild-type mice against this tissue damage. We also find drug-induced polyuria in mrp1(-/-) mice, which correlates with the presence of mrp1 protein in the urinary collecting tubules, the major site of kidney water reabsorption. Our results indicate that specific inhibitors of MRP1 used to reverse MDR, in combination with carcinostatic drugs transported by MRP1, might lead to drug-induced mucositis, (temporary) infertility, and diabetes insipidus.     

Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.     

Arachidonic acid-induced dye uncoupling in rat cortical astrocytes is mediated by arachidonic acid byproducts

Arachidonic acid (AA) induced a concentration- and time-dependent reduction in gap junction-mediated dye coupling between cultured astrocytes. The effect was greatly diminished by inhibition of cyclooxygenases and lipoxygenases. The action of a low concentration of AA (5 microM) was also prevented by Ca2+-free extracellular solution or a high concentration of melatonin, a potent free radical scavenger, but not by Nomega-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor. Thus, this effect may depend on Ca2+ influx and oxygen free radicals but not on NO generation. Cellular uncoupling induced by a high (100 microM), but not a low, AA concentration was rapidly reversed by washing with albumin containing solution. After reversal from 5 min but not 2.5 min inhibition with a high AA concentration dye coupling between astrocytes became refractory to a low concentration of AA, suggesting desensitization of the response elicited by a low concentration of the fatty acid. Dye uncoupling occurred without changes in levels and state of phosphorylation (immunoblotting and 32P-incorporation) of connexin43, the main astrocyte gap junctional protein. However, maximal cell uncoupling induced by a low (Slow action) but not by a high (Fast action) AA concentration was paralleled by a reduction in connexin43 (immunofluorescence) at cell-to-cell contacts. It is proposed that the AA-induced dye uncoupling is mediated by byproducts that induce rapid channel closure or slow removal of connexin43 gap junctions.     

Endocytosis in renal proximal tubules. Experimental electron microscopical studies of protein absorption and membrane traffic in isolated, in vitro perfused proximal tubules

The present study provides at least partly answers to some of the questions outlined in the introduction (see also Figs. 2 and 3): Endocytosis and intracellular transport of ferritin, HRP and insulin tracers (125I-insulin, native insulin and insulin-gold) was followed by use of EM-autoradiography, immunocytochemistry and cytochemistry. Proteins are internalized into endocytic vacuoles and transferred to the lysosomes for degradation. Tracers were not transferred to the Golgi apparatus. 125I-insulin is internalized by specific receptor mediated endocytosis from the apical plasma membrane, substantiating the hypothesis that specific endocytosis receptors are responsible for reabsorption of certain proteins. The binding sites are localized in endocytic invaginations and in the microvillus membrane. The binding sites in the invaginations are responsible for endocytosis, whereas the function of the microvilli binding sites is unclear, but they possess the ability to migrate in the plane of the microvillus membrane. Binding to specific binding sites and subsequent internalization of insulin takes place with high efficiency corresponding to more than 50% of the perfused load. Not all proteins are reabsorbed with high efficiency e.g. EGF which has similar molecular weight and pI is shown to be reabsorbed with substantially lower efficiency (about 4%). Binding and absorption efficiency of insulin may also change due to alterations in flow rate and perfused loads of protein: The load determines the magnitude of uptake and the flow rate determines the efficiency in binding and uptake. These changes are suggested to be caused by concomitant changes in the mean luminal concentration. The reabsorption process for insulin is efficient and of large capacity, and is only saturable (Michaelis-Menten kinetics) at very high concentrations of insulin. The proximal tubular internalization and degradation of 125I-insulin reach steady state rapidly. The processing can be described by a two-compartment model with t1/2 for transfer of 125I-insulin to lysosomes of 8.5 min and for lysosomal degradation of 72 min. 125I-PYY a linear peptide with similar molecular weight as EGF and insulin is not endocytosed but extracted with high efficiency (75% removed) by degradation by brush border peptidases and a substantial transtubular transport of TCA-precipitable PYY takes place by a paracellular route. A small vesicular transport of colloidal tracers was demonstrated constituting about 0.5% of the endocytosed amount. A method for covalently cross-linking insulin tracers to apical binding sites is described and evaluated. Recycling of apical binding sites was estimated to be very efficient and did not involve lysosomes or the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organic solutes in fluid absorption by renal proximal convoluted tubules

Proximal convoluted tubules were dissected from rabbit kidneys and perfused with artificial solutions in vitro. The effect of various organic solutes on rate of fluid absorption and transepithelial voltage was tested by removing solutes from or adding them to perfusate and/or bath. Omission of albumin from the bath caused rate of fluid absorption to descrease 33% without any change in voltage. Omission of glucose, lactate, alanine, and citrate from the bath had no effect. In contrast, when they were removed from perfusate, rate of fluid absorption fell by 45-75% (depending on whether they were replaced by NaCl or mannitol and NaCl), and voltage (normally negative in lymen) decreased to near zero. Adding glucose or alanine individually to perfusate caused a small increase in rate of fluid absorption and a relatively large increase in voltage. alpha-Methyl-D-glucoside and cycloleucine (which are transported but not metabolized) had effects similar to glucose and alanine, except that voltage changes were not as great. Phlorizin (10(-5) M in perfusate) had the same effect as removing glucose from perfusate. When glucose and alanine were added to perfusate, epithelial cell swelled significantly. Lactate and citrate also caused rate of fluid absorption to increase when they were added to perfusate, but they did not affect transepithelial voltage nor did they cause cells to swell significantly. Possible mechanisms of these effects and the role of organic solutes in fluid absorption by proximal convoluted tubules are discussed.