Effect of quercetin on hypoxic injury in freshly isolated rat proximal tubules

The bioflavonoid quercetin, which has antioxidant properties, protects renal tubular epithelial cells from oxidant-induced injury by inhibiting lipid peroxidation. We examined the effect of quercetin on hypoxia-induced injury in freshly isolated rat renal proximal tubules. Hypoxia induced rapid loss of cellular ATP, followed by functional and structural alterations measured as a decrease in tubular potassium content and sequentially by an increase in lactate dehydrogenase release. Furthermore, hypoxia increased lipid peroxidation, measured as thiobarbituric acid-reactive substances. Quercetin significantly inhibited hypoxia-induced functional and structural tubular injury in addition to lipid peroxidation but did not alter hypoxia-induced ATP depletion. These results demonstrate the potency of the bioflavonoid quercetin in protecting proximal tubules from hypoxic injury, which is independent of tubular energy metabolism and may be related to the inhibition of lipid peroxidation.     

Hypoxia induces a specific set of stress proteins in cultured endothelial cells.

Vascular endothelial cells (EC) are the initial cells within the vascular wall exposed to decreases in blood ambient oxygen concentration. The mechanisms by which they tolerate low levels of oxygen are unknown, but may parallel the response to other cellular stresses, such as heat shock. After 4-8 h of hypoxia, we found a decrease in total protein synthesis in both cultured bovine aortic and pulmonary arterial EC. SDS-PAGE and autoradiographic analysis of [35S]methionine-labeled proteins demonstrated the concomitant induction of a specific set of proteins (Mr 34, 36, 47, and 56 kD) in both cell types. These hypoxia-associated proteins (HAPs) were cell-associated and up-regulated in a time- and oxygen concentration-dependent manner. Comparison of these proteins with heat shock proteins (HSPs) demonstrated that HAPs were distinct from HSPs. EC maintained chronically in 3% O2 continued to synthesize elevated levels of HAPs, yet further up-regulated these proteins when exposed to 0% O2. The presence of five times the normal media glucose concentration did not alter the appearance of HAPs. Hypoxia sensitive renal tubular epithelial cells up-regulated no proteins corresponding to HAPs and were irreversibly damaged within 8 h of exposure to 0% O2. In vitro translation experiments demonstrated that the steady-state level of several mRNAs was higher in the anoxic EC than in normoxic EC and encoded for proteins of Mr 32, 35, 37, 40, and 48 kD that were different from proteins encoded by HSP mRNAs. The induction of HAPs during acute hypoxia and their continued synthesis in chronic hypoxia suggest that HAPs may be important in the maintenance of endothelial cell integrity under conditions of decreased ambient oxygen.     

31P nuclear magnetic resonance study of steady-state adenosine 5'-triphosphate levels during graded hypoxia in the isolated perfused rat kidney

1. A model of controlled hypoxia in the isolated perfused rat kidney has been used to compare the extent of reduction in the steady-state level of adenosine 5'-triphosphate (ATP) from that initially observed with alterations in renal function and with the development of tubular cell injury. 2. ATP depletion was observed in response to decreased total oxygen delivery even when delivery greatly exceeded consumption and the venous oxygen tension remained in excess of 150 mmHg. 3. Increases in the fractional excretion of sodium occurred progressively below an apparent threshold value of whole kidney ATP of approximately 80% of the baseline. 4. With modestly decreased oxygen delivery, cellular injury was confined to deep proximal tubule and medullary thick ascending limb of Henle's loop. Severely decreased oxygen delivery rates were associated with cellular damage spreading throughout the cortex. 5. Even the smallest reductions in whole kidney ATP were associated with morphological damage to tubular cells. The extent of reduction in whole kidney ATP was closely correlated and approximately equivalent to the calculated volume of injured cells. 6. Our results indicate that reduction in whole kidney ATP during decreased oxygen delivery is a valid marker of the extent of injurious cellular hypoxia and are consistent with the view that cellular ATP concentrations in hypoxia are markedly inhomogeneous. They support the hypothesis that specific regions of the perfused kidney become critically hypoxic and develop cellular injury while overall oxygen delivery remains high. Areas at risk include deep proximal tubule as well as the medullary thick ascending limb of Henle's loop.     

Gene Therapy by Targeted Adenovirus-mediated Knockdown of Pulmonary Endothelial Tph1 Attenuates Hypoxia-induced Pulmonary Hypertension

Serotonin is produced by pulmonary arterial endothelial cells (PAEC) via tryptophan hydroxylase-1 (Tph1). Pathologically, serotonin acts on underlying pulmonary arterial cells, contributing to vascular remodeling associated with pulmonary arterial hypertension (PAH). The effects of hypoxia on PAEC-Tph1 activity are unknown. We investigated the potential of a gene therapy approach to PAH using selective inhibition of PAEC-Tph1 in vivo in a hypoxic model of PAH. We exposed cultured bovine pulmonary arterial smooth muscle cells (bPASMCs) to conditioned media from human PAECs (hPAECs) before and after hypoxic exposure. Serotonin levels were increased in hypoxic PAEC media. Conditioned media evoked bPASMC proliferation, which was greater with hypoxic PAEC media, via a serotonin-dependent mechanism. In vivo, adenoviral vectors targeted to PAECs (utilizing bispecific antibody to angiotensin-converting enzyme (ACE) as the selective targeting system) were used to deliver small hairpin Tph1 RNA sequences in rats. Hypoxic rats developed PAH and increased lung Tph1. PAEC-Tph1 expression and development of PAH were attenuated by our PAEC-Tph1 gene knockdown strategy. These results demonstrate that hypoxia induces Tph1 activity and selective knockdown of PAEC-Tph1 attenuates hypoxia-induced PAH in rats. Further investigation of pulmonary endothelial-specific Tph1 inhibition via gene interventions is warranted.     

Mechanisms whereby exogenous adenine nucleotides improve rabbit renal proximal function during and after anoxia.

When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloride-treated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three- to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.     

人近曲肾小管上皮细胞缺氧/复氧损伤模型的建立

目的 建立一种新的人近曲肾小管上皮细胞HK-2缺氧／复氧损伤模型。方法 本实验使用人近曲HK-2。实验分为缺氧4、12和24h及缺氧24h后复氧4、12和24h组，每个实验组均设立空白对照组。采用经高温灭菌的液体石蜡覆盖法造成缺氧环境；苔盼兰摄取法进行细胞计数及检测细胞存活率，生化法检测培养基中的乳酸脱氢酶（LDH）含量。结果 人肾小管上皮细胞经过缺氧／复氧处理后，苔盼兰摄取率显著提高，细胞存活率显著下降，LDH含量升高。说明缺血-再灌注损伤导致了细胞膜的完整性破坏，甚至细胞发生了不可逆转的损伤。结论 采用液体石蜡覆盖法建立人近曲肾小管上皮细胞缺氧／复氧损伤模型，较其他制模方法操作简单、易行、可靠。     

Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 2.7.1.40) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11 +/- 0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 +/- 0.04 U/microgram DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +/- 0.13; hypoxia, 0.94 +/- 0.13 microgram enzyme protein/microgram DNA). Specific activity was not significantly different after 96 h (L2: normoxia, 261 +/- 11; hypoxia, 261 +/- 14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic O2 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to charcterizing enzymes, as well as quantitating cellular enzyme content.     

STUDIES ON THE MODE OF ACTION OF DIPHTHERIA TOXIN : I. PHOSPHORYLATED INTERMEDIATES IN NORMAL AND INTOXICATED HELA CELLS

Intracellular levels of ATP, GTP, and hexose phosphates have been determined in HeLa cells at intervals after exposure to saturating doses of diphtheria toxin. Toxin causes no significant change in the level of any of these phosphorylated intermediates either in the presence or absence of glucose over a period of at least 5 to 6 hours. It is concluded that the inhibition of protein synthesis which occurs in HeLa cells at about 2 hours after the addition of saturating doses of toxin, does not result from an effect of toxin on energy metabolism.     

Alterations of endothelial nucleotide levels by mycophenolic acid result in changes of membrane glycosylation and E-selectin expression.

The effect of the inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH), mycophenolic acid, on intracellular nucleotides and the synthesis of cellular glycoproteins was evaluated in human umbilical vein endothelial cells. A clinically attainable concentration (10 micromol/l) of mycophenolic acid decreased guanosine-5'-triphosphate (GTP) levels significantly and led to a strong elevation of uridine-5'-triphosphate (UTP), whereas intracellular adenosine-5'-triphosphate (ATP) pools remained unaffected. The staining of the endothelial cell membranes with lectins specific for fucose and mannose (Ulex europaeus- and Galanthus nivalis agglutinin, respectively) was reduced, reflecting an inhibition of fucose and mannose incorporation into endothelial glycoproteins. The surface expression of E-selectin, an important determinant for leuko-endothelial interactions decreased significantly. Guanine and guanosine prevented the actions of mycophenolic acid and reversed the drug-induced decrease in GTP and its associated effects. The findings that mycophenolic acid produces alterations in the formation of glycoproteins and in the membrane architecture are indicative of metabolic lesions induced by an agent that depresses guanine nucleotide synthesis through inhibition of IMPDH. The pronounced reduction of E-selectin surface expression on endothelial cells accompanied by changes of endothelial cell fucosylation, a prerequisite for the contact with lymphocytic L-selectin, indicates an inhibitory effect of mycophenolic acid in the rolling phase of leukocyte recruitment and strongly implies a new and additional immunosuppressive mechanism of this agent.     

Energy utilization and changes in some intermediates of glucose metabolism in normal and hypoxic rat brain after decapitation

Energy metabolism was studied in the cerebral cortex of rats during and after hypoxia induced by breathing a gas mixture of 7% O2 in N2 for 2 h. Cortical energy stores (2ATP + ADP + phosphocreatine) remained unchanged after hypoxic treatment. Lactate rose over four-fold. Pyruvate, glucose and glucose 6-phosphate concentrations also increased significantly. Metabolic activity in the cortex expressed as the utilization of high-energy phosphates 5, 10 and 30 s after decapitation was decreased by 30% after hypoxia and remained lowered for 3 h during recovery. This was accompanied by elevated glucose consumption and lactate production, suggesting that the maintenance of the energy balance after hypoxia was partly due to activation of the glycolytic pathway. During the recovery period, these metabolic abnormalities returned towards control values, but, after 6 h of recovery the high-energy phosphate utilization increased transitorily above the control values.     

Effect of MicroRNA-218 on the viability,apoptosis and invasion of renal cell carcinoma cells under hypoxia by targeted downregulation of CXCR7 expression

ObjectiveTo investigate the effect of microRNA-218 on the viability, apoptosis and invasion of renal cell carcinoma cells under hypoxia by targeted regulation of expression of chemokine receptor 7 (CXCR7).MethodsThe expression of miR-218 in renal cell carcinoma cell lines under normal and hypoxia conditions, as well in normal renal tubular epithelial cells (HK2) was measured using RT-PCR. MiR-218 mimic and NC were transfected into renal cell carcinoma cell line ACHN using Lipofectamine™ 2000. The expression of miR-218 was analyzed using RT-PCR. The viability, apoptosis, migration and invasion of the transfected cells were assayed using the MTT assay, flow cytometry and transwell assays. The expression of CXCR7 was assayed using RT-PCR and Western blot. Luciferase reporter was used to verify the downstream target of miR-218.ResultsThe expression of miR-218 was lower than in renal cell carcinoma cell lines ACHN, 769-p and Caki-1 that in HK-2. The expression of miR-218 in the renal carcinoma cell lines was lower under hypoxia than under normal oxygen conditions. The expression of miR-218 in ACHN cells under normal and hypoxic conditions was significantly increased after transfection with miR-218 mimic. Compared with NC transfected cells under normal oxygen condition, the mimic-transfected cells had reduced viability, migration ability and invasion ability, and increased apoptosis, and mimic transfected-cells under hypoxia had significantly reduced viability, migration ability and invasion ability, and increased apoptosis. Overexpression of miR-218 mimic resulted in significant reduction in the expression of CXCR7 at protein and mRNA levels under normal and hypoxic conditions. Luciferase reporter assay confirmed that CXCR7 is the target protein of miR-218.ConclusionUp-regulation of miR-218 expression in renal cell carcinoma under hypoxia can result in significant and targeted down-regulation of CXCR7 expression, which could reduce cell viability, migration and invasion ability and induce apoptosis in the cancer cells.     

Exogenous adenine nucleotides replete endothelial cell adenosine triphosphate after oxidant injury by adenosine uptake

We studied the ability of human umbilical vein endothelial cells to recover from oxidant-induced ATP depletion. When endothelial cell ATP levels were depressed to 0.93 +/- 0.14 pmol/micrograms protein (compared with 4.96 +/- 0.6 pmol/micrograms protein in control cells) by hydrogen peroxide generated with 25 mU/ml glucose-glucose oxidase over 45 minutes, ATP levels returned to 1.73 +/- 0.21 pmol/micrograms protein during a 3-hour recovery period after oxidant injury ceased. When 25 microM ATP, ADP, AMP, or adenosine was added to the recovery media, intracellular ATP was significantly (p less than 0.001) increased to greater than 4.4 pmol/micrograms cell protein for each metabolite. HPLC of supernatants from oxidant-injured endothelial cells incubated with ATP, ADP, and AMP demonstrated extracellular metabolism of the adenine nucleotides to adenosine. When adenosine transport was inhibited with dipyridamole and nitrobenzylthioinosine, recovery of intracellular ATP by exogenous ATP, ADP, AMP, and adenosine was significantly (p less than 0.001) inhibited. Such cells were intact, as demonstrated by lack of LDH release. When oxidant stress was prolonged to 90 minutes, ATP depletion was irreversible, regardless of exogenously supplied adenosine; such cells demonstrated loss of cell integrity as demonstrated by release of intracellular LDH. Our results demonstrated that exogenous adenine nucleotides enhance recovery of oxidant-induced ATP depletion through metabolism to adenosine and subsequent adenosine uptake. Prolonged oxidant injury resulted in irreversible ATP depletion and loss of cell integrity that was not altered by exogenously supplied adenosine.     

Beneficial effects of yohimbine on posthypoxic recovery of cardiac function and myocardial metabolism in isolated perfused rabbit hearts.

The present study was undertaken to elucidate the possible actions of yohimbine on cardiac function and metabolism in the hypoxic and subsequently reoxygenated myocardium. For this purpose, rabbit hearts were perfused for 20 min under hypoxic conditions, followed by 45 min reoxygenated perfusion, and their functional and metabolic alterations with and without yohimbine treatment were examined. Hypoxia induced cessation of cardiac contractile force, rise in resting tension and depletion of tissue high-energy phosphates, which were poorly recovered by subsequent reoxygenation. Hypoxia also induced release of creatine kinase and ATP metabolites from perfused hearts and increases in tissue calcium and sodium contents, which were further enhanced upon subsequent reoxygenation. When hypoxic hearts were treated with 3 to 30 microM yohimbine, several beneficial effects were observed in a concentration-dependent manner. This included enhancement of posthypoxic recovery of contractile function and suppression of the hypoxia- and reoxygenation-induced rise in resting tension. Hypoxia/reoxygenation-induced release of ATP metabolites was inhibited and restoration of myocardial high-energy phosphates enhanced. Inhibition of reoxygenation-induced rise in tissue calcium and sodium and creatine kinase release were also noted. The findings suggest that suppression of transmembrane flux of ions, substrates and enzymes during hypoxia/reoxygenation plays a role in the posthypoxic functional and metabolic recovery. Yohimbine (3-30 microM) significantly depressed the maximal stimulus frequency the left atria could follow. These results suggest a close relationship between depression in the maximal driving frequency of atria and enhancement of the posthypoxic contractile and metabolic recovery of perfused hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac surgery: myocardial energy balance, antioxidant status and endothelial function after ischemia-reperfusion.

Myocardial and endothelial damage is still a widely debated problem during the ischemia-reperfusion sequence in heart surgery. We evaluated myocardial purine metabolites, antioxidant defense mechanisms, oxidative status and endothelial dysfunction markers in 14 patients undergoing coronary artery by-pass graft (CABG). Heart biopsies were taken before aortic cross-clamping (t1), before clamp removal (t2) and 30 min after reperfusion (t3); perchloric extracts of the tissue were analyzed for glutathione, NAD, nucleotide nucleoside and base content by capillary electrophoresis (CE). In plasma samples from the coronary sinus we evaluated: nitrate and nitrite concentrations by CE, plasma glutathione peroxidase (plGPx) by ELISA, endothelin-1 (ET-1) by RIA and reactive oxygen metabolites (ROM) by colorimetric assay. During the ischemic period (t2) we observed a reduction in cellular NAD and GSH levels, as well as nitrate, nitrite and plGPx. ATP and GTP levels decreased and their catabolic products AMP, GMP, IMP, adenosine, inosine and hypoxanthine accumulated. The energy charge, ATP/ADP ratio, and nucleotide/(nucleoside + base) ratios decreased. At t3, levels of plasma ET-1 increased and monophosphate nucleotides tended to return to basal values. The energy charge did not increase but the nucleotide/(nucleoside + nucleobase) ratio recovered to some extent. Levels of nitrates plus nitrites continued to decrease. No significant variation in ROM levels was observed. Our data indicate that oxidative stress and endothelial damage are major events during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration of the normal energy balance for 30 min after reperfusion. The AMP deaminase pathway leading to IMP production is active during ischemia and adenosine is not the main compound derived from ATP break-down in the human heart. The possible role of extracorporeal circulation is also discussed.     

The beneficial effect of 2′-deoxycoformycin in renal ischemia-reperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation

Renal ischemia injures the renal tubular cell by disrupting the vital cellular metabolic machinery. Further cell damage is caused when the blood flow is restored by oxygen free radicals that are generated from xanthine oxidase. Oxygen radicals cause lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy metabolism. In the present study, the possible therapeutic usefulness of the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), during renal ischemia and reperfusion injury was investigated. The effects of DCF on renal malondialdehyde (MDA) and ATP levels were studied after 45 min ischemia and 15 min subsequent reperfusion in rat kidneys. MDA levels remained unchanged during ischemia, but increased after the subsequent reperfusion. DCF pretreatment (2.0 mg/kg i.m.) decreased MDA and increased ATP levels during the ischemia-reperfusion period. DCF exerts a dual protective action by facilitating purine salvage for ATP synthesis and inhibiting oxygen radical-induced lipid peroxidation. These results suggest that DCF therapy could be beneficial in the treatment of ischemia-reperfusion renal injuries.     

Impaired wound healing in hypoxic renal tubular cells: roles of hypoxia-inducible factor-1 and glycogen synthase kinase 3β/β-catenin signaling

Wound and subsequent healing are frequently associated with hypoxia. Although hypoxia induces angiogenesis for tissue remodeling during wound healing, it may also affect the healing response of parenchymal cells. Whether and how wound healing is affected by hypoxia in kidney cells and tissues is currently unknown. Here, we used scratch-wound healing and transwell migration models to examine the effect of hypoxia in cultured renal proximal tubular cells (RPTC). Wound healing and migration were significantly slower in hypoxic (1% oxygen) RPTC than normoxic (21% oxygen) cells. Hypoxia-inducible factor-1α (HIF-1α) was induced during scratch-wound healing in normoxia, and the induction was more evident in hypoxia. Nevertheless, HIF-1α-null and wild-type cells healed similarly after scratch wounding. Moreover, activation of HIF-1α with dimethyloxalylglycine in normoxic cells did not suppress wound healing, negating a major role of HIF-1α in wound healing in this model. Scratch-wound healing was also associated with glycogen synthase kinase 3β (GSK3β)/β-catenin signaling, which was further enhanced by hypoxia. Pharmacological inhibition of GSK3β resulted in β-catenin expression, accompanied by the suppression of wound healing and transwell cell migration. Ectopic expression of β-catenin in normoxic cells could also suppress wound healing, mimicking the effect of hypoxia. Conversely, inhibition of β-catenin via dominant negative mutants or short hairpin RNA improved wound healing and transwell migration in hypoxic cells. The results suggest that GSK3β/β-catenin signaling may contribute to defective wound healing in hypoxic renal cells and tissues.     

Comparison of the toxicity of the radiocontrast agents, iopamidol and diatrizoate, to rabbit renal proximal tubule cells in vitro

Radiographic contrast agent-induced acute renal failure is an increasingly recognized clinical event. Multiple factors have been implicated in its development. Recent experiments have demonstrated that sodium diatrizoate, a common ionic radiocontrast agent, is moderately toxic to proximal tubule cells in vitro, and that this toxicity is enhanced by hypoxia. In this study, we compare toxicities of the nonionic radiocontrast agent, iopamidol, and the commonly used ionic contrast agent, diatrizoate. Suspensions enriched in proximal tubule segments were exposed for 82.5 min to 10 or 25 mM diatrizoate or 10 or 25 mM iopamidol with or without 22.5 min or 30 min of hypoxia. Cell viability parameters, including basal and uncoupled respiratory rates, tubule cell potassium and calcium levels and cell ATP content were measured. No consistent differences in tubule viability parameters were observed between tubule suspensions exposed to 10 mM concentrations of the radiocontrast agents during either oxygenated or hypoxic conditions. Under oxygenated conditions, both 25 mM iopamidol and diatrizoate exposure produced greater metabolic alterations in renal tubules than control conditions, but the effects were not statistically significant. With concomitant hypoxia, the alterations after 25 mM diatrizoate exposure were significantly greater than those seen after exposure to 25 mM iopamidol. Iopamidol had less of a detrimental effect on renal tubule potassium content and both basal and uncoupled respiratory rates than that of diatrizoate under these conditions. Thus, diatrizoate is more toxic to rabbit renal proximal tubule cells than iopamidol in vitro, and this difference in toxicity is enhanced by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of poly(ADP-ribose) polymerase inhibition on outer medullary hypoxic damage

Poly(ADP-ribose) polymerase (PARP) activation after free-radical-induced DNA damage depletes cellular energy stores and participates in ischemia-reflow injury. We studied the potential protective effect of the water-soluble PARP inhibitor 3-aminobenzamide (3-AB) in a rat model of acute renal failure (ARF) from combined administration of radiocontrast, indomethacin and N(omega)-nitro-L-arginine methyl ester. Kidney function at 24 h was better preserved in rats treated with 3-AB as compared to control animals. However, the extent of tubular hypoxic damage was not significantly mitigated. It is concluded that PARP inhibition may attenuate renal dysfunction in this model of ARF with medullary hypoxic tubular injury even while the extent of tubular necrosis is not significantly altered. Further studies of this dyssynchrony of structure and function may provide important insights into the sequence of events that promotes renal failure after medullary injury.     

Relation of myocardial oxygen consumption and function to high energy phosphate utilization during graded hypoxia and reoxygenation in sheep in vivo.

This study investigates the relation between myocardial oxygen consumption (MVO2), function, and high energy phosphates during severe hypoxia and reoxygenation in sheep in vivo. Graded hypoxia was performed in open-chested sheep to adjust PO2 to values where rapid depletion of energy stores occurred. Highly time-resolved 31P nuclear magnetic resonance spectroscopy enabled monitoring of myocardial phosphates throughout hypoxia and recovery with simultaneous MVO2 measurement. Sheep undergoing graded hypoxia (n = 5) with an arterial PO2 nadir of 13.4 +/- 0.5 mmHg, demonstrated maintained rates of oxygen consumption with large changes in coronary flow as phosphocreatine (PCr) decreased within 4 min to 40 +/- 7% of baseline. ATP utilization rate increased simultaneously 59 +/- 20%. Recovery was accompanied by marked increases in MVO2 from 2.0 +/- 0.5 to 7.2 +/- 1.9 mumol/g per min, while PCr recovery rate was 4.3 +/- 0.6 mumol/g per min. ATP decreased to 75 +/- 6% of baseline during severe hypoxia and did not recover. Sheep (n = 5) which underwent moderate hypoxia (PO2 maintained 25-35 mmHg for 10 min) did not demonstrate change in PCr or ATP. Functional and work assessment (n = 4) revealed that cardiac power increased during the graded hypoxia and was maintained through early reoxygenation. These studies show that (a) MVO2 does not decrease during oxygen deprivation in vivo despite marked and rapid decreases in high energy phosphates; (b) contractile function during hypoxia in vivo does not decrease during periods of PCr depletion and intracellular phosphate accumulation, and this may be related to marked increases in circulating catecholamines during global hypoxia. The measured creatine rephosphorylation rate is 34 +/- 11% of predicted (P < 0.01) calculated from reoxygenation parameters, which indicates that some mitochondrial respiratory uncoupling also occurs during the rephosphorylation period.