guanosine-triphosphate and Ischemia

Apoptosis is increasingly recognized as a major mode of cell demise after ischemic injury to the kidney. The mediators of apoptotic cell death are many and include changes in intracellular pH, calcium, free radicals, ceramide, and adenosine triphosphate (ATP) depletion. Recently, we identified guanosine triphosphate (GTP) depletion as an independent trigger for apoptotic death after chemical anoxia in vitro. We further demonstrated that GTP salvage with guanosine inhibits tubular cell apoptosis after ischemic injury in vivo. This inhibition of apoptosis was accompanied by a significant protective effect on renal function. We also showed that p53 is the mediator of apoptosis in the setting of GTP depletion and ischemic injury. Indeed, salvage of GTP with guanosine prevented the ischemia-induced increase in p53 protein. Further, pifithrin-alpha, a potent and specific inhibitor of p53, inhibited apoptosis and protected renal function with a profile similar to that seen with guanosine. Finally, the protective effects of pifithrin-alpha involved both down-regulation of the transcriptional activation of Bax and a direct inhibition of p53 translocation to mitochondria. We propose that GTP depletion and activation of p53 are major inducers of apoptotic cell death after ischemic renal injury. In this setting, guanosine and pifithrin-alpha are potent inhibitors of apoptosis and are thus potentially useful in preventing and ameliorating functional injury to the ischemic kidney.

Topics: Acute Kidney Injury; Animals; Apoptosis; Guanosine Triphosphate; Humans; Ischemia; Tumor Suppressor Protein p53

Renal tubular cells die by apoptosis as well as necrosis in experimental models of ischemic and toxic acute renal failure as well as in humans with acute tubular necrosis. It is not yet possible, however, to determine the relative contribution of these two forms of cell death to loss of renal tubular cells in acute tubular necrosis. The beneficial effect of administering growth factors to animals with acute tubular necrosis is probably related to the potent antiapoptotic (survival) effects of growth factors as well as to their proliferative effects. Rapamycin inhibits both of these effects of growth factors and delays the recovery of renal function after acute tubular necrosis by inhibiting renal tubular cell regeneration and by increasing renal tubular cell loss by apoptosis. The administration of caspase inhibitors ameliorates ischemia-reperfusion injury in multiple organs including the kidney. However, the extent to which this protective effect of caspase inhibition is caused by reduced intrarenal inflammation, or by amelioration of renal tubular cell loss due to apoptosis, remains uncertain. In addition to caspase inhibition, the apoptotic pathway offers many potential targets for therapeutic interventions to prevent renal tubular cell apoptosis.

Topics: Acute Kidney Injury; Apoptosis; Caspase Inhibitors; Cell Adhesion; Cisplatin; Growth Substances; Guanosine Triphosphate; Humans; Ischemia; Kidney Tubules; Necrosis; Sirolimus

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK10 and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations.

Topics: Adenosine Triphosphate; Animals; cdc42 GTP-Binding Protein; Cytoskeletal Proteins; Detergents; Guanosine Triphosphate; Ischemia; LLC-PK1 Cells; Phosphoproteins; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Solubility; Stress Fibers; Swine

To elucidate the mechanisms of ischemia-mediated myopathy using in vitro model, changes of purine nucleotides, membrane lipid peroxidation(TBARS), intracellular calcium ([Ca2+]i)levels, generation of free radicals, and deoxyribonucleic acid (DNA) fragmentation were examined in mouse-derived C2C12 myotubes under the condition with an inhibition of glycolytic and oxidative metabolism as the ischemic condition. In purine nucleotides, intracellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations rapidly and significantly decreased after the treatment with ischemia. No remarkable differences were observed in other purine nucleotides, with the exception of inosine monophosphate (IMP) and extracellular hypoxanthine levels, both of which increased significantly during the ischemia. The lactate dehydrogenase activity in culture supernatant of C2C12 myotubes increased significantly from 2 to 4 hr after the ischemia. On the generation of free radicals, no spectrum was detected in supernatants throughout the observation period, whereas supernatant TBARS concentration increased rapidly and significantly after the ischemia. The relative intensity of [Ca2+]i significantly increased after the ischemia. On the fragmented deoxyribonucleic acid(DNA), no TUNEL positive cells was detected in C2C12 myotubes after 1 hr of the ischemia, however the positive cell percentage subsequently increased. From these results, it was suggested that the ischemic condition induced changes of membrane permeability and increase of [Ca2+]i, both of which lead to cell membrane damage, although a free radical generation was not detected. The ischemic condition also induced the release of substrate hypoxanthine for free radical generation and might initiate the apoptotic pathway in C2C12 myotubes.

Topics: Adenosine Triphosphate; Animals; Calcium; Cells, Cultured; DNA Fragmentation; Electron Spin Resonance Spectroscopy; Free Radicals; Guanosine Triphosphate; In Situ Nick-End Labeling; In Vitro Techniques; Ischemia; L-Lactate Dehydrogenase; Lipid Peroxidation; Lipid Peroxides; Mice; Muscle Fibers, Skeletal; Muscle, Skeletal; Thiobarbituric Acid Reactive Substances

Topics: Animals; Antineoplastic Agents; Apoptosis; Chromatography, High Pressure Liquid; Clinical Trials as Topic; Down-Regulation; Guanosine Triphosphate; History, 20th Century; Humans; Ischemia; Kidney Neoplasms; Leukemia; Models, Biological; Neoplasms; Ribavirin; Signal Transduction; Time Factors; Tumor Cells, Cultured

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. Recently, we linked anoxia-induced apoptosis in renal cell cultures specifically to the depletion of GTP. We therefore hypothesized that enhancing GTP repletion in vivo might protect function by reducing apoptosis in postischemic tubules. Male C57 black mice (the "I" group of animals) underwent bilateral renal artery clamp for 32 minutes to induce ischemia and then received either normal saline ("NS") or guanosine ("G"). After 1 hour of reperfusion, renal GTP levels in NS/I were reduced to nearly half of those in sham operated mice, whereas these levels were nearly unchanged in G/I mice. Morphologic examination of tubular injury revealed no significant differences between the two groups. However, there was a significant reduction in the number of apoptotic tubular cells in the medulla in the G/I group as compared with the NS/I group. At 24 hours, creatinine was significantly elevated in the NS/I group, compared to the G/I group. We conclude that guanosine protects against renal ischemic injury by replenishing GTP stores and preventing tubular apoptosis.

Topics: Animals; Apoptosis; Cell Death; Cell Line; Guanosine; Guanosine Triphosphate; Hypoxia; In Situ Nick-End Labeling; Ischemia; Kidney; Male; Mice; Mice, Inbred C57BL; Phenotype; Protein Binding; Reperfusion Injury; Swine; Time Factors

Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.

Topics: Adenosine Triphosphate; Alanine; Animals; Antibiotics, Antineoplastic; Antimycin A; Apoptosis; Cell Hypoxia; Cells, Cultured; Deoxyglucose; Enzyme Inhibitors; Guanosine; Guanosine Triphosphate; Ischemia; Kidney Cortex; Kidney Tubules, Proximal; LLC-PK1 Cells; Male; Models, Biological; Mycophenolic Acid; Necrosis; Oxidative Phosphorylation; Rats; Rats, Sprague-Dawley; Swine

The small intestine of rats was prepared according to a procedure which is taken for preservation and transplantation in clinical practice. The blood supply of the rat intestine was completely interrupted for 30 min in situ. During this period the lumen of the intestine was rinsed with Ringer-lactate solution. This ischaemic period was followed by 10 min of reperfusion. As a result a decrease in ATP, and GTP concentrations, and of the total adenine nucleotide content during the preservation period occurred. In animals pretreated with superoxide dismutase (i.v. application; superoxide dismutase preparation from human erythrocytes) an accelerated restoration of nucleotide concentrations during the reperfusion period was observed. From the beneficial effect of superoxide dismutase it can be concluded, that there is a considerable formation of active species of oxygen which disturb the energy generation by the mitochondrial respiratory chain during ischaemia/re-oxygenation.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Energy Metabolism; Guanosine Triphosphate; Intestine, Small; Ischemia; Male; Mesenteric Arteries; Perfusion; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

The observability of nucleoside triphosphate (NTP) by 31P NMR spectroscopy was studied in the isolated rat liver during hypothermic perfusion and a subsequent 4-h cold ischemia. The influence of hypothermia (4 degrees C) was examined because of its delaying effects on cell injury induced by the ischemic conditions. The viability of the liver after hypothermic ischemia was assessed by measuring the recovery of the beta-NTP resonance after reperfusion. In 4-h cold ischemic liver, recovery was found to be in the range of 90-100% and consequently NTP visibility was studied under these conditions. Because the individual purine (or pyrimidine) NTPs are not distinguishable in the liver on the basis of their 31P NMR chemical shifts, the contributions of UTP and GTP were investigated by HPLC. The changes in liver NTP content measured either by NMR on isolated liver or by HPLC after perchloric acid extraction from the same organ are not significantly different. The total NTP level in normothermic perfused liver is 7.6 +/- 0.2 mumol NTP/g liver dry wt as determined by NMR. In such a liver, ATP + GTP + UTP and ATP contents measured by HPLC are, respectively, 7.9 +/- 1.0 and 6.3 +/- 0.9 mumol/g liver dry wt. This indicates that all NTP is detected by NMR and that a 20% contribution of the signal occurs from UTP + GTP. Under 4-h cold ischemic conditions, NTP visibility remains unchanged, furthermore the UTP + GTP contribution reaches 32% of the whole NTP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Guanosine Triphosphate; Hypothermia, Induced; Ischemia; Liver; Magnetic Resonance Spectroscopy; Male; Manganese; Perfusion; Phosphorylation; Rats; Rats, Inbred Strains; Uridine Triphosphate

To assess the effects of body temperature on renal susceptibility to ischemic injury, rats were rendered acutely hypothermic (90-93 degrees F), normothermic (98-99 degrees F), or hyperthermic (101-103 degrees F) with a heat-controlled surgical board and then were subjected to 25 min of bilateral renal artery occlusion (RAO). Renal high-energy phosphates, their degradation products, and nonprotein sulfhydryl (NPSH) content were assessed at selected times during the peri-ischemic period. The severity of acute renal failure (ARF) was determined for 48 h following RAO by blood urea nitrogen (BUN) and plasma creatinine determinations and by renal histology. Ischemic ATP, ADP, AMP, GTP, GDP, UTP, and NAD levels and postischemic NPSH levels (15 min reflow) inversely correlated with temperature (P less than 0.001). BUN, creatinine concentrations (at 24 and 48 h), and histological injury (at 48 h) directly correlated with temperature (P less than 0.01). Hyperthermia in the absence of RAO had no demonstrable adverse renal effects. We conclude that hyperthermia potentiates ischemic renal injury, whereas hypothermia confers protection. These effects are associated with, and may be influenced by, temperature-induced changes in renal high-energy phosphate availability and oxidant stress during the ischemic/postischemic period.

Topics: Adenine Nucleotides; Animals; Arterial Occlusive Diseases; Blood Urea Nitrogen; Body Temperature; Creatine; Female; Guanosine Diphosphate; Guanosine Triphosphate; Hypoxanthine; Hypoxanthines; Ischemia; Kidney; NAD; Rats; Rats, Inbred Strains; Sulfhydryl Compounds; Time Factors; Uridine Triphosphate

The major pathway of purine catabolism in mouse kidney during ischemia occurs through IMP, inosine, hypoxanthine, and xanthine. Short periods of ischemia (reversible cell injury) allow a rapid return of the energy charge to control values and a rapid return of ATP and GTP to value of 60-70% of control. ATP and GTP then slowly return to control levels over the next 24 h. Long periods of ischemia (irreversible cell injury; ischemic times longer than 1 h) allow a gradual return of the energy charge to control levels. ATP, GTP or total adenine or guanine nucleotides do not return to control levels even after 24 h of reinfusion under these circumstances. We conclude that irreversibly injured kidney cells retain the ability to phosphorylate purine nucleotides, but lose the ability to restore the concentrations of the purine nucleotides to control values.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Cell Survival; Guanosine Triphosphate; Inosine Monophosphate; Ischemia; Kidney; Male; Mice; Purine Nucleotides

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Brain; Carbon Radioisotopes; Cats; Cerebrovascular Circulation; Chromatography, Ion Exchange; Formates; Guanine Nucleotides; Guanosine Triphosphate; Hypoxanthines; Inosine; Inosine Nucleotides; Ischemia; NAD; Pentosyltransferases; Phosphotransferases; Purine Nucleotides; Spectrophotometry, Ultraviolet; Time Factors; Tritium