Phospholipase A2-induced cytoprotection of proximal tubules: potential determinants and specificity for ATP depletion-mediated injury.

Addition of phospholipase A2 (PLA2) to isolated proximal tubular segments (PTS) has previously been shown to decrease hypoxic cell death without altering ATP concentrations. The study presented here was undertaken to identify determinant(s) of this protection, and to define the spectrum of injuries against which it can operate. PTS were extracted from mouse kidneys and subjected to diverse forms of injury (hypoxia/reoxygenation, antimycin A, Ca2+ ionophore, amphotericin B, FeSO4, and myohemoglobin). In subtoxic doses, addition of PLA2 significantly reduced hypoxic- and antimycin A-induced injury (percentage of lactate dehydrogenase release); however, a dose-dependent exacerbation of all other forms of injury resulted. The ability of PLA2 to mitigate hypoxic injury remained intact despite the inhibition of Na,K-ATPase (ouabain) or the inducement of cytoskeletal disruption (cytochalasin D). However, it was negated by minimally toxic amphotericin B or Ca2+ ionophore doses, indicating its dependence on preserved ionic gradients. Nevertheless, neither lowering/removing buffer Ca2+ or NaCl concentrations, nor hypertonic mannitol addition reproduced the cytoprotective effect of PLA2. PLA2 induced synergistic deacylation in hypoxic tubules, suggesting that unsaturated fatty-acid accumulation might mediate its cytoprotective effect. The fact that the addition of exogenous arachidonate, but not palmitate, to tubules protected against hypoxia, but worsened nonhypoxic forms of injury, supported this hypothesis. Since arachidonate might induce "feedback" inhibition of intracellular PLA2, the ability of an intracellular phospholipase inhibitor (ONO-RS-082; Biomol, Plymouth, PA) to blunt hypoxic damage was tested. This agent fully reproduced the cytoprotective effect of PLA2. It was concluded that: (1) PLA2-induced cytoprotection is relatively specific for ATP depletion injury; (2) it is dependent on, but not explained by, maintenance of NaCl and Ca2+ gradients; (3) it does not require Na,K-ATPase activity or cytoskeletal integrity for its expression; and (4) extracellular PLA2, via arachidonate release, may cause feedback inhibition of intracellular PLA2, thereby protecting critical intracellular targets from attack.

Iron, heme oxygenase, and glutathione: effects on myohemoglobinuric proximal tubular injury.

UNLABELLED: This study assessed the impacts of iron, heme oxygenase (HO), hydroxyl radical (.OH), and glutathione (GSH) on the initiation phase of myohemoglobinuric proximal tubular injury using a novel model system. Rhabdomyolysis was induced in rats by glycerol injection and four hours later proximal tubular segments (PTS) were isolated. They were incubated for 0 to 90 minutes either in the presence or absence of an iron chelator (deferoxamine; DFO), .OH scavengers, an .OH trapping agent (salicylate; to gauge .OH production), GSH, or catalase. In selected experiments, an HO inhibitor (Sn protoporphyrin) was given at the time of glycerol injection to assess HO's acute effects on the evolving injury. Cell death and lipid peroxidation were quantified by % LDH release and malondialdehyde (MDA) generation, respectively. PTS from normal rats served as controls. Post-glycerol PTS manifested progressive LDH release (47 +/- 2%) and 20-fold MDA increments during the incubations, whereas only 11 +/- 1% LDH release and no MDA generation was observed in the normal PTS. DFO completely prevented both parameters of glycerol-induced injury. HO inhibition exerted an acute protective effect, despite previous in vivo data suggesting that HO is a cytoprotectant. Neither .OH scavengers nor catalase mitigated post-glycerol injury, the latter correlating with reduced, not increased, .OH production. GSH slightly decreased LDH release while causing a paradoxical threefold MDA increment. The latter was iron dependent (blocked by DFO), was expressed in normal PTS, and it could be reproduced by equimolar cysteine. That GSH increased iron-dependent lipid peroxidation in a cell free system (exogenous phosphatidylcholine) indicated that GSH metabolism to cysteine was not a requirement for this reaction. IN CONCLUSION: (1) chelatable iron can fully account for heme protein-triggered proximal tubular injury; (2) HO contributes to this injury, presumably by causing iron release; (3) the heme-induced injury appears to be mediated by non-.OH oxidizing intermediates; (4) GSH can exert both anti- and pro-oxidant effects; and (5) i.m. glycerol injection, followed by proximal tubular isolation, represents a new and highly useful model for studying direct determinants of heme protein cytotoxicity.

Postischemic proximal tubular resistance to oxidant stress and Ca2+ ionophore-induced attack. Implications for reperfusion injury.

BACKGROUND: The severity of "reperfusion injury" is dependent on the extent to which the involved pathways are activated and on the degree of tissue susceptibility to them. This study was undertaken to ascertain whether preexistent ischemic proximal tubular damage (ischemic "pre-conditioning") significantly alters the expression of two purported mediators of reperfusion damage: oxidant stress and cytosolic Ca2+ loading. EXPERIMENTAL DESIGN: Male Sprague-Dawley rats underwent 35 minutes of bilateral renal arterial occlusion. Fifteen minutes or 24 hours later, the kidneys were removed, proximal tubular segments (PTS) were isolated, and their susceptibility to oxidant stress (H2O2 or FeSO4) and to cytosolic Ca2+ loading (Ca2+ ionophore, A23187) was determined. Results were contrasted to those obtained with normal PTS. Cell injury was quantified by percentage of cellular lactate dehydrogenase released. Lipid peroxidation was gauged by PTS malondialdehyde (MDA) concentrations. As an index of endogenous antioxidant defenses, PTS catalase and superoxide dismutase activities were determined. Vulnerability to lipid peroxidation is highly dependent on phospholipid unsaturated fatty content, so PTS fatty acid concentrations also were assessed. RESULTS: Although PTS harvested at 15 minutes postischemia manifested sublethal injury (increased lactate dehydrogenase release under control conditions), no increased vulnerability to the oxidant insults or to the Ca2+ ionophore was noted. By 24 hours of reflow, cytoresistance to each of the insults had developed. Postischemic PTS demonstrated no increase in basal MDA concentrations (indicating a lack of in vivo lipid peroxidation), and when challenged with H2O2 or FeSO4, significantly less MDA generation developed (vs. the normal PTS). This resistance to lipid peroxidation was not associated with increased superoxide dismutase/catalase levels or altered PTS fatty acid content. CONCLUSIONS: Sublethal ischemic proximal tubular injury does not directly predispose to oxidant stress or cytosolic Ca2+ loading, and by 24 hours postischemia, increased resistance to these insults develops. Decreased membrane susceptibility to lipid peroxidation may contribute to this result.

Phospholipase A2 activity can protect renal tubules from oxygen deprivation injury.

During hypoxic or ischemic renal tubular injury, phospholipase A2 (PLA2) induces membrane deacylation, causing fatty acid accumulation and phospholipid breakdown. Because these changes can compromise cellular integrity, PLA2 activity has been widely proposed as a critical mediator of hypoxic renal tubular injury and, hence, of ischemic acute renal failure. To explore this hypothesis, isolated rat proximal tubules were subjected to continuous oxygenation or to hypoxic injury with or without exogenous PLA2 addition (porcine or bovine pancreatic PLA2; bee or snake venom PLA2). Cell death was quantified by lactic dehydrogenase (LDH) release. Pancreatic PLA2 (0.4 unit/ml) caused no LDH release under oxygenated conditions, and it dramatically attenuated hypoxic cell death (e.g., no PLA2, 55 +/- 3% LDH release; porcine pancreatic PLA2, 22 +/- 1% LDH release; P < 0.001). Bee and snake venom PLA2 (0.4 unit/ml) were directly toxic to tubules under oxygenated conditions, and this injury was additive with that induced by hypoxia. However, when these venoms were serially diluted (removing their overt toxicity), they, too, mitigated hypoxic cell death (LDH release with PLA2, 33 +/- 2%; without PLA2, 60 +/- 1% LDH release; P < 0.001). PLA2-mediated cytoprotection was Ca2+ dependent (negated by Ca2+ chelation), and it was expressed despite worsening hypoxia-associated membrane deacylation/fatty acid accumulation (12 times) and ATP depletion. These results indicate that PLA2 activity can exert both beneficial and deleterious effects on O2-deprived renal tubules, the net result of which can be a salvaging of cells from hypoxic cell death.

Physiological pH. Effects on posthypoxic proximal tubular injury.

After O2 deprivation, tissue acidosis rapidly self-corrects. This study assessed the effect of this pH correction on the induction, and pathways, of posthypoxic proximal tubular injury. In addition, ways to prevent the resultant injury were explored. Isolated rat proximal tubular segments (PTSs) were subjected to hypoxia/reoxygenation (50/30 or 30/50 minutes) under the following incubation conditions: 1) continuous pH 7.4, 2) continuous pH 6.8, or 3) hypoxia at pH 6.8 and reoxygenation at pH 7.4 (NaHCO3 or Tris base addition). Continuously oxygenated PTSs maintained under these same pH conditions served as controls. Lethal cell injury was assessed by lactate dehydrogenase (LDH) release. pH effects on several purported pathways of hypoxia/reoxygenation injury were also assessed (ATP depletion, lipid peroxidation, and membrane deacylation). Acidosis blocked hypoxic LDH release (pH 7.4, 50 +/- 2%; pH 6.8, 6 +/- 1%) without mitigating membrane deacylation or ATP depletion. During reoxygenation, minimal LDH was released (3-5%) if pH was held constant. However, if posthypoxic pH was corrected, immediate (< or = 5 minutes) and marked cell death (e.g., 55 +/- 3% with Tris) occurred. This was dissociated from lipid peroxidation or new deacylation, and it was preceded by a depressed ATP/ADP ratio (suggesting an acidosis-associated defect in hypoxic/posthypoxic cell energetics). Realkalinization injury was not inevitable, since it could be substantially blocked by 1) posthypoxic glycine addition, 2) transient posthypoxic hypothermia, or 3) allowing a 10-minute reoxygenation (cell recovery) period before base addition. Neither mannitol nor graded buffer Ca2+ deletion conferred protection. Acute pH correction caused no injury to continuously oxygenated PTSs. Conclusions are as follows: 1) Posthypoxic "pH shock" causes virtually immediate cell death, not by causing de novo injury but, rather, by removing the cytoprotective effect of acidosis. 2) This injury can be prevented by a variety of methods, indicating a great potential for salvaging severely damaged posthypoxic PTSs.

Inorganic iron effects on in vitro hypoxic proximal renal tubular cell injury.

Iron-dependent free radical reactions and renal ischemia are believed to be critical mediators of myohemoglobinuric acute renal failure. Thus, this study assessed whether catalytic iron exacerbates O2 deprivation-induced proximal tubular injury, thereby providing an insight into this form of renal failure. Isolated rat proximal tubular segments (PTS) were subjected to either hypoxia/reoxygenation (H/R: 27:15 min), "chemical anoxia" (antimycin A; 7.5 microM x 45 min), or continuous oxygenated incubation +/- ferrous (Fe2+) or ferric (Fe3+) iron addition. Cell injury (% lactic dehydrogenase [LDH] release), lipid peroxidation (malondialdehyde, [MDA]), and ATP depletion were assessed. Under oxygenated conditions, Fe2+ and Fe3+ each raised MDA (approximately 7-10x) and decreased ATP (approximately 25%). Fe2+, but not Fe3+, caused LDH release (31 +/- 2%). During hypoxia, Fe2+ and Fe3+ worsened ATP depletion; however, each decreased LDH release (approximately 31 to approximately 22%; P < 0.01). Fe(2+)-mediated protection was negated during reoxygenation because Fe2+ exerted its intrinsic cytotoxic effect (LDH release: Fe2+ alone, 31 +/- 2%; H/R 36 +/- 2%; H/R + Fe2+, 41 +/- 2%). However, Fe(3+)-mediated protection persisted throughout reoxygenation because it induced no direct cytotoxicity (H/R, 39 +/- 2%; H/R + Fe3+, 25 +/- 2%; P < 0.002). Fe3+ also decreased antimycin toxicity (41 +/- 4 vs. 25 +/- 3%; P < 0.001) despite inducing marked lipid peroxidation and without affecting ATP. These results indicate that catalytic iron can mitigate, rather than exacerbate, O2 deprivation/reoxygenation PTS injury.

Pharmacodynamic studies of cyclosporine in marrow transplant recipients. A comparison of three assay methods.

We investigated the correlation between trough cyclosporine concentration in plasma measured by polyclonal fluorescence polarization immunoassay (FPIA) and polyclonal radioimmunoassay (RIA) or in whole blood measured by high-performance liquid chromatography (HPLC) and the risk of renal dysfunction or acute graft-versus-host disease in 29 patients undergoing allogeneic bone marrow transplantation for leukemia. The FPIA and RIA values were highly correlated (r = 0.93) and on the average CsA concentrations measured by FPIA were 1.56 times higher than those measured by RIA. Ten patients developed renal dysfunction and 10 developed grades II-IV acute GVHD. Although univariate analysis showed that plasma CsA concentrations measured by either FPIA or RIA were significantly correlated with renal dysfunction, the association was stronger with FPIA. Plasma CsA concentrations measured by FPIA but not RIA remained a significant risk factor for renal dysfunction in a multivariate relative risk model. Amphotericin therapy was significantly associated with renal dysfunction in the univariate analysis but not in the multivariate analysis. No significant associations were found between whole blood CsA or CsA M1 concentration, patients' age, gender, or CsA dose and the risk of renal dysfunction. None of the covariates analyzed significantly correlated with the development of acute GVHD. These data suggest that plasma CsA concentrations measured by nonspecific assays may more accurately correlate with renal dysfunction than whole-blood CsA concentrations measured by HPLC in marrow transplant recipients.

Evidence against increased hydroxyl radical production during oxygen deprivation-reoxygenation proximal tubular injury.

The purpose of this study was to assess whether proximal renal tubules generate excess hydroxyl radical (.OH) during hypoxia/reoxygenation or ischemia/reperfusion injury, thereby supporting the hypothesis that reactive oxygen species contribute to the pathogenesis of postischemic acute renal failure. In the first phase of the study, rat isolated proximal tubular segments (PTS) were subjected to hypoxia (95% N2- 5% CO2) for 15, 30, or 45 min, followed by 15 to 30 min of reoxygenation in the presence of sodium salicylate, a stable .OH trap. Cellular injury after hypoxia and reoxygenation was assessed by lactate dehydrogenase release; .OH production was gauged by hydroxylated salicylate by-product generation (2,3-, 2,5-dihydroxybenzoic acids (DHBA); quantified by HPLC/electrochemical detection). Continuously oxygenated PTS served as controls. Despite substantial lactate dehydrogenase release during hypoxia (8 to 46%) and reoxygenation (8 to 11%), DHBA production did not exceed that of the coincubated, continuously oxygenated control PTS. In the second phase of the study, salicylate-treated rats were subjected to 25 or 40 min of renal arterial occlusion +/- 15 min of reperfusion. No increase in renal DHBA concentrations occurred during ischemia or reperfusion, compared with that in sham-operated controls. To validate the salicylate trap method, PTS were incubated with a known .OH-generating system (Fe2+/Fe3+); in addition, rats were treated with antioxidant interventions (oxypurinol plus dimethylthiourea). Fe caused marked DHBA production, and the antioxidants halved in vivo DHBA generation. In conclusion, these results suggest that exaggerated .OH production is not a consequence of O2 deprivation/reoxygenation tubular injury.

Temperature effects on ischemic and hypoxic renal proximal tubular injury.

UNLABELLED: Body temperature (T) profoundly alters the severity of ischemic acute renal failure. Therefore, the present study evaluated T effects on: (a) in vitro proximal tubular cell killing during hypoxia/reoxygenation to assess when it exacerbates injury; (b) renal ATP losses and metabolic rate (O2 consumption) during hemorrhagic shock (55-60 mm Hg); and (c) membrane deacylation (assessed by free fatty acid, FFA, release) to determine if T modifies this pathway of ischemic renal damage. Hypoxic cell kill (45 minutes) was 20 +/- 1% 47 +/- 4%, and 61 +/- 2% at 32 degrees C, 37 degrees C, and 40 degrees C respectively (by lactate dehydrogenase release; p less than 0.001). During reoxygenation (15 minutes), minimal lactate dehydrogenase was released, irrespective of T. ATP decrements during shock were profoundly T dependent (% loss of ATP; 15%, 30%, 58% at 32.5 degrees C, 37 degrees C, and 39.5 degrees C, respectively; p less than 0.001), reflecting T-dependent increments in renal metabolic rate, not decreased O2 delivery (arterial O2 content; renal blood flow). ATP losses during shock correlated with the extent of S3 proximal tubular morphologic damage. O2 deprivation dramatically increased FFA levels both in vivo and in vitro but the increments were only slightly T dependent. In vitro, % lactate dehydrogenase release and FFA levels did not significantly correlate and bovine serum albumin, a FFA binder, conferred no protection. CONCLUSIONS: (a) T dramatically accentuates hypoxic, not reoxygenation, injury; (b) changes in membrane deacylation do not appear to underlie this effect; and (c) T has a profound impact on renal ATP losses during shock, thereby affecting the severity of ischemic renal damage.

Regional responses within the kidney to ischemia: assessment of adenine nucleotide and catabolite profiles.

UNLABELLED: Renal cortex (C) has predominantly aerobic metabolism, whereas inner medulla (IM) has both aerobic and anaerobic capacities. This study was undertaken (1) to assess how well rat IM anaerobic metabolism maintains this region's ATP content during ischemia; and (2) to determine whether regional variations in adenylate pool/catabolite responses to ischemia exist, obscuring interpretation of cellular energetics in rat studies of acute renal failure (ARF). Adenine nucleotides/catabolites were measured in rat C, IM and outer medulla (OM) after 15 and 45 min of ischemia. After 15 min, all regions showed profound ATP depletion, although the IM maintained slightly higher (by 0.23 mumol/g) absolute ATP levels than C/OM tissues (normal ATP value = 8.7 mumol/g). By 45 min, significant differences in regional ATP levels did not exist. Striking regional catabolite differences were apparent at both 15 and 45 min. Most prominent were: (1) intrarenal purine base/inosine gradients, levels falling approx. 22-50% from C to IM; and (2) preferential OM AMP/IMP/adenosine accumulation. To assess whether more homogeneous results might be found in rabbit kidney, possibly making this animal preferable to rats for studies of renal ischemia, rabbit C, OM and IM adenylate pools were analyzed after 15 min of ischemia. C vs. IM ATP differences were greater (approx. 1.3 mumol/g) and large catabolite concentration differences were still apparent. CONCLUSIONS: (1) anaerobic mechanisms support IM ATP levels during ischemia but, in terms of normal concentrations, the impact is small, particularly in the rat; and (2) marked regional differences in adenylate catabolite levels exist within ischemic kidneys. These need to be recognized when analyzing adenylate pool responses in ischemic ARF.

Isolation and identification of a novel human metabolite of cyclosporin A: dihydro-CsA M17.

A novel metabolite of cyclosporin A was observed in human blood and urine. An analytical sample of this metabolite was isolated from human urine and the structure was determined to be (8-hydroxy-6,7-dihydro-MeBMT1) cyclosporin based on the 1H-NMR, 13C-NMR, FAB-MS, and HPLC characteristics of the biological sample as well as by comparison with a synthetically derived authentic sample. The significance of this metabolite in terms of the pathway by which cyclosporin A is metabolized is discussed.

Effects of xanthine oxidase inhibition on ischemic acute renal failure in the rat.

To assess the effects of xanthine oxidase (XO) inhibition on ischemic injury, rats were pretreated with oxypurinol (OXY, 5 mg/kg) and subjected to 30 min of bilateral renal artery occlusion. OXY's effect on adenine nucleotide-nucleoside-purine base concentrations was determined at 10 and 30 min of ischemia and during reperfusion (5 and 30 min). To assess whether XO-mediated oxidant stress influences the severity of ischemic acute renal failure (IARF), the effects of 1) OXY pretreatment and 2) hypoxanthine infusion were assessed. During ischemia OXY inhibited XO activity (more than fourfold rise in hypoxanthine-xanthine ratios) and induced quantitatively trivial but significant increases in ATP and total adenine nucleotide concentrations (by 30 min). Increased OXY dosage (15 mg/kg) or allopurinol (40 mg/kg) had no greater effects. At 5 min of reflow, OXY maintained XO inhibition but did not influence adenine nucleotide levels. By 30 min of reflow, 17-20% increments in ATP-total adenine nucleotides resulted. Nevertheless, OXY did not lessen the severity of IARF (assessed by azotemia-histology at 24 h). Hypoxanthine infusion increased end-ischemic hypoxanthine concentrations by 47%, but it did not change the severity of renal damage. Conclusions include 1) OXY-allopurinol induces intrarenal XO inhibition; 2) XO inhibitors slightly increase late ischemic-reperfusion adenine nucleotide concentrations; and 3) neither XO inhibition nor intrarenal hypoxanthine loading alters the severity of IARF, suggesting that XO-mediated oxidant stress is not a critical, consistent mediator of ischemic renal injury.

Degree and time sequence of hypothermic protection against experimental ischemic acute renal failure.

The purpose of this study was to assess the degree, time sequence, and biochemical correlates of hypothermic protection against ischemic acute renal failure. Rats subjected to 40 minutes of bilateral renal artery occlusion (RAO) were made mildly hypothermic (32 degrees-33 degrees C, by cold saline peritoneal lavage) during the following time periods: 1) RAO only, 2) reperfusion only (beginning at 0, 15, 30, or 60 minutes after RAO and maintained for 45 minutes), or 3) during and after (0-45 minutes) RAO. Continuously normothermic (37 degrees C) RAO rats served as controls. The control rats developed severe acute renal failure (blood urea nitrogen [BUN], 95 +/- 4 mg/dl; creatinine, 2.2 +/- 0.1 mg/dl; and extensive tubular necrosis at 24 hours). Hypothermia confined to RAO was highly protective (BUN, 33 +/- 5 mg/dl; creatinine, 0.62 +/- 0.07 mg/dl; and minimal necrosis). Hypothermia partially preserved ischemic renal adenylate high-energy phosphate (ATP and ADP), increased AMP and inosine monophosphate concentrations, and lessened hypoxanthine/xanthine buildup (assessed at end of RAO). Hypothermia confined to the reflow period (beginning at 0, 15, and 30 minutes) was only mildly protective (e.g., BUN, 58-63 mg/dl); the degree of protection did not differ according to the time of hypothermic onset. Lowering reflow temperature to 26 degrees C had no added benefit. Hypothermia that started at 60 minutes after RAO conferred no protection. Combining ischemic and postischemic hypothermia abolished all renal failure (assessed at 24 hours). This study offers the following conclusions: Mild hypothermia can totally prevent experimental ischemic acute renal failure. Hypothermia is highly effective during ischemia, and it is mildly protective during early reflow; these benefits are additive. During early reflow, hypothermic protection is not critically time dependent. By 60 minutes of reflow, no effect is elicited; this absence of effect possibly signals completion of the reperfusion injury process. Hypothermia's protective effects may be mediated, in part, by improvements in renal adenine nucleotide content and, possibly, by decreasing postischemic oxidant stress.

Blood cyclosporine pharmacokinetics in patients undergoing marrow transplantation. Influence of age, obesity, and hematocrit.

Blood cyclosporine pharmacokinetics were studied in 85 patients aged 1-52 (median: 22) years undergoing allogeneic bone marrow transplantation for the treatment of hematologic disease. Pharmacokinetic studies were carried out during the first two weeks posttransplant after an intravenous dose of 2.1-4.4 mg/kg. Whole-blood cyclosporine concentrations were measured by high-performance liquid chromatography. Multiple stepwise regression analysis indicated that age (P less than 0.001) and hematocrit (P less than 0.05) correlated with cyclosporine clearance (CL) while steady-state volume of distribution (Vss) did not correlate with any of the factors studied. Cyclosporine CL significantly differed among nonobese patients in different age groups; patients less than or equal to 10 years old had a higher mean CL (13.1 ml/min/kg) than patients 11-20, 21-30, 31-40, or greater than 40 years old (mean CL: 8.5-10.3 ml/min/kg) (P less than 0.05). No significant differences in cyclosporine CL and Vss were observed between obese (greater than 125% ideal body weight) patients and age-matched nonobese patients. Hematocrit values (range: 24-39) were inversely correlated with cyclosporine CL, which suggests that red blood cells function as important ligands in cyclosporine binding. These results show that blood cyclosporine CL is higher in marrow transplant recipients than in solid organ transplant recipients and that these differences may be related to lower hematocrits in marrow transplant recipients compared with solid organ transplant recipients. When compared with previously published serum cyclosporine CL data, our findings suggest that age-related changes in CL are primarily related to changes in plasma protein binding and that obesity does not significantly alter cyclosporine CL and Vss.

High-performance liquid chromatographic column-switching method for two cyclosporine metabolites in blood.

Cyclosporine (CSA) is biotransformed to many metabolites which may contribute to its immunosuppressive and nephrotoxic activity. We report a rapid and sensitive, automated column-switching high-performance liquid chromatographic (HPLC) method for measuring CSA-M17 in whole blood; the method also separates CSA-M1. CSA metabolite standards were isolated by a preparative-scale HPLC method. Samples were prepared by protein precipitation with acetonitrile followed by dilution with water. CSA-M17 was initially separated on a C8 column; final separation was on a C18 column. The inter-day relative standard deviation at 50 ng/ml was 8% (n = 3). Limit of detection was 20 ng/ml.

Monitoring cyclosporin concentrations in marrow transplant recipients: comparison of two assay methods.

Renal dysfunction is the major dose-limiting toxicity associated with cyclosporin therapy. We have previously shown in patients undergoing allogeneic bone marrow transplantation that serum cyclosporin concentrations, as measured by radioimmunoassay (RIA), correlate significantly with the development of renal dysfunction. However, since the RIA measures both parent drug and metabolites, the relative role of each in the development of nephrotoxicity could not be determined. Therefore, we re-measured cyclosporin concentrations in the same serum samples by high-performance liquid chromatography (h.p.l.c.). Serum cyclosporin concentrations of less than 50, 50-100 and greater than 100 ng/ml, as measured by h.p.l.c., were considered equivalent to cyclosporin concentrations of less than 150, 150-250 and greater than 250 ng/ml, as measured by RIA. Contrary to results by RIA, cyclosporin concentrations measured by h.p.l.c. did not significantly correlate with renal dysfunction, which suggests that measurement of serum cyclosporin concentrations by h.p.l.c. provides no clinical advantage to RIA for monitoring cyclosporin concentrations to prevent renal dysfunction.

Age-dependent cyclosporine: pharmacokinetics in marrow transplant recipients.

We evaluated the effect of age on cyclosporine pharmacokinetics in 69 nonobese patients aged 10 months to 56 years (median 22 years) undergoing allogeneic bone marrow transplantation for treatment of aplastic anemia or hematologic malignancy. Cyclosporine pharmacokinetics were studied during the first 2 posttransplant weeks after an intravenous dose of 2.6 to 3.5 mg/kg. Serum cyclosporine concentrations were measured by HPLC. Cyclosporine concentration-time data were fitted to a two-compartment model with a nonlinear regression program. There was a significant inverse linear correlation between age and both total systemic clearance (CL) (r = 0.42; P less than 0.001) and volume of distribution at steady-state (Vss) (r = 0.33; P less than 0.01). Mean (+/- SE) cyclosporine CL was 82 +/- 21, 45 +/- 5, 38 +/- 9, 44 +/- 8, and 20 +/- 3 ml/min/kg and mean cyclosporine Vss was 34 +/- 11, 28 +/- 10, 15 +/- 4, 14 +/- 5, and 4.7 +/- 0.7 L/kg in patients 0 to 10 (n = 12), 11 to 20 (n = 19), 21 to 30 (n = 12), 31 to 40 (n = 17), and greater than 40 (n = 9) years old, respectively. Patients 0 to 10 years old had a significantly higher cyclosporine CL than those 11 to 40 or greater than 40 years old and also had a significantly larger Vss than those greater than 40 yrs old (P less than 0.05). Age-related differences in CL or Vss were also observed when these parameters were normalized by body surface area.(ABSTRACT TRUNCATED AT 250 WORDS)