The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH.

BACKGROUND AND PURPOSE: Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown. EXPERIMENTAL APPROACH: Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown. KEY RESULTS: ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar 'dose'-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1. CONCLUSIONS AND IMPLICATIONS: These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.

Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells.

Two H(+)-K(+)-ATPase isoforms are present in kidney: the gastric, highly sensitive to Sch-28080, and the colonic, partially sensitive to ouabain. Upregulation of Sch-28080-sensitive H(+)-K(+)-ATPase, or "gastric" H(+)-K(+)-ATPase, has been demonstrated in hypokalemic rat inner medullary collecting duct cells (IMCDs). Nevertheless, only colonic H(+)-K(+)-ATPase mRNA and protein abundance increase in this condition. This study was designed to determine whether Sch-28080 inhibits transporters other than the gastric H(+)-K(+)-ATPase. In the presence of bumetanide, Sch-28080 (200 microM) and ouabain (2 mM) inhibited (86)Rb(+) uptake (>90%). That (86)Rb(+) uptake was almost completely abolished by Sch-28080 indicates an effect of this agent on the Na(+)-K(+)-ATPase. ATPase assays in membranes, or lysed cells, demonstrated sensitivity to ouabain but not Sch-28080. Thus the inhibitory effect of Sch-28080 was dependent on cell integrity. (86)Rb(+)-uptake studies without bumetanide demonstrated that ouabain inhibited activity by only 50%. Addition of Sch-28080 (200 microM) blocked all residual activity. Intracellular ATP declined after Sch-28080 (200 microM) but recovered after removal of this agent. In conclusion, high concentrations of Sch-28080 inhibit K(+)-ATPase activity in mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.

Molecular and pathophysiologic mechanisms of hyperkalemic metabolic acidosis.

In summary, hyperkalemia may have a dramatic impact on ammonium production and excretion. Chronic hyperkalemia decreases ammonium production in the proximal tubule and whole kidney, inhibits absorption of NH4+ in the mTALH, reduces medullary interstitial concentrations of NH4+ and NH3, and decreases entry of NH4+ and NH3 into the medullary collecting duct. The potential for development of a hyperchloremic metabolic acidosis is greatly augmented when renal insufficiency with associated reduction in functional renal mass coexists with the hyperkalemia, or in the presence of aldosterone deficiency or resistance. Such a cascade of events helps to explain, in part, the hyperchloremic metabolic acidosis and reduction in net acid excretion characteristic of several experimental models of hyperkalemic-hyperchloremic metabolic acidosis including: obstructive nephropathy, selective aldosterone deficiency, and chronic amiloride administration (7.9).

Macrophagelike vacuolated renal tubular cells in the urine of a male with osmotic nephrosis associated with intravenous immunoglobulin therapy. A case report.

BACKGROUND: Osmotic nephrosis is a form of renal tubular injury that has been found in patients treated with intravenous immunoglobulin (IVIG). CASE: A 46-year-old male who had two courses of chemotherapy for acute myelogenous leukemia was found to have refractory thrombocytopenia. After IVIG (Sandoglobulin 12%, Novartis) administration (1 g/kg) for five consecutive days, the patient became oliguric and eventually anuric on the fifth dose. Hemodialysis was initiated, and urine production was noted on day 2 of hospitalization. Routine cytologic examination of fresh, voided urine showed numerous macrophagelike, bland epithelial cells with abundant, multivacuolated cytoplasm. Cytokeratin immunostain revealed positivity, thus confirming the epithelial origin of these cells. CONCLUSION: To our knowledge, this is the first such case reported in the English-language cytology literature. Awareness of a patient's clinical history may be helpful in avoiding an incorrect diagnosis. Urine cytology may be useful in obtaining an early diagnosis of osmotic nephrosis in patients receiving high-dose IVIG therapy that may eliminate the need for a renal biopsy.

H+,K+-ATPase.

The H+,K+-ATPases comprise a group of integral membrane proteins that belong to the X+,K+-ATPase subfamily of P-type cation-transporting ATPases. Although these H+,K+-ATPase isoforms share approximately 60-70% amino acid identity, they exhibit discrete kinetic and pharmacological properties when expressed in heterologous systems. HK alpha2 has been categorized by its insensitivity to Sch-28080, an inhibitor of the gastric H+,K+-ATPase, and partial sensitivity to ouabain, an inhibitor of the Na+,K+-ATPase. This functional profile contrasts with the pharmacological sensitivities ascribed to HK alpha2 in transport studies in rat isolated medullary collecting ducts perfused in vitro and in mouse medullary collecting duct cell lines. HK alpha2 mRNA and protein abundance appears to be both tissue and site-specifically upregulated in response to chronic hypokalemia. This regulatory response has been localized to the outer and inner medulla. To reconcile these expressed sensitivities to those reported in vitro in isolated tubules and cells in culture, it would be necessary to invoke modification of the pharmacologic insensitivity of the colonic H+,K+-ATPase to Sch-28080. Although a 'unique' beta-subunit has been reported recently, this beta-subunit (beta(c)) is identical at the amino acid level to the recently cloned beta3-Na+,K+-ATPase. Moreover, while HK alpha2 can assemble indiscriminately with any X+,K+-ATPase beta-subunit, HK alpha2 has been reported to assemble stably with beta1-Na+,K+-ATPase in the renal medulla and in the distal colon. It remains conceivable that subunit assembly could be tissue specific and might respond to different physiological and pathophysiological stimuli. Furthermore, recent studies have suggested that the H+,K+-ATPase is both Na+-dependent and localized to the apical membrane in the distal colon. Therefore, future studies will need to resolve these discrepancies by determining if a unique, yet undiscovered H+,K+-ATPase isoform exists in kidney, or if post-translational modifications of the alpha- and/or beta-subunits could account for these functional diversities.

Contrasting functional and regulatory profiles of the renal H+,K+-ATPases.

The H+,K+-ATPases belong to the X+,K+-ATPase subfamily of P-type cation-transporting ATPases. While these H+,K+-ATPase isoforms share approximately 60%-70% amino acid identity, they exhibit discrete kinetic and pharmacological properties. The colonic alpha isoform (HKalpha2) is insensitive to Sch-28080, an inhibitor of the gastric H+,K+-ATPase, and is sensitive to high concentrations of ouabain. This profile contrasts with the sensitivities attributed to HKalpha2 in transport studies. HKalpha2 mRNA and protein abundance appear to be both site-specifically upregulated in response to chronic hypokalemia, and have been localized to the outer and inner medulla. To reconcile expressed sensitivities with those reported in vitro in isolated tubules and cells in culture, it requires transformation of the expressed insensitivity of the colonic H+,K+-ATPase to Sch-28080. Although a "unique" beta subunit has been reported recently, this beta subunit ("betac"), is identical at the amino acid level to the recently cloned beta3-Na+,K+-ATPase. Moreover, while HKalpha2 can assemble indiscriminately with any X+,K+-ATPase beta subunit, HKalpha2 has been reported to assemble stably with beta1-Na+,K+-ATPase in the renal medulla and in the distal colon. It is conceivable that subunit assembly could be tissue-specific and might respond to different physiological and pathophysiological stimuli. Recent studies have suggested that the H+,K+-ATPase is both Na+-dependent and localized to the apical membrane in the distal colon. Future studies will be needed to resolve these discrepancies by determining if a unique, yet undiscovered H+,K+-ATPase isoform exists in the kidney, or if posttranslational modifications of the alpha and/or beta-subunits could account for these functional diversities.

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.

Peptides derived from the human transferrin receptor stimulate endosomal acidification via a Gi-type protein.

UNLABELLED: Peptides derived from the human transferrin receptor stimulate endosomal acidification via a Gi-type protein. BACKGROUND: Acidification of the endosomal compartment is a prerequisite for intracellular processing of endocytosed complexes. Endosomal acidification is accomplished by an H+-ATPase, in parallel with a Cl- conductance. Previous studies from our laboratory have demonstrated that endosomal acidification is modulated by a pertussis toxin-sensitive mechanism, suggesting that endosomal acidification could be regulated through a self-contained signal transduction pathway. This study was designed to test this hypothesis using the transferrin receptor as a model. METHODS: Synthetic peptides corresponding to a region of the cytosolic domain of the transferrin receptor and containing a KPKR sequence were used to stimulate endosomal acidification in a G-protein-dependent manner. RESULTS: Peptides activated the Gi, as evidenced by stimulation of the rate of GTPgammaS binding. A transferrin receptor peptide that lacked the KPKR sequence did not stimulate endosomal acidification and failed to promote GTPgammaS binding to Gi proteins. CONCLUSIONS: These results demonstrate that regulation of endosomal acidification can be achieved, in part, through a Gi-mediated signal transduction pathway. These findings suggest that regulation of endosomal acidification through such a pathway may facilitate intracellular processing of the transferrin receptor.

Unexpected severe hypocalcemia during continuous venovenous hemodialysis with regional citrate anticoagulation.

Citrate is known to induce acute hypocalcemia in patients undergoing liver transplantation during the anhepatic phase. We describe the case of a 71-year-old woman with fulminant hepatic failure secondary to hepatitis A, who was started on continuous venovenous hemodialysis (CVVHD) for acute renal failure. Because anticoagulation with heparin was untenable, regional anticoagulation was accomplished by trisodium citrate (46.7%) infusion. Unfortunately, severe hypocalcemia developed when citrate accumulated because of impaired hepatic metabolism. Because of chelation by citrate, the ionized calcium concentration declined to values as low as 2.72 mg/dL (normal, 4.5 to 5.6 mg/dL), whereas the total calcium concentration remained in the normal range. With an unusually high calcium chloride infusion rate via a central line (up to 140 mL/h of 10 mEq/dL CaCl2) and additional boli of CaCl2 (for a total of 190 mEq), the ionized calcium concentration could be maintained at target levels. Nevertheless, the ionized calcium concentration was maintained in the normal range, and the total calcium concentration increased to a value as high as 15 mg/dL. Thus, the total to ionized calcium ratio was 3.5:1. After 24 hours of treatment, trisodium citrate infusion was gradually reduced from 15 mL/h to 7 mL/h, and the calcium chloride infusion was decreased to 50 mL/h. Nevertheless, persistence of the elevated total to ionized calcium ratio (3:1) indicated citrate accumulation likely secondary to decreased hepatic metabolism. Using this approach, the patient was successfully maintained on CVVHD with regional citrate anticoagulation for a total of 11 days without any additional complications. We conclude that CVVHD with regional citrate anticoagulation can be used in patients with acute hepatic failure if increased CaCl2 requirements are anticipated and if citrate is infused at a lower rate compatible with decreased citrate metabolism. Citrate accumulation should be suspected in patients with an elevated total to ionized Ca++ ratio during CVVHD with citrate anticoagulation.

Dietary K+ restriction upregulates total and Sch-28080-sensitive bicarbonate absorption in rat tIMCD.

In tubules from the terminal segment of the inner medullary collecting duct (tIMCD) from rats with chronic metabolic acidosis, our laboratory has shown that bicarbonate absorption (JtCO2) is inhibited by removal of K+ from the luminal fluid or by the addition of Sch-28080 to the perfusate. The present study asked whether total and/or Sch-28080-sensitive JtCO2 is regulated by changes in systemic K+ homeostasis. Rat tIMCD tubules were perfused in vitro in symmetrical, HCO-3/CO2-buffered solutions containing 10 mM KCl + 6 mM NH4Cl. Total and Sch-28080-sensitive JtCO2 were measured in rats with varying K+ intake. In K+-replete rats, baseline JtCO2 was 2.1 +/- 0.3 pmol . mm-1 . min-1 (n = 6). In rats fed a K+-deficient diet for 3 days, JtCO2 was 5.4 +/- 0.7 pmol . mm-1 . min-1 (n = 16, P < 0. 05). To determine the mechanism for the increase in HCO-3 absorption observed with K+ restriction, the Sch-28080-sensitive component of JtCO2 was measured in each treatment group. Following the addition of Sch-28080 (10 microM) to the perfusate, a 40% reduction in JtCO2 was observed in K+-restricted rats. JtCO2 was not reduced following the addition of Sch-28080 in rats with normal K+ intake. Because Sch-28080-sensitive JtCO2 was increased in K+-restricted rats, Sch-28080-sensitive JtCO2 was studied further in tIMCD tubules from rats in this treatment group. In K+-restricted rats, JtCO2 decreased by 20% following the addition of 5 mM ouabain to the perfusate. This ouabain-induced decline in JtCO2 was observed both in the presence and in the absence of Sch-28080. We conclude that total and Sch-28080-sensitive net acid secretion is increased with dietary K+ restriction. However, since approximately 50% of JtCO2 is insensitive to both Sch-28080 and ouabain, future studies will be necessary to define other mechanisms of luminal acidification in the rat tIMCD.

Expression of HKalpha2 protein is increased selectively in renal medulla by chronic hypokalemia.

Our laboratory has demonstrated by Northern analysis that chronic hypokalemia increases HKalpha2 (i.e., alpha-subunit of the colonic H+-K+-ATPase) mRNA abundance in the rat. To determine whether the increase in mRNA correlated with an increase in HKalpha2 protein, an antibody was raised against a synthetic peptide derived from amino acids 686-698 of the HKalpha2 sequence. The anti-HKalpha2 antibody hybridized to rat distal colon membranes which migrated at approximately 100 kDa (expected mobility of HKalpha2). HKalpha2 protein was not detected in plasma membranes from rat whole kidney or stomach (100 microg) derived from control animals. The antibody was then used to investigate changes in expression of HKalpha2 in renal cortex, renal medulla, and distal colon in two pathophysiological conditions: 1) chronic hypokalemia (LK) and 2) chronic metabolic acidosis (CMA). In LK rats there was a marked, but selective, increase in the abundance of HKalpha2 protein in membranes prepared from renal medulla. Nevertheless, a corresponding increase in HKalpha2 protein abundance was not observed in membranes prepared from the distal colon of LK rats. HKalpha2 protein abundance in CMA was indistinguishable from controls. Moreover, chronic hypokalemia had no effect on expression of alpha1-Na+-K+-ATPase or HKalpha1 in kidney or distal colon under any experimental condition. Therefore, HKalpha2 protein is tissue- and site-specifically upregulated in response to chronic hypokalemia but not by CMA. Furthermore, this regulatory response is localized to the renal medulla.

The alpha-subunit of the colonic H+,K+-ATPase assembles with beta1-Na+,K+-ATPase in kidney and distal colon.

Previous experiments from our laboratory (Codina, J., Kone, B. C., Delmas-Mata, J. T., and DuBose, T. D., Jr. (1996) J. Biol. Chem. 271, 29759-29763) demonstrated that the alpha-subunit of the colonic H+, K+-ATPase (HKalpha2) requires coexpression with a beta-subunit to support H+/K+ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HKalpha2 formed stable and functional alpha.beta complexes when coexpressed with either the rat beta1-subunit of the Na+,K+-ATPase or the beta-subunit of the gastric H+,K+-ATPase, suggesting that different beta-subunits may interact with HKalpha2. The present studies tested this hypothesis by development and application of a specific antibody against HKalpha2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HKalpha2 co-precipitates with the same beta-subunit in organs known to express HKalpha2 protein. The data demonstrate that HKalpha2 assembles with beta1-Na+,K+-ATPase in the renal medulla and in distal colon.

Adaptation to low-K+ media increases H(+)-K(+)-ATPase but not H(+)-ATPase-mediated pHi recovery in OMCD1 cells.

Studies in rat and rabbit outer medullary collecting duct of inner stripe origin (OMCDis) suggest that both H(+)-ATPase and H(+)-K(+)-ATPase participate in H+ secretion. However, the relative contributions of these transporters, and, in particular, that of H(+)-K(+)-ATPase to K+ absorption have not been defined precisely. The present study was designed to delineate more clearly the response of these two transporters to hypokalemia and acidosis in a newly developed mouse OMCD1 cell line. In cells grown in normal K+ (5 mM) media, intracellular pH (pHi) recovery was similar either in the presence or absence of K+ in the perfusate (delta pHi/min = 0.014 +/- 0.001 vs. 0.017 +/- 0.003, not significant). The inhibitory effects of Sch-28080 (10 microM) and bafilomycin A1 (10 nM) on pHi recovery were evident only in the presence and absence of K+ in the perfusate, respectively. In cells grown in low-K+ (2.5 mM) media to simulate chronic hypokalemia, pHi recovery was significantly faster than in cells grown in normal K+ media (delta pHi/min = 0.045 +/- 0.01 vs. 0.014 +/- 0.001, P < 0.01) and was inhibited specifically by Sch-28080, not by bafilomycin A1. In contrast, in cells preconditioned to low pH (7.0) to simulate chronic acidosis, the enhanced pHi recovery was abolished by bafilomycin A1 but not by Sch-28080. 86Rb+ uptake, when used as a K+ congener, was inhibited by Sch-28080. The K(m) for 86Rb+ uptake (H(+)-K(+)-ATPase activity) and the 50% inhibitory concentration for Sch-28080 were 270 and 5.0 microM, respectively. These studies provide evidence that, in morphologically homogeneous OMCD1 cells, 1) both H(+)-K(+)-ATPase and H(+)-ATPase participate in pHi regulation, 2) the H(+)-K(+)-ATPase is selectively upregulated by preconditioning in low-K+ media, and 3) conversely, preconditioning in low-pH media stimulates only the H(+)-ATPase. Thus, in OMCDis, the H(+)-K(+)-ATPase and H(+)-ATPase respond selectively and independently to chronic hypokalemia and acidosis, respectively.

Mannitol-induced acute renal failure.

The osmotic diuretic mannitol may be used in diverse clinical settings, such as providing "renal protection" in patients at risk for acute renal failure, decreasing intracranial pressure in patients with intracranial trauma, and preventing the dialysis-disequilibrium syndrome. Mannitol is commonly used after cardiac catheterization, cardiovascular surgery, and exposure to intravenous contrast dyes. This study presents a case in which a long-term renal transplant recipient receiving cyclosporine therapy concomitantly developed acute renal failure after the administration of high-dose mannitol in an attempt to induce an osmotic diuresis. The diagnosis of "osmotic nephrosis" was confirmed by renal biopsy, and the condition was reversed by cessation of the agent. Studies in experimental animals indicate that cyclosporin A can potentiate the tubular toxicity of mannitol, but such an association has not been verified in humans. Numerous studies confirm the nephrotoxic potential of high-dose mannitol, especially in patients with renal insufficiency. The clinical utility of the osmolar gap in preventing mannitol nephrotoxicity is emphasized.

Acute renal failure in the 21st century: recommendations for management and outcomes assessment.

Acute renal failure (ARF) remains a common and potentially devastating disorder affecting as many of 5% of all hospitalized patients, with a higher prevalence in patients in critical care units. ARF is more frequently observed in the setting of multiorgan dysfunction syndrome (MODS) and in elderly patients with complex disease, where mortality is high. Numerous technical advances have not yet impacted favorably on this high mortality rate. This report summarizes recommendations from participants at the National Institutes of Health Conference: "Acute Renal Failure in the 21st Century," May 6 to 8, 1996, in Bethesda, MD. The focus is on categorizing recent clinically relevant developments in the field and on identification of new research initiatives to transfer a new body of knowledge derived from fundamental studies and laboratory investigation to the management of ARF in the new millennium. The development of a multicenter database through cooperative multicenter studies is advocated. Future studies should define the appropriate outcome measures to assess and emphasize the impact of hemodynamic monitoring, adjunctive agents, and adequacy and modality of renal replacement therapy on outcomes in ARF.

Effect of chronic hypokalemia on H(+)-K(+)-ATPase expression in rat colon.

Although the kidney plays the major role in the regulation of systemic K+ homeostasis, the colon also participates substantively in K+ balance. The colon is capable of both K+ absorption and secretion, the magnitude of which can be modulated in response to dietary K+ intake. The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) has been proposed as a possible mediator of K+ absorption in distal colon, but inhibitor profiles obtained in recent studies suggest that two, and perhaps more, distinct H(+)-K(+)-ATPase activities may be present in mammalian distal colon. We have developed highly specific probes for the catalytic alpha-subunits of colonic and gastric H(+)-K(+)-ATPase, alpha 1-Na(+)-K(+)-ATPase, and beta-actin, which were used in Northern analysis of total RNA from whole distal colon and stomach obtained from one of three experimental groups of rats: 1) controls, 2) chronic dietary K+ depletion, and 3) chronic metabolic acidosis. The probe for the colonic but not the gastric H(+)-K(+)-ATPase alpha-isoform hybridized to distal colon total RNA in all groups. A significant increase in colonic H(+)-K(+)-ATPase mRNA abundance was observed in response to chronic dietary K+ depletion but not to chronic metabolic acidosis. The alpha 1-isoform of Na(+)-K(+)-ATPase, which is also expressed in distal colon, did not respond consistently to either chronic dietary K+ depletion or chronic metabolic acidosis. The gastric probe did not hybridize to total RNA from distal colon but, as expected, hybridized to total stomach RNA. However, the abundance of gastric H(+)-K(+)-ATPase or Na(+)-K(+)-ATPase in stomach was not altered consistently by either chronic dietary K+ depletion or metabolic acidosis. Under the conditions of this study, it appears that the mRNA encoding the colonic alpha-isoform is upregulated by chronic dietary K+ restriction, a condition shown previously to increase K+ absorption in the distal colon.

Effective clearance of methotrexate using high-flux hemodialysis membranes.

We report the first series demonstrating effective clearance of methotrexate using acute intermittent hemodialysis with a high-flux dialyzer. The study was performed on six patients, two females and four males aged 13 to 72 years. All were patients at M.D. Anderson Cancer Center. Patients were dialyzed for 4 to 6 hours daily using a Fresenius F-80 membrane (Fresenius Inc, Walnut Creek, CA). Following the initiation of dialysis, there was a reduction in arterial and venous serum concentration of methotrexate with time. Mean plasma clearance of methotrexate during dialysis in these six patients was 92.1 +/- 10.3 mL/min. One patient who was nearly functionally anephric was studied in detail. In this patient, following a high dose of methotrexate (7.2 g/m2), approximately 63% of this dose was cleared with 6 hours of hemodialysis. With subsequent dialysis performed daily for 6 hours, the drug was cleared completely in 5.6 +/- 0.3 days (n = 7 separate methotrexate treatments). A reduction in plasma methotrexate concentration from 1,733 +/- 40 micromol/L 1 hour postinfusion to less than 0.3 micromol/L in 5 to 6 days was observed for these seven separate treatments. We conclude that significant clearance of methotrexate can be achieved with high-flux dialyzers, making methotrexate therapy a viable treatment option in patients with responsive malignancies despite the presence of renal failure.