Organic Anion Transport Inhibitor Research Articles

Nitric oxide donors induce extrusion of cyclic GMP from isolated human blood platelets by a mechanism which may be modulated by prostaglandins

In the presence of 3-isobutyl-methylxanthine (IBMX), induction of cyclic 3′,5′-guanosine monophosphate (GMP) production in human washed platelets (HWP) by nitric oxide donors (NOD) is followed by its accumulation in the surrounding medium in a time- and concentration-dependent manner. Thirty minutes incubation of HWP with 3-morpholino-sydonimine (SIN-1, 10 μM) at 37 °C resulted in a 4.6-fold increase of cyclic GMP in platelets, whereas in the extracellular medium the increase was 17.6-fold. Similar results were obtained when other NOD such as S-nitroso- N-acetylpenicyllamine (SNAP) and 3-(2-metoxy-5-chlorophenyl)oxatriazol-5-imine (GEA 3184) and the selective phosphodiesterase inhibitor, zaprinast (M&B 22948, 10 μM), were used. Probenecid (1–300 μM), an inhibitor of organic anion transport, or ouabain (1–300 μM), an inhibitor of Na + K + adenine triphosphate (ATP)-ase had no effect on cyclic GMP production or extrusion after stimulation with SIN-1. Significantly, prostaglandin A 1 (PGA 1) and prostaglandin D 2 (PGD 2) inhibited the efflux of cyclic GMP from platelets induced by SNAP (10 μM) in a concentration-dependent fashion, with an IC 50 of 63 ± 16 and 143 ± 17 μM, respectively. These studies suggest that the extrusion of cyclic GMP from human platelets after activation of soluble guanylate cyclase by NOD may contribute to the control of cyclic GMP levels in platelets with potential physiological and therapeutic consequences.

Transport characteristics of three fluorescent conjugated bile acid analogs in isolated rat hepatocytes and couplets

The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain,were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(Nϵ-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(Nϵ-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37°C with 0.3 μmol/L of FBA and 0.15 mol/L of Na +, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 ± 0.5 for CGamF, 7.2 ± 0.3 for C-NBD-L, and 13.7 ± 1.0 for CDC-NBD-L (mean, ± SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 μmol/L and was saturable. The Michaelis constant (K m) value (μmol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na +, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na + independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na +-dependent and Na + independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these non-fluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBDL was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN + or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 μmol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.

Transport characteristics of three fluorescent conjugated bile acid analogs in isolated rat hepatocytes and couplets

The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain, were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(N epsilon-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(N epsilon-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37 degrees C with 0.3 mumol/L of FBA and 0.15 mol/L of Na+, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 +/- 0.5 for CGamF, 7.2 +/- 0.3 for C-NBD-L, and 13.7 +/- 1.0 for CDC-NBD-L (mean, +/- SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 mumol/L and was saturable. The Michaelis constant (Km) value (mumol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na+, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na+ independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na(+)-dependent and Na(+)-independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these nonfluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBD-L was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN- or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 mumol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.

Inhibition of renal xenobiotic excretion by tetrachloroguaiacol: Mechanism and possible consequences

AbstractThe weak acid tetrachloroguaiacol (teCG; pKa 6.0) is the major chlorinated phenol produced during chlorine bleaching of wood pulp. Upon its release into the environment, it is persistent and markedly bioaccumulated (100‐ to 1,000‐fold). Within the organism (pH ∼ 7.4), it should be >95% ionized, making it a likely inhibitor of renal organic anion transport. Its effects on both passive membrane permeability and mediated transport processes were determined in basolateral (BLM) and luminal membrane (BBM) vesicles isolated from the rat kidney cortex. Vesicular solute uptake was measured by the rapid filtration technique at pH 7.5. Passive membrane permeability (assessed by mannitol uptake) was altered only at very high concentrations of teCG (≥ 1,000 μM). In contrast, transport of the model organic anion p‐aminohippurate (PAH) by BLM vesicles was significantly inhibited by as little as 5 μM (1.3 mg/L) teCG and totally abolished by 200 μM. Inhibition was competitive (K1 = 31.3 μM). Decreasing assay pH diminished inhibition of the PAH uptake, reflecting the reduced concentration of ionized teCG. Other membrane effects were seen at higher teCG concentrations (> 100 μM). The BLM sodium/glutarate cotransport was modestly, but significantly, stimulated by 100 to 500 μM teCG, whereas BBM sodium/glucose cotransport was inhibited by similar teCG concentrations. Thus, teCG has multiple effects on renal membrane function. However, at environmental levels (0.1 to 30 μM in plasma), its principal membrane effects would be related to changes in organic anion secretion.

Biphasic increase in cytosolic free calcium induced by bradykinin and histamine in cultured tracheal smooth muscle cells: is the sustained phase artifactual?

The effects of bradykinin (BK) and histamine on intracellular Ca2+ ([Ca2+]i) were studied in fura-2-loaded guinea-pig tracheal smooth muscle cells in culture. BK, at 10 nM, and histamine, at 100 microM, induced a rise in [Ca2+]i which was inhibited by the B2 antagonist Hoe 140 and by the H1 antagonist triprolidine, respectively. This rise in [Ca2+]i is biphasic, consisting of a rapid transient phase followed by a sustained phase. The transient phase, induced by either BK or histamine, was strongly inhibited by thapsigargin, a microsomal Ca(2+)-ATPase inhibitor, usually used to deplete certain intracellular Ca(2+)-stores. Ni2+ (4 mM) did not affect the transient phase but abolished the sustained phase when cells were stimulated by BK, further supporting the fact that the transient phase was only dependent on intracellular Ca2+ pools. The sustained phase was partially (for BK) and completely (for histamine) inhibited by 30 microM Mn2+. This effect could be completely reversed by the addition of DTPA, a cell-impermeant chelator of Mn2+, indicating that the Mn2+ exerted its effect extracellularly. The presence of 1 mM probenecid (an inhibitor of a membrane organic anion transporter that extrudes fura-2) drastically inhibited the sustained phase by more than 77% for BK and 88% for histamine. Our results suggest that the effects of BK and histamine on airway smooth muscle cells are mediated via bradykinin B2 receptors and histamine H1 receptors, respectively whose activation allows the rapid transient rise in [Ca2+]i from thapsigargin-sensitive intracellular Ca2+ pools. The sustained phase is proposed to be drastically influenced by an acceleration of fura-2 extrusion during the increase of [Ca2+]i via a probenecid-sensitive mechanism.

Cellular Transport of Drugs

The ability of drugs to enter cells and to reach an adequate concentration within the appropriate intracellular compartment may be an important determinant of the efficacy of therapy for infections due to intracellular pathogens. Antibiotics vary considerably in their ability to accumulate within cells. All solutes can be taken up by endocytosis, and some compounds reach high intracellular concentrations after crossing the plasma membrane by diffusion or by specific transport processes. Cellular organelles have properties that affect the intracellular distribution of drugs and that may account for the net accumulation of drugs within cells. An important example is the ability of acidic organelles to trap lysosomotropic weak bases. Cells also express transport proteins that may limit the intracellular accumulation of drugs by secreting them across the plasma membrane into the extracellular medium. Thus, cellular organic anion transporters can decrease the intracellular accumulation of certain antibiotics, and the multidrug-resistance transporter, or p-glycoprotein, confers resistance to antineoplastic agents. Inhibition of organic anion transport may have therapeutic value. A better appreciation of the mechanisms by which drugs accumulate within cells and within specific intracellular compartments may lead to the design of agents that reach higher concentrations at clinically relevant intracellular sites.

Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor

A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

Affinity identification of organic anion transporters in brush-border membrane vesicles from rat kidney

The inhibitory properties of bromoacetyl-p- aminohippuric acid as the affinity probe of the organic anion transport system were studied. Bromoacetylated p- aminohippurate was shown to be able to inhibit irreversibly the p- aminohippurate (PAH) uptake in brush-border membrane vesicles. The inhibition depends on both the time of treatment and the affinity probe concentration. The treatment of brush-border membrane with 1 mM bromoacetyl-p- aminohippurate for 1.5 h results in 100% irreversible inhibition of PAH transport but no changes were observed in the activity of alkaline phosphatase, γ-glutamyltranspeptidase or maltase. The affinity labelling of the organic anion transporters was performed with bromoacetyl-p- amino[ 3 H] hippuric acid . It was shown, by means of SDS-polyacrylamide gel electrophoresis, that the probe bound covalently to the brush-border membrane proteins with molecular masses of 28 kDa, 63 kDa, 98 kDa, and > 150 kDa . The data obtained with SITS and probenecide as the organic anion transport inhibitors indicate that brush-border membrane proteins of 28 kDa, 63 kDa, 98 kDa may correspond to the organic anion transport system.

Organic Anion Transport and Reabsorptive Fluid Flux in Diabetic Retinal Vessels: a Microperfusion Study

Microperfusion techniques were used to characterize in vitro the fluorescein transport and fluid movement across isolated diabetic rabbit retinal vessels. Unbranched segments of afterioles of alloxan-induced diabetic rabbits were isolated and cannulated on a microperfusion apparatus. Movement of 3 H-inulin, the reference tracer, and 14C-fluorescein, the test tracer, across the vascular walls was studied using microperfusion techniques adapted by us to study vascular permeability. The diabetic retinal vessels showed a fluid flux from bath to lumen of 3·6 ± 0·6 nl min-1 mm-1 with perfusion rates of 7·1 ± 0·3 nl min-1. This value was 43% (P ≤ 0·05) lower when compared with normal values obtained in previous work. Bath to lumen fluorescein fluxes were 2·1 ± 0·5 and 7·6 ± 1·6 × 10-12 mol min-1 mm,-1, for fluorescein concentrations of 0·87 × 10-4 and 1·5 × 10-4 M, respectively. These values are 23% (P ≤ 0·05) and 38·7% (P ≤ 0·05), respectively, lower when compared to values obtained from normal retinal vessels. Probenecid, a competitive inhibitor of organic anion transport, added to the bath, had no effect. Increasing bath concentrations of fluorescein caused increasing fluid flux from bath to lumen, and again, probenecid added to the bath has no effect. These results show a significant decrease of fluorescein transport and of net fluid flux across diabetic retinal vessel walls from outside into the lumen, suggesting that in experimental alloxan-induced diabetes there is an early, probably functional, involvement of the retinal vessels in the initial alteration of the blood-retinal barrier (BRB).

Nephrotoxicity mechanism of cis-platinum (II) diamine dichloride in mice

Male Swiss OF1 mice were injected subcutaneously with 20 mg/kg of cis-platinum (II) diamine dichloride ( cis-platin). Examination of cryostat kidney sections stained for alkaline phosphatase (APP) revealed damage to about 10, 20, 40 and 50% of the proximal tubules after 7, 24, 48 and 72 h, respectively. Pretreatment with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), (i.p. 3 mmol/kg) potentiated the tubule damage of cis-platin. In contrast, pretreatment with organic anion transport inhibitor probenecid (i.p. 3 × 0.75 mmol/kg) reduced the number of damaged tubules by approximately 80% at 72 h after cis-platin injection. Pretreatment with the γ-glutamyltranspeptidase (γ-GT) inactivator acivicin (AT-125, 50 mg/kg p.o., plus 50 mg/kg i.p.) failed to prevent cis-platin induced renal toxicity. Pretreatment with the β-lyase inactivator aminooxyacetic acid (AOAA, 2 × 100 mg/kg p.o.) and with the renal cysteine conjugate S-oxidase inhibitor methimazole (40 mg/kg i.p.) reduced the number of damaged tubules by approximately 40% and 75%, respectively in mice treated with cis-platin. The results suggest that the platinum-sulfhydryl group complexes formed are taken up by the kidney cells through an organic anion transport mechanism which is probenecid-sensitive. In the cells these complexes are stable for several hours, depending on the intracellular glutathione (GSH) level, and gradually undergo transformation to reactive metabolite(s) by renal intracellular β-lyase and S-oxidase.

Characterization of the ATP-dependent leukotriene C4 export carrier in mastocytoma cells.

The biosynthesis of leukotriene C4 (LTC4) must be followed by an export of this mediator into the extracellular space where it interacts with receptors. Using mastocytoma cells we have demonstrated the existence of a primary-active, ATP-dependent transport mediating this export of LTC4 [Schaub, T., Ishikawa, T. & Keppler, D. (1991) FEBS Lett. 279, 83-86]. The following inhibitors served to characterize further this transport system in plasma membrane vesicles from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half-maximal inhibition of the ATP-dependent LTC4 transport at 71 microM. Cyclosporin A and its non-immunosuppressive analog PSC 833 inhibited the ATP-dependent transport with Ki values of 4.5 microM and 30 microM, respectively. The LTD4 receptor antagonist 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-[(3-dimethylamino-3- oxopropyl)-thio]-methyl]thio)propanoic acid (MK 571) was the most potent competitive inhibitor of the export carrier with a Ki value of 0.8 microM. The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC4-binding membrane proteins using [3H]LTC4 as the photolabile ligand. Proteins with molecular masses of about 190 kDa and 35 kDa were predominantly labeled. In addition, a minor [3H]LTC4 labeling was observed in the molecular mass range of 100 kDa. The [3H]LTC4 labeling of the 190-kDa protein was competed for by MK 571. The labeled proteins resisted extraction from the membrane with 2% sodium taurocholate suggesting that they are integral membrane proteins. Treatment of the membrane proteins with peptide N-glycosidase F resulted in the appearance of an additional labeled polypeptide of about 140 kDa suggesting that the 190-kDa protein is a glycoprotein. Photoaffinity labeling with 8-azido[alpha-32P]ATP predominantly labeled the LTC4-binding 35-kDa protein. The [3H]LTC4-labeled 190-kDa protein showed a mean isoelectric point at pH 6.3 with a range of pH 5.8-6.7, while the 35-kDa protein had an isoelectric point at pH 6.8. Specific labeling of a 190-kDa membrane glycoprotein by the glutathione conjugate LTC4, which is competed for by a potent inhibitor of the ATP-dependent LTC4 export carrier, pinpoints its involvement in the ATP-dependent transport of LTC4 and related conjugates.

Mechanism of mobilization of renal and hepatic cadmium by dithiocarbamates in mice

To clarify the mechanism of mobilization of nenal and hepatic cadmium (Cd) by N- benzyl- D-glucamine dithiocarbamate (BGD) and N-p- hydroxymethylbenzyl- D-glucamine dithiocarbamate (HBGD) in mice exposed to Cd, the effects of pretreatment with probenecid, an organic anion transport inhibitor, or with acivicin, a γ-glutamyltranspeptidase (γ-GTP) inhibitor and ureter-ligation were investigated on the excretion and distribution of chelating agents and Cd. The renal contents of BGD and HBGD were increased by ureter-ligation and decreased by acivicin pretreatment. The mobilizing effect of BGD on the renal Cd was inhibited by probenecid pretreatment. The action of HBGD in removing Cd from the kidney was inhibited by both probenecid pretreatment and ureter-ligation. These results suggest that BGD and HBGD are mainly taken up into the renal tubular cells through the basolateral membrane which is dependent on the action of γ-GTP; that the Cd-BGD complex formed in the tubular cells is secreted by a probenecid-sensitive organic anion transport system through the basolateral membrane; and that the Cd-HBGD complex formed in the tubular cells is secreted to the tubular lumen by an organic anion transport system through the brush border membrane. Probenecid pretreatment increased the hepatic contents of BGD and HBGD and also promoted the effects of these chelating agents in removing Cd from the liver, indicating an inhibitory effect of probenecid on the glucuronidation of BGD and the secretion of HBGD from the kidney. These results suggest that BGD and HBGD are taken up into the liver and secreted from the organ to the bile by a transport system other than a probenecid-sensitive transport mechanism.

Nephrotoxicity of 4-amino-3-S-glutathionylphenol and its modulation by metabolism or transport inhibitors.

The nephrotoxicity of 4-amino-3-S-glutathionylphenol (PAP-GSH), a known metabolite of 4-amino-phenol (PAP), was determined in male Fischer 344 rats. Administration of a single dose of 40 or 60 mumol kg-1 caused a marked elevation in blood urea nitrogen and an increase in the urinary excretion of glucose, protein and gamma-glutamyltransferase (GGT). These changes were associated with histological alterations in the proximal tubule, where at the lower dose the lesion was restricted to the S3 region of the proximal tubule in the medullary rays, while at the higher dose the lesion extended to affect the S3 region in both the medullary rays and the outer stripe of the outer medulla. Studies with [35S]-PAP-GSH at 40 mumol kg-1 showed selective retention of radioactivity in the kidney, relative to other organs 24 h after dosing and that some radioactivity was covalently bound to renal proteins. Pretreatment of animals with probenecid, an inhibitor of renal organic anion transport, or aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, had little or no effect on the toxicity. In contrast, pretreatment of animals with acivicin, an inhibitor of gamma-glutamyltransferase, or co-administration of PAP-GSH with ascorbic acid almost completely protected against the nephrotoxicity. This protection was associated with a decreased concentration of radioactivity from [35S]-PAP-GSH in the kidneys and a decrease in the amount covalently bound to renal protein. Thus, the nephrotoxicity of PAP-GSH may be mediated by oxidation and further processing of the glutathione conjugate via gamma-glutamyltransferase.

Effect of ascorbic acid, acivicin and probenecid on the nephrotoxicity of 4-aminophenol in the Fischer 344 rat.

4-Aminophenol (p-aminophenol, PAP) causes selective necrosis to the pars recta of the proximal tubule in Fischer 344 rats. The basis for this selective toxicity is not known but PAP can undergo oxidation in a variety of systems to form the 4-aminophenoxy free radical. Oxidation or disproportionation of this radical will form 1,4-benzoquinoneimine which can covalently bind to cellular macromolecules. We have recently reported that a glutathione conjugate of PAP, 4-amino-3-S-glutathionylphenol, is more toxic to the kidney than the parent compound itself. In this study we have examined the distribution and covalent binding of radiolabel from 4-[ring 3H]-aminophenol in the plasma, kidney and liver of rats 24 h after dosing and related these findings to the extent of nephrotoxicity. In addition, we have examined the effect of ascorbic acid which will slow the oxidation of PAP; acivicin, an inhibitor of gamma-glutamyltransferase and hence the processing of glutathione-derived conjugates; and probenecid, an inhibitor of organic anion transport on the nephrotoxicity produced by PAP. Administration of a single dose of PAP at 458 or 687 mumol kg-1 produced a dose-related alteration in renal function within 24 h which was associated with proximal tubular necrosis. The lesion at the lower dose was restricted to the S3 proximal tubules in the medullary rays, while at the higher dose it additionally affected the S3 tubules in the pars recta region of the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the renal transport of trimethylpentanoic acid metabolites of 2,2,4-trimethylpentane in rat renal cortical slices.

2,2,4-Trimethylpentane (TMP), a nephrotoxic component of unleaded gasoline in male but not female rats, undergoes oxidative metabolism to yield 2,2,4- and 2,4,4-trimethylpentanol, pentanoic acid and 5-hydroxypentanoic acid. We have examined the effect of three of these pentanoic acid metabolites on the renal transport of the organic anion p-aminohippurate (PAH) and the organic cation tetraethylammonium (TEA) in renal cortical slices from male Fischer 344 rats. 2,4,4-Trimethylpentanoic acid, the major urinary metabolite in rats, produced a selective decrease in the accumulation of PAH without affecting TEA accumulation. Kinetic analysis showed that 2,4,4-trimethylpentanoic acid was a competitive inhibitor of the organic anion transport system, with a Ki of 4 mM. 2,4,4-Trimethyl-5-hydroxypentanoic acid also showed selective inhibition of PAH transport, while 2,2,4-trimethylpentanoic acid was less selective and reduced both PAH and TEA transport. Additional studies with radiolabeled 2,4,4-trimethylpentanoic acid showed that there was a time- and concentration-dependent accumulation of radioactivity into slices of renal cortex. However, experiments conducted at 4 degrees C and studies with metabolic inhibitors, or with an inhibitor of organic anion transport, indicated that little of the accumulated material was entering the cell. We conclude from these studies that the pentanoic acid metabolites formed from 2,2,4-trimethylpentane are not actively transported by the renal organic anion transport system. In summary, in vitro the pentanoic acid metabolites appear to bind to renal cortical tissue and thereby reduce the transport of PAH.

The effects of 2,3,5-(triglutathion- S-yl)hydroquinone on renal mitochondrial respiratory function in vivo and in vitro: Possible role in cytotoxicity

Administration of 2,3,5-(triglutathion- S-yl)hydroquinone [2,3,5-(triGSyl)HQ] to rats causes severe renal proximal tubular necrosis. Although the cellular target(s) for 2,3,5-(triGSyl)HQ is not known, substantial evidence implicates mitochondria as the primary cellular target for aliphatic S-conjugates. To determine whether mitochondria are targets for 2,3,5-(triGSyl)HQ, the in vivo and in vitro effects of this conjugate on rat renal mitochondria (RRM) were investigated. In vitro exposure of RRM to 2,3,5-(triGSyl)HQ inhibited site I-supported respiration to a much greater extent than site II-supported respiration. Inhibition of mitochondrial function, as manifested by decreases in the respiratory control ratios, were a consequence of significant elevations in state 4 respiration. Inhibition of constitutive γ-GT activity with AT-125 had no effect on the ability of 2,3,5-(triGSyl)HQ to decrease mitochondrial function. The effects of 2,3,5-(triGSyl)HQ on mitochondrial function in vivo were subsequently assessed. Shortly (0.5–2.0 hr) following administration of 2,3,5-(triGSyl)HQ (20 μmol/kg, iv) to rats, a significant elevation of state 4 respiration was observed. Thereafter (4–16 hr) state 4 respiration returned to control values and state 3 respiration became significantly depressed. A total collapse in RRM function occurred by 24 hr. The effects of 2,3,5-(triGSyl)HQ on state 4 respiration preceded significant elevations in blood urea nitrogen, which occurred at 8 hr. However, pretreatment of animals with probenecid, and inhibitor of organic anion transport, caused a significant decrease in the 2,3,5-(triGSyl)HQ-mediated elevations in state 4 respiration at 1 hr, without preventing the subsequent development of renal necrosis. In contrast, AT-125, which protected animals from 2,3,5-(triGSyl)HQ-mediated nephrotoxicity, had no effect on the early (1 hr) elevations in state 4 respiration but did prevent the later (8 hr) decreases in state 3 respiration. The data suggest that the early elevation in state 4 respiration observed in vivo is unlikely to contribute to 2,3,5-(triGSyl)HQ-mediated nephrotoxicity. The relationship between the decrease in state 3 respiration seen at later time points and the subsequent development of toxicity require further study before a cause and effect relationship can be determined.

Acute renal toxicity of thiabendazole (TBZ) in ICR mice

The acute toxic effects of thiabendazole [2-(4′-thiazolyl)benzimidazole; TBZ] on the kidneys of ICR mice were investigated. The mice were given 0, 250, 500 or 1000 mg TBZ/kg body weight by gavage (using olive oil as a vehicle), and the kidneys were subjected to pathological examination at 1, 3, 5 or 24 hr after dosing. Gross findings were slight enlargement and the presence of whitish areas (white maculae) in kidneys of treated mice at 3, 5 or 24 hr after dosing. Histological findings were desquamation of degenerated cells in proximal tubules of treated mice at 1 hr. Dilation of proximal, distal and collecting tubules was apparent in treated mice at 3, 5 and 24 hr. TBZ-induced renal injury was reduced by pretreatment with inducers of the microsomal monooxygenase system (sodium phenobarbital, β-naphthoflavone and 3-methylcholanthrene) and were enhanced by pretreatment with inhibitors of that system (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and piperonyl butoxide). The concentration of TBZ in blood at 1 or 5 hr after dosing was lower in mice pretreated with microsomal monooxygenase system inducers and was higher in those pretreated with the inhibitors, than in those given TBZ alone. These results suggest that TBZ-induced renal injury may be attributable to the parent compound rather than its metabolites. Measurement of organic ion uptake into renal slices revealed significant depression of [1- 14C]tetraethylammonium bromide (TEA) uptake in treated mice at 1 or 5 hr, whereas uptake of p-[glycyl-2- 3H]aminohippurate (PAH) was not depressed at 1 or 5 hr after dosing. The reduction in uptake of TEA is interpreted as the result of competitive suppression of the tubular transport of TEA by TBZ. TBZ-induced renal injury was reduced by organic cation transport inhibitors [N′-methylnicotinamide (NMN) or thiamine] but not by organic anion transport inhibitor [ p-(dipropylsulphamyl)benzoic acid probenecid], suggesting that the reduction of TBZ-induced renal injury is the result of competitive suppression of the tubular transport of TBZ by NMN or thiamine.

Nephrotoxicity of the 1:1 acrolein-glutathione adduct in the rat

Previous metabolic studies in rats have suggested in vivo formation of the acrolein-glutathione (acrolein-GSH) adduct following administration of the highly reactive α,β-unsaturated aldehyde acrolein. Early studies by several investigators demonstrated that similar compounds such as α,β-unsaturated aldehyde-cysteine adducts have toxic (carcinostatic) activity against Ehrlich ascites tumor cells implanted in mice. The current studies investigated the in vivo toxicity associated with the acrolein-GSH adduct in the male Sprague-Dawley rat. The 1:1 acrolein-GSH adduct was synthesized and characterized by physical-chemical methods. Rats given the acrolein-GSH adduct intravenously at 0.5 or 1 mmol/kg developed nephrotoxicity characterized by glucosuria, proteinuria, elevation in serum urea nitrogen, and gross and histologic changes of the kidney. The toxicity was not affected by pretreatment of rats with pyrazole, an alcohol dehydrogenase inhibitor; disulfiram, an inhibitor of aldehyde dehydrogenases; or probenecid, a renal organic anion transport inhibitor. Administration of a similar but nonaldehydic glutathione conjugate, S-n-propylglutathione, did not result in nephrotoxicity in the rat. The nephrotoxicity induced by the acrolein-GSH adduct was inhibited by acivicin, a γ-glutamyl-transpeptidase inhibitor. These results indicate that the acrolein-GSH adduct requires processing through the first step of the renal mercapturic acid synthesis pathway to be activated to a toxic species.

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H 2O 2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H 2O 2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H 2O 2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.

Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstarate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.