Renal Proximal Tubule Suspensions Research Articles

Sex Differences in the mRNA, Protein, and Functional Expression of Organic Anion Transporter (Oat) 1, Oat3, and Organic Cation Transporter (Oct) 2 in Rabbit Renal Proximal Tubules

Sex differences in transport of the organic anion (OA) substrate p-aminohippurate (PAH) and the organic cation (OC) substrate tetraethylammonium (TEA) have been recognized for some time. In the rat kidney, androgens up-regulate and estrogens down-regulate PAH and TEA transport, which correlate with similar changes in mRNA and protein expression for the renal basolateral membrane transporters organic anion transporter (Oat) 1 and organic cation transporter (Oct) 2. However, these sex differences are not readily demonstrated in other species. The present study characterizes the kinetics of basolateral membrane PAH, estrone sulfate (ES), and TEA uptake in renal proximal tubule (RPT) suspensions isolated from female and male rabbits to compare functional expression of transport with mRNA and protein expression for rbOat1, rbOat3, and rbOct2. Although rbOat1-rbOat3 mRNA expression exhibited developmental differences, no sex differences in mRNA levels were observed. Oat1 and Oat3 protein expression in RPT suspensions also was similar between adult female and male rabbits. In contrast, rbOct1 and rbOct2 mRNA levels did not show developmental differences, but rbOct2 mRNA expression was greater in adult male than female rabbits. However, the sex difference in rbOct2 mRNA level did not translate to rbOct2 protein expression. Importantly, functional expression of Oat1, Oat3, and Oct2 transport as measured by kinetics (J(max) and K(t)) of PAH, ES, and TEA uptake was similar between adult male and female rabbits, and correlated with rbOat1, rbOat3, and rbOct2 protein expression. Thus, unlike rodents, rabbit renal OA and OC transport does not exhibit sex differences, pointing to the need for caution in extrapolating transport-related sex differences between species.

Identification of novel targets of cephaloridine in rabbit renal proximal tubules synthesizing glutamine from alanine

Cephaloridine, which accumulates in the renal proximal tubule, is a model compound used for studying the toxicity of antibiotics towards this nephron segment. Several studies have demonstrated that cephaloridine alters renal intermediary and energy metabolism, but the mechanism by which this compound interferes with renal metabolic pathways remains incompletely understood. In an attempt to improve our knowledge in this field, we have studied the influence of cephaloridine on the synthesis of glutamine, which represents a key metabolic process involving several important enzymatic steps in the rabbit kidney. For this, suspensions of rabbit renal proximal tubules were incubated for 90 and 180 min in the presence of 5 mM alanine, an important glutamine precursor, both in the absence and the presence of 10 mM cephaloridine. Glutamate accumulation and glutamine synthesis were found to be inhibited by cephaloridine after 90 and 180 min of incubation, and cephaloridine accumulation in the renal proximal cells occurred in a time-dependent manner. The renal proximal tubule activities of alanine aminotransferase and glutamate dehydrogenase, which initiates alanine removal and releases the ammonia needed for glutamine synthesis, respectively, were inhibited to a significant degree and in a concentration-dependent manner by cephaloridine concentrations in the range found to accumulate in the renal proximal cells. Citrate synthase and glutamine synthetase activities were also inhibited by cephaloridine, but to a much lesser extent. The above enzymatic activities were not found to be inhibited when they were measured after successive dilutions of renal proximal tubules incubated for 180 min in the presence of 5 mM alanine and 10 mM cephaloridine. When microdissected segments (S1-S3) of rabbit renal proximal tubules were incubated for 180 min with 5 mM alanine with and without 5 and 10 mM cephaloridine, glutamate accumulation and glutamine synthesis were also inhibited in the three renal proximal segments studied; the latter cephaloridine-induced inhibitions observed were concentration-dependent except for glutamine in the S3 segment. These results are consistent with the view that cephaloridine accumulates and is toxic along the entire rabbit renal proximal tubule. They also demonstrate that cephaloridine interferes in a concentration-dependent and reversible manner mainly with alanine aminotransferase and glutamate dehydrogenase, which are therefore newly-identified targets of the toxic effects of cephaloridine in the rabbit renal proximal tubule.

Interaction of the metal chelator DMPS with OAT1 and OAT3 in intact isolated rabbit renal proximal tubules.

2,3-Dimercapto-1-propanesulfonic acid (DMPS) is used clinically to increase urinary excretion of heavy metals, including mercury and arsenic. We used single S2 segments and suspensions of rabbit renal proximal tubules (RPT) to test the interaction of this anionic heavy metal chelator with basolateral transporters OAT1 and OAT3. RTPCR revealed expression of both transporters in single S2 segments. [3H]PAH and 3H-labeled estrone sulfate ([3H]ES) were used as specific substrates for rbOAT1 and rbOAT3, respectively. PAH and ES were transported into nonperfused single RPT segments with Kt values of 67 +/- 20 and 3.4 +/- 1.2 microM, respectively, and into tubule suspensions with Kt values of 58 +/- 17 and 7.7 +/- 2.1 microM, respectively. Reduced DMPS (DMPSH) inhibited uptake of both substrates into single tubule segments with Kapp values of 405 +/- 49 microM (for [3H]PAH) and 320 +/- 66 microM (for [3H]ES). Oxidized DMPS (DMPSS), the prevalent form in the blood, also inhibited uptakes of [3H]PAH (Kapp of 766 +/- 190 microM) and [3H]ES (696 +/- 166 microM). Inward gradients of ES, DMPSH, and DMPSS trans-stimulated the 30-s efflux of preloaded [3H]ES across the basolateral membrane of RPT. Similarly, DMPSH, and PAH itself, trans-stimulated the 15-s efflux of [3H]PAH. In contrast, efflux of [3H]PAH was inhibited by the presence of DMPSS in the bathing medium. These data suggest that, whereas both OAT1 and OAT3 probably transport DMPSH, DMPSS transport may be limited to OAT3. This is the first evidence showing that both OAT1 and OAT3 can transport DMPS across the basolateral membrane of RPT.

Gluconeogenesis from glutamine and lactate in the isolated humanrenal proximal tubule: longitudinal heterogeneity and lack of response to adrenaline

Recent studies in vivo have suggested that, in humans in the postabsorptive state, the kidneys contribute a significant fraction of systemic gluconeogenesis, and that the stimulation of renal gluconeogenesis may fully explain the increase in systemic gluconeogenesis during adrenaline infusion. Given the potential importance of human renal gluconeogenesis in various physiological and pathophysiological situations, we have conducted a study in vitro to further characterize this metabolic process and its regulation. For this, successive segments (S1, S2 and S3) of human proximal tubules were dissected and incubated with physiological concentrations of glutamine or lactate, two potential gluconeogenic substrates that are taken up by the human kidney in vivo, and glucose production was measured. The effects of adrenaline, noradrenaline and cAMP, a well established stimulator of gluconeogenesis in animal kidney tubules, were also studied in suspensions of human renal proximal tubules. The results indicate that the three successive segments have about the same capacity to synthesize glucose from glutamine; by contrast, the S2 and S3 segments synthesize more glucose from lactate than the S1 segment. In the S2 and S3 segments, lactate appears to be a better gluconeogenic precursor than glutamine. The addition of cAMP, but not of adrenaline or noradrenaline, led to the stimulation of gluconeogenesis from lactate and glutamine by human proximal tubules. These results indicate that, in the human kidney in vivo, lactate might be the main gluconeogenic precursor, and that the stimulation of renal gluconeogenesis observed in vivo upon adrenaline infusion may result from an indirect action on the renal proximal tubule.

Gluconeogenesis from glutamine and lactate in the isolated human renal proximal tubule: longitudinal heterogeneity and lack of response to adrenaline.

Recent studies in vivo have suggested that, in humans in the postabsorptive state, the kidneys contribute a significant fraction of systemic gluconeogenesis, and that the stimulation of renal gluconeogenesis may fully explain the increase in systemic gluconeogenesis during adrenaline infusion. Given the potential importance of human renal gluconeogenesis in various physiological and pathophysiological situations, we have conducted a study in vitro to further characterize this metabolic process and its regulation. For this, successive segments (S1, S2 and S3) of human proximal tubules were dissected and incubated with physiological concentrations of glutamine or lactate, two potential gluconeogenic substrates that are taken up by the human kidney in vivo, and glucose production was measured. The effects of adrenaline, noradrenaline and cAMP, a well established stimulator of gluconeogenesis in animal kidney tubules, were also studied in suspensions of human renal proximal tubules. The results indicate that the three successive segments have about the same capacity to synthesize glucose from glutamine; by contrast, the S2 and S3 segments synthesize more glucose from lactate than the S1 segment. In the S2 and S3 segments, lactate appears to be a better gluconeogenic precursor than glutamine. The addition of cAMP, but not of adrenaline or noradrenaline, led to the stimulation of gluconeogenesis from lactate and glutamine by human proximal tubules. These results indicate that, in the human kidney in vivo, lactate might be the main gluconeogenic precursor, and that the stimulation of renal gluconeogenesis observed in vivo upon adrenaline infusion may result from an indirect action on the renal proximal tubule.

BIPHASIC EFFECTS OF DOPAMINE ON 86RUBIDIUM UPTAKE IN RAT RENAL PROXIMAL TUBULES

The mechanism(s) by which dopamine inhibits Na+ -K+ -ATPase activity in the renal proximal tubule is still controversial. We studied the short-term effects of dopamine on the sodium pump in rat renal proximal tubule suspensions with the 86Rb uptake method. Dopamine and the D1-like agonist, SKF81297, initially stimulated Na+ -K+ -ATPase activity at 5 min and subsequently inhibited it at 10 min and 20 min; the inhibition by 10 μM dopamine at 20 min was 21.3 ± 4.5 %. The inhibitory effect of dopamine on Na+ -K+ -ATPase activity was mimicked by thymeleatoxin (a classical protein kinase C [PKC] agonist) while Sp-8-CPT-cAMPS (a protein kinase A [PKA] agonist) had no effect. However, the combination of the PKC and PKA agonists mimicked the biphasic effects of dopamine and SKF81297. Rp-8-CPT-cAMPS (a PKA inhibitor), U-73122 (a phospholipase C inhibitor), or calphostin C (a PKC inhibitor), blocked the dopamine-mediated biphasic effects on Na+ -K+ -ATPase activity. It is suggested that the biphasic effects of dopamine on Na+ -K+ -ATPase activity (an initial stimulation and a subsequent inhibition) are transduced by activating both PKA and PKC through a D1-like receptor.

Toxicity And Transport Of Three Synthesized Mercury-Thiol-Complexes In Isolated Rabbit Renal Proximal Tubule Suspensions

ABSTRACTPrevious work has suggested that endogenous sulfhydryls, such as glutathione (GSH) and cysteine, are involved in the uptake and toxicity of HgCh2- To study this possibility, uptake and toxicity of synthesized Hg(SG)2, Hg(cysteinylglycine)2 [Hg(CYS-GLY)2] and Hg(CYS)2 were investigated in rabbit renal proximal tubule suspensions (RPT). The intracellular K+ was used as a toxicity indicator, and the mercury content in the tubules was measured by proton induced x-ray emission analysis. The toxicity rank order of the three synthesized mercury-thiol-complexes from the highest to the lowest was: Hg(CYS)2 > Hg(CYS-GLY)2 > Hg(SG)2- However, no significant difference among the mercury contents in the tubules exposed to these synthesized mercury-thiol-complexes was detected. Acivicin (0.25mM), an inhibitor of γ -glutamyltranspeptidase (GGT), decreased the toxicity of Hg(SG)2 in a manner that did not decrease the uptake of mercury in the tubules. This suggests that the toxicity of Hg(SG)2 requires processing to Hg(CYS-GLY)2 or Hg(CYS)2, while Hg(SG)2 may be taken up by the tubules via Na+-dependent GSH transporter since 10 mM acivicin, an inhibitor of this transporter dramatically decreased the uptake of Hg(SG)2. Organic anion transporter plays a minor role, if any, in the toxicity and uptake of Hg(SG)2 and Hg(CYS)2 since p-aminohippuric acid (PAH), an inhibitor of organic anion transporter, did not have significant effect on their uptake and toxicity. L-phenylalanine, an inhibitor of the neutral amino acid decreased the uptake of mercury, but to a lesser extent. This suggested that neutral amino acid transporter seemed to play a role, in part, in the toxicity and uptake of synthesized Hg(CYS)2- In summary, the data suggested that basolateral transport is important for the toxicity of the three synthesized mercury-thiol-complexes, and a variety of mechanisms are involved in the toxicity and uptake of these complexes in isolated rabbit RPT.

Diverse Cytoprotectants Prevent Cell Lysis and Promote Recovery of Respiration and Ion Transport

Numerous agents have been reported to prevent cell lysis. However, little information is available concerning the ability of cytoprotectants to promote the return of physiological functions. The goal of this study was to determine whether a diverse group of cytoprotectants prevent cell lysis and promote the recovery of respiration and ion transport following anoxia (60 min)/reoxygenation (60 min) in rabbit renal proximal tubule (RPT) suspensions. Cell lysis (LDH release) was determined immediately following the anoxic and reoxygenation periods. Mitochondrial function (basal respiration) and active Na+transport (ouabain-sensitive respiration) was determined after the reoxygenation period. LDH release increased to 75 ± 11% after the anoxic period and did not increase further during the reoxygenation period. LDH release in controls was 6 ± 1% and did not vary over time. Glycine (2 mM), strychnine (1 mM), nifedipine (100 μM) and niflumic acid (100 μM) added immediately prior to the anoxic period completely blocked LDH release. All cytoprotectants increased basal respiration from 39 ± 7% of controls in the anoxic samples to 65–77% of controls. Glycine, strychnine and nifedipine increased ouabain-sensitive respiration from 10 ± 3% of controls in anoxic samples to 51–77% of control. Niflumic acid did not increase ouabain-sensitive respiration. These results demonstrate that glycine, strychnine and nifedipine are ‘true’ cytoprotectants preventing both cell lysis and promoting the recovery of mitochondrial function and ion transport after an anoxic insult.

A rapid β-NADH-linked fluorescence assay for lactate dehydrogenase in cellular death

Lactate dehydrogenase (LDH) release is a common marker of cellular death. Traditionally, the fraction of LDH released has been measured using a NADH-linked UV-Vis spectrophotometric method. The limitation of this method is that samples are usually run serially and thus is time intensive. Therefore, we developed a NADH-linked LDH assay using a fluorescence plate reader that had a correlation of 0.95 with the traditional UV-Vis spectrophotometric method. Using rabbit renal proximal tubule suspensions at a concentration of 1 mg cellular protein/ml of media, the fluorescence assay can determine LDH release in 22 samples in 2 min using 12 μL of cellular homogenates and 150 μL of media. The parallel processing of samples and smaller volumes used in the fluorescence assay results in decreased analysis time and costs.

PROTEINASES IN RENAL CELL DEATH

The role of proteinases in renal proximal tubule (RPT) cellular death was examined using specific inhibitors of proteinases. Rabbit RPT suspensions were incubated with antimycin A for 1 h or tetrafluoroethyl-L-cysteine (TFEC) for 4 h in the absence or presence of the specific cysteine proteinase inhibitor L-trans-epoxysuccinyl-leucylamido (4-guanidino)butane (E-64), the serine proteinase inhibitors N-p-tosyl-L-lysine chloromethyl ketone (TLCK) or 3,4-dichloroisocoumarin (DCS), the serine and cysteine proteinase inhibitors leupeptin or antipain, or the aspartic proteinase inhibitor pepstatin. E-64 and pepstatin decreased lactate dehydrogenase (LDH) release, a marker of cell death, from RPT exposed either to antimycin A or TFEC. TLCK, DCS, leupeptin, or antipain did not decrease antimycin A- or TFEC-induced cell death. Bromohydroquinone- or t-butylhydroperoxide-induced cell death was not decreased by any of the proteinase inhibitors. Loss of lysosomal membrane potential, indicated by neutral red release, occurred prior to the onset of antimycin A-induced cell death. Extensive inhibition of lysosomal cathepsins B and L by E-64 was correlated with cytoprotection. However, E-64 was only protective after some cell death had occurred. These results suggest that lysosomal cysteine and aspartic proteinases, but not serine proteinases, play a role in RPT cell death induced by antimycin A or TFEC. The observation that E-64 was only protective after some cell death had occurred suggests that lysosomal cathepsins are released from dying cells and subsequently attack the remaining viable cells.

Lack of Correlation between para-Aminophenol Toxicity in Vivo and in Vitro in Female Sprague—Dawley Rats

The present study was designed to test the hypothesis that para-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.

Dopamine causes stimulation of protein kinase C in rat renal proximal tubules by activating dopamine D 1 receptors

Although it is suggested that in the renal proximal tubules, dopamine D 1 receptor activation causes inhibition of Na +/K + ATPase via a phospholipase C and protein kinase C coupled pathway, the direct stimulation of protein kinase C by dopamine has not been reported. The present study was designed to examine the effects of dopamine and selective dopamine D 1 receptor and dopamine D 2 receptor agonists on protein kinase C activity. The renal proximal tubule suspensions were obtained from male Sprague-Dawley rats. The tubules were incubated separately with dopamine and fenoldopam in the presence or absence of dopamine D 1 receptor antagonist, SCH 23390 ([(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine]). The protein kinase C activity was measured by using a kinase target peptide, conjugated to a fluorescent molecule in water. The amino acid sequence of this peptide is, Proline-Leucine-Serine-Arginine-Threonine-Leucine-Serine-Valine-Alanine-Alanine-Lysine (PKSRTLSVAAK). We found that dopamine and fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol] produced concentration-dependent increases in protein kinase C activity, which was blocked by SCH 23390. However, the dopamine D 2 receptor agonist, bromocriptine [(5′α)-2-bromo-12′-hydroxy-2′-(1-methyl-ethyl)-5′-(2-methylpropyl) ergo-taman-3′,6′,18-trione] failed to stimulate protein kinase C activity at all the concentrations tested. These results provide direct evidence that dopamine stimulates protein kinase C activity via activation of dopamine D 1 receptors.

Tetrapentylammonium (TPeA): slowly dissociating inhibitor of the renal peritubular organic cation transporter

The efflux of tetraethylammonium (TEA) from suspensions of rabbit renal proximal tubules is completely blocked by 500 μM tetrapentylammonium (TPeA) in the extracellular medium. The basis of this trans-inhibition of TEA transport by TPeA was examined in tubule suspensions. At TPeA concentrations < 10 μM, efflux of TEA was reduced by ∼ 50%, whereas at concentrations > 10 μM, TPeA reduced efflux an additional 50% to produce a near complete block of TEA efflux. Increasing concentrations of TPeA from 0–500 μM were found to produce a biphasic, concentration-dependent trans-inhibition of TEA efflux from tubule suspensions suggesting that TPeA may block efflux by binding to both a high and low affinity TPeA binding site. The trans-inhibition of TEA efflux by TPeA at low concentrations (< 10 μM) may result from a slow carrier turnover when TPeA is bound to the carrier site. To determine whether the inhibitory effectiveness of TPeA was also associated with its slow dissociation from the carrier site, the effect of a 10 s preincubation with 1 μM TPeA on TEA uptake was examined. The uptake of TEA by tubules preincubated for 10 s with TPeA was reduced by ∼ 30–50% compared to control tubules not preincubated with TPeA. A 10 s preincubation with 150 μM unlabeled TEA had no effect on TEA uptake compared to control tubules not preincubated with TEA. When the 10 s preincubation with 1 μM TPeA was followed by a 10 min recovery period, TEA uptake returned to control levels, indicating that the prolonged inhibition was reversible. This prolonged inhibition of TEA uptake after a 10 s preincubation with 1 μM TPeA, as suspected, may arise from a slow dissociation of TPeA from the OC transporter following a rapid association to the binding site. TPeA inhibition of TEA uptake into tubules was competitive in nature with a K i of 1 μM. The ability of TEA to compete with TPeA for binding to the carrier suggests that the binding of TPeA to the carrier can be displaced by large concentrations of TEA. These observations suggest that the interactions of TPeA, and perhaps similarly large hydrophobic OCs, with the OC transporter are complex.

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats.

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.

Rabbit renal proximal tubule suspensions as a model for nephrotoxicity evaluation of native or in situ metabolized beta-lactam antibiotics.

Rabbit renal proximal tubule suspensions have proved a convenient model for nephrotoxicity studies, particularly for direct nephrotoxins such as Cephaloridin (Olivier et al., 1992). In order to evaluate the ability of this model to metabolize certain drugs to nephrotoxic compounds, we used another B-lactam antibiotic, Imipenem, known to be nephrotoxic through renal metabolism via hydrolysis by dehydropeptidase I (DHP) (Birnbaum et al., 1985). The use of Cilastatin, an inhibitor of this enzyme, should confirm this metabolic pathway.

Mechanism of cisplatin nephrotoxicity in rat renal proximal tubule suspensions

The effect of the nephrotoxic antineoplastic agent, cisplatin, on isolated rat renal proximal tubule suspensions was examined. [ 195mPt]Cisplatin (31.7 μ m) uptake was progressive over a 5-hr incubation period to a maximum value of 8.39 nmol/mg protein. The addition of the metabolic inhibitors KCN and iodoacetic acid reduced cisplatin uptake at the later time points. The organic cation tetraethylammonium (1 m m) reduced [ 195mPt]cisplatin (31.7 μ m) uptake to a greater extent than the organic anion p-aminohippuric acid (1 m m) suggesting preferential use of the cationic transport system by cisplatin. Biochemical perturbations observed included increased release of the lysosomal enzyme N-acetyl-β- d-glucosaminidase as well as a time- and concentration-dependent increase in lipid peroxidation. Cisplatin inhibited both nystatin-stimulated and ouabain-sensitive respiration (Qo 2) after only 15 min of treatment. The biochemical changes observed were correlated with morphological alterations in the mitochondria. The results suggest that inhibition of oxidative phosphorylation may be a primary event in cisplatin-induced nephrotoxicity with later changes in lipid peroxidation and enzyme leakage.

Arachidonic acid release in renal proximal tubule cell injuries and death.

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 microM FCCP), the oxidant tert-butyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 microM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-14C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A2 activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.

Rabbit renal proximal tubule suspensions as a model for nephrotoxicity evaluation of native or in situ metabolized b-lactam antibiotics

The leaves of Catha edulis Vahl. Endl., known as Khat, are widely used as a “natural amphetamine” stimulant in Africa and in the Arabian Peninsula. In recent years, there has been an increase in the amounts exported to the USA, Australia and Europe. Although the dried, powdered form of the young and mature leaves, as well as samples from different geographical origins, have similar physical appearance, they have not been proven to exhibit equipotent stimulatory activity. In this study, powdered young and mature leaves were differentiated using vibrational spectroscopy, which included mid-infrared (MIR) and near-infrared (NIR) spectroscopy and hyperspectral imaging (HSI), and ultra performance liquid chromatography-mass spectrometry (UPLC–MS), in combination with chemometric data analysis. Samples originating from Ethiopia or South Africa could also be distinguished using chemometric models constructed from HSI data. Principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA) was successfully applied to classify powdered Khat samples according to their geographical origin and level of maturity. The OPLS-DA model constructed from the MIR and UPLC–MS data displayed the ability to clearly distinguish between young and mature leaves, as illustrated by the model statistics (R2Xcum and Q2cum > 80% and R2Y > 90%). Shortwave infrared hyperspectral imaging could distinguish Khat samples from two distinct geographical regions with good model statistics (R2Xcum = 0.91 and Q2cum = 0.92) and discriminate between the maturity levels (R2Xcum = 0.85 and Q2cum = 0.80). These models could prove valuable as rapid techniques for identifying confiscated Khat in forensic environments.

Strychnine and Glycine Protect Renal Proximal Tubules from Various Nephrotoxicants and Act in the Late Phase of Necrotic Cell Injury

We have previously shown that strychnine mimics the cytoprotective properties of glycine in renal proximal tubule (RPT) suspensions exposed to antimycin A (AA). The aims of this study were to determine whether the cytoprotective properties of strychnine applied to various types of nephrotoxicants and to examine the temporal aspects of the cytoprotection of glycine and strychnine. Tubular release of LDH activity was used as a marker of cell death. Glycine (2 mM) or strychnine (1 mM) added 5 min prior to the toxicant decreased LDH release in rabbit RPT suspensions exposed to 25 μM tetrafluoroethyl-L-cysteine (TFEC), 10 μM HgCl2, 0.5 mM t-butyl hydroperoxide (TBHP), or 0.2 mM bromohydroquinone (BHQ) for 4 hr, or 2 mM sodium cyanide (NaCN) for 2 hr. The relative rank order of effectiveness of glycine and strychnine was NaCN = TFEC > BHQ > DCVC ⪢ TBHP > HgCl2. The temporal aspects of strychnine and glycine protection were examined by exposing RPT to either AA or TFEC for 1 or 3 hr, respectively, and then adding either 1 mM glycine or 1 mM strychnine. Glycine and strychnine decreased LDH release in AA-treated RPT at 1.25 and 2 hr and TFEC-treated RPT at 4 hr. In addition, when RPT exposed to AA or TFEC and treated with strychnine or glycine were washed at either 1 or 4 hr, protection was eliminated at later time points. When glycine was added to RPT treated with either PCBC, TFEC, or DCVC 5 min prior to or 30, 60, 120, and 180 min following toxicant addition, LDH release was reduced at all time points. These results demonstrate that strychnine and glycine protect RPT from a variety of diverse nephrotoxicants, strychnine and glycine do not need to be present at the time of toxic insult, strychnine and glycine cytoprotection is reversible, and strychnine and glycine act in the late phase of necrotic cell injury.

Peritubular organic cation transport in isolated rabbit proximal tubules.

The physiological characteristics of peritubular organic cation transport were examined by measuring the transport of the organic cation tetraethylammonium (TEA) in rabbit renal proximal tubule suspensions and isolated nonperfused rabbit renal proximal tubules. Peritubular organic cation transport in both single S2 segments and suspensions of isolated renal proximal tubules was found to be a high-capacity, high-affinity, carrier-mediated process. For tubule suspensions, the maximal capacity of the carrier for TEA (Jmax) and the concentration of TEA at 1/2 Jmax (Kt) (1.49 +/- 0.21 nmol.min-1.mg dry wt-1 and 131 +/- 16 microM, respectively), did not differ significantly from those measured in single S2 segments (Jmax, 1.16 +/- 0.075 nmol.min-1.mg dry wt-1; Kt, 108 +/- 10 microM). In addition, the pattern of inhibition of peritubular TEA transport by long-chain n-tetraalkylammonium compounds (n = 1-5) was both qualitatively and quantitatively similar in single S2 segments and tubule suspensions, exhibiting an increase in inhibitory potency with increasing alkyl chain length. For example, in tubule suspensions, apparent Michaelis constants for inhibition of TEA uptake ranged from 1.3 mM for tetramethylammonium (TMA) to 0.8 microM for tetrapentylammonium (TPeA). To determine whether these compounds were substrates for the peritubular organic cation transporter, their effect on the efflux of [14C]TEA from tubule suspensions was examined. A concentration of 0.5 mM of the short-chain tetraalkyls TMA or TEA increased the efflux of [14C]TEA (i.e., trans-stimulated) from tubules in suspension. The longer-chain tetraalkyls tetrapropylammonium, tetrabutylammonium, and TPeA all decreased the efflux of [14C]TEA from tubules in suspension; TPeA completely blocked efflux.(ABSTRACT TRUNCATED AT 250 WORDS)