Pieter J. Boogaard

Toxicity of the cysteine-S-conjugates and mercapturic acids of four structurally related difluoroethylenes in isolated proximal tubular cells from rat kidney: Uptake of the conjugates and activation to toxic metabolites

Isolated proximal tubular cells from rat kidney were incubated with the cysteine-S-conjugates and corresponding mercapturates of the potent nephrotoxicants tetrafluoroethylene (TFE), chlorotrifluoroethylene (CTFE), 1,1-dichloro-2,2-difluoroethylene (DCDFE) and 1,1-dibromo-2,2-difluoroethylene (DBDFE). Toxicity of these S-conjugates was determined by their ability to inhibit alpha-methylglucose uptake by the cells. The cytotoxicity of the cysteine-S-conjugates and mercapturates of TFE and CTFE was similar, but the cysteine-S-conjugates of DCDFE and DBDFE were more toxic than their mercapturates. The cytotoxicity of the conjugates decreased in the following order TFE approximately CTFE greater than DCDFE greater than DBDFE, which is the same as observed in vivo. Inhibition of renal cysteine-S-conjugate beta-lyase by aminooxyacetic acid alleviated the cytotoxicity of both the cysteine-S-conjugates and the mercapturic acids of the four haloethylenes. The cytotoxicity of the mercapturates, but not of the cysteine-S-conjugates, could be reduced by probenecid, suggesting that the cysteine-S-conjugates are transported by a different carrier system than the mercapturates. The deacetylation of the mercapturates of TFE and CTFE in the cells was much higher than that of the mercapturates of DCDFE and DBDFE. The cysteine-S-conjugates of DCDFE and DBDFE were N-acetylated by the cells whereas the other cysteine-S-conjugates were not (TFE) or only marginally (CTFE) N-acetylated. The observed differences in cytotoxicity may be explained by differences in (1) the balance between acetylation/deacetylation by the cells, (2) the conversion rate of the S-conjugates to toxic metabolites by renal beta-lyase and (3) the transport into the proximal tubular cells.

Renal proximal tubular cells in suspension or in primary culture as in vitro models to study nephrotoxicity

The kidney forms a frequent target for xenobiotic toxicity. The complex biochemical mechanisms underlying nephrotoxicity are best studied in vitro provided that reliable and relevant in vitro models are available. Since most nephrotoxicants affect primarily the cells of the proximal tubules (PTC), much effort has been directed towards the development of in vitro models of PTC. This review focuses on the preparation of PTC and the use of these cells. Discussed are important criteria such as the viability (survival time) of the cells and the parameters to assess toxicity. Recent studies have shown that isolated PTC in suspension are especially suitable for studies on the biochemical mechanisms of acute nephrotoxicity, whereas PTC in primary culture may be used to investigate mechanisms of nephrotoxic damage at very low concentrations, upon prolonged exposure. PTC cultured on porous filter membranes provide new possibilities to study toxicity in relation to cell and transport polarity. Primary cell cultures of human PTC have been set up. Although a further characterization of these systems is needed, recent data indicate their usefulness.

Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity: I. An improved method for preparation of proximal tubular cells and their functional characterization by α-methylglucose uptake

Rat renal proximal tubular cells were isolated by successive EGTA and collagenase perfusions and purified by filtration and isopycnic centrifugation. The method is rapid and provides a much higher fraction of proximal tubular cells (90-95%) than comparable methods. The yield of viable (97 +/- 3%) cells is high (30 X 10(6) cells/g kidney). The intracellular ATP was 16 +/- 2 nmol/mg protein and remained essentially constant for at least 3 hr. The cells were characterized for transport of organic ions and glucose. Glucose transport was studied by alpha-[14C]methylglucose uptake; apparent Km and Vmax values were 3.4 +/- 0.5 mM and 4.1 +/- 0.9 nmol/min.mg protein, respectively. This transport could almost be completely inhibited by phloridzin, indicating that the uptake is mediated by the brush border glucose carrier.

Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure.

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbeccos modified Eagles medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.

4-methylthiobenzoic acid reduces cisplatin nephrotoxicity in rats without compromising anti-tumour activity

Administration of 4-methylthiobenzoic acid (MTBA) (100 mg/kg) strongly reduced cisplatin nephrotoxicity (7.5 mg/kg, 20 min after MTBA) in rats as determined by histopathology and blood urea nitrogen. Anti-tumour activity against a colonic adenocarcinoma, CC 531, that was implanted in rats, was unaffected by MTBA pretreatment. Studies with isolated renal proximal tubular cells (PTC) demonstrated that preincubation of the cells with MTBA diminished cisplatin nephrotoxicity in vitro as it did in vivo. Preincubation of the PTC with probenecid completely abolished the protective effect of MTBA against cisplatin toxicity. These data indicate that MTBA is actively transported into the PTC. The mechanism of action of MTBA was investigated by NMR studies which showed that cisplatin and cis-diamminediaquaplatinum(II), its hydrolysis product, reacted with the methylthio-sulphur. We suggest that MTBA after selective accumulation in the kidney inactivates cisplatin intracellularly by nucleophilic attack of the methylthio-sulphur to the Pt-moiety. Since MTBA shows no acute toxicity in the rat, even if administered at very high doses, it may be useful to suppress the nephrotoxic side effects of cisplatin anti-tumour therapy.

Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity: II. α-Methylglucose uptake as a sensitive parameter for mechanistic studies of acute toxicity by xenobiotics

Many nephrotoxic agents exert their effect primarily on the cells of the proximal tubules. We isolated these cells and investigated whether the uptake of alpha-methylglucose (alpha-MG) could serve as a parameter to assess effects of nephrotoxins on the functional integrity of the cells. Agents that are acutely nephrotoxic in vivo, CD2+, Hg2+, UO22+, p-aminophenol, and bis-2,3-dibromopropylphosphate, inhibited alpha-MG uptake at low concentrations. Most agents that exert their effect in vivo with delay or only when used chronically (gentamicin, cephaloridine, phenacetin, and acetaminophen) inhibited alpha-MG uptake only at much higher concentrations; cisplatin, however, inhibited alpha-MG uptake at a low concentration. S-(1,1-Difluoro-2,2-dichloroethyl)-L-cysteine and its N-acetyl derivate, two metabolites of the nephrotoxic agent 1,1-dichloro-2,2-difluoroethylene, inhibit alpha-MG uptake. Aminooxyacetic acid, which prevents the formation of the ultimate toxic metabolite by inhibition of beta-lyase, abolished almost completely the toxicity of both compounds. The nephrotoxic conjugate of hexachlorobutadiene, S-(1,2,3,4,4-pentachlorobutadienyl)-glutathione, also inhibited alpha-MG uptake. The selective inhibitor of gamma-glutamyltranspeptidase, anthglutin, completely prevented this inhibition. These results indicate that the uptake of alpha-methylglucose by isolated proximal tubular cells from rat kidney is a useful parameter for the study of nephrotoxicity, since in vitro results reflect acute nephrotoxicity as observed in vivo.

The role of metallothionein in the reduction of cisplatin-induced nephrotoxicity by Bi3+-pretreatment in the rat in vivo and in vitro

Nephrotoxicity induced by cisplatin (CDDP) was reported to be reduced by Bi3(+)-pretreatment, which selectively induces renal metallothionein (MT). In the present study renal MT had increased to 250% of control in rats that received bismuth subnitrate (50 mumol/kg/day, orally) for 8 days. In vitro experiments demonstrated that the reduction of CDDP-induced toxicity is a renal effect: in proximal tubular cells (PTC) isolated from Bi3(+)-treated rats the toxicity of CDDP, and also of HgCl2, CdCl2 and p-aminophenol, was reduced as compared to PTC from untreated rats. In contrast to the reduction in CDDP, Hg2+ and Cd2+ toxicity, the reduction in p-aminophenol toxicity cannot be explained by the metal-binding properties of MT. MT was reported to act as a free radical scavenger, which may explain our observation since p-aminophenol toxicity is thought to be a consequence of the generation of oxygen radicals. In vivo experiments showed that the overall renal Pt-content as well as the Pt bound to renal MT is lower in Bi3(+)-pretreated rats than in untreated rats, 24 hr after administration of CDDP (12 mg/kg), suggesting that the reduction in nephrotoxicity is not due to increased binding of Pt2+ to renal MT. Renal superoxide dismutase (SOD) activity was increased in rats that had only received CDDP. Such a rise in SOD may result from peroxidative damage caused by exposure to CDDP. The fact that SOD was not elevated in rats that received Bi3+ prior to CDDP suggests that (i) peroxidation contributes to CDDP-induced nephrotoxicity and (ii) the anti-oxidant properties of MT are responsible for the reduction of this toxicity.

NEPHROTOXICITY OF HALOGENATED ALKENYL CYSTEINE-S-CONJUGATES

In 1916 a relationship was postulated between the occurrence of aplastic anaemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene. The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found. Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity. The haloalkenyl cysteine-S-conjugates (Cys-S-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloalkenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity will be outlined.

Alpha-Methylglucose Uptake by Isolated rat Kidney Proximal Tubular Cells as a Parameter for Cell Integrity in Vitro

A large number of nephrotoxins cause damage to the proximal tubule in vivo. Many typical in vivo tubular cell functions are conserved in freshly isolated rat kidney cells (Ormstad et al., 1981; Jones et al., 1979). An advantage of the use of isolated cells as compared to the in vivo experiment is that it permits a defined quantitatively and qualitatively cellular environment, which allows study of the relationship between the concentration of a nephrotoxin, exposure time and effect. Extra-renal effects can also be excluded, thus isolated tubular cells are very suitable to study the effects of nephrotoxins which exert their effect directly at the tubular site.

026 Dimensional flow cardiovascular magnetic resonance: two-centre, 1.5t, phantom and in-vivo validation study

Background Validation of four-dimensional (4D) flow CMR accelerated acquisition methods is needed to make them more robust for clinical applications.1 Our aim was to compare three widely-used acceleration methods in 4D flow CMR: 4D segmented fast-gradient-echo (4D- turbo-field-echo, 4D-TFE), 4D non-segmented gradient-echo with echo-planar imaging (4D- EPI) and 4D-k-t Broad-use Linear Acquisition Speed-up Technique accelerated TFE (4D-k-t BLAST). Methods CMR was performed in two institutions on identical 1.5T systems. Acceleration methods were compared in static/pulsatile phantoms (Figure 1) and 25 volunteers. In volunteers, the CMR protocol included: cines, 2D phase contrast (PC) at the aortic valve (AV) and mitral valve (MV) and three whole-heart free-breathing (no respiratory motion correction) 4D flow CMR pulse sequences. Field-of-view, slices, phases (30), voxel size and VENC were the same for each subject. In volunteers, net acquisition time for each 4D flow sequence was recorded, as well as a visual grading of image quality on a four-point scale: 0, no artefacts to 3, non-evaluable. Abstract 026 Figure 1 Illustration to demonstrate static and pulsatile flow phantom setup. Results For the pulsatile phantom experiments, the mean error against the reference flow by time beaker measurements for 4D-TFE was 4.9%±1.3%, for 4D-EPI 7.6%±1.3%u2009and for 4D-k-t BLAST 4.4%±1.9%. In vivo, acquisition time was shortest for 4D-EPI at 7u2009min59s±2u2009min30s. 4D- EPI and 4D-k-t BLAST had minimal artefacts, while for 4D-TFE, 40% of AV and MV assessments were non-evaluable because of phase dispersion artefacts. Peak velocity assessment using 4D-EPI demonstrated best correlation to 2D PC (AV: r=0.78, p<0.001; MV: r=0.71, p<0.001). Coefficient of variability (CV) for net forward flow (NFF) volume was least for 4D-EPI (7%) (2D PC:11%, 4D-TFE: 29%, 4D-k-t BLAST: 30%, respectively) (Figure 2, 3). Abstract 026 Figure 2 Bland Altman analysis and scatter plots for the assessment of peak velocity using all the acceleration methods. Abstract 026 Figure 3 Scatter plots of net forward flow (NFF) through the mitral and aortic valve to investigate consistency between all the four methods. Conclusion Of the three 4D flow CMR methods tested, 4D-EPI demonstrated the least susceptibility to artefacts, good image quality, modest agreement with the current reference standard for peak intra-cardiac velocities and the highest consistency of intra-cardiac flow quantifications. Competing interests The authors declare that they have no competing interests. Acknowledgement We thank Gavin Bainbridge, Caroline Richmond, Margaret Saysell and Petra Bijsterveld for their invaluable assistance in recruiting and collecting data for this study. Funding Sources This work was supported by the British Heart Foundation [FS/10/62/28409 to S.P.] and Dutch ZonMw [Project Number: 104003001 to J.W]. References . Dyverfeldt P, Bissell M, Barker AJ, Bolger AF, et al. 4D flow cardiovascular magnetic resonance consensus statement. J Cardiovasc Magn Reson2015;17:72.