Use of isolated kidney cells for study of drug metabolism

doi:10.1016/0006-2952(79)90378-2

Biochemical Pharmacology

Volume 28, Issue 6, 15 March 1979, Pages 929-935

https://doi.org/10.1016/0006-2952(79)90378-2 Get rights and content

Abstract

Isolated kidney cells were prepared from rat kidneys using a recirculating perfusion system with collagenase. The preparation was rapid and provided a high yield of intact metabolically active kidney cells predominantly of tubular origin. The respiration rate was 2.7 μmol O₂/hr per 10⁶ cells and was not stimulated by ADP. GSH content was 28.9 nmol/10⁶ cells and did not decline during 2 hr of incubation. Cytochrome P450 content was 0.064 nmol/10⁶ cells. The cells were characterized for their drug metabolizing activity using paracetamol as substrate. The rate of formation of the glucuronide and sulfate derivatives was linear for 2 hr, but slower than previously reported for rat liver cells. In contrast to incubations with liver cells, no glutathione conjugate was detected. However, formation of both cysteine and N-acetylcysteine derivatives was observed. The rate of formation of total sulfhydryl conjugates was about 50 per cent of that reported for liver cells when expressed on a cytochrome P450 basis. These studies establish the reliability and utility of this cell preparation as a model system for the study of drug metabolism by the kidney.

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The role of biotransformation and oxidative stress in 3,5-dichloroaniline (3,5-DCA) induced nephrotoxicity in isolated renal cortical cells from male Fischer 344 rats
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All chemicals used were the highest purity available and were purchased from Sigma–Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Untreated male rats were anesthetized with pentobarbital (75 mg/kg, ip) and isolated renal cortical cells (IRCC) were obtained using the collagenase perfusion method of Jones et al. (1979). Cell viability was initially determined by trypan blue (2% w/v) exclusion and lactate dehydrogenase (LDH) release.
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4-Amino-2-chlorophenol: Comparative in vitro nephrotoxicity and mechanisms of bioactivation
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For each compound, the highest purity level available was used. Untreated rats were anesthetized (pentobarbital sodium, 75 mg/kg, ip) and isolated rat renal cortical cells (IRCC) were obtained via the collagenase perfusion method of Jones et al. [28]. Initial cell viability was typically 85–95% as determined by trypan blue (2% w/v) exclusion and initial lactate dehydrogenase (LDH) release (∼5–10%).
Chlorinated anilines are nephrotoxicants both in vivo and in vitro. The mechanism of chloroaniline nephrotoxicity may occur via more than one mechanism, but aminochlorophenol metabolites appear to contribute to the adverse in vivo effects. The purpose of this study was to compare the nephrotoxic potential of 4-aminophenol (4-AP), 4-amino-2-chlorophenol (4-A2CP), 4-amino-3-chlorophenol (4-A3CP) and 4-amino-2,6-dichlorophenol (4-A2,6DCP) using isolated renal cortical cells (IRCC) from male Fischer 344 rats as the model and to explore renal bioactivation mechanisms for 4-A2CP. For these studies, IRCC (∼4 × 10⁶ cells/ml) were incubated with an aminophenol (0.5 or 1.0 mM) or vehicle for 60 min at 37 °C with shaking. In some experiments, cells were pretreated with an antioxidant or cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), peroxidase or cyclooxygenase inhibitor prior to 4-A2CP (1.0 mM). Lactate dehydrogenase (LDH) release served as a measure of cytotoxicity. The order of decreasing nephrotoxic potential in IRCC was 4-A2,6-DCP > 4-A2CP > 4-AP > 4-A3CP. The cytotoxicity induced by 4-A2CP was reduced by pretreatment with the peroxidase inhibitor mercaptosuccinic acid, and some antioxidants (ascorbate, glutathione, N-acetyl-l-cysteine) but not by others (α-tocopherol, DPPD). In addition, pretreatment with the iron chelator deferoxamine, several CYP inhibitors (except for the general CYP inhibitor piperonyl butoxide), FMO inhibitors or indomethacin (a cyclooxygenase inhibitor) failed to attenuate 4-A2CP cytotoxicity. These results demonstrate that the number and ring position of chloro groups can influence the nephrotoxic potential of 4-aminochlorophenols. In addition, 4-A2CP may be bioactivated by cyclooxygenase and peroxidases, and free radicals appear to play a role in 4-A2CP cytotoxicity.
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Control and NPX rats were age-matched for all studies. Isolated renal cortical cells were obtained by collagenase perfusion [30]. PT cells were enriched from the cortical cells by Percoll density-gradient centrifugation [31] and the PT cells were then placed into primary culture [32].
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Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-l-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.
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Use of isolated kidney cells for study of drug metabolism

Abstract

Biochem. biophys. Res. Commun.

Biochem. Pharmac.

Meth. Enzym.

Analyt. Biochem.

J. Chromat.

Meth. Enzym.

Meth. Enzym.

J. biol. Chem.

Archs. Biochem. Biophys.

J. biol. Chem.

Toxic. appl. Pharmac.

Am. J. Physiol.

Adv. Enzymol.

Hoppe-Seyler's Z. physiol. Chem.

J. Cell Biol.