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 Dulbecco's modified Eagle's 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.
Boogaard
P J
PJ
Division of Toxicology, Leiden University, The Netherlands.
Zoeteweij
J P
JP
van Berkel
T J
TJ
van't Noordende
J M
JM
Mulder
G J
GJ
Nagelkerke
J F
JF
D000111:Acetylcysteine; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017402:Chlorofluorocarbons; D003545:Cysteine; D005978:Glutathione; D006846:Hydrocarbons, Halogenated / Q000633:toxicity; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008757:Methylglucosides / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D017402:Chlorofluorocarbons; D006846:Hydrocarbons, Halogenated; D008757:Methylglucosides; C027020:methylglucoside; C054365:1,1-dichloro-2,2-difluoroethylene; C016304:chlorotrifluoroethylene; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine