Isolation and characterisation of human proximal tubular cells derived from kidney cortical segments.
1. Human renal proximal tubular cells (HPTC) were isolated by collagenase digestion and purified following filtration and isopycnic Percoll density centrifugation. This method used cortical tissue obtained from surgical nephrectomies and was both rapid and simple, providing a preparation of cells with high viability (> 93 +/- 3%) and recovery (16 +/- 7 x 10(6) cells g-1 of cortical tissue). 2. Characterisation of the isolated cells showed that, in terms of morphology, enzyme profile, transport systems and hormonal responsiveness, they were > 95% proximal tubular. The transport systems obeyed Michaelis-Menten kinetics, with the kinetic parameters of the glucose transport system (Km = 2.5mM, Vmax = 7.7 nmol min-1 mg-1 protein) suggesting a higher proportion of PT cells originating from the S1-S2 segment of the nephron. Isolated HPTC also maintained levels of reduced glutathione (GSH) (11.9 +/- 3.2 nmol mg-1 protein) and exhibited cytochrome P450-dependent activity, levels of spectrally determined P450 being 0.22 +/- 0.07 nmol mg-1 protein. 3. These results demonstrate the isolation of a viable and functioning homogeneous preparation of HPTC from cortical tissue, with potential for use in short term pharmacological, physiological and toxicological studies.
McLaren
J
J
Department of Medicine, University of Aberdeen, Foresterhill, UK.
Whiting
P
P
Simpson
J
J
Hawksworth
G
G
D000410:Alanine Transaminase / Q000378:metabolism; D018826:CD13 Antigens / Q000378:metabolism; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport; D007672:Kidney Cortex / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D010088:Oxidoreductases / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D005723:gamma-Glutamyltransferase; D000410:Alanine Transaminase; D018826:CD13 Antigens; D017364:Collagenases; D005978:Glutathione