Isolation and characterization of rat renal proximal tubular cells

Abstract

The nephrotoxicity of several drugs and environmental chemicals is confined to the proximal tubule (PT) of the kidney. The availability of PT cells in suspension provides a model to investigate mechanisms of PT damage. Previous studies have used either a heterogeneous suspension of cells isolated from the kidney (Ormstad et al., 1981), a cell population enriched for PT cells by the use of selective media (Detrisac i't al., 1984) or an established cell line, with little similarity to PT cells. Kidney cortices from Sprague-Dawley rats (250-300 g) were chopped into 2 3mm fragments, washed in Hank's salt solution. then incubated in Hank's salt solution containing 0.1 % (w/v) collagenase and 2.4 m ~ C a ' + for 30 min at 37°C. The cortical fragments were then filtered through 7Spm mesh and washed three times using Hank's salt solution containing Ca'+. A Percoll density gradient was prepared (starting density 1.044 g/ml, 300 m o w ) and centrifuged at 2OOOOg. 4' C for 3Omin. This produced a gradient ranging from 1.010 to 1.060g/ml. After centrifugation three main bands appeared at densities 1.040 g/ml (A), 1.058 g/ml (B) and 1.060 g/ml (C). The cells or tubular fragments in each band were characterized morphologically using light and electron microscopy. A contained mostly single PT cells ( > 80%) with some smaller cells present. B contained a mixture of single cells, glomeruli and tubular fragments, whilst C contained > 90% PT fragments and some single PT cells. The tubular fragments in C were characterized by open lumina, prominent microvilli and numerous primary and secondary lysosomes. Enzymes of intermediary metabolism were used as markers for PT and distal tubular (DT) cells. FructoseI ,6-bisphosphatase (FBP. EC 4.1.2.13) (Allen & Blair, 1972) is confined to the PT, whilst hexokinase (HK, EC 2.7.1 . I ) (Schmidt et al., 1975) is found in the DT and phosphoglucosisomerase (PGI. EC 5 . 3 . I .9) occurs in both the PT and DT (Guder & Ross, 1984). In homogenates prepared from either cortex or medulla, FBP was 3-fold higher in the cortex, whereas HK was 3-fold higher in the medulla (Table I) . PGI activity was