Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Abstract

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serumfree medium (Medium RK-1). A hormone-deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and Ta. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and Ta, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors.Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated a-methylglucoside (a-MG) against a concentration gradient. However, little or no a-MG accumulation was observed in the absence of Na ÷. Metabolic inhibitor studies also indicated that a-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K ÷ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM c~-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na÷-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and y-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.Primary kidney-cell culture is valuable for studying renal functions in vitro (1). Although several established kidney-ceU lines have been used to study the mechanism of transepithelial solute transport by the kidney (2, 3,4,5,6), investigations using such cell lines have several limitations. First, the available cell lines may 0nly resemble particular kidney-cell types to a limited extent. Secondly, only a limited number of established kidney-ceU lines are available for study. Consequently, culture systems are also needed that closely resemble cells in a number of nephron segments (7). Primary kidney-cell culture provides an excellent means to resolve these problems (8).Previous studies with primary-kidney cultures have been, however, impeded by the presence of serum in the tissueculture medium (9). The transporting epithelial cells in primary-kidney cultures maintained in serum-supplemented medium, have been overgrown by fibrobl...

Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-κB signal pathways in renal proximal tubule cells

“…PTCs have a number of differentiated typical functions of the renal proximal tubules, including a polarized morphology, as well as a distinctive proximal tubule transport and hormone response (9,17). It was previously reported that the ATPincreased cell proliferation in primary cultured PTCs is consistent with the results obtained from intact renal tissue (23,24).…”

“…The primary rabbit PTC cultures were prepared by the method reported by Chung et al (9). PTCs were grown in a DMEM-F-12 medium (GIBCO-BRL, Gaithersburg, MD) containing 15 mM HEPES and 20 mM sodium bicarbonate (pH 7.4).…”

Oxidant‐Induced Cell Death in Renal Epithelial Cells: Differential Effects of Inorganic and Organic Hydroperoxides

“…Rabbit proximal tubular cells were isolated by the method of Brendel & Meezan (1975) and prepared for cultures according to the method of Chung et al (1982) with some modifications from adult male New Zealand white rabbits as described previously (Kim et al 1998).…”

Preincubation in acid medium increases Na/H antiporter activity in cultured renal proximal tubule cells.

“…Renal proximal tubule cells were isolated from the kidneys of young male rabbits and placed in primary culture using an approach similar to that described (6)(7)(8)(9) (5 ,ug/ml), 50 nM hydrocortisone, transferrin (35 pug/ml), 29 nM sodium selenite, 20 AM ethanolamine, 1.5 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 ,ug/ml). Fetal bovine serum was added to 3% (vol/vol) to the culture medium for the first 3 days to allow better cell attachment.…”