No abstract

This paper reports the preparation and describes the properties of three renal tubular cell lines derived using SV40 infection of primary cultures of rabbit kidney cortical cells, enriched in proximal cells. RC.SV1 was initially derived from cultures grown in the presence of fetal calf serum exhibiting a low degree of proximal differentiation. The cells were subsequently adapted to grow in serum-free hormonally defined medium and display basic properties of proximal tubule cells including well-developed apical microvilli, strong expression of brush-border hydrolases, Na+-coupled glucose uptake, and increased cyclic AMP production when exposed to PTH. The other two cell lines were derived from cultures in serum-free hormonally defined medium and propagated in the same medium. They are characterized by some common properties including rare and short microvilli, low expression of apical hydrolases, and low or undetectable Na+-dependent glucose uptake, but differ by their abilities to respond by an increase in cAMP to various hormonal stimuli. RC.SV2 cells are sensitive to calcitonin and to a lesser extent to isoproterenol and PTH, suggesting that they may originate from the thick ascending limb of Henle's loop and the bright portion of the distal tubule. RC.SV3 responds essentially to isoproterenol and arginine vasopressin, suggesting a more distal origin (late distal and initial collecting tubule). Emergence of distal cell lines from cultures exhibiting proximal characteristics may be related to distal cell overgrowth as suggested by analysis of growth kinetics and increased Na+/H+ exchanger activity in RC.SV2 compared with RC.SV1.

Abstract. To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33°C) and restrictive (39.5°C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5°C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33°C respond only to isoproterenol (ISO, 10 -6 M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5°C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10 -7 M dDarginine vasopressin (dDAVP/ basal: 18.2, apparent Ka: 5 x 10 -9 M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2-type AVP receptors (,050 fmol/10 ~ cells) which are undetectable when SV40 genome is activated (33°C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5°C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells.