Maintenance of proximal and distal cell functions in SV40‐transformed tubular cell lines derived from rabbit kidney cortex

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.

Heat Shock Protein gp96 Interacts with Protein Phosphatase 5 and Controls Toll-like Receptor 2 (TLR2)-mediated Activation of Extracellular Signal-regulated Kinase (ERK) 1/2 in Post-hypoxic Kidney Cells

“…Briefly, the kidneys were rapidly removed under sterile conditions, and primary cultures of RTECs were prepared using previously described methods (8,33). Kidneys were injected using fine needles with modified defined medium (DM) as described (33) supplemented with 0.1% collagenase (Roche Diagnostics GmBH).…”

“…Thin kidney slices were then minced and incubated in the same collagenase-supplemented DM for 45 min at 37°C. Kidney slices were gently dispersed using sterile needles, and then poured through 100-and 40-m nylon gauzes, as described (33). The resulting cell suspension was then seeded on chambered coverglass slides or collagencoated 48-well trays (ϳ 2 ϫ 10 4 cells per well), and grown at 37°C in a 5% CO 2 -95% air atmosphere in the same modified defined DM medium.…”

Matrix Metalloproteinase 2 (MMP2) and MMP9 Are Produced by Kidney Collecting Duct Principal Cells but Are Differentially Regulated by SV40 Large-T, Arginine Vasopressin, and Epidermal Growth Factor

“…To analyze the effects of temperature per se irrespective of the functional status of SV40 large-T oncogene on metalloproteinase synthesis, we used as control a rabbit collecting duct cell line of the same cell type origin (RC.SV3), previously established in our laboratory after infection of renal cells with the wild-type SV40 strain LP (23). This cell line was studied under the same culture conditions as RC.SVtsA58 and analyzed at 33 and 39.5°C.…”

The Functional c.-2G>C Variant of the Mineralocorticoid Receptor Modulates Blood Pressure, Renin, and Aldosterone Levels

“…Rabbit RCSV3 cells derived from a kidney cortical collecting duct 14 (kindly provided by Prof P. Ronco, Hôpital Tenon, Paris, France) were grown as described previously 15 and transfected using lipofectamine 2000 (Invitrogen) with 0.25 g of pcDNA3 plasmid containing either MR-2G or MR-2C, 0.625 g of a GRE2_TATA_luc reporter plasmid plasmid 16 and 0.25 g of pSV␤gal. The day after transfection, steroids were added at different concentrations, and 48 hours after transfection, luciferase and ␤-galactosidase activities were assayed using the Dual-Light System and the Galacton-Plus Substrate (Applied Biosystems).…”