Within the brush border (BB) of proximal tubule cells (PTC), the intermicrovillar area connecting the base of the microvilli constitutes a distinct microdomain characterized by the presence on the cytoplasmic side of the membrane of an extensive clathrin coat (1). Kerjaschki and Farquhar (2) and Chatelet et al. (3) have shown that a 330-kD glycoprotein (gp), initially described as the nephritogenic antigen of Heymann's nephritis, is concentrated on the luminal aspect of these areas, at variance with other BB proteins such as maltase (4), which are distributed over the entire surface of the microvilli. To our knowledge, gp330 is the only membrane gp confined to the intermicrovillar domain (IMVD) . It is also expressed in coated pits of glomerular epithelial cells, pneumocytes type II, epididymis, and epithelial cells of the visceral yolk sac (VYS) (5, 6), but its function remains unknown . This paper describes a 280-kD BB protein, defined by mAbs raised against rat renal BB, which is only found in the IMVD of the PTC and on the apical domain of epithelial cells of the VYS . When injected to pregnant rats, the mAbs induce embryonic resorption and/or fetal abnormalities, suggesting that the 280-kD protein plays a key role in endocytosis-related cell function. Materials and MethodsAntibodies. The two mAbs reported in this study (F5/75, an IgGI with a pl of 6.8-7.6, and F5/46, an IgGI with a pl of 8.1-8.5) were obtained by immunization of mice against rat BB (7) . They were analyzed in relation to mAb F1/12 and polyclonal antibodies specific for gp330 (8), and mAb F2/180 reactive with the 300-kD maltase .Characterization of the 280-kD Antigen. The antigen identified by mAb F5/75 and F5/46 on tubular BB was identified by immunoprecipitation ofradiolabeled BB membrane vesicles (BBMV) (7) and immunoadsorption techniques . Purified mAbs F5/75 and F5/46, as well as mAbs F1/12 and F2/180, were individually coupled to Sepharose 4B (Pharmacia Fine Chemicals, Velizy, France) . All steps involving antigenic preparations and immunoadsorption were carried out in the presence of 1 mM PMSF and 2 mM benzamidine . BBMV (7,8) and yolk sacs dissected at 19 d of gestation were washed extensively in PBS and solubilized in 1 % Triton X100 . 1-2 ml of each immunoabsorbent was incubated with either 20 mg of solubilized BBMV, or with the material from 10 yolk sacs.

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.

Histiotrophic nutrition is essential during the peri-implantation development in rodents, but little is known about receptors involved in protein and lipid endocytosis derived from the endometrium and the uterine glands. Previous studies suggested that cubilin, a multiligand receptor for vitamin, iron, and protein uptake in the adult, might be important in this process, but the onset of its expression and function is not known. In this study, we analyzed the expression of cubilin in the pre- and early post-implantation rodent embryo and tested its potential function in protein and cholesterol uptake. Using morphological and Western blot analysis, we showed that cubilin first appeared at the eight-cell stage. It was expressed by the maternal-fetal interfaces, trophectoderm and visceral endoderm, but also by the future neuroepithelial cells and the developing neural tube. At all these sites, cubilin was localized at the apical pole of the cells exposed to the maternal environment or to the amniotic and neural tube cavities, and had a very similar distribution to megalin, a member of the LDLR gene family and a coreceptor for cubilin in adult tissues. To analyze cubilin function, we followed endocytosis of apolipoprotein A-I and HDL cholesterol, nutrients normally present in the uterine glands and essential for embryonic growth. We showed that internalization of both ligands was cubilin dependent during the early rodent gestation. In conclusion, the early cubilin expression and its function in protein and cholesterol uptake suggest an important role for cubilin in the development of the peri-implantation embryo.