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

Mechanisms underlying the pathophysiology of minimal change nephrotic syndrome (MCNS), the most frequent of glomerular diseases in children, remain elusive, although recent arguments suggest that T cell dysfunction may be involved in the pathogenesis of this disease. Recently, we reported that activated T cells of these patients display a down-regulation of IL-12R β2 chain, suggesting an early commitment toward Th2 phenotype. In this study, we show that the short form of the proto-oncogene c-maf, a known activator of the IL-4 gene, is highly induced in MCNS T cells during relapse, where it translocates to the nuclear compartment and binds to the DNA responsive element. Unexpectedly, the nuclear localization of c-maf did not promote the IL-4 gene transcription in relapse. Using several approaches, we show in this study that RelA blunts IL-4 induction in T cells during the relapse in these patients. We demonstrate that the ex vivo inhibition of proteasome activity in T cells from relapse, which blocks NF-κB activity, strongly increases the IL-4 mRNA levels. Overexpression of c-maf in T cells induces a high level of IL-4 promoter-driven luciferase activity. In contrast, coexpression of c-maf with NF-κB RelA/p50, or RelA, but not p50, inhibits the c-maf-dependent IL-4 promoter activity. Finally, we demonstrated that, in T cell overexpressing RelA and c-maf, RelA expelled c-maf from its DNA binding site on IL-4 gene promoter, which results in active inhibition of IL-4 gene transcription. Altogether, these results suggest that the involvement of c-maf in Th2 commitment in MCNS operates through IL-4-independent mechanisms.