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Previous studies have identified two high-molecular weight (280 and 330 kd) glycoproteins expressed by coated pits of the proximal renal tubule and yolk sac and have further established that, in vivo, antibodies to gp280 but not to gp330 induce fetal malformations. In the present study, we report the effect of these antibodies on the endocytic process by yolk sac visceral epithelial cells of rat embryos explanted at day 10 of gestation. Antibodies to gp280 markedly altered development of the yolk sac and embryo, induced malformations, inhibited by 40% the uptake of [14C] sucrose and perturbed the intracellular traffic of internalized proteins. Under control conditions, rat immunoglobulin G present in the culture medium was immunolocalized in lysosomes of epithelial cells, whereas in the presence of antibody, it was detected in small vesicles scattered through the apical cytoplasm. Alterations of the endocytic pathway were confirmed by experiments analyzing the uptake of peroxidase added to the medium for 2 to 60 minutes. The initial compartments of endocytosis visualized by peroxidase were increased in size and abnormal in shape and the transfer of the internalized peroxidase to the lysosomal compartment was delayed. In contrast, antibodies to gp330 had a minimal effect on embryonic development and did not induce fetal malformations. Endocytosis was only modestly altered; uptake of [14C] sucrose was decreased by 25%, and only minor modifications of the intracellular transit of peroxidase could be detected. We suggest that the key role of anti-gp280 antibodies is via trapping of the target antigen in the early endocytic compartment thus preventing its normal function in lysosomal transfer.

We have previously reported the production of monoclonal antibody (MAb) 1BE12, which recognizes a glycoprotein in breast-cancer cells. In the present work, 1BE12 reactivity was tested by immunohistochemistry in bladder carcinoma (92 cases) and in non-tumoral bladder samples (15 cases). In 71% of bladder tumors, more than 30% of cells were intensely stained by 1BE12. The percentage of reactive cells was higher in cancers invading the muscle than in more superficial tumors (p = 0.039). In non-tumoral bladder, immuno-staining, when present, was usually confined to the superficial layers with a low number of cells stained (less than 30%) in 13/15 cases. Slot blots, performed on urine samples from 43 bladder-cancer patients and 21 healthy controls, were quantified by densitometry scanning. We found higher optical density (OD) values in urine from muscle-invasive-cancer patients than in urine from more superficial tumors and healthy controls, with a significantly different distribution (p = 0.005). The urinary antigen was detected by immunoblotting with 1BE12 as high-molecular-weight species (greater than 150 kDa). The reactive glycoprotein could thus be purified by immunoaffinity and FPLC filtration from the perchloric-acid-soluble fraction of urine from patients with invasive bladder carcinoma. The availability of purified antigen will allow us to quantitate our assay, in order to evaluate its potential use as a prognostic indicator in bladder-cancer patients.

An immunoultrastructural study has been undertaken for analysis of the extrarenal localization of two brush border (BB) glycoproteins of 90 kd and 330 kd expressed by glomeruli. In previous studies on renal tissue, it has been shown that gp 330 is restricted to coated pits and BB intermicrovillar invaginations, whereas gp 90 is diffusely distributed on epithelial cell membranes and in addition expressed on glomerular endothelial cells. At variance from observations made in the kidney, gp 330 and gp 90 are mainly expressed on different epithelial cell types: gp 330 is found on the coated pits of the epithelium lining the epididymis and on the entire surface of the microvilli of Type II pneumocytes, whereas gp 90 is detected on the gut brush border and the biliary pole of hepatocytes; gp 90 is also found on endothelia lining all distal capillary beds studied in heart, lung, liver, and spleen. These observations thus indicate that gp 90 is expressed by a wide variety of cell types, all associated with high membrane turnover. Conversely, gp 330 has a restricted distribution at the organ, cellular and subcellular level and may be related with a defined system of receptor-mediated endocytosis.

The authors have previously reported the production of monoclonal anti-brush border antibodies defining two glycoprotein (gp) antigens of 90 and 330 kd which are also expressed on glomerular cells and may thus be of significance for in situ formation of immune complexes. The 330-kd antigen is involved in Heymann's nephritis, whereas the 90-kd antigen induces transient glomerular immune deposits. In this report, the authors describe the renal ultrastructural localization of the two antigens, which are both detectable on brush border and glomerular epithelial cells but assume contrasting patterns: gp 90 is diffusely present on the cell membranes, whereas gp 330 is concentrated in the intermicrovillar region and within the coated pits. Their localization within proximal tubular cells, on the membrane of intracytoplasmic vesicles, suggests that they are involved in endocytosis. In addition, gp 90 is expressed on endothelial cells lining glomerular capillaries. These observations may help us to understand the formation and different kinetics of immune glomerular deposits.

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.