Podocalyxin in rat platelets and megakaryocytes.

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent Mr of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

Morphologic changes suggesting abnormal renal differentiation in congenital nephrotic syndrome.

Retrograde differentiation (or dedifferentiation) has recently been proposed as a pathogenetic mechanism involved also in various renal diseases. Here we studied whether evidence of these mechanisms can be found in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF). These patients show isolated massive proteinuria but no primary symptoms from any other organ systems. For the analysis we used antibody markers of early (fibronectin, stem cell factor, Wilms' tumor gene product, cytokeratin) and later (laminin, midgestation and kidney, heparin binding growth-associated molecule) stages of nephron differentiation as well as for apoptosis (acridine orange staining), rescue from apoptosis (anti-Bcl-2 antibodies) and cell proliferation (antibodies to proliferating cell nuclear antigen). In the peritubular spaces atypically organized areas were found which appeared positive with markers of low stages of differentiation, but neither abnormal cell proliferation nor activation of the apoptotic pathway could be detected. As morphologic signs of abnormal tissue organization, we found clusters of tightly compacted and large glomeruli corresponding to the size of two to three normal glomeruli. However, all individual glomerular cell compartments (mesangial, endothelial, visceral epithelial cells) appeared balanced in relative cell numbers. Together these results may indicate abnormal early mesenchymoepithelial tissue interaction leading to excessive and poorly organized formation of glomeruli. This could be causally related also to the serious functional immaturity of CNF kidneys presented as isolated proteinuria.

Tyrosine phosphorylation of p72syk induced by anti-9-O-acetyl GD3 antibodies in human peripheral blood mononuclear cells.

We have previously shown that a subpopulation of peripheral blood CD4+ T-cells are strongly positive for the ganglioside 9-O-acetyl GD3. Treatment of peripheral blood mononuclear cells (PBMC) with monoclonal anti-9-O-acetyl GD3 antibodies (MoAb 27A) induced a dose-dependent proliferative response in the cells. Here we show that binding of 27A IgG to PBMC induces rapid tyrosine phosphorylation of p72syk and mobilization of phosphoinositides. Equal phosphorylation was not seen after stimulation of PBMC with anti-GD3 antibodies (MoAb R24) despite the apparent similar cell surface distribution of the two gangliosides. This suggests that the O-acetyl modification enhances the signal transduction mediated by GD3 ganglioside.

Decrease of glomerular disialogangliosides in puromycin nephrosis of the rat.

Puromycin aminonucleoside nephrosis (PAN) is a model for human minimal change nephropathy induced in rats by injection of puromycin. In PAN, defective sialylation of a major sialoprotein of podocytes, podocalyxin, has been demonstrated and the consequent decrease of anionic charge suggested as a causative factor for increased glomerular permeability and proteinuria. Whether defective sialylation is a general feature of PAN affecting also glomerular glycosphingolipids is not known. We have shown that rat glomeruli are rich in disialogangliosides GD3 and O-acetyl GD3, the functions of which are not known. Here, we made a sequential analysis of the glomerular gangliosides, especially of GD3 and its O-acetyl derivative in acute PAN using immunohistochemical and biochemical techniques and compared the results with another rat model of glomerular disease, Heymann nephritis. The prominent immunohistochemical finding was the almost total disappearance of glomerular O-acetyl GD3 and a substantial decrease of its precursor GD3 peaking at 10 days after injection of puromycin. Segmental areas lacking these gangliosides remained in glomeruli still at 30 days after injection. The response was dose dependent. Semiquantitative analysis by thin layer chromatograms showed that O-acetyl GD3 was decreased by 41% already at 3 days and by 60% at 10 days after injection of puromycin. Also GD3, the immediate precursor of O-acetyl GD3, was decreased by 20 and 19%, respectively, at 3 and 10 days after injection. At 3 days after injection, overt proteinuria had not started. At these times, no other changes were observed in the glomerular gangliosides. The decrease of glomerular GD3 and O-acetyl GD3 indicates a decrease of GD3 synthase activity and perhaps of O-acetyltransferase activity in PAN nephrosis. As these changes preceded the overt proteinuria, they may have a causal relationship to it. In the glomeruli of Heymann nephritic rats, no similar changes were seen, suggesting that the sialylation defect is not due to proteinuria but is a consequence of targeted puromycin action on cells.

Characterization of a rat glomerular visceral epithelial cell line.

Cultures of glomerular epithelial cells (GEC) are currently used to identify important cellular and molecular mechanisms involved in the pathogenesis of renal diseases. However, there is still controversy in the literature as to the visceral or parietal origin of cultured GEC. Our aim was to firmly establish the nature of a GEC cell line. The reactivity of cultured GEC was investigated with a large panel of mono- and polyclonal antibodies by using immunofluorescent techniques and compared with literature data on the in vivo expression of these antigens on podocytes. In addition, the podocyte specific 5A (podocalyxin), 13A and 27A (9-O-acetylated GD3) antigen expression was investigated in immuno-overlay experiments with isolated gangliosides and in immunoprecipitations with metabolically labelled cells. In general, immunoreactivities between cultured GEC and literature data on GEC in vivo expressions were similar. Important podocyte epitopes in vivo were expressed by cultured GEC such as podocalyxin, gp330 and the 13A antigen. Cultured GEC however differed from their in vivo counterparts in their expression of keratin-18, their lack of expression of pp44 and no detectable immunohistological expression of the ganglioside 9-O-acetylated GD3. Interestingly, the podocyte-specific epitope 9-O-acetylated GD3 was detected by the 27A antibodies in immuno-overlays of isolated GEC gangliosides. Moreover, by using the 27A antibody, we were able to precipitate the podocyte-specific 103-kD protein from 35S-methionine metabolically labelled GEC. From our immunohistological data together with the detectability of the 27A antigen we conclude that the cell line we use very probably originates from glomerular visceral epithelial cells.

O-acetyl GD3 ganglioside in human peripheral blood T lymphocytes.

O-acetyl GD3 ganglioside is a cell surface molecule of some neural, neural crest and renal cells. Here we show, by using mAbs specific for O-acetyl GD3 (clone 27A) and flow-cytometric, biochemical or immunological techniques, that it is also expressed at high intensity level on the surface of 49.6% (median) of the CD3+ cells (T lymphocytes), at medium level in 16.2% of the CD16+ (natural killer) cells, at very low level in 51.9% of CD14+ cells (monocytes) and in 6.9% of CD20+ cells (B lymphocytes), but not in other human blood cells. Of the CD4+ or CD8+ cells, 52.6 or 36.5% respectively were 27A+. Furthermore, 81.6% of the CD45RO+ lymphocytes carried the O-acetyl GD3 ganglioside. It was not detected in the thymus, although its immediate precursor, the GD3 ganglioside, was present in the medullary thymocytes, suggesting that O-acetyltransferases are regulated by maturation events taking place in the periphery. The anti-O-acetyl GD3 antibodies induced a strong mitogenic response in cultured peripheral blood mononuclear cells, but not in purified T cells. However, in combination with phorbol myristate acetate the antibodies induced proliferation also in purified T cells, suggesting that protein kinase C priming is needed for this effect. This and the restricted expression of O-acetyl GD3 suggest a functional role for this ganglioside in T cell subpopulations.

Nephron segment and cell-type specific expression of gangliosides in the developing and adult kidney.

Despite the increasing knowledge of the role of gangliosides in normal and diseased tissues, little is known of the presence, distribution and functions of these molecules in the kidney. In this study we analyzed the main gangliosides of isolated glomeruli and cortical, medullary and papillary fractions of the human, rat and bovine kidneys biochemically. In addition, we used immunohistochemistry to visualize the distribution of GM1/GM2, GD2, GD3 and O-acetyl GD3 gangliosides along the nephron. Furthermore, we explored the species specific expression of gangliosides by comparing those from the rat, bovine and human kidney, and studied the pattern of ganglioside expression during development. In glomeruli, cortical tubuli, medullae and papillae, a relatively simple pattern of main gangliosides was observed as revealed by thin layer chromatographic (TLC) analysis in each species studied. Furthermore, considerable changes in the glomerular gangliosides during maturation were observed, with a complex type of gangliosides predominating during the fetal age and with a preference to more simple precursors upon maturation. Interestingly, the immunohistochemical detection revealed a distinct pattern of ganglioside compartmentation to various nephron segments or cell types. These findings provide a basis for studying the role of segment- and cell type-specific gangliosides for local functions.

A cell-type specific ganglioside of glomerular podocytes in rat kidney: an O-acetylated GD3.

We recently described a monoclonal antibody (clone 27A) that detected a membrane antigen specific for glomerular podocytes in adult rat kidney. After binding in vivo, the antibodies induced rapid changes in the foot processes. Here we show that in other rat tissues the antigen is detectable only in cells of adrenal medulla, in some cells of neural or neural crest origin, and in 1 to 5% of the cells of a rat pheochromocytoma cell line PC-12. Attempts to isolate the antigen revealed that it is an acidic, sialic acid containing lipid, as shown by thin layer chromatography and immuno-overlay techniques. Further characterization of the gangliosides extracted from rat glomeruli, bovine kidney, rat adrenal glands, or from PC-12 cells by ion exchange, thin layer, and gas liquid chromatography identified the antigenic lipid as a modified disialosyllactosylceramide (GD3). The results of mild alkaline treatment or periodate oxidation of the antigenic ganglioside, as well as chemical O-acetylation studies of standard gangliosides, showed that the modified ganglioside is O-acetylated, most probably at the 9-carbon of its terminal sialic acid residue. To our knowledge this is the first report of cell-type specific expression of gangliosides in the kidney.