Antibodies to glycolipids activate complement and promote proteinuria in passive Heymann nephritis.

Passive Heymann nephritis is an experimental rat model of human membranous nephropathy induced by injection of antisera against crude renal cortical fractions such as Fx1A or rat tubular microvilli. This results in the formation of subepithelial immune deposits, the activation of the C5b-9 membrane attack complex of complement, and severe proteinuria. While the formation of immune deposits is attributed to in situ immune complex formation with antibodies specific for the gp330-Heymann nephritis antigenic complex (HNAC), activation of complement and proteinuria appear to be caused by at least one additional antibody species present in anti-Fx1A sera. We have separated by affinity absorption polyspecific antisera against Fx1A and rat microvilli into one IgG fraction directed specifically against microvillar proteins (anti-Fx1A-prot) and another IgG fraction specific for glycolipids (ant-Fx1A-lip) of tubular microvilli. When injected into rats, the anti-Fx1A-prot fraction induced immune deposits but failed to activate complement or produce proteinuria, similar to results obtained with affinity-purified anti-gp330 IgG. When the antibodies of the anti-Fx1A-lip fraction were injected alone they did not bind to glomeruli. By contrast, when the IgGs specific for the Fx1A-prot fraction (or for gp330-HNAC) were combined with those directed against the Fx1A-lip glycolipid preparation, immune deposits were formed, in situ complement activation was observed, and also proteinuria was induced. It is concluded that within anti-Fx1A and anti-microvillar sera there are at least two IgG fractions of relevance for the development of PHN: one directed against the gp330-HNAC complex which is responsible for the development of immune deposits, and a second specific for glycolipid antigen(s) which activate(s) the complement cascade.     

The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1.

Nephrosis induced in rats by puromycin aminonucleoside treatment (PAN) results in the apical displacement of the glomerular filtration slit membrane by newly formed, intercellular occluding-type junctions. Similar changes can also be induced by acute kidney perfusion with protamine sulfate (PS). We have investigated the molecular nature of these altered junctions using an antibody to ZO-1, a protein found exclusively in tight junctions. Immunoblotting demonstrates ZO-1, a 225-kd band, in glomerular extracts of normal, PAN-, and PS-treated rats. By immunofluorescence, ZO-1 was localized at the base of podocytes outlining the capillary loops of glomeruli from all three experimental groups. At the electron microscope level, using immunoperoxidase or immunogold labeling, ZO-1 was concentrated along the cytoplasmic surfaces of the slit diaphragms of normal rats. In PAN or PS rats, it was concentrated along both the newly formed occluding-type junctions and the remaining slit diaphragms. When podocalyxin (the major membrane sialoprotein of the podocyte) was similarly localized, it was found exclusively apical to the displaced slit membrane. Based on morphology and the presence of ZO-1, the altered junctions seen in PAN and PS rats appear to represent bona fide tight junctions. Their rapid (15-minute) induction in PS-treated rats suggests that on neutralization of the cell surface charge by polycation perfusion, discontinuous tight junctions form from a preexisting pool of junctional proteins. These findings raise the possibility that glomerular hydraulic conductivity may be regulated in part by regulating the relative patency and width of the filtration slits through focal tight junction assembly.     

Podocalyxin in human haematopoietic cells

Podocalyxin-like protein (PCLP) is a sialomucin-type membrane protein structurally related to CD34 and endoglycan. It was first described in glomerular podocytes and endothelial cells. In mice, PCLP is present in haemangioblasts, and in both chicken and mice it is a marker of early haematopoietic stem cells and lineage-restricted haematopoietic progenitors. Its expression decreases during differentiation of haematopoietic cells. Of mature blood cells, only chicken and rat thrombocytes express PCLP protein. PCLP expression in human haematopoietic cells has not been studied. Here we demonstrate PCLP mRNA in human CD34+ cells, in lineage committed erythroid, megakaryocyte and myeloid progenitors, in K562 leukaemia cells, and in peripheral blood leucocytes. The mRNA expression level was higher in developing cells than in mature leucocytes. By Northern blotting and cDNA sequencing, the haematopoietic and renal PCLP mRNAs were identical. Of the mobilized CD34+ cells, 28% (mean; range 14-61%) expressed PCLP protein and the majority of PCLP+ cells were CD117+. Almost all of the K562 cells expressed PCLP protein. Surprisingly, PCLP protein was not detected in any mature blood cells. These results suggest that human PCLP may be a valuable marker for a subset of haematopoietic stem cells.     

Selective loss of podoplanin protein expression accompanies proteinuria and precedes alterations in podocyte morphology in a spontaneous proteinuric rat model

To evaluate changes during the development of proteinuria, podocyte morphology and protein expression were evaluated in spontaneously proteinuric, Dahl salt-sensitive (Dahl SS) rats. Dahl SS rats on a low-salt diet were compared with spontaneously hypertensive rats (SHR) at age 2, 4, 6, 8, and 10 weeks. Blood pressure, urinary protein excretion, urinary albumin excretion, and podocyte morphology were evaluated. In addition, the expression of 11 podocyte-related proteins was determined by analyzing protein and mRNA levels. In Dahl SS rats, proteinuria became evident around week 5, increasing thereafter. SHR rats remained non-proteinuric. Dahl SS rats showed widespread foot process effacement at 10 weeks. At < or =8 weeks, expression and distribution of the podocyte proteins was similar between the two strains, except for the protein podoplanin. At 4 weeks, podoplanin began decreasing in the glomeruli of Dahl SS rats in a focal and segmental fashion. Podoplanin loss increased progressively and correlated with albuminuria (r = 0.8, P < 0.001). Double labeling experiments revealed increased expression of the podocyte stress marker desmin in glomerular areas where podoplanin was lost. Dahl SS rats did not show podoplanin gene mutations or decreased mRNA expression. Thus, podocyte morphology and the expression and distribution of most podocyte-specific proteins were normal in young Dahl SS rats, despite marked proteinuria. Our study suggests that decreased expression of podoplanin plays a role in the decrease of glomerular permselectivity.     

Lymphatic precollectors contain a novel, specialized subpopulation of podoplanin low, CCL27-expressing lymphatic endothelial cells

Expression of the lymphoendothelial marker membrane mucoprotein podoplanin (podo) distinguishes endothelial cells of both blood and lymphatic lineages. We have previously discovered two distinct subpopulations of lymphatic endothelial cells (LECs) in human skin that were defined by their cell surface densities of podoplanin and were designated LEC^podo-low and LEC^podo-high. LEC^podo-low is restricted to lymphatic precollector vessels that originate from initial LEC^podo-high-containing lymphatic capillaries and selectively express several pro-inflammatory factors. In addition to the chemokine receptor protein Duffy blood group antigen receptor for chemokines, these factors include the constitutively expressed chemokine CCL27, which is responsible for the accumulation of pathogenic CCR10⁺ T lymphocytes in human inflammatory skin diseases. In this study, we report that CCR10⁺ T cells accumulate preferentially both around and within CCL27⁺ LEC^podo-low precollector vessels in skin biopsies of human inflammatory disease. In transmigration assays, isolated CCR10⁺ T lymphocytes are chemotactically attracted by LEC^podo-low in a CCL27-dependent fashion, but not by LEC^podo-high. These observations indicate that LEC^podo-low-containing precollector vessels constitute a specialized segment of the initial lymphatic microvasculature, and we hypothesize that these LEC^podo-low-containing vessels are involved in the trafficking of CCR10⁺ T cells during skin inflammation.