Abstract. Podocalyxin is the major sialoprotein in the glycocalyx of glomerular podocytes. Here we report on its extraglomerular localization, using a monospecific antibody which was obtained by affinity purification of IgG on nitrocellulose transfers of glomerular podocalyxin. By indirect immunofluorescence, podocalyxin was found in the blood vessels of several organs (lung, heart, kidney, small intestine, brain, pancreas, aorta, the periportal blood vessels in liver, and the central arteries of follicles of the spleen, but not in the endothelia that line the sinusoids of the latter organs). By immunoelectron microscopy--using immunogold conjugates in diffusion Cpre-embedding") and surface Cpostembedding") procedures--podocalyxin was localized on the luminal membrane domain of endothelial cells, in a patchy distribution. The presence of podocalyxin was confirmed in SDS extracts of lung tissue by immunoblotting.We conclude that (a) podocalyxin is a widespread component of endothelial plasma membranes, (b) it is restricted to the luminal membrane domain, and (c) it is distributed unevenly on the endothelial cell surface.

Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynoneal-lysine adducts in glomeruli of PHN rats by immunofluoresence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by -85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria.These findings are consistent with the premise that ROSinduced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with

Reactive oxygen species (ROS) have been implicated in the production of glomerular damage in passive Heymann nephritis (PHN), an experimental form of membranous nephropathy with neutrophil-independent proteinuria. Immunohistochemistry with monoclonal antibodies specific for cytochrome b558 (a major component of the oxidoreductase complex of the respiratory burst in stimulated neutrophilic granulocytes) showed that this enzyme is localized within visceral glomerular epithelial cells (GECs) in a dense, granular pattern in rats with PHN and proteinuria. By immunoelectron-microscopy, the cytochrome was found in membrane vesicles within the GEC and also extraceliularly on the GEC membranes facing the glomerular basement membrane (GBM). By immunoblotting, cytochrome bsm was detected in highest concentration in lysates of isolated glomeruli from proteinuric rats. By contrast, only traces were found in normal glomeruli by immunohistochemistry. Depletion of complement abolished the expression of the cytochrome. Using an ultrastructural cerium-H202 histochemistry technique, the functional activity of the glomerular ROS-generating system was demonstrated exclusively in proteinuric PHN, where H202 was found in highest concentration within the GBM. These results provide evidence that in rats with PHN and proteinuria, the GECs express and externalize respiratory-burst enzymes that generate ROS in a manner similar to neutrophilic granulocytes, which could then lead to glomerular damage.Passive Heymann nephritis (PHN) is an experimental model of human membranous nephropathy that is induced in rats by injection of antibodies directed against crude fractions of kidney cortex (Fx1A) (1). There is evidence that the subepithelial immune deposits in PHN are formed in situ from immune complexes of the membrane glycoprotein (gp) complex gp330/44 kDa, which is present in glomerular visceral epithelial cells (GEC) and from circulating antibody (2-4).

MethodsGlomerular visceral epithelial cells are endowed with a sialic acid-rich surface coat (the "glomerular epithelial polyanion"), which in rat tissue contains the sialoprotein podocalyxin. We have identified a major membrane sialoprotein in human glomeruli that is similar to rat podocalyxin in its sialic acid-dependent binding of wheat germ agglutinin and in its localization on the surface of glomerular epithelial and endothelial cells, as shown by immunoelectron microscopy, using the monoclonal antibody PHM5. Differences in the sialoproteins of the two species are indicated by the discrepancy of their apparent molecular weights in sodium dodecyl sulfate gels, by the lack of cross reactivity of their specific antibodies, and by the lack of homology of their proteolytic peptide maps. It is therefore possible that the human glomerular sialoprotein and rat podocalyxin are evolutionarily distinct, but have similar functions.

Several recent studies have focused on similarities between glomerular podocytes and neurons because the two cells share a specialized cytoskeletal organization and several expression-restricted proteins, such as nephrin and synaptopodin. In neurons, the small guanosine triphosphatase Rab3A and its effector rabphilin-3A form a complex required for the correct docking of synaptic vesicles to their target membrane. Because rabphilin-3A binds in neurons to cytoskeletal proteins also important for podocyte homeostasis, and the complex rabphilin-3A-Rab3A has been demonstrated in neurons and neuroendocrine cells, the aim of our work was to investigate their possible expression and regulation in podocytes. Normal kidneys from mouse, rat, and human were studied by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to evaluate the expression of Rab3A and rabphilin-3A. Double-staining immunohistochemistry and immunogold electron microscopy were then used to precisely localize the two proteins at the cellular and subcellular levels. Rab-3A and rabphilin-3A regulations in disease were then analyzed in growth hormone-transgenic mice, a well established model of focal and segmental glomerulosclerosis, and in human biopsies from proteinuric patients. Our results demonstrated that rabphilin-3A and Rab3A are present in normal mouse, rat, and human kidneys, with an exclusively glomerular expression and a comma-like pattern of positivity along the glomerular capillary wall, suggestive for podocyte staining. Co-localization of both molecules with synaptopodin confirmed their presence in podocytes. By immunogold electron microscopy both proteins were found around vesicles contained in podocyte foot processes. Their expression was increased in growth hormonetransgenic mice compared to their wild-type counterpart, and in a subset of biopsies from proteinuric patients. Our data, demonstrating the presence of two synaptic proteins in podocytes, further supports similarities between cytoskeletal and vesicular organization of podocytes and neurons. The altered expression observed in mouse and human proteinuric diseases suggests a possible role for these molecules in glomerulopathies. Glomerular podocytes, the last barrier of glomerular filtration, are highly specialized branched cells provided with interdigitating foot processes that externally cover the entire glomerular capillary surface. Recent advances in cell biology and genetics have expanded our knowledge about these cells, especially through the identification of several functionally important specific podocyte proteins. 1 Some of these proteins, such as nephrin, GLEPP-1 (glomerular epithelial protein-1), synaptopodin, and the amino acid transporters CAT3 (cationic amino acid transporter-3) and EAAT2 (excitatory amino acid transporter-2), have been found to be specifically shared by podocytes and neurons, 2-5 two process-bearing cells that, although derived from different embryological layers, have several features in common, 6 such as a hi...