Biogenesis of podocalyxin--the major glomerular sialoglycoprotein--in the newborn rat kidney

The appearance and distribution of podocalyxin on the glomerular epithelium (podocytes) during glomerular development was determined in the newborn rat kidney using specific monoclonal and affinity-purified polyclonal antibodies. Kidneys from 2-day-old rats were perfusion-fixed and processed for immunofluorescence or immunoperoxidase localization or immunogold labeling on ultrathin frozen sections. Podocalyxin first appeared on the apical surfaces of the presumptive podocytes of the S-shaped body above the level of the junctional complexes that connect the cells at this stage. The latter consist of a shallow occluding zonule and a deeper adhering zonule. Early in the capillary loop stage, when the urinary spaces open and the junctional complexes migrate from the apex to the base of the cells, labeling for podocalyxin extended along the lateral plasmalemma above the migrating junctions. In the maturing glomerulus when the foot processes form and the occluding and adhering junctions give way to developing slit diaphragms, podocalyxin was found along all newly-opened surfaces above the occluding junctions or slit membranes. No labeling was found below the latter. Podocalyxin was also detected intracellularly throughout the entire exocytotic pathway--i.e., in the rough endoplasmic reticulum and perinuclear cisternae, in Golgi cisternae and associated vesicles, and in carrier vesicles presumably en route to the cell surface. It is concluded that 1) podocalyxin is synthesized at a high rate in the differentiating podocyte; 2) its distribution is restricted to the apical plus lateral plasmalemmal domain facing the urinary spaces above the migrating junctions; 3) its time of appearance and distribution during glomerular development are identical to that reported earlier for epithelial polyanion; and 4) its synthesis and insertion into the podocyte plasmalemma is closely coupled to the development of the foot processes and filtration slits.     

The human cancer and stem cell marker podocalyxin interacts with the glucose-3-transporter in malignant pluripotent stem cells

Podocalyxin, an integral plasma membrane cell-adhesion glycoprotein, is a marker of human pluripotent and multipotent stem cells. Podocalyxin is also a marker of many types of cancers and its expression correlates with an aggressive and poor-prognosis tumor phenotype. The function of podocalyxin in stem cells and malignant cells is unknown. Protein sequence data obtained from purified podocalyxin protein isolated from embryonal carcinoma cancer stem cells reveals peptide sequence data for the glucose-3-transporter. Protein-precipitation experiments of embryonal carcinoma protein extracts identify a podocalyxin/glucose-3-transporter protein complex. Cell imaging studies demonstrate co-localization of podocalyxin and glucose-3-transporter and confirm the interaction in vivo. Finally, siRNA podocalyxin-knockdown experiments show decreased expression levels of the glucose-3-transporter. These findings suggest a novel interaction of the glucose-3-transporter and the cell-adhesion protein podocalyxin. In pluripotent stem cells and in human cancer disease, podocalyxin may function in part to regulate and maintain the cell surface expression of the glucose-3-transporter.     

Identification of endoglycan, a member of the CD34/podocalyxin family of sialomucins

CD34 and podocalyxin are structurally related sialomucins, which are expressed in multiple tissues including vascular endothelium and hematopoietic progenitors. These glycoproteins have been proposed to be involved in processes as diverse as glomerular filtration, inhibition of stem cell differentiation, and leukocyte-endothelial adhesion. Using homologies present in the cytoplasmic tails of these proteins, we have identified a novel member of this family, which we designate endoglycan. This protein shares a similar overall domain structure with the other family members including a sialomucin domain, but also possesses an extremely acidic amino-terminal region. In addition, endoglycan contains several potential glycosaminoglycan attachment sites and is modified with chondroitin sulfate. Endoglycan mRNA and protein were detected in both endothelial cells and CD34(+) bone marrow cells. Thus, CD34, podocalyxin, and endoglycan comprise a family of sialomucins sharing both structural similarity and sequence homology, which are expressed by both endothelium and multipotent hematopoietic progenitors. While the members of this family may perform overlapping functions at these sites, the unique structural features of endoglycan suggest distinct functions for this molecule.     

Endothelial cell membranes contain podocalyxin--the major sialoprotein of visceral glomerular epithelial cells

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 ("pre-embedding") and surface ("postembedding") 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.     

Human embryonal carcinoma tumor antigen,Gp200/GCTM-2, is podocalyxin

We previously characterized a peanut agglutinin-binding tumor antigen, gp200, a surface membrane glycoprotein expressed on human embryonal carcinoma, a malignant stem cell of testicular tumors. Gp200 is remarkably similar to another embryonal carcinoma antigen, GCTM-2, a cell differentiation marker that is also detected in blood of testis cancer patients, yet neither molecular identity is known. We now report the identity of gp200 as podocalyxin. Protein sequence results of gp200 peptides match with podocalyxin sequence. Furthermore, two distinct monoclonal antibodies, specific for podocalyxin, react positively with gp200. Therefore, gp200 is a testicular tumor form of podocalyxin, a surface membrane glycoprotein that was originally discovered as a scaffolding extracellular matrix protein of kidney podocyte cells. Podocalyxin is also expressed on subsets of hematopoietic cells where it has a putative function as a cell adhesion protein. This is the first report of podocalyxin expression on malignant cells.     

Expression of caveolin-1 and podocalyxin in rat lungs challenged with 2-kDa macrophage-activating lipopeptide and Flt3L

Caveolin-1 is one of the important regulators of vascular permeability in inflamed lungs. Podocalyxin is a CD34 protein expressed on vascular endothelium and has a role in podocyte development in the kidney. Few data are available on the expression of caveolin-1 and podocalyxin in lungs challenged with Toll-like receptor 2 (TLR2) agonists such as mycoplasma-derived macrophage activating lipopeptide or with immune modulators such as Fms-like tyrosine kinase receptor-3 ligand (Flt3L), which expands dendritic cell populations in the lung. Because of the significance of pathogen-derived molecules that act through TLR2 and of the role of immune modulators in lung physiology, we examine the immunohistochemical expression of caveolin-1 and podocalyxin in lungs from rats challenged with a 2-kDa macrophage-activating lipopeptide (MALP-2) and Flt3L. Normal rat lungs expressed caveolin-1 in alveolar septa, vascular endothelium and airway epithelium, especially along the lateral borders of epithelial cells but not in alveolar macrophages. MALP-2 and Flt3L decreased and increased, respectively, the expression of caveolin-1. Caveolin-1 expression seemed to increase in microvessels in bronchiole-associated lymphoid tissue (BALT) in Flt3L-challenged lungs but not in normal or MALP-2-treated lungs. Podocalyxin was absent in the epithelium and alveolar macrophages but was present in the vasculature of control, Flt3L- and MALP-2-treated rats. Compared with control and MALP-2-treated rats, Flt3L-treated lungs showed greater expression of podocalyxin in BALT vasculature and at the interface of monocytes and the endothelium. These immunohistochemical data describing the altered expression of caveolin-1 and podocalyxin in lungs treated with MALP-2 or Flt3L encourage further mechanistic studies on the role of podocalyxin and caveolin-1 in lung inflammation.     

Transmembrane collagen XVII is a novel component of the glomerular filtration barrier

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.     

Glycoprotein hyposialylation gives rise to a nephrotic-like syndrome that is prevented by sialic acid administration in GNE V572L point-mutant mice

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparent myopathies or motor dysfunctions. However, they had a short lifespan and exhibited renal impairment with massive albuminuria. Histological analysis showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte foot process morphologies in the kidneys. Glycan analysis using several lectins revealed glomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin, which is one of important molecules for the glomerular filtration barrier. Administering Neu5Ac to the mutant mice from embryonic stages significantly suppressed the albuminuria and renal pathology, and partially recovered the glomerular glycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observed in these mutant mice resulted from impaired glomerular filtration due to the hyposialylation of podocyte glycoproteins, including podocalyxin. Furthermore, it was possible to prevent the nephrotic-like disease in these mice by beginning Neu5Ac treatment during gestation.     

Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton

Podocytes of the renal glomerulus are unique cells with a complex cellular organization consisting of a cell body, major processes and foot processes. Podocyte foot processes form a characteristic interdigitating pattern with foot processes of neighboring podocytes, leaving in between the filtration slits that are bridged by the glomerular slit diaphragm. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells or pericytes. Mutations affecting several podocyte proteins lead to rearrangement of the actin cytoskeleton, disruption of the filtration barrier and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context.     

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.     

Neph-Nephrin proteins bind the Par3-Par6-atypical protein kinase C (aPKC) complex to regulate podocyte cell polarity

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their foot processes and the interposed slit diaphragm. So far, very little is known about the guidance cues and polarity signals required to regulate proper development and maintenance of the glomerular filtration barrier. We now identify Par3, Par6, and atypical protein kinase C (aPKC) polarity proteins as novel Neph1-Nephrin-associated proteins. The interaction was mediated through the PDZ domain of Par3 and conserved carboxyl terminal residues in Neph1 and Nephrin. Par3, Par6, and aPKC localized to the slit diaphragm as shown in immunofluorescence and immunoelectron microscopy. Consistent with a critical role for aPKC activity in podocytes, inhibition of glomerular aPKC activity with a pseudosubstrate inhibitor resulted in a loss of regular podocyte foot process architecture. These data provide an important link between cell recognition mediated through the Neph1-Nephrin complex and Par-dependent polarity signaling and suggest that this molecular interaction is essential for establishing the three-dimensional architecture of podocytes at the kidney filtration barrier.     

Glomerular endothelium: a porous sieve and formidable barrier

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.     

Requirement of podocalyxin in TGF-beta induced epithelial mesenchymal transition

Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeleton assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype.     

The fine structure of gill 'podocytes' in Panulirus argus (crustacea)

A cell type structurally resembling the podocyte of the renal glomerulus is situated in the gill of the crustacean Panulirus argus. These cells adjoin the medial septum of the gill filament and invariably face the efferent haemolymph channel. The basal cell surface is produced into a series of regular ridges, between which are inserted elongated cell processes, together constituting a palisade that includes narrow slits (250 A or more in width) resembling the filtration pores between the foot process of the glomerular epithelium. In each instance, the slit is traversed by a diaphragm which in the crustacean 'podocyte' is ca. 30 A in width and contiguous with the outer leaflet of the unit membrane limiting the cell. Numerous coated vesicles originate from the cell surface beneath the diaphragms. The possible role of these cells in detoxification by withdrawal of materials from the circulation is discussed.     

Podocalyxin Regulates Murine Lung Vascular Permeability by Altering Endothelial Cell Adhesion

Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin and endoglycan) as vascular endothelial cell markers, there is remarkably little known of their vascular function. Podocalyxin (gene name Podxl), in particular, has been difficult to study in adult vasculature as germ-line deletion of podocalyxin in mice leads to kidney malformations and perinatal death. We generated mice that conditionally delete podocalyxin in vascular endothelial cells (Podxl^ΔEC mice) to study the homeostatic role of podocalyxin in adult mouse vessels. Although Podxl^ΔEC adult mice are viable, their lungs display increased lung volume and changes to the matrix composition. Intriguingly, this was associated with increased basal and inflammation-induced pulmonary vascular permeability. To further investigate the etiology of these defects, we isolated mouse pulmonary endothelial cells. Podxl^ΔEC endothelial cells display mildly enhanced static adhesion to fibronectin but spread normally when plated on fibronectin-coated transwells. In contrast, Podxl^ΔEC endothelial cells exhibit a severely impaired ability to spread on laminin and, to a lesser extent, collagen I coated transwells. The data suggest that, in endothelial cells, podocalyxin plays a previously unrecognized role in maintaining vascular integrity, likely through orchestrating interactions with extracellular matrix components and basement membranes, and that this influences downstream epithelial architecture.     

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.     

Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish,Mice and Humans

Background

Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration.

Methods and Findings

We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus.

Conclusions

Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.     

The Structure of Anuran Podocyte—Determined by Ecology?

Abstract The podocytes of ten frog species with different habitat preference were investigated by scanning electron microscopy. The visceral epithelium within these species shows considerable variation in the branching mode of the cellular processes, in the number of pedicels and in the form of cell bodies. The presence of various podocyte cell forms within anurans of one family (e.g. within Ranidae and Discoglossidae) indicates that podocytic structure is not manifested phylogenetically. The complexity of processes and pedicel numbers are high in glomeruli of terrestrial and semiterrestrial frogs but low in aquatic and semiaquatic animals. Consequently, podocyte structure is (a) correlated with environmental conditions and (b) plays an important role in osmoregulation. Furthermore, since podocytes are suggested to serve as stabilizers of glomerular vessels, the cells of the visceral epithelium provide the structural basis for regulation of glomerular filtration rate, e.g. for glomerular intermittency.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

The ultrastructure of the renal corpuscle, the neck segment, the proximal tubule and the intermediate segment of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) was examined by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and freeze-fracture technique. The glomerular filter apparatus consists of the podocyte epithelium, a distinct basement membrane, a subendothelial space and the capillary endothelium. Emanating from the podocyte cell body, several long primary processes encircle neighboring capillaries. The short slender foot processes originating from the primary processes interdigitate with those from other primary processes, thereby forming the meandering filtration slit. Thick bundles of microfilaments are found in the primary processes, but absent in the foot processes. The basement membrane consists of a lamina rara externa and a rather thin lamina densa (50 nm thickness). The wide subendothelial space contains abundant microfibrils, a few collagen fibrils and many thin processes of mesangial cells. The endothelium is flat and fenestrated (compared to mammals displaying relatively few fenestrations); some of the fenestrations are bridged by a diaphragm. The glomerular mesangium is made up of the mesangial cells and a prominent mesangial matrix containing microfibrils and collagen fibrils. The cells of the neck and intermediate segments display numerous cilia with their microtubules arranged in the typical 9 + 2 pattern. The basal bodies of the cilia are attached to thick filaments with a clear crossbanding pattern of 65 nm periodicity. The proximal tubule is composed of cells typical for this segment (PT cells) and light cells lacking a brush border (bald-headed cells). The PT cells measure 10-25 micron in height and 15-30 micron in width and do not interdigitate at their lateral borders with each other. Their basolateral cell membrane is amplified by many folds projecting into lateral intercellular spaces and into basal recesses. The brush border is scarce and composed of loosely arranged short microvilli.