Podocytes in glomerulus of rat kidney express a characteristic 44 KD protein

We describe a new monoclonal antibody (MAb) directed against glomerular visceral epithelial cells (podocytes), generated by immunization with isolated rat kidney glomeruli. In immunoblotting experiments this MAb (IgG1 subclass) reacted with a 44 KD protein. In cryostat sections of normal rat kidney the MAb stained glomerular podocytes; therefore, we called the antigen pp44 (podocyte protein 44 KD). On 0.5-micron cryostat sections the signal could be more precisely ascribed to the podocyte foot processes, whereas the cell bodies appeared virtually unreactive. On ultra-thin frozen sections pp44 was found within the cytoplasm of podocyte foot processes at their origin from their parent processes. The podocyte cell membrane was not labeled. All other parts of the nephron were unreactive. An additional but weaker immunoreaction was found in the arterial endothelium; the endothelia of other vessels (peritubular capillaries, veins) were negative. In human kidney anti-pp44 revealed the same staining pattern as in rat kidney. The expression of pp44 was also studied in newborn rat kidney. The early stages of glomerular development (renal vesicle, S-shaped body) were negative. pp44 first appeared during the capillary loop stage, i.e., when formation of podocyte foot processes commences. In comparing the present results with published data, pp44 is clearly different from other antigens thus far described in podocytes. From the results of this investigation we conclude that pp44 represents a novel cytoplasmic protein of podocytes. Our data suggest a cytoskeletal role for pp44 in preserving the complex architecture of podocytes. This idea is confirmed by the simultaneous appearance of foot processes and anti-pp44 immunoreactivity during glomerular development.     

Podocytes in metanephric organ culture express characteristic in vivo phenotypes

 Podocytes outgrown from isolated glomeruli in vitro have failed to express fully differentiated in vivo phenotypes. In an attempt to determine whether podocytes in metanephric culture accomplish terminal differentiation, as observed in vivo, we investigated expression of their characteristic phenotypic features in rat metanephric organ cultures using immunohistochemistry and electron microscopy. Rat metanephroi were harvested on embryonic day 12.5 and cultured on transmembrane filters for 9 days. Morphological examination revealed two maturation stages when the podocytes resembled those of the S-shaped body stage and maturational stages of glomeruli in vivo. Electron microscopy revealed that, firstly podocytes lost their intercellular contacts and, simultaneously, the tight junctions shifted into close proximity to cell bases, followed by foot process development. Immunohistochemistry demonstrated that the tight junction protein, ZO-1, and specific podocytic markers, pp44, 5-1–6, podocalyxin and vimentin were expressed in a cell maturity-dependent manner, as observed in newborn rat kidneys. Furthermore, glomerular basement membrane components, collagen type IV and laminin, were expressed in the glomerular center. Our findings that cell maturity-dependent expression of structural and functional phenotypes in podocytes in metanephric culture was the same as that observed in developing kidneys in vivo indicate that podocyte differentiation during glomerulogenesis may be operated by an intrinsic property, such as programmed cell fate. Furthermore, these highly differentiated podocytes in vitro may provide clues that will help to establish a podocyte culture system. Accepted: 26 February 1997     

Microtubule Associated Protein 1b (MAP1B) Is a Marker of the Microtubular Cytoskeleton in Podocytes but Is Not Essential for the Function of the Kidney Filtration Barrier in Mice

Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated cytoskeleton is requisite for their integrity. Although much knowledge has been gained on the organization of cortical actin networks in podocyte’s foot processes, less is known about the molecular organization of the microtubular cytoskeleton in primary processes and the cell body. To gain an insight into the organization of the microtubular cytoskeleton of the podocyte, we systematically analyzed the expression of microtubule associated proteins (Maps), a family of microtubules interacting proteins with known functions as regulator, scaffold and guidance proteins. We identified microtubule associated protein 1b (MAP1B) to be specifically enriched in podocytes in human and rodent kidney. Using immunogold labeling in electron microscopy, we were able to demonstrate an enrichment of MAP1B in primary processes. A similar association of MAP1B with the microtubule cytoskeleton was detected in cultured podocytes. Subcellular distribution of MAP1B HC and LC1 was analyzed using a double fluorescent reporter MAP1B fusion protein. Subsequently we analyzed mice constitutively depleted of MAP1B. Interestingly, MAP1B KO was not associated with any functional or structural alterations pointing towards a redundancy of MAP proteins in podocytes. In summary, we established MAP1B as a specific marker protein of the podocyte microtubular cytoskeleton.     

Claudin-6 localized in tight junctions of rat podocytes

Tight junctions rarely exist in podocytes of the normal renal glomerulus, whereas they are the main intercellular junctions of podocytes in nephrosis and in the early stage of development. Claudins have been identified as tight junction-specific integral membrane proteins. Those of podocytes, however, remain to be elucidated. In the present study, we investigated the expression and localization of claudin-6 in the rat kidney, especially in podocytes. Western blot analysis and RT-PCR revealed that the neonatal kidney expressed much higher levels of claudin-6 than the adult kidney. Immunofluorescence microscopy showed intense claudin-6 staining in most of the tubules and glomeruli in neonates. The staining in tubules declined distinctly in adults, whereas staining in glomeruli was well preserved during development. Claudin-6 in glomeruli was distributed along the glomerular capillary wall and colocalized with zonula occludens-1. The staining became conspicuous after kidney perfusion with protamine sulfate (PS) to increase tight junctions in podocytes. Immunoelectron microscopy showed that immunogold particles for claudin-6 were accumulated at close cell-cell contact sites of podocytes in PS-perfused kidneys, whereas a very limited number of immunogold particles were detected, mainly on the basal cell membrane and occasionally at the slit diaphragm and close cell-cell contact sites in normal control kidneys. In puromycin aminonucleoside nephrosis, immunogold particles were also found mainly at cell-contact sites of podocytes. These findings indicate that claudin-6 is a transmembrane protein of tight junctions in podocytes during development and under pathological conditions.     

Characterization of an endothelial protein in the developing rabbit kidney

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.     

Clathrin and clathrin-accessory proteins in rat kidney cortex epithelia

Several vectorial transport routes in mammalian cells involve clathrin and associated proteins. In kidney epithelia urine production requires numerous transport processes. However, only little is known about the distribution of clathrin and its associated proteins in this organ in situ. We now report on the presence and distribution of clathrin and its accessory proteins AP1, AP2, Eps15, Epsin, CALM and Clint/EpsinR in the epithelia of the rat kidney cortex using immunoblotting, immunofluorescence and immuno-electron microscopy. Our data show that all investigated proteins are ubiquitously present in rat kidney cortex epithelia, however, with distinct distribution patterns. In the renal corpuscle, podocytes showed the most conspicuous labelling. Clathrin, AP2 and CALM were highly expressed in foot processes, while AP1 was primarily localized in the cell body. In the proximal tubule all proteins were present in dots along the plasma membrane and most conspicuous below the brush border. However, clathrin and AP2 co-localized in vesicle subtypes distinct from those containing clathrin and AP1. In the distal tubule and in the cortical collecting duct all proteins were found in the apex of the cells; however, AP1 and Clint/EpsinR showed additional staining in perinuclear dots. The occurrence and distribution of the investigated proteins in kidney epithelia are discussed with respect to their possible involvement in the functions of the specific nephron segment.Preliminary results were presented at the 28th Annual Meeting of the German Society for Cell Biology, March 16th–19th 2005, Heidelberg, Germany.     

Identification and characterization of a novel tight junction-associated family of proteins that interacts with a WW domain of MAGI-1

The membrane-associated guanylate kinase protein, MAGI-1, has been shown to be a component of epithelial tight junctions in both Madin-Darby canine kidney cells and in intestinal epithelium. Because we have previously observed MAGI-1 expression in glomerular visceral epithelial cells (podocytes) of the kidney, we screened a glomerular cDNA library to identify the potential binding partners of MAGI-1 and isolated a partial cDNA encoding a novel protein. The partial cDNA exhibited a high degree of identity to an uncharacterized human cDNA clone, KIAA0989, which encodes a protein of 780 amino acids and contains a predicted coiled-coil domain in the middle of the protein. In vitro binding assays using the partial cDNA as a GST fusion protein confirm the binding to full-length MAGI-1 expressed in HEK293 cells, as well as endogenous MAGI-1, and also identified the first WW domain of MAGI-1 as the domain responsible for binding to this novel protein. Although a conventional PPxY binding motif for WW domains was not present in the partial cDNA clone, a variant WW binding motif was identified, LPxY, and found to be necessary for interacting with MAGI-1. When expressed in Madin-Darby canine kidney cells, the full-length novel protein was found to colocalize with MAGI-1 at the tight junction of these cells and the coiled-coil domain was found to be necessary for this localization. Because of its interaction with MAGI-1 and its localization to cell-cell junctions, this novel protein has been given the name MAGI-1-associated coiled-coil tight junction protein (MASCOT).     

Actin filament organization of foot processes in rat podocytes.

The foot processes of podocytes possess abundant microfilaments and modulate glomerular filtration. We investigated the actin filament organization of foot processes in adult rat podocytes and the formation of the actin cytoskeletal system of immature podocytes during glomerulogenesis. Electron microscopy revealed two populations of actin cytoskeletons in foot processes of adult podocytes. One is the actin bundle running above the level of slit diaphragms and the other is the cortical actin network located beneath the plasmalemma. Immunogold labeling for actin-binding proteins demonstrated that alpha-actinin and synaptopodin were localized in the actin bundle, whereas cortactin was in the cortical actin network. Immunofluorescence labeling for actin-binding proteins in immature podocyte showed that alpha-actinin was localized at the level of the junctional complex, whereas cortactin was distributed beneath the entire plasmalemma. Synaptopodin was first observed along the basal plasmalemma from the advanced S-shaped body to the capillary loop stage. We conclude that foot processes have specialized actin filamentous organization and that its establishment is associated with the expression and redistribution of actin-binding proteins during development.     

Coiled-coil intermediate filament stutter instability and molecular unfolding

Intermediate filaments (IFs) are the key components of cytoskeleton in eukaryotic cells and are critical for cell mechanics. The building block of IFs is a coiled-coil alpha-helical dimer, consisting of several domains that include linkers and other structural discontinuities. One of the discontinuities in the dimer's coiled-coil region is the so-called 'stutter' region. The stutter is a region where a variation of the amino acid sequence pattern from other parts of the alpha-helical domains of the protein is found. It was suggested in earlier works that due to this sequence variation, the perfect coiled-coil arrangement ceases to exist. Here, we show using explicit water molecular dynamics and well-tempered metadynamics that for the coil2 domain of vimentin IFs the stutter is more stable in a non-alpha-helical, unfolded state. This causes a local structural disturbance in the alpha helix, which has a global effect on the nanomechanics of the structure. Our analysis suggests that the stutter features an enhanced tendency to unfolding even under the absence of external forces, implying a much greater structural instability than previously assumed. As a result it features a smaller local bending stiffness than other segments and presents a seed for the initiation of molecular bending and unfolding at large deformation.     

Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1

Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression. Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.     

The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene

We have cloned and sequenced a cDNA encoding an intermediate filament protein (IF) from the planarian Dugesia japonica named DjIFb. The deduced amino acid sequence of DjIFb has similarity to those of protostomic IFs and lamins, supporting a previous hypothesis that the protostomic IFs, including DjIFb, are evolutionarily closer to lamins than to vertebrate cytoplasmic IFs. In addition, analysis of the exon/intron organization revealed that 8 out of 10 introns of DjIFb were coincident in their position, even in the codon phase, with those of the non-neuronal IF of the snail Helix aspersa. This suggests that the Platyhelminthes are not the most primitive Bilateria but instead are evolutionarily close to the Mollusca. The DjIFb gene was expressed in particular cells, probably a kind of adhesive gland cell, which were present in the marginal region encircling the planarian body. The localization of DjIFb protein suggests that it plays an important role in the secretion of an adhesive substance. The specific expression pattern of the DjIFb gene enabled us to monitor how the body margin forms during planarian regeneration.     

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.     

NSAIDs acutely inhibit TRPC channels in freshly isolated rat glomeruli

Using a novel approach for analysis of TRPC channel activity, we report here that NSAIDs are involved into regulation of TRPC channels in the podocytes of the freshly isolated decapsulated glomeruli. Fluorescence and electron microscopy techniques confirmed the integrity of podocytes in the glomeruli. Western blotting showed that TRPC1, 3 and 6 are highly expressed in the glomeruli. Single-channel patch clamp analysis revealed cation currents with distinct TRPC properties. This is the first report describing single TRPC-like currents in glomerular podocytes. Furthermore, our data provide a novel mechanism of NSAIDs regulation of TRPC channels, which might be implicated in maintaining the glomerular filtration barrier.     

The VacA toxin of Helicobacter pylori identifies a new intermediate filament-interacting protein

The VacA toxin produced by Helicobacter pylori acts inside cells and induces the formation of vacuoles arising from late endosomal/lysosomal compartments. Using VacA as bait in a yeast two-hybrid screening of a HeLa cell library, we have identified a novel protein of 54 kDa (VIP54), which interacts specifically with VacA, as indicated by co-immunoprecipitation and binding experiments. VIP54 is expressed in cultured cells and many tissues, with higher expression in the brain, muscle, kidney and liver. Confocal immunofluorescence microscopy with anti-VIP54 affinity- purified antibodies shows a fibrous pattern typical of intermediate filaments. Double label immunofluorescence performed on various cell lines with antibodies specific to different intermediate filament proteins revealed that VIP54 largely co-distributes with vimentin. In contrast to known intermediate filament proteins, VIP54 is predicted to contain approximately 50% of helical segments, but no extended coiled-coil regions. The possible involvement of this novel protein in interactions between intermediate filaments and late endosomal compartments is discussed.     

The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors

Mutations in ric-3 (resistant to inhibitors of cholinesterase) suppress the neuronal degenerations caused by a gain of function mutation in the Caenorhabditis elegans DEG-3 acetylcholine receptor. RIC-3 is a novel protein with two transmembrane domains and extensive coiled-coil domains. It is expressed in both muscles and neurons, and the protein is concentrated within the cell bodies. We demonstrate that RIC-3 is required for the function of at least four nicotinic acetylcholine receptors. However, GABA and glutamate receptors expressed in the same cells are unaffected. In ric-3 mutants, the DEG-3 receptor accumulates in the cell body instead of in the cell processes. Moreover, co-expression of ric-3 in Xenopus laevis oocytes enhances the activity of the C.elegans DEG-3/DES-2 and of the rat alpha-7 acetylcholine receptors. Together, these data suggest that RIC-3 is specifically required for the maturation of acetylcholine receptors.     

Anion exchanger 1 in red blood cells and kidney: Band 3's in a pod

The bicarbonate/chloride exchanger 1 (AE1, Band 3) is abundantly expressed in the red blood cell membrane, where it is involved in gas exchange and functions as a major site of cytoskeletal attachment to the erythrocyte membrane. A truncated kidney isoform (kAE1) is highly expressed in type A intercalated cells of the distal tubules, where it is vital for urinary acidification. Recently, kAE1 has emerged as a novel physiologically significant protein in the kidney glomerulus. This minireview will discuss the known interactions of kAE1 in the podocytes and the possible mechanisms whereby this important multispanning membrane protein may contribute to the function of the glomerular filtration barrier and prevent proteinuria.