The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.     

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.     

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60-80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.     

Ultrastructural study of the glomerular slit diaphragm in fresh unfixed kidneys by a quick-freezing method.

In normal kidneys fixed by perfusion with tannic acid and glutaraldehyde, glomerular slit diaphragms have been reported to consist of highly ordered and isoporous substructures with a zipper-like configuration. We have re-evaluated the ultrastructure of fixed or unfixed glomeruli using quick-freezing and deep-etching (QF-DE) and freeze-substitution (QF-FS) methods. In the fixed slit diaphragms, zipper-like substructures were often observed by the QF-DE method. In contrast, in fresh unfixed glomeruli the slit diaphragms mainly consisted of non-porous substructures. The slit diaphragms were more widely opened in the fixed glomeruli, as examined by the QF-FS method. These results suggest that the foot processes shrink during tissue preparation by conventional methods with chemical fixatives, and that the broadening of slit diaphragms and zipper-like substructures are formed by the pulling apart of adjacent foot processes due to shrinkage.     

Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys

The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1α were detected in the cerebrum, but only one form of neurexin-1α was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.     

Early glomerular filtration defect and severe renal disease in podocin-deficient mice

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.     

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte β1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin β1 (podocin-Cre β1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre β1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin β1 activity in epithelial cells. To further explore whether integrin β1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte β1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.     

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.     

Ultrastructural study of human glomerular capillary loops with IgA nephropathy using quick-freezing and deep-etching method

Immunoglobulin A (IgA) nephropathy shows great variability regarding the histological features of the lesions of human renal glomeruli. In the present study, the quick-freezing and deep-etching (QF-DE) method was used to analyze the glomerular ultrastructure of biopsied kidney tissues from children with IgA nephropathy. Biopsied renal tissues were routinely prepared for light microscopy, immunofluorescence microscopy, conventional electron microscopy, and replica electron microscopy. The three-dimensional ultrastructure of glomeruli of the kidney was clearly observed by using the QF-DE method. Three layers of glomerular basement membranes, i.e., middle, inner and outer layers, were clearly detected in the replica electron micrographs. The middle layer was 343.0+/-24.2 nm (n=20) in width and formed polygonal meshwork structures. We also observed slit diaphragms, electron-dense mesangial deposits, and increased amounts of mesangial matrix and foot process effacement. Many delicate filaments were found to be distributed from the apical to the bottom portions between neighboring foot processes. The ultrastructural difference between the replica electron micrographs and conventional electron micrographs was found to be especially marked in the appearance of foot processes and connecting filaments between the neighboring foot processes. The examination of extracellular matrix changes, as revealed at high resolution by the QF-DE method, gave us some morphofunctional information relevant to the mechanism of proteinuria with IgA nephropathy.     

Avian Podocytes,Which Lack Nephrin,Use Adherens Junction Proteins at Intercellular Junctions

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.     

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Electron microscopic study of glomerular filtration barrier in the rat kidney embedded in polymerized glutaraldehyde-urea.

Embedding kidney in polymerized glutaraldehyde-urea favors the retention of glycoprotein matrix of the cell coat and the basement membrane of the glomeruli. The basement membrane appears as a single layer with uniform amorphous matrix. Thick glycoprotein coat covers the whole surface of prodocytes and their foot processes. In areas other than the slits and the portion of the foot processes which touch on the basement membrane, the coat is a continuous layer with an average thickness of 490 A. In the slits between the foot processes of podocytes there is an actual fusion of glycoprotein coats; the average width of the slit is 415 A. The glycoprotein 'plugs' in the slit may be a significant portion of the glomerular filtration barrier against macromolecules, together with the basement membrane and the slit diaphragms.     

The Adaptor Protein Grb2 Is Not Essential for the Establishment of the Glomerular Filtration Barrier

The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.     

The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1), which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target.     

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.     

The Extracellular Matrix Protein SC1/Hevin Localizes to Multivesicular Bodies in Bergmann Glial Fibers in the Adult Rat Cerebellum

SC1 is an extracellular matrix molecule prominent in the mammalian brain. In the cerebellum, SC1 localizes to Bergmann glial cells and perisynaptic glial processes that envelop synapses in the molecular layer. In the present study, confocal microscopy revealed a punctate distribution of SC1 along Bergmann glial fibers that colocalized with the intermediate filament GFAP when fibers were viewed in cross-section. Immunoelectron microscopy showed that the punctate SC1 pattern corresponded to the localization of SC1 in multivesicular bodies situated within Bergmann glial fibers. The pattern of SC1 localization was not disrupted following hyperthermia or pilocarpine-induced status epilepticus. The present study suggests that SC1 protein may reach its destination in perisynaptic glial processes and glial endfeet by transport along Bergmann glial fibers in multivesicular bodies and that this process is preserved following stress.     

Nephrin expression is increased in anti-Thy1.1-induced glomerulonephritis in rats

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3-7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.     

A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier

The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.