Diseases of the glomerular filter of the kidney are a leadingcause of end-stage renal failure. Recent studies have emphasizedthe critical role of the slit diaphragm of podocytes for thesize-selective filtration barrier of the kidney and revealednovel aspects of the mechanisms that lead to proteinuria, inboth inherited and acquired diseases (1–4). Several criticalstructural protein components of the slit diaphragm have beenidentified. Recently, it has been speculated that these slitdiaphragm proteins, in addition to their structural functions,participate in common signaling pathways. This review focuseson what is known about signaling at the slit diaphragm. It providesa snapshot of our current understanding of the signaling propertiesof slit diaphragm proteins and projects a framework for furtherstudies necessary to delineate the function and dynamics ofthe slit diaphragm protein complex and the pathogenesis of nephroticsyndrome.
Ultrafiltration of plasma in the renal glomeruli is a majorfunction of the kidney. The glomerular filter through whichthe ultrafiltrate has to pass consists of three layers: thefenestrated endothelium, the intervening glomerular basementmembrane, and the epithelial podocyte foot processes. This filtrationbarrier behaves as a size-selective sieve restricting the passageof macromolecules on the basis of their size, shape, and charge(5–7). Although the glomerular filter is a primary targetof a large number of progressive disorders that lead to chronicrenal insufficiency, until recently, little was known aboutthe importance of podocytes for establishing the size-selectivefiltration barrier of the kidney. The recent description ofgene defects in hereditary nephrotic syndrome resulting in theloss of podocyte proteins has dramatically changed this situation.Together with data from various animal models, these studieshave unraveled important aspects of the pathogenesis of proteinurickidney diseases and challenged our understanding of the glomerularfilter (1,8–10).
Podocytes are the visceral epithelial cells of the kidney glomerulus(11–13). They elaborate long, regularly spaced, interdigitatedfoot processes that completely enwrap the glomerular capillaries.Interdigitating podocyte foot processes form an 40-nm-wide filtrationslit and are connected by a continuous membrane-like structurecalled the slit diaphragm. When podocytes are injured, the intercellularjunctions and cytoskeletal structure of the foot processes arealtered and the cell takes on an "effaced" phenotype. Typicalslit diaphragm structures disappear and proteinuria develops(10). Because there is no case of nephrotic syndrome withoutmajor changes in podocyte morphology, it has been speculatedfor many years that the slit diaphragm is an important componentof the glomerular filter (14).
A milestone in glomerular research was the cloning of nephrinby the Tryggvason group in 1998 together with its localizationto the slit diaphragm of podocytes (15–18). This worksuggested that nephrin is a critical structural component ofthe slit membrane and bridges the distance between interdigitatingpodocyte foot processes (3,9,19). Nephrin, encoded by NPHS1,the gene mutated in congenital nephrotic syndrome of the Finnishtype, is a transmembrane adhesion protein of the Ig superfamily(Figure 1). Both humans and mice lacking nephrin are born withouttypical slit diaphragms and exhibit severe podocyte abnormalitiesand massive proteinuria already in utero (20,21).
Figure 1. Nephrin and neph1 are adhesion proteins of the Ig superfamily. They contain carboxy terminal cytoplasmic tails that can be phosphorylated at tyrosine residues (red y’s). Phosphotyrosine motifs of these proteins serve as docking sites for intracellular signaling adaptors.
Recently, several additional components of the slit diaphragmhave been identified (22–25). Mutations in the genes encodingfor these proteins cause severe podocyte changes and nephroticsyndrome. NPHS2, the gene responsible for a steroid-resistantform of nephrotic syndrome, was cloned by the Antignac groupand encodes for podocin, a stomatin family membrane protein(23). Podocin almost exclusively localizes to the slit diaphragmof podocytes and interacts with the cytoplasmic tail of nephrin(26–28). Gene disruption in mice results in a severe congenitalnephrotic syndrome (29). The cytoplasmic multi-adaptor proteinCD2AP was cloned by the Shaw laboratory as a CD2-interactingprotein in lymphocytes (30). CD2AP also interacts with nephrinand localizes to the cytoplasmic face of the slit diaphragm(31). It is interesting that mice completely lacking CD2AP dieof massive proteinuria 6 wk after birth, suggesting a criticalrole for CD2AP in slit diaphragm function (22). Moreover, CD2APhaploinsufficiency seems to be linked to glomerular diseasesusceptibility both in mice and in humans, further supportingthe critical role of CD2AP for the integrity of the glomerularfilter (32,33). Donoviel et al. (24) identified neph1, anotherIg superfamily protein (Figure 1) that localizes to the slitand causes congenital nephrotic syndrome in knockout mice. Neph1is a member of a family of Ig adhesion molecules that are expressedin podocytes and interact with podocin (34). Recently, the adhesionprotein and member of the protocadherin superfamily of proteinsFAT1 was shown to localize to the slit membrane of podocytes(35). Targeted deletion of the fat1 gene in mice results innephrotic syndrome and podocyte changes that resemble nephrinmutation (25).
The identification of these slit diaphragm proteins suggestedthat these proteins may serve important structural functionsat the filtration slit necessary for establishing and maintainingan intact glomerular filter. However, accumulating evidencesuggests that slit diaphragm proteins do not only serve structuralfunctions setting up a size- and charge-selective filtrationbarrier but also may participate in common signaling pathwaysnecessary to maintain the functional integrity of podocytes(27,36–38). This model proposes that the slit diaphragmprotein complex is a highly dynamic protein complex that recruitssignal transduction components and initiates signaling to regulatecomplex biologic programs in the podocyte, such as regulationof cytoskeletal rearrangements, polarized sorting and endocytosis,cell differentiation and suppression of proliferation, mechanotransduction,or podocyte viability.
Proof of Principle: Nephrin and Neph1 Are Signaling Proteins
Signal transduction is the process of converting extracellularsignals into cellular responses. Transmission of an extracellularsignal involves transmembrane proteins that have domains onboth sides of the plasma membrane (Figure 2). The basic principleof signaling is that ligand binding on the extracellular sideconverts the receptor from an inactive to an active form. Theprocess is called signal transduction because a signal has ineffect been transduced across the membrane. In general, signaltransduction systems are assembled through protein–proteininteractions (39). This means that signaling events that areinitiated at the plasma membrane control the reversible assemblyof multiprotein complexes to regulate intracellular processes.Much of this regulation is achieved through attachment of phosphategroups to (phosphorylation) or cleavage of phosphate (dephosphorylation)from serine, threonine, or tyrosine residues of crucial proteinsubstrates (40). Phosphorylation of these residues creates bindingsites for modular phosphoprotein-binding domains such as Srchomology 2 and phosphotyrosine-binding domains in the case ofphosphotyrosine or 14-3-3, WW domains, forkhead-associated domains,and, tentatively, WD40 repeats and leucine-rich regions in thecase of phosphoserine/phosphothreonine. Thus, phosphorylationconnects proteins with their upstream kinases and downstreameffectors to form multiprotein complexes (40,41).
Figure 2. The slit diaphragm of podocytes is a specialized cell junction with signaling properties. The slit diaphragm connects interdigitating foot processes of the podocyte (FP) and is an essential part of the glomerular filter of the kidney. Slit diaphragm proteins (nephrin and neph1) recruit cytoplasmic adaptor proteins to initiate signal transduction events that lead to the regulation of complex biologic programs.
Nephrin and neph1 are adhesion proteins and members of the Igsuperfamily (Figure 1). In addition to their extracellular domainsthat are part of the slit membrane connecting interdigitatingfoot processes and a single transmembrane domain, these proteinscontain a short cytoplasmic tail with a number of tyrosine residuesthat are predicted to serve as target for dynamic phosphorylationin vivo (27,34). Intriguing is that tyrosine phosphorylationat the cytoplasmic face of the slit diaphragm has been observedmany years before the cloning of nephrin or the descriptionof signaling properties of slit diaphragm proteins (42). Inagreement with this finding, it has recently been shown thatnephrin and neph1 are phosphorylated on tyrosine residues invitro and in vivo (28,36,37,43–45). Furthermore, recentdata suggest that tyrosine phosphorylation of the cytoplasmictail of these proteins seems to be tightly regulated (37,38).
What is the protein (kinase) responsible for tyrosine phosphorylationof nephrin and neph1? A recent study demonstrated that the Srcfamily nonreceptor protein tyrosine kinase Fyn directly bindsto the cytoplasmic tail of nephrin and mediates nephrin phosphorylationin vitro and in vivo (43). It is interesting that targeted deletionof fyn in mice with or without co-deletion of the tyrosine kinasegene yes resulted in the loss of nephrin tyrosine phosphorylation,a severe podocyte dysfunction, foot process effacement, andproteinuria, suggesting that these abnormalities may be at leastpartially related to altered nephrin phosphorylation (43,46).
Moreover, it could be shown that nephrin and neph1 are signalingproteins and able to stimulate transcriptional activation ina model system (27,37). Nephrin and neph1 activate the transcriptionfactor AP-1 via the stimulation of a mitogen-activated proteinkinase module. Direct interaction of the tyrosine-phosphorylatedcytoplasmic tail of nephrin with podocin dramatically facilitatednephrin signaling, whereas inhibition of tyrosine phosphorylationof the cytoplasmic tail of nephrin abrogated nephrin-mediatedsignal transduction (27). It is interesting that podocin-mediatedaugmentation of nephrin signaling required the direct interactionof the carboxy-terminal cytoplasmic domain of podocin with thecytoplasmic tail of nephrin (27,38). By contrast, in anotherstudy, neph1 signal transduction was augmented by direct interactionof the cytoplasmic tail of neph1 with the multiadaptor and scaffoldingprotein ZO-1 (37). Although derived from a model system, thesestudies provided the proof of principle that slit diaphragmproteins can initiate signal transduction.
Intracellular Signaling Adaptors: The Functional Slit Diaphragm Complex Assembles through Dynamic Protein–Protein Interactions
Signal transduction from cellular receptors requires intracellularadaptor proteins (Figure 3). Adaptor proteins are noncatalyticpolypeptides that contain one or more protein interaction modulesthat mediate protein interactions (47). As described above,many of the protein interactions are regulated through phosphorylationand dephosphorylation of crucial protein and lipid substrates(40,48).
Figure 3. Signaling at the slit diaphragm. Hypothetical signaling pathways involved in the regulation of podocyte biology and glomerular function. AKT, AKT/protein kinase B; Cbl, Casitas B-lineage lymphoma; Cort, cortactin; Endo, endophilin; MAPK, mitogen-activated protein kinase family; Syn, synaptojanin; TRP, hypothetical TRP channel.
By screening phosphotyrosine-binding proteins for their interactionwith the tyrosine-phosphorylated cytoplasmic tail of nephrin,the protein p85 was the first SH2 domain-containing proteinto be identified that binds to the tyrosine-phosphorylated cytoplasmictail of nephrin in vivo (36). p85 is the regulatory subunitof class Ia phosphoinositide 3-OH (PI3) kinase. PI3 kinase activityis responsible for the phosphorylation of lipids at the innerleaflet of the plasma membrane (49). Recruitment of the regulatoryp85 subunit to the cytoplasmic tail of nephrin induced the activationof the p110 catalytic subunit, which converts the membrane lipidphosphatidylinositol-4,5-biphosphate to phosphatidylinositol-3,4,5-triphosphate.Thus, lipids at the cytoplasmic side of the filtration slitare phosphorylated by nephrin-activated PI3 kinase, which mayinitiate a cascade of events in the podocyte foot process (36).
Signaling proteins with pleckstrin-homology (PH) domains accumulateat sites of PI3 kinase activation by directly binding to thesephosphorylated lipids. These proteins themselves regulate avariety of crucial cellular programs such as cell survival,actin cytoskeletal dynamics, endocytosis, and cell metabolism(49). Of particular interest in the podocyte is the PH domain–containingserine-threonine kinase AKT (36). Other PH domain proteins thatare activated by PI3 kinase and could play a role in podocytebiology include GDP-GTP exchange factors for Rac and ARF6 andprotein tyrosine kinases of the Bruton’s tyrosine kinase(Btk) and Tec family. Binding of PI3 kinase–generatedphospholipids to the PH domain of AKT leads to the translocationand activation of AKT. Among a wealth of effects, AKT activityhas been found to be required for the growth factor–dependentsurvival of a wide variety of cell types ranging from fibroblaststo neurons by blocking apoptosis (50). Consequently, nephrin-mediatedactivation of PI3 kinase and AKT activity has been shown toinhibit podocyte apoptosis and to increase the threshold forpodocyte cell death induced by apoptotic stimuli (36).
However, nephrin is not the only slit diaphragm protein to associatewith p85 and activate PI3 kinase. Recently, it was demonstratedthat CD2AP directly interacts with p85 (36). Together with nephrin,CD2AP strongly activates PI3 kinase in podocytes. Targeted disruptionof the cd2ap gene dramatically reduces AKT activity in podocytesand is associated with an increased susceptibility to podocyteapoptosis (36). These findings are particularly interestingin the context of the pathogenic steps that lead to glomerulosclerosis(51–54). Podocyte death and podocyte depletion have beenproposed as hallmarks of both primary and secondary forms ofglomerulosclerosis for many years and are now considered a keystep in the development of progressive renal disease (51,55–57).Thus, the structural and functional integrity of the slit diaphragmproteins and signaling at the slit diaphragm may be requiredfor the inhibition of apoptosis and for cell survival in podocytes.In support of this hypothesis, cd2ap+/– heterozygous miceare haploinsufficient and develop severe glomerular changesat 9 mo of age with a histologic pattern similar to that inhuman focal segmental glomerulosclerosis (32,33).
Although the underlying mechanism of CD2AP-mediated PI3 kinaseactivity at the molecular level is not completely understood,recent evidence suggests that by directly interacting with severaltarget proteins, CD2AP may orchestrate PI3 kinase effectorsto amplify efficient PI3 kinase signaling (T. Benzing, unpublisheddata). In analogy to CIN85, CD2AP may also be involved in therecycling and endocytosis of transmembrane receptors (e.g.,nephrin), thereby regulating signal transduction from the slitdiaphragm (32,58). An additional function of the PI3 kinasedownstream effector AKT is to repress collagenase expressionand to induce the synthesis of laminin and type IV collagenchains (59,60); both are key components of the glomerular basementmembrane. Because basement membrane abnormalities are involvedin the development of several forms of proteinuria, it is temptingto speculate that AKT activation may contribute to the synthesisand/or maintenance of an intact glomerular basement membrane.These data further support the concept that signal transductionat the slit diaphragm is critical for podocyte function, viability,and integrity of the glomerular filter.
It’s All about Location: Signaling at the Slit Diaphragm Needs a Lipid Raft Surrounding
As mentioned above, nephrin-mediated signaling is facilitatedby the direct interaction with the NPHS2 gene product podocin(27). Podocin, a member of the stomatin protein family witha short amino terminal domain, a transmembrane region, and acytosolic carboxy terminal domain, is exclusively expressedin podocytes of the developing and mature glomeruli and predictedto form a membrane-associated hairpin-like structure with theN- and C-terminal domains facing the cytosolic side of the slitdiaphragm (26). A functional interaction of nephrin with podocinwas indirectly confirmed in patients with hereditary nephroticsyndrome. In an interesting study, Koziell et al. (61) couldshow a functional interrelationship between NPHS1 and NPHS2,the genes encoding for nephrin and podocin. However, until recently,the question remained how podocin by directly interacting withnephrin may facilitate nephrin signaling. Some light has beenshed on the mechanism through which podocin can augment nephrinsignaling by a recent study that demonstrated that podocin-mediatedrecruitment of nephrin to so-called lipid raft microdomainsof the plasma membrane is required for efficient nephrin signaling(38).
Multiple pieces of evidence suggest that the plasma membraneof the filtration slit has a special lipid composition (28,43,44,62).Lipid rafts are specialized microdomains of the plasma membranewith a unique lipid content and a concentrated assembly of signaltransduction molecules (63). Rafts have been proposed to formplatforms for many important cellular processes, such as polarizedsorting of membrane proteins and signal transduction (64–66).Mundel and co-workers (28,44) could show that podocin is a lipidraft–associated protein at the filtration slit. Subsequently,Huber et al. (38) demonstrated that podocin serves to recruitnephrin into lipid raft microdomains. Lipid raft targeting ofnephrin was required for the proper initiation of nephrin signaling.Disease-causing podocin mutations failed to target nephrin torafts and lost their ability to augment nephrin signal transduction(38). Thus, not only the protein components but also a correctlipid composition of the plasma membrane at the slit diaphragmis important for proper signal transduction at the filtrationslit.
Talking about the Ligands: What Can We Learn from Model Organisms?
As detailed above, the basic principle of signal transductionis that ligand binding on the extracellular side converts thereceptor (in our case, nephrin) from an inactive to an activeform. Consequently, an important question is, "What could bethe ligand that binds to the extracellular domain of the proteinsat the slit diaphragm inducing the activation of signaling cascadesas described above?" Early work suggested that nephrin may engagein homophilic interactions (16). Moreover, cross-linking ofnephrin molecules was sufficient to induce an increase in tyrosinephosphorylation of the cytoplasmic tail of nephrin (45). Althoughsubsequent studies confirmed homophilic binding of nephrin molecules(67–69), these homophilic interactions may not be theonly interactions occurring in vivo. Recently, neph1 was shownto serve as a binding partner of the extracellular domain ofnephrin (67,69,70). Although these interactions were surprisinglypromiscuous, these data suggested that cis and trans interactionsinvolving at least nephrin and neph1 Ig superfamily proteinsare required to set up an intact slit diaphragm structure andinduce efficient signal transduction. The identification ofneph1 led to the description of a family of neph proteins inhumans and mice (neph1, neph2, neph3/filtrin), all expressedin the podocyte (34,71). Whether these proteins act as additionalligands of nephrin at the filtration slit is not clear to date.
These data are strongly supported by experimental results derivedfrom various model organisms. Nephrin and neph proteins canbe found in different species ranging from worm and fly to miceand humans. Although the fruit fly Drosophila melanogaster andthe nematode C. elegans lack structures comparable to a mammaliankidney glomerulus, some cells engage in specialized cell–cellcontacts that closely resemble the specialized cell junctionof podocytes. The Drosophila homologues of nephrin (hibris [hbs]and sticks-and-stones [sns]) interact with homologues of mammalianneph1 (dumbfounded [duf] and roughest/irreC [rst]) (72–74)to engage in intimate cell junctions that are required for myoblastfusion during embryonic muscle development. During skeletalmuscle development and regeneration after injury, mononucleatedmyoblasts fuse to form multinucleated muscle fibers. This processof myoblast fusion is amenable to genetic dissection in thefly, in which muscle formation involves a well-defined temporospatialsequence of events that are remarkably conserved also in mammalianmyogenesis. Recognition and fusion require the formation ofintense cell–cell contacts, so-called prefusion complexes,that are based on neph and nephrin proteins and comparable tothe slit diaphragm cell junctions of podocytes (75–77).Loss of one of the nephrin or neph homologues in this systemdisrupts the cellular program required for myoblast fusion.Intriguing is that in vivo mapping of the functional proteindomains required for myoblast fusion revealed that the intracellulardomain mediates their activity (77), suggesting that intactsignal transduction is required for the proper function of theseproteins.
In agreement with this concept, syg-1, the C. elegans homologueof neph3, has been shown to be required as a guidepost signalin neuronal synapse formation in C. elegans (78), a processthat also requires tightly regulated signal transduction events.Thus, from these data, the concept is emerging that membersof the neph and nephrin protein families form specialized celljunctions that are involved in pathfinding, orientation, formationof prefusion complexes, and the intimate organization of cellextensions in various cells and organisms, including the mammaliankidney. All of these programs are strictly dependent on intracellularsignaling networks.
Hanging on a Scaffold: The Role of Actin Cytoskeleton and PDZ Domain Proteins in the Organization of Signaling Proteins at the Filtration Slit
Although many of the key proteins of the slit diaphragm havenow been identified, the fundamental question remains how thedifferent proteins are organized at this specialized cell junction.Given the fluid nature of plasma membranes, restriction of podocyteproteins to the slit diaphragm protein complex at the base ofthe filtration slit requires anchoring mechanisms. Obviously,this restriction is a critical prerequisite for the maintenanceof a sieve as well as efficient signal transduction at the filtrationslit. In addition to the function of podocin, which recruitsnephrin into specialized lipid raft microdomains of the plasmamembrane at the slit diaphragm, two crucial mechanisms seemto be important for the assembly of slit diaphragm components.First, it has been shown that slit diaphragm proteins are associatedwith the cortical actin cytoskeleton in the podocyte in vitroand in vivo (79–81). This interaction seems at least inpart to be mediated by CD2AP (82). Each foot process is equippedwith a microfilament-based contractile apparatus composed ofactin, myosin-II, -actinin, talin, paxillin, and vinculin (83–85).The slit diaphragm protein complex is linked to the actin cytoskeletonand seems to modify actin dynamics. CD2AP has been shown toassociate with WASp, the Arp2/3 complex, the actin-capping proteinCAPZ, and cortactin, proteins involved in actin filament assembly(86–88). Actin fiber formation is a highly dynamic processthat is governed by continuous assembly and disruption of filaments.It is well known that the dynamics of the submembranous actinmeshwork of podocytes is profoundly changed in effaced footprocesses (1). The current belief is that loss of the organizedstructure of the actin cytoskeleton in podocytes is a prerequisitefor podocyte foot process effacement and proteinuria. Thesefindings assign a central role to the submembranous actin cytoskeletonfor maintaining the stability of the podocyte’s shapeand the integrity of the slit diaphragm protein complex. Moreover,it seems that loss of the integrity of the slit diaphragm complexhas a dramatic effect on the actin cytoskeleton leading to footprocess effacement and proteinuria (1). Work by the Shimizu,Salant, and Chugh groups showed that injection of antisera directedagainst the extracellular domains of nephrin or neph1 resultsin rapidly developing proteinuria, disorganization of the actincytoskeleton, and foot process effacement (70,89–91).The critical importance of an intact submembranous actin skeletonis highlighted by the fact that mutations in the actin-bundlingprotein -actinin-4 have been detected in an autosomal dominanthereditary form of focal segmental glomerulosclerosis (92).Patients with mutations in the ACTN4 gene as well as mice withtargeted disruption of ACTN4 or transgenic overexpression ofa mutated form of -actinin-4 display effaced podocyte foot processesand a steroid-resistant form of focal segmental glomerulosclerosis(93,94). Although a critical role of signaling at the slit diaphragmfor active actin remodeling and the maintenance of the podocytestructure is highly conceivable, little is known about the effectorsof slit diaphragm proteins involved in transmitting these signalsto the actin meshwork (95,96). It is interesting that -actinin-4has been shown to bind to densin-180 in the postsynaptic densityin neurons (97). Densin-180 has recently been localized to theslit diaphragm of podocytes by Holthöfer et al. (98) andmay provide one important link of slit diaphragm proteins tothe actin cytoskeleton.
The second anchoring mechanism that restricts podocyte proteinsto the slit diaphragm protein complex is binding to scaffoldingproteins. The first of these scaffolding proteins localizedto the cytoplasmic face of the filtration slit by Farquhar andcolleagues (42,99) was a protein called zonula occludens 1 (ZO-1).ZO proteins are membrane-associated multidomain proteins thatare usually localized at sites of intercellular junctions. Theycontain several protein interaction modules, namely three PDZdomains, a Src homology 3 (SH3) domain, and a guanylate kinase(GUK) domain (100). PDZ domains are protein-binding modulesthat recognize short peptide motifs within their protein targets(101). In most cases, the last three to five residues at theextreme carboxy terminus of a transmembrane protein representthe target sequence. Genetic evidence from invertebrate systemsdemonstrates a role for ZO proteins in facilitating signal transduction,and evidence from vertebrate systems demonstrates a structuralrole in organizing transmembrane protein complexes (102,103).In a recently published functional study, it was shown thatZO-1 directly binds to the cytoplasmic tail of neph proteins(37). This interaction was mediated by the first PDZ domainof ZO-1 and the last three amino acids of the carboxy terminaldomain of neph proteins and induced a dramatic increase in tyrosinephosphorylation of the cytoplasmic tail of neph1. Thus, in additionto clustering neph proteins, ZO-1 alters the phosphorylationstate of Neph1 and its ability to induce signal transduction.It has been suggested that ZO-1 in addition to its signalingfunction interacts with the actin cytoskeleton and componentsof the paracellular seal (104). Although the functional implicationsof ZO-1/actin association have not yet been established in vivo,ZO-1 could link Neph1 and its associated binding proteins tothe actin cytoskeleton and contribute to the organization ofthe foot processes of podocytes. It is interesting to note thatthe interaction of the cytoplasmic tail of neph1 with PDZ domainproteins seems to be regulated by phosphorylation of a criticalthreonine residue (C. Reinhardt, T.B. Huber, T. Benzing, unpublisheddata), which suggests that the specificity for PDZ domain bindingat the slit diaphragm is regulated by signaling events.
How can we put all of this together? In this article, I havesummarized data suggesting that signaling at the slit diaphragmis critical for the regulation of podocyte function, maintenanceof foot process structure, and possibly podocyte survival (Figure 3).Although many aspects of this concept have yet to be addressedin various animal models, recent studies clearly indicate thatnephrin and neph proteins are signaling proteins. However, theregulation of dynamics and kinetics of signaling is completelyunclear. The recent generation of mouse strains that allow forpodocyte-specific gene targeting will greatly aid the studyof signaling in vivo (105–107).
It is reasonable to speculate that the slit diaphragm proteincomplex is highly dynamic. Protein components of the complexare endocytosed, recycled, and returned back to the cell surface.In this regard, it is conceivable that nephrin or neph1 moleculesthat enter the complex may be dephosphorylated and inactive.Interaction with their respective ligand at the slit diaphragmmay then induce tyrosine phosphorylation and initiation of signaling.This could be a way of sensing repair and turnover of slit diaphragmcomponents. In conjunction with ion channels, this system couldalso serve to monitor pressure at the filtration slit (108,109).It is interesting to note that the closest homologue of podocinin C. elegans, a stomatin family protein called MEC-2, is involvedin mechanosensation in the worm (110). Thus, it is temptingto speculate that podocin has similar functions in the mammaliankidney.
An interesting question is whether podocyte foot processes areable to regulate the width of the filtration slit, slit diaphragmpermeability, and glomerular ultrafiltration. Although experimentaldata are almost completely lacking, it seems that this couldbe the case. The slit diaphragm is a robust structure with afairly constant width. However, it is increasingly appreciatedthat the width may vary and that the slit diaphragm has to bepartially elastic and highly dynamic in nature (3,111). In additionto acting as a molecular sieve, it has been suggested that theslit diaphragm may impose substantial resistance to liquid flowacross the glomerulus (112). Thus, fine tuning of the widthof the slit diaphragm may have an impact on the hydraulic resistanceand thus contribute to the regulation of glomerular ultrafiltrationand permeability of the filter.
It is clear that the slit diaphragm is not the only membranedomain where signaling takes place in the podocyte. Recent experimentalevidence suggests a critical role for integrin signaling atthe sole of the podocyte foot process (113), for growth factorssuch as vascular endothelial growth factor (114) or basic fibroblastgrowth factor (115), protein tyrosine phosphatases (116), andchemokines (117,118) in podocyte development and glomerularpathophysiology.
However, it is clearly more and more appreciated that (1) theslit diaphragm protein complex does not only serve as staticmolecular sieve but rather is a highly dynamic functional proteincomplex and (2) signaling at the slit diaphragm may be criticalfor maintaining the integrity of the podocyte architecture andthe function of the glomerular filter of the kidney. A betterunderstanding of the mechanisms involved in signal transductionat the slit diaphragm will definitively provide valuable insightsinto important aspects of glomerular diseases in general. Progressin research on podocyte biology and signaling will greatly helpto improve our understanding of the pathophysiology not onlyof inherited but also of the more common acquired diseases,namely minimal change disease, primary focal segmental glomerulosclerosis,and membranous or diabetic nephropathy. From this perspective,at some point patients will benefit from the progress that hasbeen made in the exciting field of podocyte cell biology andsignal transduction.
Acknowledgments
Our work is supported by the Deutsche Forschungsgemeinschaft(BE 2212).
I thank members of my laboratory for helpful discussions andTobias B. Huber and many others for great experimental work.Thanks to Wolfgang Kühn and Gerd Walz for critically readingthe manuscript and to Jörg Benzing for help with the artwork.I apologize to those whose work is not described here becauseof space limitations.
Miner JH: A molecular look at the glomerular barrier. Nephron Exp Nephrol 94: e119–e122, 2003[CrossRef][Medline]
Brenner BM, Hostetter TH, Humes HD: Molecular basis of proteinuria of glomerular origin. N Engl J Med 298: 826–833, 1978[Medline]
Brenner BM, Hostetter TH, Humes HD: Glomerular permselectivity: Barrier function based on discrimination of molecular size and charge. Am J Physiol 234: F455–F460, 1978
Rennke HG, Venkatachalam MA: Glomerular permeability of macromolecules. Effect of molecular configuration on the fractional clearance of uncharged dextran and neutral horseradish peroxidase in the rat. J Clin Invest 63: 713–717, 1979
Antignac C: Genetic models: Clues for understanding the pathogenesis of idiopathic nephrotic syndrome. J Clin Invest 109: 447–449, 2002[CrossRef][Medline]
Tryggvason K, Ruotsalainen V, Wartiovaara J: Discovery of the congenital nephrotic syndrome gene discloses the structure of the mysterious molecular sieve of the kidney. Int J Dev Biol 43: 445–451, 1999[Medline]
Mundel P, Shankland SJ: Podocyte biology and response to injury. J Am Soc Nephrol 13: 3005–3015, 2002[Free Full Text]
Kriz W, Lemley KV: The role of the podocyte in glomerulosclerosis. Curr Opin Nephrol Hypertens 8: 489–497, 1999[CrossRef][Medline]
Kreidberg JA: Podocyte differentiation and glomerulogenesis. J Am Soc Nephrol 14: 806–814, 2003[Free Full Text]
Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003[Abstract/Free Full Text]
Rodewald R, Karnovsky MJ: Porous substructure of the glomerular slit diaphragm in the rat and mouse. J Cell Biol 60: 423–433, 1974[Abstract/Free Full Text]
Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome. Mol Cell 1: 575–582, 1998[CrossRef][Medline]
Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestila M, Jalanko H, Holmberg C, Tryggvason K: Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci U S A 96: 7962–7967, 1999[Abstract/Free Full Text]
Holthofer H, Ahola H, Solin ML, Wang S, Palmen T, Luimula P, Miettinen A, Kerjaschki D: Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney. Am J Pathol 155: 1681–1687, 1999[Abstract/Free Full Text]
Holzman LB, St John PL, Kovari IA, Verma R, Holthofer H, Abrahamson DR: Nephrin localizes to the slit pore of the glomerular epithelial cell. Kidney Int 56: 1481–1491, 1999[CrossRef][Medline]
Tryggvason K: Unraveling the mechanisms of glomerular ultrafiltration: Nephrin, a key component of the slit diaphragm. J Am Soc Nephrol 10: 2440–2445, 1999[Free Full Text]
Holmberg C, Antikainen M, Ronnholm K, Ala Houhala M, Jalanko H: Management of congenital nephrotic syndrome of the Finnish type. Pediatr Nephrol 9: 87–93, 1995[CrossRef][Medline]
Putaala H, Soininen R, Kilpelainen P, Wartiovaara J, Tryggvason K: The murine nephrin gene is specifically expressed in kidney, brain and pancreas: Inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet 10: 1–8, 2001[Abstract/Free Full Text]
Shih NY, Li J, Karpitskii V, Nguyen A, Dustin ML, Kanagawa O, Miner JH, Shaw AS: Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 286: 312–315, 1999[Abstract/Free Full Text]
Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, Antignac C: NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24: 349–354, 2000[CrossRef][Medline]
Donoviel DB, Freed DD, Vogel H, Potter DG, Hawkins E, Barrish JP, Mathur BN, Turner CA, Geske R, Montgomery CA, Starbuck M, Brandt M, Gupta A, Ramirez-Solis R, Zambrowicz BP, Powell DR: Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN. Mol Cell Biol 21: 4829–4836, 2001[Abstract/Free Full Text]
Ciani L, Patel A, Allen ND, Ffrench-Constant C: Mice lacking the giant protocadherin mFAT1 exhibit renal slit junction abnormalities and a partially penetrant cyclopia and anophthalmia phenotype. Mol Cell Biol 23: 3575–3582, 2003[Abstract/Free Full Text]
Roselli S, Gribouval O, Boute N, Sich M, Benessy F, Attie T, Gubler MC, Antignac C: Podocin localizes in the kidney to the slit diaphragm area. Am J Pathol 160: 131–139, 2002[Abstract/Free Full Text]
Huber TB, Kottgen M, Schilling B, Walz G, Benzing T: Interaction with podocin facilitates nephrin signaling. J Biol Chem 276: 41543–41546, 2001[Abstract/Free Full Text]
Schwarz K, Simons M, Reiser J, Saleem MA, Faul C, Kriz W, Shaw AS, Holzman LB, Mundel P: Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 108: 1621–1629, 2001[CrossRef][Medline]
Roselli S, Heidet L, Sich M, Henger A, Kretzler M, Gubler MC, Antignac C: Early glomerular filtration defect and severe renal disease in podocin-deficient mice. Mol Cell Biol 24: 550–560, 2004[Abstract/Free Full Text]
Dustin ML, Olszowy MW, Holdorf AD, Li J, Bromley S, Desai N, Widder P, Rosenberger F, van der Merwe PA, Allen PM, Shaw AS: A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94: 667–677, 1998[CrossRef][Medline]
Shih NY, Li J, Cotran R, Mundel P, Miner JH, Shaw AS: CD2AP localizes to the slit diaphragm and binds to nephrin via a novel C-terminal domain. Am J Pathol 159: 2303–2308, 2001[Abstract/Free Full Text]
Kim JM, Wu H, Green G, Winkler CA, Kopp JB, Miner JH, Unanue ER, Shaw AS: CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science 300: 1298–1300, 2003[Abstract/Free Full Text]
Wolf G, Stahl RA: CD2-associated protein and glomerular disease. Lancet 362: 1746–1748, 2003[CrossRef][Medline]
Sellin L, Huber TB, Gerke P, Quack I, Pavenstadt H, Walz G: NEPH1 defines a novel family of podocin interacting proteins. FASEB J 17: 115–117, 2003[Abstract/Free Full Text]
Inoue T, Yaoita E, Kurihara H, Shimizu F, Sakai T, Kobayashi T, Ohshiro K, Kawachi H, Okada H, Suzuki H, Kihara I, Yamamoto T: FAT is a component of glomerular slit diaphragms. Kidney Int 59: 1003–1012, 2001[CrossRef][Medline]
Huber TB, Hartleben B, Kim J, Schmidts M, Schermer B, Keil A, Egger L, Lecha RL, Borner C, Pavenstadt H, Shaw AS, Walz G, Benzing T: Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol 23: 4917–4928, 2003[Abstract/Free Full Text]
Huber TB, Schmidts M, Gerke P, Schermer B, Zahn A, Hartleben B, Sellin L, Walz G, Benzing T: The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1. J Biol Chem 278: 13417–13421, 2003[Abstract/Free Full Text]
Huber TB, Simons M, Hartleben B, Sernetz L, Schmidts M, Gundlach E, Saleem MA, Walz G, Benzing T: Molecular basis of the functional podocin-nephrin complex: Mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains. Hum Mol Genet 12: 3397–3405, 2003[Abstract/Free Full Text]
Kurihara H, Anderson JM, Farquhar MG: Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes. Am J Physiol 268: F514–F524, 1995
Verma R, Wharram B, Kovari I, Kunkel R, Nihalani D, Wary KK, Wiggins RC, Killen P, Holzman LB: Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin. J Biol Chem 278: 20716–20723, 2003[Abstract/Free Full Text]
Simons M, Schwarz K, Kriz W, Miettinen A, Reiser J, Mundel P, Holthofer H: Involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm. Am J Pathol 159: 1069–1077, 2001[Abstract/Free Full Text]
Lahdenpera J, Kilpelainen P, Liu XL, Pikkarainen T, Reponen P, Ruotsalainen V, Tryggvason K: Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases. Kidney Int 64: 404–413, 2003[CrossRef][Medline]
Yu CC, Yen TS, Lowell CA, DeFranco AL: Lupus-like kidney disease in mice deficient in the Src family tyrosine kinases Lyn and Fyn. Curr Biol 11: 34–38, 2001[CrossRef][Medline]
Pawson T: Protein modules and signalling networks. Nature 373: 573–580, 1995[CrossRef][Medline]
Cantley LC: The phosphoinositide 3-kinase pathway. Science 296: 1655–1657, 2002[Abstract/Free Full Text]
Downward J: Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol 10: 262–267, 1998[CrossRef][Medline]
Kriz W, Lemley KV: The role of the podocyte in glomerulosclerosis. Curr Opin Nephrol Hypertens 8: 489–497, 1999
Rennke HG: How does glomerular epithelial cell injury contribute to progressive glomerular damage? Kidney Int Suppl 45: S58–S63, 1994[Medline]
Kim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, Holzman L, Kershaw D, Wiggins R: Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int 60: 957–968, 2001[CrossRef][Medline]
Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, Bottinger EP: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 108: 807–816, 2001[CrossRef][Medline]
Elger M, Kriz W: Podocytes and the development of segmental glomerulosclerosis. Nephrol Dial Transplant 13: 1368–1373, 1998[Free Full Text]
Fries JW, Sandstrom DJ, Meyer TW, Rennke HG: Glomerular hypertrophy and epithelial cell injury modulate progressive glomerulosclerosis in the rat. Lab Invest 60: 205–218, 1989[Medline]
Kriz W, Gretz N, Lemley KV: Progression of glomerular diseases: Is the podocyte the culprit? Kidney Int 54: 687–697, 1998[CrossRef][Medline]
Li X, Talts U, Talts JF, Arman E, Ekblom P, Lonai P: Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane. Proc Natl Acad Sci U S A 98: 14416–14421, 2001[Abstract/Free Full Text]
Mawal-Dewan M, Lorenzini A, Frisoni L, Zhang H, Cristofalo VJ, Sell C: Regulation of collagenase expression during replicative senescence in human fibroblasts by Akt-forkhead signaling. J Biol Chem 277: 7857–7864, 2002[Abstract/Free Full Text]
Koziell A, Grech V, Hussain S, Lee G, Lenkkeri U, Tryggvason K, Scambler P: Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration. Hum Mol Genet 11: 379–388, 2002[Abstract/Free Full Text]
Sorensson J, Fierlbeck W, Heider T, Schwarz K, Park DS, Mundel P, Lisanti M, Ballermann BJ: Glomerular endothelial fenestrae in vivo are not formed from caveolae. J Am Soc Nephrol 13: 2639–2647, 2002[Abstract/Free Full Text]
Simons K, Toomre D: Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1: 31–39, 2000[CrossRef][Medline]
Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, Lisanti MP: Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol 19: 7289–7304, 1999[Free Full Text]
Kurzchalia TV, Parton RG: Membrane microdomains and caveolae. Curr Opin Cell Biol 11: 424–431, 1999[CrossRef][Medline]
Gerke P, Huber TB, Sellin L, Benzing T, Walz G: Homodimerization and heterodimerization of the glomerular podocyte proteins nephrin and NEPH1. J Am Soc Nephrol 14: 918–926, 2003[Abstract/Free Full Text]
Khoshnoodi J, Sigmundsson K, Ofverstedt LG, Skoglund U, Obrink B, Wartiovaara J, Tryggvason K: Nephrin promotes cell-cell adhesion through homophilic interactions. Am J Pathol 163: 2337–2346, 2003[Abstract/Free Full Text]
Barletta GM, Kovari IA, Verma RK, Kerjaschki D, Holzman LB: Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers. J Biol Chem 278: 19266–19271, 2003[Abstract/Free Full Text]
Liu G, Kaw B, Kurfis J, Rahmanuddin S, Kanwar YS, Chugh SS: Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability. J Clin Invest 112: 209–221, 2003[CrossRef][Medline]
Ihalmo P, Palmen T, Ahola H, Valtonen E, Holthofer H: Filtrin is a novel member of nephrin-like proteins. Biochem Biophys Res Commun 300: 364–370, 2003[CrossRef][Medline]
Dworak HA, Charles MA, Pellerano LB, Sink H: Characterization of Drosophila hibris, a gene related to human nephrin. Development 128: 4265–4276, 2001[Abstract/Free Full Text]
Strunkelnberg M, Bonengel B, Moda LM, Hertenstein A, de Couet HG, Ramos RG, Fischbach KF: rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila. Development 128: 4229–4239, 2001[Abstract/Free Full Text]
Ramos RG, Igloi GL, Lichte B, Baumann U, Maier D, Schneider T, Brandstatter JH, Frohlich A, Fischbach KF: The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein. Genes Dev 7: 2533–2547, 1993[Abstract/Free Full Text]
Ruiz-Gomez M, Coutts N, Price A, Taylor MV, Bate M: Drosophila dumbfounded: A myoblast attractant essential for fusion. Cell 102: 189–198, 2000[CrossRef][Medline]
Bour BA, Chakravarti M, West JM, Abmayr SM: Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev 14: 1498–1511, 2000[Abstract/Free Full Text]
Artero RD, Castanon I, Baylies MK: The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling. Development 128: 4251–4264, 2001[Abstract/Free Full Text]
Shen K, Bargmann CI: The immunoglobulin superfamily protein SYG-1 determines the location of specific synapses in C. elegans. Cell 112: 619–630, 2003[CrossRef][Medline]
Yuan H, Takeuchi E, Salant DJ: Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton. Am J Physiol Renal Physiol 282: F585–F591, 2002[Abstract/Free Full Text]
Saleem MA, Ni L, Witherden I, Tryggvason K, Ruotsalainen V, Mundel P, Mathieson PW: Co-localization of nephrin, podocin, and the actin cytoskeleton: Evidence for a role in podocyte foot process formation. Am J Pathol 161: 1459–1466, 2002[Abstract/Free Full Text]
Welsch T, Endlich N, Kriz W, Endlich K: CD2AP and p130Cas localize to different F-actin structures in podocytes. Am J Physiol Renal Physiol 281: F769–F777, 2001[Abstract/Free Full Text]
Lehtonen S, Zhao F, Lehtonen E: CD2-associated protein directly interacts with the actin cytoskeleton. Am J Physiol Renal Physiol 283: F734–F743, 2002[Abstract/Free Full Text]
Ichimura K, Kurihara H, Sakai T: Actin filament organization of foot processes in rat podocytes. J Histochem Cytochem 51: 1589–1600, 2003[Abstract/Free Full Text]
Kobayashi N: Mechanism of the process formation; podocytes vs. neurons. Microsc Res Tech 57: 217–223, 2002[CrossRef][Medline]
Drenckhahn D, Franke RP: Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man. Lab Invest 59: 673–682, 1988[Medline]
Hutchings NJ, Clarkson N, Chalkley R, Barclay AN, Brown MH: Linking the T cell surface protein CD2 to the actin-capping protein CAPZ via CMS and CIN85. J Biol Chem 278: 22396–22403, 2003[Abstract/Free Full Text]
Lynch DK, Winata SC, Lyons RJ, Hughes WE, Lehrbach GM, Wasinger V, Corthals G, Cordwell S, Daly RJ: A Cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton. J Biol Chem 278: 21805–21813, 2003[Abstract/Free Full Text]
Badour K, Zhang J, Shi F, McGavin MK, Rampersad V, Hardy LA, Field D, Siminovitch KA: The Wiskott-Aldrich syndrome protein acts downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote formation of the immunological synapse. Immunity 18: 141–154, 2003[CrossRef][Medline]
Narisawa M, Kawachi H, Oite T, Shimizu F: Divalency of the monoclonal antibody 5-1-6 is required for induction of proteinuria in rats. Clin Exp Immunol 92: 522–526, 1993[Medline]
Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shia M, Shimizu F, Salant DJ: Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin. J Clin Invest 104: 1559–1566, 1999[Medline]
Kawachi H, Abrahamson DR, St John PL, Goldstein DJ, Shia MA, Matsui K, Shimizu F, Salant DJ: Developmental expression of the nephritogenic antigen of monoclonal antibody 5-1-6. Am J Pathol 147: 823–833, 1995[Abstract]
Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ, Mathis BJ, Rodriguez-Perez JC, Allen PG, Beggs AH, Pollak MR: Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 24: 251–256, 2000[CrossRef][Medline]
Michaud JL, Lemieux LI, Dube M, Vanderhyden BC, Robertson SJ, Kennedy CR: Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4. J Am Soc Nephrol 14: 1200–1211, 2003[Abstract/Free Full Text]
Kos CH, Le TC, Sinha S, Henderson JM, Kim SH, Sugimoto H, Kalluri R, Gerszten RE, Pollak MR: Mice deficient in alpha-actinin-4 have severe glomerular disease. J Clin Invest 111: 1683–1690, 2003[CrossRef][Medline]
Yuan H, Takeuchi E, Taylor GA, McLaughlin M, Brown D, Salant DJ: Nephrin dissociates from actin, and its expression is reduced in early experimental membranous nephropathy. J Am Soc Nephrol 13: 946–956, 2002[Abstract/Free Full Text]
Saran AM, Yuan H, Takeuchi E, McLaughlin M, Salant DJ: Complement mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy. Kidney Int 64: 2072–2078, 2003[CrossRef][Medline]
Walikonis RS, Oguni A, Khorosheva EM, Jeng CJ, Asuncion FJ, Kennedy MB: Densin-180 forms a ternary complex with the (alpha)-subunit of Ca2+/calmodulin-dependent protein kinase II and (alpha)-actinin. J Neurosci 21: 423–433, 2001[Abstract/Free Full Text]
Ahola H, Heikkila E, Astrom E, Inagaki M, Izawa I, Pavenstadt H, Kerjaschki D, Holthofer H: A novel protein, densin, expressed by glomerular podocytes. J Am Soc Nephrol 14: 1731–1737, 2003[Abstract/Free Full Text]
Schnabel E, Anderson JM, Farquhar MG: The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium. J Cell Biol 111: 1255–1263, 1990[Abstract/Free Full Text]
Hung AY, Sheng M: PDZ domains: Structural modules for protein complex assembly. J Biol Chem 277: 5699–5702, 2002[Free Full Text]
Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC: Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275: 73–77, 1997[Abstract/Free Full Text]
Mitic LL, Van Itallie CM, Anderson JM: Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. Am J Physiol Gastrointest Liver Physiol 279: G250–G254, 2000[Abstract/Free Full Text]
Fanning AS, Anderson JM: PDZ domains: Fundamental building blocks in the organization of protein complexes at the plasma membrane. J Clin Invest 103: 767–772, 1999[Medline]
Fanning AS, Ma TY, Anderson JM: Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1. FASEB J 16: 1835–1837, 2002[Abstract/Free Full Text]
Eremina V, Wong MA, Cui S, Schwartz L, Quaggin SE: Glomerular-specific gene excision in vivo. J Am Soc Nephrol 13: 788–793, 2002[Abstract/Free Full Text]
Moeller MJ, Kovari IA, Holzman LB: Evaluation of a new tool for exploring podocyte biology: Mouse nphs1 5' flanking region drives LacZ expression in podocytes. J Am Soc Nephrol 11: 2306–2314, 2000[Abstract/Free Full Text]
Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB: Two gene fragments that direct podocyte-specific expression in transgenic mice. J Am Soc Nephrol 13: 1561–1567, 2002[Abstract/Free Full Text]
Goodman MB, Schwarz EM: Transducing touch in Caenorhabditis elegans. Annu Rev Physiol 65: 429–452, 2003[CrossRef][Medline]
Goodman MB, Ernstrom GG, Chelur DS, O’Hagan R, Yao CA, Chalfie M: MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation. Nature 415: 1039–1042, 2002[CrossRef][Medline]
Kriz W, Kretzler M, Provoost AP, Shirato I: Stability and leakiness: Opposing challenges to the glomerulus. Kidney Int 49: 1570–1574, 1996[Medline]
Smithies O: Why the kidney glomerulus does not clog: A gel permeation/diffusion hypothesis of renal function. Proc Natl Acad Sci U S A 100: 4108–4113, 2003[Abstract/Free Full Text]
Kreidberg JA, Donovan MJ, Goldstein SL, Rennke H, Shepherd K, Jones RC, Jaenisch R: Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development 122: 3537–3547, 1996[Abstract]
Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, Quaggin SE: Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 111: 707–716, 2003[CrossRef][Medline]
Floege J, Kriz W, Schulze M, Susani M, Kerjaschki D, Mooney A, Couser WG, Koch KM: Basic fibroblast growth factor augments podocyte injury and induces glomerulosclerosis in rats with experimental membranous nephropathy. J Clin Invest 96: 2809–2819, 1995
Wharram BL, Goyal M, Gillespie PJ, Wiggins JE, Kershaw DB, Holzman LB, Dysko RC, Saunders TL, Samuelson LC, Wiggins RC: Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate. J Clin Invest 106: 1281–1290, 2000[Medline]
Banas B, Wornle M, Berger T, Nelson PJ, Cohen CD, Kretzler M, Pfirstinger J, Mack M, Lipp M, Grone HJ, Schlondorff D: Roles of SLC/CCL21 and CCR7 in human kidney for mesangial proliferation, migration, apoptosis, and tissue homeostasis. J Immunol 168: 4301–4307, 2002[Abstract/Free Full Text]
Huber TB, Reinhardt HC, Exner M, Burger JA, Kerjaschki D, Saleem MA, Pavenstadt H: Expression of functional CCR and CXCR chemokine receptors in podocytes. J Immunol 168: 6244–6252, 2002[Abstract/Free Full Text]
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[Abstract][Full Text]
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Introduction
Proof of Principle: Nephrin...
40-nm-wide filtration slit and are connected by a continuous membrane-like structure called the slit diaphragm. When podocytes are injured, the intercellular junctions and cytoskeletal structure of the foot processes are altered and the cell takes on an "effaced" phenotype. Typical slit diaphragm structures disappear and proteinuria develops (10). Because there is no case of nephrotic syndrome without major changes in podocyte morphology, it has been speculated for many years that the slit diaphragm is an important component of the glomerular filter (14). 
-actinin, talin, paxillin, and vinculin (83–85). The slit diaphragm protein complex is linked to the actin cytoskeleton and seems to modify actin dynamics. CD2AP has been shown to associate with WASp, the Arp2/3 complex, the actin-capping protein CAPZ, and cortactin, proteins involved in actin filament assembly (86–88). Actin fiber formation is a highly dynamic process that is governed by continuous assembly and disruption of filaments. It is well known that the dynamics of the submembranous actin meshwork of podocytes is profoundly changed in effaced foot processes (1). The current belief is that loss of the organized structure of the actin cytoskeleton in podocytes is a prerequisite for podocyte foot process effacement and proteinuria. These findings assign a central role to the submembranous actin cytoskeleton for maintaining the stability of the podocyte’s shape and the integrity of the slit diaphragm protein complex. Moreover, it seems that loss of the integrity of the slit diaphragm complex has a dramatic effect on the actin cytoskeleton leading to foot process effacement and proteinuria (1). Work by the Shimizu, Salant, and Chugh groups showed that injection of antisera directed against the extracellular domains of nephrin or neph1 results in rapidly developing proteinuria, disorganization of the actin cytoskeleton, and foot process effacement (70,89–91). The critical importance of an intact submembranous actin skeleton is highlighted by the fact that mutations in the actin-bundling protein 
