Nectins and nectin-like molecules (Necls) are structurally related transmembrane proteins primarily involved in cell adhesion. Nectins and afadin, the adaptor or anchoring protein, stabilize the epithelium and endothelium and establish apical-basal polarity of epithelial cells, independently or in cooperation with other cell adhesion molecules. Necls facilitate cell–cell communication implicated in cell movement and proliferation, immune responses, and cancer cell phenotypes. Necls interact with nectins and specific ligands at cell–cell contacts, whereas Necls associate with integrin αvβ3 and growth factor receptors on the same cell surface. Besides their roles in cell adhesion, nectins regulate the activities of Rho family small G proteins which play critical roles in maintaining the apical junctions of epithelial cells through reorganization of the actin cytoskeleton. Since mice lacking the Rho GDP-dissociation inhibitor (GDI)α show massive proteinuria and degeneration of renal epithelial cells, nectins and other cell adhesion molecules may play roles in the structural and functional aspects of renal diseases. Here we summarize our knowledge of nectins and Necls and discuss cell adhesion biology in the kidney.

In multicellular organisms, cell adhesion plays important roles in various cellular processes including morphogenesis, differentiation, proliferation, and migration [1, 2]. The nectin and nectin-like molecule (Necl) superfamily has recently emerged as an immunoglobulin (Ig)-like family that participates in calcium-independent cell adhesion [3]. They function as components of the epithelial junctional complexes, receptors for virion entry, tumor suppressors, and molecules involved in immune reactions. Based on their biochemical similarities, we have proposed the nectin and Necl families, with four and five members, respectively, in order to promote comprehensive understandings of these molecules and provide insights into their biology and pathology [3].

The apical junctions, namely tight junctions and adherens junctions, are sites of mechanical cell attachment regulated by dynamic changes in the actin cytoskeleton. Particularly, adherens junctions have prototypic roles for stabilizing the epithelium and establishing apical-basal polarity of epithelial cells. These functions have been elucidated by studies on nectins and cadherins that are two major cell adhesion molecules at adherens junctions. Nectin-based cell adhesion and cadherin-based cell adhesion function both individually and in combination to establish and maintain the mature apical junctions. Components clustered at the junctional apparatus possess signaling capabilities that provide the means to integrate changes in morphology and gene expression during tissue and organ development [4]. Rho family small G proteins are important signal transducers downstream of nectins and cadherins, and regulate epithelial and endothelial barrier functions through regulation of both the actin cytoskeletal organization and the integrity of intercellular junctions [5,6,7,8,9]. Cell contact and subsequent signaling are thus coupled at the apical junctions, representing the structural and functional aspects involved in the morphogenesis of multicellular organisms.

Specialized structures composed of endothelial cells, podocytes, and renal tubular cells play critical roles in glomerular and tubular functions. Genetic and biochemical studies have made important contributions to our knowledge of normal glomerular filtration and the mechanisms of proteinuria. Dysregulation of podocyte proteins is thought to play a key role in the development of the nephropathy since studies on hereditary proteinuria syndromes have led to the identification of proteins involved in the development and function of the glomerular filtration barrier [10]. Some components of the podocyte slit diaphragm are reportedly defective in humans: Nephrins, Neph-1 and Neph-2, are transmembrane proteins with extracellular IgG-like motifs, FAT-1 and FAT-2 are transmembrane proteins with extracellular cadherin-like motifs, and podocin is an integral membrane protein with its N- and C-terminals located intracellularly. These proteins are thus thought to form a complex that contributes to the structure of the slit diaphragm, connects the diaphragm to the intracellular actin cytoskeleton, and participates in signaling related to turnover of the glomerular filter. However, physiological regulation of their functions and pathological mechanism in causing renal disorders remain largely unknown because of the lack of suitable experimental conditions.

Studies on signalings initiated by the engagement of nectins and cadherins provide some insights into the formation of the kidney-specific apparatus and maintenance or turnover of renal cell adhesion. Protein phosphorylation mediated by c-Src and activation of Rho family small G proteins are supposed to be involved in normal kidney functions through remodeling of the actin cytoskeleton. Consistently, mice lacking Rho GDIα show prominent proteinuria and progressive degeneration of renal epithelial cells. Here we review recent studies on the nectin and Necl systems and discuss the roles of the apical junctions in glomerular filtration and tubular function.

All of the nectins have an extracellular region of three Ig-like domains, a transmembrane region, and a cytoplasmic tail region (fig. 1). The extracellular domain of nectins first binds to form cis-dimers on the surface of the same cells, and then promotes cell–cell contacts by forming homophilic or heterophilic trans-dimers in a calcium-independent manner. Heterophilic interactions have been detected between nectin-2 and nectin-3, between nectin-1 and nectin-3, and also between nectin-1 and nectin-4 (fig. 1). Importantly, heterophilic trans-dimers form stronger cell–cell attachment than homophilic trans-dimers [11, 12], which actually determines the type of cell adhesion. Their cytoplasmic domains directly interact with the actin-binding protein afadin [13, 14] and the cell polarity protein Par-3, and indirectly interact with annexin II [15, 16], IQGAP1 [17, 18], and other actin-binding proteins. Namely, nectins play a dual role in promoting adhesion between homotypic cells and between heterotypic cells in contrast to cadherins that exclusively promote adhesion between homotypic cells.

Necls are characterized by possessing domain structures similar to those of nectins, but lack the ability to interact with afadin (fig. 1) [3, 19]. The first Ig-like domain plays an essential role in calcium-independent homophilic and heterophilic trans-interactions [20]. Each member of Necls, except for Necl-5, forms homophilic trans-dimers whereas all members form heterophilic trans-dimers with other Necl members. They also show heterophilic interaction with nectins, specific ligands, and viruses. These possible combinations of their interactions are shown (fig. 1) [8, 9]. For example, Necl-1 shows homophilic and heterophilic cell–cell adhesion activity with Necl-2, nectin-1, and nectin-3, but not with Necl-5 or nectin-2 [21]. On the other hand, Necl-5 forms homophilic cis- dimers but not homophilic trans- dimers. Necl-5-based cell adhesions are not stable but easily reconstructed to the binding between Necl-5 and nectin-3 and subsequently converted to that between nectin-1 and nectin-3, suggesting the role in the quick fixing of cell contact that is programmed to achieve the remodeling process. Although Necl-5 is grouped with Necls because of the lack of ability to bind afadin [19, 22], Necl-5 is phylogenetically closer to the nectin family than the Necl family [19, 23], and could be a subfamily distinct from nectins and Necls. Thus Necls form a family with more versatile functions than nectins.

Nectins are expressed ubiquitously in epithelial cells of various organs including the kidney, where they localize to adherens junctions and are thought to play a regulatory role in cadherin-based adherens junction formation [13, 24, 25]. In contrast, Necls are expressed in a variety of cells where they localize to the leading edges of moving cells or the basolateral plasma membrane in polarized cells [19, 26]. Therefore, Necls appear to play physiological roles except for the formation of adherens junctions, although both nectins and Necls mediate homotypic as well as heterotypic cell–cell interaction [8, 9, 26].

Nectins Recruit Adherens Junction and Tight Junction Components to Establish the Apical Junctions in MDCK Cells

Nectins and cadherins participate in the organization of adherens junctions in epithelial sheets (fig. 2). Nectins are composed of four members (table 1) whereas cadherins form a large superfamily with over 80 members of classical cadherins and non-classical cadherins [27]. As the cytoplasmic tails of cadherins bind the catenin complex, those of nectins directly bind afadin as well as Par-3 [13, 28]. Importantly, anchoring proteins such as catenins, afadin, and Par-3 cooperatively contribute to cluster cadherins and nectins. Thus, nectins play roles in forming mature adherens junctions that are strong yet easily remodeled.

Nectin-based cell–cell adhesions act independently and cooperate with cadherin-based cell–cell adhesions. In MDCK cells, nectins initiate cell–cell adhesion and then recruit cadherins to the nectin-based cell–cell adhesion sites to establish mature adherens junctions. Nectins need to interact with annexin II and IQGAP1 to establish adherens junctions in MDCK cells [16, 18, 29]. Nectins further promote formation of tight junctions by recruiting JAM-A, claudin-1, and occludin [30]. On the other hand, nectins are physically associated with integrin αvβ3 [31] and PDGF receptor through their extracellular domains to cooperatively regulate the formation of adherens junctions in MDCK cells [31, 32] and cell survival. Thus, nectins play roles for establishing apical junctional complex, as well as for promoting ‘cross-talk’ between cell–cell junctions and cell–matrix adhesions [9,33,34,35].

Signalings from Nectins Promote Apical Junction Formation

When cells contact each other via binding of cell adhesion molecules, signals are transduced to strengthen the linkage between the apical junctions and the actin cytoskeleton. Interactions to form trans-dimers of nectins firstly activate c-Src. Subsequent signalings cause the activation of Rap1, Cdc42 and Rac, which promote the formation of adherens junctions mediated by the IQGAP1-dependent remodeling of the actin cytoskeleton (fig. 2) [18,36,37,38,39,40,41]. In addition, the afadin and activated Rap1 complex interacts with p120ctn to strengthen the binding between p120ctn and E-cadherin [42, 43]. Furthermore, the cell polarity proteins, Par-3 and Par-6, and atypical protein kinase C (PKC) that form a ternary complex could be implicated in the assembly of adherens junctions [28,44,45,46]. These cell polarity proteins and afadin could play cooperative roles in the formation of adherens junctions and tight junctions. Consequently, nectins induce elaborate protein interactions and signalings to establish mature adherens junctions in two ways: direct interactions with F-actin-binding proteins as well as indirect organization of Cdc42- and Rac-mediated signalings (fig. 2) [9].

Signalings Mediated by Afadin

Except for the ability to link nectins to the actin cytoskeleton, afadin plays additional roles in cell survival and growth in non-epithelial cells. Afadin knockdown experiments by RNA interference show the downregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling in NIH3T3 cells with cell contacts. Afadin supports PDGF-induced activation of PI3K and the anti-apoptotic Akt signaling. Akt promotes cell survival by phosphorylating and thereby inactivating several pro-apoptotic proteins including BAD and Forkhead transcription factors [47]. Consistently, embryonic stem cells lacking afadin show increased apoptosis.

On the other hand, part of afadin translocates to the leading edge of NIH3T3 cells without cells contacts. The formation of leading edge structures and cell movement involve activation of Rapl and Rac mediated by PDGF signaling. Afadin binds SHP-2 tyrosine phosphatase to promote interaction between SHP-2 and PDGF receptor, as well as enhances phosphatase activity of SHP-2. Afadin appears to elaborately regulate the dephosphorylation process of PDGF receptor and thus modify the PDGF-Ras signaling. The PDGF receptor also recruits Rap1 to activate Rac1, resulting in the formation of leading edge structures and cell movement. Notably, these mechanisms are in close relation to Necl-5-mediated contact inhibition of NHI3T3 cells as described below. Afadin-mediated signalings may thus participate in the signaling process of developmental morphogenesis and remodeling of epithelial sheets.

Functions of Necl-1

Necl-1 is specifically expressed in the adult and fetal brain and in neurogenic cells [3, 21, 48]. Necl-1 binds membrane-associated guanylate kinase family members such as Dlg3/MPP3, Pals2, and CASK [21, 49] and modulates the reorganization of the actin cytoskeleton through protein 4.1 [23]. Dlg3/MPP3 functions to organize cell–cell junctions and mediates tumor suppression [50], while Pals2 is involved in the localization of other transmembrane proteins [26]. Necl-1 is recruited to the nectin-1 and nectin-3-based cell–cell adhesion in the process of synapse formation at puncta adherentia junctions in the hippocampus. Puncta adherentia junctions act as mechanical adhesion sites of neuronal cells, participating in part in the formation of synaptic junctions that regulate neurotransmission. After assembled to the primordial synapses, Necl-1 may be translocated to the axonal shafts where Necl-1 may function to maintain axon bundle formation [51]. Moreover, Necl-1 mediates segregation of synapses from neighboring axons, preventing neurotransmitters from diffusing and stimulating neighboring synapses [51]. Thus, the ability of Necl-1 to interact with nectin-1 and nectin-3 suggests involvement in the process of remodeling synapses in a neural activity-dependent manner [3, 8].

Functions of Necl-2

Necl-2 is expressed in the brain, testis, gallbladder, liver and pancreas, but not in fibroblasts or endothelial cells [26, 52]. Necl-2 mediates homotypic and heterotypic interactions with nectin and Necl family members, thus inducing cell adhesion [26, 53, 54]. Necl-2 is also known as a tumor suppressor gene TSLC1, which inactivates tumors in nude mice [55] and is frequently inactivated in many non-small cell lung cancers (NSCLCs) [55, 56]. Restoration of Necl-2 expression strongly suppresses the metastasis of human NSCLC cells from the spleen to the liver in nude mice [57]. Although the mechanism of tumor-suppressing activity is not fully studied, reducing Necl-2 activity may cause a disruption in cell polarity and adhesion, thus resulting in neoplastic growth (fig. 3). The cytoplasmic domain of Necl-2 contains two important motifs, a protein 4.1-binding motif at the juxtamembrane region and a PDZ-binding domain at the C-terminus, and recruits intracellular adaptors [48, 52, 58]. One of the protein 4.1 family molecules DAL-1 acts to anchor Necl-2 to the actin cytoskeleton, and plays a crucial role in the tumor suppression mediated by Necl-2 [57]. DAL-1 is shown to be downregulated in lung cancer, and restoration of DAL-1 expression to a normal level in lung cancer cells significantly suppressed cell growth in vitro. Colocalization of Necl-2 and DAL-1 at the cell–cell attachment site depends on the integrity of the actin cytoskeleton [57]. On the other hand, Necl-2 binds Dlg3/MPP3 and Pals2. Pals2 is known to bind Lin-7 that regulates the proper localization of Let-23 [59], the homolog of mammalian epidermal growth factor receptor that regulates vulval induction in Caenorhabditis elegans. Accordingly, Necl-2 may play tumor-suppressing roles by regulating growth factor receptors through Pals2 in mammals.

Necl-2 binds the class-I MHC-restricted T-cell-associated molecule CRTAM [60,61,62,63], which play pivotal roles in antiviral immunity. In addition, the coordinate expression of Necl-2 and CRTAM in the cerebellum strongly suggests an important role in the function of Purkinje neurons as well [64].

Functions of Necl-5

Cell biological studies have suggested that Necl-5 functions in cell adhesion [19, 22, 65, 66], migration [67,68,69], and proliferation [70]. Necl-5 functions as a cell adhesion molecule through trans-interaction with nectin-3 [19, 22]. Experiments conducted in L fibroblast cells show that this trans-dimerization promotes recruitment of E-cadherin to nectin-3 in the initial stages of cell–cell contact [66]. Necl-5 colocalizes with αvβ3 integrin-containing membrane microdomains [22] and is functionally associated with integrin αvβ3 in regulating cell motility in the absence of vitronectin [67]. Furthermore, Necl-5 promotes the transition from the G1 to the S phase of the cell cycle in NIH3T3 cells [70], accompanied by an enhancement of cell proliferation, activation of the Ras-Raf-MEK-ERK signaling pathway, and an upregulation of cyclin D2 and cyclin E [70]. Necl-5 is associated with Sprouty2 that inhibits the PDGF-Ras signaling for proliferation, and thereby prevents Sprouty2 from acting as a negative growth regulator [71,72,73]. Necl-5 is thus proposed to play unique roles in the ‘cross-talk’ between cell–matrix adhesions and cell–cell junctions [9].

Necl-5 regulates contact inhibition of cell movement and proliferation [74] (fig. 4). The role of Necl-5 has recently been reviewed in detail [8, 9]. It is believed that Necl-5 makes a ternary complex with integrin αvβ3 and the PDGF receptor at the leading edges of migrating cells [67]. When Necl-5 forms trans-dimers with nectin-3 at cell–cell contacts in NIH3T3 cells, Necl-5 undergoes downregulation from the cell surface, resulting in a reduction in cell proliferation and movement [75]. Since the signalings from both integrin αvβ3 and PDGF receptor are attenuated as a consequence of Necl-5 downregulation, Necl-5 could mediate growth arrest of cells cultured to confluence [75]. Consistently Necl-5 expression is also tightly regulated in response to changes in cell density, which is observed in NIH3T3 cells and liver epithelial cells, but not in the transformed cells [76, 77]. Hence Necl-5 explains part of the mechanisms of contact inhibition of cells in culture.

The rodent ortholog of Necl-5 (Tage4/PVR/CD155) was identified in rats as a tumor-specific marker of hepatocellular carcinoma [78,79,80] and colon carcinoma [81, 82], while Necl-5 is known as a poliovirus receptor/CD155 mediating the entry of human poliovirus [83]. In contrast to the low-level expression of Necl-5 in most adult organs, Necl-5 is overexpressed in various types of cancer cells [80,84,85,86,87] and a wide range of transformed cell lines [19, 80, 81, 84, 88]. Since Necl-5 is also expressed by hepatoblasts in the liver from embryonic day 12 to 14 [80], Necl-5 appears to be an onco-fetal protein that functions during development and plays a role in the progression of cancer. Interestingly, Necl-5 acts as an immediate–early response gene during liver regeneration [77]. It remains an unanswered question, however, whether enhanced expression of Necl-5 results in disruption of adherens junctions that is one of the hallmarks of cancer cells exhibiting malignant transformation.

Necl-5 binds DNAX accessory molecule-1 (DNAM-1/CD226) [89] that is highly expressed in leukocytes and platelets. The interaction between DNAM-1 and Necl-5 induces the cytotoxic activities of NK cells [89], regulates monocyte transmigration through endothelial cell junctions [90], and suppresses multinucleated osteoclast formation [91]. Since DNAM-1 is also expressed in platelets, interactions of tumor cells expressing Necl-5 with platelets may favor formation of tumor emboli and subsequent metastatic events. Necl-5 is actually involved in experimental metastasis when tumor cells are injected into the tail vein and colonies in the lung are scored [92]. Although the molecular mechanism of Necl-5 involvement in these studies is not clearly defined, the ligand stimulation of Necl-5 by DNAM-1 may trigger some physiological functions between the cells expressing each transmembrane protein, while it may suppress other cell fusion events.

Physiological Roles of Nectins

The phenotype of knockout mice tends to develop in selective tissues or cell lineages where the gene of interest plays an essential function that is not readily compensated by related proteins. Indeed, this is also the case for the deficiency of nectins. Knockout mice lacking each member of nectins are viable because the function of each member is redundant within the nectin family or compensated by other cell adhesion proteins. However, characteristic phenotypes develop if the interactions of nectins are unique and indispensable (table 1). One example is presented by knockout studies on nectin-1 and nectin-3 indicating that heterophilic trans-interactions between nectin isoforms play important roles in morphogenesis. Both nectin-1-deficient and nectin-3-deficient mice show microphthalmia associated with a separation of the apex–apex adhesion between the pigment and non-pigment epithelia of the ciliary body [93]. Microphthalmia is a phenotype of a developmental defect of the vitreous body because of malfunction of the ciliary body. Another example is presented by knockout studies on nectin-2 and nectin-3 [94, 95]. Both nectin-2-deficient and nectin-3-deficient mice show male-specific infertility and are defective in the later steps of sperm morphogenesis [96, 97], exhibiting distorted nuclei and abnormal distribution of mitochondria. Heterophilic interactions are essentially required between nectin-2 on the Sertoli cell side and nectin-3 on the spermatid side. Accordingly, lacking nectin-2 or nectin-3 impairs the structure of Sertoli cell–spermatid junctions by dislocalization of nectin-3 or nectin-2, respectively. It is of note that mice independently lacking two different genes show exactly the same phenotypes, which further emphasizes the significance of heterophilic interactions between nectin isoforms in vivo.

Cell–cell junctions and contacts are essential for neuronal cell migration, axon bundle formation and plasticity of synapses in the brain [21, 98]. Nectins are involved in forming puncta adherentia junctions at synapses of a certain set of neurons [99]. Nectin-1 and nectin-3 are asymmetrically localized at the synapses of hippocampal neurons, forming heterophilic trans-dimers on the presynaptic and postsynaptic sides [100]. At the synapses in the CA3 area of the hippocampus, the number of puncta adherentia junctions is decreased in both nectin-1-deficient and nectin-3-deficient mice [101]. Furthermore, an abnormal trajectory of mossy fibers is detected at the stratum lucidum of the hippocampus in these mice, possibly as a result of impaired puncta adherentia junctions. The mechanism of abnormal trajectories is further studied in hippocampal pyramidal neurons in culture. Nectin-1 is preferentially localized in axons whereas nectin-3 is present in both axons and dendrites in these neurons. The absence of nectin-1 loosens the contacts between axons and dendritic spines, suggesting that the axon-biased localization of nectin-1 and its trans-interaction with nectin-3 is critical for the ordered association of axons and dendrites [102]. These studies clearly elucidated the roles of nectins for organization of the central nervous system.

Clinical studies on nectin-1 have revealed important roles in facial and skin morphogenesis in humans. Mutations of the nectin-1 gene cause disorders, Zlotogora-Ogur syndrome and Margarita island ectodermal dysplasia, characterized by cleft lip/palate, syndactyly, mental retardation, and ectodermal dysplasia [103, 104]. Renal impairment has not been reported in these syndromes. The absence of nectin-1 in mice also results in a phenotype similar to that in humans; the mice show a clear defect in skull bone development. Further studies are currently underway.

Signalings from nectins are involved in gene expression. An obvious example is loricrin, an abundant protein of the cornified cell layer of the skin. Nectin-1 activates Rap1-ERK signaling to upregulate loricrin expression in keratinocytes. Consistently, mice lacking nectin-1 show a selective reduction in loricrin protein levels of the skin [105]. Therefore, nectin-1-mediated signalings actually induce gene expression although the whole bio-informatic profiles are not elucidated.

The mechanisms underlying these phenotypes are consistent with data obtained by biochemical and cell biological studies. It is believed that knockout studies on nectins disclosed the heterophilic interactions between nectin isoforms which play important roles in the morphogenesis of the eyes, brain, skin, and testes. However, mice lacking nectins show no morphological change in the renal corpuscles and proximal and distal convoluted tubules in the kidney where multiple types of nectins are expressed. This is because the function of a nectin isoform is compensated by redundant expression of other isoforms and, by no means, because nectins play no definite role in the kidney. On the contrary, the roles of nectin associated with an elaborate back up system may be more important than those of genes whose defects instantly lead to hereditary syndromes of proteinuria. Nectins actually play crucial roles in cell adhesion and formation of junctions in MDCK cells and are involved in mouse spermatogenesis which is one of urogenital phenotypes, and these observations are not totally irrelevant to properties of normal epithelial cells in the kidney.

Physiological Roles of Afadin

Afadin is the only cytoplasmic protein known to anchor nectins to adherens junctions [14]. Afadin-deficient mice show embryonic lethality and developmental defects at stages around gastrulation (E7.5), including disorganization of the ectoderm, impaired migration of the mesoderm, loss of somites, and mixed amorphous structures derived from the ectoderm and mesoderm [106]. When afadin is conditionally inactivated using the Cre/loxP system, the absence of afadin significantly reduces the capacity of maintaining cell–cell contacts, leading to fluid leakage or apoptosis induced by extracellular stress. Hydrocephalus is the main phenotype of mice lacking afadin, which is observed at E16.5 when the expression of Cre is driven by the nestin promoter. Ependymal cells of cerebral ventricles are disorganized and the stenosis of the aqueduct is prominent between the third and fourth ventricles. The mice lacking an isoform of atypical PKCλ in the brain also show hydrocephalus similar to that observed with mice lacking afadin [107], suggesting that the afadin and aPKC/Par-3/Par-6 complex may be commonly involved in the machinery required for the formation of the cerebral ventricle.

In addition, mice lacking afadin specifically in the colon are obtained by intercrossing with the villin-Cre mice. These mice show the dislocalization of nectin-2 from the apical junction to the basolateral membrane domain of the epithelial cells. Although the histological appearance of the colon epithelium appears to be mostly intact, mice lacking afadin have greater sensitivity than do the wild-type mice for apoptosis of the epithelial cells when challenged with dextran sulfate sodium. Thus, the functional integrity of the apical junctions of the colon lacking afadin may be impaired although nectins and afadin are not critically required for the morphogenesis and maintenance of the colon epithelium. It is intriguing to examine the outcome of conditional knockout of afadin in endothelial cells of the glomerulus, podocytes or tubular epithelial cells.

Biological Significance of Rho Family Proteins

Rho family small G proteins, including Rho, Rac, and Cdc42, regulate the reorganization of the actin cytoskeleton and the integrity of adhesion complexes [108, 109]. The roles of Rho family proteins are well documented: Rho induces stress fibers, Rac promotes lamellipodia, and Cdc42 promotes filopodia in cells in culture. As with epithelial and endothelial cells, Rac supports the formation of intercellular junctions and enhances the endothelial barrier [110,111,112], while Cdc42 is necessary for maintaining epithelial polarity and promoting the formation of tight junctions [113]. Activation of RhoA results in the increase in endothelial permeability [114]. Reportedly, MDCK cells overexpressing both the dominant active and negative mutants of RhoA, Rac1, and Cdc42 show disruptions in epithelial gate function and distinct morphological alterations in apical and basal filamentous actin pools [115]. These cytoskeleton-dependent activities of Rho family proteins are essential for epithelial and endothelial cells to assemble or disassemble cell–cell contact sites.

Rac and Cdc42 are activated by nectin and cadherin engagement. Activated Rac and Cdc42 are spatially confined to the outer margins in extending cell contact zones. E-Cadherin engagement recruits the Arp2/3 complex for actin assembly at the cell surface to nucleate branched actin filaments in lamellipodia [116]. Interestingly, Rho family proteins regulate both cortactin and N-WASP proteins, major actin nucleation-promoting factors for Arp2/3 activity. Whereas Rac promotes cortactin recruitment in motile cells [117], N-WASP is normally auto- inactivated but stimulates Arp2/3 complex-mediated actin polymerization when bound to activated Cdc42 and phosphatidylinositol-4,5-biphosphate [118]. Furthermore, formins have a domain for binding RhoA and RhoC and form dimers to promote elongation of actin filaments. These actin nucleators and their regulators thus reorganize the actin cytoskeleton in response to the Rho family protein-mediated signaling.

With the exception of Rac2, knockout studies on each member of the Rho family proteins are not very informative due to embryonic lethality. However, their regulators are good targets for gene disruption because they are not always essential for the viability of knockout mice. Rho family proteins serve as a molecular switch for transducing signals by exchanging the GTP- and GDP-bound states, which are regulated by guanine-nucleotide exchange proteins, GDP dissociation inhibitors (GDIs), and GTPase-activating proteins [119]. For example, a deficiency in Rho GDIα, which is the isomer ubiquitously expressed in mouse tissues, leads to a less deleterious phenotype than a deficiency in its substrate small G protein itself.

Disruption of the Rho GDIα gene in mice results in selective defects in the kidneys and reproductive organs [120]. Notably, Rho GDIα-deficient mice develop prominent proteinuria mimicking nephrotic syndrome, leading to death due to renal failure within a year [119]. Histologically, degeneration of tubular epithelial cells and dilatation of distal and collecting tubules are prominent in the kidneys. The absence of Rho GDIα leads to the selective activation of Rac1 and Cdc42 in the kidneys, whereas RhoA activity is not affected [Fujita T, unpublished data]. In addition, Rho GDIα-deficient mice show an increased permeability of pulmonary vasculature because of tight junction disruption caused by sustained activation of RhoA but not Rac1 or Cdc42 [121]. Importantly, inhibition of Rho kinase by its inhibitor Y-27632 can diminish the increased microvascular permeability in the lungs of Rho GDIα-deficient mice. Therefore, Rho GDIα differentially regulates the activities of Rho family proteins in vivo. Such unbalanced regulations of Rho family proteins may lead to the junctional disassembly and disruption of the endothelial or epithelial barrier, even though the precise mechanism remains to be elucidated.

Pathogenesis of Massive Proteinuria in Rho GDIα-Deficient Mice

Rho GDIα deficiency would affect the glomerulus causing proteinuria in two ways (fig. 5). One possibility is that activated Rho increases the permeability of glomerular endothelial cells possessing fenestration with a relatively large gap. Another possibility is that activated Rac and Cdc42 cause disruption of intercellular junctions between the podocytes in the glomerulus. Studies on hereditary proteinuria syndromes show that nephrin, Neph-1, Neph-2, and podocin are crucial for the normal development and function of the kidneys, and that the podocyte slit diaphragm plays important roles in establishing the filtration characteristics of the glomerulus, preventing leakage of serum albumin [10]. In the rat nephritis model, exposure of visceral glomerular epithelial cells to complement initially alters the balance of Rho family proteins, with the increased RhoA activity and with the decreased Rac1 or Cdc42 activity [122]. Concerted activities of Rho family proteins are probably required for the physiological function of the podocyte slit diaphragm although this possibility has not been thoroughly investigated. On the other hand, Rho GDIα-deficient mice show prominent histological changes, disruption of the apical junctions and actin cytoskeleton, which are readily detected by electron microscopy in tubular epithelial cells. Decrease in adhesion strength between renal tubular cells may be in parallel with an increase in paracellular permeability (fig. 5). Therefore, massive proteinuria in Rho GDIα-deficient mice could be attributed to impaired glomerular filtration as well as leaky barrier function of tubular epithelial cells or the impaired reabsorption of urine.

Studies on nectins and Necls provide us with significant insights into the molecular mechanisms that are essential for establishing and maintaining the structure and function of adherens junctions. Adherens junctions control epithelial cell polarity while focal adhesions tend to inhibit cell migration. This balance should be elaborately regulated by ‘cross-talk’ between integrins and nectins or Necls. Necls form a family with more versatile functions than nectins: Necl-1 acts as an inducer of synaptic junctions in the brain, Necl-2 acts as a tumor suppressor protein and a signal for immune surveillance, and Necl-5 acts as a tumor initiator promoting cell growth. Their roles appear to be crucial for the differentiation and morphogenesis of many tissues including the kidneys.

The Rho family proteins are key regulators to understand cell adhesion biology in the kidney. However, whether or not podocin, nephrin, and related molecules initiate Rac and Cdc42 signaling remains unknown, and even such possibilities have not yet been examined. On the other hand, engagement of nectins and cadherins initiates Rac and Cdc42 signaling, resulting in reorganization of the actin cytoskeleton through actin nucleators such as formins, Arp2/3 comoplex, N-WASP, and WAVE. Thus, nectins and Necls play critical roles in establishing the apical junctions of MDCK cells although it remains largely unclear how they play roles in the kidney. They may have the potential to develop a new field of cell adhesion biology in the kidney.

Approaches to restoring adherens junctions, cell–cell communication, and cell–matrix communication are emerging as a new therapeutic strategy for proteinuria. Aberrant immune responses or hormonal events are likely to have deleterious effects in the kidneys, leading to disruption of apical junctions of the glomerulus or renal tubular cells. Therapeutic targets might be the molecules involved in pathways affecting the adhesive properties of nectins and Necls. c-Src is a candidate because it regulates both the disruption of adherens junctions and focal-adhesion turnover [123]. Other candidates are Rho family small G proteins and transcriptional repressor proteins, such as snail transcriptional factor, slug, and Twist, that downregulate E-cadherin gene expression [124]. Restoring RhoA activity or antagonizing Rac activity appears to be effective to reduce the proteinuria [122]. Whether these drug therapies improve the excess remodeling of the actin cytoskeleton observed in renal injury remains unanswered.

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