*Division of Nephrology, Albert Einstein College of Medicine, Bronx, New York; and Division of Nephrology, University of Washington, Seattle, Washington.
Correspondence to Dr. Stuart J. Shankland, Division of Nephrology, University of Washington, Box 356521, Seattle, WA 98195. Phone: 206-543-3792; Fax: 206-685-8661;E-mail: stuartjs{at}u.washington.edu
The visceral glomerular epithelial cell, also called podocyte,is a terminally differentiated cell that lines the outer aspectof the glomerular basement membrane (GBM). It therefore formsthe final barrier to protein loss, which explains why podocyteinjury is typically associated with marked proteinuria. Indeed,all forms of nephrotic syndrome are characterized by abnormalitiesin the podocyte. In this review, we will provide an update ofthe known functions of recent podocyte-specific proteins andfocus on the slit diaphragm (SD) and the mechanisms underlyingfoot process (FP) flattening and how the podocyte responds toinjury.
Podocyte FP are not static, but rather contain a contractilesystem similar to that seen in pericytes. This contractile apparatusis composed of actin, myosin-II, -actinin-4, talin, and vinculin(1). The actin filament bundles form arches between adjacentFP of the same podocyte (2). Figure 1 is a schema of our currentunderstanding of the molecular composition of the cytoskeletonin podocyte FP. Importantly, the actin filaments are connectedto the underlying GBM at focal contacts via an 31 integrin complex(3,4). The bends of the actin filament arches appear to be connecteddirectly to the microtubules of the major processes (own unpublishedresults). FP are anchored to the GBM via 31 integrin (5) anddystroglycans (6,7). Neighboring FP are connected by a cell-celljunction, the glomerular SD, which represents the main sizeselective filter barrier in the kidney (8–10). The SDis thought to be a modified adherens junction (11) that is composedof a growing number of proteins, including nephrin (12–14),P-cadherin, CD2AP (15–18), ZO-1 (19), FAT (20), podocin(16,21), and possibly Neph1 (see reference 22 and below). Inaddition to the contractile proteins described above, we havereported the association of synaptopodin with the actin filamentsin FP (23). Synaptopodin is the first member of a novel classof proline-rich proteins (24) and, like -actinin-4, interactswith the tight junction protein MAGI-1 (25) that is also expressedin podocytes (26).
Figure 1. Molecular anatomy of the podocyte foot process (FP) actin cytoskeleton. This schematic shows two adjacent podocyte FP with the interposed slit diaphragm (SD) complex. The localization of NEPH-1 at the SD and its heterophilic interaction with nephrin remain to be established. The actin cytoskeleton is the common downstream pathway and receives input form three podocyte domains: the apical domains, the lateral SD-containing domain, and the basal domain of the FP sole plate, which links the podocyte to the GBM. Interference with any of the three domains will ultimately cause FP effacement and proteinuria/nephritic syndrome. -act4, -actinin-4; 31, 31 integrin; -DG, -dystroglycan; -DG, -dystroglycan; Na(+)/H(+)ERF2, Na(+)/H(+) exchanger regulatory factor 2; P, paxillin; P-cad, P-cadherin; Synpo, synaptopodin; T, talin; V, vinculin.
The basolateral portion of the foot processes represents thecenter of podocyte function and is defined by three membranedomains: the apical membrane domain, the SD protein complex,and the basal membrane domain or sole plate (27). The submembranousregions of all compartments are connected to the FP actin cytoskeleton,e.g., on the apical membrane domain, podocalyxin associateswith the actin cytoskeleton through interactions with ezrinand the actin cytoskeleton via Na+/H+-exchanger regulatory factor2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1)domains and an ERM-binding region (28,29). The FP actin cytoskeletonis highly dynamic and ultimately determines the structural maintenanceof the filtration slits as demonstrated in the acute PS/heparinmodel by several groups (30–32). Interference with oneof the three domains eventually leads to changes in the actincytoskeleton from coordinated stress fibers into a dense network(33) with fusion of podocyte FP and obliteration of filtrationslit. Proteins regulating or stabilizing F-actin are thereforeof critical importance for sustained function of glomerularfiltration (29,33–38).
Podocyte -actinin-4 induction precedes FP effacement in experimentalnephrotic syndrome (36), and the podocalyxin/NHERF2/ezrin/actininteractions are disrupted in pathologic conditions associatedwith changes in FP (29). The -actinin-4 molecule is a novelmember (39) of the actin family of actin-filament cross-linkingproteins and has an important function in podocytes. Mutationsin the ACTN4 gene encoding -actinin-4 have been demonstratedby Pollak and colleagues in an autosomal dominant form of focalsegmental glomerular sclerosis (FSGS) (see related article byPollak in this issue of JASN [40]) and underscore the exquisiterole of the actin cytoskeleton in short-term and long-term regulationof podocyte structure (41,42).
Podocytes are injured in many forms of human and experimentalglomerular disease, including minimal change disease, FSGS,membranous glomerulopathy, diabetes mellitus, and lupus nephritis(8,27). Independent of the underlying disease, the early eventsare either characterized by alterations in the molecular compositionof the SD without visible changes in morphology or, more obviously,by a reorganization of the FP structure with fusion of filtrationslits and apical displacement of the SD (8,37,43). On the basisof recent progress in the molecular pathology of podocytes,four major causes can be identified that lead to FP effacementand proteinuria: (1) interference with the SD complex and itslipid rafts; (2) interference with the GBM or the podocyte-GBMinteraction (6,7,44–49); (3) interference with the actincytoskeleton and its associated protein -actinin-4 (36,41);and (4) interference with the negative apical membrane domainof podocytes, e.g., neutralization of negative cell surfacecharges (28–30,50).
In addition to the SD as the major site of glomerular permselectivity(see below), glomerular filtration is also regulated on thelevel of the GBM (51–53). In a comprehensive and elegantstudy comparing mice lacking either the alpha3 chain of typeIV collagen, the major constituent of glomerular basement membrane,the LMX1B transcription factor, or nephrin, Hamano et al. (53)showed that defects induced by proteins of GBM lead to an insidiousplasma protein leak, whereas the defects induced by SD proteinslead to a precipitous plasma protein leak. Finally, there isevidence in FSGS and in idiopathic nephrotic syndrome in ratsthat podocyte damage may be caused by circulating albuminuricfactors (54,55). As depicted in Figure 1, the -actinin moleculecan interact with components of the integrin complex at theGBM and with the -catenin molecule of the SD complex. Hence,-actinin may link these two different compartments of the FPtogether, thereby providing a molecular explanation for theobservation that the actin cytoskeleton serves as the "commonfinal pathway" organizing FP effacement independent of the originalunderlying site or cause of podocyte damage. From a clinicalpoint of view, it is important to note that these early structuralchanges in podocyte morphology, such as substructural alterationsin SD composition or FP effacement, have to be reversed withina certain period of time to prevent development of severe andprogressive glomerular damage (43,56–58) (also see below).
The SD: A Dynamic Site ofGlomerular Permselectivity
In mature podocytes, the SD represents the only cell-cell contactbetween podocytes. The SD represents a tiny membrane bridgingthe 30- to 40-nm-wide filtration slit. In 1974, Rodewald andKarnowsky (59) showed that the SD is made up of rodlike unitsconnected in the center to a linear bar forming a zipper-likeappearance, but its molecular composition and anchorage in theFP remained unknown. The normal SD function is crucial to maintainingthe integrity of the FP (8–10). The recent discovery ofseveral novel SD proteins and their mutation analysis, includingnephrin (60), CD2AP (17,61), podocin (62), and the nephrin homologueneph1 (22), have shed light on the pathogenesis of proteinuriaand emphasized the critical role of the SD in maintaining thenormal function of the glomerular filtration barrier. However,the mechanisms regulating the structural changes that occurduring FP effacement are still largely unknown. A fuller understandingof the molecular basis of glomerular kidney disease requireselucidation of the relationship between SD proteins and themaintenance of FP structure.
The Critical Role of Nephrin in Maintaining the Glomerular Filtration Barrier
Twenty-five years after the hallmark finding by Rodewald andKarnovsky (59), the discovery of the transmembrane protein nephrinas a major component of the SD complex by Tryggvason and others(12–14) provided a seminal progress in podocyte biology.Mutation analysis of the nephrin gene, NPHS1, by positionalcloning elucidated the underlying genetic defect in congenitalnephrotic syndrome of the Finnish type as causative for FP effacementin this disease (60). Similarly, injection of anti-nephrin antibodyin animals induced substructural alterations of the SD withreduction of permselectivity and consecutive proteinuria (63).The inactivation of the nephrin gene in mice by homologous recombinationresulted in reduction of visible SD, severe proteinuria, andpartial FP effacement (53,64), Similarly, nephrin TRAP micealso lack SD and show fibrotic glomeruli as well as cystic tubularlesions (65). Nephrin is a large (1241–amino acid, 185-kD)transmembrane molecule with Ig-like domains. N-linked glycosylationis critical for the plasma membrane localization of nephrin(66). Its predicted structure and biochemical properties, aswell as electron microscopy studies, suggested that nephrinmay form dimers through homophilic interactions across the filtrationslit (67). However, several groups have failed to show sucha homophilic interaction and the nephrin homologue in drosophila,hibris, was found to form heterophilic interactions with a proteincalled dumbfounded but not homophilic interaction with otherhibris molecules (68,69).
Nephrin may also contribute functional properties to the SD,perhaps by participating in a protein complex in which interferencewith any of the components may lead to functional destabilizationof the SD and consequent FP effacement and proteinuria (seebelow). Although the causal role of nephrin in congenital nephroticsyndrome of the Finnish type is now well established, its functionalrole in acquired forms of nephrotic syndrome remains to be established.Several studies have reported a modulation or correlation ofnephrin expression with levels of proteinuria, including puromycinaminonucleoside (PAN) (70), diabetes (71,72), and minimal changedisease (MCD) (73). The latter study showed that MCD is associatedwith disruption of the SD. At this point, it is too early toconclude whether the changes are causal or secondary, but arecent study analyzing the recurrence of nephrotic syndromein kidney grafts of patients with congenital nephrotic syndromeof the Finnish type has shed some light on this issue (74).This study showed that circulating anti-nephrin antibodies mighthave a pathogenic role in the development of heavy proteinuriain kidney grafts of NPHS1 patients with Fin-major/Fin-majorgenotype (74).
A Rapidly Expanding List of Proteins that Comprise the SD
At the intracellular insertion site of the SD, the adapter proteinCD2AP has been localized (17,18,76), which was originally discoveredas a protein interacting with the CD2 receptor in T lymphocytes(77). CD2AP is critical for orchestrating the so-called immunologicsynapse between B cells and T cells (77,78) but has gained anunexpected important role in podocyte cell biology, becauseCD2AP knockout mice die several weeks after birth with FP effacementand nephrotic syndrome (75). CD2AP interacts with nephrin viaa novel C-terminal domain (18) and is also capable of associatingwith the actin cytoskeleton (79). The latest putative componentof the SD complex is NEPH-1, a homologue of nephrin, which wasdiscovered using retrovirus-mediated mutagenesis (22). The homozygousknockout mice of NEPH-1 show FP effacement (22). Whether NEPH-1interacts with nephrin is not yet known, but in the light ofthe absence of a nephrin-nephrin homophilic interaction andthe similar phenotype of both knockouts, a heterophilic interactionbetween nephrin and NEPH-1 appears plausible.
The role of other molecules that are associated with the SDawaits clarification. ZO-1 has long been known to localize tothe intracellular site of insertion of the SD. It interactswith the actin cytoskeleton (80) and may also participate insignaling events through tyrosine phosphorylation (81). Of noteis that the redistribution of ZO-1 was associated with the developmentof proteinuria in spontaneously proteinuric MWF rats, althoughthe podocyte FP were normal and SD preserved in these animals(82). P-cadherin (11) and FAT (20), which are widely expressedcadherin superfamily proteins, define the SD as a modified adherensjunction and may provide structural support to this specializedcell-cell contact. Interestingly, the expression of ZO-1, P-cadherin,and FAT is not altered in nephrin null mice (53).
Podocin is a new member of the stomatin family of hairpin-likeintegral membrane proteins with intracellular N- and C-termini.Podocin is encoced by the NPHS2 gene, which is mutated in autosomalrecessive, steroid-resistant nephrotic syndrome (62). Stomatinis present as high-order oligomers in erythrocyte lipid rafts,where it has a scaffolding function (83). Podocin localizesto the SD (16,21), accumulates there in an oligomeric form inlipid rafts and associates via its C-terminus with CD2AP andnephrin (16). Further studies revealed direct interaction ofpodocin and CD2AP (16). Hence, podocin may act as a scaffoldingprotein, serving in the structural organization of the slitdiaphragm and the regulation of its filtration function. Invitro co-expression studies showed that podocin facilitatednephrin signaling via AP-1 in HEK cells (84), but the relevanceof this finding for podocytes has not yet been demonstrated.
Involvement of Lipid Rafts in Functional Organization of the SD
Lipid rafts are specialized membrane domains enriched in cholesterol,glycosphingolipids, and GPI-anchored proteins (85). By compartmentalizingcell membranes, they recruit and cluster membrane proteins ina selective and dynamic fashion. Hereby, they provide molecularframeworks for numerous cell biologic processes, such as exocytosisand endocytosis, cell adhesion, and signal transduction events(86–88). Recent work from our lab established that lipidraft microdomains are critical for the dynamic functional organizationof the SD (89). We have shown that nephrin associates with lipidrafts and co-immunoprecipitates with a podocyte-specific 9-O-acetylatedganglioside (89). The in vivo injection of an antibody againstthis ganglioside causes morphologic changes of the filtrationslits resembling FP effacement. In this model, nephrin translocatedto the apical pole of the narrowed filtration slits and underwenttyrosine phosphorylation (89).
Previous studies have described a role for tyrosine phosphorylationin the assembly and disassembly of the slit diaphragm (81).So far, it is unclear which kinases are involved in regulatingthese events, but the genetic inactivation of the src familykinase fyn caused proteinuria in mice (90). Interestingly, asa double-acylated molecule, fyn, has a high affinity for lipidrafts (91,92). Hence, it is intriguing to speculate that fynis involved in regulating the dynamics of the SD complex. Insummary, the last 4 yr have been extremely fruitful in providingextensive information on the molecular composition of the SDand have opened up new avenues to understanding podocyte function(8,27). From a clinical perspective, it is exciting that thereare novel experimental data that may link the salutary effectsof angiotensin-converting enzyme (ACE) inhibitors and angiotensinreceptor blockers drugs to changes in the composition of theSD (93–97).
As discussed above, podocytes are the target of many forms ofinjury, including antibodies to podocyte membrane antigens (membranousnephropathy, minimal change disease) (98), hemodynamic injury(reduced nephron number, diabetes, metabolic diabetes) (99–101),gene mutations (nephrin, -actinin, CD2AP; see review by Pollakin this issue [40]), protein overload states (102), toxins (NSAIDS,adriamycin) (103), infections (HIV) (see review by Ross andKlotman in this issue [104]), and unknown causes (idiopathicFSGS) (105). Moreover, in secondary forms of FSGS, such as afterloss of nephron number, hypertension, and tubulointerstitialdisease, podocytes are also injured (106). However, regardlessof the type of renal injury, loss of podocyte number contributesto the development of glomerulosclerosis (see below).
There is a growing body of experimental and clinical literatureshowing that podocyte number is a critical determinant for thedevelopment of glomerulosclerosis and that a decrease in podocytenumber leads to progressive renal failure. For example, Wigginsand colleagues (107) recently showed that glomerulosclerosiscorrelated with podocyte loss during the normal physiologicaging process in rats. A single injection of PAN, a podocytetoxin, causes a marked decrease in podocyte number in rats.Kim et al. (107) showed that repeated injections of PAN furtheraugmented podocyte loss and that the regions devoid of podocytesdeveloped glomerulosclerosis. However, glomerulosclerosis wasonly initiated when podocyte number decreased by 10 to 20%.Moreover, there was a significant correlation between the decreasein podocyte number and the development of glomerulosclerosis,because the authors showed that increased podocyte loss withrepeated PAN injections correlated with scarring (107). Krizand colleagues (108) showed that a decrease in podocyte numberin the Masugi nephritis model also contributed significantlyto the development of renal failure.
One of the first studies to show that a decrease in podocytenumber also correlated with disease progression in human diseasewas performed by Meyer and colleagues (109). They showed thata decrease in podocyte number in type II diabetic Pima Indianscorrelated closely with those patients who had microalbuminuria,the earliest manifestation of diabetic nephropathy. Moreover,they showed that the decrease in podocyte number was more pronouncedin patients with more advanced nephropathy (109). In contrastto the decrease in podocyte number, mesangial and glomerularendothelial cell number remained normal. More recently, Steffeset al. (110) showed a similar paradigm in patients with typeI diabetic nephropathy. Taken together, these important studiesshowed that a decrease in podocyte number is a significant predictorof disease progression in diabetic nephropathy. Finally, Lemleyand coworkers (111) recently showed that despite injury to themesangial cell in IgA nephropathy, a decrease in podocyte numbercorrelated significantly with reduced renal function and globalglomerulosclerosis.
Mechanism Underlying Glomerulosclerosis after a Decrease in Podocyte Number
The mechanism(s) underlying the development of glomerulosclerosisfollowing a decrease in podocyte number has been proposed byKriz, Rennke, and others (56,112–115). Because podocytesare located on the outer aspect of the glomerular basement membrane,one of the functions of podocytes is to provide a tensile supportto the underlying glomerular capillary loop, by opposing thehydrostatic capillary pressure (115,116). It is the belief ofmost authorities that there is a finite number of podocytes/glomerulusand that individual podocytes cover a specific area of GBM.Thus if podocyte number is decreased, there are insufficientpodocytes to cover that specific area of basement membrane.The sequence of events in the development of glomerulosclerosisare as follows (56,113). First, podocyte loss, and the inabilityto replace those lost because of a lack of proliferation (seebelow), results in a localized "bare" or denuded GBM at thatsite. Second, the lack of tensile support normally providedby podocytes (117) is lost in the area of denudation and leadsto the outward bulging of the capillary loop (due to hydrostaticcapillary pressures). Because many forms of glomerular diseasesare associated with increased intraglomerular hydrostatic capillarypressure, this process is further augmented. Third, the "expanding"capillary loop causes the denuded basement membrane to abuton Bowman’s capsule, leading to synechia formation, whichSchwartz and Lewis (105) have shown is the first committed stepto the development of FSGS. Finally, inspissated proteins andhyalinosis develop in the capillary loops, and progressive scarringensues.
Because a decrease in podocyte number (podocytopenia) underliesglomerulosclerosis, recent studies have focused on the causesunderlying podocytopenia (Figure 2). The etiology of podocytopeniaincludes apoptosis, detachment, and the inability or lack ofpodocytes to proliferate; each will be discussed below.
Figure 2. Causes of podocytopenia. After injury, podocytes can undergo apoptosis or detachment or fail to proliferate. These events lead to a decrease in podocyte number (podocytopenia), which contributes to the development of progressive glomerulosclerosis. The mechanisms underlying podocytopenia are being elucidated. Apoptosis results from increased transforming growth factor– (TGF-), angiotensin II, reactive oxygen species (ROS), and a decrease in the cyclin-dependent kinase (CDK) inhibitors p21 and p27. The 31 integrin is most likely to be critical in podocyte detachment from the underlying glomerular basement membrane (GBM). In contrast to other glomerular cells, podocytes do not typically proliferate in response to injury and cannot replace those lost by apoptosis and detachment. The inability to proliferate is secondary to increased levels of the CDK-inhibitors p21, p27, and/or p57.
Apoptosis
Cell number reflects the balance between an increase in cellnumber due to proliferation, and a decrease in cell number dueto apoptosis (programmed cell death). Although earlier studiesfailed to document significant podocyte apoptosis (118), recentstudies have shown that podocytes undergo apoptosis in glomerulardisease (119). One explanation for the earlier difficulty indetecting podocyte apoptosis is that apoptotic podocytes arelikely flushed out in the urine, making it technically difficultto detect these cells. However, apoptosis has recently beenshown in podocytes with human glomerular disease (V. D’Agati,personal communication). Moreover, Wiggins and colleagues (107)have clearly demonstrated podocyte apoptosis after toxic injuryin the PAN model. Bottinger and colleagues (119) recently showedthat apoptosis is increased in TGF- transgenic mice, which leadsto a decrease in podocyte number and glomerulosclerosis. Follow-upstudies by Bottinger’s group (120) showed that TGF- inducedpodocyte apoptosis was mediated by specific Smad pathways, andwe have recently shown that TGF- induced podocyte apoptosisis augmented in the absence of the CDK-inhibitors p21 and p27(unpublished data).
Recent studies have further examined the mechanisms underlyingpodocyte apoptosis. Singhal and colleagues (121) showed thatangiotensin II (AngII) induces apoptosis in cultured rat podocytes.This effect was dose- and time-dependent. AngII-induced apoptosiswas reduced by blocking either the subtype I or II receptorsand was completely prevented when both receptors were inhibited.AngII-induced apoptosis was in part TGF-–dependent. TheSmad signaling pathways underlying TGF-–induced podocyteapoptosis have recently been delineated by Bottinger and coworkers(119). Other mediators of podocyte apoptosis have been shown.For example, puromycin induces podocyte apoptosis in culture,which is mediated through reactive oxygen species (122). Takentogether, these studies show that apoptosis increases in podocytesunder certain circumstances and contributes to the loss of cellnumber. Future studies are now focusing on understanding thepathways mediating this process.
Detachment
A second mechanism underlying a decrease in podocyte numberis detachment of cells from the underlying GBM (Figure 2). Indeed,studies by Hara and colleagues (123–125) showed that cellsobtained in the urine of patients with various glomerular diseasesstained positive for the podocyte marker, podocalyxin. Similarresults have been shown in PAN model of podocyte injury in rats(107). We have recently asked if podocytes detaching are viableor only apoptotic, as has been discussed above. Our data showthat in the passive Heymann nephritis model of membranous nephropathyand in the streptozotocin model of diabetic nephropathy in rats,podocytes were readily detected in the urine, identified byimmunostaining with podocyte-specific antibodies, such as nephrin,podocin, and Glepp-1. When these cells obtained in the urinewere resuspended in tissue culture media and plated onto tissueculture dishes and grown under cell culture conditions, theyadhered to tissue culture plates (75). The vast majority ofadherent cells were podocytes. Moreover, there was an increasein podocyte cell number during the first days in culture. Theseresults suggest that a fraction of podocytes detaching fromthe GBM in experimental membranous and diabetic nephropathyare viable and that they may have proliferative potential underthese conditions. Future studies need to be directed towardbetter understanding the mechanisms of podocyte detachment,especially the role of specific integrins, such as the 31 integrin(126) or dystroglycans (6).
Lack of Proliferation
A decrease in podocyte number has also been shown to be consequentto a lack of appropriate proliferation after injury in thiscell type (113). As a result, after cell loss (by detachmentand/or apoptosis), the inability to proliferate prevents therestoration of normal podocyte number (118). This contrastswith mesangial and glomerular endothelial cells, which readilyproliferate in response to many forms of injury (127). Thereis a large body of literature showing that podocyte proliferationcorrelates closely with its state of differentiation, whichmay provide important clues into the mechanisms underlying thelack of proliferation (128). During glomerulogenesis, presumptiveand immature podocytes proliferate and are actively engagedin the cell cycle (129). However, during the critical S-phaseof kidney development, podocytes exit the cell cycle to takeon a terminally differentiated and quiescent phenotype, whichis required for their highly specialized function.
Proliferation is governed at the level of the cell cycle bycell cycle regulatory proteins (130). To proliferate, cyclinsmust bind to and activate partner cyclin-dependent kinases (CDK).In contrast, CDK are inactivated by CDK-inhibitors, includingp21, p27, and p57 (131). Thus the balance of cyclin-CDK complexesand CDK-inhibitors determines if cells proliferate or are quiescent.In both mice and humans, immunostainings for p27 and p57 areabsent in immature proliferating podocytes during the S-shapedstage of glomerular development. However, podocyte differentiationcoincides with a marked increase in the expression of the CDK-inhibitorsp27 and p57 in podocytes (132,133). This differential expressionof CDK-inhibitors persists in normal podocytes. However, theCDK-inhibitors p21, p27, and p57 alone are not required fornormal glomerular development, because the kidneys from thesenull mice are histologically normal (134–136).
The passive Heyman nephritis (PHN) model has many similaritiesto human membranous nephropathy, and it is induced by the administrationof an antibody directed against the Fx1A antigen on the ratpodocyte (137). We began by asking if podocytes are capableof increasing cyclins and CDK required for proliferation. AfterC5b-9–induced injury in PHN rats, protein levels for cyclinA and CDK2 increase (118), suggesting that the lack of podocyteproliferation may be due to a cell cycle inhibitor(s), ratherthan a failure to engage the cell cycle per se. Indeed, thelevels of the CDK-inhibitors p21 and p27 increase specificallyin podocytes after complement-dependent injury in PHN rats (118).Furthermore, the CDK-inhibitors limit podocyte proliferationby binding to and inhibiting specific cyclin-CDK complexes.
A key role for p21 and p27 in limiting the proliferative responseof podocytes has been confirmed in studies utilizing specificCDK-inhibitor null mice. The administration of an anti-glomerularantibody to induce experimental podocyte injury caused markedpodocyte de-differentiation in p21-/- (135) and p27-/- (134)mice compared with control wild-type mice receiving the sameantibody, and this was accompanied by increased podocyte proliferation.Glomerular extracellular matrix protein accumulation was alsoincreased in diseased p21 and p27-/- mice, and this was accompaniedby a significant decrease in renal function (134,135). The roleof the CDK-inhibitor p57 remains enigmatic due to the lack ofa viable knockout mouse (138). However, podocyte protein levelsfor p57 are decreased in PHN, and in anti-glomerular antibodydisease in the mouse, loss of expression localizes predominantlyin proliferating podocytes (136).
Although the vast majority of human podocyte diseases are notassociated with proliferation, podocyte proliferation does occurin idiopathic collapsing glomerulopathy and HIV-associated nephropathy(see review by Ross and Klotman in this issue [104]). In thesediseases, there is increased expression of cyclin A and Ki-67and a reduction in p27 and p57 in cells that are proliferating(139,140). In contrast, CDK-inhibitors do not decrease in humandiseases characterized by the absence of podocyte proliferation(membranous nephropathy, MCD and FSGS). Taken together, thesestudies show that the CDK-inhibitors p21, p27, and p57 havea critical role in determining the outcome of diseases of podocytesand limit proliferation by reducing DNA synthesis (Figure 2).
Abnormalities in Podocyte Mitosis.
Studies have unequivocally shown podocyte polyploidy in experimentalmembranous nephropathy (141,142). Polyploidy is defined as anincrease in DNA content and is seen histologically as multinucleatedcells. These observations suggest that podocytes can undergomitosis but that there is either an abnormality in the completionof mitosis and/or in cytokinesis (cell division). When culturedpodocytes are exposed to sublytic C5b-9 attack, a variety ofsignaling pathways are activated, including JNK, phospholipases,calcium, and MAPK cascades (143–145). Sublytic C5b-9 attackalso causes cells to engage the cell cycle in vitro and in vivo.However, our data suggested a delay and/or inhibition of podocytesentering mitosis (146). To test the possibility that this observationcould be due to a defect in the G2/M checkpoint, cultured podocyteswere exposed to antibody with and without a complement source.Sublytic C5b-9 injury caused a marked increase in the cell cycleinhibitor p53, and this was also accompanied by an increasein p21. This was accompanied by a delayed entry into mitosis.An increase in p53 and p21 was also shown in vivo in the PHNmodel of C5b-9 induced podocyte injury.
Follow-up studies showed that sublytic C5b-9 induced DNA damagein podocytes in vitro and in vivo. Moreover, C5b-9 increasedthe levels of checkpoint kinase-1 and -2 protein levels, whichhave been shown to arrest cells at G2/M. Taken together, theseresults suggest that the reduction in podocyte mitosis aftersublytic C5b-9 induced injury is due to DNA damage.
Mechanical Stretch Reduces Podocyte Proliferation.
Glomerular disease is initiated by specific types of injuryto individual glomerular cell types. However, regardless ofthe inciting injury, studies have shown that the common pathwayto progressive glomerular scarring is an increase in intraglomerularcapillary pressure, also known as glomerular hypertension (147).Indeed, lowering intraglomerular pressure with ACE inhibitorsand/or angiotensin receptor blockers reduces the progressionof glomerular diseases, including diabetic nephropathy (94).One of the consequences of increased intraglomerular pressureis increasing mechanical stretch on resident glomerular celltypes (148). Studies have shown that applying mechanical stretchto glomerular cells is a useful model to study the effect ofstretch on these cell types. Applying mechanical stretch tocultured mesangial cells activates a variety of signaling pathwaysand leads to increased proliferation (149). In contrast, mechanicalstretch decreases podocyte proliferation, and the decrease incell number was not due to apoptosis (150).
Recent studies have shown that when podocyte were grown in serum(a source of growth factors), stretch decreased the levels ofcyclins D1, A and B1 and cdc2 in cultured podocytes (150). Moreover,in cultured mouse podocytes, stretch also increases the levelsof specific CDK-inhibitors. Stretch caused an early increasein p21, followed by an increase in p27 at 24 h and a delayedincrease in p57 at 72 h (150). In contrast to the growth arrestseen in wild-type cells exposed to stretch, p21Cip1 -/- podocytesexposed to stretch continued to proliferate. These results showthat a role for CDK-inhibitors in limiting the podocyte’sproliferative capacity after stretch, and may explain in partwhy podocytes do not proliferate in states of increased intraglomerularpressure.
The studies discussed above show that podocytes typically tryto maintain their differentiated, specialized, and quiescentphenotype at all costs, even to the detriment of renal function.The inability to readily proliferate and replace those lostdue to apoptosis or detachment results in a "nude" basementmembrane, which leads to glomerulosclerosis. The notion thatpodocytes undergo "compensatory" hypertrophy to cover the "nude"areas has been proposed. However, with time, podocyte hypertrophyis detrimental, and this uncompensated state leads to scarring.Nagata and coworkers (151) recently showed that podocyte hypertrophywas mediated by specific CDK-inhibitors.
In summary, our understanding of podocyte biology has increasedsignificantly in the past few years, and we are learning aboutnew proteins that are specifically expressed in this cell typeand may underlie certain diseases that we previously classifiedas "idiopathic." The molecular mechanisms leading to podocyteeffacement are now better understood, as is the response toinjury. As more investigators continue to focus on podocytes,it is likely that future therapeutic targets will be identified,which will improve the renal survival of patients with podocytediseases.
Acknowledgments
This work was supported by Public Health Service grants (DK34198,DK52121, DK51096, DK56799, and DK57683), George M. O’BrienKidney Center Grant (DK47659), and the Juvenile Diabetes ResearchFoundation. Due to space limitations, a number of interestingpublications could not be mentioned herein.
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Introduction
Molecular Anatomy of the...
-actinin-4, talin, and vinculin (1). The actin filament bundles form arches between adjacent FP of the same podocyte (2). Figure 1 is a schema of our current understanding of the molecular composition of the cytoskeleton in podocyte FP. Importantly, the actin filaments are connected to the underlying GBM at focal contacts via an 
1 integrin complex (3,4). The bends of the actin filament arches appear to be connected directly to the microtubules of the major processes (own unpublished results). FP are anchored to the GBM via 
