I. INTRODUCTION

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The podocyte is a most spectacular cell type. Its location, its architecture, and its relevance are unique. Almost ignored in renal research for decades, since the mid 1990s, there has been an outset in podocyte research worldwide. However, still today not a single function defined in classic physiological terms can be solidly assigned to the podocyte. We suppose that it functions as a specific pericyte counteracting the high transmural distending forces permitting the high-pressure perfusion of glomerular capillaries, but we do not have any direct evidence. We suppose that the podocyte is crucially involved in establishing the specific permeability properties of the glomerular filter, but we do not know the details. We suppose that the podocyte is responsible for the continuous cleaning of the filter, but we know very little about how this function is carried out.

On the other hand, the evidence accumulates that failure of the podocyte decisively accounts for the initiation of progressive renal diseases, as well as for the maintenance of the progression to end-stage renal failure. With the prolongation of our life expectancy, the incidence of chronic renal failure rises dramatically along with an enormous increase of the burden to healthcare budgets worldwide to provide the expensive renal replacement therapies including dialysis and transplantation. Therefore, all research efforts are welcomed to reach a better understanding of this cell type and to develop rules on how to protect podocytes from injury. We hope that this review will stimulate research in this field.

	II. DEVELOPMENTAL ASPECTS

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A nephron of the permanent kidney develops from the mesenchymal metanephric blastema by induction through the ureteric bud. The tips of the branching ureteric (collecting) ducts induce the clustering of individual mesenchymal cell aggregates that convert to an epithelial phenotype. Such a cell aggregate represents a nephronanlage that undergoes many mitotic cycles and differentiation stages, connects with the duct, and subsequently generates a nephron. In the very beginning, this process centers around the development of the glomerulus and is generally divided into the following stages: vesicle, comma and S-shaped, glomerular capillary loop, and maturing glomerulus (54, 171, 396).

The vesicle is the first epithelial structure consisting of polarized cells and is surrounded by a basement membrane. On one side it joins with the ureteric bud, and a continuous lumen is formed between the vesicle and the duct. On the opposite side a cleft appears within the growing nephronanlage, producing a comma-shaped or S-shaped profile (depending on the section plane). Figure 1 shows a rat kidney S-shaped body. The lip beneath this cleft is established by a prominent crescent-shaped layer of epithelial cells which ultimately differentiate into podocytes.

View larger version (83K): [in this window] [in a new window]   Fig. 1. Rat kidney, S-shaped body is shown. In this stage of development, the podocyte precursor cells are arranged in a crescent-shaped layer of epithelial cells. The cells broadly attached to each other laterally. Mitotic figures are frequently encountered (B). The basal aspect of the cells faces the vascular cleft (asterisk) that contains precursor cells of the mesangium and endothelial cells. Apically the podocytes protrude into the future Bowman's space (BS). A: light microscopy, magnification ×~5,000. B: transmission electron microscopy, ×~13,000.

The podocyte precursor cells are simple, polygonal cells. They vividly multiply. During this early stage of glomerular development, the presumptive podocytes are connected by apical junctions. Structurally, these junctions resemble tight junctions, which express ZO-1 (401) but also desmosomal proteins (135). The expression of podocalyxin and of the cell junction protein ZO-1 commences at this stage (401).

As podocytes enter the subsequent capillary loop stage, they begin to establish their characteristic complex cell architecture, including the formation of foot processes and of a slit membrane. At this stage, desmosomal proteins disappear (135), ZO-1 protein migrates from its apical to a basal location where the slit membrane develops (192, 402). In conjunction with the appearance of the slit membrane-associated proteins, nephrin (192), podocin (49) and CD2AP (256) are expressed. This phenotypic conversion is associated with the loss of mitotic activity (302) (see sect. IV) and accompanied by the expression of several other specific proteins, including the actin-associated protein synaptopodin (293), the major surface protein podocalyxin (402), a podocyte-specific membrane protein tyrosine phosphatase, glomerular epithelial protein 1 (Glepp 1) (484), and the final intermediate filament protein vimentin (302).

A large number of transcription factor genes have been identified that are involved in the induction of the renal vesicle and subsequent nephrogenesis. (The following summary excludes those genes that most likely play their major role earlier in ureteric bud branching; for more comprehensive information, see Reference 54.) The Pax-2 gene, a mammalian homeobox gene, encodes for a transcription factor that is essential for the conversion of cells of the metanephric mesenchyme to the renal vesicle. Gene knock-out experiments show that in the absence of this factor no formation of the vesicle occurs (98, 376, 465). The further differentiation of these early epithelial cells and their maturation to podocytes is then correlated with the decrease of PAX-2 and the rise of WT-1 expression, suggesting that WT-1 may negatively regulate PAX-2 expression (381). Downregulation of PAX-2 appears as a prerequisite to allow podocyte differentiation that is governed by WT-1. Podocyte precursor cells in the vesicle and subsequent stages strongly express the WT-1 protein, and this expression remains a specific marker of podocytes during the entire ontogeny and in the adult (294, 296). Dominant mutations in WT-1 are associated with the Denys-Drash and Frasier syndromes, manifested by glomerulopathy, mesangial sclerosis, and male pseudohermaphroditism (29, 273).

Lmx-1b, a Lim homeobox gene, is first expressed in podocyte precursor cells of the S-shaped body and continues to show this expression throughout nephrogenesis (59). Homozygous Lmx-1b mutant mice have podocyte and glomerular basement membrane (GBM) abnormalities at birth, demonstrating an essential role for glomerular development (59). Mutations of the human Lmx-1b gene are responsible for the nail-patella syndrome, which is frequently associated with glomerulopathy (99).

Pod-1, a basic helix-loop-helix protein like WT-1, is expressed in podocytes during glomerular development and appears to be involved in differentiation of this cell type (355).

The structural prominence of the podocyte in early nephrogenesis is emphasized by findings pointing to a central role of this cell type in regulating the development of the entire renal corpuscle. The main players in this process appear to be angiogenic factors. The development of the glomerular capillaries and mesangium starts from cells that are found within the cleft above the podocyte layer in the comma-shaped body. These cells are derived from surrounding mesenchymal cells including sprouts from existing vessels (392, 396).

There is increasing evidence that several signaling systems are involved in this recruitment and differentiation process. First the vascular epidermal growth factor (VEGF)-flk-1 axis becomes active. VEGF is expressed in the podocyte precursor cells of the comma-shaped body, and its receptor flk-1 (VEGFR2) is found on endothelial cells in the cleft and adjacent mesenchyme (211, 469), suggesting that VEGF initiates the penetration of endothelial sprouts into the cleft. Indeed, studies using genetic and immunological strategies to block VEGF in vivo (138, 211) have confirmed that VEGF is indispensable for glomerular capillary growth. Surprisingly, after completion of nephrogenesis, podocytes continue to express VEGF; here, the factor has been postulated to play a role in maintenance of endothelial fenestrae (112). Very recently, experiments blocking the VEGF expression after birth have shown that within a few weeks glomerular capillaries disappear and the tuft collapses (211).

Podocytes seem not to express VEGFR1 or VEGFR2 receptors, but they express neuropilin-I, a potential coreceptor for the VEGFR2 receptor. Thus podocytes may bind VEGF, but the functional significance of the expression of neuropilin I on podocytes is not yet clear (158).

In concert with VEGF, angiopoietins (85) play a major role in glomerular capillary development (497). Renal endothelial cells, including those within the cleft of the nephronanlage, express the TIE-1 receptor; its ligands are ANG I and ANG II. Recent evidence suggests that ANG I is derived from podocytes and ANG II from mesangial cells. Both bind to the same receptor (Tie-1), but ANG II fails to activate it; hence, ANG II is an inhibitor of ANG I (497, 511, 512).

A further important regulatory system in early glomerular capillary development is represented by the Eph/ephrin family of membrane receptors and counterreceptors (493). Ephrin-B2 expression is first prominent in the podocyte progenitor cells adjacent to the vascular cleft, and a corresponding receptor, Eph-B4, is expressed on endothelial cells (449), suggesting that cell-to-cell interactions may play an important role in glomerular microvascular assembly.

Subsequent to their recruitment, endothelial cells start to produce platelet-derived growth factor (PDGF)-BB, and the PDGF receptor  becomes expressed by mesangial precursor cells. The function of this axis is required for proliferation and assembly of glomerular capillaries and mesangium (42, 253, 440). Transforming growth factor (TGF)-1 actions are also implicated in this process of stabilizing glomerular vasculature (258).

After establishment of a glomerular vasculature, signaling events in the opposite direction appear necessary for the final maturation of podocyte function. Production of GBM components by podocytes and its maturation are marked by the replacement of laminin-1 with laminin-11 (consisting of -5/-2/-1 chains) as well as by the replacement of -1, -2 chains of type IV collagen by collagen -3, -4, and -5 (IV) chains characteristic for the mature GBM (282, 284). Recent work from grafting experiments (447) suggests that factors emerging from endothelial cells mediate the switch to laminin-11 [and possibly also to collagen -3, -4, -5 (IV)] production in podocytes. As shown in several mutant mouse models (73, 160, 282, 285, 314), failure of these changes are all associated with severe structural (podocyte, GBM) and functional (protein leakage) injuries.

	III. STRUCTURE OF PODOCYTES

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Podocytes are highly differentiated cells. They have a voluminous cell body, which bulges into the urinary space. The cells give rise to long primary processes that extend toward the capillaries to which they affix by numerous foot processes (including the most distal portions of primary processes). The foot processes of neighboring podocytes regularly interdigitate, leaving between them meandering filtration slits that are bridged by an extracellular structure, known as the slit diaphragm. The filtration slits are the site of convective fluid flow through the visceral epithelium. Figure 2 shows the urinary side of the capillary wall, which is covered by the highly branched podocytes.

View larger version (196K): [in this window] [in a new window]   Fig. 2. Scanning electron micrograph of normal rat glomerular capillaries. The urinary side of the capillary wall is covered by the highly branched podocytes. Rat kidney, magnification ×~6,000.

Podocytes are polarized epithelial cells with a luminal or apical and a basal cell membrane domain. The latter corresponds to the sole plates of the foot processes, which are embedded into the GBM. The border between the basal and luminal membranes is represented by the slit diaphragm. The luminal membrane and the slit diaphragm are covered by a thick surface coat that is rich in sialoglycoproteins, including podocalyxin, podoendin, and others, which are responsible for the high negative surface charge of the podocytes (173, 395) (for details, see sect. XIIB). Frequently, the apical surface gives rise to a few fingerlike protrusions that float within Bowman's space; under inflammatory conditions, those processes may be dramatically increased in number and length (see below). The basal cell membrane, i.e., the membrane covering the soles of the foot processes, mediates the affixation to the GBM (see below); it regularly contains coated pits (201). Both membranes, apical and basal, are heterogeneous with respect to their lipid composition; both contain densely distributed cholesterol-rich domains (331, 386), corroborating the finding that specific membrane proteins of podocytes are obviously arranged in rafts (409, 432).

The cell body contains a prominent nucleus, a well-developed Golgi system, abundant rough and smooth endoplasmic reticulum, prominent lysosomes, and many mitochondria. In contrast to the cell body, the cell processes contain only a few organelles. The density of organelles in the cell body indicates a high level of anabolic as well as catabolic activity. In addition to the work necessary to sustain the structural integrity of these complexly shaped cells in the adult, most, if not all, components of the GBM are synthesized by podocytes.

A well-developed cytoskeleton accounts for the unique shape of the cells and the maintenance of the processes. Figure 3 shows the organization of the cytoskeleton of podocyte processes. In the cell body and the primary processes, microtubules and intermediate filaments, such as vimentin and desmin, dominate, whereas microfilaments, in addition to a thin cortex of actin filaments beneath the cell membrane (97), are densely accumulated in the foot processes. Here they are part of a complex contractile apparatus (97). As seen from reconstruction studies (388), the microfilaments form loop-shaped bundles, with their limbs running along the longitudinal axis of the foot processes. The bends of these loops are located centrally at the transition to the primary processes and may readily be connected to the microtubules by the microtubule-associated protein  (389) (Fig. 3). Peripherally, the actin bundles appear to be anchored in the dense cytoplasm associated with the cell membrane of the soles of foot processes (241) (for details, see below).

View larger version (37K): [in this window] [in a new window]   Fig. 3. Cytoskeleton of podocyte processes, shown in several schemes. A: cross section through a glomerular capillary loop. The foot processes, which arise from primary processes (2 are depicted in cross section) and cover the outer aspect of the glomerular basement membrane, contain a complete, actin-based contractile apparatus (shown in red). The primary processes contain longitudinally arranged bundles of microtubules (shown in green). Details of the arrangement of cytoskeletal elements in podocyte processes are seen in B-D. B: view from above. C: section of foot processes parallel to and, in D, perpendicular to, the longitudinal axis of foot processes (C corresponds to the w-x line, D to the y-z line). Two major processes (1 in white, 1 in yellow) with their foot processes are shown. The actin filaments (red) of foot processes form continuous loops that end in the foot process sole plates. Centrally, at their bend, they are in close association with microtubules (green) that run longitudinally in the major processes. The microtubule-associated protein tau (shown in blue) has been localized along the bends of the microfilament bundles and is suggested to mediate connections between the microtubules and the actin filament bundles. [Modified from Sanden (388).]

The filtration slits have a constant width of ~30-40 nm (133, 374) and are bridged by the slit diaphragm. Based on transmission electron microscopy findings in chemically fixed, tannic acid-stained material, Rodewald and Karnovsky (374) have published a model of the substructure of the slit membrane. It consists of rodlike units, connected in the center to a linear bar, together forming a zipperlike pattern. The rectangular pores have the approximate size of an albumin molecule. Thus, in an en face view, the slit membrane has the width and appearance similar to an adherent junction.

	IV. PODOCYTE CELL CYCLE CONTROL

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As a consequence of the high degree of differentiation of podocytes, it was postulated that they, in analogy to neurons, are unable to proliferate (229). An inability to repopulate a damaged glomerulus with functional podocytes was in good agreement with the progressive ultrastructural lesions seen in podocytes during filtration barrier failure.

Systematic analysis of the cell cycle regulatory molecules in podocytes revealed indeed a tight control of cell cycle quiescence, in sharp contrast to the proliferative capacity of the neighboring mesangial cells (419). An escape of the podocyte from cell cycle blockade results in a disruption of glomerular architecture followed by a rapid decline of renal function, as demonstrated by the deleterious course of collapsing and human immunodeficiency virus (HIV) nephropathy (31, 299, 415).

In this section we discuss the origin of the proliferation block during glomerulogenesis and describe the cell cycle regulation profile in the intact glomerulus and in disease states with and without podocyte proliferation.

The deleterious consequences of uncontrolled cell proliferation or death for every multicellular organism have resulted in an evolutionary conserved, tightly regulated control mechanism for cell cycle proliferation and apoptosis. Cell proliferation is divided into discrete steps of the cell cycle, with the progression into the next step occurring in a sequential and synchronized manner. Quiescent, nondividing cells in the G₀ phase enter the cycle with the G₁ transition, progressing through the DNA duplicating step in the S phase. After the G₂ phase, mitosis occurs in the M phase. After completion of the cycle, cells can enter into the next round of duplication or into the resting G₀ phase. The cell cycle progression is regulated by cyclin and cyclin-dependent kinase (CDK) complexes. CDK activity is controlled by cyclin-dependent kinase inhibitors (CKIs). Each step in the cell cycle is initiated and controlled by a specific set of cyclins, CDKs, and CKIs. However, the p21Cip/Kip family of CKIs, including p27 and p57, can inhibit various cyclin-CDK complexes and are therefore responsible for induction and maintenance of cell cycle quiescence (for an introduction to cell cycle regulation from a nephrologists perspective, please refer to Ref. 419). Entry into the cell cycle can have three consequences for a cell: 1) cell proliferation, if cyclin expression is followed by repression of the corresponding CKIs; 2) cellular hypertrophy, if the cell cycle entry with increased protein synthesis is not followed by DNA synthesis and cell division is blocked as a consequence of an increase in CKIs leading to a G₁/S arrest; and 3) cellular apoptosis as the abortive default pathway of cell cycle progression. The balance of these three pathways determines the net effect on cell number and size in a given tissue.

An overview of the control elements of the cell cycle relevant for this review is given in Figure 4.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Schematic diagram of cell cycle regulation. Progression from G0 to M phase is advanced by cyclins and cyclin-dependent kinases (CDK). CDK inhibitors can block further progression, leading to podocyte hypertrophy. Only elements relevant for this review are shown.

In the S-shaped body state of glomerulogenesis, the "presumptive" podocytes still express markers of a proliferative tissue including proliferating cell nuclear antigen (PCNA) and Ki-67 together with cyclin A and B1 (300). With transition to the capillary loop state, a fundamental phenotype switch occurs with induction of mesenchymal intermediate filaments (see sect. IX), induction of a series of mature podocyte markers (see sect. II), and the development of foot-process interdigitation. In parallel to these changes, the disappearance of cell cycle promoters and a reciprocal upregulation of the cell cycle inhibitors CKI p27 and p57 occurs (32, 70, 297, 300), coinciding with the proliferation arrest of podocytes seen in mature glomerulus. These observations are consistent with the notion of cell cycle quiescence induced by an upregulation of CKI, like p27 and p57, in podocytes as a prerequisite for terminal differentiation. The knock-out of the CKI p57 mice was shown to induce a less differentiated podocyte phenotype, providing the first experimental evidence for involvement of CKIs in podocyte differentiation (513).

In the mature glomerulus, podocytes have a low level of DNA synthesis and do not readily proliferate under normal conditions nor in a wide variety of renal diseases (232, 239, 335, 371). The inability of podocytes to undergo proliferation in most adult diseases is most likely the consequence of a robust expression or even upregulation of the CKI inhibitors p21, p27, and p57 with disease progression.

Shankland et al. (416) were able to show in a Heyman nephritis model that podocytes do upregulate cyclin A and CDK2 in response to immune-mediated damage. However, a parallel induction of CKI p21 and p27 could effectively blocked entry into the next steps of the cell cycle. According to the above-stated paradigm, this should result in cellular hypertrophy, which is indeed a key finding in the response of podocytes to glomerular damage (see sect. XIII for a detailed discussion). Studies examining the response of rat podocytes to sublytic concentration of complement as an in vitro model of membranous nephropathy showed an augmentation of growth factor-induced DNA synthesis in response to C5b-9 (418). C5b-9 resulted in an increase in the S phase cyclin A and CDK2 and a decrease in CKI p27, but not p21. Furthermore, the M phase proteins cyclin B and cdc2 were repressed. This cell cycle profile led to an increased cellular DNA content without cell proliferation, confirming the above-mentioned in vivo observations of an arrest in podocytes at the G₂/M phase (418). The critical role of the CKI p21 and p27 for a quiescent podocyte phenotype could be demonstrated in p21 and p27 knock-out mice. In these mice, glomerular damage not only allowed podocytes to enter the cell cycle and increase DNA content but also to complete cell division, resulting in a increased cell number in Bowman's space (330). The cells in the Bowman's space had lost differentiated podocyte marker (WT-1 and GLEPP-1), and the animals showed an aggravated disease course (208).

The alterations seen in podocyte damage in the p21 and p27 / mice resembled the human glomerular diseases with podocyte proliferation, namely, HIV nephropathy (31), collapsing glomerulopathy (298, 472), and the cellular variant of focal-segmental glomerulosclerosis (80). Intact human podocytes differ from murine podocytes in that they do not express p21 but show a robust signal for p27 and p57 (70, 300, 415).

In a systematic analysis of podocyte marker in HIV and collapsing nephropathy, Barisoni et al. (31) showed a severe dysregulation of the cellular phenotype in proliferating podocytes with a loss of podocyte process structure accompanied by a loss of WT-1, Glepp-1, podocalyxin, CALLA, C3b receptor, and synaptopodin. In parallel, an induction of the proliferation marker Ki-67 could be observed (31). In nonproliferative minimal change or membranous nephropathy, none of the above markers was altered compared with healthy controls (32). A comparable podocyte phenotype can be induced in HIV-1 transgenic mice, directly implicating HIV in podocyte dysregulation. Conditionally immortalized podocytes from the HIV-1 transgenic mice showed increased proliferation without contact inhibition. This model system should allow a detailed functional dissection of the podocyte dysregulation by HIV (405).

In comprehensive studies of CKIs in human disease, p27 and p57 levels remained unchanged in minimal change disease and membranous nephropathy (32, 415), blocking podocyte proliferation despite DNA synthesis (25). In collapsing glomerulopathy, cellular focal segmental glomerulosclerosis (FSGS), and HIV nephropathy, p27, p57, and cyclin D1 were lost in areas of podocyte proliferation. A marked increase of the proliferation marker Ki-67 and, surprisingly, of the CKI p21 was seen in the podocyte proliferative diseases. The role of p21 under these conditions is not clear; however, it has recently been recognized that low-level p21 induction can enhance proliferation (341), and a transient p21 increase was seen during human glomerulogenesis in podocyte progenitor cells (300). That podocyte proliferation in collapsing nephropathy is a rapidly deleterious and not a regeneratory pathway has been emphasized in a recent clinicopathological follow-up study (247).

Growth factors and cell matrix interactions have been reported to influence podocyte behavior (see sects. XI and XIII). Several studies implicate an activation of the TGF- pathway in podocyte damage in vivo and have shown its antiproliferative effects in vitro (9, 123, 131, 417, 494). A peculiar finding highlighting the unique cell cycle regulation of podocytes in the kidney is the generation of binucleated podocytes after systemic application of basic fibroblast growth factor (bFGF) in vivo. These polyploid podocytes had to go through a full cell cycle including mitosis but appear unable to undergo cytokinesis (125, 234). This cell cycle arrest could also explain contradictory conclusions reached on the ability of podocytes to proliferate in response to cytokines (125).

In analogy to neurons, one could speculate that the complex cytoskeleton effectively inhibited the completion of cell division. For reentry into the cell cycle, a deconstruction of the highly structured cytoskeletal organization in podocytes would be required. However, loss of the actin filaments in the foot processes would abolish glomerular permselectivity. Podocyte cell cycle quiescence appears therefore to be a prerequisite for a functional glomerulus.

The third option during cell cycle progression is the entry into the apoptotic pathway of cell removal. Loss of podocytes correlates closely with the degree of progression in type II diabetic Pima Indians, with fewer podocytes per glomerulus in the rapidly progressing group compared with slow progressors (252, 279, 336, 445). In puromycin-induced nephrosis in rats, a close correlation between the degree of glomerular damage and the reduction of podocyte number per glomerulus was observed (209). Because detachment of cells is able to induce cell death, a process termed anoikis (129), disruption of podocyte-GBM interaction could be a critical final event in podocyte damage (see sects. VIII and IX). Hara and co-workers (156, 157, 305) have indeed detected cells positive for podocyte marker in the urine of a variety of renal diseases. Interestingly, a reduction in urinary podocyte excretion was found with improved glycemic control in early diabetic nephropathy (304).

In the first study of the molecular mechanism of apoptosis induction in podocytes, Schiffer et al. (398) could unequivocally demonstrate a wave of apoptosis in the early stages of glomerulosclerosis in TGF-1 transgenic mice. TGF- and the downstream TGF- signaling molecule Smad7 were able to induce apoptosis in cultured podocytes. TGF- required p38 mitogen-activated protein (MAP) kinase and caspase-3, whereas Smad7 blocked the nuclear translocation of NF-kappaB. Involvement of CKI inhibitors in podocyte apoptosis can be concluded from the increased incidence of podocyte apoptosis in glomerulonephritis in p21 / mice (208). As these mice exhibit significant podocyte proliferation, the activated proapoptotic pathway may be quite different from that seen in cell death in quiescent podocytes.

In summary, podocytes in the adult kidney are unable to undergo regenerative proliferation to compensate for a loss of podocytes or an increase in GBM surface area. As a consequence of the strict block in cell division, podocytes can progress to the cell cycle and can even undergo nuclear division in a variety of glomerular diseases, but not cell division. The CKIs p21, p27, and p57 appear to be responsible for this G₁/S transition block, inducing the considerable podocyte hypertrophy seen in progressive renal failure.

An escape of podocytes from the strict cell cycle control is not a regenerative, but rather a disastrous event, causing rapid glomerular destruction.

	V. CELL CULTURE OF PODOCYTES

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In the past, due to the lack of available specific morphological and immunological markers for cultured podocytes, it was uncertain whether outgrowing glomerular cells were of a visceral or parietal origin. One important question was to address whether podocytes would be seriously damaged or survive at all after the culturing of glomeruli and which structural changes of podocytes might occur in situ. By using transmission and scanning electron microscopy, Norgaard (316) investigated the effect of culturing isolated glomeruli on podocyte morphology. Just 8 h after glomerular culture, a broadening of podocyte foot processes, slendering of the primary and secondary foot processes, and the appearance of many microvilli occurred. Thereafter, slit pores became narrowed and were often occluded by a tight junction. Structures resembling slit diaphragms displaced from the basement membrane occurred. After 2 days, retraction of foot processes was completed and slit pores disappeared. Thereafter, podocytes rounded up and were in contact with each other and found in a single-layered epithelium. In addition, podocytes showed a decreased iron staining, indicating a reduction of their anionic proteins on their cell surfaces. Thus it has been suggested that the phenotype of podocytes in culture resembles the phenotype of the podocytes in the fetal glomerulus and that the morphological changes are similar to those seen in injured podocytes (316, 317). Subsequent studies have shown that the first cells to grow out of the glomerulus were of epithelial origin (224).

Two early outgrown glomerular cell types have been identified in several studies: a small, polyhedral ciliated cell which grows in colonies with the cells joined by junctional complexes (41, 78, 224, 276) and a second very large, often multinucleated cell (317, 506). According to the structural resemblance with glomerular cells in situ, some authors have suggested that the first cell type is derived from the parietal epithelium of Bowman's capsule, whereas the second is derived from the visceral epithelium (56, 317, 506).

However, polyhedral-shaped cultured glomerular epithelial cells have been shown to react with megalin, 27 A, and Fx1A fraction, antibodies which recognize antigens in the glomerulus that are only expressed by podocytes (65, 78, 276). In addition, in a rat podocyte cell line and a simian virus 40 (SV40)-transformed human podocyte cell line, which show a cobblestone appearance, several podocyte-specific antigens have been detected, indicating that cells were of visceral origin (19). In contrast, a lack of expression of podocyte markers has been found in early cultured glomerular epithelial cells (169). By analyzing the pattern of several podocyte-specific antigens on cultured glomerular epithelial cells, two groups came to the conclusion that most if not all glomerular-derived cells with a polygonal appearance were of parietal origin (169, 505). Some of these contrasting findings in morphology and antigen presentation of glomerular-derived cells might be due to the different techniques and conditions used for culturing glomeruli. Another explanation for the different observations is the fact that podocytes change their characteristics in cell culture. Reiser and co-workers (294) observed that glomerular-derived proliferating cells in the first cell culture passage exhibit a cobblestone appearance and that they express WT-1 and O-acetylated ganglisoide, specific markers of podocytes in vivo, but not synaptopodin, a marker of podocyte foot processes in vivo. Four days after reaching confluency, the cobblestone cells began to converge into large arborized cells, which developed processes. These arborized cells exhibited WT-1 and O-acetylated ganglisoide, and they also showed a marked expression of synaptopodin, indicating that these cells possess markers of podocyte foot processes (294). Very recently, the morphology and expression of podocyte markers from cellular outgrowths from descapsulated glomeruli, encapsulated glomeruli, and tubular fragments have been reinvestigated. Cells outgrown from decapsulated glomeruli tend to become elongated to as much as 100-200 µm and possess long thin cytoplasmatic processes, which often overgrow neighboring cells. These cells stained strongly with antibodies against WT-1, synaptopodin, and podocalyxin but not with an antibody against nephrin. In contrast, cells outgrown from encapsulated glomeruli had a cobblestone appearance, and they exhibited lamellipodia. WT-1 and synaptopodin were not detected in cells from tubular fragments, but a weak staining of WT-1 and synaptopodin was detected in pan cadherin positive parietal cells. Thus it has been suggested that parietal epithelial cells might transdifferentiate into podocytes or that alterations of parietal epithelial cells in culture may occur (503). Interestingly, 27-kDa heat shock protein (hsp27), P-31 antigen, and vimentin, proteins which are only expressed in podocytes in vivo, are also expressed in cultured parietal epithelial cells and even in cultured tubular epithelial cells. In outgrowing cells from encapsulated glomeruli, large irregularly shaped cells could be also observed after ~6 days, but their staining pattern was identical to cobblestone cells. Therefore, the authors suggested that these cells might have been converted from parietal epithelial cells (503).

Differentiated podocytes in primary culture show little proliferative activity, and thus it is difficult to propagate a cloned podocyte cell line or to perform experiments that require a large number of cells. In addition, some contamination by other glomerular cell types cannot be excluded in early cell culture passages. Therefore, a conditionally immortalized podocyte cell line has been propagated from a transgenic mouse expressing a temperature-sensitive SV40 large T antigen (295). Cells that are grown under nonpermissive conditions, i.e., at 37°C, stop growing and exhibit many morphological and immunologic properties of differentiated podocytes, i.e., they are arborized and express synaptopodin (295). Very recently, a conditionally immortalized human podocyte cell line has been established by transfection with a temperature-sensitive SV40-T gene. At the permissive temperature of 33°C, these cells grew in a cobblestone morphology. Differentiated human podocytes that were grown at 37°C expressed markers of differentiated podocytes in vivo, including nephrin; podocin; CD2AP and synaptopodin; ZO-1; -, -, and -catenin; and P-cadherin. Thus this cell model seems to be a good in vitro tool for studying human podocyte biology (387). Figure 5 shows undifferentiated and differentiated, conditionally immortalized human podocytes expressing the slit membrane proteins P-cadherin, podocin, and nephrin.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Cell culture of podocytes. Conditionally immortalized human podocytes in culture are shown. Left panels: cells grown under permissive conditions (at 33°C). Right panels: cells grown under nonpermissive conditions (at 37°C). Only differentiated podocytes under permissive conditions express the slit membrane proteins P-cadherin, nephrin, and podocin (for details, see sects. V and VII). (From Saleem M. A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. J Am Soc Nephrol 13: 630-638, 2002. Copyright 2002 Lippincott Williams & Wilkins.)

In conclusion, at this point there is no doubt that podocytes with an arborized phenotype can be propagated in primary culture and that glomerular cells that first show a cobblestone appearance can convert into arborized cells which then express markers of podocytes and even of the slit diaphragm in vivo. There is the possibility that parietal cells can convert into arborized cells. However, compared with immortalized podocytes in cell culture, these cells seem to express WT-1 and synaptopodin much weaker, and they do not express nephrin (503). Further studies have to show whether these cells, compared with podocytes in culture, may be able to express other markers of podocytes to study the process of transdifferentiation of these cells. It is easier, compared with the investigation of the podocytes in situ, to investigate biological functions of podocytes in vitro. However, it is obvious that like other cell types, podocytes in monoculture cannot mimic completely the complex in vivo characteristics of podocytes. Many cellular functions change during culturing of cells, and therefore, results obtained from podocytes in culture have to be interpreted with care.

	VI. GENE EXPRESSION ANALYSIS OF PODOCYTES

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The interest in mRNA expression analysis in glomeruli has been fueled by the rapid developments in molecular biology. These novel techniques could offer information about nature and prognosis of disease processes activated in podocytes. The real-time polymerase chain reaction (PCR) allows highly accurate template quantification from minimal tissue samples (67, 119). The cDNA array technology displays gene expression patterns of thousands of mRNAs in a single reaction, but requires considerable amounts of starting material (11).

Reverse transcription (RT) PCR-based approaches have been used for some time for gene expression analysis of microdissected specimen in human and experimental renal disease (51, 351). Several technical problems including limitation in mRNA quantity, available quantification systems, and biopsy population could be recently overcome allowing for high-throughput analysis of a series of cDNAs (68). Also, laser microdissection and RNA expression analysis have been shown to be feasible on glomerular cross section of frozen or Formalin-fixed material, detecting, i.e., WT-1 and synaptopodin as podocyte-specific cDNAs (67, 68). A summary of a current protocol applicable for routine application in a multicenter setting is described in Figure 6.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Gene expression analysis in microdissected human glomeruli. From a freshly taken renal biopsy specimen, a cortical part is conserved in an RNase inhibitor solution. Glomeruli are obtained by manual or laser-assisted microdissection; RNA is isolated, reverse transcribed, and analyzed by real-time RT-PCR or cDNA array hybridization after linear amplification. Expression profiles can be correlated with clinical parameters and histological alterations. For detailed descriptions, see Cohen and co-workers (67, 68).

The above expression analysis of microdissected glomeruli still contains glomerular endothelial and mesangial cells. The same holds true for a series of experimental systems developed to study the cellular functions of podocytes including whole animal experiments, transfilter organ culture systems, kidney cortex slices in culture, and isolated glomeruli (see above). The selective analysis of glomerular epithelium-derived cells is only possible in cell culture, but even with the latest technologies questions concerning dedifferentiation phenomena remain (see above). As a consequence of these technical limitations, our understanding of the podocyte cell biology in vivo remains incomplete. As an alternative approach, an in vivo single-cell analysis has been established (404).

The amount of RNA in a single cell is estimated to be 0.1-1 pg and is difficult to handle experimentally. A modification of the PCR was used by Lambolez et al. (245a) for single-cell RT-PCR analysis of cultured neurons. This method can only be applied to tissue allowing access to single cells. The unique glomerular anatomy with the podocytes residing on the outside of the GBM makes this cell type an ideal target for single-cell RT-PCR. Podocytes can be aspirated selectively under visual control with a micropipette preventing any contamination from nearby tissue. With the application of a modified real time RT-PCR, a quantitative approach to podocyte-specific gene expression has been demonstrated (226, 404). A schematic representation of the experimental protocol is given in Figure 7. Combinations of this technique with immunhistology and in situ hybridization should prove advantageous to investigate patterns of gene expression and elucidate the roles of specific genes in podocyte function.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Schematic of single podocyte RT-PCR. Single podocytes are aspirated by negative pressure applied to the interior of the micropipette; RNAs of a single podocyte are reverse transcribed and quantified using real-time RT-PCR technology. For detailed descriptions, see Schroeppel et al. (404) and Kretzler et al. (226).

	VII. GLOMERULAR FILTRATION BARRIER AND SLIT MEMBRANE

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Podocytes form a tight network of interdigitating cellular extensions, called foot processes, which are bridged by so-called "slit diaphragms." Figure 8A shows a view of a podocyte in situ, and Figure 8B shows the composition of the glomerular filter including the porous endothelium, the GBM, and the podocyte foot processes with the interposed slit diaphragm. The filtration barrier is freely permeable by water and small solutes, but to a large extent, the size selectivity of the filtration barrier for proteins is represented by the slit diaphragms of podocytes. There is a good body of evidence that the podocyte slit diaphragm resembles an adherens-like intercellular junction. On grazing sections the slit diaphragm has morphological features reminiscent of adherens junctions, including the presence of a wide intercellular gap and a central dense line (374). Moreover, the slit membrane arises from a tight junction during glomerular development, and the tight junction zonula adherens-associated zonula occludens protein ZO-1 is concentrated along the cytoplasmic surface of the slit diaphragm (401). Our understanding of the molecular structure of the slit diaphragm has been greatly improved in the last few years. Several molecules, including ZO-1 (401), nephrin (379), CD2AP (424), FAT (183), and P-cadherin (368), have all been shown to be expressed within the slit diaphragm, and some of those molecules play a major role for its integrity. A schematic drawing of the molecular equipment of the podocyte foot processes is shown in Figure 9.

View larger version (117K): [in this window] [in a new window]   Fig. 8. A: low-power view of a podocyte in situ. The cell body is connected to the capillary wall by processes that finally split into the interdigitating foot processes on the outer surface of the glomerular basement membrane. In the bottom left, grazing section hits the slit membrane (star). Near the nucleus a characteristic membrane-bound inclusion body (asterisk) is visible; its relevance is unknown. B: the glomerular filter consists of three components: porous endothelium, glomerular basement membrane, and podocyte foot processes with the interposed slit membrane. The endothelial pores are not bridged by a diaphragm. Figure is transmission electron microscopy from a rat. Magnification: A, ×~11,000; B, ×~48,000.

View larger version (85K): [in this window] [in a new window]   Fig. 9. Schematic drawing of the molecular equipment of the podocyte foot processes. Cas, p130Cas; Cat, catenins, CD, CD2-associated protein, Ez, ezrin, FAK, focal adhesion kinase, ILK, integrin-linked kinase; M, myosin; N, NHERF2; NSCC, nonselective cation channel; PC, podocalyxin; S, synaptopodin; TPV, talin, paxillin, vinculin; U, utrophin; z, ZO-1. See text for further explanations. [Modified from Endlich et al. (106).]

ZO-1 is a 225-kDa protein that is localized at the cytoplasmatic face of intercellular junctions. ZO-1 is a member of the membrane-associated guanylate kinase (MAGUK) protein family. ZO-1 may play a role in organizing signal transduction and transmembrane protein complexes. ZO-1 contains three copies of the PDZ domain. It is concentrated in slit diaphragms and has been demonstrated to interact with the components of cell-cell junctions (occludin, -catenin, and ZO-2) and cytoskeletal networks (spectrin, F-actin) (287).

ZO-1 is localized in the podocyte foot processes where it is expressed precisely and continuously at the points of insertion of the slit diaphragms into the lateral cell membrane. During development, ZO-1 appears early at a time when the apical junction complexes between podocytes are composed of a kind of atypical intercellular junction. ZO-1 is connected with these junctions during the time they migrate down the lateral cell surface. These junctions disappear and are replaced by slit diaphragms. It has been suggested that the slit diaphragm is a variant of the tight junction (401). In mice, ZO-1 and nephrin are closely colocalized in the mature glomerulus, but it has been suggested that they may arrive at their final positions from opposite directions (191). In humans, ZO-1 is first expressed in the late S-shaped bodies simultaneously with nephrin. ZO-1 has been detected at the basal margin of the developing podocytes, but also on the lateral surfaces as strings and dots. Like nephrin, ZO-1 is located especially in junctions with ladderlike structures, but in contrast to nephrin, the expression of ZO-1 was more abundant at the intercellular junctions during development (380). In fetal NPHS1 kidneys with Fin-major/Fin-major genotype, expression of ZO-1 did not change, indicating that proteinuria must not ultimately result in an altered expression of ZO-1 (380). However, injection of an antibody against nephrin resulted in a progressive decrease of ZO-1 protein expression in podocytes as early as 1 h after antibody injection. After 5 days, ZO-1 could not be detected in these proteinuric rats. This suggests that this particular antibody against nephrin may induce additional alterations of the signaling pathways in podocytes, leading to a downregulation of ZO-1 protein (193).

B.  Nephrin

1.  Expression of nephrin

NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type, an autosomal recessive disease affecting ~1:10,000 newborns in Finland (205). A total of 50 mutations have been reported so far. The Fin-major (2-bp deletion in exon 2 that results in a frameshift and introduces a stop codon within the same exon) and the Fin-minor (nonsense mutation in exon 26 resulting in a stop at Arg-1109) mutations are the two most commonly found mutations (>90% of all Finnish patients with congenital nephrotic syndrome; NPHS1) (38). The respective gene product of NPHS1, nephrin, a 180-kDa transmembrane protein, is exclusively expressed by glomerular podocytes within the kidney and predominantly localized to the glomerular slit diaphragm (205, 379). N-linked glycosylation has been shown to be important for nephrin folding and thereby plasma membrane localization (500). Mutations of the NPHS1 gene are characterized by massive proteinuria which starts already in utero. Patients with a congenital nephrotic syndrome of the Finish type are treated with early nephrectomy and renal transplantation, but ~20% show recurrence of nephrotic syndrome. The reason for this phenomenon seems to be an increased antibody titer against nephrin in these patients (485).

During development, beginning with early capillary loop stage glomeruli, nephrin expression in mice has been demonstrated on or near intercellular junctions between forming foot processes of podocytes (364). Nephrin could neither be detected in primitive nephric structures, such as comma- and S-shaped bodies, nor in lateral junction complexes between immature podocytes. Nephrin expression has been detected in the earliest slit diaphragm regions between adjacent cells, suggesting that localization of nephrin corresponds with the first appearance of the definitive slit diaphragm during development (96, 132, 364). In humans, nephrin mRNA was first detected in late S-shaped bodies of 13- to 23-wk human fetal kidneys. Immunoelectron microscopy studies show that nephrin is present in junctions with ladderlike structures between the differentiating podocytes. In NPHS1 mutant glomeruli, filamentous ladderlike structures and slit diaphragms are missing, whereas ZO-1 and P-cadherin, two other components of the slit diaphragm, are expressed normally. Thus formation of the ladderlike structures, as well as the slit diaphragms, seemed to be dependent on the expression of nephrin, but early junctions between the developing podocytes at the capillary loop stage were normal. Therefore, nephrin is not needed for the early development and migration of junction complexes at the S-shaped and capillary loop stages (380). On the basis of the structure of the glomerular podocyte slit diaphragm and the electron microscopic localization of nephrin, it was suggested that the NH₂-terminal six immunoglobulin repeats of nephrin form interdigitating zipperlike homophilic interactions. Whether nephrin participates in homophilic interactions remains to be established (379).

In addition to its expression in podocytes, nephrin was also detected in different regions of the brain and in the pancreas (354). In the pancreas, nephrin has been shown to be expressed in the -cells of the islets of Langerhans (338).

Electron microscopic examination of NPHS1 kidneys reveals a thinner lamina densa of the GBM than in controls, but no other structural abnormality of the GBM has been detected so far (22). Inactivation of NPHS1 in mice leads to an immediate massive proteinuria and edema after birth, causing death within 1 day. The kidneys of NPHS1-deficient mice showed effacement of podocyte foot processes and the absence of the slit diaphragm (354).

Nephrin knock-out mice showed a normal structure of the glomerular basement membrane and a normal expression of the glomerular basement membrane proteins type IV collagen and laminin. The expression of podocyte-specific proteins, such as ZO-1, P-cadherin, and FAT, were not changed in nephrin knock-out mice. In the latter study it has been also demonstrated that ₃-collagen IV knock-out mice were normal until week 4, with the exception of sporadic ultrastructural defects in the glomerular basement membrane and a lack of ₃-, ₄-, and ₅-chains of type IV collagen and alterations in the laminin content of the GBM. In contrast, up until week 4, these mice did not show any change of the molecular structure of the slit diaphragm. However, the inception of proteinuria starting at week 5 in ₃-collagen knock-out mice was associated with slit diaphragm alterations, podocyte effacement, and a significant reduction in the expression of nephrin. Thus a decreased expression of nephrin correlates with a loss of glomerular filter integrity (151, 354).

Injection of the monoclonal antibody (MAb) 5-1-6, which recognizes the extracellular domain of nephrin, induces proteinuria (464). Interestingly, after injection of MAb 5-1-6, focal areas of effacement occurred, suggesting that alterations of nephrin are not accompanied by structural changes of the podocytes. It may be speculated that MAb 5-1-6 leads to activation of signaling pathways, resulting in the disturbance of the cytoskeletal architecture of the podocytes (464). After injection of the antibody, the glycosyl matrix of the podocytes remained intact, but the number of anionic sides expressed by heparan sulfate as well as carboxyl groups had decreased (130).

Because a decrease of nephrin expression has been suggested to be associated with proteinuria, studies have been conducted to explore whether nephrin expression is altered in glomerular diseases. With the use of RT-PCR in isolated glomeruli, it has been shown that mRNA expression of nephrin is decreased in glomeruli from patients with minimal change nephropathy and membranous nephropathy (132). Moreover, in membranous nephropathy, minimal change nephropathy, and FSGS, but not in IgA nephropathy, a reduced nephrin staining and a granular redistribution of nephrin has been reported. In membranous nephropathy, granular deposits of nephrin were colocalized with the extracellular immune deposits (96). A changed expression of nephrin has also been reported in experimental animal models such as puromycin aminonucleoside nephrosis, mercuric chloride-treated rats, as well as after injection of an antibody against nephrin, in which the pattern of nephrin IF staining shifted from epithelial/linear to granular (170, 262, 263, 464). During Heymann nephritis, nephrin dissociates from actin, and its expression is reduced in early stages of this experimental membranous nephropathy (510).

In contrast, by using in situ hybridization and immunohistochemistry, Patrakka et al. (345) found neither a change of nephrin mRNA expression nor a decrease or change of the distribution pattern of nephrin in glomeruli from pediatric patients suffering from proteinuria caused by minimal change nephrosis, FSGS, and membranous nephropathy. The authors state that immunohistochemistry and in situ hybridization may not be optimal methods for studying the role of nephrin in proteinuric renal diseases, because both methods may fail to detect small alterations in the amount or distribution of nephrin (345).

Controversial findings have been reported on the expression of nephrin in diabetic nephropathy. In the streptozotocin model of the rat and the nonobese diabetic mouse, mRNA expression of nephrin increased up to twofold during several weeks of follow-up. Glomeruli from diabetic animals showed an additional nephrin localization, i.e., a weaker reactivity at the epithelial aspect while a more intracellular localization was observed (1). In contrast, others have found a decrease in both gene and protein expression of nephrin in streptozotocin diabetic rats (48). The controversial finding might, at least in part, be explained by the fact that nephrin expression was investigated at different time points after induction of diabetes in the studies (4, 8, and 16 wk vs. 32 wk; Refs. 1, 48). In vitro studies show that preformed immune complexes, i.e., agIgG4 and other agents like membrane attack complex (MAC), tumor necrosis factor (TNF), and puromycin, which are known to change the cytoskeletal arrangements in podocytes, induce a redistribution and loss of nephrin from the cell surface of cultured human podocytes. Cytochalasin B, which disorganizes microfilaments, prevents nephrin redistribution on the surface of podocytes (96).

At this point, little is known about the signaling pathways involved in the regulation of nephrin expression. Phorbol 12-myristate 13-acetate has been reported to increase the nephrin expression in A293 cells, indicating that nephrin expression is controlled by protein kinase C (486).

The function of nephrin in slit diaphragm maintenance is poorly understood. Structural analysis suggests that nephrin is a member of the immunoglobulin superfamily (IgCAM). It has an extracellular portion containing eight Ig motifs and one type III-fibronectin domain. The intracellular domain contains eight tyrosine residues, suggesting that nephrin may act as a signaling adhesion molecule (205).

Genetic evidence indicates that CAMs of this immunoglobulin superfamily are important for activity-dependent synapse formation at the neuromuscular junction in Drosophila. They have also been implicated in synaptic remodeling during learning in Aplysia (264, 268). CAMs are also signaling molecules which are involved in modulating cell-cell and cell-extracellular matrix (ECM) interactions, and they induce signal transduction events, which are crucial for cell adhesion, motility, cell growth, and survival (430). Evidence for the hypothesis that nephrin acts as a signaling molecule was raised from mutation analysis studies. A frequent point mutation, a C to T substitution at nt3325 in the NPHS1 gene, results in a nonsense mutation that leads to the deletion of the COOH-terminal 132 of 155 residues of the intracellular domain. The deletion of most of the cytoplasmic domain results in foot process effacement and proteinuria, indicating the importance of this domain (205).

It has recently been suggested that nephrin is associated in an oligomerized form with lipid rafts. Nephrin-containing rafts could be immunoisolated with a 27A antibody which stains a podocyte-specific 9-O-acetylated GD3 ganglioside. After injection of the antibody, foot process effacement and proteinuria occurred, and this was associated with nephrin dislocation to the apical segments of the narrowed filtration slits. In parallel, tyrosine phosphorylation of nephrin was found (432). It has been shown that nephrin and CD2AP are associated with glomerular cell actin. Nephrin seems to traverse a relatively cholesterol-poor region of the podocyte plasma membrane. In addition, a small pool of actin-associated nephrin and CD2AP resides in lipid rafts, possibly in the cholesterol-rich apical region of the podocyte foot processes (509).

Nephrin has been shown to trigger phosphorylation of p38 and c-Jun, which was augmented by podocin. Dominant-negative mutants of small G proteins (Cdc42, Rac1, RhoA) and protein kinases that activate JNK (MKK4) and p38 (MKK3, MKK6) inhibited the nephrin-mediated AP-1 activation. A dominant-negative MEK1 had no effect on the nephrin-mediated AP-1 activation. This suggests that nephrin stimulates p38 and JNK but not ERK1/ERK2. Therefore, nephrin acts as a signaling molecule that can activate MAP kinase cascades (177). Cells overexpressing podocin and nephrin showed an increase in AP-1 activation. Podocin, but not CD2AP, increased nephrin-mediated AP-1 activation to ~40-fold without affecting nephrin protein levels. Truncation of nephrin at amino acid 1160 resulted in the binding of only marginal amounts of podocin, indicating that a sufficient interaction between podocin and nephrin requires the COOH-terminal 81 amino acids of nephrin. This nephrin mutation still promotes a high level of AP-1 activation, but in contrast to wild-type nephrin, podocin failed to augment the AP-1 activation triggered by mutant nephrin. Thus interaction with podocin might stabilize nephrin or recruit nephrin to lipid rafts and thereby increase nephrin signaling in podocyte foot processes (177).

Transgenic manipulation of the podocyte might allow the study of podocyte function in vivo. The nephrin promoter has been considered a good candidate for directing the expression of transgenes in a podocyte-specific manner. A 1.25-kb DNA fragment from the human nephrin promoter and 5'-flanking region has been recently identified. The fragment, which includes the predicted initiation codon and immediate 5'-flanking region of nephrin, directs podocyte-specific expression in vivo. Using this promoter fragment could allow the study of podocyte functions in vivo and help to identify transacting factors that are required for podocyte-specific expression (496). In addition, a 8.3-kb and a 5.4-kb fragment containing the 5'-flanking promoter sequence of nephrin were recently identified and characterized, and mice transgenic for both constructs were generated. NSPH1 transgene showed an expression with a high penetrance in the podocytes and brain (288). Very recently, a glomerular-specific Cre-recombinase transgenic murine line under the control of the NPHS1 promoter was generated, and a successful Cre-mediated excision of a "floxed" transgene specifically in podocytes has been demonstrated in vivo. This murine founder line represents a very good tool for the manipulation of the expression of genes in podocytes in vivo (111).

Very recently, a transmembrane protein containing five extracellular immunoglobulin-like domains structurally related to human nephrin was identified in the mouse. The encoding protein is called NEPH1. NEPH1 is expressed widely and was found within the kidney in podocyte foot processes. Mutation of the NEPH1 locus resulted in effacement of podocyte foot processes and proteinuria, but mice showed no edema. NEPH1 knock-out mice showed a high perinatal lethality, and all mice died between 3 and 8 wk of age. Beyond podocyte foot process effacement, 3-wk-old Neph1 knock-out mice had diffuse mesangial hypercellularity and increased mesangial matrix. It has been suggested that NEPH1 plays a role in cell-cell interactions. NEPH1 may interact with other NEPH1 proteins or it may be the ligand for nephrin (95).

Podocin is a ~42-kDa protein. It is predicted to form a membrane-associated hairpin-like structure with a cytosolic NH₂- and COOH-terminal domain that is typical for stomatinlike proteins and caveolins (49). Very recently, podocin has been shown to be localized on podocyte foot process membrane at the insertion site of the slit diaphragm. It accumulates in an oligomeric form in lipid rafts of the slit diaphragm, and in vivo studies demonstrate that it interacts via its COOH-terminal domain with CD2AP and with nephrin (408). In neurons of Caenorhabditis elegans, mutations of a stomatin homolog MEC-2, a highly homologous protein to stomatin, have been shown to link mechanosensory channels and the microtubule cytoskeleton of the touch receptor neurons, suggesting that stomatin is a molecular link in a stretch-sensitive system (172). Further studies should address whether mechanically gated ion channels are connected to the cytoskeleton of the podocytes and whether podocin links these ion channels to the cytoskeleton of podocytes.

CD2-associated protein (CD2AP) was initially shown as an SH₃-containing protein that binds to the cytoplasmic domain of CD2 and enhances CD2 clustering. It anchors CD2, a T cell and natural killer cell membrane protein which facilitates T-cell adhesion to antigen-presenting cells. CD2AP has a molecular weight of 80 kDa. It contains an actin-binding site located at the NH₂ terminus, a proline-rich region, and three SH₃ domains, one of which interacts with CD2 (103). In developing podocytes at the capillary loop stage, CD2AP is detected slightly later than nephrin (256). Within the glomerulus, CD2AP is only expressed in the podocyte, but it is also found in collecting duct cells and some proximal tubular cells (256). CD2AP knock-out mice develop proteinuria and kidney failure. Glomeruli from 1-wk-old animals show loss of foot process integrity in some glomeruli. At 2 wk of age, almost all podocytes were affected, and in many glomeruli, mesangial matrix deposits occur. This indicates that disease progression in CD2AP knock-out mice begins with podocyte injury leading to a consecutive damage of the mesangium. By 4 wk, glomeruli are sclerotic with increased deposits and distended capillary loops and mice die at the age of 6-7 wk. Coimmunoprecipitation of CD2AP by a nephrin fusion protein indicates that both proteins are associated (424). However, in contrast, in 293T kidney cells used as an overexpression system, neither nephrin nor podocin interacted with CD2AP, which could be due to a lack of a podocyte-specific adapter in this system (177).

Developing CD2AP knock-out mice at first exhibit normal foot processes and slit diaphragms and show no alteration in nephrin expression, suggesting that CD2AP is neither necessary for the correct localization of nephrin at the slit diaphragm nor for the development of intact foot processes. On the other hand, in adult glomeruli of CD2AP knock-out mice, nephrin was not detectable in most glomeruli (256). In the laminin ₂-chain knock-out mouse model of nephrotic syndrome, extensive foot process effacement was associated with aberrant clustering of CD2AP in podocytes. This suggests that CD2AP indeed plays a role in the maintenance of the slit diaphragm (256). Mutations of the human Lmx-1b gene are responsible for the nail-patella syndrome, which is frequently associated with glomerulopathy (99). Podocytes from Lmx-1b knock-out mouse retained a cuboidal shape and did not form foot processes and slit diaphragms. These podocytes showed a reduced expression of the ₄-chain of collagen IV and the slit diaphragm proteins CD2AP and podocin. In addition, there are Lmx-1b binding sites in the putative regulatory regions of both CD2AP and NPHS2. Thus a reduced expression of proteins associated with foot processes and the glomerular slit diaphragm may contribute to the nephropathy associated with the nail-pat