Podocyte Differentiation and Glomerulogenesis

This review will concentrate on the assembly of the glomerulus and the differentiation of the podocyte during that process. The differentiation of the vascular component of the glomerulus will not be emphasized, as it has been the subject of several recent reviews (1–4), except in discussing the influence of the podocyte on the development of the glomerular vasculature. Glomerulogenesis Our understanding of the morphologic features of glomerular assembly, and indeed of the development of the nephron and the entire kidney, owe much to the work of Edith Potter and her colleague Vitoon Osathanondh during the mid 1960s. In a seminal series of articles that are today overly neglected, they provided much of understanding of how the kidney as an entire organ is assembled (5–10). With regard to the glomerulus, it was shown how it emerges from one end of the S-shaped body during the development of the nephron (10). In understanding this process, and indeed understanding the development of the nephron in its entirety, it is essential to convert two-dimensional pictures into a three-dimensional understanding. This is especially difficult as the nephron is not a fully symmetrical structure on all axes. With these caveats in mind, the following is a description of glomerular development. As shown in Figures 1A and 1B, at one end of the S-shaped body a layer of columnar epithelial cells is present, which represents the future visceral epithelial cells or podocytes. The basal aspect of these cells rests on the future glomerular basement membrane (GBM); on the other side of this basement membrane is a cleft between the podocytes and the cells, which will contribute to the tubular portion of the nephron. The cells that will contribute to the glomerular capillaries, i.e., endothelial and mesangial cells, migrate into this cleft. The origin of these cells is a subject of ongoing research and, as mentioned above, has been discussed in recent reviews (1–4). On the other side of this patch of future podocytes, overlying their apical surface, is a lining of thin cells that will become the parietal epithelium, also known as Bowman’s capsule. Figure 1. : Development of the glomerulus. (A and B) show two different sections through an S-shaped body. In both cases, the early capillary loop is observed within the glomerular cleft and the thin lining of cells that will comprise Bowman’s capsule is adjacent to the apical side of the podocytes. In panel B, the cells that will comprise the proximal tubule are also noted. Panels C and D show how the podocyte layer begins to form a “cup” around the capillary bundle and how the capillary bundle remains attached to the outside vasculature. Panel E shows a more mature glomerulus, by which time the podocytes have migrated around the capillary loops. The capillary loops are still wider than they will be in a fully mature glomerulus. Panel F is an electron micrograph showing the podocyte layer at the point where most lateral cell attachments have been lost, except at the basal end of the lateral membrane, where the slit-diaphragm complex will be assembled in mature podocytes. A thin GBM is present at this stage. P, podocyte; C, capillary loop; BC, Bowman’s capsule; PT, proximal tubule.The development of the glomerulus is a dynamic process involving the expansion of the original capillary component into a plexus of six to eight individual loops, and the concomitant migration of the podocytes to be distributed around these loops (Figure 1, C and D). Although cause and effect relationships are not known, the capillary bundle displaces the layer of future podocytes as it expands. This layer of future podocytes, in turn, form a “pocket” surrounding the capillary bundle (Figure 1D), which maintains contact with the outside vasculature through an arterial and venous supply that will become part of the glomerular stalk. As the primitive podocytes form this pocket, the GBM remains a constant barrier between the epithelial and capillary components. The podocytes themselves do not remain a columnar epithelium. As they form this pocket, they begin to lose their lateral cell-cell attachments to each other (Figure 1F), but they remain attached to the GBM such that they no longer resemble a traditional epithelial cell. During this time, they also begin to migrate around the capillary loops, so that they no longer form a continuous uniform patch of cells. By this time, the glomerulus can be recognized as a discrete structure apart from the remainder of the nephron. It is during this phase of glomerular development and podocyte maturation that foot processes begin to form (Figure 1E). Early pictures of foot process assembly suggest that it begins with the selective detachment of podocytes from the GBM. However, the appearance of mature podocytes, with foot processes extending a significant distance from the main cell body, are also suggestive of a process whereby cytoplasmic extensions resembling filopodia extend themselves as a scaffolding around the capillary loops. Foot Process Assembly The extent to which foot process assembly reflects selective cell detachment versus cell extension and migration is not known; indeed, it is difficult to determine, given our inability to observe this process in living cells. One informative observation bearing on this issue is that adjacent foot processes are derived from different podocytes. Cells that begin as adjacent epithelial cells end up as cells with isolated cell bodies but interdigitated foot processes. It is possible that this situation arises entirely by extension of foot processes from cells that have initially dissociated from each other. However, another possibility is suggested by recent work examining cell-cell contact in keratinocytes (11). In this case, which is not generalizable to all epithelial cells, cell-cell contact is initiated through the extension of interdigitated filopodia between two cells (11). In the case of keratinocytes, these filopodia resolve into a conventional cell-cell junction mediated by cadherins. These findings in keratinocytes suggest two possible hypotheses for the assembly of foot processes. In one model, foot process assembly begins when podocytes are still maintaining cadherin-mediated adhesions along their lateral membranes (Figure 2B). In this case, adjacent cells would extend interdigitating filopodial-type extensions along their basal aspects, where they are attached to the GBM. Then, these filopodial extensions would be maintained as foot processes, while the remainder of the cell loses its cadherin-mediated adhesion such that appear as independent cell bodies. In the alternate model, podocytes first entirely dissociate from each other and then extend filopodial-like extensions that interdigitate to form foot processes (Figure 2A). Present observations appear to favor the model depicted in Figure 2B, as it is not normal to observe capillaries in developing glomeruli whose outer walls are not associated with an extension from a podocyte, implying that poodcytes or mesangial cells always fully encompass the capillary loops. However, the author is unaware of any studies that directly assess these hypotheses. A necessary component of both models is a phase in which there is partial dissociation of the initial cell body from the GBM, in places where it was originally fully attached along its entire basal membrane. Figure 2. : Two models of foot process formation. These are apical views from “on top” of the podocytes. For clarity, only two adjacent podocytes are depicted in both cases. (A) Two primitive podocytes that have separated from each other begin to extend processes that meet and become interdigitated. For simplicity, only one major extension is shown, although several would normally extend from the full circumference of the podocyte. (B) Two podocytes, which begin as columnar epithelial cells, always remain attached to each other along the basal end of their lateral membranes. Their common cell-cell junction is remodeled from a simple linear one to one that interdigitates and that contains the slit-diaphragm complex. The heavy lines represent the cell body of the podocyte, which loses attachment to other podocytes, except at the cell-cell junction between foot processes.Maturation of the GBM The maturation of the GBM has been the subject of several recent reviews (12,13) and is briefly dealt with here. The earliest epithelial cells of the nephron mainly express laminin-1. As soon as it is possible to define a nascent GBM, i.e., the basal lamina underlying the layer of future podocytes, a shift in laminin expression to isoforms containing the α4 subunit is observed (14–16). Upon further maturation of the GBM, there is a second shift to the expression of α5 and β2 subunits, which are components of laminin 11, and this continues to be the major laminin isoform in the mature GBM (14–16). There is also a shift in the expression of type IV collagen (17). The early nephron mainly expresses the α1 (IV) and α2 (IV) subunits (17); upon maturation of the GBM, there is a shift to α3, α4, and α5 (IV) subunits. Other major components of the GBM are nidogen (entactin) and heparan sulfate proteoglycans, most notably agrin (13). Slit-Diaphragm The slit-diaphragm (SD) has also been the subject of many recent reviews (18–20). This is a structure observed by electron microscopy between adjacent foot processes, which is a major component of the protein barrier between the circulation and Bowman’s space. The SD has, in recent years, been the focus of intense study, as mutations in several genes encoding protein components of the SD have been found to be the cause of various forms of childhood kidney disease (21). The first of these was nephrin; mutations in the NPHS1 gene that encodes nephrin are responsible for the Finnish form of congenital nephrotic syndrome (22). Nephrin is a transmembrane protein localized to the SD (23–25) that was originally thought to provide the structural link between two foot processes, without which it is not possible to maintain the foot process structure. Recent work has suggested a more complicated model. First, mice containing a targeted mutation in the nephrin gene were surprisingly capable of assembling foot processes, although the SD structure itself was not apparent (26–28). These mice suffered from heavy proteinuria during the neonatal period and died within a few days, with foot process effacement observed before death. These results confirm that the SD itself is a major component of the protein barrier and that the GBM by itself is not sufficient. Second, they indicate that there must be other structural molecules besides nephrin responsible for the initial assembly of foot processes. Recently, additional members of the nephrin family, neph1 to neph3, have been identified (29,30). Neph1-deficient mice also develop heavy proteinuria (30), indicating that neph1 is also an important component of the protein barrier. Two other proteins have also been identified as important components of the SD: podocin and CD2-AP (18,31–34). Podocin, the product of the NPHS2 locus, was identified as the product of a gene involved in autosomal recessive steroid-resistant nephrotic syndrome (35). Podocin is a transmembrane protein that interacts with nephrin and neph1 (29,31). CD2-AP, originally identified as an important protein in lymphocytes, was found to also be important in the kidney when CD2-AP knockout mice developed glomerular disease (36). CD2AP is also associated with the SD. Podocin, CD2AP, and nephrin all appear to be present in a complex within lipid rafts (31,37), which may also be associated with the cytoskeleton (38). Foot processes can be assembled in the absence of nephrin; it is therefore necessary to formulate new hypotheses about the role of the SD complex in foot process assembly. As alluded to above, it is possible that Neph1 is partially redundant with nephrin. Alternatively, it is possible that the driving forces responsible for the cytoskeletal reorganization involved in foot process assembly are entirely anchored at the adhesion apparatus linking the podocyte to the GBM. Finally, it is possible that there are additional molecules at the SD involved in cytoskeletal reorganization. Two additional adhesion molecules have been described at the SD: P-cadherin and a large proto-cadherin named FAT (39,40). Finding P-cadherin at the SD suggests that this structure or some subset of its components may bear some relationship to adherens junctions found in more traditional epithelial cells. However, the importance of P-cadherin at the SD is obscured by the failure to find significant renal dysfunction in P-cadherin knockout mice, although their kidneys have not been closely examined (41). FAT is a large proto-cadherin, containing a much larger extracellular domain than traditional cadherins (42,43). It was first defined as a tumor suppressor gene in drosophila (44). Its function in mammalian cells is unknown. ZO-1 is a protein most commonly associated with tight junctions, but it is also sometimes associated with adherens junctions (45,46). ZO-1 is found at the cytoplasmic face of the SD (47), providing another indication that the SD complex may functionally resemble an epithelial cell-cell junction. The observation that cell junctional proteins are found at the SD may have implications for the assembly of foot processes. Podocytes begin as columnar epithelial cells, and adjacent foot processes are always from different podocytes; it is possible that the SD complex begins as a cell-cell junction between adjacent epithelial cells and is conserved as these cells otherwise dissociate from each other. However, this model does not explain how the extensive interdigitation would occur to achieve mature foot processes. Podocyte Differentiation Podocytes are distinguished as a separate population from other cells in the developing nephron during the transition from the S-shaped body to the distinct glomerulus. At this time, they are expressing podocyte-specific markers such as WT1 and nephrin (22,48,49), which are not expressed in other cells of the nephron (in the case of nephrin) or whose expression pattern becomes restricted to podocytes (e.g., WT1). Little is known about the molecular genetic basis for segmentation of the nephron, particularly which genes act to define the podocyte versus tubular lineages. There is speculation that genes generally involved in segmentation, such as Hox genes or members of the Notch family, may be involved, but there is no published evidence of a role for any of these genes in segmentation of the mammalian nephron. However, in the development of the frog pronephros, inhibition of the Notch signaling pathway expands the domain of Wt1 expression (50), the latter usually associated with development of the glomus. Thus, this provides support for the of the Notch signaling pathway in segmentation of the nephron. It be that of the of the mammalian nephron, of segmentation will be a difficult as it is not to the various within the of a developing It is therefore that our understanding of segmentation will further studies in and before this can be in mammalian in Podocytes The in podocytes and WT1 is a and protein first identified as a tumor suppressor gene for a tumor of the kidney observed in in WT1 is expressed in the at the of kidney but as the S-shaped expression of WT1 becomes restricted to the podocyte where it is maintained at than in developing glomeruli in WT1 from renal the Wt1 knockout is not informative about a role for WT1 in podocyte differentiation or It must also be although there is on the molecular function of the of many genes that are much of this work has not been informative of a role for WT1 in podocytes. recent studies have to more on the role of WT1 in podocytes. The WT1 gene has two major forms of WT1 There are several such that at distinct are possible although there is on distinct of with different The first in between the domain of WT1 and the to express WT1 containing are normal that isoforms containing do not have a major role in kidney development or The other is not a separate but features two distinct at the end of such that can be in between and The the structure of the its results from mutations that the of the WT1 a mesangial within the the forms of WT1 found in maintain the to with proteins through the it is that the associated with are to a A recent from our involved the of mice that expressed a mutation of WT1 in podocytes Podocyte as from the expression of GBM, and cytoskeletal by this but there was development of the glomerular capillaries and expression of on capillary endothelial cells These results suggested that WT1 mainly be responsible for expression of that development of the glomerular than the differentiation of the podocyte A was in a recent an kidney cell which suggested that WT1 may expression of a podocyte cell protein that is thought to be involved in maintaining between podocytes to In the study, expression in glomeruli of mice A recent by was more to which also mutations in which to syndrome results from mutations that an to express the form of the form of targeted mutations to the Wt1 gene that in the inability to express the or forms of mutations in glomerular although mice only to express the isoform a more that there may be an or for the isoform during glomerular development containing one normal and one only the with developing several the expression of a component of the was in these knockout mice in to studies with the WT1 these studies the of the Wt1 and the in in a full understanding of its function during podocyte is a mutations of which are responsible for a that may glomerular disease is expressed in podocytes, and recent studies involving knockout mice assembly of podocyte foot processes and expression of CD2AP, and the and α4 of type IV collagen in glomeruli have been found in the of these genes indicating a major role for in podocyte-specific gene known as and is a expressed early in kidney development and in podocytes of S-shaped bodies in podocytes of In knockout mice, glomerular development at the capillary loop In most the podocytes remain as cells that have their lateral cell-cell attachments but remain fully to the GBM without any foot processes. the for in podocyte differentiation and glomerular development is before the time when podocytes would normally begin around the capillary loops and assembling foot processes. At present, there is no on possible genes in podocytes. is a domain of the expressed in podocytes of capillary loop glomeruli It has been shown to have an important role in segmentation of the is maintained in mature podocytes. There is a failure of foot process in podocytes, to podocytes of have their lateral cell-cell attachments is expressed in podocytes, indicating that is more or in a separate pathway from There is a in expression of podocin and nephrin in although this does by for the glomerular encodes a expressed in the developing kidney, other in the developing It is expressed in the as the nephron expression to be restricted to the podocytes, to mice are generally but some renal The of podocytes in these kidneys has not been Podocyte Two major adhesion are involved in between the podocyte and the GBM, and the complex. is the major expressed by podocytes although of are also The importance of in podocyte differentiation first apparent when the knockout of the gene in an inability to foot processes, and neonatal mice do not more than a few most to glomerular dysfunction The GBM itself is a role for in maintaining the structural of the GBM. the portion of the podocyte in contact with the GBM fully a simple role of an adhesion is not for as this suggest that podocyte more to an adjacent extracellular This is with findings in other cells which suggest a role for is a for and the latter of which is a major component of the GBM studies on also as a for and but it is thought that mainly as a for these other isoforms of laminin in is expressed early in podocyte before the transition from to It is not known with interacts before expression of it may with or with of that are to in the GBM. It also be that the of to is described in it is possible that the for is not as in the in a of the family, is not always with in podocytes A tight between and has been which also to isoforms of to a on In appear to have a role in cell-cell adhesion than in the of the in podocytes is not is known about the role of the complex in podocytes. The knockout results in early it to podocytes. However, mice have been published that may be with mice expressing in podocytes to this Other is a cell protein found on the of podocytes It is to have a role in maintaining podocyte cell through and expression of in cells to cell with this possibility in are to foot processes or and are during the first a present on the apical cell of podocytes mice are but have and are more to in a model Podocyte It is that cytoskeletal is the basis for foot process assembly, although our understanding of this process is at its earliest is an protein found in podocytes and in a restricted population of cells in the function in foot process assembly or other of podocyte cytoskeletal is not is an isoform of an expressed in podocytes first to in the of podocyte when mutations in the gene were identified in a form of It is not these mutations normal podocyte but then to disease in Podocytes and the of Podocytes express that have the to glomerular development. they express and which may the development of the glomerular capillaries is generally involved in capillary and with the and maturation of capillaries There is a body of evidence that is not only involved in the but also in the of glomerular capillaries and that failure to maintain the capillary may be a major component of glomerular disease this additional is a of the Notch of signaling molecules that are involved in cell is expressed in podocytes; in mice, there is a failure to develop the normal glomerular vasculature 1, a of was expressed in the adjacent endothelial mesangial cells, providing additional support for the that glomerular vascular development on from podocytes There is much to be about podocytes and their with adjacent cells. Although there are many genetic and models in which foot process assembly is or our understanding of the molecular processes that foot processes is still in its In it will be important to the of the SD complex and GBM adhesion apparatus in foot process assembly. It will also be important to additional of the expressed in podocytes, as as there are additional podocyte differentiation that are to be whose expression pattern may podocyte differentiation structural molecules involved in foot process as as that capillary development. of important in podocyte differentiation as it to the segmentation of the nephron, i.e., during the of the and S-shaped is the of glomerular cells and from the proximal as as that the tubule The recent of that podocyte-specific gene and podocyte cells lines will future in the is by the and the of The author and and for This review is to the of Edith Potter