Among all of the capillaries in the body, the glomerulus is arguably the most unusual and important, if not the most aesthetically interesting. In this chapter, we review the morphogenesis of this unique capillary, discuss the origins of its cells and extracellular matrices, and describe some of the primary regulatory events that occur during glomerular development.
Among all of the capillaries in the body, the glomerulus is arguably the most unusual and important, if not the most aesthetically interesting. In this chapter, we review the morphogenesis of this unique capillary, discuss the origins of its cells and extracellular matrices, and describe some of the primary regulatory events that occur during glomerular development.
Formation of the permanent, metanephric kidney begins at embryonic day 11 in mice, day 12 in rats, and during the 4th-5th week of gestation in humans. As the ureteric bud projects from the mesonephric duct and enters the metanephric anlage, mesenchymal cells condense around the bud’s advancing tip. Soon thereafter, the condensed mesenchyme converts to an epithelial phenotype and proceeds through a developmental sequence of nephric structures, which are termed vesicle, comma-, and S-shaped, developing capillary loop, and maturing glomerulus stages. Bud tip stimulation of mesenchymal cell induction and aggregation, conversion to epithelium, and glomerular and tubule differentiation occur repeatedly until the full complement of nephrons has developed. Nephrogenesis concludes ~1 week after birth in rodents, and during the 34th gestational week in humans.
At the inception of the vesicle stage of nephron development, the aggregated mesenchymal cells near the ureteric bud tips convert to a cluster of epithelial cells (vesicle), and begin assembling a basement membrane matrix containing collagen type IV, laminin, and basement membrane proteoglycans around the basal surface of the vesicle. As development progresses through the comma- and then S-shaped stages, a groove (vascular cleft) forms in the lower aspect of the vesicle, into which endothelial precursor cells (angioblasts) migrate ( Figure 26.1 ). Two epithelial layers can be distinguished beneath the vascular cleft: visceral epithelial cells (which ultimately differentiate into podocytes); and parietal epithelial cells (which will become the thin epithelium lining Bowman’s capsule of the mature nephron). Epithelial cells above the vascular cleft ultimately develop into proximal, Henle’s loop, and distal tubule epithelium. During the S-shaped phase of nephron development, the distal segment fuses with the same ureteric bud branch tip that initially induced the nephric structure, so that the lumen of the forming nephron is now continuous with that of the developing collecting system. Continued growth and branching of the ureteric bud leads to the induction of new mesenchymal aggregates, and glomerulo- and tubulogenesis continues until the full complement of nephrons is achieved.
Vascular Clefts of Comma- and S-Shaped Stage
With the progressive invasion and differentiation of endothelial cells, the developing capillary endothelium assembles a subendothelial basement membrane matrix. Similarly, the developing podocyte cell layer assembles a subepithelial basement membrane, so that two distinct basal laminae can be seen between the endothelial and epithelial cells ( Figure 26.2 ). As nephrons develop further, these two basement membranes layers merge to form the glomerular basement membrane (GBM), with endothelial cells lining its inner surface, and podocytes adherent to its outer surface.
As glomerular capillary loops begin to form, the endothelial cells gradually flatten and become extensively fenestrated ( Figures 26.3 and 26.4 ). Initially, the fenestrations are spanned by diaphragms, but these structures soon disappear. The epithelial podocytes, which originally were columnar with apical junctional complexes, also begin to flatten and begin sending out basolateral cytoplasmic projections that interdigitate with similar projections from neighboring cells ( Figure 26.3 ). As glomeruli mature, these projections go on to develop into the podocyte pedicels or foot processes ( Figures 26.3 and 26.4 ). The apical junctional complexes migrate basolaterally between these cellular projections and, although the mechanism is not fully-understood (see below), convert into the slit diaphragm complex between foot processes ( Figure 26.4 ). Metabolic labeling studies, histochemical and immunohistochemical techniques, and inter-species transplantation experiments have all shown that both the endothelium and epithelium are actively synthesizing glomerular basement membrane (GBM) proteins at this time.
Here, capillary loop diameters expand, and endothelial and podocyte cell layers differentiate further until the fully mature glomerular morphology is achieved. Unfused basement membranes are rarely seen in maturing glomeruli. On the other hand, complex, irregular projections of basement membrane are commonly found beneath podocytes at this stage, particularly in areas where foot processes are broad and their formation into relatively narrow pedicels is still incomplete. In vivo labeling studies have shown that these subepithelial basement membrane segments are somehow spliced or inserted into the existing fused GBM, possibly to provide the additional GBM material necessary for the inflating capillaries. Shortly after the initial, vascular cleft stage, and continuing into maturing glomeruli, modification and remodeling of the GBM occurs, with the appearance of new basement membrane protein isoforms and the disappearance of earlier species. As discussed later, however, we do not understand how these events are regulated at either the gene or protein level. Additionally, once the glomerulus is fully mature ( Figure 26.4 ), matrix synthesis and cell morphogenesis virtually halt (except for some poorly-understood activities responsible for GBM “maintenance” or “turnover”), and how these processes are downregulated is also not known.
Origin of the Glomerular Endothelium
Although compelling evidence has accumulated showing that nephron epithelial cells, including the visceral and parietal epithelium of Bowman’s capsule, all derive originally from the metanephric mesenchyme, the origin of vascular endothelial cells during kidney organogenesis has been more difficult to understand. Studies conducted nearly 30 years ago convincingly showed that cells of extrarenal origin grew into the metanephros and established the microvasculature, including the glomerular capillary tufts. These studies involved the grafting of embryonic, avascular mouse or quail kidney rudiments onto avian chorioallantoic membranes. After culturing in ovo , the kidney grafts contained glomerular endothelial and mesangial cells stemming from host chorioallantoic tissues, therefore signifying the ingress of vessel progenitors from sites outside the kidney.
Contrary to the results discussed above, several lines of evidence from a number of more recent experiments have shown that the metanephros contains its own pool of endothelial progenitors (angioblasts) capable of vascularizing nephrons in vivo . The first clues about the existence of these intrinsic metanephric angioblasts came from transplantation studies between mice and rats. For example, when E12 mouse kidneys are grafted into anterior eye chambers of rats, the vascular and glomerular basement membranes that develop within the grafts after transplantation are almost entirely of mouse (graft) origin. Similarly, when E12 kidneys are transplanted under kidney capsules of adult ROSA26 mice (which bear a ubiquitously expressed LacZ reporter gene useful as a cell lineage marker), all of the microvascular and glomerular endothelial cells within grafts are derived from the engrafted kidney, not from the host. Furthermore, when kidneys from E12 ROSA26 mice are grafted into the nephrogenic renal cortices of newborn wild-type hosts, endothelial cells stemming from the grafts can be seen integrating into host vasculature. In additional experiments, when embryonic kidneys from Flk1 (VEGF receptor-2)–LacZ heterozygous mice are grown under routine organ culture conditions, Flk1-LacZ-positive microvessels do not develop in vitro , despite the extensive formation of metanephric tubules and avascular glomerular epithelial tufts. When these same cultured kidneys are then transplanted into anterior eye chambers of wild-type host mice, the grafts develop microvessels and vascularized glomeruli lined by Flk1-LacZ-expressing cells, indicating again that the endothelium originates from the engrafted kidney itself, and not from the host. Several other research groups have reached similar conclusions independently. For example, when avascular metanephroi from E11 Tie1/LacZ transgenic mice are transplanted into newborn wild–type hosts, widespread expression of Tie1/LacZ is found within glomeruli developing within grafts. Others have immunolocalized putative angioblasts in the metanephric mesenchyme of prevascular embryonic rat kidney.
Although current evidence shows that the embryonic kidney contains a pool of angioblasts capable of establishing the glomerular endothelium, whether these progenitors originate initially from outside the metanephric blastema or instead stem directly from metanephric mesenchyme is not yet clear. Nevertheless, immunolabeling experiments in developing rat kidney shows that endothelial, as well as mesangial, cell precursors share common markers during glomerulogenesis (RECA-1 and Thy1.1, respectively), suggesting that they may indeed derive from metanephric mesenchyme. Other immunolocalization and transplantation experiments have shown that juxtaglomerular cells in developing kidney also originate from metanephric mesenchyme, although they appear to stem from a different lineage than endothelial and mesangial cells.
Endothelial Cell Recruitment and Differentiation
Mechanisms controlling vascular development are highly complex and involve several different transcription factors, cell-cell and cell-matrix interactions, and many membrane receptor–ligand signaling cascades. Although our knowledge of these systems in a variety of vascular beds has improved dramatically during the past several years, many key questions regarding temporal and spatial controls still persist. With respect to the formation of glomerular capillaries, the process can be considered to progress through four interrelated events: (1) angioblast survival, proliferation and differentiation into endothelium; (2) glomerular endothelial cell recruitment; (3) initial assembly of the glomerular capillary tuft and associated mesangium; and (4) glomerular capillary stabilization and maturation.
Among all of the mechanisms involved with development of the systemic vasculature, signals evoked by binding of VEGF to its cellular receptors, VEGFR-1 and VEGFR-2, are singularly critical. Mice with homozygous Vegfa gene deletions die by E9.5 with severe vascular deficits. Remarkably, Vegfa heterozygote mutants also succumb by E12 with vascular phenotypes, indicating that a single Vegfa allele is insufficient to direct normal vascular development. Homozygous (but not heterozygous) Vegfr1 and Vegfr2 mutants die at mid-gestation, due to failure of endothelial differentiation and vessel integrity, respectively.
Developing podocytes are key sources of VEGF, and its secretion and binding to angioblasts bearing VEGF receptors may initiate their recruitment into the vascular cleft of comma- and S-shaped nephrons, which is the initial site of glomerulogenesis. Because Vegfa and Vegfr2 knockout mice die with vascular phenotypes before glomerulogenesis commences, the precise role of this ligand-receptor pair in mediating glomerular endothelial development has been difficult to analyze fully. Nevertheless, and underscoring the importance of the VEGF signaling system, injection of VEGF-blocking antibodies into developing mouse kidney cortex inhibits glomerular capillary formation in vivo . With the advent of cell selective and/or inducible gene deletion technologies, additional evidence for the importance of podocyte-derived VEGF has been obtained. For example, homozygous deletion of Vegfa selectively in podocytes (obtained in bi-transgenic mice carrying nephrin-cre recombinase and floxed Vegfa alleles) results in animals which die perinatally with non-vascularized glomeruli. Heterozygous deletion of Vegfa causes no evident phenotype initially. By 2.5 weeks of age, however, mice become proteinuric, and glomeruli contain swollen endothelial cells and hyaline deposits similar to those seen in patients with pre-eclampsia. By contrast, overexpression of the VEGF 164 isoform specifically in podocytes leads to collapsing glomerulopathy and death at ~5 days of age. When Vegfa is selectively deleted in podocytes of adult mice (using a Tet-On conditional expression model), mice become severely proteinuric and hypertensive, and glomeruli resemble those of humans with thrombotic microangiopathy (mesangiolysis, endothelial swelling, red cell fragmentation, and fibrin deposition). Clearly, the cellular controls for maintaining VEGF protein expression within an optimal range are critically important for the appropriate establishment and maintenance of the glomerular capillary.
Regulation of Endothelial Development
Hypoxia-Inducible Transcription Factors (HIFs)
Transcription of VEGF and VEGFR genes is activated by hypoxia-inducible transcription factors (HIFs), which consist of heterodimers of HIF α- and β-subunits. Under normal oxygen concentrations, the HIF α-subunit undergoes prolyl hydroxylation, binding to von Hippel Lindau protein (VHL), polyubiquitination, and proteasomal degradation. In hypoxia, the prolyl hydroxylase enzyme is inhibited, and HIF α chain degradation is avoided. Hypoxia-stabilized HIF α/β heterodimers bind to hypoxia-responsive elements (HREs) located in promoter/enhancer regions of inducible genes, many of which are proteins expressed in response to hypoxic stress. For example, erythropoietin, transferrin, VEGF, VEGFR1, and VEGFR2 are among the more than 70 distinct genes known to be transcriptionally activated by HIFs.
There are at least three distinct HIF α- and two β-subunits known at present, making a variety of different HIF isoforms possible. Because HIF stabilization is enhanced in cells experiencing subnormal oxygen tensions, such as those in rapidly growing tissues, robust VEGF and VEGFR synthesis commonly occurs during organogenesis. Increased VEGF/VEGFR signaling stimulates mitosis in endothelial progenitor cells, phosphorylation of the antiapoptotic kinases Akt/PKB and focal adhesion kinase (FAK), and upregulation of the survival factors Bcl2 and A1. In time, these events can lead to the creation of new blood vessels, which can then provide appropriate levels of oxygen specifically to the formerly hypoxic tissue sites.
Renal HIF Expression
The expression patterns for several of the different HIF α- and β-subunits have been documented in developing human, rat, and mouse kidney using in situ hybridization and immunohistochemistry. In general, both HIF-1 and HIF-2α are found in glomeruli, with specific immunolocalization of HIF-2α protein to immature podocytes (which are rich sources of VEGF). HIF-2α is also expressed by developing vascular endothelial cells in the kidney (most of which express VEGFR-2), whereas HIF-1α is found in cortical and medullary collecting duct epithelium. HIF-1α and HIF-2α protein are undetectable in fully mature glomeruli. Mice with a global deletion of Hif2α show no defects in glomerular development or function, and no deficits in VEGF or Flk1 expression. Interestingly, HIF-1β is apparently ubiquitously expressed by all cells in the kidney, but HIF-2β distribution is greatly restricted during development and, in mice, becomes confined to nuclei in cells of the thick ascending limb of Henle’s loop.
The selective expression of certain HIF isoforms in different tissue compartments of developing kidney may reflect the coordinated regulation of different sets of HIF target genes. Importantly, individuals with mutations in VHL, a key protein in mediating HIF α chain degradation, and thereby reducing expression of HIF target genes, are prone to developing hemangioblastomas and clear cell-renal cell carcinomas. Some studies have shown that HIF-1α and HIF-2α had differential and sometimes antagonistic effects on the growth of clear cell-renal cell carcinomas, with HIF-1α retarding and HIF-2α promoting tumor growth. These findings provide further evidence for differential effects of different HIF isoforms, and call for more studies examining the expression of HIF and HIF gene targets in the developing kidney. Surprisingly, when Vhl is selectively deleted in podocytes, glomerular vascularization patterns are not affected, and kidneys develop normally. On the other hand, mice become proteinuric by 4 weeks of age, and there is ectopic deposition of collagen (α1) 2 α2(IV) in peripheral loop GBMs, and upregulation of an ancient oxygen-binding protein, neuroglobin, specifically in podocytes.
Once glomeruli are vascularized and fully mature, podocytes still continue VEGF synthesis. Likewise, expression of Flk1 is also maintained by glomerular endothelial cells of mature kidneys. VEGF-Flk1 signaling in glomeruli therefore probably exerts functions extending well beyond those needed for mobilization of angioblasts and initial formation of the capillary tuft. For example, in co-cultures of epithelial cells with endothelium, epithelial-derived VEGF has been shown to induce fenestrae formation in the endothelium. When Vegfr2 is inducibly deleted in adult mice, podocytes appear normal, but there is loss of viable glomerular endothelial cells. Perhaps the continued expression of VEGF by podocytes and Flk1 by glomerular endothelial cells in vivo is necessary for maintenance of the highly-differentiated state seen in the endothelium.
Other Growth Factor/Receptors
Beyond VEGF and VEGFR, several other growth factor-receptor signaling systems important for vessel development systemically are also crucial for glomerular capillary formation, including the Tie/angiopoietin and PDGFR/PDGF families. Developing glomerular endothelial cells express Tie-2, and and one of its ligands, angiopoietin-1, is important for vascular organization and remodeling. Another Tie-2 ligand, angiopoietin-2, may mediate vascular integrity and permeability. The coordinated expression of these two angiopoietins may therefore regulate the maturation and stabilization phases of glomerular development (reviewed in ). Additionally, Tie-2 and at least some members of the angiopoietins contain defined HREs in their promoters, making their transcriptional regulation by hypoxia/HIFs seem likely. Similarly, an HRE is found in the PDGFB gene promoter, although this may not necessarily be responsive to hypoxia. During early glomerular development, PDGFB protein is expressed by podocytes. This may be important for the glomerular recruitment of immature mesangial cells, which express the PDGFB receptor, PDGFRβ. In later developmental stages, both PDGF and PDGFRβ expression becomes confined to the mesangium, which may provide autocrine signals required for mesangial cell proliferation and/or maturation (see below).
Like other developing vessels, at least some neuronal axon guidance receptors and ligands are also found in developing glomeruli. For example, neuropilin-1 (Np1), which is a co-receptor with VEGFR2 for VEGF 164 (but lacks a cytoplasmic signaling domain), immunolocalizes to glomerular endothelial cells. Semaphorins-3A and -3F, which are ligands for Np1, have been found on podocytes, suggesting that semaphorin-Np1 signaling between podocytes and endothelium may help pattern glomerular morphogenesis. One study, however, has also reported that Np1 is expressed by podocytes in vivo . Recent experiments also showed that podocyte-derived VEGF may act as an autocrine survival factor for cultured podocytes in vitro . Additionally, these same studies found an upregulation of VEGFR2 in cultured podocytes, suggesting that VEGF/VEGFR2 signaling is important not only for glomerular capillary formation and maintenance, but also for podocyte differentiation.
Other receptor-ligand signaling systems probably crucial for glomerular capillary formation include members of the Eph/ephrin receptor/counter-receptor families. Specifically, the receptor tyrosine kinase EphB1 and its ligand, ephrin-B1, which itself is also a transmembrane protein receptor, are both expressed in similar distribution patterns in developing kidney microvasculature. Although the precise roles for Eph/ephrin signaling in the glomerulus are still uncertain, knockout mice display lethal vascular phenotypes, including defects in vessel patterning, sprouting, and remodeling (reviewed in ). Reciprocal gradients of Eph and ephrin protein concentrations have been identified in the developing brain, where they appear to direct accurate neuronal patterning in the visual system. Perhaps analogous events take place in the developing glomerulus, where spatial signals conveyed between endothelial cells help target them to correct microanatomical domains.
Development of the Mesangium
Fundamentals regarding the development of the intercapillary mesangium, as well as the origin and recruitment of mesangial cell progenitors, are still largely unresolved issues in glomerular biology. Nevertheless, we have known for some time that PDGFB and its receptor, PDGFRβ, are both expressed by mesangial cells of mature glomeruli. Additionally, studies in developing kidney have shown that immature podocytes produce PDGFB which may help recruit mesangial cell progenitors expressing PDGFRβ into glomeruli. Later, podocyte expression of PDGF declines, and the synthesis of both PDGF and PDGFRβ becomes confined to the mesangial cells, perhaps to promote their proliferation or maturation. Gene deletion studies in mice have conclusively shown an absolute requirement for PDGFB/PDGFRβ signaling. Null mutants for either genes die perinatally, with massive hemorrhaging systemically. Importantly, glomeruli in these mutants entirely lack mesangial cells and consist of one or only a few large, swollen capillary loops.
Interestingly, once the glomerulus has fully matured, there appears to be a small population of extraglomerular mesangial cells capable of completely repopulating the glomerulus if the intraglomerular mesangium becomes severely injured. These mesangial reserve cells reside in the juxtaglomerular apparatus, and are distinct from renin-secreting cells, macrophages, vascular smooth muscle cells, and endothelial cells. In an anti-Thy-1 model of proliferative glomerulonephritis in rats, these extraglomerular mesangial reserve cells migrate into the glomerulus and entirely restore the depleted intraglomerular mesangium. Alternatively, some studies suggest that bone marrow hematopoitic stem cells can be a source of mesangial cells. Perhaps additional studies based on these fascinating observations can shed more light on the origin and development of mesangial cells during glomerulogenesis. Similarly, much more work needs to be done on the assembly and maintenance of the mesangial matrix. Although this matrix undergoes morphologic and compositional changes throughout glomerulogenesis, it has not yet been the topic of thorough study. Whether the mesangial matrix is produced exclusively by mesangial cells or whether glomerular endothelial cells and podocytes also contribute components, are also not understood. On the other hand, and as discussed later, considerable progress has been made in understanding the assembly of the GBM (see below).
Factors Regulating Podocyte Differentiation
Determination of podocyte identity and regulation of podocyte differentiation are fundamental aspects of glomerulogenesis about which much remains to be learned. Although the podocyte transcription factors described below have been shown to be crucial for proper differentiation, exactly how these regulators interact to orchestrate the complex acquisition of the podocyte phenotype remains a mystery. Studies aimed at defining the entire glomerular transcriptome have been very useful for defining what genes may play important roles in podocyte determination, differentiation, and function; however, we have only just begun to gain an understanding of how the podocyte achieves its unique and important properties through interacting pathways.
The protein that likely has one of the earliest roles in podocyte differentiation is Wilms tumor 1 (WT1), a zinc-finger protein that is involved in both transcription and RNA processing. WT1 is expressed at the initial stages of nephrogenesis, when it has been shown to regulate such developmentally important genes as Bmp7 , Six2 , and Sall1 . But once glomerulogenesis begins, there is a dramatic increase in the level of WT1 in podocyte precursors, and podocytes continue to express WT1 throughout life (reviewed in ). One potentially important function of WT1 is to downregulate expression of Pax2 in the immature podocytes of S-shape stage nephrons, as artificial overexpression of Pax2 widely, including in podocytes, causes severe nephrotic syndrome. In addition, WT1 has been shown to regulate a number of genes, including NPHS1, the gene encoding nephrin (discussed below). This provides a clear mechanism whereby alteration in WT1 function directly affects glomerular function.
Several lines of investigation reveal important roles for WT1 in promoting proper podocyte differentiation (reviewed in ). Most notable is that heterozygous mutations that affect the zinc-finger structure of WT1 cause Denys–Drash syndrome in humans. This rare disease is characterized by proteinuria, nephrotic syndrome, diffuse mesangial sclerosis, and ESRD. Expression of mutant WT1 forms associated with Denys–Drash syndrome in transgenic mice, either globally or specifically in podocytes, causes various glomerular and podocyte defects that are consistent with the human pathology. Moreover, heterozygous mutations in WT1 that affect the normal pattern of WT1 RNA alternative splicing to generate the so-called +KTS and −KTS isoforms of WT1, cause Frasier syndrome. The renal component of this disease includes proteinuria that begins in early childhood and progresses to FSGS, but the course to ESRD is slower than observed in Denys–Drash syndrome. Mice engineered to express only the +KTS or the −KTS isoforms develop severe podocyte and glomerular defects, further emphasizing the important role that WT1 and these specific splice variants play in glomerular development and function.
Lmx1b, a LIM-homeodomain protein, is another transcription factor expressed in podocytes that is affected in human disease. Heterozygous mutations in LMX1B cause Nail–Patella syndrome, an autosomal dominant disease with skeletal abnormalities, nail hypoplasia, and variably penetrant nephropathy associated with accumulation of fibrillar material in the GBM that appears to be collagen type III. Although Lmx1b +/− mice do not exhibit a phenotype, Lmx1b −/− mice die shortly after birth with abnormalities in dorsal limb structures, including absence of nails and patellae, abnormal glomeruli, attenuated podocyte maturation with lack of normal slit diaphragms, and tubular protein casts. Analysis of gene expression in Lmx1b −/− podocytes has revealed decreases in collagen α3 and α4(IV), podocin, and CD2AP, which are all known to be important for proper glomerular filtration. The reduced expression in Lmx1b −/− mice suggests a basis for the partially penetrant nephropathy in LMX1B +/− humans, but gene expression studies in affected individuals do not support this hypothesis. Thus, exactly how LMX1B haploinsufficiency causes nephropathy in humans remains a mystery. If there really is no reduction in the expression of relevant podocyte genes, then perhaps there is a lack of repression, either direct or indirect, of genes injurious to glomerular function, as previously proposed.
Pod1, a basic helix-loop-helix transcription factor also known as epicardin, capsulin, and Tcf21, is highly expressed early during kidney development in condensing metanephric mesenchyme and in stromal cells. In the developing nephron, Pod1 is expressed in podocyte precursors at the S-shaped stage, and persists in adult podocytes. The kidney phenotype in Pod1 −/− mice, which die at birth due to heart and lung defects, is complex, but it is clear that there are fewer glomeruli associated with defects in ureteric bud branching. In addition, there is a striking arrest of glomerular development at the capillary loop stage, and podocytes fail to mature properly, elaborating only rudimentary foot processes. Gene expression profiling studies of Pod1 −/− glomeruli revealed 3986 genes expressed differently than in wild-type glomeruli, demonstrating that Pod1 has profound effects on gene expression.
Kreisler/Mafb is a basic domain leucine zipper transcription factor expressed in podocytes from the capillary loop stage onwards. Mice that are homozygous for a point mutation affecting the kreisler DNA-binding domain die within 24 hours of birth. The podocytes of these mice fail to extend foot processes or establish slit diaphragms, and there is a significant reduction in expression of nephrin, podocin, and CD2AP. Interestingly, whereas Pod1 is expressed in kreisler −/− podocytes, kreisler is not expressed in Pod1 −/− podocytes. This suggests the existence of a transcriptional hierarchy in which Pod1 may activate expression of kreisler.
Foxc2 is a member of the forkhead/winged-helix family of transcription factors. During nephrogenesis, Foxc2 is first expressed in a subset of cells in the comma-shaped body and then is expressed in developing podocytes at the S-shaped and capillary loop stages, and more weakly at maturity. Foxc2 −/− mice exhibit small kidneys and reduced numbers of glomeruli with ballooned capillaries, suggesting a defect in adhesion of mesangial cells (which are present) to the glomerular basement membrane (GBM). Ultrastructural analyses revealed that podocytes fail to extend processes or assemble slit diaphragms, and endothelial cells fail to become fenestrated, suggesting an arrest of differentiation of these two cell types. Gene expression profiling studies of Foxc2 −/− glomeruli showed reductions in a number of known podocyte genes, such as kreisler/Mafb, Nphs2 (podocin), and Podxl1 (podocalyxin), although others, such as Nphs1, Wt1, and Cd2ap, were not affected. Interestingly, in the Xenopus pronephros, the combined knockdown of WT1 and Foxc2 resulted in the loss of all podocyte marker gene expression.
Notch2 is expressed in developing podocytes from as early as the comma-shaped stage. Although the total absence of Notch2 in mice results in embryonic lethality at E11.5 before glomerulogenesis begins, homozygosity for a hypomorphic Notch2 allele allows survival until 24 hours after birth. These latter Notch2 −/− mice exhibit small kidneys with cortical vascular lesions. Some glomeruli arrested before the capillary loop stage and were not vascularized, whereas others became vascularized but had ballooned capillaries due to an absence of mesangial cells. In addition, genetic interaction studies demonstrated that Jagged1, a Notch ligand, is required for proper Notch2 signaling during glomerulogenesis. Additional studies using a conditional Notch2 allele showed that Notch2 (but not Notch1) is required for acquisition of podocyte and proximal tubule cell fates.
Formation of the Slit Diaphragm Complex
In mature glomeruli, the slit diaphragm (SD) represents the only known connection between adjacent podocytes and podocyte foot processes. The SD spans the space between the interdigitated foot processes ( Figure 26.4 ), and has been proposed to play a crucial role in glomerular filtration by serving as the major barrier to albumin, although some data do not support this view. The SD has also been shown to mediate important signaling events that are responsible for maintaining podocyte survival and differentiation, suggesting that it plays dual roles in ensuring proper glomerular function. But interestingly, mutant mice without slit diaphragms due to lack of nephrin have dramatic leakage of albumin into the urine, but they do not have major alterations in podocyte survival or gene expression.
Slit Diaphragm Components
Nephrin and the Neph Family
The explosion of research into SD composition and function began with the identification of the nephrin gene ( NPHS1 ), as mutated in congenital nephrotic syndrome of the Finnish type, and the nephrin protein as an integral component of the SD ( Figure 26.5 ). Nephrin is an immunoglobulin superfamily member containing eight extracellular Ig-like domains, a fibronectin type III domain, a single-pass transmembrane domain, and a cytoplasmic tail of ~155 amino acids, including several tyrosines that have been predicted to be important for its function, perhaps by interacting with actin-binding Nck adapter proteins. Humans and mice lacking nephrin never establish SDs, demonstrating the importance of nephrin for SD formation. A group of three related Ig superfamily molecules that interact with nephrin, Neph1-3, are also found at the SD, as well as in other tissues, and it is clear that at least Neph1 is required for maintenance of the filtration barrier. Interestingly, nephrin interacts directly with the Neph proteins ( Figure 26.5 ), and disturbing their association in vivo causes proteinuria.
Further evidence for the importance of nephrin-Neph interactions come from the model organisms Drosophila (fruitflies) and C. elegans (nematodes), which have conserved homologs of nephrin and Neph proteins. In both these invertebrates, a nephrin homolog on one cell interacts with a Neph homolog on another cell to promote a developmental event. Amazingly, all three mouse Neph proteins can partially compensate for the nematode Neph-like protein SYG-1. Together with data from mouse, these results suggest that nephrin-Neph interactions are conserved across phyla.
ZO-1 and Podocin
Before the identification of nephrin, zonula occludens-1 (ZO-1), an epithelial tight junction protein, was shown to be localized to the cytoplasmic face of the SD. ZO-1 has recently been shown to interact with Neph1–3, which may serve to anchor it to the SD. In turn, podocin, whose encoding gene ( NPHS2 ) is mutated in familial steroid resistant nephrotic syndrome, appears important for anchoring nephrin and the Neph family to the SD ( Figure 26.5 ). Podocin is a hairpin-shaped integral membrane protein that interacts with the cytoplasmic tails of nephrin and Neph family members, and is required to target nephrin to lipid raft microdomains, which have been proposed to be crucial for SD organization and function.
Consistent with the notion that the SD is a modified adherens junction, two cadherins, P-cadherin and the FAT1 protocadherin, have been localized to the SD. P-cadherin was found to associate with ZO-1 and the catenins at the SD, but the functional importance of P-cadherin at the SD has yet to be demonstrated, and humans with mutations in the P-cadherin gene ( CDH3 ) are not reported to have kidney disease. FAT1, an extremely large member of the cadherin family, is involved in regulating actin cytoskeleton dynamics and cell polarization. Mice lacking FAT1 die within two days of birth, and their podocytes lack SDs and significant foot process formation. This underscores the importance of FAT1 in the establishment or maintenance of SDs. A model of the SD that can accommodate the very large extracellular domain of FAT1 has not yet been presented, but FAT1 may somehow contribute to the central density of the SD that is apparent in ultrastructural analyses.
CD2-associated protein (CD2AP) is a modular cytoplasmic docking protein that interacts with multiple slit diaphragm components, including nephrin and podocin, as well as with the actin cytoskeleton ( Figure 26.5 ). Mice lacking CD2AP exhibit congenital nephrotic syndrome with progressive loss of SDs and foot processes, and they die at 6–7 weeks of age. In addition, CD2AP haploinsufficiency has been associated with renal disease in both mice and humans, and a patient homozygous for a premature stop codon mutation (R612X) lacking the final 28 amino acids of 639 total exhibited proteinuria, FSGS, and kidney failure by 3 years of age. CD2AP is thought to be an adaptor that forms a bridge between the SD and the cytoskeleton, but it also appears to play a role in the trafficking of endocytic vesicles. Although CD2AP is very widely expressed, it seems to be required only in the podocyte ; this could be due to compensation by a paralog of CD2AP, CIN85, although CIN85 activity in podocytes appears to be detrimental.
That mice lacking CD2AP are able to form normal foot processes and SDs initially suggests the existence of other adaptor proteins in podocytes that might play an earlier role in organizing SDs and linking them to the cytoskeleton. Indeed, the Nck adaptor proteins (Nck1 and Nck2), which contain three Src homology 3 (SH3) domains and one SH2 domain, bind to phosphotyrosine-containing motifs in the cytoplasmic tail of nephrin via their SH2 domains. In addition, clustering of nephrin-Nck complexes reorganized the actin cytoskeleton in transfected cells. Importantly, genetically-engineered mice lacking Nck1 and Nck2 in podocytes failed to elaborate foot processes or assemble SDs, and inducible deletion of Nck1 and Nck2 in adult podocytes rapidly leads to proteinuria and glomerulosclerosis. These exciting findings establish a potentially pivotal role for the Nck adaptor proteins in organizing and maintaining SDs, and suggest one mechanism whereby the Fin minor mutation in NPHS1, which results in a nephrin protein lacking most of its cytoplasmic tail and all of the intracellular tyrosines, causes congenital nephrotic syndrome.
TRPC6 is a member of the transient receptor potential family of cation channel proteins, and is associated with the SD. Mutations in TRPC6 have been shown to cause autosomal dominant FSGS in several different families, and this was associated with increased calcium current amplitude in some cases. Calcium flux mediated by TRPC6 could have an important role in regulating podocyte and SD homeostasis, perhaps by regulating actin dynamics and motility and calcium-sensitive transcription factors such as those in the NFAT family.
Slit Diaphragm Assembly Mechanisms
As mentioned earlier, the slit diaphragm bears some relationship to adherens junctions, and ZO-1 immunolocalizes to the cytoplasmic domains on the lateral surfaces of podocytes adjacent to the extracellular diaphragm spanning the slit pore. The synthetic patterns of all of the SD components during glomerular development have not yet been studied in detail. However, nephrin and podocin first appear during the late S-shaped and early capillary loop stages of nephron development, which is when foot process interdigitation first occurs, simultaneous with the first ultrastructural appearance of SDs. Also occurring at this stage of development is the apical-to-basal translocation of protein complexes involved in cell polarity, an event crucial for proper slit diaphragm and foot process formation. The precise sequence of SD assembly, including how the linkages between the ectodomains of SD proteins are made, as well as connections with the internal cytoskeleton and associated elements, are not known. Similarly, the extent to which the SD may be dynamic and undergo modification with age is poorly-understood. Nevertheless, an increasing number of studies have shown that the SD does not merely represent a static component of the glomerular filtration barrier, but it also functions to influence podocyte behavior.
Signaling at the Slit Diaphragm
The first evidence that the SD complex may have a signaling role came from studies of nephrin activity in cultured cells. Transfection of cells with nephrin increased activator protein-1 (AP1)-mediated transcriptional activation, and this effect on intracellular signaling was augmented by cotransfection with podocin, to which nephrin binds. These authors later showed that podocin recruits nephrin to lipid rafts, which are cholesterol-enriched plasma membrane microdomains associated with high concentrations of signaling proteins and high levels of signaling activity. Furthermore, as alluded to above, nephrin contains multiple tyrosines in its cytoplasmic tail. Some of these can be phosphorylated by the Src-family tyrosine kinase, Fyn, and clustering of nephrin by Fyn increases phosphorylation. Phosphorylation of nephrin both augments its interaction with podocin and allows Nck adaptor proteins to bind its tail and influence the organization of the adjacent actin cytoskeleton. Consistent with this, Fyn −/− mice have variably coarsened or effaced foot processes. In addition, trans-heterozgyous Fyn +/− ; Cd2ap +/− mice exhibit marked proteinuria and FSGS, and CD2AP can be immunoprecipitated with Fyn from glomerular lysates. These findings demonstrate that Fyn-mediated signaling likely depends upon interactions with CD2AP.
Nephrin-mediated signaling has also been shown to involve stimulation of phosphoinositide 3-OH kinase (PI3K). Nephrin and CD2AP interact with the p85 regulatory subunit of PI3K, recruit PI3K to the plasma membrane and, in conjunction with podocin, stimulate PI3K activation of AKT signaling in podocytes. This is proposed to regulate the behavior of podocytes and protect them from detachment–induced apoptosis (anoikis), which suggests that proper signaling at the SD is important for maintaining podocyte health.
Neph family proteins, like nephrin, have cytoplasmic tails containing tyrosines that can be phosphorylated. Also like nephrin, Neph1 can induce AP-1-mediated transcriptional activation. ZO-1, which binds to the cytoplasmic tail of Neph proteins, enhances tyrosine phosphorylation of Neph1 and augments AP-1 activation. As ZO-1 can also interact with the actin cytoskeleton, one can envisage how the podocin-CD2AP-nephrin-Neph-ZO-1 complex can activate signaling in podocytes and provide stability to the SD via linkage to actin ( Figure 26.5 ). Interfering with any of these interactions likely leads to abnormal podocyte behavior and proteinuria; in this regard, the actin cytoskeleton in podocyte foot processes also has specialized components. These include α-actinin-4, which is mutated in autosomal dominant FSGS, and synaptopodin, which regulates α-actinin’s actin bundling activity and binds to CD2AP. That trans-heterozygous Synpo +/− ; Cd2ap +/− mice develop proteinuria and FSGS-like lesions underscores the importance of the SD-actin cytoskeleton linkage in maintaining podocyte health, structure, and function. Further support comes from a study showing that the action of cyclosporine A in reducing proteinuria has to do with a protective effect on synaptopodin and on the podocyte’s actin cytoskeleton.