INVITED REVIEW
Molecular regulation of nephron endowment

Amander T. Clark and John F. Bertram

Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3168, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
MOLECULAR REGULATION OF...
MOLECULAR REGULATION OF...
DETERMINANTS OF NEPHRON...
NEPHRON ENDOWMENT IN HOMOZYGOUS...
ESTIMATING NEPHRON NUMBER IN...
CONCLUDING REMARKS
REFERENCES

Recent data suggests that the number of nephrons in normal adult human kidneys ranges from ~300,000 to more than 1 million. There is increasing evidence that reduced nephron number, either inherited or acquired, is associated with the development of essential hypertension, chronic renal failure, renal disease in transitional indigenous populations, and possibly the long-term success of renal allografts. Three processes ultimately govern the number of nephrons formed during the development of the permanent kidney (metanephros): branching of the ureteric duct in the metanephric mesenchyme; condensation of mesenchymal cells at the tips of the ureteric branches; and conversion of the mesenchymal condensates into epithelium. This epithelium then grows and differentiates to form nephrons. In recent years, we have learned a great deal about the molecular regulation of these three central processes and hence the molecular regulation of nephron endowment. Data has come from studies on cell lines, isolated ureteric duct epithelial cells, isolated metanephric mesenchyme, and whole metanephric organ culture, as well as from studies of heterozygous and homozygous null mutant mice. With accurate and precise methods now available for estimating the total number of nephrons in kidneys, more advances in our understanding of the molecular regulation of nephron endowment can be expected in the near future.

kidney; development; stereology


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
MOLECULAR REGULATION OF...
MOLECULAR REGULATION OF...
DETERMINANTS OF NEPHRON...
NEPHRON ENDOWMENT IN HOMOZYGOUS...
ESTIMATING NEPHRON NUMBER IN...
CONCLUDING REMARKS
REFERENCES

UNTIL RECENTLY, it was generally accepted that the normal human adult kidney contained approximately 1 million nephrons. However, in a landmark study which utilized recently developed unbiased stereological sampling and counting methods, it was shown that the number of nephrons in 37 normal human kidneys ranged from ~300,000 to more than 1 million, more than a threefold range (58). This study demonstrated for the first time the enormous range in nephron number in human kidneys, and provided concrete support for the intriguing concept of nephron endowment, which argues that low nephron number, either acquired or inherited, is associated with the development of essential hypertension, chronic renal failure, renal disease in transitional indigenous populations, and possibly the long-term success of renal allografts (7-9, 48).

The past decade has witnessed great advances in our understanding of the molecular regulation of kidney development, including nephron development. These studies have not so much been inspired by the theory of nephron endowment, but more by the fact that the developing kidney is an ideal organ in which to study many of the fundamental mechanisms of developmental biology. As described below, the development of the permanent kidney, or metanephros, involves epithelial tubule elongation, tubule branching, cell condensation, mesenchymal-to-epithelial conversion, and angiogenesis, as well as the development and differentiation of numerous specialized cell types. Three of these processes ultimately govern the number of nephrons formed: growth of the ureteric duct (which involves both elongation of the duct as well as branching), mesenchymal cell condensation, and conversion of the mesenchymal cell condensate into epithelium. In this review, we discuss the key molecules known to be involved in these three critical events. In each case, consideration is given to the particular experimental strategies that were used to identify the roles of specific molecules. These strategies have included localization (temporal and spatial) of ligands and their receptors, in vitro models (culture of renal epithelial lines, isolated ureteric epithelium, isolated metanephric mesenchymal cells, metanephric mesenchyme, metanephric organ culture, and urogenital blocks), and in vivo studies (heterozygous and homozygous null mutant mice). We show that although much is still to be learned about the molecular regulation of nephron endowment, some molecules clearly play a primary role in nephron endowment while others play an ancillary role. Furthermore, the levels of certain molecules also appear critical to establishment of adequate nephron endowment. The concept of gene dosage and nephron development/endowment has thus emerged.


    DEVELOPMENT OF THE METANEPHROS
TOP
ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
MOLECULAR REGULATION OF...
MOLECULAR REGULATION OF...
DETERMINANTS OF NEPHRON...
NEPHRON ENDOWMENT IN HOMOZYGOUS...
ESTIMATING NEPHRON NUMBER IN...
CONCLUDING REMARKS
REFERENCES

The principal morphological events involved in metanephric development, as distinct from the molecular events, have been understood for many years and are illustrated in Fig. 1. During vertebrate development, the permanent kidney is generated by the interaction of two tissue components: the epithelial ureteric bud and the metanephric mesenchyme. The ureteric bud branches from the Wolffian duct and invades the metanephric mesenchyme. Thereafter, the key event in nephrogenesis is the reciprocal inductive interaction between these two tissues. The metanephric mesenchyme induces the ureteric bud to grow and branch to form the collecting ducts. At the tips of the branches of the ureteric duct, the mesenchyme is induced to form a mesenchymal condensate. Following this the condensate converts into an epithelial vesicle.


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Fig. 1.   Schematic representation of metanephric development, from ureteric duct induction (A) to mesenchymal induction (B), condensation (C), vesicle (D), comma-shaped body (E), S-shaped body (F), uriniferous tubule (G), and growth and differentiation (H). White diamond-shaped cells (A-C) are mesenchymal cells, while black diamond-shaped cells (A-E) are mesenchymal cells predetermined to form glomerular endothelial cells. Arrows in A indicate the inductive signal sent from the mesenchyme to the ureteric duct epithelium inducing ureteric duct branching (B). Open arrows in B indicate the reciprocal signals sent from the ureteric duct epithelium to the mesenchyme inducing condensation (C). Solid black tubes (B-F) represent invading blood vessels that contribute to the renal vasculature, exclusive of glomerular capillaries. Dotted lines in G represent the afferent and efferent arterioles that connect the glomerular capillaries to the remainder of the renal vasculature. Although the full details of vascular development remain unclear in the metanephros, here are illustrated recent findings that suggest the origin of the glomerular vasculature is different from that of the remainder of the renal vasculature.

Nephron development from the epithelial vesicle involves a number of stages. First, the vesicle develops into a comma-shaped body. This develops into an S-shaped body. At this stage, the future renal corpuscle and proximal and distal tubules can be identified, with the lower limb of the S-shaped body going on to form the renal corpuscle. At about this stage, the S-shaped body fuses with the ureteric duct, and the lumina of these structures become continuous. A so-called uriniferous tubule has now formed. The renal corpuscle continues to develop with development of the glomerular tuft of capillaries and differentiation of glomerular epithelial cells (podocytes). At the same time, the nephron tubule elongates and differentiates along its length.

At this stage also, the medulla of the kidney begins to form, with growing nephron tubules and the interstitium now apparent. Nephrogenesis is complete in humans by birth (34), whereas in rats it is not complete until approximately postnatal days 8-10 (57). This postnatal period of kidney development in rodents provides a window of opportunity for in vivo manipulation studies, which to date has been little utilized by metanephric researchers.

Although these morphological events have been well characterized, we mention recent studies that address cell lineage and determination in the intermediate mesoderm. The intermediate mesoderm ultimately forms the urogenital system in the embryo. The invading ureteric duct epithelial cells are clearly predetermined cells of the collecting ducts observed in the adult kidney. Clonal studies have recently identified that the metanephric mesenchyme is also capable of contributing cells to the elongating ureteric duct, at least in vitro (69). At the same time, signals from the tips of the ureteric branches induce adjacent mesenchymal cells in the nephrogenic zone to aggregate and form a condensate. One report has suggested that the condensed mesenchymal cells, which are the recipients of the inductive signal, are in fact immediate descendants of ureteric bud epithelia (30). This report suggests that progenitor cells of the ureteric bud undergo an epithelial-to-mesenchymal transition to form a mesenchymal population of nephron progenitors. These mesenchymal cells are then induced to condense and convert into the epithelial vesicle of the nephron. Apart from the metanephric mesenchyme, which differentiates into nephron epithelia, a second compartment has been identified in the metanephric mesenchyme that forms renal vasculature progenitor cells or angioblasts (16). For many years it was thought that the renal vasculature was entirely derived from ingrowth of vessels from outside the kidney, but recent results from several groups suggest that at least some of the renal vasculature, including the glomerular tuft of capillaries, is derived from metanephric mesenchymal cells (16, 35, 99). A third compartment in the metanephric mesenchyme was recently identified as putative stromal cells, which express the transcription factor BF-2 (29).

The relationships between these cell lineages remain relatively unexplored. However, the importance of normal tissue interaction between each compartment and their relationship to nephron number was clearly demonstrated in the BF-2 homozygous null mutant mice. Abnormalities in stromal differentiation induced through the "knock in" of a lacZ cassette at the BF-2 locus saw the number of nephrons formed in -/- mice reduced by 75% compared with wild-type mice. Although BF-2 was never expressed in the ureteric duct or nephron epithelia, a reduction in branch point initiation in the ureteric duct was observed (see Branch Initiation below), and abnormalities in nephron differentiation occurred. Ultimately these events resulted in decreased nephron number.

In this review, we focus on three main developmental events in the regulation of nephron number. These are ureteric duct branching, mesenchymal condensation, and conversion of the condensate into an epithelial vesicle. First, the branching pattern of the ureteric which involves interaction of the ureteric duct with mesenchymal cells expressing BF-2 establishes the basic histoarchitecture of the kidney. Moreover, the number of nephrons that are formed in the kidney is critically dependent on the extent of ureteric duct branching. Second, mesenchymal condensation only occurs at predetermined sites adjacent to the ureteric duct, as no blind-ended nephrons exist in the normal adult kidney. Finally, only induced mesenchymal condensates convert to nephron epithelia. Here, we are not suggesting a single branch-single condensate-single nephron relationship. Instead, the molecular signals that drive ureteric duct branching and architecture would ultimately determine the positional information for mesenchymal condensation and nephron growth.


    MOLECULAR REGULATION OF GROWTH AND BRANCHING OF THE URETERIC DUCT
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ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
MOLECULAR REGULATION OF...
MOLECULAR REGULATION OF...
DETERMINANTS OF NEPHRON...
NEPHRON ENDOWMENT IN HOMOZYGOUS...
ESTIMATING NEPHRON NUMBER IN...
CONCLUDING REMARKS
REFERENCES

Growth of the ureteric duct involves branching as well as branch elongation. Unfortunately, the literature often refers to ureteric duct growth as involving "tubulogenesis." However, "tubulogenesis" implies that a tube has formed from a structure that was not originally a tube, as occurs for example in the formation of the nephron tubule. Although tubulogenesis is commonly observed in cultured kidney epithelial cells [for example, Madin-Darby canine kidney epithelial cells (MDCK cells)], it does not occur in the growth of the ureteric duct. Thus, although cell culture can provide a simple system for analyzing tubule growth, we must acknowledge that it is not an exact paradigm of ureteric duct growth in vivo. Moreover, we suggest it is desirable to consider tubule elongation and branching as separate events, likely to be regulated by different molecules. Separate consideration of the molecular regulation of these processes follows.

Branch Initiation

Many growth factors and oncogenes including hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha ), and c-met, as well as extracellular matrix (ECM) molecules, have been implicated in ureteric duct branch initiation in vitro. However, the most convincing evidence to date for a ligand/receptor complex initiation in ureteric bud branching concerns the protooncogene c-ret and its ligand glial cell line-derived neurotrophic factor (GDNF).

The c-ret protooncogene encodes a receptor tyrosine kinase with a Mr of 150 and 170 kDa (76). In developing metanephroi, c-ret mRNA is initially expressed throughout the growing ureteric epithelium, but expression soon becomes restricted to the tips of the ureteric buds (62). The c-ret homozygous null mutant mice have significant metanephric as well as enteric nervous system abnormalities (85, 86). The metanephroi of these animals are either absent or rudimentary. Those metanephroi that do form demonstrate severe dysplasia, reduced nephron number, reduced ureteric branches, and no recognizable medulla, cortex, or nephrogenic zone. The addition of phosphorothioated antisense oligonucleotides complementary to c-ret to mouse metanephric organ culture results in limited ureteric duct branching (45). Culture of mouse metanephroi with c-ret antisense together with either TGF-alpha or insulin-like growth factor I results in partial recovery of the branching epithelial phenotype. In this study, Liu et al. (45) also demonstrated that associated with a decrease in c-ret translation was a decrease in the translation of certain ECM molecules, most notably heparan sulfate proteoglycan (HSPG), laminin, and collagen type IV.

The c-ret ligand was recently identified to be GDNF (22, 76). GDNF is structurally related to the TGF-beta superfamily. In situ hybridization demonstrates that GDNF mRNA is expressed in mesenchyme surrounding the tips of the ureteric bud branches (22, 70). Like c-ret, GDNF homozygous null mutant mice demonstrate complete renal agenesis and no enteric nervous system (55, 67, 81). However, unlike heterozygous c-ret (+/-) mice, which are apparently normal, heterozygous GDNF (+/-) mice have varying degrees of renal abnormalities (67). These alternate phenotypes ultimately lead to the discovery of new GDNF family receptors designated GDNF family receptor-alpha (GDNFR-alpha ) and GDNFR-beta (82). GDNFR-alpha is expressed in condensing mesenchyme, nephrons, and ureteric duct tips (76). In contrast, GDNFR-beta is expressed in the uninduced metanephric mesenchyme and renal pelvis (82). In situ binding of 125I-labeled GDNF in the metanephros was found only at the ureteric duct tips, suggesting the site of signal transduction involves GDNF and GDNFR-alpha in ureteric duct tips only. Placement of GDNF-soaked beads next to the Wolffian duct of urogenital ridges in vitro induces ureteric bud formation on the Wolffian duct (76). Furthermore, in these experiments it was interesting to note that GDNF-soaked beads placed on urogenital ridge explants were also capable of inducing ectopic ureteric bud branches from the Wolffian duct outside the metanephric field (76). The buds that formed were always orientated toward the GDNF source. Growth of isolated ureteric buds in hanging drop cultures results in cell dispersal and apoptosis. In contrast, culture of ureteric bud hanging drop cultures in the presence of exogenous GDNF increases ureteric epithelial cell adhesiveness and promotes the formation of a basal lamina (76). Therefore, downstream effects of GDNF and c-ret appear to involve formation of an ECM and basal lamina around the advancing ureteric duct epithelium. Most recently, Pepicelli et al. (64) also demonstrated that ureteric duct branching and growth orientation is mediated by GDNF. In this study, mouse metanephric organ culture with GDNF-soaked beads resulted in ureteric duct mitogenesis. In contrast, isolated ureteric duct hanging drop cultures (76) demonstrated no role for GDNF in the proliferation of ureteric duct epithelial cells. These differences are obviously due to the presence or absence of the feedback loops between the metanephric mesenchyme and the ureteric duct epithelium. The most recent study went on to demonstrate that Wnt-11 is a potential target of GDNF/c-ret signaling (64). As previously described, BF-2 homozygous null mutant mice have decreased numbers of ureteric duct branch points. Furthermore, disruption in BF-2 signaling in the metanephric mesenchyme, presumably in the developing stroma, disrupts c-ret expression in the ureteric duct (29). This was associated with an 8- to 16-fold reduction in the number of ureteric branches and consequently a decrease by 75% in the number of nephrons formed (29).

With metanephric development and ureteric duct growth, changes in the synthesis and distribution of ECM molecules become apparent. The invading ureteric bud is surrounded by a delicate basal lamina composed of collagen type IV, laminin, and proteoglycans, in particular HSPG (25, 84). These molecules are synthesized and secreted by the ureteric duct epithelium. Of particular relevance to ureteric duct branching is the initial concentrated expression of proteoglycans at the tip of the ureteric duct (43). Proteoglycan molecules consist of a core protein containing O-glycosidic linkages and glycosaminoglycan (GAG) side chains. The GAG side chains appear to regulate the activity of the proteoglycan by sequestering growth factors or presenting growth factors such as fibroblast growth factors (FGFs) to their cell surface receptors (15). Removal of sulfated GAGs from metanephric rudiments inhibits both ureteric duct branching and elongation (17). Furthermore, treatment of these sulfated GAG-deprived kidneys with GDNF induced primitive ureteric bud branches that are incapable of lengthening (76). These studies demonstrate the importance of GDNF in branch initiation. However, this study also highlights the point that other undefined molecules are involved in ureteric duct growth which are not rescued with GDNF stimulation in GAG-depleted metanephroi.

Integrins are transmembrane molecules composed of a heterodimer between one alpha -subunit and one beta -subunit. Initially, integrins were thought to provide anchorage sites between cells and the ECM. It is now known that integrins play a role in cell migration, vasculogenesis, wound repair, and development. The alpha 8-subunit is expressed by mesenchymal cells surrounding the ureteric duct as well as the condensing mesenchymal cells at the ureteric bud tip (56). The alpha 8-integrin subunit always forms a heterodimer with the beta 1-subunit and serves as a receptor for fibronectin, vitronectin, and tenascin. However, these ligands do not colocalize with the alpha 8-subunit in the metanephros, suggesting the presence of an unidentified ligand (56). Homozygous alpha 8 null mutant mice have renal agenesis and dysgenesis. In over half of live births, the ureteric duct and kidneys do not form. In the 40% of cases where a ureter has formed, its elongation from the Wolffian duct to the metanephric mesenchyme is delayed, and branching is limited (56). It is interesting to note that heterologous mesenchymes such as lung parenchyma and submandibular mesenchyme do not support the growth of the ureteric duct (76), even though the branching and lengthening program of the ureteric duct is intrinsic to the duct itself. The alpha 8 knockout mice demonstrate the importance of the relationship between the ureteric duct, basal lamina, and mesenchyme for appropriate ureteric duct architecture.

Bone morphogenetic protein-4 (BMP-4) has recently been shown to have a significant effect on tubule growth and branching in the developing lung (3). BMP-4 is expressed at the tips of the bronchial buds and is believed to promote branching at this point. When BMP-4 is misexpressed at the distal tips of lung epithelial buds using SP-C enhancer/promoter, branching morphogenesis is affected (3). In the metanephros, BMP-4 mRNA is expressed in a subpopulation of mesenchymal cells surrounding the ureteric duct, the collecting ducts, and the ureteric duct leading up to the nephrogenic zone (20). As the location of BMP-4 receptors is currently unknown, it is difficult to predict whether BMP-4 acts on ureteric duct branching or elongation. BMP-4 homozygous null mutant mice die at gastrulation (97). Of interest, BMP-4 (+/-) heterozygous mice backcrossed onto the C57BL/6 genetic background demonstrate cystic kidneys (21). Therefore, this result, like that for GDNF heterozygous mice, supports the important emerging concept of gene dosage in renal development. This concept is discussed below (see NEPHRON ENDOWMENT IN HOMOZYGOUS AND HETEROZYGOUS NULL MUTANT MICE).

Branch Elongation

Once a ureteric duct branch point has formed, the epithelial tubule buds, elongates around the branch point, and lengthens into the nephrogenic zone. To date, no molecule has been identified that solely regulates branch elongation, since the molecules found to regulate branch elongation also appear to play a role in branch initiation. Thus a redundant network of factors appears to exist. These molecules are discussed in Downstream Molecules in Ureteric Duct Branching and Elongation below.

Inhibition of Branch Initiation and Elongation

Associated with the molecules that positively regulate ureteric duct branch initiation and elongation are the molecules that inhibit these processes. The TGF-beta superfamily is a large superfamily of molecules closely related in terms of amino acid homology, molecular processing, and receptor interactions. The TGF-beta superfamily encompasses the TGF-beta family, BMPs, activins, inhibins, and Mullerian inhibitory substance. All these growth factors signal through serine/threonine kinase receptors.

TGF-beta 1 mRNA is highly expressed in the nephrogenic zone as well as in all regions of the ureteric duct (13). TGF-beta type II receptor, the principal TGF-beta family signaling receptor, is also expressed on the ureteric duct and in nephrogenic zone mesenchyme (11, 12). Early experiments with MDCK cells demonstrated that TGF-beta 1 inhibited tubulogenesis and branching (83). However, it is now known that MDCK cells are extremely susceptible to TGF-beta 1, which makes interpretation of these results difficult. Unlike MDCK cells, murine inner medullary collecting duct cell lines (mIMCD-3 cells) are less susceptible to the inhibitory effects of TGF-beta 1 (79). Growth of mIMCD-3 cells in the presence of Swiss 3T3-conditioned media results in tubulogenesis and branching (79). When TGF-beta 1, TGF-beta 2, or TGF-beta 3 is added at a critical concentration, mIMCD-3 cells form long straight tubules without branch points. At higher concentrations, branching and elongation are totally inhibited (79). Furthermore, the activity of TGF-beta was associated with a reduction in HGF expression by mIMCD-3 cells, a molecule that promotes branching in this system.

In rat metanephric organ culture, addition of excess TGF-beta 1 inhibits growth of the ureteric duct (14, 72). Further studies using metanephric organ culture have demonstrated that TGF-beta 1 has no morphological effect on nephron (as distinct from ureteric) tubule growth (70) or glomerular formation (14). However, ureteric duct growth and survival of nephrogenic zone mesenchyme are adversely effected (14). In some cases, long straight ureteric duct tubules were observed with no apparent branch points (14, 72). In other instances, ureteric duct growth was totally inhibited. Whole mount immunohistochemistry against cytokeratin 8 has demonstrated that in the presence of TGF-beta 1, the ureteric duct forms umbrella-like arcades rather than dichotomous branches (70). In combination, these results suggest that TGF-beta potency is dependent on the presence of other growth factors, ECM molecules, regulators of TGF-beta activity, and of course the presence of receptor complexes in the ureteric duct epithelium. Of interest, TGF-beta 1 homozygous null mutant mice demonstrate no abnormal renal phenotype (41). In comparison, type II receptor null mutants (59) die at embryonic day 9.5 (E9.5) due to abnormalities in vasculogenesis and placental development. The redundancy of TGF-beta family members in branching morphogenesis may explain in part the absence of an abnormal renal phenotype in the TGF-beta 1 knockouts.

Piscione et al. (68) recently analyzed the role of BMP-2 and BMP-7 in branching morphogenesis using mIMCD-3 cells and mouse metanephric organ culture. BMP-2 was found to inhibit branching and elongation of both mIMCD-3 cells and ureteric duct epithelium in vitro. In comparison, BMP-7 at low concentrations promoted branching of mIMCD-3 cells, whereas at high concentrations it completely inhibited branch initiation and elongation in mIMCD-3 cells. Using BMP-7-coated beads, Piscione et al. (68) found that ureteric duct branching closest to the bead was inhibited, whereas further from the bead, and therefore lower concentrations of BMP-7, branching morphogenesis was promoted. Therefore, the effect of TGF-beta 1 and BMP-7 on branch initiation and elongation are dependent upon the local concentration of growth factor, cell type, and the extracellular environment. BMP-2 homozygous null mutant mice die before metanephric development begins (100). BMP-7 knockout mice (19, 36, 47) die of renal failure and have severe abnormalities of the kidneys, eyes, and skeleton. In metanephroi, the ureter branches once or twice, and nephron development begins. In these animals, comma-shaped bodies and S-shaped bodies form normally, and glomerulogenesis occurs in these first generations of nephrons. After E14.5 (19), ureteric duct branch initiation and nephrogenesis stops, and renal architecture becomes disorganized. As a result BMP-7 -/- mice are born with small kidneys and reduced nephron endowment (19, 36, 47). Why ureteric duct branch initiation and nephrogenesis begins normally and then stops is unknown.

Downstream Molecules in Ureteric Duct Branching and Elongation

HGF is highly expressed in the nephrogenic zone mesenchyme of developing metanephroi as well as in mesenchymal cell condensates. Its receptor, c-met, is expressed in the ureteric duct epithelium and in early nephron structures (98). Fusion protein experiments in which the intracellular domain of c-met is fused to the extracellular domain of trkA demonstrate that intracellular signaling through c-met, regardless of the ligand bound in the extracellular domain, induces tubulogenesis and branching in MDCK cells (75). Furthermore, growth of MDCK cells in three-dimensional type I collagen gels with serum and HGF results in the formation of a branched tubular structure from cysts (53, 54, 83). Epithelial cysts also form when HGF-neutralizing antibody is added to mouse metanephroi in culture (98). From this evidence, it is surprising that HGF knockout and c-met knockout mice have no renal abnormalities (94). Taken together, these results demonstrate that HGF and c-met are capable of inducing tubule formation and branching in vitro, but most likely act together with other molecules including EGF and TGF-alpha through the EGF receptor in ureteric duct growth.

Culture of mouse metanephroi in the presence of EGF results in a slight increase in DNA synthesis (10). However, it is not known whether this increase in cell proliferation occurs primarily in the ureteric duct or in other cell populations. Therefore, the contribution of EGF-stimulated cellular hyperplasia to ureteric duct elongation is unclear. Culture of MDCK cells in type I collagen gels in the presence of EGF results in the formation of tubular structures with little or no branching (71).

TGF-alpha appears to play an intermediate role between branch initiation and elongation. When rat metanephroi are cultured in serum-free chemically defined media, they secrete TGF-alpha but not EGF into the media (71). Addition of TGF-alpha neutralizing antibodies to rat metanephroi in culture severely inhibits ureteric epithelial growth by activity through the EGF receptor (2). Thus, during in vitro metanephric ureteric duct growth, it appears that TGF-alpha , not EGF, acts primarily through the EGF receptor.

The EGF receptor is expressed throughout the ureteric duct and is also found in mesenchymal cell condensates, comma-shaped bodies, S-shaped bodies, glomeruli, nephron tubules, and interstitial cells (63). UB cells (a ureteric duct cell line) have been used to examine the role of the EGF receptor in ureteric duct branching. When signal transduction through the EGF receptor was inhibited using tyrophostin AG1478 (78), tubulogenesis, branching, and elongation were inhibited. Addition of tyrophostin AG1478 together with HGF to UB cells resulted in almost complete inhibition of elongation and branching after 24 h. However, with increased time in culture (5 days), tubule elongation and branching was observed regardless of the presence of the inhibitors (78).

The matrix metalloproteinases (MMPs) are a large family of zinc-dependent matrix degrading enzymes. This family includes the interstitial collagenases, stromeolysins, and type IV collagenases. In mouse metanephroi, Western blotting was used to demonstrate that the metanephros expresses both MMP-2 and MMP-9 (44). Inhibition of MMP-9 using both a neutralizing antibody or its natural inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1) results in severe inhibition of ureteric duct growth (44). However, these results do not demonstrate whether MMP-9 plays a role in ureteric branch initiation, branch elongation, or both. It can be speculated that the MMPs would be required for localized digestion of ECM at the leading edge of the elongating ureteric duct tip.

Overexpression of MMP-3 in the mammary gland of mice leads to increased branch number (92). To date, there have been no studies on the role of MMP-3 in the regulation of ureteric duct growth.

Cell Birth and Death in Generating Ureteric Duct Architecture

The classic microdissection studies of Osathanondh and Potter (60, 61) showed that the initial branches of the ureteric duct are lost with formation of the renal pelvis. As no blind-ended nephrons or ureteric ducts are thought to exist in the adult kidney, the ureteric bud branches and nephrons that form in the region that becomes the pelvis are presumably removed via apoptosis. Thus formation of the renal pelvis involves a balance between cell birth and cell death in the ureteric duct.

Although the molecular regulation of cell birth and death in the collecting duct epithelium are unknown, recent data suggest a possible role for the homeobox gene Emx-2 in this process. Emx-2 is a vertebrate homolog of Drosophila head gap gene empty spiracles (ems) (87). The Emx-2 gene was first isolated from an 8-wk human embryo cDNA library (87). In mouse metanephroi, Emx-2 is found in metanephric tubules. When this gene is knocked out, there is a complete absence of kidneys, ureter, gonads, and genital tract (52). Heterozygous null mutant mice appear normal. However, in the homozygous null mutants, the Wolffian duct, ureteric bud, and metanephric mesenchyme form. This suggests that Emx-2 is not involved in specification of the intermediate mesoderm to renal bias. Explant cultures of Emx-2 -/- metanephric mesenchyme in the presence of wild-type ureteric bud epithelia results in normal metanephric growth. In contrast, coculture of wild-type metanephric mesenchyme with Emx-2 -/- ureteric bud results in apoptosis of the ureteric bud epithelial cells. Therefore, these experiments demonstrate that the loss of Emx-2 activity in the ureteric duct induces apoptosis of the ureteric duct epithelium, whereas loss of Emx-2 in the metanephric mesenchyme has no apparent effect on nephron induction.


    MOLECULAR REGULATION OF MESENCHYMAL CONDENSATION
TOP
ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
MOLECULAR REGULATION OF...
MOLECULAR REGULATION OF...
DETERMINANTS OF NEPHRON...
NEPHRON ENDOWMENT IN HOMOZYGOUS...
ESTIMATING NEPHRON NUMBER IN...
CONCLUDING REMARKS
REFERENCES

The signal that initiates the first morphological event in nephron formation, namely mesenchymal cell condensation, comes only from the ureteric duct in vivo. Significantly, this signal is only transmitted from the tips of the ureteric duct branches, not along the entire length, hence the relationship (albeit not a one-to-one relationship) between ureteric duct branching and nephron number.

Heterologous tissues including embryonic spinal cord, embryonic brain, and submandibular mesenchyme can mimic the effect of the ureteric duct as a mesenchymal condensate inducer in vitro (84). This rescues the mesenchymal cells from a death fate and promotes epithelial formation and nephrogenesis.

Maintenance

The Wilms' tumor gene (WT-1) belongs to the zinc finger gene family. In the developing mouse metanephros, WT-1 is expressed in uninduced mesenchyme, the stem cells of the nephrogenic zone, condensates, and glomerular podocytes (1). Once the mesenchyme is induced to condense, WT-1 expression is upregulated. Recent studies suggest that upregulation of WT-1 in condensates is regulated by FGF-2 (see Migration/Proliferation below). When the WT-1 gene is knocked out, the metanephric mesenchyme forms, but then a wave of apoptosis occurs. The metanephrogenic mesenchyme, which initially forms, is incapable of being induced to condense (40). These findings suggest that WT-1 acts for a very brief time in nephrogenesis and potentially makes the mesenchymal cell population receptive to the inductive signal sent from the ureteric duct. Of interest, WT-1 activity is not only required in the mesenchyme of the metanephros but also in the caudal region of the mesonephros (the second primitive kidney) (77). This gradient of WT-1 expression in the intermediate mesoderm may prove crucial in the positioning of the metanephros during organogenesis. Unfortunately, the molecules that act downstream in the WT-1 pathway are ill-defined, and therefore their role in condensation of metanephric mesenchyme cannot be fully ascertained at this time.

Migration/Proliferation

The initial inductive molecular signal sent from the ureteric bud tips to the adjacent metanephric mesenchymal cells is not known, although signaling appears to require multiple feedback loops between the ureteric duct and metanephric mesenchyme. One of the earliest morphological events in mesenchymal cell condensation is the migration of the induced mesenchymal cells away from the duct (84). Mesenchymal cell proliferation then ensues.

Pax-2 is the earliest known marker of mesenchymal condensation. In mouse metanephroi, Pax-2 is expressed in early mesenchymal condensates. Expression persists in vesicle and comma- and S-shaped bodies (18). When Pax-2 antisense oligonucleotides are added to metanephric culture, ureteric branching is significantly limited (73). However, this inhibition of ureteric duct branching is potentially a consequence of the inhibition of condensate formation.

Pax-2 homozygous null mutant mice demonstrate no mesonephric, metanephric, or genital development. The ureteric duct and the nephron tubules do not form (93). Although this finding demonstrates a significant role for Pax-2 in renal development, it does not provide any information on the roles of Pax-2 in either ureteric duct or nephron development. A recent review (42) addressed the phenotype of Danforth's short tail syndrome. This involves the development of hypoplastic metanephroi in which invasion of the ureteric bud is inhibited (66). In these mice, mesenchymal cell condensation does not occur, and there is no expression of Pax-2. Activity of Pax-2 is therefore necessary prior to or during the migration of the mesenchyme away from the ureteric duct and its subsequent proliferation (84).

FGFs constitute a large family of molecules that interact through a number of receptor tyrosine kinases. The interactions of these growth factors with their specific high-affinity receptors are critically dependent upon sequence-specific HSPGs. The receptor mechanics of the FGFs are complex and have been well reviewed (15). Of the twelve FGF family members, FGF-2 appears to be involved in the condensation of mesenchyme at the ureteric bud tips (65). However, FGF-2 alone is incapable of promoting mesenchyme to epithelial conversion (65). The activity of FGF-2 in condensation appears to involve the activation of downstream signaling molecules such as sonic hedgehog (39).

Recently, Karavanova and coworkers (37) demonstrated that isolated metanephric mesenchymes grown in the presence of FGF-2 and ureteric bud-conditioned media supplemented with TGF-alpha develop past the condensate stage. The condensed mesenchyme undergoes mesenchymal-to-epithelial conversion and nephron-specific epithelial differentiation. Therefore, once condensation is initiated by FGF-2, further nephron development is aided by secreted factors from the ureteric duct. The findings of Perantoni et al. (65) regarding the role of FGF-2 in condensation are supported by recent findings from our own laboratory. We have found that addition of specific heparan sulfate GAGs to rat metanephric culture inhibits ureteric duct growth (elongation and branching), as well as mesenchymal cell proliferation and condensation (5). No mesenchymal-to-epithelial conversion is observed. These GAGs are known to specifically direct FGF-2 to its signaling receptor. On the other hand, incubation of rat metanephroi with both the GAG and exogenous FGF-2 leads to marked mesenchymal cell proliferation. Again, however, ureteric duct growth was severely inhibited and no epithelialization was observed. The mesenchymal cells were found adjacent to all surfaces of the ureteric epithelium. When the GAG was added to rat metanephric culture following formation of condensates and comma-shaped bodies (day 2), some mature glomeruli were observed by day 5. This indicates that FGF-2 was less important during the later stages of nephrogenesis. These results are difficult to interpret in isolation but do indicate that expression of FGF-2 in the correct location and in the correct concentration is necessary, either directly or indirectly, for mesenchymal cell condensation.

BF-2 homozygous null mutant mice were found to have ectopic expression of c-ret in the lengths of ureteric duct. This was also associated with ectopic expression of mesenchymal condensates and abnormally large mesenchymal condensates (29).


    MOLECULAR REGULATION OF MESENCHYMAL-TO-EPITHELIAL CONVERSION
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Although we are considering mesenchymal condensation and epithelial conversion as separate events, they in fact occur simultaneously. For example, with migration and proliferation of the induced mesenchymal cells away from the ureteric duct, changes in cell-to-cell adhesion, ECM composition, and expression of epithelial markers become apparent. These changes are associated with the formation of morphologically recognized epithelia in comparison to the mesenchymal phenotype of the remaining cells in the nephrogenic zone.

Formation of a Basal Lamina

Metanephric mesenchymal cells are surrounded by an ECM, which rapidly changes in composition upon induction. Uninduced mesenchymal cells are surrounded by collagen type I and type III and fibronectin (23, 24). On the cell surface, uninduced mesenchymal cells express nerve cell adhesion molecule (NCAM), WT-1, syndecan, and a variety of integrins (24, 74). With induction, collagen types I and III are rapidly lost and are replaced by collagen type IV, laminin alpha -chain in association with beta  and beta 2, and HSPG (23, 24).

Cell Polarization/Adhesion

The Wnt family of molecules constitutes a large family of 16 closely related cysteine-rich secreted molecules. Wnt-1 is capable of conferring nephron-inducing activity to fibroblastic cell lines (31). Furthermore, coculture of cells expressing Wnt-1 with mouse metanephric mesenchyme results in mesenchymal-to-epithelial conversion. However, Wnt-1 is not expressed in the metanephros (96).

Instead, a second member of the Wnt family (Wnt-4) appears to be involved in nephrogenesis. This molecule is expressed in condensing mesenchyme and comma- and S-shaped bodies (90). This places Wnt-4 in the appropriate location to act in the conversion of condensed mesenchyme to epithelium. Further evidence for a role for Wnt-4 in mesenchymal-to-epithelial conversion came with the generation of homozygous null mutant Wnt-4 mice (90). Although the mice developed metanephric mesenchyme, this mesenchyme was incapable of undergoing epithelial conversion.

A recent study by Kispert et al. (38) using transfilter organ culture demonstrated that Wnt-4 is required only for conversion of the mesenchymal condensate to epithelia and not for later stages of nephrogenesis. Furthermore, Wnt-4 activity is dependent on cell contact and the presence of sulfated proteoglycans (38). These results along with the lack of epithelial tubules in the Wnt-4 homozygous null mutant mice suggest that Wnt-4 plays a critical role in the conversion of mesenchyme to epithelium, in the metanephros. Interestingly, Wnt-4 is not produced by the tips of the ureteric duct epithelium and instead acts as a mesenchymal-derived factor for inducing epithelialization.

Laminins are large glycoproteins composed of three chains forming a crucifix composed of one alpha -chain and two beta -chains. As mentioned previously, laminin forms a major component of all basal laminae in the adult and developing kidney (24, 25). In the metanephros, laminin alpha 2 is expressed in the undifferentiated mesenchyme (88). However, with induction and condensation, the mesenchymal cells stop producing laminin alpha 2 and begin to synthesize laminins beta 1 and gamma 1. The laminin alpha -chain (laminin alpha 1) subsequently does not appear in the mesenchymal condensate until epithelial polarization (88).

Expression of the alpha 6beta 1 integrin correlates with the expression of laminin alpha 1 in polarizing mesenchymal cells as they form epithelia (89). In isolated metanephric mesenchymes, the alpha 6-integrin is upregulated with mesenchymal polarization and epithelial formation. Furthermore, disruption of alpha 6 activity prevents tubulogenesis in this system (89). A second integrin involved in mesenchymal-to-epithelial conversion is alpha 8 (56). Previously, this integrin was discussed with reference to ureteric duct branching (56). However, alpha 8 knockout mice also reveal roles for this integrin in mesenchymal-to-epithelial conversion. Analysis of the metanephroi that form in knockout alpha 8 mice revealed that alpha 8 plays no role in mesenchymal cell condensation, as the markers Pax-2 and the low-affinity nerve growth factor receptor p75NTR were expressed. Furthermore, these condensed mesenchymal cells had migrated away from the ureteric duct. However, in the majority of cases, the conversion of condensing mesenchymal cells to epithelium was defective (56).


    DETERMINANTS OF NEPHRON ENDOWMENT
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ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
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Despite the substantial progress that has been made in recent years in our understanding of the molecular regulation of nephron development, our understanding of the mechanisms that determine whether a normal human kidney develops 300,000 nephrons or more than 1 million nephrons remains largely unclear. Considering the foregoing discussion of the molecular regulation of ureteric duct branch initiation and lengthening, mesenchymal cell condensation, and mesenchymal-to-epithelial conversion, it is clear that disturbances to any of these events may lead to the development of a kidney with reduced nephron endowment. Sakurai and Nigam (80) recently estimated that a 2% decrease in the "efficiency" of ureteric bud branching would manifest itself as an ~50% reduction in the "normal" number of nephrons after 20 generations of branching. A complete understanding of the molecular regulation of branch initiation and growth will provide precise information on just how this process of branching is achieved "efficiently" in the developing kidney. The mass of undifferentiated metanephric mesenchyme can also be hypothesized to regulate nephron endowment. Identification of the factors that regulate proliferation, migration, and survival of these cells will be crucial in fully understanding the molecular regulation of nephron endowment. The relationship between the degree of apoptosis in the metanephros and nephron endowment remains poorly defined (51).

In recent years the contribution of nongenetic determinants to the regulation of nephron endowment has generated considerable interest. Of particular social importance, in utero exposure of the rat fetus to the aminoglycoside antibiotic gentamicin results in nephron deficit (26, 49). In subsequent metanephric organ culture experiments, gentamicin was shown to restrict nephron endowment through inhibition of ureteric duct branch point formation and lengthening (27, 28). It remains to be determined whether gentamicin inhibits a specific molecular pathway in ureteric duct growth or acts as a more general inhibitor of cellular activity. A reduction in nephron number has also been linked to intrauterine growth retardation (IUGR) in humans and animals (33, 34, 46, 50, 57). The molecular events by which IUGR manifests itself in a reduction in nephron number have not been explored but undoubtedly warrant further investigation. For a fuller consideration of IUGR and nephron endowment, see Merlet-Benichou et al. (51).


    NEPHRON ENDOWMENT IN HOMOZYGOUS AND HETEROZYGOUS NULL MUTANT MICE
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As described above, renal developmental abnormalities have been described in numerous homozygous null mutant mice including GDNF, c-ret, BMP-7, alpha 8-integrin, Emx-2, WT-1, Pax-2, BF-2, and Wnt-4 knockout mice. In many of these animals, the renal phenotype is so severe that little, if any, information can be gained about the roles of these molecules in nephron endowment. Knockout of other molecules, including the TGF-beta type II receptor and BMP-2, leads to embryonic death before the commencement of metanephric development, so again the role of these molecules in regulation of nephron endowment cannot be determined using homozygous null mutations.

However, quantitative determination of nephron number in heterozygous null mutant mice may provide a powerful in vivo strategy for determining the effects of gene dosage on nephron endowment. While the majority of heterozygous null mutant mice have been reported to have no renal abnormalities, in none of these studies has nephron endowment been accurately determined. In addition, estimation of nephron number in knockout mice without any phenotype may also prove worthwhile. Moreover, in very few studies have mice been studied into older age, and such parameters as systemic blood pressure, creatinine clearance, and proteinuria have rarely been determined. A number of heterozygous nulls have been reported to have renal abnormalities, including GDNF (55, 67, 81) and BMP-4 (21) heterozygotes. Piscione et al. (68) have shown in vitro that the effects of BMP-7 are dependent on its local concentration within the developing kidney tissue. These in vivo and in vitro findings suggest that critical concentrations of certain molecules are required for normal nephrogenesis. Therefore, are critical concentrations of certain molecules required for normal nephron endowment? Determination of nephron endowment in these animals and the relationships to gene dosage appears worthwhile.

Accurate information about nephron endowment is impossible to obtain from a routine qualitative histological analysis of kidney sections, but precise and unbiased stereological methods have been developed in recent years for estimating the total number of nephrons in kidneys. These methods are described in some detail below. Correlation of nephron number with quantitative data on levels of specific mRNAs provides a powerful approach to assessing the effects of gene dosage on nephron endowment.

The stereological methods described below can also be used to estimate total nephron number in homozygous or heterozygous null mutants crossed onto different backgrounds. Recently, for example, Dunn et al. (21) reported that BMP-4 heterozygous mutants have renal defects when bred on a C57BL/6 background. In comparison, when BMP-4 heterozygous mice are bred on a 129 × Black Swiss background the kidney defects are not consistent between individuals. They concluded that strain-dependent differences in the activity of genes encoding extracellular proteins that bind and inactivate BMP-4, BMP receptors, downstream effectors, or proteins with partially overlapping functions may cause local variations in active BMP protein levels at critical stages during kidney development. Clearly, sophisticated questions can be asked using these powerful genetic approaches. Again, however, stereological analysis is required to ascertain the effects on nephron endowment.


    ESTIMATING NEPHRON NUMBER IN VIVO AND IN VITRO
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In recent years, unbiased stereological methods have been developed for estimating the total number of glomeruli, and thereby nephrons, in kidneys. The most commonly employed method involves counting glomeruli using the physical disector (correctly spelled with 1 "s") principle (91) in a known fraction of the kidney. This is known as the physical disector/fractionator strategy and has been used to count nephrons in many laboratory animals, as well as in humans (4, 58). A significant advantage of the physical disector/fractionator combination compared with traditional methods for counting glomeruli, is that glomeruli are sampled uniformly: small glomeruli have the same chance of being counted as large glomeruli. Moreover, no knowledge or assumptions of glomerular size, size heterogeneity, shape, orientation, or location in the kidney is required. In practical terms, the method involves successively sampling the kidney (slices, sections, fields in sections) and then viewing pairs of adjacent sections with a pair of matching light microscopes. Glomeruli sampled by an unbiased counting frame in the first microscope that are not present in the adjacent section are counted. Total nephron number is calculated as this sum multiplied by the inverses of the successive sampling fractions. Full details of the method are given in Bertram (4) and Bertram et al. (6). A similar but not identical method was used by Hinchliffe et al. (32-34), who studied the effects of IUGR on renal development.

The physical disector/fractionator combination can also be used to count glomeruli in developing metanephroi and in metanephroi following organ culture. When dealing with such small organs, however, it is not possible, or necessary, to first cut them into slices. Rather, the organs can be embedded whole in paraffin or methacrylate, and a known fraction of sections can then be mounted for glomerular counting. Indeed, in the early stages of development, or following metanephric organ culture, given that so few glomeruli are present, glomeruli should be counted in all sections. This is not laborious when methacrylate sections are used, because one typically uses 20-µm sections.

The stereological methods described above involve identifying and counting glomeruli, and thereby nephrons, in histological sections stained with routine dyes (hematoxylin and eosin, periodic acid-Schiff). Possibly a better approach, particularly when studying developing organs, is to count structures that express specific differentiation markers. Such markers could be identified using histochemistry or immunohistochemistry. For example, sections immunostained for Pax-2 or WT-1 could be used to count condensing mesenchyme and glomeruli, respectively, with stereology. Lectin histochemistry combined with image analysis has been used to quantitatively assess glomerular development and ureteric branching in cultured metanephroi (95).


    CONCLUDING REMARKS
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ABSTRACT
INTRODUCTION
DEVELOPMENT OF THE METANEPHROS
MOLECULAR REGULATION OF GROWTH...
MOLECULAR REGULATION OF...
MOLECULAR REGULATION OF...
DETERMINANTS OF NEPHRON...
NEPHRON ENDOWMENT IN HOMOZYGOUS...
ESTIMATING NEPHRON NUMBER IN...
CONCLUDING REMARKS
REFERENCES

In this review, we have focused on some key molecules that appear to be important in the molecular regulation of nephron formation. Although many of the details of the molecular program required to generate and build a nephron are known, in a global sense, precisely what determines whether a human kidney develops 300,000 nephrons or 1,000,000 nephrons remains unknown. Clearly, IUGR and maternal malnutrition influence fetal weight, kidney size, and nephron number. The dosage of specific genes may also be shown to be significant. Presumably, however, the development of the first nephron is driven and regulated by the same molecules as those required to build the last nephron. When the molecular processes that govern these three major events are determined, a fuller understanding of the molecular regulation of nephron endowment will presumably be closer at hand.


    ACKNOWLEDGEMENTS

Our research on metanephric development is supported by grants from the National Health and Medical Research Council of Australia. Amander Clark is the recipient of an Australian Postgraduate Award. Ian Miatke provided expert assistance with the preparation of Fig. 1.


    FOOTNOTES

Current address of A. T. Clark: Dept. of Pathology, Baylor College of Medicine, Houston, Texas, 77030.

Address for reprint requests and other correspondence: J. F. Bertram, Dept. of Anatomy, Monash Univ., Clayton, Victoria 3168, Australia (E-mail: J.Bertram{at}med.monash.edu.au).


    REFERENCES
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CONCLUDING REMARKS
REFERENCES

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