Kidney Development: Regulatory Molecules Crucial to Both Mice and Men

Molecular Genetics and Metabolism 71, 391–396 (2000) doi:10.1006/mgme.2000.3072, available online at http://www.idealibrary.com on

MINIREVIEW Kidney Development: Regulatory Molecules Crucial to Both Mice and Men Carlton M. Bates 1 Children’s Research Institute, Children’s Hospital, Columbus, Ohio 43205; and Division of Nephrology, Department of Pediatrics, College of Medicine & Public Health, Ohio State University, Columbus, Ohio 43210 Received August 1, 2000

teric bud epithelia, were found to be necessary and sufficient for in vitro kidney formation (1). The ureteric bud forms as an evagination of the caudal portion of the Wolffian or mesonephric duct at mouse embryonic day 11.5 (E11.5) or at about the fifth week of gestation in humans (2). The bud then grows into the metanephric mesenchyme, a density of cells adjacent to the mesonephric duct. The mesenchyme induces the ureteric bud to branch successively (see Fig. 1). In turn, each ureteric bud branch tip signals local areas of mesenchyme to condense and eventually converts into epithelium of the nephron, i.e., glomerular and tubular epithelium (see Fig. 1). The nephron then fuses with the ureteric bud, which forms the collecting duct, pelvis, and ureter. Vascular components of the kidney may develop from metanephric mesenchymal cells in situ (3) or may migrate into the blastema from precursors outside the kidney (4). Scientists began deciphering molecular control of kidney development in the 1990s. Although many factors are expressed in the embryonic kidney, mouse gene targeting studies began revealing which were crucial for normal renal development. Also, manipulation of mouse and rat kidney explants with mRNA antisense oligomers has identified other factors important for renal organogenesis. At present, the Kidney Developmental Database website lists more than 40 genes that participate in rodent renal organogenesis (URL addresses http://golgi.ana.ed.ac.uk/kidhome. html and http://www.ana.ed.ac.uk/anatomy/database/ kidbase/kidhome.html). Since most of these regulatory

Although the study of embryonic kidney development began in the 1950s, three decades passed until scientists began identifying the molecular controls of renal organogenesis. Most of these advances have come from mouse gene targeting and rodent kidney explant manipulation. Translation of the rodent data to human congenital kidney disease has only just begun. The activities of those regulatory molecules proven to be used in common appear remarkably similar in mouse and human renal development. Examples of these genes include glial cell line-derived neurotrophic factor (GDNF), RET, PAX2, Wilms tumor suppressor (WT1), and components in the renin–angiotensin pathway. Other factors that participate in mouse renal organogenesis, such as N-Myc, may later be proven important in human kidney development. © 2000 Academic Press Key Words: kidney; development; human; rodent; GDNF; RET; PAX2; WT1; angiotensin; N-Myc.

BACKGROUND ON KIDNEY DEVELOPMENT In the 1950s, Clifford Grobstein demonstrated that embryonic kidney rudiments could be excised and grown in culture, recapitulating development in vivo, except that the explants were avascular (1). Two tissues derived from the intermediate mesoderm, the metanephric mesenchyme and the ure1 Address correspondence to the author at Children’s Research Institute, 700 Children’s Drive, Columbus, OH 43205. Fax: (614) 722-2716. E-mail: [email protected].

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FIG. 1. Mouse kidney explant immunostained against the branching ureteric bud in green (arrow) and developing glomeruli from the metanephric mesenchyme in red (arrowheads) (unpublished from C.B.).

molecules have only recently been described in rodents, their relevance to human kidney development is largely speculative. Abnormalities in those few that have been proven important in both mice and men, however, result in strikingly similar defects. The remainder of this review will describe examples of these regulatory molecules including GDNF and its receptor RET, PAX2, WT1, and components of the renin–angiotensin pathway (Table 1). N-Myc, recently identified as a regulatory molecule in mouse renal development, will be discussed as an example of a gene that may later be found to be important in humans [for more complete reviews on the molecular control of kidney development, see (5) and (6)]. REGULATORY MOLECULES IN KIDNEY DEVELOPMENT Ret is a receptor tyrosine kinase expressed in the mouse embryonic kidney as well as portions of the central, peripheral, and enteric nervous system (7). In developing renal structures, Ret is initially located on the mesonephric duct and the early ureteric bud, and later its expression is restricted to ureteric bud branch tips (7). Mice with a c-ret null mutation demonstrated complete renal agenesis or severe dysplasia, secondary to defective ureteric bud branching (8). Nonrenal manifestations included enteric ganglia agenesis with intestinal obstruction (8) and sympathetic ganglia defects (9). The Ret ligand was later identified as glial cell

line-derived neurotrophic factor, a transforming growth factor-␤ family member (10,11). In the kidney, GDNF is expressed in the metanephric mesenchyme adjacent to the ureteric bud tips (10,11). Mice with mutations in GDNF demonstrated defects similar to c-ret-deficient mice, including kidney aplasia and dysplasia, sympathetic ganglia defects, and intestinal obstruction (12,13). Interestingly, mice heterozygous for the GDNF mutation occasionally demonstrated unilateral or bilateral kidney dysplasia (12,13). Although defects in RET-GDNF signaling have been linked with many nonrenal, human diseases, information on kidney defects is only now emerging. Constitutive overexpression of RET due to somatic rearrangements (no mouse model available) is associated with some papillary thyroid carcinomas (14). Deletions or mutations with decreased activity in RET and more rarely in GDNF are associated with autosomal dominant forms of Hirschprung’s disease, consistent with the mouse data (14,15). In addition, germline ret mutations may cause multiple endocrine neoplasia (MEN) type 2A, MEN type 2B, and familial medullary thyroid carcinoma (14). Although less is known about the role of RET and GDNF in the human kidney, a recent report documents unilateral kidney aplasia in mother and son with a germline RET mutation (16). Both have familial medullary thyroid cancer and the son was diagnosed with Hirschprung’s disease as an infant (16). Thus, the human kidney defects and Hirschprung’s disease associated with GDNF and RET mutations are highly reminiscent of the abnormalities in mice. Pax2 is a paired-box DNA-binding protein expressed in developing mouse kidney and nervous tissues including the optic stalk, midbrain– hindbrain junction, and spinal cord (17). In the kidney, Pax2 is detected in the caudal mesonephric duct, the ureteric bud, and later in mesenchymal condensates induced by the bud (18). As the condensates convert into nephron epithelia, Pax2 expression is suppressed, most likely by WT1 (18,19). Pax2 may also be regulated by other factors such as FGF-8 and Pax6 (17). Candidate targets of Pax2 activity include Wt1 (20), Pax5 (21), Pax6, and itself (17). In addition to optic nerve and inner ear defects (22), Pax2 null mice do not develop kidneys (23). Their caudal mesonephric ducts degenerate and ureteric buds do not form; without stimuli from the buds, rudimentary metanephric mesenchymes quickly disappear (23). As with GDNF, mice heterozygous for Pax2 mutations often demonstrate

KIDNEY DEVELOPMENT IN MICE AND MEN

kidney hypoplasia (23). Conversely, unregulated expression of Pax2 in mice is associated with cystic, hyperproliferative mouse kidney phenotypes (24). As in mice, both over- and underexpression of PAX2 are associated with human diseases. PAX2 haploinsufficiency may lead to renal– coloboma syndrome, an autosomal dominant disease characterized by kidney, optic nerve, and retinal defects (25). The renal abnormalities associated with PAX2 deficiency in humans include bilateral hypoplasia, unilateral aplasia, glomerular and interstitial fibrosis, and vesicoureteric reflux (25). In addition to colobomas, other nonrenal manifestations of reductions in PAX2 levels include hearing and central nervous system defects (25). Persistent PAX2 expression is also associated with human renal disorders. Human fetal and infantile multicystic dysplastic kidneys demonstrate strong PAX2 staining in proliferating dysplastic tubules and cyst epithelium (of ureteric bud origin) and only faint expression in less than 10% of control collecting ducts (26). PAX2 is also persistently expressed in other proliferative kidney diseases such as autosomal dominant polycystic kidney disease, renal cell carcinoma, and Wilms tumor (24). Thus, the spectrum of human renal (and nonrenal) disorders associated with both over- and underexpression of PAX2 parallels the abnormalities in mice. WT1, Wilms tumor suppressor, is a transcription factor detected primarily in developing mouse renal and gonadal structures and transiently in mesothelium around the heart and gut, ventral spinal cord, and brain (27). In the developing kidney, Wt1 is expressed at low levels in the early, undifferentiated metanephric mesenchyme, at intermediate levels in the early condensing mesenchyme, and at higher, sustained levels (into adulthood) in the glomerular podocytes (27). WT1 has alternative splice forms that function differently in vitro. For example, one variant induces tumorigenesis in culture while the another suppresses neoplastic transformation (28). Cotransfection studies suggest targets of WT1 activity including Pax2 (negative regulation), insulin-like growth factor-2, insulin-like growth factor-1, platelet-derived growth factor-A chain, transforming growth factor-␤, and itself (29). Like Pax2 mutants, Wt1 knockout mice do not form ureteric buds and have only transient metanephric mesenchymes (30). In contrast to Pax2 mutants, however, Wt1 mutant mesonephric ducts are intact and can be stimulated by wild-type mesenchyme to sprout ureteric buds (29,30). Further, wild-

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type ureteric buds and artificial stimuli such as spinal cord could not induce Wt1 mutant mesenchymes, confirming that the metanephric mesenchyme was defective (29). The nonrenal abnormalities displayed by Wt1 null mice included complete gonadal dysgenesis and minor defects in heart, lung, and mesothelial structures (30). Human WT1 mutations usually affect the kidneys and gonads as predicted by the mouse models. WT1 disruption is linked to some cases of sporadic Wilms tumor, which arises from undifferentiated rests of mesenchyme, the aberrant renal tissue in mouse Wt1 mutants (31). WT1 disruption is responsible for many of the features of WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) resulting from an 11p13 deletion (31). In addition to Wilms tumor, patients with WAGR often display abnormalities in their external genitalia and urinary tract (31). [PAX6, another gene deleted in WAGR syndrome, is likely responsible for lens malformations (31).] Mutations in WT1 are also associated with Deny’s Drash syndrome (DDS), a sporadic triad of pseudohermaphroditism, kidney disease, and Wilms tumor (31). Frasier syndrome, similar to DDS, but arising from mutations in different WT1 isoforms, is characterized by renal disease, XY gonadal dysgenesis, and gonadal tumors (31). Consistent with the expression pattern of WT1 in mice, patients with Deny’s Drash and Frasier syndrome usually develop abnormalities in their glomerular epithelium, leading to diffuse mesangial sclerosis and focal and segmental sclerosis, respectively (31). On occasion, WT1 mutations are associated with cases of diffuse mesangial sclerosis or focal sclerosis in the absence of Deny’s Drash or Frasier syndrome (31,32). In addition to regulating intravascular volume and blood pressure in the adult, the renin–angiotensin pathway participates in prenatal development. All of the components of the renin–angiotensin system (angiotensinogen, renin, angiotensin-converting enzyme, and the angiotensin II receptors) are expressed in the embryonic rodent kidney (33). Although mice and humans have a single angiotensin II type 2 (AT 2) receptor, mice have two isoforms of the AT 1 receptor (AT 1A and AT 1B) while humans have a single isoform (33). Angiotensin II type 1A receptor gene (Agtr1A) and Agtr1B single knockouts each demonstrated no phenotype; however, mice with combined mutations developed many abnormalities. The double knockout mice were small and hypotensive, and their kidneys demonstrated paren-

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TABLE 1 Molecule

Mouse

Human

GDNF gene GDNF protein RET gene RET protein PAX2 gene PAX2 protein Wilms tumor suppressor gene Wilms tumor suppressor protein Angiotensin II receptor gene

GDNF GDNF c-ret Ret Pax2 Pax2 Wt1 WT1 Agtr1A Agtr1B Agtr2 AT 1A AT 1B AT 2 N-myc N-Myc

GDNF GDNF RET RET PAX2 PAX2 WT1 WT1 AT 1

Angiotensin II receptor protein

N-MYC gene N-MYC protein

AT 2 AT 1 AT 2 N-MYC N-MYC

chymal disturbances including vascular thickening, smaller inner medullas, poorly maturing glomeruli, tubular dilatation, and interstitial fibrosis (34,35). This phenotype was recapitulated in mice with targeted deletions of angiotensinogen and angiotensinconverting enzyme (35). In contrast, mice with a disruption of Agtr2 were hypertensive and demonstrated abnormalities in ureteric bud formation resulting in (often unilateral) multicystic dysplastic kidneys, hypoplastic kidneys, ureteropelvic junction stenosis, hydronephrosis, and megaureter (34,36). In humans, use of angiotensin-converting enzyme inhibitors in pregnant females has resulted in many fetal kidney abnormalities, including renal dysplasia and anuria (37). Other manifestations include intrauterine growth retardation, pulmonary hypoplasia, oligohydramnios, calvarial hypoplasia, persistent ductus arteriosus, and even death (37). Many of these findings are similar to the abnormalities in mice with mutations in angiotensinogen, renin, angiotensin-converting enzyme, and the combined Agtr1A and 1B genes. Human genetic correlates for the mouse mutations, however, have yet to be described. In contrast, polymorphisms in the human AT 2 receptor gene have been linked with ureteric bud anomalies in humans. An AT 2 receptor allele with a 1332A⬎G substitution was detected in 77% of German and 74% of American males with ureteropelvic junction obstruction or multicystic dysplastic kidneys versus 42% of both German and American controls (36). Another study documented the 1332A⬎G transition in 75% of males with primary obstructive megaureter as opposed to 42% of con-

trols (38). Thus, as in mice, human AT 2 receptor gene alterations correlate with ureteric bud developmental errors. N-myc is among those genes not yet proven to participate in human kidney development. N-Myc is a transcription factor expressed in the developing central nervous system, peripheral nervous system, intestine, heart, lung, and kidney (39). In embryonic kidneys, N-myc is expressed in induced mesenchymal condensates around ureteric bud tips, although its expression fades as the mesenchyme converts into the epithelium of the nephron (39). In mice, two N-myc mutations were generated, including a null (N) allele (39) and a “leaky” or hypomorphic (H) allele (40). Mice with an NN or NH genotype die of cardiac defects in utero on day 1 or of renal organogenesis on days 3– 4; thus, data of N-Myc activity in the developing kidney were limited (39,40). Those with an HH genotype died of lung hypoplasia with reportedly normal kidneys shortly after birth (40). To describe the effects of the N-myc mutations on the mouse kidney, our laboratory combined the use of embryonic kidney explants and in vivo analysis (when possible). We determined that N-Myc deficiency causes dose-dependent decreases in ureteric bud branching and glomerular numbers, leading to hypoplastic kidneys (41). Relative to controls, N-myc mutant kidneys demonstrated substantial decreases in proliferation as opposed to increases in apoptosis (41). Although a human correlate has not yet been found, N-MYC mutations could be responsible for some cases of renal hypoplasia, especially when associated with hypoplastic lungs as in the Potter sequence. CONCLUSIONS In summary, researchers have made many advances in understanding the molecular control of rodent kidney development. Only a small number of these genes have been proven to be important in humans; however, alterations in these genes often result in defects remarkably similar to those in mice. As the research continues, it is likely that more of these mouse mutations will find human correlates. ACKNOWLEDGMENTS The research conducted at the Center for Developmental Biology at the University of Texas, Southwestern Medical Center in Dallas and the Children’s Research Institute in Columbus, Ohio was supported by the National Institutes of Health (Grant 5-K08DK02571-02) (C.B.), the Texas National Kidney Foundation

KIDNEY DEVELOPMENT IN MICE AND MEN (C.B.), and the Excellence in Education Foundation Support for Developmental Biology (Luis F. Parada). 17.

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