Sunder Sims-Lucas and Carlton M. Bates
The process of renal morphogenesis provides a basis for understanding the causes of kidney aplasia, dysplasia, and hypoplasia, which together are the second leading cause of chronic renal insufficiency in children. It also may provide insight into congenital nephron underendowment, which may not present as overt renal disease in childhood but has been linked with adult-onset diseases such as hypertension and end-stage renal failure.1,2
OVERVIEW OF RENAL MORPHOGENESIS
The permanent mammalian kidney or meta-nephros forms following the development of transient excretory precursors, the pronephros and the mesonephros. The pronephros, the first urinary system in vertebrates, appears at 22 days of human embryogenesis in the cervical region (Fig. 464-1).3 Each pronephric nephron consists of a simple glomus that filters the blood through tubular epithelial cords called nephrotomes.4 While the pronephros is not functional in mammals and eventually regresses, it must form or there will be complete renal (metanephric) agenesis.5 The mesonephros, the second urinary system in vertebrates, appears at about the fourth week of human gestation, immediately caudal to the pronephric ducts (Fig. 464-1).3 Unlike the pronephros, the mesonephros is functional in mammals during embryogenesis. While most of the mesonephros ultimately regresses, portions of the mesonephric duct in males form the vas deferens, the seminal vesicle, and part of the epididymis, while some mesonephric tubules persist as testicular efferent ductules. In females, the mesonephros usually degenerates completely due to the presence of müllerian inhibitory substance.
FIGURE 464-1. Mammalian embryonic kidney development. Mammalian kidney development proceeds in a craniocaudal direction beginning with formation of the pronephros, then the mesonephros, and finally the metanephros, or the adult kidney. Although transitory, the pronephros and mesonephros are necessary for development of the metanephros. The metanephros begins when the ureteric bud invades the metanephric mesenchyme.
The metanephros, the definitive kidney in mammals, first appears in the fifth week of human gestation in the region of the hind-limb (Fig. 464-1).3 Paired densities of mesenchyme, termed metanephric mesenchyme, secrete molecules that induce an epithelial outgrowth from the mesonephric duct called the ureteric bud, which then invades the mesenchyme. Once these 2 tissues make contact, they begin a series of reciprocal signaling events leading to formation of the meta-nephric kidney. The mesenchymal cells induce the ureteric duct to elongate and branch fairly dichotomously (Fig. 464-2) to ultimately form the collecting ducts, pelvis, and ureter. Signals from the ureteric bud tips in turn stimulate the mesenchymal cells to condense and then convert into nephron epithelia (Fig. 464-3). Surrounding the developing nephrons and ureteric bud tree is stromal mesenchyme, which develops into the interstitial tissues of the adult kidney. There is also an extensive vascular network that develops in the metanephric kidney.
DEVELOPMENT OF THE METANEPHRIC KIDNEY
Much of the genetic programming of the pronephros and mesonephros mimic that of the metanephros, and absence of the mesonephros leads to metanephric agenesis. Each step of metanephric formation and the molecular signals regulating these steps are outlined in the following sections. Most of these molecules have been characterized in mice, given the ability to manipulate genes globally or in specific tissues such as the kidney.6-11
URETERIC BUD OUTGROWTH
Ureteric bud outgrowth from the mesonephric (wolffian) duct is one of the earliest stages of metanephric kidney development. The bud initially appears as an evagination from the wolffian duct that then elongates and makes direct contact with the surrounding metanephric mesenchyme. Under normal circumstances, there is only 1 ureteric bud induction site per wolffian duct. The metanephric mesenchyme secretes glial cell line–derived neurotrophic factor (GDNF) signals that bind to RET (a receptor tyrosine kinase) and GDNF family receptor alpha-1 (GFRα1, a coreceptor), both of which are present in the wolffian duct.93-98 Recent studies have also begun to identify human renal abnormalities associated with GDNF/RET mutations.101,102
FIGURE 464-2. Ureteric bud branching. Whole-mount image of an embryonic mouse kidney explant, dissected at 11.5 days gestation and grown for 3 days in culture. Note the green fluorescent protein labeling the ureteric bud tree. As expected, most branching has been bifud, although there is an example of lateral branching (arrow) and trifud branching (arrowhead). Image = 40× magnification.
URETERIC BUD BRANCHING MORPHOGENESIS
Once the ureteric bud makes contact with the metanephric mesenchyme, it receives additional signals to begin branching. This process has been most extensively studied in the mouse metanephros, which develops in a manner analogous to the human kidney (see Fig. 464-2).104 Usually, tips of the ureteric bud proliferate extensively until they form an ampulla, which then bifurcates into 2 new branches. Other branch patterns include trifud branching, in which 3 branches form from one ampulla, and lateral branching, in which a single branch develops directly from a trunk segment. Studies from human specimens suggest that there are approximately 15 generations of rapid, early branching. After this, the 10th- to 15th-generation branches elongate extensively, leading to the formation of the medullary collecting ducts, necessary for concentration of urine. Toward the end of metanephric development, the many cortical ureteric bud tips induce multiple nephrons that connect to single collecting ducts in “arcades,” while others give rise to lateral branches connected to individual nephrons. The initial ureteric trunk develops into the ureter, and the earliest-generation ureteric branches form the pelvis and calices.
FIGURE 464-3. Metanephric nephron formation. Hematoxylin and eosin–stained section of a 1-day-old postnatal mouse kidney. Ureteric bud tips (T) induce formation of condensing mesenchyme (CM). The condensed mesenchyme then converts into an epithelial renal vesicle (R). The vesicles undergo conformational changes, becoming comma-shaped bodies (C), S-shaped bodies (S), and then mature glomeruli (G) and nephron tubules. Note the directional axis of metanephric nephron development, with less mature elements at the outer cortex. Image = 40× magnification.
METANEPHRIC MESENCHYME FORMATION
Prior to ureteric bud outgrowth, the meta-nephric mesenchyme appears as paired densities of tissues in the region of the hindlimb, which then leads to ureteric bud outgrowth. Many signaling molecules have been identified as crucial for normal mesenchymal formation and function. One example is WT1 (Wilms tumor antigen), a transcription factor expressed initially in the undifferentiated metanephric mesenchyme and later strongly in glomerular podocytes.115Moreover, Wt1 homozygous knockout embryonic mice fail to form kidneys because of defective metanephric mesenchymal tissues that cannot induce ureteric buds.47,116 Another crucial transcription factor is PAX2, which is expressed in many renal tissues including the nephric duct, ureteric bud, early metanephric mesenchyme, and condensed mesenchyme.117 In addition to direct effects on the nephric ducts, Pax2 homozygous knockout mice fail to form kidneys because of abnormalities in the meta-nephric mesenchyme.44,45
MESENCHYMAL CONDENSATION AND INDUCTION
Shortly after the metanephric mesenchyme forms, it undergoes apoptosis in the absence of ureteric bud tissues. At each terminal ureteric bud tip, local areas of metanephric mesenchyme initially condense and then undergo a conversion from mesenchyme into epithelial vesicles. Wnt9b acts as the inducer from the ureteric bud that stimulates Wnt4 in the condensed mesenchyme, leading to formation of renal vesicles and nephron epithelium.18
NEPHRON FORMATION (NEPHROGENESIS)
After formation of epithelial vesicles, the future nephrons undergo conformational changes progressing from comma-shaped bodies to S-shaped bodies and ultimately to adult nephrons (see Fig. 464-3). At the S-shaped stage, the lower clefts of the S begin differentiating into glomerular podocytes that are invaded by developing endothelial cells and mesangial cells (see Chapter 465). Distal to the lower cleft, other segments of the nephron begin to form, including the proximal tubules, thin descending and thick ascending limbs of the Henle loop, and the distal convoluted tubules. The most distal end of the nephron, called the connecting segment, then fuses with the nascent ureteric bud–derived collecting ducts. There is a directional axis of meta-nephric nephron development, with earliest nephrons formed in the juxtamedullary cortex and the last formed at the outer cortex. In some mammals (such as rodents), nephrogenesis does not finish until after birth, while in humans no new nephrons develop after about 34 to 36 weeks of gestation.124 At birth, most humans possess between 600,000 and 1,100,000 nephrons per metanephric kidney (although the actual range is approximately 0.2 to 2 million).125
GLOMERULAR DEVELOPMENT
Glomerular development has been examined extensively.127 Glomeruli consist of podocytes, endothelial cells, and mesangial cells surrounded by parietal epithelial cells of the Bowman capsule. All of the former 3 cell types send reciprocal signals to one another (much like the ureteric bud and metanephric mesenchyme), ending with a tight association between the mature cell types. As podocytes mature, they develop several projections, called foot processes, that interdigi-tate with other podocyte foot processes. Ultimately, the basal lamina of podocytes fuses with those of the closely apposed endothelial cells to form the glomerular basement membrane. In addition, podocytes develop a specialized cell-cell junction between adjacent foot processes called the slit diaphragm, which is a key component of the glomerular filtration barrier.
DEVELOPMENT OF THE RENAL STROMA
Until recently, the developing kidney was thought to be composed exclusively of ureteric bud tissues and nephrogenic mesenchyme all derived from intermediate mesoderm. It is now known that there is another metanephric tissue lineage (or lineages) known as renal stromal mesenchyme.135 The origin of the renal stroma is unknown, but data suggest that it is derived from neural crest.135 While the renal stroma was initially thought to provide a structural framework in which meta-nephric development could occur, it has recently been shown to actively regulate both ureteric bud branching and nephron development. Elegant studies have shown how signaling through retinoic acid receptors a1 and b2 in the cortical stroma are necessary for sustained expression of RET and proper branching in the ureteric bud.58,136
VASCULAR DEVELOPMENT IN THE METANEPHROS
At early stages of renal development, endothelial cell precursors form an invading microvessel that tracks along with the ureteric bud trunk (but is not initially attached to the aorta).137 Eventually, these labeled cells differentiate into mature endothelial cells within the entire renal vasculature, including arterioles and capillaries.137 At the S-shaped stage of development, a single looped capillary invades the lower glomerular cleft; this initial capillary later divides into several loops within the glomerulus.127 Furthermore, as the glomerular endothelial cells become apposed to podocytes, they contribute components of the glomerular basement membrane. They also develop fenestrations or slits across from podocyte slit diaphragms. Immature mesangial cells share characteristics and express markers similar to developing mesenchyme-derived renal arteriolar smooth muscle cells such as α smooth-muscle actin and renin.138,139 Ultimately, mature mesangial cells provide support for glomerular tufts.
There are ever-increasing numbers of molecules that are known to regulate renal vascular development. Within the glomerulus, podocytes express molecules that stimulate migration and differentiation of developing endothelial cells, including vascular endothelial growth factors A and C, angiopoietin 1, and ephrin B2, all of which bind to their respective receptors on the endothelial cells.146-149 In addition, vascular endothelial growth factor (VEGF)-A expression is necessary for survival and differentiation of mesangial cells.150 Platelet-derived growth factor receptor (PDGFR) signaling has also been shown to be crucial for mesangial cell development, as deletion of either Pdgfrb(in mesangial cells) or the ligand Pdgf results in absence of mesangial cells.41,151-154 Finally, α5 laminin is essential for proper attachment of mesangial cells to the glomerular basement membrane.155