To bud or not to bud: the RET perspective in CAKUT

Abstract

Congenital anomalies of the kidneys or lower urinary tract (CAKUT) encompass a spectrum of anomalies that result from aberrations in spatio-temporal regulation of genetic, epigenetic, environmental, and molecular signals at key stages of urinary tract development. The Rearranged in Transfection (RET) tyrosine kinase signaling system is a major pathway required for normal development of the kidneys, ureters, peripheral and enteric nervous systems. In the kidneys, RET is activated by interaction with the ligand glial cell line-derived neurotrophic factor (GDNF) and coreceptor GFRα1. This activated complex regulates a number of downstream signaling cascades (PLCγ, MAPK, and PI3K) that control proliferation, migration, renewal, and apoptosis. Disruption of these events is thought to underlie diseases arising from aberrant RET signaling. RET mutations are found in 5-30 % of CAKUT patients and a number of Ret mouse mutants show a spectrum of kidney and lower urinary tract defects reminiscent of CAKUT in humans. The remarkable similarities between mouse and human kidney development and in defects due to RET mutations has led to using RET signaling as a paradigm for determining the fundamental principles in patterning of the upper and lower urinary tract and for understanding CAKUT pathogenesis. In this review, we provide an overview of studies in vivo that delineate expression and the functional importance of RET signaling complex during different stages of development of the upper and lower urinary tracts. We discuss how RET signaling balances activating and inhibitory signals emanating from its docking tyrosines and its interaction with upstream and downstream regulators to precisely modulate different aspects of Wolffian duct patterning and branching morphogenesis. We outline the diversity of cellular mechanisms regulated by RET, disruption of which causes malformations ranging from renal agenesis to multicystic dysplastic kidneys in the upper tract and vesicoureteral reflux or ureteropelvic junction obstruction in the lower tract.

Figure 1. RET structure and signaling system

(A) Illustration shows RET gene structure with 20 exons (solid boxes) and the putative two major RET isoforms: short RET 9 (1072 aa) or long RET 51 (1114 aa). The different isoforms are generated by alternative splicing and differ only in the number of aa residues in the cytosolic tail. RET 9 has nine amino acids at the carboxyl terminus that are different than the 51 terminal residues in RET 51. The extracellular domain consists of 4 cadherin motifs. The key tyrosine (Y) residues in the cytoplasmic domain are labeled. Note that the RET 51 isoform has an additional tyrosine residue Y1096. (B) Schematic shows RET signaling components using RET 51 as an example. GFRα1, the essential co-receptor of RET, is a glycophosphatidylinositol linked protein present on cell surface in the epithelia or in the mesenchyme. Two molecules of GDNF, ligand for RET signaling, bind to homodimers of Gfrα1 which then recruit two RET molecules in the cell membrane. Formation of this hexameric complex induces autophosphorylation of specific tyrosine residues in the intracellular kinase domain that serve as docking sites for the intracellular adaptors and activates the indicated downstream signaling cascades. Asterisks on SHC denote that other adaptors including SHC, IRS1–2, FRS2, DOK 1–6, and ENIGMA can also bind to the multi-docking Y1062 site. Activated SHC can either trigger the PI3K-AKT pathway or the ERK-MAPK pathway depending on the adaptor complex recruited. RET 51 has the additional residue Y1096 which can also activate the PI3K-AKT pathway. (aa = amino acids; UB = ureteric bud; PLCγ = phospholipase Cγ; PKC = protein kinase C)

Figure 2. RET, GDNF, and GFRα1 expression during murine kidney development

Schematic illustrates Ret, Gdnf, and Gfrα1 expression at the indicated time points during kidney development in the mouse. Ret and Gfrα1 proteins are expressed along the entire WD whereas Gdnf is expressed in the adjacent MesM at E9.5 (corresponds to approximately 3 weeks in humans). At E10.5 the site of UB induction shows high Gfrα1 and Ret expression compared to that in the anterior WD. Note that Gfrα1 is also expressed in the MesM and MesT. The Wolffian duct attaches to the cloaca by E10.5 and the MesM begins to degenerate. At E11.5, and during subsequent branching high expression of Ret and Gfrα1 is present in the UB tips. Gfrα1 and Gdnf are also expressed in the MetM and activate Ret signaling in the UB. The common nephric duct (CND) has high Ret and Gfrα1 expression and undergoes apoptosis to ensure proper ureter migration and insertion into the bladder. WD = Wolffian duct; MesM = mesonephric mesenchyme; UB = ureteric bud; MesT = mesonephric tubules; MetM = metanephric mesenchyme.

Figure 3. Role of specific Ret tyrosine residues and downstream signaling cascades in kidney development

Schematic showing Ret9 intracellular structure with 3 major tyrosine phosphorylation docking sites and their downstream targets. Loss of Y1015 signaling in either isoform of Ret prevents recruitment of PLCγ causing severe renal dysplasia with multiplexed kidneys, UVJ obstruction, megaureter, VUR, and gonadal dysgenesis. Y1062 is a multiadaptor docking site (*, only SHC shown, see text for other binding proteins). Loss of Y1062 in Ret9 abrogates MAPK and PI3K signaling and causes bilateral renal agenesis. The loss of signaling through the Y981 docking site affects recruitment of SRC resulting in reduced MAPK activity and causes an incompletely penetrant megaureter. Note that in the Ret51 isoform (not shown) the additional Y1096 site provides redundancy and normal kidney formation occurs when only Y981 or Y1062 are mutated. UVJ = ureterovesical junction; VUR = vesicoureteral reflux.

Figure 4. Gene and pathways associated with the RET signaling complex

The schematic depicts the major direct and indirect regulators of RET-GFRα1-GDNF signaling (upstream) and those that are affected by RET-GFRα1-GDNF signaling (downstream). In the upstream panel, red indicates inhibitors and green indicates stimulators or activators of RET signaling. In the downstream panel, proteins in red inhibit GDNF-RET activity and those in green are activated or are positively regulated by GDNF-RET either at transcription or posttranslational level.