New congenital anomalies of the kidney and urinary tract and outcomes in Robo2 mutant mice with the inserted piggyBac transposon

Abstract

Background: Disruption of ROBO2 in humans causes vesicoureteral reflux (VUR)/congenital anomalies of the kidney and urinary tract (CAKUT). PiggyBac (PB) is a DNA transposon, and its insertion often reduces-but does not eliminate-gene expression. The Robo2 insertion mutant exhibited non-dilating VUR, ureteropelvic junction obstruction (UPJO) not found in reported models. We studied the incidence and outcomes of VUR/CAKUT in this mutant and explored the relationship between Robo2 gene expression and the occurrence and severity of VUR/CAKUT. Methods: The urinary systems of newborn mutants were evaluated via Vevo 770 micro-ultrasound. Some of the normal animals-and all of the abnormal animals-were followed to adulthood and tested for VUR. Urinary obstruction experiments were performed on mice with hydronephrosis. The histology of the kidney and ureter was examined by light microscopy and transmission electron microscopy. Robo2 PB/PB mice were crossed with Hoxb7/myr-Venus mice to visualize the location of the ureters relative to the bladder. Results: In Robo2 PB/PB mice, PB insertion led to an approximately 50 % decrease in Robo2 gene expression. The most common (27.07 %, 62/229) abnormality was non-dilating VUR, and no statistically significant differences were found between age groups. Approximately 6.97 % displayed ultrasound-detectable CAKUT, and these mice survived to adulthood without improvement. No severe CAKUT were found in Robo2 PB/+ mice. The refluxing ureters showed disorganized smooth muscle fibers, reduced muscle cell populations, intercellular edema and intracytoplasmic vacuoles in smooth muscle cells. Both UPJ and UVJ muscle defects were noted in Robo2 PB/PB mice.Conclusions: Robo2 PB/PB mice is the first Robo2-deficient mouse model to survive to adulthood while displaying non-dilating VUR, UPJO, and multiple ureters with blind endings. The genetic background of these mutants may influence the penetrance and severity of the CAKUT phenotypes. VUR and other CAKUT found in this mutant had little chance of spontaneous resolution, and this requires careful follow-up. We reported for the first time that the non-dilated refluxing ureters showed disorganized smooth muscle fibers and altered smooth muscle cell structure, more accurately mimicking the characteristics of human cases. Future studies are required to test the role of Robo2 in the ureteric smooth muscle.

Intrauterine low-protein diet aggravates developmental abnormalities of the urinary system via the Akt/Creb3 pathway in Robo2 mutant mice

“…Robo2 mutant FVB/NJ mice were used. PB was inserted into the first intron of the Robo2 gene (Ensembl ID: ENSMUSG00000052516, chr: 16.74, 379, 623) (24). Hoxb7-mVenus fluorescent plasmid mice were donated by Dr. Frank Costantini (Department of Genetics and Development, Columbia University).…”

“…We subsequently found that Robo2 PB/PB and Robo2 PB/ϩ mice manifested multiple CAKUT phenotypes, including duplicated kidney, vesicoureteric reflux, ureteropelvic junction obstruction, and renal hypoplasia/dysplasia. Although Robo2 PB/ϩ mice also presented various CAKUT phenotypes, their incidence rates were much lower than those of Robo2 PB/PB mice, suggesting that the incidence of CAKUT is closely related to the level of Robo2 expression in mouse models (24).…”

“…Based on the findings of our previous studies (24,42), we postulate that the adverse environment of an intrauterine lowprotein diet may increase the incidence of CAKUT in Robo2 mutant mouse offspring through a common downstream signaling pathway or a synergistic effect. Therefore, in the present study, we established an intrauterine low-protein diet Robo2 mutant mouse model to observe the postnatal urinary malformations and early stage kidney developmental abnormalities so as to investigate whether an intrauterine low-protein diet can increase the incidence of CAKUT in Robo2 mutant mice and the underlying mechanism.…”

Cellular and molecular determinants of normal and abnormal kidney development

“…Generation of heterozygous and mosaic mutant mice with reduced Robo2 resulted in VUR (W. Lu, van Eerde, et al, 2007), a phenotype associated with disorganized smooth muscle fibers and altered smooth muscle cell structure within the ureters (J. Liu, Sun, Shen, Wu, & Xu, 2016). Further VUR causing mutations have been found in a range of other genes associated with UB specification and outgrowth, including EYA1, GATA3, HOXA13, PAX2, ROBO2, SIX1, and SALL1 (Hwang et al, 2014;W.…”

“…ROBO2 is part of the SLIT2‐ROBO2 signaling which plays an important role in normal UB induction and induction of nephrogenesis by negatively regulating GDNF/RET activity (Grieshammer et al, ). Generation of heterozygous and mosaic mutant mice with reduced Robo2 resulted in VUR (W. Lu, van Eerde, et al, ), a phenotype associated with disorganized smooth muscle fibers and altered smooth muscle cell structure within the ureters (J. Liu, Sun, Shen, Wu, & Xu, ). Further VUR causing mutations have been found in a range of other genes associated with UB specification and outgrowth, including EYA1 , GATA3 , HOXA13 , PAX2 , ROBO2, SIX1, and SALL1 (Hwang et al, ; W. Lu, van Eerde, et al, ; Sanyanusin et al, ; Schimmenti et al, ; Weber et al, ).…”