Antenatal Hydronephrosis and VUR in Robo2 Mice Figure 1. Noninvasive detection of antenatal hydronephrosis and duplex kidney in Robo2 mouse embryos. The outline of the kidneys depicted in ultrasound images were marked by red dot line. (A) Schematic diagram of ultrasound detection of mouse embryonic kidney in pregnant mice; 4 schematic embryos inside a pregnant mouse were shown; red square represented the ultrasound scanhead. (B,C) Normal E18.5 mouse embryonic kidneys detected by Vevo 770 ultrasound (B) and H&E histology confirmation (C). (D,E) Ultrasound detection of antenatal hydronephrosis (asterisks) in an E18.5 Robo2 homozygous embryo (D) and H&E histology confirmation (E) showing bilateral dysplastic kidney with hydronephrosis (asterisks). (F–J) Ultrasound detection of prenatal duplex kidney and upper pole hydronephrosis with hydroureter in an E18.5 Robo2 mosaic embryo. Antenatal hydronephrosis (asterisks) in the upper pole of right kidney (rk) of an E18.5 Robo2 mosaic embryo detected with B-mode ultrasound in transverse plane (F), ventral coronal plane (G) and dorsal coronal plane (H). A normal left kidney (lk) was also detected in the transverse plane (F) and ventral coronal plane (G). The hydronephrosis (asterisks) in the upper pole of right kidney (rk) was connected to an enlarged hydroureter (hu) in (G). A dorsal coronal plane (H) showing upper and lower right duplex kidney (divided by an artificial white dot line) as well as upper pole hydronephrosis (asterisks). (I) Gross structure of the urinary tract system from the same E18.5 Robo2 mosaic embryo examined by ultrasound in (F–H) showing enlarged right duplex kidney with upper pole hydronephrosis (asterisks) connected to an enlarged hydroureter (hu). The right duplex kidney and upper pole hydronephrosis (asterisks) in this E18.5 Robo2 mosaic embryo was confirmed by H&E histology (J); a white arrow depicting internalized nephrogenic zone (renal cortex) separating upper and lower duplex right kidney. Scale bars, 1.0 mm. Abbreviation: b, urinary bladder; k, kidney; lk, left kidney; rk, right kidney; p, renal pelvis; sp, spine; st, stomach. doi:10.1371/journal.pone.0024763.g001 procedure enabled us to follow the antenatal hydronephrosis progression noninvasively in the same Robo2-deficient mouse from embryo to neonate, a period with major physiological and functional changes during kidney and urinary tract development. To determine the natural history of disease progression of antenatal hydronephrosis in Robo2 mice, we identified 67 E18.5 embryos from the eight pregnant mice of Robo2 heterozygous matings noted above and followed them to postnatal week six by ultrasound (Fig. 3). The anteroposterior renal pelvic diameter of each mouse was used as a marker to measure progression of hydronephrosis (Fig. 3A). Among these 67 E18.5 embryos, 29 (43%) developed antenatal hydronephrosis, and of these, 25 (37%) were bilateral and four (6%) were unilateral. Three (5%) embryos had a duplex kidney with upper pole hydronephrosis (one bilateral and two unilateral), and five (7%) embryos developed combined unilateral hydronephrosis and contralateral duplex kidney (with upper pole hydronephrosis). After birth, 20 (80%) mice with bilateral antenatal hydronephrosis died within 48 hours (Fig. 3B and Table S3). Only two (8%) mice with bilateral antenatal hydronephrosis survived to 6 weeks and died in week 9 with small body size (Fig. 3C) and renal scarring suggesting reflux nephropathy (Fig. 3D). However, all four (100%) mice with unilateral antenatal hydronephrosis survived (Table S3) together with three (100%) mice with duplex kidney and upper pole hydronephrosis. We then monitored the renal pelvic diameters of seven surviving mice with antenatal hydronephrosis (two bilateral, four unilateral, one with combined antenatal hydronephrosis and duplex kidney – total nine kidney units with antenatal hydrone- phrosis) to postnatal week 6. The degree of hydronephrosis progressed rapidly with no spontaneous resolution (Fig. 3A) and the most significant enlargement of renal pelvis occurred in the first two weeks of postnatal life (Fig. 3E and Table S4). Since mouse nephrogenesis does not cease until 2 weeks after birth, this result supports the idea that severe hydronephrosis is especially detrimental to the developing kidney [26,27]. Interestingly, micro- ultrasound follow-up of three mice with duplex kidney (one bilateral and two unilateral – total four duplex kidney units with upper pole hydronephrosis) showed spontaneous resolution of the upper pole hydronephrosis (Figure S4). These results suggest that severe progressive antenatal hydronephrosis caused by Robo2 mutations is a significant risk factor for postnatal death. Hydronephrosis in Robo2 mutant mice is caused by ureterovesical junction dilatation and vesicoureteral reflux The phenotype of antenatal hydronephrosis and nephropathy can be caused by either urinary tract obstruction or reflux, both PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24763