Antenatal Hydronephrosis and VUR in Robo2 Mice Figure 2. Noninvasive detection of postnatal hydronephrosis and cystic renal dysplasia phenotype in Robo2 newborn mice. (A) Ultrasound scanhead position (arrow) and a newborn mouse under anesthesia. (B,C) Representative ultrasound and gross structure images of wild- type normal newborn kidneys. (D,E) Representative ultrasound and gross structure images of Robo2 homozygous newborn kidneys with hydronephrosis (asterisks); an ideographic anterior to posterior (ARP) plane in (C,E) depicting B-mode ultrasound scanning. (F–H) Cystic renal dysplasia marked by multiple hypoechoic dark area (asterisks) were shown in a Robo2 mosaic newborn (F) and a homozygous newborn (G). H&E histology of cystic renal dysplasia from a Robo2 homozygous newborn was showed in (H). Scale bars, 1.0 mm. Abbreviation: b, urinary bladder; k, kidney; sp, spine; u, ureter. doi:10.1371/journal.pone.0024763.g002 of which elevate the pressure in the upper urinary tract. We have previously showed that Robo2 mosaic mice developed abnormally located ureterovesical junctions (UVJs) [4]. To determine if these abnormally located UVJs in Robo2-deficient mice lead to VUR or urinary obstruction, we performed noninvasive echo-enhanced ultrasonic cystography with a microbubble ultrasound contrast agent (UCA) in four 6-week old Robo2 female mosaic mice that had been diagnosed with unilateral antenatal hydronephrosis and four age-matched wild-type controls. The reason to choose female mice for this experiment is a technical one since there is a low success rate of urethral catheterization in adult male mice [28]. We and others have recently tested the feasibility of this microbubble method in inbred wild-type mice with low-grade non-dilating VUR without hydronephrosis by 3D reconstruction [29]. In order to determine if Robo2 mosaic mice developed high- grade dilating VUR with free flow of microbubble inside the mouse urinary tract without 3D reconstruction, we used the innovative FunnelCath urethral catheter for mice. This infusion friendly mouse catheter has a thin distal segment for easy entry into the mouse urethra and can be visualized inside the mouse bladder by ultrasonic scan (Figure S5). After infusion of Option (a FDA-approved microbubble UCA) into the urinary bladder (Fig. 4, Video S1 and Video S2), we found that all (4/4, 100%) Robo2 mosaic mutant mice with hydronephrosis displayed VUR (Fig. 4A–H, Video S3). Sequential images showed that the microbubble UCA flowed retrogradely from the bladder into the dilated ureter (Fig. 4F) and was seen in the renal pelvis (Fig. 4G, Video S3) about 3 seconds after the infusion of the microbubble UCA into the bladder, which suggests a low pressure reflux. The microbubble UCA was washed out from renal pelvis quickly (as fast as 18 seconds) after it appeared (Figure S6, Video S4), suggesting that no obstruction was present in the upper urinary tract of Robo2 mutant mice. None of four (4/4, 100%) control mice displayed urinary reflux after Option microbubble infusion into the bladder (Fig. 4H). The reflux phenotype was confirmed in Robo2 mosaic mice by infusing methylene blue into the bladder (Fig. 4I–L), an open abdominal invasive method that is the current gold standard to evaluate VUR in mice [30,31]. These results indicate that loss of Robo2 indeed causes urinary reflux rather than urinary obstruction as previously thought [4] and the Robo2 mosaic mouse represents an excellent animal model for congenital high-grade dilating VUR. To determine if there is a causal relationship between VUR and hydronephrosis and if the process of hydronephrosis formation in Robo2 adult mice is active or a residual effect of antenatal hydronephrosis, we performed ultrasound-guided percutaneous aspiration of the unilateral hydronephrotic kidney in three Robo2 mosaic mutant mice (Fig. 5A–C, Video S5 and Video S6). Interestingly, we noted rapid re-accumulation of urine in the renal pelvis within 24 hours (Fig. 5D). To determine the source of the urine that reconstituted the unilateral hydronephrosis, we injected methylene blue into the renal pelvis of the contralateral unaffected kidney (Fig. 5E) and found that urine flowed into the dilated renal pelvis through the bladder and enlarged ureterovesical junction (Fig. 5E,F). In addition to further demonstrating the VUR phenotype, these results suggest that unilateral hydronephrosis in Robo2 mice is exacerbated by the retrograde flow of urine from the contralateral functional kidney through an enlarged ureterovesical junction, which may further impair the growth, development and function of an already dysplastic kidney. PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24763