Antenatal Hydronephrosis and VUR in Robo2 Mice Figure 5. Causal relationship of hydronephrosis and VUR in Robo2 mosaic mice. Unilateral hydronephrosis were exacerbated by the retrograde flow of urine from the contralateral functional kidney. (A–D) Ultrasound-guided percutaneous aspiration of urine from the right renal pelvis of a Robo2 mosaic mouse with severe unilateral hydronephrosis: (A) right renal pelvis with hydronephrosis before ultrasound-guided needle insertion; (B) ultrasound-guided needle (arrow) insertion; (C) collapsed right renal pelvis after ultrasound-guided needle aspiration; (D) reappeared right hydronephrosis one-day after aspiration. p, pelvis; k, kidney. (E,F) Methylene blue dye tracing showing retrograde flow of urine refluxing into the right renal pelvis from contralateral functional left kidney after methylene blue injection into the left renal pelvis; asterisk in (E) marked the methylene blue injection site in the left renal pelvis; arrows indicated the direction of urine flow that was labeled by methylene blue. (F) High magnification of boxed ureterovesical junction region in panel (E) showing abnormal enlarged right ureteral orifice (arrowhead) which disrupted the anti-reflux mechanism and allowed ureteral reflux to occur. doi:10.1371/journal.pone.0024763.g005 following the closure of the external urethral sphincter during bladder filling [40,41]. This active ostial-valve function of the ureterovesical junction allows coordinated opening and closure of the ureteral orifice, which represents a boundary area between the low pressure urinary segment in the upper urinary tract (i.e. the ureter and kidney) and the highly variable pressure segment in the lower urinary tract (i.e. the bladder and urethra). Together, the active and passive anti-reflux mechanisms act as a one-way valve allowing ejection of the bolus of urine into bladder lumen when its pressure is low and preventing retrograde urine reflux when the bladder pressure is high [40,41]. Our present study provides strong evidence that Robo2 is critical for maintaining both active and passive anti-reflux mechanisms. First, low pressure retrograde urine flow was clearly demonstrated in Robo2 mutant mice via noninvasive microbubble contrast agent enhanced urosonography, a safe, reliable and radiation-free imaging modality that has been widely used in clinical medicine to detect VUR in children [42]. Second, both ultrasound-guided percutaneous aspiration and dye tracing studies demonstrated that postnatal Robo2 mice with unilateral hydronephrosis had an incompetent anti-reflux mechanism and developed urinary reflux by the retrograde flow of urine from the contralateral functional kidney through enlarged ureteral orifice. In VUR patients, this condition is often referred to as vesicoureteral reflux dysplasia (VURD) syndrome [43,44,45]. The term of VURD was initially introduced to describe persistent unilateral reflux and renal dysplasia in boys with posterior urethral valves (PUVs) [44]. However, we have neither identified PUVs in Robo2 mutant mice nor observed any signs of PUV-associated bladder outlet obstruction, which are characterized by bladder dilatation and bladder wall thickening with PUV-specific ultrasound finding (i.e. bladder neck keyhole sign) in male gender only [46]. Instead, most Robo2 mutant mice developed golf-hole like ureteral orifice dilatation in both male and female gender with normal bladder volume and thin bladder wall. These results suggest that VURD may also happen in children with non-obstructive uropathy like primary dilating VUR. The third evidence supporting a role for Robo2 in the anti-reflux mechanism is the golf-hole like ureteral orifice dilatation detected by both micro-ultrasonography and Hoxb7-GFP reporter gene in Robo2 mutant mice with hydrone- phrosis. This ureteral orifice dilatation could disrupt both active and passive anti-reflux mechanisms and lead to urinary reflux. Since the dilatation of ureterovesical junction structure in Robo2 mutant mice was too severe to be corrected by postnatal trigone or ureteral maturation and remodeling, we found no spontaneous resolution of hydronephrosis in Robo2 mutant mice with congenital golf-hole like wide-open ureteral orifice detected at birth. Interestingly, similar ureterovesical junction defects of bilateral wide-open ureteral orifices and absence of intravesical ureteral segments have already been found in a patient with dilating VUR and Robo2 gene disruption [4]. The golf-hole like ureteral orifice dilatation has also been recognized in high-grade dilating VUR patients with no spontaneous resolution [47]. Lastly, in addition to its expression in early metanephric mesenchyme, Robo2 (and its ligand Slit2) is expressed in the developing ureterovesical junction. Malformed and disorganized ureteral muscle fiber architecture in Robo2 homozygous mutant embryos could also lead to attenuation of the trigone and dysfunction of active valve mechanism of ureterovesical junction, which is formed by intercalation of ureteral muscle fibers and bladder muscle during development PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24763