Noninvasive Assessment of Antenatal Hydronephrosis in Mice Reveals a Critical Role for Robo2 in Maintaining Anti-Reflux Mechanism Hang Wang1,3, Qinggang Li1,4, Juan Liu1, Cathy Mendelsohn 2 , David J. Salant1, Weining Lu1* 1 Renal Section, Department of Medicine, Boston University Medical Center, Boston, Massachusetts, United States of America, 2 Department of Urology, Columbia University, New York, New York, United States of America, 3 Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China, 4 Department of Nephrology, PLA General Hospital, Beijing, China Abstract Antenatal hydronephrosis and vesicoureteral reflux (VUR) are common renal tract birth defects. We recently showed that disruption of the Robo2 gene is associated with VUR in humans and antenatal hydronephrosis in knockout mice. However, the natural history, causal relationship and developmental origins of these clinical conditions remain largely unclear. Although the hydronephrosis phenotype in Robo2 knockout mice has been attributed to the coexistence of ureteral reflux and obstruction in the same mice, this hypothesis has not been tested experimentally. Here we used noninvasive high- resolution micro-ultrasonography and pathological analysis to follow the progression of antenatal hydronephrosis in individual Robo2-deficient mice from embryo to adulthood. We found that hydronephrosis progressed continuously after birth with no spontaneous resolution. With the use of a microbubble ultrasound contrast agent and ultrasound-guided percutaneous aspiration, we demonstrated that antenatal hydronephrosis in Robo2-deficient mice is caused by high-grade VUR resulting from a dilated and incompetent ureterovesical junction rather than ureteral obstruction. We further documented Robo2 expression around the developing ureterovesical junction and identified early dilatation of ureteral orifice structures as a potential fetal origin of antenatal hydronephrosis and VUR. Our results thus demonstrate that Robo2 is crucial for the formation of a normal ureteral orifice and for the maintenance of an effective anti-reflux mechanism. This study also establishes a reproducible genetic mouse model of progressive antenatal hydronephrosis and primary high- grade VUR. Citation: Wang H, Li Q, Liu J, Mendelsohn C, Salant DJ, et al. (2011) Noninvasive Assessment of Antenatal Hydronephrosis in Mice Reveals a Critical Role for Robo2 in Maintaining Anti-Reflux Mechanism. PLoS ONE 6(9): e24763. doi:10.1371/journal.pone.0024763 Editor: Saverio Bellusci, Childrens Hospital Los Angeles, United States of America Received May 11, 2011; Accepted August 17, 2011; Published September 20, 2011 Copyright: 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants R01DK078226 (to WL), a subcontract of R01HD060050 (to WL), and was also supported in part by Research Grant #6-FY08-34 from the March of Dimes Foundation (to WL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wlu@bu.edu Introduction About one percent of human fetuses have congenital anomalies of the kidney and urinary tract (CAKUT), a family of birth defects comprising kidney anomalies such as antenatal hydronephrosis, duplex kidney and renal dysplasia, and ureteric defects such as vesicoureteral reflux (VUR) and urinary obstruction [1]. Although CAKUT is a complex genetically heterogeneous developmental disorder with variable phenotype, it can be caused by mutations in a single gene that controls early kidney and urinary tract development [2,3,4]. Antenatal hydronephrosis occurs in more than 1% of all pregnancies and is one of the most common CAKUT abnormalities detected on prenatal ultrasonography [5]. Antenatal hydronephrosis can be caused by a wide spectrum of renal and urological conditions ranging from transient dilatation of the fetal kidney collecting system that resolves spontaneously after birth to clinically significant urinary tract obstruction or VUR that leads to renal failure [6,7]. As many as 1% children have VUR characterized by the retrograde flow of urine from the bladder to the ureter and kidney [8]. VUR is also the cause of 15–20% of cases of antenatal hydronephrosis [9,10,11]. Despite the high incidence of antenatal hydronephrosis and VUR in the pediatric population, the natural history, causal relationship, developmental origins and genetic basis of these clinical conditions remain ill-defined [12,13,14]. ROBO2 is a member of the immunoglobulin superfamily and encodes a cell adhesion molecule involved in axonal guidance and neurogenesis [15,16]. It is a receptor for SLIT2 ligand [17], and SLIT2-ROBO2 signaling acts as a chemorepulsive guidance cue to control axon pathfinding and neuron migration during nervous system development [15]. Slit2-Robo2 signaling is also crucial for normal development of the kidney and urinary collecting system. Expression of Robo2 in the metanephric mesenchyme and Slit2 in the ureteric bud is necessary for normal ureteric budding and reciprocal induction of nephrogenesis. Thus, mouse knockouts that lack Slit2 or Robo2 develop supernumerary ureteric buds, duplex kidneys, hydroureter and renal dysplasia [4,18]. Recently, we and others have also found that human ROBO2 is disrupted in a VUR patient with a chromosome translocation [4], and point mutations of ROBO2 have been identified in VUR patients from several unrelated families [4,19]. Conventional Robo2 homozygous knockout mice die within 48 hours after birth due to severe abnormalities of the urinary tract PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24763