kidney fused with a normal lower pole kidney with undilated renal pelvis. Micro-ultrasound analysis of postnatal CAKUT phenotype in newborn and adult mice The same Vevo 770 micro-ultrasound imaging system and Vevo Compact Anesthesia System used for pregnant mice above were used for mice after birth. For newborn mice, a special small head mask was used to introduce the same inhalant anesthesia of mixed oxygen and isoflurane. We used the same ultrasound transducer RMV-708 scanhead on newborn, one week and two weeks old mice. Another ultrasound transducer RMV-704 scan- head (40 MHz center frequency with frequency band between 20 MHz to 60 MHz, 6 mm focal length and axial resolution of 40 mm) was used on mice after two weeks old. The normal kidney structure and CAKUT phenotype were detected relatively easily under ultrasound B-mode imaging in mice after birth because we could adjust the position of anesthetized mouse body to allow the best view of renal pelvis and adjacent structures. The largest anteroposterior renal pelvic diameter of each mouse was obtained by Vevo 770 analytic software line measurement tool. Two- dimensional videos of mouse urinary tract and bladder were recorded in B-mode during a 300-frame image recording phase (also called cine loop in Vevo 770 analytic software). Three- dimensional video was obtained from serial transverse 2D images along the entire mouse urinary tract in the caudal-cephalic direction (i.e. ultrasound imaging began from the bladder, moving upper to the ureter, and finally reaching the kidney region), followed by reassembly using Vevo 770 analytic software. Micro-ultrasound detection of high-grade dilating VUR in CAKUT mice by urethral catheterization and microbubble ultrasound contrast agents A polyurethane FunnelCath mouse catheter (Instech Solomon, PA, # PUFC-C30-10) was used for urethral catheterization in adult female mice. This FunnelCath urethral catheter (tapers from 3Fr proximal end to 1.2Fr distal end) has a distal thin segment of 1.2 French gauge (ID: 0.23 mm; OD: 0.41 mm) which can easily enter the external orifice of mouse urethra without injury to the urethral inner epithelium lining. This catheter also has a proximal thick segment of 3 French gauge (ID: 0.66 mm; OD: 1.07 mm) with a Luer-Taper end which can be connected to a syringe or pump for controlled ultrasound contrast agents (or methylene blue) infusion. After mice were anesthetized, the external orifice of urethra was sterilized with povidone-iodine. The distal thin end of FunnelCath mouse catheter was lubricated with sterile mineral oil and inserted gently into the external urethra orifice. The catheter was advanced slowly inside urethra without force until it reached the bladder when urine was drained and visible inside the catheter. The catheter was typed down on the mouse handling platform before the proximal thick end was connected to a syringe filled with microbubble ultrasound contrast agents. Microbubbles are gas filled small bubbles (,1–10 mm in diameter) encapsulated with a solid shell [53]. They have been widely used as contract agents for ultrasound imaging in clinical medicine [54] including diagnosis of VUR in children [42,55]. Option (Perflutren Protein-Type A Microspheres Injectable Suspension, USP) is a FDA-approved ultrasound microbubble contract agent (GE Healthcare, NDC 0407-2707-03). The microbubbles in Option suspension have an albumin shell and perflutren gas core with mean diameter ranging 3.0–4.5 mm and have been used for many indicated ultrasound imaging procedures [56]. Each mL of Optison contains 5.0–8.06108 microbubbles. Antenatal Hydronephrosis and VUR in Robo2 Mice We diluted the Option microbubbles 20 fold to 2.5–4.06107/ml with 16PBS for retrograde urosonography to evaluate VUR in mice. A syringe infusion pump (Harvard Apparatus, Holliston, MA) was used to infuse diluted microbubbles into the mouse bladder through the urethra catheter at a perfusion rate of 100 ml/ min. Coupled with 1.2 French gauge thin urethral catheter, this low speed infusion rate would infuse microbubbles into the bladder without increasing the bladder pressure significantly. Overflow microbubbles will leak out from mouse bladder through external urethra orifice without inducing artificial VUR in wild-type control mice (Fig. 4H). To detect urinary reflux in Robo2 mice, Vevo 770 micro- ultrasound system was operated under the control of Vevo Contrast Mode software (VisualSonics, # VS-11639). The RMV- 704 scanhead was used to detect microbubble flow movement in the urinary tract before and after contrast agent bladder infusion to detect any retrograde urine flow in the ureter and renal pelvis. In order to visualize urine flow marked by microbubble contrast agents, two ultrasound imaging serial videos were captured under Vevo Contrast Mode software: the first baseline background imaging serial video for the mouse urinary tract before micro- bubble infusion and a second microbubble enhanced imaging serial video of the same mouse urinary tract after microbubble bladder infusion. Each ultrasound imaging serial video contains 800 individual ultrasound image frame data acquired and stored digitally as a cine loop in Vevo 770 system memory. A final contrast-mode imaging serial video of mouse urinary tract was then digitally constructed by overlaying and comparing baseline background data acquired before microbubble infusion and microbubble enhanced data acquired after microbubble bladder infusion. The appearance and movement of microbubble signal inside the mouse ureter or renal pelvis after microbubble bladder infusion indicated VUR. The microbubble signal could be rendered in either gray-scale (Figure S5, Figure S6, Video S1, and Video S4) or in green-scale (Fig. 4A–H, Video S2, Video S3). Some tissue with strong echogenicity might also be marked as green in the green-scale setting without a real microbubble signal (Fig. 4A–D). These false green signals could be subtracted when compared with baseline background data. A microbubble method assessing low-grade non-dilating VUR in inbred wild-type mice has been described elsewhere [29]. Confirmation of VUR in Robo2 mutant mice by methylene blue cystogram VUR in Robo2 mutant and control mice were confirmed according to previously published methods [57,58]. A 27-gauge needle attached via tubing to a syringe filled with 10% methylene blue in saline was inserted into the mouse bladder. The syringe was raised vertically from 30 to 150 cm in relation to the position of the mouse at a rate of 30 cm/min. The hydrostatic pressure (cm-H2O) at which micturition or reflux occurs was recorded. The experiment was stopped when there was no reflux at 150 cm for 10 second. VUR confirmation was also performed by infusing 10% methylene blue into the mouse bladder through a mouse urethra catheter at a perfusion rate of 100 ml/min using a Harvard Apparatus syringe infusion pump. Overflow methylene blue leaked out from the mouse bladder through the external urethra orifice without VUR in wild-type control mice. Ultrasound-guided percutaneous needle aspiration treatment of severe hydronephrosis in mice Adult mice with unilateral hydronephrosis were anesthetized with isoflurane on the VisualSonics Vevo Compact Anesthesia PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24763