John H. Makari

Directed Differentiation of Bone Marrow Derived Mesenchymal Stem Cells Into Bladder Urothelium

PURPOSE We have previously reported that embryonic rat bladder mesenchyma has the appropriate inductive signals to direct pluripotent mouse embryonic stem cells toward endodermal derived urothelium and develop mature bladder tissue. We determined whether nonembryonic stem cells, specifically bone marrow derived mesenchymal stem cells, could serve as a source of pluripotent or multipotent progenitor cells. MATERIALS AND METHODS Epithelium was separated from the mesenchymal shells of embryonic day 14 rat bladders. Mesenchymal stem cells were isolated from mouse femoral and tibial bone marrow. Heterospecific recombinant xenografts were created by combining the embryonic rat bladder mesenchyma shells with mesenchymal stem cells and grafting them into the renal subcapsular space of athymic nude mice. Grafts were harvested at time points of up to 42 days and stained for urothelial and stromal differentiation. RESULTS Histological examination of xenografts comprising mouse mesenchymal stem cells and rat embryonic rat bladder mesenchyma yielded mature bladder structures showing normal microscopic architecture as well as proteins confirming functional characteristics. Specifically the induced urothelium expressed uroplakin, a highly selective marker of urothelial differentiation. These differentiated bladder structures demonstrated appropriate alpha-smooth muscle actin staining. Finally, Hoechst staining of the xenografts revealed nuclear architecture consistent with a mouse mesenchymal stem cell origin of the urothelium, supporting differentiated development of these cells. CONCLUSIONS In the appropriate signaling environment bone marrow derived mesenchymal stem cells can undergo directed differentiation toward endodermal derived urothelium and develop into mature bladder tissue in a tissue recombination model. This model serves as an important tool for the study of bladder development with long-term application toward cell replacement therapies in the future.

Temporal-Spatial Protein Expression in Bladder Tissue Derived From Embryonic Stem Cells

PURPOSE Identifying developmental proteins could lead to markers of bladder progenitor cells, which could be used to investigate bladder diseases. We recently reported a novel embryonic stem cell model in which to study differential protein expression patterns during bladder development. Differential and temporal expressions of the endodermal proteins known as forkhead box (Foxa1 and Foxa2) were observed. In the current study we further delineated these protein expression patterns. MATERIALS AND METHODS Epithelium was removed from the underlying mesenchyma from embryonic day 18 rat bladders. Heterospecific recombinant xenografts were created by combining embryonic stem cells plus embryonic bladder mesenchyma and placed beneath the renal capsule of mouse hosts. Grafts were harvested at 16, 18, 21, 28, 35 and 42 days, and evaluated with hematoxylin and eosin, trichrome staining, and immunohistochemistry for uroplakin, smooth muscle alpha-actin, p63, Foxa1, Foxa2 and androgen receptor. RESULTS At 16 days uroplakin was detectable and it seemed to correlate with the loss of Foxa2, while Foxa1 remained at all time points. Androgen receptor was first noted in stroma at day 16. It localized to urothelial nuclei at day 21 and was undetectable at 42 days. Adjacent to the urothelium alpha-smooth muscle actin was seen on day 16 and it was localized in bundles to the periphery of the graft at later time points. Staining for basilar urothelium with p63 confirmed basilar orientation at all time points. CONCLUSIONS We report the temporal spatial expression of various genes in early bladder development. This suggests that some proteins may be potential markers of bladder progenitor cells. Characterizing these markers may potentially identify bladder progenitor cells that have been directed toward a lineage path destined to become urothelial cells. Ultimately these multipotential progenitor cells could be isolated and used to study and treat diseases that affect the bladder.

The need for additional procedures in patients undergoing proximal hypospadias repairs as reported in the pediatric health information system database.

PURPOSE Using administrative data from freestanding pediatric hospitals in the United States, we characterized the frequency and type of additional procedures required in patients undergoing proximal hypospadias repair in a larger cohort than in published case series across multiple surgeons and institutions. MATERIALS AND METHODS A search of the Pediatric Health Information System (PHIS) database by CPT code between January 1, 2005 and June 30, 2010 identified patients undergoing 1 or 2-stage repair for proximal hypospadias. Patient records with inconsistent coding or the suggestion of an alternate pathological condition were excluded from study. A forward query to June 30, 2011 identified additional hypospadias related interventions by CPT codes. RESULTS We identified 1,679 patients from a total of 37 hospitals. Potential followup was 1 to 6.5 years. One-stage repair was performed in 85.7% of patients at a median age of 10 months. In patients undergoing 2-stage repair the median age at initial repair was 10 months and the median interval between stages was 6 months. Of all patients 26.2% required 1 or more additional interventions beyond definitive repair. Of all additional interventions 84.0% were open, 7.2% were endoscopic treatment for stricture, 0.4% were combined endoscopic and open interventions, and 8.4% were endoscopic evaluation. The median interval from definitive repair to the first intervention was 9 months. CONCLUSIONS These data indicate that more than a quarter of patients who underwent proximal hypospadias repair at pediatric hospitals required additional intervention(s) after what was thought to be definitive repair. These data help create a broader context in a contemporary cohort of patients treated with proximal hypospadias repair.

Nine Year Retrospective Review of Surgical Treatment of Vesicoureteral Reflux: Comparison of Three Approaches

Objective: To analyze our nine-year experience in the surgical management of vesicoureteral reflux (VUR) with open ureteral reimplantation, robotic reimplantation, and endoscopic correction with Deflux. Methods: We retrospectively reviewed all patients undergoing surgical intervention for primary VUR at our institution between 2001 and 2010. Treatment success was defined as complete resolution of VUR on postoperative voiding cystourethrography. Surgeries were performed by four pediatric urologists. All robotic reimplantations were performed by a single surgeon. Categorical comparisons were made using Pearson’s Chi-Square or Fisher’s Exact test, and continuous variables were compared using Mann-Whitney U. Results: One hundred eighty-three patients (287 ureters) were included. Fourteen patients underwent robotic surgery, while the open surgery and Deflux cohorts included 93 and 76 patients, respectively. Due to the significantly smaller sample size of the robotic cohort, statistical comparisons were made only between the open surgery and Deflux cohorts. Postoperative VUR resolution rate was 100% (open), 85% (robotic), and 78.4% (Deflux). Open reimplantation had a significantly higher VUR resolution rate than Deflux (p<0.001). Overall, 13.9% of patients developed contralateral reflux, with no significant differences between the open and Deflux cohorts. Conclusions: In this study, we found significantly higher success rates with open reimplantation versus Deflux. While robotic reimplantation had high success rates and short hospital stays, the smaller sample size limited statistical comparison of this modality to open surgery or Deflux. We continue to enroll patients into a prospective series of all VUR procedures at our institution, which will result in more robust comparisons.

Mass in failed renal allograft: Questions

We present an 11-year-old girl with a history of end-stage kidney disease secondary to congenital nephrotic syndrome, managed on nightly peritoneal dialysis, who presented with abdominal pain. The initial diagnosis of congenital nephrotic syndrome was suspected prenatally owing to elevated maternal serum alpha-fetoprotein levels with a normal 46XX karyotype. Upon birth, the infant was found to be edematous, hypoalbuminemic, and hypothyroid. The working diagnosis was Finnish type congenital nephrotic syndrome. Genetic studies were not obtained because they were unavailable at the time. The patient underwent deceased donor renal transplantation in 2004. After suspected traumatic injury to the allograft in 2006, the patient subsequently developed a massive capsular leak from the graft and profound anasarca. Despite attempted repair, the allograft sustained permanent damage, which eventually progressed over the next 2 years to end-stage renal disease, ultimately requiring nightly peritoneal dialysis. The patient was managed on minimal immunosuppression at that time, taking prednisolone 3 mg every other day. The patient presented in the fall of 2012 with a new complaint of abdominal pain. An extensive workup was carried out, including an abdominal and renal ultrasound, which revealed no structural abnormalities. The working diagnosis was drain pain from the peritoneal dialysis catheter or positional pain. The pain subsided, and in early winter 2013, the patient re-presented with similar complaints. Physical examination findings included several well-healed abdominal scars from past surgeries, including gastrostomy tube placement, peritoneal dialysis catheter placement, and renal transplantation. She had diffuse tenderness to palpation of her abdomen. The patient’s peritoneal dialysis catheter was in place without erythema or exudate. Her renal allograft was palpable. She did not reveal any lymphadenopathy. The remainder of the physical examination was unremarkable. The patient underwent an extensive workup. Laboratory investigation revealed a normal white blood cell count at 8.2× 103/μL. Creatinine was stably elevated at 11.9 mg/dL. Electrolytes were otherwise within normal limits. C-reactive protein was elevated at 1.71 mg/dL. Cytomegalovirus (CMV) DNA, quantitative PCR was negative at less than 500 copies/mL. Epstein–Barr virus (EBV) DNA, quantitative, was negative at less than 200 copies/mL. Initial renal ultrasound showed a complex mass adjacent to the kidney measuring 8.0×4.2 cm, which had not been visualized 5 months previously. Computed tomography (CT) scan delineated a hypodense tissue mass infiltrating the renal sinus and periureteral space (Fig. 1). It was also noted that the renal vasculature appeared splayed on multiple images. The answers to these questions can be found at http://dx.doi.org/10.1007/ s00467-014-2936-y. M. Riemenschneider (*) : C. D’Alessandri-Silva Department of Nephrology, Connecticut Children’s Medical Center, 282 Washington Street, Hartford, CT 06106, USA e-mail: mriemenschneider@connecticutchildrens.org

Mass in failed renal allograft: Answers

Inflammatory myofibroblastic tumor (IMT), also termed inflammatory pseudotumor or pseudosarcomatous fibromyxoid tumor, is a rare, typically benign tumor consisting of spindle cell proliferation [1–5]. IMT was originally reported in the lung, and has subsequently been reported in multiple extrapulmonary locations [2, 6]. Renal IMT is a rare entity, and even fewer reports present cases of renal allograft IMT [4, 7]. It is most often a benign condition, although the differential diagnosis includes malignant processes. The differential diagnosis includes renal cell carcinoma, inflammatory fibrosarcoma , myxo i d l e i omyo s a r c oma , Wi lms ’ t umo r , xanthogranulomatous pyelonephritis, and plasma cell granuloma, amongst others [2, 4, 8]. The pathogenesis is unknown and remains controversial [2, 3, 5]. Presentation can be insidious and nonspecific. Fever, malaise, abdominal discomfort, and weight loss are common presenting complaints [1, 5, 6]. Laboratory evaluation can also be nonspecific. Findings can include an elevated erythrocyte sedimentation rate, microcytic anemia, thrombocytosis, leukocytosis or hypergammaglobulinemia [1, 6]. Many pediatric clinicians will choose ultrasound as their first line of imaging when approaching a nonspecific abdominal complaint. Sonographically, IMT may appear as a spectrum of echogenicity, either hypoor hyperechoic lesions. Their margins may be illor well-defined [8]. In the case that we report, ultrasound findings were nonspecific in elucidating the composition of the mass: solid tumor versus hematoma. Newer ultrasound techniques with enhanced power Doppler interrogation and concomitant contrast agents may improve visualization of intratumoral vascularity [9]. On computed tomography (CT), the more frequent radiological appearance of renal pseudotumor reveals an ill-defined, homoor heterogeneous mass with hypovascularity [4]. Delayed contrast enhancement has been reported, and is thought to be secondary to accumulation in the fibrotic tissues [9]. Park et al. reported that IMT may appear as a hypo-, isoor hyperdense mass on contrast-enhanced CT [8]. In our case, CT confirmed hypodense tissue seen infiltrating the entire renal sinus and the periureteral space of the transplanted kidney. This refers to the article that can be found at http://dx.doi.org/10.1007/ s00467-014-2928-y M. Riemenschneider (*) : C. D’Alessandri-Silva Department of Nephrology, Connecticut Children’s Medical Center, 282 Washington Street, Hartford, CT 06106, USA e-mail: mriemenschneider@connecticutchildrens.org

MP44-01 A COMPARISON OF PRACTICE PATTERNS IN THE MANAGEMENT OF VESICOURETERAL REFLUX BETWEEN PEDIATRIC UROLOGISTS AND PEDIATRIC NEPHROLOGISTS

INTRODUCTION AND OBJECTIVES: Vesicoureteral reflux (VUR) is often diagnosed and managed by both Pediatric Urologists (PU) and Pediatric Nephrologists (PN), yet little data exists comparing practice patterns between these two subspecialties. We sought to describe practice variation between PU and PN as it pertains to VUR. METHODS: An e-mail invitation was sent to 675 SPU and 753 ASPN members. Survey Monkey was used to obtain demographic and practice pattern data for commonly encountered clinical scenarios relating to VUR and urinary tract infection (UTI). Data from non-PU/PN responders and from those answering fewer than 37 of the 39 questions (95%) was excluded from analysis. Statistical analysis was performed using SPSS 17.0. Categorical variables were compared using Chisquare test or Fisher’s Exact Test. Pair-wise comparisons were made using the Bonferroni adjustment. RESULTS: 255 (18%) physicians responded; of these, 4 (2%) were non-PU/PN and 48 (19%) answered 95% of survey questions, resulting in a final cohort of 203 respondents (133 PU, 70 PN). Age and practice setting were similar; more non-US PU than PN responded to the survey (20% vs. 7%, P<0.05). 98% of PU and 83% of PN responded that they would evaluate a child with a single febrile UTI with imaging (p<0.001). 91% of PU and 99% of PN would evaluate a child with a multiple afebrile UTIs with imaging (p<0.001). Independent of the type of UTI, PU favor using KUB and DMSA more frequently than PN, whereas PN prefer renal sonogram or VCUG. 78% and 74% of PU were somewhat/very likely to consider deflux or reimplant for breakthrough infection, versus 60% and 28% of PN (p<0.05). 91% of PU vs. 76% of PN believe that treating bowel and bladder dysfunction (BBD) alone may lead to resolution of VUR (p<0.01). BBD screening by PU (58%) involved history and other modalities; PN rely most often on history alone (60%). 58% of urologists are somewhat/very likely to screen siblings with sonogram versus 40% of nephrologists (p<0.01). PU and PN were equally concerned with the emergence of resistant bacterial organisms when making decisions regarding prophylactic antibiotics (PN 72% vs. PU 69%). CONCLUSIONS: Minimal consensus exists between PU and PN in the diagnosis and management of VUR. PU use more diverse imaging in diagnosis and are more likely to pursue surgical intervention for breakthrough UTIs than PN. More aggressive evaluation for BBD by PU than PN may be related to belief in the impact BBD on VUR resolution. Further investigation is necessary to determine if practice variation is related to training, society guidelines, or other factors.

460 INCIDENCE OF REPEAT DEXTRANOMER/HYALURONIC ACID COPOLYMER INJECTION AMONG PEDIATRIC HEALTH INFORMATION SYSTEM HOSPITALS

During follow up, VUR disappeared spontaneously in 7, while antireflux surgery was performed in 7. Mean systolic blood pressure (BP) and mean diastolic BP at final visit were 108 mmHg (127 to 89) and 66mmHg (89 to 41). According to Guideline by Japanese Society of Hypertension, while none exceeded standard level of systolic BP, two patients slightly exceeded standard level of diastolic BP. No significant proteinuria was detected in all patients. Estimated GFR was evaluated in all of 10 patients over 10 years old at final visit and in 7 of 17 patients under 10 during follow up. CKD stage was 1 in all these 17 patients. CONCLUSIONS: Significant clinical findings related to UDRPL detected at less than 1 year old was rarely observed during a mean of 10 years follow up in this study. Accordingly, UDRPL seems to be a benign condition. No specific attention would be necessary during follow up in patients with UDRPL on one side and no or limited scar on the other on the initial nuclear renal scan.