30th ESPU Congress - Lyon, France - 2019

S3: BASIC RESEARCH 2

Moderators: Luke Harper (France), Katherine Herbst (USA)

ESPU Meeting on Wednesday 24, April 2019, 15:50 - 16:50


15:50 - 15:53
S3-1 (PP)

EARLY LIFE VOIDING DYSFUNCTION CAUSES LUTS IN ADOLESCENCE THROUGH ALTERATIONS OF BLADDER NEURONAL PATHWAYS

Nao IGUCHI 1, Anna MALYKHINA 1 and Duncan WILCOX 2
1) University of Colorado, Aurora, USA - 2) 2Children's Hospital Colorado, Aurora, USA

PURPOSE

Bladder dysfunction in early life contributes to lifelong urinary tract symptoms and kidney failure. Despite its recognition, the underlying mechanism was not well understood. Voiding in neonatal mice depends on the perigenital-bladder reflex triggered by their mother licking the perineum until voluntary bladder control emerges around 3-week-old. The aim of this study was to examine the effects of early life voiding perturbation including urinary retention and disturbing normal voiding cycle induced by neonatal maternal separation (NMS) using a murine model.

MATERIAL AND METHODS

Newborn mouse pups were divided into control and NMS groups after birth. NMS pups were removed from their dam and housed individually (6h per day) from postnatal day 2 to 14 days. Pups in the control group stayed with their dam all the time. Effects of NMS on bladder function were assessedin vitroby detrusor contractility studies as well as by evaluation of gene expression in the bladder at 6-week-old.

RESULTS

NMS caused a significant decrease cholinergic receptor-mediated detrusor contractility besides an increase in purinergic-mediated contractility compared to control group. Gene expression studies revealed that a downregulation of M2 and M3 muscarinic receptors alongside of an upregulation of a purinergic receptor, P2x1, specifically expressed in detrusor muscle cells.

CONCLUSIONS

Our results provide evidence that early life bladder dysfunction can affect the bladder development and alter the molecular mechanisms that control bladder function later stages in life. Developing a selective anti-purinergic therapy would provide an alternative treatment for patients who do not respond to antimuscarinic drugs.


15:53 - 15:56
S3-2 (PP)

BLADDER FIBROSIS: EARLY MOLECULAR CHANGES AFTER 24-HOUR COMPLETE URETHRAL OBSTRUCTION

Yutao LU 1, Rikke NØRREGAARD 2, Jens C. DJURHUUS 2 and L. Henning OLSEN 1
1) Aarhus University, Department of Urology & Department of Clinical Medicine, Aarhus, DENMARK - 2) Aarhus University, Department of Clinical Medicine, Aarhus N, DENMARK

PURPOSE

Acute complete bladder outlet obstruction (BOO) can be seen in pediatric patients. The aim of this study is to elucidate the molecular changes in the bladder after acute infravesical obstruction.

MATERIAL AND METHODS

Sixty male and female mice were randomly divided into Control, Sham and BOO groups with 10 mice in each group. Urethral obstruction was achieved by 6-0 suture tying around mid-urethra. In the Sham group, only skin incision was made without further dissection. The bladder tissue was harvested 24 hours after the procedure. Western blot and QPCR analysis were performed for related fibrosis markers. Colonic tissue was analysed to evaluate a possible systemic reaction due to the surgical stress.

RESULTS

Our data indicated upregulation of fibrotic markers (TGF-beta, pSMAD2/3, pSMAD1/5, alpha-SMA), and downregulation of the anti-fibrotic protein (bone mophogenic protein-7, (BMP-7)) in both BOO male and female mice.  Fibronectin protein showed a tendency to increase in BOO male mice while it was significantly decreased in female BOO mice comparing to the Control group. Gender differences concerning histological damage and BMP-7 expression distribution were also observed in BOO group. In addition, the sham group showed increased bladder weight, TGF-beta expression, combined with BMP-7 downregulation compared to the control group. In colonic tissue, TGF-beta and interleukin-1 did not show significant differences between groups.

CONCLUSIONS

Acute BOO induces series of early molecular changes, including a significant fibrotic reaction. The gender differences observed in this study needs further investigation. Single skin incision seems to cause molecular changes in the bladder.


15:56 - 15:59
S3-3 (PP)

★ POTENTIAL BENEFICIAL EFFECT OF TOCOTRIENOLS ON BLADDER DYSFUNCTION DUE TO PARTIAL BLADDER OUTLET OBSTRUCTION

Nao IGUCHI 1, M.Irfan DÖNMEZ 1, Anna MALYKHINA 1 and Duncan WILCOX 2
1) University of Colorado, Aurora, USA - 2) Children's Hospital Colorado, Aurora, USA

PURPOSE

Bladder dysfunction due to posterior urethral valves is a major issue that contributes to renal impairment of those patients. Only available medication is symptomatic use of anticholinergics. Hypoxia inducible factor (HIF) pathway has been shown to be involved in bladder dysfunction caused by partial bladder outlet obstruction (PBOO). In this study, we aimed to evaluate if tocotrienols (minor ingredients of vitamin E and HIF inhibitor) has beneficial effects on bladder function following PBOO in a murine model.

MATERIAL AND METHODS

PBOO was surgically created by ligation of the bladder neck in 3-week-old male mice. Animals were divided into sham or PBOO+Soy bean oil (SBO, vehicle) and sham or PBOO+tocotrienol enriched palm oil extract (Toco) by daily oral administration from 0 to 14 days post-surgery. Bladder function was examined in vitro by physiological tests, and in vivo by weekly void spot assays (VSA). Also, bladder histology to examine the morphological changes among all groups of animals.

RESULTS

PBOO induced fibrosis in the bladder as previously described, while the bladders from Toco group was significantly less fibrotic than SBO group following PBOO. VSA showed significant increases in the number of small voids in PBOO groups than in sham groups. PBOO+Toco group did not show a progressive increase in small voids as observed in PBOO+SBO group. Detrusor contractility in PBOO+Toco mice showed less severe decreases compared to PBOO+SBO group.

CONCLUSIONS

Results of our study indicate that oral tocotrienol treatment may help slowing down the progression of bladder dysfunction caused by PBOO.


15:59 - 16:02
S3-4 (PP)

THE NOTION OF OBSTRUCTIVE UROPATHY IN CONTEXT OF TELOCYTES AND TGF B1 INTERPLAY IN ITS PATHOGENESIS

Michal WOLNICKI 1, Veronika ALEKSANDROVYCH 2, Krzysztof GIL 3, Janusz SULISLAWSKI 4, Barbara DOBROWOLSKA-GLAZAR 4, Ireneusz HONKISZ 4 and Rafal CHRZAN 4
1) University Children Hospital, Paediatric Urology, Krakow, POLAND - 2) JAGIELLONIAN UNIVERSITY MEDICAL COLLEGE, DEPARTMENT OF PATHOPHISIOLOGY, Krakow, POLAND - 3) JAGIELLONIAN UNIVERSITY MEDICAL COLLEGE, Krakow, POLAND - 4) JAGIELLONIAN UNIVERSITY MEDICAL COLLEGE, DEPARTMENT OF PEDIATRIC UROLOGY, Krakow, POLAND

PURPOSE

Newly discovered interstitial cells – telocytes are presumably involved in the pathogenesis of obstructive uropathies (OU). This study was conducted to assess the density of telocytes related to collagen formation in the ureter and to urinary TGF b1 concentrations in this group of patients.

MATERIAL AND METHODS

This prospective study was approved by the local Ethical Committee. The samples were taken from 35 surgically treated patients due to UPJ obstruction. Control Group (CG) are 22 patients with non-obstructive disease of the urinary tract-mainly with renal tumors. Tissue specimens were immunolabeled, using specific telocytes markers: c-kit, CD34 and PDGFRα. The semiquantitative analysis of telocytes density was performed, using a simple scale with one, two or three pluses. TGF b1 concentration was evaluated in a urine sample taken from the bladder before and after surgery. TGF b1 concentration was evaluated in a urine sample from the bladder before and after surgery. Routine histology was performed for collagen deposits analysis. Student T test and ANOVA test (analysis of variance) were used for statistic with p<0.001 and a=0,05 considered significant.

RESULTS

Density of telocytes was decreased (one plus) in OU group compared to CG group (three pluses). The TGF b1 in bladder urine before surgery ranged from 4.8 up to 180 pg/ml (median 11.5 pg/ml) and decreased after surgery (median - 4.6 pg/ml). In control group TGF b1 concentrations never excided 4.6 pg/ml. The ratio of collagen and smooth muscle was altered in OU patients (%): collagen 40 ± 10; muscle fibers 40 ± 10,  in CG group (%): collagen  32 ± 10; muscle fibers (%) 54 ± 12.

CONCLUSIONS

A declined density of telocytes accompanied hydronephrosis development. The elevation of TGF b1 correlated with collagen accumulation. Both factors contributed to fibrotic transformation in OU patients.


16:02 - 16:14
Discussion
 

16:14 - 16:17
S3-5 (PP)

NORMAL AND HYDRONEPHROTIC MORPHOLOGICAL PROFILE OF THE PELVIS IN CHILDREN OF THE FIRST THREE YEARS OLD

Nikolay KHVATYNETS 1 and V.V. ROSTOVSKAYA 2
1) G.N. Speransky City Children's Hospital № 9, Surgical center of child urology and andrology, Moscow, RUSSIAN FEDERATION - 2) I.M. Sechenov First Moscow State Medical University, (Sechenov University), Moscow, RUSSIAN FEDERATION

PURPOSE

To investigate the age-related dynamics of the morphometry of pelvis in children under 3 y.o. in normal and with hydronephrosis

MATERIAL AND METHODS

We compared two groups: normal autopsy (n=20) and specimens of patients with UPJO (n=20). Histology was analyzed using methods of morphometry and photocolorimetry with the calculation of the conjunctive tissue-muscle coefficient (CMC)

RESULTS

Normally 0-6 m.o. the thickness of the lamina muscularis 139±33.2μm(p=0.05) occured due to myocyte hyperplasia, from 12-24 m.o. - 275.0±48.1μm(p=0.28) occured due to myocyte hypertrophy; the submucosa growth increased to 102,5±3,5μm(p=0,28) and 182,0±19,8μm(p=0,68) at the same periods. At the age of 6-12 and 24-36 m.o. the optical density in the muscular layer increased from 226.17 to 241.88, in the submucosa - from 231.09 to 247.50.
The thickness of the submucosa in children with UPJO to the date of birth was make up to 188.5 ± 85.9 μm (p=0.83). The lamina muscularis (229.0 ± 68.4 μm (p=0.84)) was 1.8 times thicker than normal.
At the age of 0-24 m.o. the growth of the lamina muscularis (570.7 ± 214.6 μm) occured due to hypertrophy of myocytes. СMC grew up to 1.21 ± 0.04 (р>0,05). In the group aged 24-36 m.o. fibrosis increased, so the volume of muscle fibers (336 ± 2.8μm (р=0,56)) decreased

CONCLUSIONS

Features of morphogenesis of pelvis in normal are the immaturity of myocytes and the intensive development of histo-structures at the age 0-6 and 12-24 m.o. UPJO inhibits the processes of maturation and differentiation of the pelvis


16:17 - 16:20
S3-6 (PP)

GENE EXPRESSION PROFILE IN BILATERAL OBSTRUCTIVE UROPATHY IN THE FETAL SHEEP MODEL

Alexander SPRINGER 1, Klaus KRATOCHWILL 2, Helga BERGMEISTER 3, Johann HUBER 4, Isabel SOBIESZEK 5, Markus UNTERWURZACHER 6 and Christoph AUFRICHT 2
1) Medical university Vienna, Paediatric Urology, Vienna, AUSTRIA - 2) Medical University Vienna, Department of Pediatrics, Vienna, AUSTRIA - 3) Medical University Vienna, Division of Biomedical Research, Vienna, AUSTRIA - 4) Section Ruminants, Education and Research Farm, University of Veterinary Medicine Vienna, Vienna, AUSTRIA - 5) Medical University Vienna, Department of Pediatrics and Adolescent Medicine, Division of Pediatric Nephrology and Gastroenterology, Vienna, AUSTRIA - 6) Medical University Vienna, Christian Doppler Laboratory for Molecular Stress Research in Peritoneal Dialysis, Vienna, AUSTRIA

PURPOSE

Bilateral fetal obstructive uropathy is a leading cause of loss of renal function. In many patients therapeutic options are limited to renal replacement therapy or kidney transplantation. To gain new knowledge and define future research targets we studied the gene expression profile of the cellular responses to obstruction in an ovine model of fetal bilateral ureteral obstruction (BUO).

MATERIAL AND METHODS

BUO was created by ligation of the urethra and urachus in six sheep fetuses at the 60th day of gestation (GA). Kidneys were harvested 80 days GA (n=2), 100 days GA (n=3) and 120 days GA (n=1). For transcriptomics profiling total RNA was extracted from bilateral renal biopsies. Affymetrix® microarrays (Ovigene Gene 1.1 ST) were used following the manufacturer’s protocol. Biostatistical analysis was performed using Ingenuity® Pathway Analysis (IPA®): Canonical Pathways, Upstream Regulators, Diseases and Bio Functions, Tox Functions, Regulator Effects, Networks, Ingenuity Toxicity Lists, Analysis Ready Molecules.

RESULTS

This analysis is BUO at 80 days GA versus 100 days GA: 367 genes were significantly expressed (p<0.01, 2-fold changes). IPA® diseases and biofunctions included embryonic development, cell death and survival, stress respone in thekidney, renal oxidative stress. IPA® tox functions included glomerular injury, nephrosis, renal tubule injury, and interstitial injury. Key genes identified in the analysis were: WNT11, BMP2, UPK (amongst others).

CONCLUSIONS

Transcriptiomis and bioinformatics were able to identify a distinct genetic fingerprint of BUO in mid-term gestation. Further analysis will enable us to categorize the genetic profile of a “dying kidney” over time and with the opportunity of defining potential new biomarkers or even novel therapeutic targets.


16:20 - 16:23
S3-7 (PP)

THE ENROLLMENT OF MIF G173C AND TNF-Α G308A GENES POLY MORPHISM BIOMARKERS IN MULTICYSTIC DYSPLASTIC KIDNEY DEVELOPMENT IN CHILDREN

Mohammed ABOUD 1 and Manal KADHIM 2
1) Ministry of Health, Pediatric surgery, Al Qadisiya, IRAQ - 2) College of Medicine Al Qadisiya University, Microbiology and Clinical Immunology, Al Qadisiya, IRAQ

PURPOSE

Multicystic dysplastic kidney (MCDK) is a developmental irregularity that is composed of non-functioning cysts. It is the most documented renal cystic complication in children and occurs in one of every 4300 live births. Aim: The present study was conducted to evaluate the correlation of MIF G173C and TNF-α G308A genes polymorphism with MCDK in children. 

MATERIAL AND METHODS

Thirty-eight patients diagnosed with MCDK (28 male and 10 female), and 30 apparently healthy individuals were enrolled as a control. The genotypes of the MIF G-173C and TNF-α G308A gene were determined by PCR–restriction fragment length polymorphism (RFLP) The serum levels of TNF-α and IL-13 were detected by ELISA technique. An expert statistical analysis was sought. 

RESULTS

Three genotypes at MIF G-173C locus were detected, GG, GC and CC with band sizes 100 pb, 100/263 pb and 363 pb respectively. For TNF-αG308A GG, GA and AA with band sizes 87 pb, 87/107 pb and 107 pb respectively. The CC genotype has obviously suggested a strong correlation with MCDK (OR of 3.6 and Etiologic Fraction (EF) of 0.619). In contrast, the GG genotype had a rather preventive role (Protective Fraction (PF) of 0.140 and low OR 0.794). TNF-α showed a strong association at genotypic level (OR = 14.671), as well as at allelic level (OR = 2.002), which demonstrates that this may be one of the risk factors for developing MCDK. 

CONCLUSIONS

CC genotype with MIF 173C allele polymorphism and AA genotype with TNF- α -G308A were mainly expressed among MCDK patients and susceptibility of their correlation to these anomalies might be prospected.


16:23 - 16:26
S3-8 (PP)

★ WHOLE EXOME SEQUENCING IDENTIFIES KIF26B, LIFR AND LAMC1 MUTATIONS IN FAMILIAL VESICOURETERAL REFLUX

Zsuzsa INGULF BARTIK 1, Ulla SILLÉN 2, Tommy MARTINSSON 3, Anna DJOS 3, Anna LINDHOLM 4 and Susanne FRANSSON 3
1) Queen Silvia Children's Hospital, Dept. Paediatric Surgery, Gothenburg, SWEDEN - 2) Queen Silvia Children's Hospital, Dept. Paediatric Surgery, Paediatric Uronephrologic Centre, Gothenburg, SWEDEN - 3) Inst. Biomedicine, Dept. Pathology and Genetics, Gothenburg, SWEDEN - 4) County Hospital Ryhov, Dept. Paediatrics, Jönköping, SWEDEN

PURPOSE

Vesicoureteral reflux (VUR) is a common urological problem in children and its hereditary nature is well recognised. However, despite decades of research, the aetiological factors are poorly understood and the genetic background has only been elucidated in the minority of cases. The aim of this study was to explore the molecular aetiology of primary hereditary VUR.

MATERIAL AND METHODS

We performed whole-exome sequencing in 13 large families with at least three affected cases. A large proportion of our study cohort had congenital hypodysplasia in addition to VUR. Variants with an allele frequency above 1% in known databases, such as the SweGen dataset, 1,000 Genomes, Exome Aggregation Consortium or Genome Aggregation Database were discarded. Sanger sequencing was used to verify significant WES findings as well as for segregation analysis for family members.

RESULTS

This high-throughput screening revealed 23 deleterious heterozygous variants in 19 genes associated with VUR or nephrogenesis. Sanger sequencing and segregation analysis in the entire families confirmed the findings in three genes in three families: frameshift LAMC1 variant and missense variants of KIF26B and LIFR genes. SALL1, ROBO2 and UPK3A gene variants, predicted to be deleterious, were excluded by segregation analysis.

CONCLUSIONS

In all, we demonstrate likely causal gene mutation in 23% of the families. Whole-exome sequencing technology in combination with a segregation study of the whole family is a useful tool when it comes to understanding pathogenesis and improving molecular diagnostics of this highly heterogeneous malformation.


16:26 - 16:29
S3-9 (PP)

UROTHELIAL CELLS SEEDING ONTO BLADDER ACELLULAR MATRIX FOR BLADDER TISSUE ENGINEERING

Massimo GARRIBOLI 1, Koichi DEGUCHI 2, Ellie PHYLACTOPOULOS 3, Paolo DE COPPI 4 and Paola BONFANTI 3
1) Evelina London Children's Hospital - Guy's and St Thomas NHS Foundation Trust / University College London Great Ormond Street Institute of Child Health, Paediatric Nephro-Urology / Stem Cell and Regenerative Medicine Section, London, UNITED KINGDOM - 2) University College London Great Ormond Street Institute of Child Health, Stem Cell and Regenerative Medicine Section, London, UNITED KINGDOM - 3) University College London Great Ormond Street Institute of Child Health / The Francis Crick Institute, London, UK, Stem Cell and Regenerative Medicine Section, London, UNITED KINGDOM - 4) University College London Great Ormond Street Institute of Child Health and Great Ormond Street Hospital, Stem Cell and Regenerative Medicine Section, London, UNITED KINGDOM

PURPOSE

We have previously presented a dynamic decellularisation protocol for generating a porcine-derived bladder acellular matrix (BAM) that can be used for bladder tissue-engineering.

We demonstrated that BAM retains the ultrastructural characteristic, is stronger and more compliant then native tissue and has angiogenetic potential.

We aimed to investigate his potential biocompatibility and define the best protocol for urothelial cells seeding.

MATERIAL AND METHODS

Normal Human Urothelial (NHU) cells were harvested from culture and seeded (1x106cells x 1cm2) onto BAM either before or after expansion and initial differentiation on flask. Differentiation and stratification was obtained replacing medium with KSFMc+5% adult bovine serum (ABS) for 5 days and increasing Calcium concentration.

The cellular coverage and proliferation of NHU cells were analysed at day 3–5–7–14 using Vybrant ® MTT Cell Proliferation Assay Kit. Trans-epithelial electrical resistance (TER) was measured to assess barrier function. Reseeded BAMs were fixed for Histological analysis (hematoxylin and eosin) and immunostaining (CK13/CK14 and Ki67).

RESULTS

MTT assay revealed that NHU cells adhered and proliferated on the BAM reaching almost complete coverage when seeded on the proliferation phase while scarce coverage was identified when cells were seeded after differentiation. MTT quantification revealed 5-to-7 days of expansion phase was optimal for maximum coverage by proliferating NHU cells.

Histology analysis demonstrated multilayered epithelial cells with cellular polarity. TER test showed significant difference between seeded and unseeded BAM (mean 131.4 Ω.cm2and 1133 Ω.cm2, p=0.017). CK14 and CK13 staining showed specific co-localisation in keeping with urothelial stratification while ki67 was down-represented as expected in differentiated cells

CONCLUSIONS

BAM obtained after decellularisation of a porcine bladder is biocompatible and has potential to support in vivo urothelial maturation. The best timing for cells seeding is during the proliferation phase.


16:29 - 16:50
Discussion