5th Joint Meeting of ESPU-SPU - Virtual

S3: BASIC RESEARCH 3

Moderators: Nicolas Kalfa (France)

ESPU-SPU Meeting on Thursday 23, September 2021, 15:10 - 15:52


15:10 - 15:13
S3-1 (PP)

ACUTE BLADDER OUTLET OBSTRUCTION INDUCES GENDER-DEPENDENT KIDNEY INJURY

Yutao LU 1, Rikke NØRREGAARD 2, Jens C DJURHUUS 2 and L Henning OLSEN 3
1) Aarhus University Hospital-Skeiby, Urology, Aarhus, DENMARK - 2) Aarhus University, Department of Clinical Medicine, Aarhus, DENMARK - 3) Aarhus University Hospital-Skeiby, Department of Clinical Medicine, Department of Urology, Aarhus, DENMARK

PURPOSE

Gender-dependent bladder remodelling was found after one day bladder outlet obstruction. In this study we investigated the changes in the kidneys secondary to the acute obstruction with the hypothesis that also in the kidneys we can observe gender-dependent changes.

MATERIAL AND METHODS

Thirty-six male and female mice were randomly divided into Control, Sham and BOO groups. A suture was tied around the proximal urethra to develop a 24-hour total obstruction. In the Sham group, a skin incision was made without dissection, whereas the mice in the Control group did not undergo an initial procedure. Western blots analysis of the kidney cortex samples was performed for fibronectin, α-Smooth Muscle Actin(α-SMA), and Gremlin. Quantitative PCRs for Transforming Growth Factor β (TGF-β), Bone Morphogenetic Protein-7 (BMP-7), Tumour Necrosis Factor-α (TNF-α), Interleukin-1 β (IL-1 β), Monocyte Chemoattractant Protein-1 (MCP-1) were also performed. The blood samples were also collected to determine plasma Na+, K+, Urea, Creatine and Osmolarity level.

RESULTS

In both genders BMP-7 was downregulated, whereas only the female exhibited upregulated TGF-β. BOO resulted in a significant upregulation of Gremlin protein level in male mice. Fibronectin upregulation was detected in both genders, and α-SMA showed no significant changes in both genders. Concerning kidney function, BOO induced significantly elevated plasma K+ and Osmolarity in both genders, while only the males developed an increased plasma creatinine level. Sham-operation induced decreased urea level in male and decreased creatinine level in the female.

CONCLUSIONS

Acute complete bladder outlet obstruction induced kidney injury in both genders. These differences suggest that the gender-dependent bladder remodelling after BOO with clear differences in volume capabilities should be considered as one of the factors besides the sex hormones leading to a different kidney injury response between genders.


15:13 - 15:16
S3-2 (PP)

USING THREE DIMENSIONAL IMAGING TO UNDERSTAND SPATIOTEMPORAL DYNAMICS OF BLADDER LYMPHATIC DEVELOPMENT

Kevin CAO 1, Daniyal JAFREE 2, Dale MOULDING 3, Navroop JOHAL 4, Paul WINYARD 2 and David LONG 2
1) UCL Great Ormond Street Institute of Child Health, Urology, London, UNITED KINGDOM - 2) UCL Great Ormond Street Institute of Child Health, Nephro-urology, London, UNITED KINGDOM - 3) UCL Great Ormond Street Institute of Child Health, Imaging, London, UNITED KINGDOM - 4) Great Ormond Street Hospital for Children, Urology, London, UNITED KINGDOM

PURPOSE

Lymphatics play a crucial role in the removal of extracellular fluid from organs, however little is known about the bladder lymphatic network forms and matures. Our aim in this research is to visualise the initiation and maturation of the bladder lymphatic network in the mouse at cellular resolution using a novel three-dimensional imaging pipeline coupled with quantitative analysis.

MATERIAL AND METHODS

We optimised the iDISCO method of wholemount imaging for the urinary tract organs (Renier et al. Cell. 2014 6;159(4):896-910), reporting previously on the role of the lymphatic network in kidney development and polycystic disease (Jafree et al. Elife.2019 6;8:e48183). This technique enables three-dimensional imaging of immuno-labelled whole-organs. After labelling with lymphatic markers (Lyve-1, Prox-1) as well as a blood vascular markers (endomucin), we imaged both male and female wild-type (CD1) mice from embryonic day 14.5 to postnatal day 11 using single and two-photon confocal microscopy. Lymphatic network visualisation and analytics performed digitally.

RESULTS

Lymphatic endothelial cells are seen at the earliest stages of bladder formation at e14.5. As the organ grows, these early, discontinuous vessel tracts follow the path of the established blood vasculature, proceeding in a neck-to-dome direction expanding and maturing to become several prominent lymphatic trunks by the end of pregnancy. Interestingly, a Prox-1-only stained cell cluster is also seen at early stages of bladder development and disappears by birth, which we speculated may be progenitor lymphatic cells arising de novo within the bladder.

CONCLUSIONS

We present through images, the first description of the spatiotemporal dynamics of fetal bladder lymphatic formation at cellular resolution.


15:16 - 15:19
S3-3 (PP)

DEVELOPMENT OF A NEW FETAL CYSTOSCOPE BASED ON AN ANATOMICAL STUDY OF THE FETAL BLADDER FOR LOWER URINARY TRACT OBSTRUCTION.

Nicolas VINIT 1, Jérome SZEWCZYK 2, Jacques BATTAGLIA 2, Thomas BLANC 1 and Yves VILLE 3
1) Necker-Enfants Malades Hospital, Department of Pediatric General Surgery and Urology, APHP - EA7328, Paris University, Paris, FRANCE - 2) Sorbonne University, Institut des Systèmes Intelligents et de Robotique, Paris, FRANCE - 3) Necker-Enfants Malades Hospital, Department of Obstetrics, Fetal Medicine and Surgery - EA7328, Paris University, Paris, FRANCE

PURPOSE

Fetal cystoscopy (FC) was developed to relieve prenatal bladder outlet obstruction and reduce postnatal morbidity in congenital lower urinary tract obstruction (LUTO). Inadequacy of the current cystoscope with anatomical constraints of the fetal bladder is a limiting factor, responsible for failed procedures and added morbidity. The aim of this project was to develop a new fetal cystoscope based on an anatomical study of the fetal bladder.

MATERIAL AND METHODS

Forty-six Magnetic Resonance Imaging of male fetuses (17 LUTO at 28.1 weeks of gestation (WG) [17.3-35] and 29 controls at 29.9 WG [21.9-35]) were reviewed. Bladder-neck angle (BNA) and bladder volume were measured. Angle values were compared between groups using Mann-Whitney's test. Development of the device was based on the constraints deduced from the anatomical study. An experimental model of LUTO bladder was created using a 3D-printed silicone LUTO bladder.

RESULTS

BNA was higher in LUTO fetuses: 127° [102-162] against 111° [89-157], p<0.01. Angulation of the scope was deduced from the BNA and therefore ranged between 18° and 78° (mean 53°). Concentric preformed tubes were deemed the most adequate technology for the cystoscope to adopt the range of angles necessary for obstacle visualization. A rotor motor was added to the device to allow safe positioning in the fetus' bladder. A 1.2-millimeter camera was used as optic system. The experimental model of LUTO bladder was validated and proof of concept was obtained.

CONCLUSIONS

This new fetal cystoscope should help overcome current technical difficulties encountered during FC.


15:19 - 15:28
Discussion
 

15:28 - 15:31
S3-4 (PP)

H-IPSE, A PARASITE-DERIVED CANDIDATE DRUG FOR BLADDER PAIN, LOCALIZES WITHIN UROTHELIAL CELLS THROUGH CLATHRIN-MEDIATED MECHANISMS AND TARGETS CELLS OUTSIDE OF THE UROTHELIUM

Olivia LAMANNA 1, Evaristus MBANEFO 1, Kenji ISHIDA 1, Franco FALCONE 2, Theodore JARDETZKY 3, Luke PENNINGTON 3 and Michael HSIEH 1
1) Children's National Hospital, Urology, Washington, USA - 2) University of Giessen, Giessen, GERMANY - 3) Stanford University, Stanford, USA

PURPOSE

IPSE (IL-4 Inducing Principle from Schistosoma mansoni Eggs) has significant therapeutic potential. We have shown IPSE alleviates bladder inflammation and pain triggered by ifosfamide and resiniferatoxin (an agonist of the capsaicin receptor). IPSE binds to DC-SIGN and the mannose receptor, suggesting it may have specific cellular receptors. As an infiltrin, IPSE translocates into host cell nuclei to alter transcription. We hypothesized that IPSE mediates its transcriptional effects following uptake by specific cell types. Our objective was to characterize which cell types internalize H-IPSE (the Schistosoma haematobium ortholog of IPSE) and determine if this occurs by clathrin- and/or caveolin-mediated endocytosis.

MATERIAL AND METHODS

H-IPSE variants H03 and H06 were conjugated to Alexa-488 fluorophore and flow cytometry was used to quantify internalization. H03 was incubated with urothelial, endothelial, immature dendritic, hepatocyte, and neuronal cell lines for 24hrs. Urothelial cells were pre-treated with filipin (inhibiting caveolin-mediated endocytosis) and/or chlorpromazine (inhibiting clathrin-mediated endocytosis) before treatment with H03 or H06. 

RESULTS

The percentage of cells positive for intracellular H03 was 78, 28, 38, 40, and 7 percent, respectively. When cells were pre-treated with chlorpromazine, the proportion of cells positive for IPSE was similar to that of untreated cells, suggesting IPSE utilizes mainly clathrin-dependent mechanisms.

CONCLUSIONS

Despite our prior findings regarding IPSE’s therapeutic effects on capsaicin receptor-mediated pain, our findings indicate that IPSE may be inefficiently taken up by capsaicin receptor expressing neurons and may be inducing its effects despite inefficient uptake, or through an alternative pathway. These observations reveal important principles relevant to understanding the therapeutic properties of IPSE.


15:31 - 15:34
S3-5 (PP)

CONSTIPATION INDUCES DETRUSOR OVERACTIVITY ALONG AN INCREASED SEROTONIN SENSITIVITY IN MURINE BLADDERS

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

PURPOSE

This study aimed to investigate the pathological mechanisms underlying the bladder bowel dysfunction (BBD). BBD is a very common pediatric condition that describes a spectrum of lower urinary tract symptoms associated with constipation. BBD is commonly associated with vesicoureteral reflux and recurrent urinary tract infections. The presence of inflammation and bladder hypertrophy in constipated children has been reported, however, the mechanisms remain largely unclear.

MATERIAL AND METHODS

Juvenile male mice underwent surgery to reduce external anal sphincter opening to induce constipation. Sham operated mice served as control. At 4 days post-op, bladder function was examined in vivo by urinary pattern and urodynamic studies as well as in vitro physiological evaluation of bladder strips. Bladder histology and molecular studies were also conducted.

RESULTS

A significant decrease in fecal output alongside a significant enlargement of colons were detected in the experimental group. Constipation caused detrusor overactivity indicated by both in vivo (urinary frequency and an increase in non-void contractions) and in vitro (an increased spontaneous contractions using bladder strips). Gene expression studies showed excitatory serotonin receptors (Htr2a and Htr2c) were upregulated by 1.5-fold in the bladders. Serotonin caused a marked increase in spontaneous activities (2.5- and 8.8-fold in the frequency and the amplitude, respectively) of bladder strips from constipation group compared to the control group.

CONCLUSIONS

Functional constipation caused bladder overactivity which was exacerbated by serotonin exposure in mice at a young age. Our results suggest that an upregulation of excitatory serotonin receptors in the bladder may play a role in pathology of BBD in childhood.


15:31 - 15:34
S3-5 (PP)

THE ROLE OF GALECTIN-3 IN THE REMODELING OF BLADDER IN RAT MODEL OF SPINAL CORD INJURY

Sang Woon KIM, Yong Seung LEE and Sang Won HAN
Yonsei University College of Medicine, Department of Urology, Seoul, REPUBLIC OF KOREA

PURPOSE

Galectin-3 belongs to the galectin family of mammalian lectins and is characterized by a carbohydrate recognition domain that has affinity for β-galactosides. Galectin-3 mediates cell–cell and cell–matrix interactions and there is mounting evidence demonstrating its key role in inflammatory and fibrotic processes. In this study,we investigate the role of Galectin-3 in bladder fibrosis and whether it is a relevant therapeutic target in bladder remodeling by assessing galectin-3 expression in urine and bladder with rat model of spinal cord injury (SCI).

MATERIAL AND METHODS

The spinal cord was transected at the level of T8-9 in female Sprague-Dawley rats (200-250g), which were divided into three groups; A: Sham, B: SCI, C: SCI with treatment (galectin-3 inhibitor, modified citrus pectin, MCP) groups, respectively. MCP was orally administered (2ml/kg/d) after spinal cord transection. Cystometrogram was conducted 1 week and 4 weeks after SCI in each group. We evaluated urodynamic parameters and bladder tissue remodeling factors.

RESULTS

Non-voiding contractions (NVCs) during the storage phase of cystometrograms tended to be increased in all two SCI groups (B and C) with a significant reduction in treatment group (C) versus B. Bladder compliance was improved, and intercontraction intervals were increased in group C compared with group B. In two groups with SCI (B and C), the expression of galectin-3 and other fibrosis markers (a-SMA, collagen III) was increased compared to group in A while decreased expression of all fibrosis markers was noticed in group C (MCP treatment). In group C, the reduction of AKT signaling pathway was demonstrated with treatment of MPC for 4 weeks. The urine galectin-3 level was increased in SCI groups with a reduction of group C versus group B by ELISA.

CONCLUSIONS

The expression of galectin-3 was increased in SCI groups with other fibrosis markers of bladder. Galectin-3 inhibitor comparably improve bladder capacity in rat, by downregulating galectin-3 and reducing bladder fibrosis.


15:34 - 15:37
S3-6 (PP)

A GLOMERULUS-ON-A-CHIP TO MODEL RENAL AUTOIMMUNE DISEASES

Astgik PETROSYAN 1, Paolo CRAVEDI 2, Valentina VILLANI 1, Roger DE FILIPPO 1, Laura PERIN 1 and Stefano DA SACCO 1
1) Children's Hospital Los Angeles, Urology, Los Angeles, USA - 2) Mount Sinai, Nephrology, New York, USA

PURPOSE

Primary membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults worldwide.  MN involves the deposition of auto-antibodies against podocyte-expressed antigens in the glomerular subepithelial space, causing podocyte injury and initiating renal damage leading to kidney failure in one third of patients. The study of mechanisms responsible for MN pathogenesis is challenged by the lack of in vitro systems that recapitulate human disease. We have developed a novel glomerulus-on-a-chip system (GOAC) using human primary podocytes   human glomerular endothelial cells (GEC) in combination with OrganoPlates and assessed the functional response to human MN serum.

MATERIAL AND METHODS

Human podocytes were seeded on microfluidic chips with hGEC. Immunofluorescence and WB were performed for podocyte, endothelial and GBM markers. Barrier selective-permeability was investigated.  Chips were cultured with serum from MN patients or healthy individuals. Functional response was assessed by albumin permeability assay. IgG/IgG4 deposition was assessed by immunofluorescence while mechanisms of action were explored by Western Blotting and immunostaining.

RESULTS

This system recapitulates salient characteristics and functions of the in vivo glomerular filtration barrier (GFB). The GOAC is permeable to inulin and impermeable to albumin. When exposed to the serum of subjects with MN, the chip displayed IgG and complement C3 deposition on the podocytes and loss of permselectivity to albumin to an extent comparable to urinary protein loss in respective patients. Moreover, we have found evidence suggesting that changes in the ILK/MAPK/SNAIL signaling pathway might contribute to podocyte damage during MN pathogenesis.

CONCLUSIONS

We have successfully developed a glomerulus-on-a-chip system that closely mimics the GFB structure and provides a powerful tool for studying renal regenerative and disease mechanisms in proteinuric diseases, toxicity effects and could inform the discovery of new drugs. This system will increase our ability to individualize treatments, thus ultimately benefiting patients affected by renal failure.


15:34 - 15:37
S3-6 (PP)

SYSTEMIC VINCRISTINE INDUCES AN INCREASE IN FUNCTIONAL BLADDER CAPACITY AND A REDUCTION OF BLADDER COMPLIANCE IN JUVENILE MICE

Naoko IGUCHI 1, Duncan WILCOX 2, Alonso CARRASCO 2 and Nicholas COST 2
1) University of Colorado Denver, Surgery, Aurora, USA - 2) Children's Hospital of Colorado, Urology, Aurora, USA

PURPOSE

In order to explore if and how vincristine (VCR) treatment in childhood impacts the lower urinary tract, we investigated the bladder and detrusor function in mice following systemic vincristine treatment. VCR is one of the most common chemotherapy agents used in pediatric patients and is well known to cause peripheral neuropathy, however, no studies conducted to evaluate the impact of this possible peripheral neuropathy on bladder function.

MATERIAL AND METHODS

Juvenile female mice received an intraperitoneal injection of 0.5mg/kg of VCR twice per week for 4 weeks.  This dosing and administration have been published as a murine equivalent to a cycle of vincristine exposure in humans.  Animals in the control group received the same volume of saline at the same schedule. Bladder and detrusor functions were assessedin vivoby voiding behavior and urodynamic studies in unanesthetized, unrestrained condition as well as in vitro physiological studies using the bladder strips at 1-month after the last treatment.  

RESULTS

The VCR-treated group were observed to have increased urinary frequency compared to the control group at 2-weeks post-treatment, while it became less prominent at 1-month post-treatment. No significant changes in detrusor contractility and spontaneous activity were observed following VCR treatment. Cystometry studies revealed significant increases in the functional bladder capacity and maximum intravesical pressure at voiding in the VCR-treated group compared to the control group (Table). 

Urodynamics comparison between the control and VCR groups *p<0.05, †p<0.001

Capacity

(μl)

Void

(μl)

Pves Max

(mmHg)

Non-void

contractions

Control 133 ± 8 135 ± 10 14.0 ± 0.7 0.2 ± 0.1
VCR 188 ± 9† 207 ± 8† 17.5 ± 0.7* 0.0 ± 0.0

CONCLUSIONS

Our results suggest that systemic VCR exposure in childhood may impact lower urinary tract function and a need for follow-up urological assessment of the patients receiving anti-neoplastic, systemic VCR.


15:37 - 15:40
S3-7 (PP)

CYP21A2 EXPRESSION AND CORTISOL PRODUCTION IN A HUMAN ADRENAL CELL LINE

Kiersten CRAIG 1, Jun YAO 2, Dix POPPAS 3 and Yariv HOUVRAS 4
1) New York Presbyterian, Urology, New York, USA - 2) Weill Cornell Medicine, Urology, New York, USA - 3) Weill Cornell Medicine/ Komansky Children's Hospital, Pediatric Urology, New York, USA - 4) Weill Cornell Medicine, Surgery, New York, USA

PURPOSE

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by loss of function mutations or deletions affecting the CYP21A2 gene. Current management requires life-long exogenous steroid treatment, mineralocorticoid replacement, and often surgery. Most patients with CAH harbor loss of function mutations that only partially reduce the 21-hydroxylase activity raising the possibility that medical therapy designed to increase CYP21A2 gene transcription would translate into clinical benefit. We previously performed a chemical genetic screen with zebrafish and found that Forskolin, an adenylyl cyclase analog, increases cyp21a2 transcription in zebrafish. We used a human adrenal cell line, H295R, to determine if similar affects can be seen in human cells. 

MATERIAL AND METHODS

H295R cells were treated with Forskolin and DMSO (control). Quantitative Polymerase Chain Reaction (qPCR) was used to detect the fold change in transcription of CYP21A2 after 12 hours of treatment. Cortisol enzyme linked immunosorbent assay was also used to quantify cortisol production in the supernatant of treated cells after 48 hours.

RESULTS

CYP21A2 expression was 1.54 fold higher following Forskolin treatment than DMSO at 12 hours. Cortisol levels in the supernatant of Forskolin treated cells were 1.56 and 1.60 fold higher thanDMSO and baseline cells, respectively.

CONCLUSIONS

Forskolin increases cyp21a2 expression and cortisol production in the H295R cell line. Our studies raise the possibility that it may be possible to repurpose select drugs such as cAMP analogs to improve treatment of patients with CAH.


15:40 - 15:52
Discussion