ESPU Meeting on Wednesday 8, June 2022, 13:15 - 14:10
13:15 - 13:18
S01-1 (OP)
Astgik PETROSYAN 1, Paola AGUIARI 1, Charmi DEDHIA 1, Xiaogang HOU 1, Geremy CLAIR 2, Yi HUANG 3, Eun Ji CHUNG 3, Roger DE FILIPPO 1, Stefano DA SACCO 1 and Laura PERIN 1
1) Children's Hospital Los Angeles, Urology, Los Angeles, USA - 2) PNNL, Richland, USA - 3) University of Southern California, Los Angeles, USA
BACKGROUND
In Alport Syndrome (AS), disruption of COL4 network leads to podocyte depletion and progressive kidney failure. Using different tools (glomerulus-on-a-chip, GOAC, COL4-defective podocytes, FUCCI mice, proteomics, nanoparticles, and spatial transcriptomics) we have identified miR-193a as possible specific disease mechanism in AS podocytes.
MATERIAL AND METHODS
Podocytes from amniotic fluid of AS patients (AS-POD) were seeded with human glomerular endothelial cells in a barrier-free system to generate AS-GOAC. Healthy primary podocytes were used as control; analysis was performed by confocal microscopy, proteomics, WB, and permselectivity. miR-193a studies were performed using mimics and inhibitors. Proteomics studies in AS mouse podocyte and spatial transcriptomics accompanied with in-situ hybridization in human AS biopsies were performed to validate our data.
RESULTS
AS-POD phenotype was confirmed by RNA-seq and WB. AS-GOAC presents impaired permselectivity, and proteomics revealed a distinctive AS signature. Mouse and human podocyte data confirmed upregulation of miR-193a (and modulation of its targets-WT1, osteopontin, VEGF) specifically in podocytes. miR-193a inhibition, delivered with innovative nanoparticles specifically in podocytes, restores podocyte-GBM interactions and prevents podocyte cycle re-entry, thus preventing podocyte loss. Results were confirmed in AS mouse glomeruli and human AS biopsies.
CONCLUSIONS
Using different tools, we studied the effects of a defective GBM at the podocyte level. Specifically, we established that targeted delivery of miR-193a inhibitor shows beneficial effects on podocyte function by modulating cell-GBM interaction and by regulating podocyte cell cycle. miR-193a inhibition in podocytes might re-establishing glomerular filtration barrier homeostasis, thus representing a therapeutic target in AS settings.
13:18 - 13:21
S01-2 (OP)
Qi ZHANG 1, Kelly BUDGE 2, Astgik PETROSYAN 1, Paola AGUIARI 1, Roger DE FILIPPO 1, Laura PERIN 1, Paolo CRAVEDI 2 and Stefano DA SACCO 1
1) Children's Hospital Los Angeles, Urology, Los Angeles, USA - 2) Mount Sinai, Nephrology, New York, USA
BACKGROUND
Primary membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults worldwide due to the deposition of anti-podocyte-antibodies against in the glomerular subepithelial space. While complement deposition is thought to play a crucial pathogenic role, the exact effector mechanism of complement in MN is unclear due to the lack of in vitro and in vivo systems that recapitulate human disease. We have developed a novel glomerulus-on-a-chip system (GOAC) using human primary podocytes and glomerular endothelial cells (GEC) and assessed functional response to human MN serum, role of Membrane-attack-complex (MAC) formation and C3a/C3aR1 signaling in MN pathogenesis.
MATERIAL AND METHODS
GOACs were cultured with serum containing either anti-PLA2R+ or THSD7A+ MN patients; sera from healthy individuals were used as control. Functional response was assessed by albumin permeability assay to evaluate permselectivity. Role of MAC and C3a/C3aR1 signaling pathway was assessed by immunofluorescence and functional analysis while mechanisms of action were explored by PCR arrays, Western Blotting and immunostaining. Results were confirmed in vitro using podocytes on which C3aR1 was silenced and in vivo using THSD7A induced MN in balb/c mice.
RESULTS
Following exposure to sera from MN patients, we have confirmed deposition of human lgG on podocytes and formation of MAC complex, accompanied by albumin leakage. MAC inhibition did not prevent albumin leakage while GOAC supplemented with C3aR1 antagonists as well as GOAC using podocytes in which C3aR1 was silenced were able to prevent glomerular filtration damage and albumin leakage. Efficacy of C3aR1 antagonists in preventing proteinuria was confirmed in vivo, substantiating our findings.
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. Using a combination of in vitro and in vivo models, we showed that C3a/C3aR signaling plays a dominant role in complement-mediated MN pathogenesis.
13:21 - 13:24
S01-3 (OP)
Yucheng GE, Wenying WANG, Ruichao ZHAN, Yukun LIU, Chen NING, Jun LI, Jun LI and Ye TIAN
Beijing Friendship Hospital, Capital Medical University, Urology, Beijing, CHINA
PURPOSE
To study the phenotypic characteristics of primary hyperoxaluria type 3 (PH3) hotspot mutation (HOGA1 c.834G>A) and the relationship between genotype and phenotype by constructing a mouse model of PH3 hotspot mutation in China.
MATERIAL AND METHODS
Cas9/sgRNA and targeting vector were microinjected into mouse zygotes to construct PH3 model with HOGA1 c.834G > A mutation. Urine samples were collected from mice aged 3 and 6 months for calcium ion and oxalic acid concentration detection.
RESULTS
Homozygous mutant model mice excreted more oxalate in urine than wild-type. Urinary oxalate was detected in 6-month-old model mice, but the results were not statistically significant.In the detection results of urinary calcium, the urinary calcium concentration in the homozygous mutation heterozygous mutation group was significantly higher than that in the wild type group, while the difference in the urinary calcium concentration between the homozygous and heterozygous mutation groups was not statistically significant. When genotypes were identical, urinary calcium concentrations were higher in 6-month-old mice than in 3-month-old mice. In addition, homozygous mutant mice showed crystalluria and a significant increase in kidney size.
CONCLUSIONS
These data suggested that hotspot mutant mice excrete more oxalic acid and calcium in the urine and may have phenotypes such as crystalline urine and significantly increased kidney size compared with wild-type mice. This animal model is not only a useful model for studying the pathogenesis and pathology of PH3 in China, but also a valuable tool for the development and evaluation of innovative drugs and therapies.
13:24 - 13:27
S01-4 (OP)
Roberto LOPES 1, Lucas Henrique SILVA 2, Marcos MELLO 1, Francisco DÉNES 1, Lydia YAMAGUCHI 2, Massuo KATO 2 and Marina TAVARES 2
1) Pediatric Urology Unit, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, Urology, São Paulo, BRAZIL - 2) Instituto de Química, Universidade de São Paulo, Chemistry, São Paulo, BRAZIL
PURPOSE
Management of asymptomatic patients with obstructive hydronephrosis (HN) frequently poses a challenge. This prospective case-control study aimed to investigate urine samples of children with ureteropelvic junction obstruction (UPJO) using gas chromatography-mass spectrometry (GS-MS) based metabolomics to analyze if metabolite proteins are useful to discriminate obstructive HN to non-obstructive HN.
MATERIAL AND METHODS
Seventy-four patients were enrolled in the study. Urinary samples were collected as well as clinical characteristics (hydronephrosis grade, differential renal function and t1/2) were measured in the following age-matched groups: 26 children with obstructive HN at initial diagnosis (preoperative); 22 children with non-obstructive HN; and 26 children without any urinary tract condition, as the control group. Results were assessed statistically using for homogenous groups, a one-way analysis of variances (ANOVA) and for nonparametric groups, Mann–Whitney test or Kruskal–Wallis test was performed. ROC curves were performed.
RESULTS
Metabolomics pro1ling revealed distinct expression patterns between obstructive HN to non-obstructive HN. Random forest analysis of urine samples suggested discriminators speci1c to obstructive HN; these include 4-hydroxyphenylacetic acid, Furoyglycine, Gluconic acid, N-acetyl-L-aspartic acid and Theitol. All these urinary metabolomics-based analytes had an AUC above 0.80 (Abstract Table), suggesting strong potential clinical value as a diagnostic signature.
Table
|
Molecular Features |
p-value (ROC) |
AUC |
|
4-hydroxyphenylacetic acid |
0.04 |
0.900 |
|
Furoyglycine |
0.01 |
0.807 |
|
Gluconic acid |
0.009 |
0.813 |
|
N-acetyl-L-aspartic |
0.0002 |
0.900 |
|
Theitol |
0006 |
0.807 |
CONCLUSIONS
These promising 1ndings suggest that analyses of urinary metabolites using gas chromatography-mass spectrometry (GS- MS) can discriminate obstructive from non-obstructive HN with excellent accuracy.
13:42 - 13:45
S01-5 (OP)
Martin SIDLER 1, Kj AITKEN 2, A. SCHROEDER 3, Priyank YADAV 2, Rosanna WEKSBERG 2 and Darius BAGLI 2
1) Hospital for Sick Children, Toronto, Ontario, CANADA & Klinikum Stuttgart, Paediatric and Neonatal Surgery, Stuttgart, GERMANY - 2) Hospital for Sick Children, Toronto, CANADA - 3) Klinik und Poliklinik für Urologie und Kinderurologie, University Medical Center, Mainz, GERMANY & Hospital for Sick Children, Toronto, CANADA
INTRODUCTION
The dysfunction and histopathology of partial bladder outlet obstruction (PBO) often remains even after ablation of the obstruction as a chronic obstructive bladder disease (COBD). COBD displays specific gene expression changes, leading to persistent changes suggestive of epigenetics. Our objectives were to examine epigenetic mechanisms of persistent dysregulation of KCNB2 smooth muscle cells (SMC) in vitro and in COBD.
MATERIAL AND METHODS
In female Sprague-Dawley rats, 6 week PBO was performed, ligating around the urethra and a 0.9 mm rod (rod is then removed) to establish a uniform obstruction. COBD is created by removal of obstructing suture for COBD after 6 weeks. Then in COBD 6 weeks later (n=8), plus PBO 6 week controls(n=8) and sham 12 week controls(n=6), voiding, residual volumes, bladder and body weights were recorded. qPCR for KCNB2 RNA and ChIP/PCR for histone-H3 lysine trimethylation(H3K27me3, generated by EZH2 activity) were performed on the bladders. Phenotype was analysed by immunostaining and western in SMC on disorganized collagen matrix(DC) vs organized collagen(OC) matrices on KCNB2 and EZH2 expression+/- KCNB2 inhibitor(STX) or EZH2 inhibitor(UNC1999).
RESULTS
DC vs. NC enhanced EZH2 expression and diminished KCNB2. STX increased SMC numbers, proliferation and decreased SMC marker expression, p<0.01. UNC1999 augmented KCNB2 expression. PBO and COBD decreased KCNB2 expression vs. sham (p<0.05) associated with pathophysiology. H3K27me3 was increased at two KCNB2 promoter regions in COBD but not PBO (>10-fold above sham levels,p<0.01).
CONCLUSIONS
Persistently altered H3K27me3 associates with pathophysiology in COBD. The matrix regulation of EZH2 activity suggests that matrix and epigenetics are regulate loss of SMC KCNB2 in COBD.
13:45 - 13:48
S01-6 (OP)
Tariq Osman ABBAS 1, Aisha ALHALABI 2, Mohammed HASSAN 2, Huseyin YALCIN 3 and Cristian PABLO PENNISI 4
1) Sidra Medicine, Aalborg University, Urology, Doha, QATAR - 2) Qatar University, Centre for Advanced Materials, Doha, QATAR - 3) Qatar University, Biomedical Research Centre, Doha, QATAR - 4) Aalborg University, Regenerative Medicine Research Group, Department of Health Science and Technology, Aalborg, DENMARK
PURPOSE
Although several surgical techniques were introduced to manage the failed hypospadias repair, this is considered a challenging clinical problem where scarring and hypovascular tissues exist particularly in repeat surgeries. Chorioallantoic membrane (CAM) is used in tissue engineering to test growth factors and biocompatibility materials and test these factors' capacity to provoke the formation of new blood vessels from established vasculature.
MATERIAL AND METHODS
The scaffolds were fabricated using layering electrospinning technique with Chitosan/PEO (60:40) and Polylactic acid (PLA) 10% and were first examined in terms of their physical, mechanical, and biological performances. To compare differently fabricated scaffolds in terms of vascularization, the constructs were tested in the CAM assay with subsequent assessment of the vascular development and biocompatibility. Theses scaffold were compared to small intestinal submucosa (SIS).
RESULTS
Vascularization induced by Chitosan Based Multilayered Scaffold led to a vessel distribution more homogenous and highly dense adjacent to the constructs, while SIS scaffold enhanced vessel density at a significantly lower extent (vessel junctions 1.2 ± 0.23 versus 2.1 ± 0.22, respectively P value 0.005). Inadequate angiogenesis and epithelialization make urethral regeneration using conventional tissue-engineered grafts a significant challenge.
CONCLUSIONS
Electrospun Chitosan Based Multilayered Scaffold shows potential to function as a tissue-engineered urethra graft with excellent angiogenesis to maintain epithelialization and urethral regeneration in ovo. The CAM assay is an easy and cheap screening tool for the angiogenic properties of such scaffolds.
13:48 - 13:51
S01-7 (OP)
Martin KAEFER 1, Paul WINCHESTER 2, Millissia BEN MAAMAR 3, Daniel BECK 3, Eric NILSSON 3, Rosalia MISSERI 1, Benjamin WHITTAM 1, Richard RINK 1 and Michael SKINNER 3
1) Riley Hospital for Children, Pediatric Urology, Indianapolis, USA - 2) Riley Hospital for Children, Neonatology, Indianapolis, USA - 3) Washington State University, Reproductive Biology, Pullman, USA
PURPOSE
The heritability of hypospadias is suggested to exceed 50% yet recent genome wide associations have found only 9% of cases to be explained by genomic variants. This suggests that environmental factors play a significant role. We have previously demonstrated a significant correlation between hypospadias severity and pesticide use. Animal models have demonstrated that pesticides result in altered DNA methylation in exposed offspring. Environmental factors may therefore play a significant role. We hypothesize that the epigenetic signatures differ depending on severity of hypospadias.
MATERIAL AND METHODS
Following IRB approval discarded foreskin samples were prospectively collected from 30 patients undergoing hypospadias correction and age matched controls undergoing routine circumsion. Level of hypospadias severity was classified based on location of the urethral meatus following correction of chordee (distal, midshaft, proximal). Epigenetic DNA methylation was examined with methylated DNA immunoprecipitation (MeDIP) with a MeDIP sequencing protocol examining greater than 95% of the genome.
RESULTS
A total of 15 distal, 9 midshaft and 6 proximal hypospadias samples were accrued. This epigenome-wide association study (EWAS) clarified differential DNA methylation regions (DMRs) in all hypospadias patients. A combination analysis of all distal, midshaft, and proximal hypospadias had 734 DMRs at p1e-05 edgeR and false discovery rate (FDR) p < 0.05. Distal hypospadias had 2725 DMRs , while midshaft hypospadias had 95 DMRs and proximal cases had 22 DMRs.
CONCLUSIONS
Epigenetic alterations were found in hypospadias, with reproducible differences noted between boys with distal, midshaft and proximal phenotypes. Paradoxically, more severe forms of hypospadias demonstrated fewer DMRs suggesting that cell populations responsible for severe phenotypic expression may no longer be present in remaining foreskin tissue, perhaps due to apoptosis. These observations suggest that altered epigenetics are involved in the etiology of hypospadias. Future work to clarify the effect of epigenetic alteration on gene expression will improve our understanding of this complex disorder.
13:51 - 13:54
S01-8 (OP)
Mohamed HASSAAN 1, Ahmed ELKASHEF 2, Abdelwahab HASHEM 2, Amira AWADALLA 3, Nashwa BARAKAT 1, Fathy EL-SEDDAWY 4 and Mahmoud ABDEL-MABOUD 4
1) Urology and Nephrology Center, Mansoura University, Egypt, Mansoura, EGYPT - 2) Urology and Nephrology Center, Mansoura University, Egypt, Department of Urology, Mansoura, EGYPT - 3) Center of Excellence for Genome and Cancer Research, Urology and Nephrology Center, Mansoura University, Egypt, Mansoura, EGYPT - 4) Faculty of Veterinary Medicine, Zagazig University, Egypt, Department of General Surgery, Zagazig, EGYPT
PURPOSE
Urethroplasty is a complex procedure that depends on functional wound healing to be successful. Excessive inflammation and fibrosis post urethroplasty may lead to recurrent urethral stricture. Dexpanthenol (DXP) has been used for wound healing for a long time. Therefore, we evaluated the effect of intraurethral DXP on urethral healing, inflammation and fibrosis after urethroplasty in rats.
MATERIAL AND METHODS
Fifty-four male Sprague–Dawley rats were randomly allocated into 3 groups (18 rats each); control, urethroplasty and DXP groups. Urethroplasty was performed by creating a 0.8-cm vertical midline urethral incision with a microknife, then closing the urethra and overlying corpus spongiosum using 7-0 PDS. Saline solution 0.9% and DXP (500 mg/kg) was then administered intraurethrally once daily till sacrification to urethroplasty and DXP groups, respectively. 6 rats from each group were sacrificed on the postoperative days 4, 8 and 15. Penectomy was performed and the penises were then sent for routine histopathological examination, interleukin (IL)-6, transforming growth factor (TGF)-ß1, ß-catenin, vascular endothelial growth factor (VEGF) and collagen I & III relative gene expression and assessment of malondialdehyde (MDA) and nitric oxide (NO) activity.
RESULTS
There was significant improvement in inflammation and fibrosis scores after intraurethral DXP application for 8 and 15 days. There was also significant up-regulation of VEGF, down-regulation of IL-6, TGF-ß1, ß-catenin and collagen I & III expression and decrease in collagen I/III ratio, after 4, 8 and 15 days of intraurethral DXP administration. DXP group showed significant reduction in MDA and NO activity on days 4, 8 and 15. Apart from histopathology and collagen I/III ratio, intraurethral application of DXP up to 15 days significantly provided more beneficial effects.
CONCLUSIONS
Administration of intraurethral DXP might minimize urethral fibrosis and scarring post urethroplasty.