ESPU Meeting on Wednesday 17, April 2024, 13:15 - 14:15
13:15 - 13:18
S01-1 (OP)
Qi CHILDREN'S HOSPITAL LOS ANGELES 1, Jack WETZELS 2, Coralien VINK 2, Paolo CRAVEDI 3, Laura PERIN 1 and Stefano DA SACCO 1
1) Children's Hospital Los Angeles, Los Angeles, USA - 2) Radboud University Nijmegen Medical Centre, Nijmegen, NETHERLANDS - 3) Mount Sinai New York, New York, USA
PURPOSE
Primary membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults due to the deposition of anti-podocyte antibodies in the glomerular subepithelial space. Several podocyte proteins have been identified as targets of these autoantibodies, with PLA2R1 and THSD7A making up about 80-85% of the total. We investigated the specific effect of anti-PLA2R and anti-THSD7A on glomerular cells using single cell transcriptomics on a human glomerulus-on-a-chip (GOAC) system.
MATERIAL AND METHODS
GOACs were generated using human primary podocytes and glomerular endothelial cells and cultured with serum containing anti-PLA2R+ or anti-THSD7A+ antibodies for 72 hours. Sera from healthy individuals were used as a control. Three samples from different individuals were used for each group. Albumin leakage assay was performed on GOAC to confirm injury. Single cell RNA-seq analysis (scRNA-seq) with a depth of 30K reads/cell was performed on isolate cells. Downstream analyses were done using UMAP, gene and pathway enrichment, and intra- and inter-cluster comparative transcriptomics.
RESULTS
Exposure to anti-PLA2R and anti-THSD7A sera from MN patients confirmed injury on the GOAC as shown by onset of albumin leakage. scRNAseq analysis showed robust activation of the complement pathway in both cohorts, as expected. Preliminary analysis also showed different activation of injury pathways in both human podocytes and human glomerular endothelial cells.
CONCLUSIONS
In conclusion, we have successfully performed scRNAseq data on human cells obtained from GOACs exposed to patients’ sera. This combination of technologies will help us unravel the glomerular mechanisms of injury in MN and provide potential new targets for the treatment of nephropathies and other glomerular diseases.
13:18 - 13:21
S01-2 (OP)
Marie BOUSQUET 1, Marie-Emilie LEBACHELIER DE LA RIVIERE 2, Sarah AMAR 1, Manon GAYET 1, Virginie MAILLARD 2, Aurélien BINET 3, Fabrice GUERIF 4 and Sébastien ELIS 2
1) Gatien de Clocheville Hospital - University Hospital of Tours, Pediatric Surgery Unit, Tours, FRANCE - 2) INRAE - University of Tours, PRC - BINGO, Nouzilly, FRANCE - 3) University Hospital of Poitiers, Pediatric Surgery Unit, Poitiers, FRANCE - 4) Bretonneau Hospital - University Hospital of Tours, Reproductive Médecine and Biology Unit, Tours, FRANCE
PURPOSE
Bisphenol A (BPA) is known as an endocrine disruptor and has been prohibited since 2015. The increasing use of structural analogs of BPA, including bisphenol S (BPS), raises concerns about the adverse effects they can share with BPA on human reproduction.
The first aim of this study was to describe the potentially negative effects of bisphenols on the quality of human embryos produced in vitro for medically assisted reproduction (MAR). The second one, was to investigate the individual factors of variability in the effects of BPS on steroidogenesis.
MATERIAL AND METHODS
Human granulosa cells (HGC) and follicular fluid (FF) were collected from oocyte punctures from women undergoing MAR from January 2019 to April 2023. First, we measured the concentrations of 4 bisphenols in the FF of each patient (mass spectrometry). The "exposed population" included patient with at least one positive dosage. Secondly, we individually cultured HGC at different BPS concentrations and then assessed in vitro steroidogenesis by measuring progesterone and estradiol concentrations (ELISA test). Finally, a correlative study was carried out after gathering patients' clinical data and embryos' characteristics.
RESULTS
We included 368 patients.
Exposure of follicular fluids to bisphenols represented 18.8% of the study population, with mainly BPS (52.5%).
Embryo rate was higher in the exposed population (p<0,05). Bisphenols' concentrations in FF were negatively correlated with the number of "high-quality embryos" (R=-0.35 ; p<0,05).
In vitro, BPS decreased progesterone and estradiol secretions by HGC (p<0,001). Progesterone secretion by HGC seemed to be more inhibited in lean individuals (R=0.33 ; p<0,05).
CONCLUSIONS
This suggests potentially negative effects of BPS on human reproduction. Firstly, BPS alters ovarian steroidogenesis in vitro. Secondly, because of its estrogenic properties, BPS may promote embryonic development in vitro to the detriment of embryonic quality, with potentially deleterious consequences for the future child.
13:21 - 13:24
S01-3 (OP)
Sarah AMAR 1, Aurélien BINET 2, Ophélie TETEAU 3, Alice DESMARCHAIS 3, Pascal PAPILLIER 3, Marlène Z. LACROIX 4, Virginie MAILLARD 5, Fabrice GUERIF 6 and Sébastien ELIS 5
1) Gatien de Clocheville Hospital - University Hospital of Tours, Paediatric Surgery Unit, Tours, FRANCE - 2) University Hospital of Poitiers, Pediatric Surgery Unit, Poitiers, FRANCE - 3) PRC, INRAE, CNRS, Tours University, IFCE, Nouzilly, FRANCE - 4) Therapeutic Innovations and Resistance (INTHERES), Toulouse University, INRAE, ENVT, Toulouse, FRANCE - 5) PRC, INRAE, CNRS, Tours University, IFCE, PRC, Nouzilly, FRANCE - 6) University Hospital of Tours, Reproduction Biology Unit, Tours, FRANCE
PURPOSE
Deleterious effects of endocrine disruptors (ED) on fertility, sexual behaviors and sexual differentiation have been proven in various species. In human, a growing number of studies show a link between ED and the increasing incidence of genital malformations and reduced fertility.
Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA) and main BPA replacement in the plastics industry. Previous studies have shown BPS to exhibit similar effects on reproduction in different species. BPS reportedly alters steroidogenesis in bovine granulosa cells. However few studies exist in humans.
We aimed to study in vitro effects of BPS on primary human granulosa cells (HGC), playing an essential role in folliculogenesis and organogenesis, particularly genital organogenesis.
MATERIAL AND METHODS
Luteinised HGC collected from 59 women undergoing in vitro fertilization procedure were cultured for 48h in the presence or absence of BPS (10nM to 50µM). BPS exposure was investigated by assessing follicular fluids for its metabolites (BPS-glucuronide or BPS-G) by mass spectrometry. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted were examined for oestradiol and progesterone secretions, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling.
RESULTS
BPS-G were found in 18.6% of patients. 10 or 50µM of BPS induced a 16% (p=0.0059) and 64% (p<0.0001) decrease, respectively, in progesterone secretion. Fifty µM BPS decreased oestradiol secretion by 46% (p<0.0001). Ten µM BPS tended to reduce CYP11A1 protein expression by 37% (p=0.0947). Fifty µM BPS increased ERRγ expression. No effects on cell proliferation or viability.
CONCLUSIONS
BPS appears to exert deleterious effects on HGC, and further studies are needed to investigate the underlying mechanisms. The disruption of steroidogenesis in the context of organogenesis is probably not without consequences via this pathway. The effects of BPS were observed after only 48h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.
13:35 - 13:38
S01-4 (OP)
Yu DING, Haojie CHEN, Wenfeng PAN, Yichen HUANG and Fang CHEN
Shanghai Children's Hospital, Urology, Shanghai, CHINA
PURPOSE
The formation of hypospadias is affected by prenatal androgen levels. However, the precise role and timing of genetic factors in this process remain unknown.
MATERIAL AND METHODS
Using whole exome sequencing analysis, we identified DNAH8 as the a new risk gene for hypospadias and established a DNAH8 KO mouse model. Morphological techniques were employed to analyze urogenital development and male differentiation in fetal mice. Furthermore, we utilized ELISA and RT-qPCR to assess testosterone levels and gene expression in steroid biosynthesis pathways. Proteomics and single-cell RNA sequencing combining with immunohistochemical staining were applied to analyze the testicular and external genital cells in fetal mice.
RESULTS
We found that DNAH8 KO mice exhibit hypospadias and male differentiation anomalies due to local effects of DNAH8 loss and systemic effects of testicular hormone deficiency. DNAH8 knockout results in a decreased number of fetal Leydig cells (FLCs) in the testes, accompanied by reduced expression of Hedgehog pathway and steroidogenic genes. Notably, the absence of DNAH8 retards the differentiation of steroid progenitor cells into FLCs, leading to a decline in prenatal testosterone levels, decreased cell population in the distal glans of the external genitalia, and ultimately affecting the proper formation of the urethra.
CONCLUSIONS
DNAH8 play key roles in testosterone production during the androgen-sensitive window, providing significant insights into the underlying pathogenesis of hypospadias as well as genetic counseling.
13:38 - 13:41
S01-5 (OP)
Zhongzhong CHEN 1, Jiaoru YANG 2, Fuying LAN 1, Yiqing LYU 1 and Pin LI 2
1) Shanghai Children's Hospital, School of Medicine, Shanghai Jiao Tong University, Department of Urology, Shanghai, CHINA - 2) Shanghai Children's Hospital, School of Medicine, Shanghai Jiao Tong University, Department of Endocrinology, Shanghai, CHINA
PURPOSE
Androgen insensitivity syndrome (AIS) is a rare genetic male reproductive disorder characterized by mutations in the androgen receptor (AR) gene. AIS patients typically exhibit clinical features such as hypospadias and cryptorchidism. Despite the description of approximately 600 mutations within four functional domains of theARgene in AIS, the precise genotype-to-phenotype relationship between AR and AIS, as well as between hypospadias and cryptorchidism, remains unclear.
MATERIAL AND METHODS
For a comprehensive exploration of the potential genetic role of AR in complete (CAIS), partial (PAIS) and mild AIS (MAIS), we initially investigate the distribution of different types of AR mutations in AIS using the public database. Subsequently, we conducted AR gene sequencing in 56 AIS patients exhibiting diverse outcomes enrolled from 2015 to 2022, categorizing each variant into groups such as missense, stop codon, insertion, deletion, duplication, and others
RESULTS
The distribution of various types ofARmutations in AIS indicates that mutations with a more substantial effect size are more commonly observed in individuals with CAIS than in those with PAIS and MAIS (p < 0.001), as reported in the androgen receptor mutations database. Individuals with high-impact AR mutations are more susceptible to developing cryptorchidism (p < 0.05) compared to other mutation types, but there is no significant difference in the development of hypospadias (p > 0.05).
CONCLUSIONS
The severity of AIS can vary widely and can be considered as an AR dosage-dependent condition. These findings provide new insights into the genetic role ofARin the development of AIS, hypospadias, and cryptorchidism.
13:41 - 13:44
S01-6 (OP)
Nao IGUCHI 1, Ali TEIMOURI 2, Duncan WILCOX 3, Anna MALYKHINA 2 and Nicholas COST 4
1) University of Colorado School of Medicine, Surgery/Urology, Aurora, USA - 2) University of Colorado School of Medicine, Aurora, USA - 3) Children's Hospital of Colorado, Aurora, USA - 4) Children's Hospital Colorado, Aurora, USA
PURPOSE
To examine the long-term effects of systemic doxorubicin (DOX) exposure during childhood on lower urinary tract (LUT) function using a juvenile murine model. DOX plays a pivotal role in the treatment of pediatric cancers, while it has a wide range of side effects including cardiotoxicity and nephrotoxicity. In our recent study, childhood cancer survivors with a history of DOX and/or vincristine exposure reported a significantly increased rate of LUT symptoms compared to a control cohort. Hence, DOX and vincristine exposure in childhood requires consideration of urological complications and long-term side effects.
MATERIAL AND METHODS
After IACUC approval, DOX (3mg/kg/week) was administered intravenously to CD-1 mice for six consecutive weeks (10-17-year-old in human). Control mice received saline similarly. Five weeks after the treatment (early twenties human age), the LUT impacts of DOX exposure were evaluated by bladder histology and physiological recording using bladder strips.
RESULTS
DOX caused induced a significant decrease in the growth ratio, while the bladder weight was compatible between the two groups in both sexes. Bladder histology showed an increased detrusor layer ratio to total area in DOX-treated mice compared to control group, especially in males (69 ± 2 vs. 64 ± 1%, p=0.0514). Relative to the control group, the bladder strips from DOX-treated females showed a marked decrease in maximal contractile force produced by depolarization of myocytes (0.46 ± 0.05 vs. 0.30 ± 0.03g/mg, p=0.010) as well as by nerve-excitation (EFS, p<0.001) and cholinergic receptor activation (p<0.001). On the other hand, no significant differences in the contractility were observed between the two groups of male mice. These results suggest that DOX treatment in early age affected bladder muscle physiology at later age, especially in females.
CONCLUSIONS
DOX exposure in childhood induces LUT dysfunction as a long-term effect. Prospective monitoring for LUT dysfunction could benefit childhood cancer survivors who received DOX.
13:55 - 13:58
S01-7 (OP)
Julia KOIVULA 1, Niklas Alarik PAKKASJÄRVI 2 and Satu KUURE 3
1) University of Helsinki, Helsinki Institute of Life Science and STEMM, Helsinki Institute of Life Science (HiLIFE) ja STEMM, Research Program Unit, Faculty of Medicine, Helsinki, FINLAND - 2) New Children's Hospital Helsinki University, Paediatric urology, Helsinki, FINLAND - 3) University of Helsinki, Helsinki Institute of Life Science (HiLIFE) ja STEMM, Helsinki Institute of Life Science (HiLIFE) ja STEMM, Research Program Unit, Faculty of Medicine, Helsinki, FINLAND
PURPOSE
The regulation differences between normal tissue-residing stem cells and Wilms tumor (WT) stem cells remain elusive. We previously demonstrated that the MAPK/ERK pathway is pivotal in regulating nephron progenitor self-renewal and collecting duct progenitor maintenance. Given indications from past research linking MAPK/ERK activity with WT through IGF2 overexpression mediation, our current study seeks to elucidate the signaling cascades and cellular transitions of renal progenitors during both development and tumorigenesis.
MATERIAL AND METHODS
We isolated renal progenitor cells from MAPK/ERK-deficient mouse embryonic kidneys (n=4/genotype) and subjected them to bulk RNA sequencing. We then conducted differential gene expression analysis to identify aberrant transcripts between wild type and MAPK/ERK-deficient progenitor cells. Further, we employed multiple pathway analyses to contrast these transcriptional alterations with those observed in WT samples.
RESULTS
GO and KEGG pathway analyses revealed several shared pathways with contrasting expression patterns in WT and MAPK/ERK-deficient renal progenitors, including cell cycle regulation. Notably, we identified novel pathways linked to extracellular matrix and chromatin regulation. Extracellular matrix-related changes (395 transcripts) were detected in both nephron progenitors and WT, suggesting that WT transformation involves dysregulation of extracellular matrix, possibly downstream of the MAPK/ERK pathway. Interestingly, chromatin related alterations (142 transcripts) were divergent in WT and nephron progenitors (p<0.05).
CONCLUSIONS
Renal progenitor cells and WT exhibit divergent signaling pathways. These disparities encompass novel pathways linked to extracellular matrix and chromatin regulation. Identification of these will in the future provide means for accurate diagnostics, enabling risk-stratified treatment options.
13:58 - 14:01
S01-8 (OP)
Astgik PETROSYAN, Valentina VILLANI, Paola AGUIARI, Min MADHI, Matthew THORNTON, Brendan GRUBBS, Roger DE FILIPPO, Kevin LEMLEY, Gregory SHACKLEFORD, Anat ERDREICH-EPSTEIN, Stefano DA SACCO and Laura PERIN
Children's Hospital Los Angeles, Los Angeles, USA
PURPOSE
Wilms Tumor (WT) is the most common pediatric renal cancer. Growing evidence links WT to aberrant nephrogenesis. While studies highlighted the genetic complexity of WT, little is known about the molecular mechanisms that regulate WT development. Here we report that uncommitted nephrogenic progenitors (NPs) expressing SIX2 and CITED1 (the master regulators of renal development) present characteristics of cancer stem cells (CSCs) and are the ones driving WT. We have also studied the role of integrins in these NPs in regulating WT development.
MATERIAL AND METHODS
WT and human fetal kidney (hFK) samples were histologically analyzed, digested to single-cell suspension, incubated with Smartflare-probe to isolate SIX2+CITED1+ cells, and processed for RNA-seq, single-cell RNA-seq and spatial transcriptomics. Xenografts of WT-NPs and hFK-NPs were generated and tumor formation was assessed. Analyses of mechanisms that regulate self-renewal vs. differentiation were performed in vitro and in vivo. Knockdown with miREs against SIX2 and CITED1 was performed on WT-NPs and processed for RNA-seq.
RESULTS
By comparing NPs from different WT subtypes and NPs from hFK we identified that cells expressing SIX2 and CITED1 fulfill CSC criteria, reliably recapitulating WT in transplantation studies. We showed that self-renewal vs. differentiation of SIX2+CITED1+ WT CSCs is regulated by the interplay between integrins ITGB1 and ITGB4. WT transplantation studies show that blocking ITGB1 or ITGB4 leads to higher number of SIX2+CITED1+ cells in the xenografts. Knockdown of SIX2 and CITED1 increased expression of kidney differentiation markers LHX1, WNT7B, PODXL, MECOM, reduced expression of nephrogenic markers MEOX1, TMEM100, EYA1, MAYFB, and increased expression of ITGB1, ITGB4, and LAMA5.
CONCLUSIONS
These studies define SIX2+CITED1+ cells as the nephrogenic CSCs of WT, where ITGB1 and ITGB4 interplay may play a role in self-renewal vs. differentiation and serve as a potential target for new strategies to treat WT.
14:01 - 14:04
S01-9 (OP)
Osanna KOSOYAN, Sargis SEDRAKYAN, Stefano DA SACCO and Laura PERIN
Children's Hospital Los Angeles, Los Angeles, USA
PURPOSE
We have identified in cells co-expressing SIX2 and CITED1 the cancer stem cells (CSC) that regulate Wilms tumor (WT) development. Our comparative in vitro and in vivo analysis highlighted that mitochondrial dysregulation plays a critical role in modulating biology of these CSC. In this work, we have performed transcriptomics analysis and mechanistic in vitro studies to assess impact of mitochondria function on WT stem cell biology
MATERIAL AND METHODS
SIX2+CITED1+ expressing CSC were isolated using smartflare probes from favorable and unfavorable WT and human fetal kidney (hFK) samples and processed for bulk and scRNA-seq. Differential gene expression and GO ontology analysis were performed to identify differences across samples. Data generated were further confirmed by bulk RNAseq on WT-CSC in which SIX2 or CITED1 were either knocked down or overexpressed, by Spatial Transcriptomics analysis, as well as by in vitro experiments and in vivo WT xenografts.
RESULTS
scRNAseq analysis revealed key differences in gene expression involved in mitochondrial function and dynamics including fission (DRAP1, DNM2, MFF and MEIF2) and fusion (MFN1/2 and OPA1) along with changes in the normal metabolomic and oxidative state. In addition, knocking down SIX2 or CITED1 in WT-NP confirmed their critical role in regulating mitochondrial dynamics and influencing their proliferative capacity and self-renewal. Spatial transcriptomics analysis further confirmed our findings, indicating the presence of mitochondrial and metabolic dysregulation in WT-CSC.
CONCLUSIONS
These data for the first time identify mitochondrial changes as a possible driver in regulating WT formation and progression, in addition to offering potential target for the discover of new WT treatments