IPSC-induced podocytes from a BORS patient with EYA1 gene mutation showed glucocorticoid-resistant and cytoskeletal rearrangement

IPSC-induced podocytes from a BORS patient with EYA1 gene mutation showed glucocorticoid-resistant and cytoskeletal rearrangement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IPSC-induced podocytes from a BORS patient with EYA1 gene mutation showed glucocorticoid-resistant and cytoskeletal rearrangement Guanyu Li, Di Lu, Liujing Xu, Shumin Zhou, Jiayi Zhang, Lijuan Wu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5591319/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The primary cause of branchio-oto-renal syndrome (BORS) is mutations in the EYA1 gene. This study aimed to explore the impact and underlying mechanisms of EYA1 mutations on podocyte injury. We collected clinical and genetic data from a 4-year-old girl diagnosed with BORS and her family. Induced pluripotent stem cells (iPSC) were derived from peripheral blood mononuclear cells of both the patient and healthy individuals, which were differentiated into podocytes in vitro. RNA-seq was used to analyze differentially expressed genes in both groups. Here, the proband, along with his brother and mother, exhibited symptoms of BORS. WES analysis identified a heterozygous splicing variant at the EYA1 locus: c.1050 + 5G > A, inherited from his mother. The proband was initially glucocorticoid-resistant. After tacrolimus treatment, his urine protein/creatinine ratio significantly improved. Compared to healthy individuals, patient-derived podocytes displayed increased motility and pronounced cytoskeletal rearrangement. Dexamethasone was ineffective in ameliorating the pathological damage induced by puromycin aminonucleoside in patient-derived podocytes. RNA-Seq results indicated significant downregulation of cell adhesion molecule signaling pathway expression in patient-derived podocytes compared to healthy controls. In BORS patients with EYA1 mutations, podocytes exhibit cytoskeletal reorganization and enhanced motility in vitro while showing resistance to steroid treatment-indicating a unique damage response that warrants further investigation. Branchio-oto-renal syndrome Induced pluripotent stem cells Podocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The Branchio-oto-renal syndrome (BORS) is a genetic disorder that follows an autosomal-dominant inheritance pattern and was first reported in 1975 by Melnick et al [1]. This syndrome is clinically characterized by hearing loss, ear malformations, and additional symptoms such as branchial cleft abnormalities and renal dysplasia. It is designated Branchio-oto syndrome (BOS) in the absence of renal anomalies. The incidence of BOS/BORS is approximately 1 in 40,000 within the general population and represents about 2% of profound hearing loss cases in children [2]. Significant heterogeneity exists in clinical presentations among individuals or family members, with variable manifestations and severity [3-4]. Deafness serves as the most prominent feature among BOS/BORS patients, primarily manifesting as sensorineural, conductive, or mixed hearing loss alongside defects in external, middle, and inner ear structures as well as branchial fistulae or cysts [5]. BORS is recognized as the most prevalent syndrome type among deaf children [6]. Notably, around 65% of BOR patients exhibit renal developmental abnormalities ranging from mild delays to complete kidney agenesis [7]. The pathogenesis of BORS remains poorly understood. Recent studies have indicated that EYA1, SIX1, and SIX5 are implicated in the development of BORS [8-9]. EYA1 is the most frequently identified pathogenic gene associated with BORS, accounting for nearly 40% of cases [10]. Located on chromosome 8q13.3 in humans, the EYA1 gene comprises 16 exons encoding a peptide of 559 amino acids [8]. It belongs to the EYA family and serves as the human homolog of the Drosophila eya gene. The other three related genes within this family (EYA2, EYA3, and EYA4) also play significant roles in the development of human ear structures, kidneys, and other organs [11]. Currently, there is no evidence supporting mutations in any of these three genes among BOS/BORS patients. EYA1 plays a crucial role in regulating the development of organs in both vertebrates and invertebrates, particularly in the early development of ear, kidney and other organs. It acts as a transcriptional co-activator by forming EYA1-SIX1 transcriptional activation complexes and multiple EYA1 regulatory networks (EYA1-Noth, SNAI2-EYA1-SIX1, DACH-EYASIX, etc.) [12-15]. Knockout of EYA1 in murine embryos have deleterious effects on the early development of multiple organs, including the ear, kidney, and skeleton [12]. In addition, the EYA1 gene plays an important role in the development of pharyngeal organs (thyroid, parathyroid, and thymus), the cardiovascular system, and craniofacial structures [16-17]. Recent studies have demonstrated that Eya1 is expressed in the tail bud nephric duct and metanephric mesenchyme of mice, playing a continuous role in the formation of functional units throughout kidney development [18]. Eya1-Six2 has been shown to interact with the SWI/SNF chromatin remodeling complex and is involved in both inducing and maintaining cell fate during renal unit development, with Brg1 serving as an upstream regulator of EYA1 [19]. However, the functional impact of EYA1 on podocytes remains to be fully elucidated. This study reports a 4-year-old girl with BORS who presented with hearing impairment, a solitary kidney, and primary hormone-resistant nephrotic syndrome. The patient exhibited primary hormone resistance. Induced pluripotent stem cells (iPSCs) derived from peripheral blood were successfully differentiated into podocytes for functional analysis, including assessments of cell motility and cytoskeletal rearrangements. We treated the differentiated iPSC-podocytes from both patients and healthy individuals with glucocorticoids and CNIs, measuring differential gene expression. Studies have shown that patients carrying EYA1 mutations display aberrant expression of cell adhesion signaling molecules, contributing to compromised podocyte function and the onset of proteinuria. This elucidates the molecular basis underlying the patient’s proteinuria. Materials and methods Clinical Pedigree Analysis The study enrolled a child diagnosed with BORS at our center, who underwent a comprehensive family history assessment and detailed physical examination. The degree of hearing loss was evaluated using pure-tone audiometry. Brain computed tomography was performed to assess the morphology of the middle and inner ear structures. Serum creatinine, urea levels, and renal ultrasound were conducted to screen for any renal abnormalities. Written informed consent was obtained from all participants or their guardians prior to participation in the study. The project received approval from the ethics committee (No.247A01). All procedures adhered to the Declaration of Helsinki. Gene Analysis The KAPA library construction kit and NimbleGen custom probe capture kit were employed for DNA extraction, fragmentation, library preparation, targeted enrichment, and sequencing. The resulting FASTQ files obtained from sequencing were aligned to the reference genome (GRCh37) using BWA tools. Subsequently, Samtools and Picard were utilized for format conversion, sequence duplication removal, rearrangement analysis, and base quality correction to generate high-quality alignment results in BAM format. Finally, ANNOVAR was applied for database annotation of mutation sites. Mutations within exons and their adjacent ±10 bp introns (including point mutations as well as deletions and insertions within 20 bp) of relevant genes were analyzed. To validate candidate variants identified through whole-exome sequencing, polymerase chain reaction amplification followed by Sanger sequencing was performed. Induced pluripotent stem cells were guided to differentiate into podocytes. Peripheral blood mononuclear cells from the proband were utilized for human induced pluripotent stem cells (hiPSC) induction (GSPHi001-A) [20]. Healthy individuals matched for age and gender with the proband, who do not exhibit nephropathy, were selected as the control group. The podocyte induction method was adapted from Qian T et al [21]. The iPSCs were expanded in mTeSR Medium to achieve 80%-90% confluency in T25 cm 2 culture flasks. Subsequently, the cells were dissociated using 0.5 mM EDTA at 37℃ for 5 minutes and subcultured onto Matrigel-coated 6-well plates at a density of 2×10 5 cells per well, supplemented with Y-27632 (Abcam, Cat No. Ab120129). The medium was refreshed daily and cultured for four days until a cell density of 80% to 90% was reached. On day zero, mTeSR Medium was replaced with podocyte medium 1 (DMEM/Ham F-12, 1% MEMNEAA, 0.5% GlutaMAX, 0.1 mM β-mercaptoethanol, 6 μM CHIR99021, 100 U/ml penicillin, and 0.1 mg/ml streptomycin) at a volume of 4 ml/well. On day one, the culture medium was changed to another fresh aliquot of podocyte medium at the same volume per well. On day two, podocyte medium 2 (hESFM containing B27 at a concentration of 2%, supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin) was added after removing podocyte medium one; again, at a volume of 4 ml/well. Podocyte medium two was changed daily thereafter. On day six, podocyte medium two was discarded and the cells were digested using ACCUTASE at 37℃ for 2-3 minutes before being resuspended in podocyte medium two and transferred to two Matrigel-coated 6-well plates at a density of 2×10 5 cells/well with a total volume of 2 ml/well supplemented with Y-27632; this media change continued daily as well. By day nine the cells were split again at a ratio of 1:6 while continuing to change podocyte medium two every other day thereafter. Finally, on day sixteen, the process of podocyte differentiation was completed when the cells were switched to maintenance culture medium (DMEM/Ham F-12 containing10% FBS,100 U/ml penicillin, and0 .1 mg/ml streptomycin) at an adjusted volume of 2ml /well. Immunofluorescence Differentiated induced podocytes at day 16 were seeded onto Matrigel-coated slides, fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. To identify the induced podocytes, primary antibodies against Podocin (Proteintech, Cat No. 20384-1-AP, diluted at a ratio of 1:50), WT-1 (R&D Systems, Cat No. AF5729, diluted at a ratio of 1:50), and synaptopodin (Abcam, Cat No. ab224491, diluted at a ratio of 1:50) were incubated overnight at 4℃ Subsequently, secondary antibodies (Thermo Fisher) were applied and incubated for two hours at 4℃. Cytoskeletal staining of the induced podocytes was performed by incubating with anti-FITC-phalloidin (diluted at a ratio of 1:30) for sixty minutes at 4℃. Finally, nuclei were stained using DAPI (Thermo Fisher; diluted to 1:10,000) prior to observation via confocal microscopy (Leica SP8; Germany). Albumin uptake assay Differentiated induced podocytes at day 16 were seeded onto Matrigel-coated slides, and FITC-BSA (2 mg/ml, Sangon Biotech, Cat No. D111074) diluted in DMEM/F12 to a concentration of 0.2 mg/ml was added. The podocytes were incubated at 37℃ in the dark for 1 hour. Cells incubated with the same concentration of albumin at 4℃served as a control. After discarding the treatment solution, the cells were rinsed three times with ice-cold PBS and fixed with 4% paraformaldehyde (PFA). Nuclei were stained with DAPI (1:100) and visualized. Treatment of induced podocytes Podocytes that had differentiated by day 16 were utilized in this study. The podocytes were cultured under adherent conditions for 2 days and divided into three groups: normal control, puromycin aminonucleoside (PAN) treatment, and dexamethasone pretreatment. The PAN treated group was exposed to 100 μg/mL PAN for 48 hours, while the dexamethasone pretreatment group received a one-hour incubation with dexamethasone (1μM) prior to PAN (100μg/mL) treatment (PAN+DEX). Each group consisted of three replicates. Podocytes that had differentiated by day 16 were utilized in this study. The podocytes were cultured under adherent conditions for 2 days and divided into three groups: normal control, puromycin aminonucleoside (PAN) treatment, and dexamethasone pretreatment. The puromycin aminonucleoside-treated group was exposed to 100 μg/mL puromycin aminonucleoside for 48 hours, while the dexamethasone pretreatment group received a one-hour incubation with dexamethasone (1μM) prior to puromycin aminonucleoside (100μg/mL) treatment. Each group consisted of three replicates. Transcriptome sequencing The podocytes were induced by treatment with Trizol, and subsequently total RNA was extracted. To ensure the quality of the RNA, degradation and contamination were monitored using 1.5% agarose gels. Additionally, the purity of the RNA was assessed using a nanometer Spectrophotometer ®. The concentration of the RNA was measured using the Qubit® RNA Detection Kit in the Qubit® 3.0 fluorometer. Furthermore, to evaluate its integrity, an Agilent Bioanalyzer 6000 system equipped with an RNA Nano 100 assay kit was employed. These experimental procedures were conducted by Zhejiang Annoroad Biotechnology Co., LTD., which utilized pruning techniques for bulk processing raw readings and mapping them to the UCSC genome through Hisat10 (v1.2.2) with default parameters settings. The edgeR algorithm was employed for differential analysis, with fold change ≥1.0 and P -value ≤0.05 serving as the thresholds. The differentially expressed genes were subjected to Gene Ontology Enrichment Analysis, with annotation conducted primarily from three perspectives: biological process (BP), cellular component (CC), and molecular function (MF). ClusterProfiler was utilized for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Wound healing assay Podocytes that had differentiated by day 16 were utilized in this study. Induced podocytes were seeded onto Matrigel-coated 12-well plates. After a 6-hour incubation, artificial wounds were generated using pipet tips and the scratched monolayers were washed twice with PBS to remove floating cells. Serum-free medium was added and the cells were cultured in a 37°C, 5% CO2 incubator. Images of each group at 0h, 24h, and 48h were captured using an inverted microscope (Leica DMi8, Germany). Each group consisted of three replicates and the percentage of migrating cells was calculated. Three independent experiments were performed. Quantitative calculation of actin stress fibers To quantitatively analyze changes in the cellular structure of induced podocyte actin [22], different phalloidin staining patterns were categorized into four distinct groups for scoring. Type A: more than 90% of the cell area was occupied by thick cables; type B: at least two thick cables running beneath the nucleus with fine cables filling the remaining cell area; type C: lacking thick cables but exhibiting some presence of thin cables; type D: showing no visible cables in the central region of the cell. Four random visual fields were selected for analysis, encompassing all cells within each field to determine. All cells within these fields were independently counted by two individuals to ascertain the percentage composition of each category. The experiment was replicated four times. Statistics All data are presented as mean ± SD. Statistical analysis was performed using 2-tailed Student’s t test or one-way ANOVA. Statistical analyses were carried out using GraphPad Prism 9.0 software. All difference analyses were considered statistically significant if P was < 0.05. Results Clinical representation of the family The proband was a female patient who first presented in December 2015 at the age of 4 years with bilateral lower limb edema. She exhibited symptoms of massive proteinuria and hypoalbuminemia, leading to a preliminary diagnosis of nephrotic syndrome. Despite receiving a standard dose of steroids (2 mg/kg/d) for 8 weeks, her symptoms did not improve. Further examination revealed a 24-hour urine protein level of 69.1 mg/kg, serum albumin concentration of 27.7 g/L, and cholesterol concentration of 6.3 mmol/L. The diagnosis was corrected as primary steroid resistant nephrotic syndrome. therefore, tacrolimus (0.1 mg/kg/d) was added to her treatment regimen. Blood drug concentrations of tacrolimus were regularly monitored while glucocorticoid use was reduced. Details regarding the treatment and outcomes are shown in Fig. 1 . During follow-up, the proband gradually developed hearing impairment without any visible external ear abnormalities; it was noted that her mother had severe hearing loss and her elder brother had external ear malformations accompanied by hearing impairment, whereas the father did not exhibit any relevant phenotypes. Based on the family history, whole-exome sequencing was performed. Next-generation sequencing identified a heterozygous splicing variant (c.1050+5G>A) at the EYA1 gene locus in the proband (II-2), her brother (II-1), and their mother (I-1), as illustrated in the pedigree diagram ( Fig. 2A ). This mutation was confirmed by Sanger sequencing across all affected family members ( Fig. 2B ). The dbscSNV software predicts that this mutation may impact splicing functionality (PP3). Table 1 summarizes previously reported pathogenic or likely pathogenic splicing mutations of EYA1. Two pathogenic variants have been documented adjacent to the 1050th nucleotide base (c.1050+1G>T, c.1051-2A>G). Considering the clinical phenotype of the proband, it is plausible that this variant is pathogenic. The proband (II-2) and her elder brother (II-1) exhibited bilateral preauricular sinuses during the physical examination ( Fig. 2C ). Their mother (I-2) also presented with a right preauricular sinus ( Fig. 2C ). Bilateral microtia was observed exclusively in II-1. Both II-1 and II-2 were diagnosed with sensorineural hearing loss, as identified by pure-tone audiometry ( Fig. 2D ). The CT scan did not reveal any significant abnormalities in the middle ear of II-1, II-2, or I-2 ( Fig. 2E ). I-1 is the proband’s father, who did not exhibit any relevant clinical phenotypes but passed away unexpectedly due to a traffic accident. The latest family follow-up status is summarized in Table 2 as of February 2024. Induced pluripotent stem cells differentiate into podocytes There has been an increasing number of reports regarding the use of iPSCs for in vitro differentiation into podocytes, providing valuable avenues to investigate the mechanisms underlying rare diseases. In this study, we established a proband-derived iPSC line (GSPHi001-A) from the peripheral blood mononuclear cells of the proband [20]. A diagram illustrating the induction and differentiation of podocyte is provided ( Fig. 3A ). By day 16, successful differentiation of iPSCs into podocytes was achieved, allowing for clear observation of both the nuclei and foot processes associated with these differentiated podocytes ( Fig. 3B ). Immunofluorescence analysis revealed the presence of podocin in both the cytoplasm and nucleus, as well as predominant WT1 expression within the nuclei of differentiated cells at day 16 ( Fig. 3C ). Additionally, staining for synaptopodin demonstrated irregular morphologies among induced cells ( Fig. 3D ). The immunofluorescence analysis confirmed the expression of podocin, WT1, and synaptopodin proteins in all induced podocytes. Transcriptomic data indicated that stem cell-related genes MANOG and SOX2 were expressed at low levels while mature podocyte-specific genes PODXL, PDPN, and SYNPO exhibited elevated expression levels ( Fig. 3E ). To further evaluate the functionality of the induced podocytes, we assessed their capacity to endocytose albumin on day 16 under temperature-dependent conditions. At 37℃ these podocytes displayed fluorescent signals within their vesicles; conversely, at 4℃ they failed to uptake albumin due to inhibited endocytosis ( Fig. 3F ). These results validate that iPSCs are capable of generating physiologically functional podocytes. The podocytes derived from the proband’s induced iPSCs exhibited reduced adhesion properties. We focused on the genes that were differentially expressed between hiPSC-derived podocytes from the proband (P-hiPSC-podocytes) and hiPSC-derived podocytes from healthy individuals (H-hiPSC-podocytes). Following RNA transcriptome sequencing of both P-hiPSC-podocytes and H-hiPSC-podocytes without any intervention. A volcano plot was generated to visualize these differentially expressed genes ( Fig. 4A ). The downregulated and differentially expressed genes are primarily enriched in biological pathways associated with epithelial cell development, cell junction assembly, morphogenesis of the embryonic epithelium, tight junction assembly, and inner ear development ( Fig. 4B ). The aberrant signaling pathways involved in inner ear development exhibit a consistent correlation with the auditory impairment characteristics observed in patients with BORS. KEGG enrichment analysis revealed significant downregulation of differentially expressed genes, particularly within the Hippo signaling pathway, tight junctions, signaling pathways regulating pluripotency of stem cells, and cell adhesion molecules ( Fig. 4C ). The hippo signaling pathway is known to play a crucial role in kidney development [23], and the downregulation of this signaling pathway may have influenced the differentiated development of the solitary kidney in the patient. KEGG enrichment analyses suggest a significant reduction in the tight junction integrity and adhesion properties of the P-hiPSC-podocytes, which may compromise the filtration barrier between podocytes, thereby facilitating the development of proteinuria. The Gene Ontology enrichment analysis of upregulated differentially expressed genes predominantly highlighted pathways associated with actin filament bundling, cytoskeletal motor activity, and microtubule motor function ( Fig. 4D ). These functions facilitate cellular mitosis, endocytosis, exocytosis, and motility. PAX8 is associated with parietal epithelial cells, PAX2 pertains to renal progenitor cells, and both PECAM1 and FLT1 are linked to endothelial cells; notably, the expression levels of these genes are all relatively low. Expression levels of the EYA1 gene were significantly reduced in P-hiPSC-podocytes compared to H-hiPSC-podocytes. However, the genes EYA1, SIX1, and SIX5 closely associated with BORS alongside SIX2, which plays a critical role in maintaining progenitor cells, exhibited only weak expression. ( Fig. 4E ). The effect of PAN treatment on t he podocytes derived from h iPSCs Puromycin aminonucleoside (PAN) is a cytotoxic agent, regularly employed to induce focal segmental glomerulosclerosis in rat models. Furthermore, this compound can be utilized in vitro to provoke podocyte injury and establish nephrotic cell models. In this study, podocytes were treated with PAN for establishing a nephropathy model, and the alterations in gene expression of podocytes following PAN treatment were analyzed. Volcano plots depicting the differentially expressed genes in the H-hiPSC-podocytes group ( Fig. 5A ) and P-hiPSC-podocytes group ( Fig. 5B ) both prior to and following PAN. The KEGG pathway enrichment analysis demonstrated that the differentially expressed genes with upregulation in H-hiPSC-podocytes and P-hiPSC-podocytes subsequent to PAN treatment were preponderantly affiliated with pathways related to formation of neutrophil extracellular traps ( Fig. 5C ). Neutrophil extracellular traps are generated during active cell death, a process that is distinct from both apoptosis and necrosis [24-25]. Following treatment with PAN, the induced podocytes exhibited indications of cell death, however, no substantial evidence of apoptosis was detected. Glucocorticoids do not improve the cell adhesion and cytoskeletal organization genes in podocytes of the BORS patient. The proband exhibited resistance to steroid therapy. To evaluate the similarity between iPSC-derived podocytes and the proband. Gene Set Enrichment (GSEA) was conducted to assess cell adhesion-related gene set scores. GSEA results showed that the score in H-hiPSC-podocytes treated with PAN significantly decreased compared to untreated controls ( Fig. 6A ). However, dexamethasone pretreatment markedly increased this score after PAN exposure ( Fig. 6B ), suggesting it can improve the impaired adhesive capacity of H-hiPSC-podocytes induced by PAN ( Fig. 6C ). Conversely, P-hiPSC-podocytes under similar conditions, dexamethasone pretreatment does not alleviate the reduced adhesive ability of P-hiPSC-podocytes caused by PAN ( Fig. 6D-F ). Disruption of the cytoskeleton significantly affects both the morphology and function of foot processes, compromising the integrity of the glomerular filtration barrier [26]. GSEA revealed significant alterations in cytoskeleton-related gene sets in H-hiPSC-podocytes both prior to and following PAN treatment ( Fig. 7A ), with pronounced changes observed after dexamethasone pretreatment ( Fig. 7B ). Dexamethasone pretreatment mitigated the cytoskeletal damage induced by PAN in H-hiPSC-podocytes ( Fig. 7C ). As anticipated, notable alterations were detected in P-hiPSC-podocytes post-puromycin treatment ( Fig. 7D ); however, dexamethasone pretreatment did not influence the puromycin nephropathy model ( Fig. 7E-F ). At a genetic level, this observation highlights an intriguing phenomenon regarding the proband’s insensitivity to hormonal regulation. Disease phenotype of proband-derived podocytes We aimed to investigate whether P-hiPSC-podocytes exhibit disease-specific characteristics. Podocytes induced on day 16 were used for validation. The previous gene enrichment analysis indicated downregulation of pathways related to podocyte adhesion and an upregulation of pathways linked to cellular motility. To assess the migratory capacity, a wound healing assay was performed. At 24 and 48 hours, the gap width in P-hiPSC-podocytes showed significant reduction ( Fig. 8A ). Quantitative analysis revealed that P-hiPSC-podocytes exhibited a higher cell migration rate ( Fig. 8B ). These findings suggest that podocytes derived from the proband displayed reduced adhesive capacity while demonstrating greater motility compared to those from healthy individuals. Actin dynamics in podocytes are crucial for cellular morphology, motility, and intracellular transport [27-28]. We employed phalloidin staining to analyze actin structures in hiPSC-derived podocytes. Typically, podocyte cytoskeletons exhibit type A or B structures, while type C or D indicates abnormalities ( Fig. 8C ). Untreated H-hiPSC-podocytes and P-hiPSC-podocytes preserved their cellular polarity through a well-organized cytoskeletal architecture. Quantitative analysis revealed that types A and B predominated in both groups ( Fig. 8D ). After PAN treatment, both H-hiPSC-podocytes and P-hiPSC-podocytes predominantly exhibited types C and D of the cytoskeleton, accompanied by a clear disruption in stress fiber polarity ( Fig. 8E ). Prior to PAN exposure, dexamethasone pretreatment led to a notable reduction in the proportions of type C and D within H-hiPSC-podocytes. Importantly, dexamethasone failed to restore aberrant skeletons in P-hiPSC-podocytes, underscoring hormone resistance. In H-hiPSC-podocytes, type A did not return to baseline levels observed without treatment, suggesting that dexamethasone was insufficient to fully restore damage inflicted on the podocyte cytoskeleton by PAN ( Fig. 8F ). Podocytes from the proband exhibited clinical characteristics consistent with steroid-resistant nephropathy. Discussion It is well-established that patients with BORS frequently present with renal malformations; however, there is a lack of studies investigating the characteristics and mechanisms of renal injury in these individuals. Moreover, despite being the primary pathogenic gene associated with BORS, reports on the impact of EYA1 on podocyte function are limited. In this study, we examined renal injury features in a family affected by BORS carrying an EYA1 mutation and explored potential cellular injuries in vitro using induced pluripotent stem cell-derived podocytes from the patient. Our findings provide novel insights into iPSC-derived podocytes from BORS patients harboring EYA1 mutations, which exhibit enhanced motility compared to those from healthy subjects. Furthermore, we observed significant glucocorticoid resistance in podocytes carrying EYA1 mutations. Collectively, our results demonstrate that the EYA1 mutation directly influences podocyte function by increasing cell motility and inducing cytoskeletal rearrangement unresponsive to glucocorticoid intervention. RNA-seq data revealed differential expression of several adhesion-related genes in podocytes derived from patients with EYA1 mutations. The findings elucidate potential effects and signaling targets of EYA1 on podocyte function, offering valuable directions for future research into the mechanistic underpinnings of kidney injury associated with EYA1. Current reports indicate that the predominant clinical phenotypes in individuals with BORS and EYA1 gene mutations include deafness (98.5%), preauricular fovea (83.6%), branchial arch abnormalities (68.5%), renal abnormalities (38.2%), and external ear anomalies (31.5%) [29]. Investigations into EYA1's role in kidney development have shown that conditional inactivation of Eya1 leads to loss of Six2 expression and premature epithelialization of renal progenitor cells, underscoring the essential coordination among Eya1, Six2, and Myc for nephron precursor proliferation and nephrogenesis [30]. Although infrequently reported, proteinuria has been observed to varying degrees in three recent BORS patients with EYA1 gene mutations [31]. This finding further underscores the necessity of enhancing proteinuria detection, including microalbuminuria assessment, for early identification of kidney damage among BORS patients. In this family, despite the proband’s mother and brother having deafness and preauricular fistula, no structural kidney abnormalities were identified. However, the proband exhibited nephrotic-level proteinuria and a solitary kidney. Whole exome sequencing revealed that the EYA1 gene mutation was inherited from the mother as a heterozygous splicing variant. These findings demonstrate significant heterogeneity in how mutations at the same site of EYA1 can influence renal phenotype within one family. Over 8 years of follow-up, both the patient's mother and brother gradually developed proteinuria while their glomerular filtration rates progressively declined. Our results underscore the necessity of closely monitoring renal function among patients with BORS and conducting timely screenings for kidney damage. Currently, the underlying mechanism by which the EYA1 gene contributes to proteinuria and renal function decline remains unclear. The involvement of EYA1 in glomerular podocyte injury has not been previously reported. In this study, we aimed to induce in vitro differentiation of podocytes using established iPSCs derived from peripheral blood mononuclear cells obtained from the patient [20]. Induced podocytes at day 16, we observed the expression of key podocyte proteins including podocin, WT-1, and Synaptopodin. Additionally, the induced podocytes exhibited multiple protrusions with tight junctions between cells and aligned cytoskeletal fibers demonstrating significant polarity alignment, consistent with previous findings by Haynes JM et al. [32]. It is widely recognized that the pathogenesis of podocyte injury primarily involves extensive fusion of foot processes. In vitro, this fusion leads to increased cell motility and cytoskeletal rearrangement. This study aimed to investigate whether patient-derived podocytes could recapitulate clinical manifestations in vitro. Our findings demonstrated that iPSC-derived podocytes from patients with EYA1 mutations exhibited typical morphological characteristics and specific protein expression associated with mature podocytes while also displaying enhanced cell motility and cytoskeletal rearrangement. These insights suggest that EYA1 mutations may impair the proper differentiation of nephron progenitors into functional podocytes and disrupt normal cellular functions. Notably, the observed functional abnormalities in cultured iPSC-derived podocytes corresponded well with the clinical phenotype of massive proteinuria, thereby providing an ideal cellular model for future investigations into the molecular mechanisms underlying podocyte injury. Furthermore, these findings underscore the potential utility of drug screening using renal organoid cultures as a promising avenue for therapeutic development. Unfortunately, due to the presence of a solitary kidney in this patient, renal pathology data were unavailable. However, a renal biopsy on a child with EYA1 gene mutation BORS by Lin et al. revealed partial fusion of podocyte foot processes [31], consistent with our previously reported in vitro findings. Therefore, it is crucial to emphasize the importance of renal biopsies in children with proteinuria and EYA1 gene mutations to enhance our understanding of the pathological mechanisms underlying renal injury associated with this condition. The patient in this study exhibited clinical resistance to steroids, and we observed that the iPSC-derived podocytes from this patient demonstrated significant resistance to dexamethasone following puromycin aminonucleoside treatment. This finding is consistent with the clinical characteristics of steroid-resistant nephropathy. A critical question raised by this study is whether EYA1, a key transcriptional regulator, plays a role in hormone resistance mechanisms. However, our RNA-Seq data did not provide definitive evidence for regulatory relationships between EYA1 and known pathogenic genes associated with steroid-resistant nephropathy. Our results indicate that mutant podocytes exhibiting abnormal expression of adhesion and cytoskeletal signaling pathway genes persist even after glucocorticoid treatment. Future studies should focus on elucidating the potential role of EYA1 mutations in steroid-resistant patients to further clarify the underlying mechanisms. In summary, this study was directed towards probing into the potential ramifications and signaling targets of EYA1 gene mutations on podocyte injury. Our discoveries illuminate the underlying mechanism of proteinuria in patients afflicted with EYA1 gene mutations, furnishing indispensable data for clinical practice, especially for non-nephrologists, to optimize kidney damage screening in individuals with BORS. Furthermore, it is of paramount importance to explore the participation of the EYA1 gene in steroid-resistant nephropathy and its underlying mechanisms. This in vitro disease model employing mature hiPSC-derived podocytes holds promise for promoting the development and application of novel therapeutics. Abbreviations BORS branchio-oto-renal syndrome BOS Branchio-oto syndrome hiPSC Human induced pluripotent stem cells H-hiPSC-podocytes hiPSC-derived podocytes from the healthy individual P-hiPSC-podocytes hiPSC-derived podocytes from the propositus GSEA Gene Set Enrichment Analysis GO Gene Ontology Enrichment Analysis KEGG Kyoto Encyclopedia of Genes and Genomes PAN puromycin aminonucleoside DEX dexamethasone PAN+DEX pretreated with dexamethasone Declarations Acknowledgements We extend our sincere gratitude to the proband and her family for their invaluable support of the publication. Author contributions GL, DL, LX, ML and XG designed and supervised research. GL, DL, LX, SZ JZ and LW performed the experiments. GL, DL, LX, LS and LW analyzed data; XL and ZM supervised the project. GL and ML wrote the draft of the manuscript. GL and XG proofread the manuscript. All authors read and approved the final manuscript. Funding This research was supported by grants from Science and Technology Program of Guangzhou (Grant No. 202102010222). Data availability The source data and any additional information in this paper will be shared upon reasonable request. Consent for publication All authors read and approved the submission and final publication. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Guangzhou Women and Children’s Medical Center (No.247A01). Informed consent in written form was acquired from the patients as well as family members. References Melnick M et al (1975) Autosomal dominant branchiootorenal dysplasia. Birth defects original article series, 11(5), 121–128. 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The 1050th nucleotide mutation in the coding region of the EYA1 gene retrieved from ClinVar is determined to be pathogenic . Name Condition(s) Clinical significance (Last reviewed) Review status NM_000503.6(EYA1): c.1051-2A>G Melnick-Fraser syndrome Pathogenic (Apr 15, 2022) criteria provided, single submitter NM_000503.6(EYA1): c.1050+1G>T Branchiootic syndrome 1, Melnick-Fraser syndrome Pathogenic (Aug 27, 2021) criteria provided, multiple submitters, no conflicts Table 2. Clinical data of the pedigree. Patient Gender Age Urinary albumin/Urine creatinine (mg/μmol) Serum creatinine (μmol/L) Blood urea nitrogen (mmol/L) eGFR (ml/min/1.73m 2 ) II-2 female 12Y 14.27 118 8.56 46 II-1 male 25Y 0.95 96.10 5.25 94.1 I-2 female 46Y 43.04 81 7.61 75 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 05 Feb, 2025 Reviewers agreed at journal 23 Dec, 2024 Reviewers invited by journal 18 Dec, 2024 Editor assigned by journal 09 Dec, 2024 First submitted to journal 05 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5591319","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391967978,"identity":"1aa6e21a-e507-4e23-bc1d-084a9510b0b8","order_by":0,"name":"Guanyu Li","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Guanyu","middleName":"","lastName":"Li","suffix":""},{"id":391967979,"identity":"7efc49b7-30f4-4650-9e51-cc450e4ddc4a","order_by":1,"name":"Di Lu","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical 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06:43:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5591319/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5591319/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72372452,"identity":"ec48773e-670d-4d08-9b0d-0702b069caea","added_by":"auto","created_at":"2024-12-26 07:54:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":448541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration depicting the treatment timeline of the proband.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/18d8e0e7d69705f262a0a932.png"},{"id":72372667,"identity":"0b7db7b8-bbb0-4cc5-b6c4-5d1426b7f877","added_by":"auto","created_at":"2024-12-26 08:02:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6293905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phenotype of the members within the family\u003c/strong\u003e. \u003cstrong\u003e(A) \u003c/strong\u003eThe Sanger sequencing map of the family revealed that I-1 exhibited the wild type, while I-2, II-1, and II-2 displayed mutant alleles (shaded portion), with a c.1050+5G\u0026gt;A mutation in the EYA1 gene. (\u003cstrong\u003eB\u003c/strong\u003e) Genealogical diagram. (\u003cstrong\u003eC\u003c/strong\u003e)Medical photographs show branchial fistulas and preauricular fistulas in II-2, II-1, and I-2 (indicated by arrows), as well as microtia in II-1. (\u003cstrong\u003eD\u003c/strong\u003e)Pure tone audiometry for II-2 and II-1. (\u003cstrong\u003eE\u003c/strong\u003e) Brain CT scans were performed on II-2, II-1, and I-2.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/7260ba4e3471340c1397b132.png"},{"id":72372669,"identity":"b95cc257-19fb-4873-98fa-d933341c3c87","added_by":"auto","created_at":"2024-12-26 08:02:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15918063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe hiPSC were induced to undergo differentiation into podocytes in an in vitro setting\u003c/strong\u003e. \u003cstrong\u003e(A) \u003c/strong\u003eSchematic illustration of the differentiation of hiPSCs into podocytes. \u003cstrong\u003e(B) \u003c/strong\u003eBright-field imaging was used to observe the differentiation of hiPSCs into podocytes on days 0, 2, 13 and 16. Podocytes induced from hiPSCs of the proband (P-hiPSC-podocytes) and those from a healthy individual (H-hiPSC-podocytes). Double immunofluorescence staining was performed for Podocin (red) and WT1 (green) (\u003cstrong\u003eC\u003c/strong\u003e), as well as for WT1 (red) and Synaptopodin (green) (\u003cstrong\u003eD\u003c/strong\u003e), Scale bar:25μm. (\u003cstrong\u003eE\u003c/strong\u003e) Expression levels of NANOG, SOX2, and PODXL genes in the transcriptome. (\u003cstrong\u003eF\u003c/strong\u003e)FITC-labelled albumin is shown in green on a merged DAPI image. 4 °C was used as a control to prevent endocytosis. Scale bars, 25 µm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/39f5de64ef67bdc45eb7b99e.png"},{"id":72372457,"identity":"380a7a23-92a7-4079-ab5b-f0446df7374a","added_by":"auto","created_at":"2024-12-26 07:54:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3492362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of hiPSC-derived podocytes\u003c/strong\u003e. Podocytes induced from hiPSCs of the proband (P-hiPSC-podocytes) and those from a healthy individual (H-hiPSC-podocytes). The volcano plot illustrates the differential gene expression between untreated P-hiPSC-podocytes and H-hiPSC-podocytes (\u003cstrong\u003eA\u003c/strong\u003e), highlighting down-regulated genes identified through GO enrichment (\u003cstrong\u003eB\u003c/strong\u003e) and KEGG enrichment analysis(\u003cstrong\u003eC\u003c/strong\u003e), as well as up-regulated genes associated with GO enriched pathways(\u003cstrong\u003eD\u003c/strong\u003e). Biological process (BP), cellular component (CC), and molecular function (MF). Heatmap depicting the expression of genes (\u003cstrong\u003eE\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/33fc65970a949a9b7d64e4c4.png"},{"id":72372454,"identity":"55e27b61-fff7-4621-a7a5-07c2da81a9da","added_by":"auto","created_at":"2024-12-26 07:54:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2214023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of podocytes subjected to puromycin aminonucleoside treatment. \u003c/strong\u003eVolcano plots of H-hiPSC-podocytes prior to and following puromycin aminonucleoside treatment(\u003cstrong\u003eA\u003c/strong\u003e). Volcano plots of P-hiPSC-podocytes, created before and after puromycin treatment(\u003cstrong\u003eB\u003c/strong\u003e). KEGG analysis of upregulated differentially expressed genes in podocytes from groups H-hiPSC-podocytes and P-hiPSC-podocytes following puromycin aminonucleoside treatment. H_CON: Untreated H-hiPSC-podocytes. H_PAN: H-hiPSC-podocytes were treated with puromycin. P_CON: Untreated P-hiPSC-podocytes.\u003cstrong\u003e \u003c/strong\u003eP_PAN: P-hiPSC-podocytes were treated with puromycin.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/a2b9bec4460b9193893730e0.png"},{"id":72372460,"identity":"52a58c70-46ef-4b1d-8e44-dfc3a1525e91","added_by":"auto","created_at":"2024-12-26 07:54:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6616554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene Set Enrichment Analysis of genes implicated in cellular adhesion\u003c/strong\u003e. GSEA was conducted on H-hiPSC-podocytes treated with puromycin aminonucleoside (PAN) and pretreated with dexamethasone (PAN+DEX), resulting in the expression scores of adhesion related genes \u003cstrong\u003e(A-C)\u003c/strong\u003e. H_CON: Untreated H-hiPSC-podocytes. H_PAN: H-hiPSC-podocytes were treated with puromycin. H_PAN+DEX: H-hiPSC-podocytes were pretreated with dexamethasone prior to puromycin aminonucleoside treatment. GSEA was conducted on P-hiPSC-podocytes treated with PAN and PAN+DEX, resulting in the expression scores of adhesion related genes \u003cstrong\u003e(D-F)\u003c/strong\u003e. P_CON: Untreated P-hiPSC-podocytes. P_PAN: P-hiPSC-podocytes were treated with puromycin. P_PAN+DEX: P-hiPSC-podocytes were pretreated with dexamethasone prior to puromycin aminonucleoside treatment.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/d6d05a0edf545d179d20007e.png"},{"id":72372462,"identity":"82ca193d-a205-48a5-8b72-c4f77ea92529","added_by":"auto","created_at":"2024-12-26 07:54:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5315884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene Set Enrichment Analysis of genes implicated in the cytoskeleton.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGSEA was performed on H-hiPSC podocytes treated with puromycin aminonucleoside (PAN) and pretreated with dexamethasone (PAN+DEX), yielding expression scores for adhesion-related genes (\u003cstrong\u003eA-C\u003c/strong\u003e). H_CON: Untreated H-hiPSC-podocytes. H_PAN: H-hiPSC-podocytes were treated with puromycin. H_PAN+DEX: H-hiPSC-podocytes were pretreated with dexamethasone prior to puromycin aminonucleoside treatment. GSEA was performed on P-hiPSC podocytes treated with PAN and PAN+DEX, yielding expression scores for adhesion-related genes (\u003cstrong\u003eD-F\u003c/strong\u003e). P_CON: Untreated P-hiPSC-podocytes. P_PAN: P-hiPSC-podocytes were treated with puromycin. P_PAN+DEX: P-hiPSC-podocytes were pretreated with dexamethasone prior to puromycin aminonucleoside treatment.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/835ac0fcc9f30b57e2cbf141.png"},{"id":72372474,"identity":"aeaf4208-945a-40e5-8f3e-f73a8dd4b36f","added_by":"auto","created_at":"2024-12-26 07:54:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3178704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of the induced podocyte phenotype\u003c/strong\u003e. Scratch healing assay was performed on P-hiPSC-podocytes and H-hiPSC-podocytes (\u003cstrong\u003eA\u003c/strong\u003e), followed by quantitative analysis of the proportion of cells with scratch healing (\u003cstrong\u003eB\u003c/strong\u003e), n=3, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, scale bar:100μm. Type A: more than 90% of the cell area was occupied by thick cables; type B: characterized by at least two thick cables running beneath the nucleus with fine cables filling the remaining cell area; type C: lacking thick cables but exhibiting some presence of thin cables; type D: showing no visible cables in the central region of the cell. Four distinct cytoskeletal patterns were observed in P-hiPSC-podocytes stained with phalloidin(\u003cstrong\u003eC\u003c/strong\u003e), scale bar:25μm. Quantitative analysis was performed on the phalloidin staining of untreated P-hiPSC-podocytes and H-hiPSC-podocytes(\u003cstrong\u003eD\u003c/strong\u003e), as well as those treated with puromycin aminonucleoside (PAN) for 48 hours(\u003cstrong\u003eE\u003c/strong\u003e) or pretreated with dexamethasone prior to puromycin aminonucleoside treatment (PAN+DEX) for 48 hours (\u003cstrong\u003eF\u003c/strong\u003e). n= 4，**\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/859f69665fd658cdd4cef4c9.png"},{"id":72374392,"identity":"9f4e16d5-ce57-49b3-a986-430ef8f94b6c","added_by":"auto","created_at":"2024-12-26 08:11:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40868280,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5591319/v1/a366c40f-eaaf-4eeb-b855-106761a1f682.pdf"}],"financialInterests":"","formattedTitle":"IPSC-induced podocytes from a BORS patient with EYA1 gene mutation showed glucocorticoid-resistant and cytoskeletal rearrangement","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Branchio-oto-renal syndrome (BORS) is a genetic disorder that follows an autosomal-dominant inheritance pattern and was first reported in 1975 by Melnick et al [1]. This syndrome is clinically characterized by hearing loss, ear malformations, and additional symptoms such as branchial cleft abnormalities and renal dysplasia. It is designated Branchio-oto syndrome (BOS) in the absence of renal anomalies. The incidence of BOS/BORS is approximately 1 in 40,000 within the general population and represents about 2% of profound hearing loss cases in children [2]. Significant heterogeneity exists in clinical presentations among individuals or family members, with variable manifestations and severity [3-4]. Deafness serves as the most prominent feature among BOS/BORS patients, primarily manifesting as sensorineural, conductive, or mixed hearing loss alongside defects in external, middle, and inner ear structures as well as branchial fistulae or cysts [5]. BORS is recognized as the most prevalent syndrome type among deaf children [6]. Notably, around 65% of BOR patients exhibit renal developmental abnormalities ranging from mild delays to complete kidney agenesis [7].\u003c/p\u003e\n\u003cp\u003eThe pathogenesis of BORS remains poorly understood. Recent studies have indicated that EYA1, SIX1, and SIX5 are implicated in the development of BORS [8-9]. EYA1 is the most frequently identified pathogenic gene associated with BORS, accounting for nearly 40% of cases [10]. Located on chromosome 8q13.3 in humans, the EYA1 gene comprises 16 exons encoding a peptide of 559 amino acids [8]. It belongs to the EYA family and serves as the human homolog of the Drosophila eya gene. The other three related genes within this family (EYA2, EYA3, and EYA4) also play significant roles in the development of human ear structures, kidneys, and other organs [11]. Currently, there is no evidence supporting mutations in any of these three genes among BOS/BORS patients.\u003c/p\u003e\n\u003cp\u003eEYA1 plays a crucial role in regulating the development of organs in both vertebrates and invertebrates, particularly in the early development of ear, kidney and other organs. It acts as a transcriptional co-activator by forming EYA1-SIX1 transcriptional activation complexes and multiple EYA1 regulatory networks (EYA1-Noth, SNAI2-EYA1-SIX1, DACH-EYASIX, etc.) [12-15]. Knockout of EYA1 in murine embryos have deleterious effects on the early development of multiple organs, including the ear, kidney, and skeleton [12]. In addition, the EYA1 gene plays an important role in the development of pharyngeal organs (thyroid, parathyroid, and thymus), the cardiovascular system, and craniofacial structures [16-17].\u003c/p\u003e\n\u003cp\u003eRecent studies have demonstrated that Eya1 is expressed in the tail bud nephric duct and metanephric mesenchyme of mice, playing a continuous role in the formation of functional units throughout kidney development [18]. Eya1-Six2 has been shown to interact with the SWI/SNF chromatin remodeling complex and is involved in both inducing and maintaining cell fate during renal unit development, with Brg1 serving as an upstream regulator of EYA1 [19]. However, the functional impact of EYA1 on podocytes remains to be fully elucidated.\u003c/p\u003e\n\u003cp\u003eThis study reports a 4-year-old girl with BORS who presented with hearing impairment, a solitary kidney, and primary hormone-resistant nephrotic syndrome. The patient exhibited primary hormone resistance. Induced pluripotent stem cells (iPSCs) derived from peripheral blood were successfully differentiated into podocytes for functional analysis, including assessments of cell motility and cytoskeletal rearrangements. We treated the differentiated iPSC-podocytes from both patients and healthy individuals with glucocorticoids and CNIs, measuring differential gene expression. Studies have shown that patients carrying EYA1 mutations display aberrant expression of cell adhesion signaling molecules, contributing to compromised podocyte function and the onset of proteinuria. This elucidates the molecular basis underlying the patient\u0026rsquo;s proteinuria.\u003c/p\u003e\n"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eClinical Pedigree Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study enrolled a child diagnosed with BORS at our center, who underwent a comprehensive family history assessment and detailed physical examination. The degree of hearing loss was evaluated using pure-tone audiometry. Brain computed tomography was performed to assess the morphology of the middle and inner ear structures. Serum creatinine, urea levels, and renal ultrasound were conducted to screen for any renal abnormalities. Written informed consent was obtained from all participants or their guardians prior to participation in the study. The project received approval from the ethics committee (No.247A01). All procedures adhered to the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe KAPA library construction kit and NimbleGen custom probe capture kit were employed for DNA extraction, fragmentation, library preparation, targeted enrichment, and sequencing. The resulting FASTQ files obtained from sequencing were aligned to the reference genome (GRCh37) using BWA tools. Subsequently, Samtools and Picard were utilized for format conversion, sequence duplication removal, rearrangement analysis, and base quality correction to generate high-quality alignment results in BAM format. Finally, ANNOVAR was applied for database annotation of mutation sites. Mutations within exons and their adjacent \u0026plusmn;10 bp introns (including point mutations as well as deletions and insertions within 20 bp) of relevant genes were analyzed. To validate candidate variants identified through whole-exome sequencing, polymerase chain reaction amplification followed by Sanger sequencing was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduced pluripotent stem cells were guided to differentiate into podocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood mononuclear cells from the proband were utilized for human induced pluripotent stem cells (hiPSC) induction (GSPHi001-A) [20]. Healthy individuals matched for age and gender with the proband, who do not exhibit nephropathy, were selected as the control group. The podocyte induction method was adapted from Qian T et al\u0026nbsp;[21]. The iPSCs were expanded in mTeSR Medium to achieve 80%-90% confluency in T25 cm\u003csup\u003e2\u003c/sup\u003e culture flasks. Subsequently, the cells were dissociated using 0.5 mM EDTA at 37℃ for 5 minutes and subcultured onto Matrigel-coated 6-well plates at a density of 2\u0026times;10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells per well, supplemented with Y-27632 (Abcam, Cat No. Ab120129). The medium was refreshed daily and cultured for four days until a cell density of 80% to 90% was reached. On day zero, mTeSR Medium was replaced with podocyte medium 1 (DMEM/Ham F-12, 1% MEMNEAA, 0.5% GlutaMAX, 0.1 mM \u0026beta;-mercaptoethanol, 6 \u0026mu;M CHIR99021, 100 U/ml penicillin, and 0.1 mg/ml streptomycin) at a volume of 4 ml/well. On day one, the culture medium was changed to another fresh aliquot of podocyte medium at the same volume per well. On day two, podocyte medium 2 (hESFM containing B27 at a concentration of 2%, supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin) was added after removing podocyte medium one; again, at a volume of 4 ml/well. Podocyte medium two was changed daily thereafter. On day six, podocyte medium two was discarded and the cells were digested using ACCUTASE at 37℃ for 2-3 minutes before being resuspended in podocyte medium two and transferred to two Matrigel-coated 6-well plates at a density of 2\u0026times;10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells/well with a total volume of 2 ml/well supplemented with Y-27632; this media change continued daily as well. By day nine the cells were split again at a ratio of 1:6 while continuing to change podocyte medium two every other day thereafter. Finally, on day sixteen, the process of podocyte differentiation was completed when the cells were switched to maintenance culture medium (DMEM/Ham F-12 containing10% FBS,100 U/ml penicillin, and0 .1 mg/ml streptomycin) at an adjusted volume of 2ml /well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferentiated induced podocytes at day 16 were seeded onto Matrigel-coated slides, fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. To identify the induced podocytes, primary antibodies against Podocin (Proteintech, Cat No. 20384-1-AP, diluted at a ratio of 1:50), WT-1 (R\u0026amp;D Systems, Cat No. AF5729, diluted at a ratio of 1:50), and synaptopodin (Abcam, Cat No. ab224491, diluted at a ratio of 1:50) were incubated overnight at 4℃ Subsequently, secondary antibodies (Thermo Fisher) were applied and incubated for two hours at 4℃. Cytoskeletal staining of the induced podocytes was performed by incubating with anti-FITC-phalloidin (diluted at a ratio of 1:30) for sixty minutes at 4℃. Finally, nuclei were stained using DAPI (Thermo Fisher; diluted to 1:10,000) prior to observation via confocal microscopy (Leica SP8; Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlbumin uptake assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferentiated induced podocytes at day 16 were seeded onto Matrigel-coated slides, and FITC-BSA (2 mg/ml, Sangon Biotech, Cat No. D111074) diluted in DMEM/F12 to a concentration of 0.2 mg/ml was added. The podocytes were incubated at 37℃ in the dark for 1 hour. Cells incubated with the same concentration of albumin at 4℃served as a control. After discarding the treatment solution, the cells were rinsed three times with ice-cold PBS and fixed with 4% paraformaldehyde (PFA). Nuclei were stained with DAPI (1:100) and visualized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of induced podocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePodocytes that had differentiated by day 16 were utilized in this study. The podocytes were cultured under adherent conditions for 2 days and divided into three groups: normal control, puromycin aminonucleoside (PAN) treatment, and dexamethasone pretreatment. The PAN treated group was exposed to 100 \u0026mu;g/mL PAN for 48 hours, while the dexamethasone pretreatment group received a one-hour incubation with dexamethasone (1\u0026mu;M) prior to PAN (100\u0026mu;g/mL) treatment (PAN+DEX). Each group consisted of three replicates.\u003c/p\u003e\n\u003cp\u003ePodocytes that had differentiated by day 16 were utilized in this study. The podocytes were cultured under adherent conditions for 2 days and divided into three groups: normal control, puromycin aminonucleoside (PAN) treatment, and dexamethasone pretreatment. The puromycin aminonucleoside-treated group was exposed to 100 \u0026mu;g/mL puromycin aminonucleoside for 48 hours, while the dexamethasone pretreatment group received a one-hour incubation with dexamethasone (1\u0026mu;M) prior to puromycin aminonucleoside (100\u0026mu;g/mL) treatment. Each group consisted of three replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe podocytes were induced by treatment with Trizol, and subsequently total RNA was extracted. To ensure the quality of the RNA, degradation and contamination were monitored using 1.5% agarose gels. Additionally, the purity of the RNA was assessed using a nanometer Spectrophotometer \u0026reg;. The concentration of the RNA was measured using the Qubit\u0026reg; RNA Detection Kit in the Qubit\u0026reg; 3.0 fluorometer. Furthermore, to evaluate its integrity, an Agilent Bioanalyzer 6000 system equipped with an RNA Nano 100 assay kit was employed. These experimental procedures were conducted by Zhejiang Annoroad Biotechnology Co., LTD., which utilized pruning techniques for bulk processing raw readings and mapping them to the UCSC genome through Hisat10 (v1.2.2) with default parameters settings. The edgeR algorithm was employed for differential analysis, with fold change \u0026ge;1.0 and \u003cem\u003eP\u003c/em\u003e-value \u0026le;0.05 serving as the thresholds. The differentially expressed genes were subjected to Gene Ontology Enrichment Analysis, with annotation conducted primarily from three perspectives: biological process (BP), cellular component (CC), and molecular function (MF). ClusterProfiler was utilized for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePodocytes that had differentiated by day 16 were utilized in this study. Induced podocytes were seeded onto Matrigel-coated 12-well plates. After a 6-hour incubation, artificial wounds were generated using pipet tips and the scratched monolayers were washed twice with PBS to remove floating cells. Serum-free medium was added and the cells were cultured in a 37\u0026deg;C, 5% CO2 incubator. Images of each group at 0h, 24h, and 48h were captured using an inverted microscope (Leica DMi8, Germany). Each group consisted of three replicates and the percentage of migrating cells was calculated. Three independent experiments were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative calculation of actin stress fibers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantitatively analyze changes in the cellular structure of induced podocyte actin [22], different phalloidin staining patterns were categorized into four distinct groups for scoring. Type A: more than 90% of the cell area was occupied by thick cables; type B: at least two thick cables running beneath the nucleus with fine cables filling the remaining cell area; type C: lacking thick cables but exhibiting some presence of thin cables; type D: showing no visible cables in the central region of the cell. Four random visual fields were selected for analysis, encompassing all cells within each field to determine. All cells within these fields were independently counted by two individuals to ascertain the percentage composition of each category. The experiment was replicated four times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean \u0026plusmn; SD. Statistical analysis was performed using 2-tailed Student\u0026rsquo;s\u003cem\u003e\u0026nbsp;t\u0026nbsp;\u003c/em\u003etest or one-way ANOVA. Statistical analyses were carried out using GraphPad Prism 9.0 software. All difference analyses were considered statistically significant if \u003cem\u003eP\u0026nbsp;\u003c/em\u003ewas \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical representation of the family\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proband was a female patient who first presented in December 2015 at the age of 4 years with bilateral lower limb edema. She exhibited symptoms of massive proteinuria and hypoalbuminemia, leading to a preliminary diagnosis of nephrotic syndrome. Despite receiving a standard dose of steroids (2 mg/kg/d) for 8 weeks, her symptoms did not improve. Further examination revealed a 24-hour urine protein level of 69.1 mg/kg, serum albumin concentration of 27.7 g/L, and cholesterol concentration of 6.3 mmol/L. The diagnosis was corrected as primary steroid resistant nephrotic syndrome. therefore, tacrolimus (0.1 mg/kg/d) was added to her treatment regimen. Blood drug concentrations of tacrolimus were regularly monitored while glucocorticoid use was reduced. Details regarding the treatment and outcomes are shown in \u003cstrong\u003eFig. 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eDuring follow-up, the proband gradually developed hearing impairment without any visible external ear abnormalities; it was noted that her mother had severe hearing loss and her elder brother had external ear malformations accompanied by hearing impairment, whereas the father did not exhibit any relevant phenotypes. Based on the family history, whole-exome sequencing was performed.\u003c/p\u003e\n\u003cp\u003eNext-generation sequencing identified a heterozygous splicing variant (c.1050+5G\u0026gt;A) at the EYA1 gene locus in the proband (II-2), her brother (II-1), and their mother (I-1), as illustrated in the pedigree diagram (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). This mutation was confirmed by Sanger sequencing across all affected family members (\u003cstrong\u003eFig. 2B\u003c/strong\u003e). The dbscSNV software predicts that this mutation may impact splicing functionality (PP3). \u003cstrong\u003eTable 1\u003c/strong\u003e summarizes previously reported pathogenic or likely pathogenic splicing mutations of EYA1. Two pathogenic variants have been documented adjacent to the 1050th nucleotide base (c.1050+1G\u0026gt;T, c.1051-2A\u0026gt;G). Considering the clinical phenotype of the proband, it is plausible that this variant is pathogenic.\u003c/p\u003e\n\u003cp\u003eThe proband (II-2) and her elder brother (II-1) exhibited bilateral preauricular sinuses during the physical examination (\u003cstrong\u003eFig. 2C\u003c/strong\u003e). Their mother (I-2) also presented with a right preauricular sinus (\u003cstrong\u003eFig. 2C\u003c/strong\u003e). Bilateral microtia was observed exclusively in II-1. Both II-1 and II-2 were diagnosed with sensorineural hearing loss, as identified by pure-tone audiometry (\u003cstrong\u003eFig. 2D\u003c/strong\u003e). The CT scan did not reveal any significant abnormalities in the middle ear of II-1, II-2, or I-2 (\u003cstrong\u003eFig. 2E\u003c/strong\u003e). I-1 is the proband\u0026rsquo;s father, who did not exhibit any relevant clinical phenotypes but passed away unexpectedly due to a traffic accident. The latest family follow-up status is summarized in \u003cstrong\u003eTable 2\u003c/strong\u003e as of February 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduced pluripotent stem cells differentiate into podocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere has been an increasing number of reports regarding the use of iPSCs for in vitro differentiation into podocytes, providing valuable avenues to investigate the mechanisms underlying rare diseases. In this study, we established a proband-derived iPSC line (GSPHi001-A) from the peripheral blood mononuclear cells of the proband [20]. A diagram illustrating the induction and differentiation of podocyte is provided (\u003cstrong\u003eFig. 3A\u003c/strong\u003e). By day 16, successful differentiation of iPSCs into podocytes was achieved, allowing for clear observation of both the nuclei and foot processes associated with these differentiated podocytes (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). Immunofluorescence analysis revealed the presence of podocin in both the cytoplasm and nucleus, as well as predominant WT1 expression within the nuclei of differentiated cells at day 16 (\u003cstrong\u003eFig. 3C\u003c/strong\u003e). Additionally, staining for synaptopodin demonstrated irregular morphologies among induced cells (\u003cstrong\u003eFig. 3D\u003c/strong\u003e). The immunofluorescence analysis confirmed the expression of podocin, WT1, and synaptopodin proteins in all induced podocytes. Transcriptomic data indicated that stem cell-related genes MANOG and SOX2 were expressed at low levels while mature podocyte-specific genes PODXL, PDPN, and SYNPO exhibited elevated expression levels (\u003cstrong\u003eFig. 3E\u003c/strong\u003e). To further evaluate the functionality of the induced podocytes, we assessed their capacity to endocytose albumin on day 16 under temperature-dependent conditions. At 37℃ these podocytes displayed fluorescent signals within their vesicles; conversely, at 4℃ they failed to uptake albumin due to inhibited endocytosis (\u003cstrong\u003eFig. 3F\u003c/strong\u003e). These results validate that iPSCs are capable of generating physiologically functional podocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe podocytes derived from the proband\u0026rsquo;s induced iPSCs exhibited reduced adhesion properties.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe focused on the genes that were differentially expressed between hiPSC-derived podocytes from the proband (P-hiPSC-podocytes) and hiPSC-derived podocytes from healthy individuals (H-hiPSC-podocytes). Following RNA transcriptome sequencing of both P-hiPSC-podocytes and H-hiPSC-podocytes without any intervention. A volcano plot was generated to visualize these differentially expressed genes (\u003cstrong\u003eFig. 4A\u003c/strong\u003e). The downregulated and differentially expressed genes are primarily enriched in biological pathways associated with epithelial cell development, cell junction assembly, morphogenesis of the embryonic epithelium, tight junction assembly, and inner ear development (\u003cstrong\u003eFig. 4B\u003c/strong\u003e). The aberrant signaling pathways involved in inner ear development exhibit a consistent correlation with the auditory impairment characteristics observed in patients with BORS. KEGG enrichment analysis revealed significant downregulation of differentially expressed genes, particularly within the Hippo signaling pathway, tight junctions, signaling pathways regulating pluripotency of stem cells, and cell adhesion molecules (\u003cstrong\u003eFig. 4C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe hippo signaling pathway is known to play a crucial role in kidney development\u0026nbsp;[23], and the downregulation of this signaling pathway may have influenced the differentiated development of the solitary kidney in the patient. KEGG enrichment analyses suggest a significant reduction in the tight junction integrity and adhesion properties of the P-hiPSC-podocytes, which may compromise the filtration barrier between podocytes, thereby facilitating the development of proteinuria. The Gene Ontology enrichment analysis of upregulated differentially expressed genes predominantly highlighted pathways associated with actin filament bundling, cytoskeletal motor activity, and microtubule motor function\u0026nbsp;(\u003cstrong\u003eFig. 4D\u003c/strong\u003e). These functions facilitate cellular mitosis, endocytosis, exocytosis, and motility.\u003c/p\u003e\n\u003cp\u003ePAX8 is associated with parietal epithelial cells, PAX2 pertains to renal progenitor cells, and both PECAM1 and FLT1 are linked to endothelial cells; notably, the expression levels of these genes are all relatively low. Expression levels of the EYA1 gene were significantly reduced in P-hiPSC-podocytes compared to H-hiPSC-podocytes. However, the genes EYA1, SIX1, and SIX5 closely associated with BORS alongside SIX2, which plays a critical role in maintaining progenitor cells, exhibited only weak expression. (\u003cstrong\u003eFig. 4E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of PAN treatment on t\u003c/strong\u003e\u003cstrong\u003ehe podocytes derived from h\u003c/strong\u003e\u003cstrong\u003eiPSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePuromycin aminonucleoside (PAN) is a cytotoxic agent, regularly employed to induce focal segmental glomerulosclerosis in rat models. Furthermore, this compound can be utilized in vitro to provoke podocyte injury and establish nephrotic cell models. In this study, podocytes were treated with PAN for establishing a nephropathy model, and the alterations in gene expression of podocytes following PAN treatment were analyzed. Volcano plots depicting the differentially expressed genes in the H-hiPSC-podocytes group (\u003cstrong\u003eFig. 5A\u003c/strong\u003e) and P-hiPSC-podocytes group (\u003cstrong\u003eFig. 5B\u003c/strong\u003e) both prior to and following PAN. The KEGG pathway enrichment analysis demonstrated that the differentially expressed genes with upregulation in H-hiPSC-podocytes and P-hiPSC-podocytes subsequent to PAN treatment were preponderantly affiliated with pathways related to formation of neutrophil extracellular traps (\u003cstrong\u003eFig. 5C\u003c/strong\u003e). Neutrophil extracellular traps are generated during active cell death, a process that is distinct from both apoptosis and necrosis [24-25]. Following treatment with PAN, the induced podocytes exhibited indications of cell death, however, no substantial evidence of apoptosis was detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlucocorticoids do not improve the cell adhesion and cytoskeletal organization genes in podocytes of the BORS patient.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proband exhibited resistance to steroid therapy. To evaluate the similarity between iPSC-derived podocytes and the proband. Gene Set Enrichment (GSEA) was conducted to assess cell adhesion-related gene set scores. GSEA results showed that the score in H-hiPSC-podocytes treated with PAN significantly decreased compared to untreated controls (\u003cstrong\u003eFig. 6A\u003c/strong\u003e). However, dexamethasone pretreatment markedly increased this score after PAN exposure (\u003cstrong\u003eFig. 6B\u003c/strong\u003e), suggesting it can improve the impaired adhesive capacity of H-hiPSC-podocytes induced by PAN (\u003cstrong\u003eFig. 6C\u003c/strong\u003e). Conversely, P-hiPSC-podocytes under similar conditions, dexamethasone pretreatment does not alleviate the reduced adhesive ability of P-hiPSC-podocytes caused by PAN (\u003cstrong\u003eFig. 6D-F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eDisruption of the cytoskeleton significantly affects both the morphology and function of foot processes, compromising the integrity of the glomerular filtration barrier [26]. GSEA revealed significant alterations in cytoskeleton-related gene sets in H-hiPSC-podocytes both prior to and following PAN treatment (\u003cstrong\u003eFig. 7A\u003c/strong\u003e), with pronounced changes observed after dexamethasone pretreatment (\u003cstrong\u003eFig. 7B\u003c/strong\u003e). Dexamethasone pretreatment mitigated the cytoskeletal damage induced by PAN in H-hiPSC-podocytes (\u003cstrong\u003eFig. 7C\u003c/strong\u003e). As anticipated, notable alterations were detected in P-hiPSC-podocytes post-puromycin treatment (\u003cstrong\u003eFig. 7D\u003c/strong\u003e); however, dexamethasone pretreatment did not influence the puromycin nephropathy model (\u003cstrong\u003eFig. 7E-F\u003c/strong\u003e). At a genetic level, this observation highlights an intriguing phenomenon regarding the proband\u0026rsquo;s insensitivity to hormonal regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisease phenotype of proband-derived podocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe aimed to investigate whether P-hiPSC-podocytes exhibit disease-specific characteristics. Podocytes induced on day 16 were used for validation. The previous gene enrichment analysis indicated downregulation of pathways related to podocyte adhesion and an upregulation of pathways linked to cellular motility. To assess the migratory capacity, a wound healing assay was performed. At 24 and 48 hours, the gap width in P-hiPSC-podocytes showed significant reduction (\u003cstrong\u003eFig. 8A\u003c/strong\u003e). Quantitative analysis revealed that P-hiPSC-podocytes exhibited a higher cell migration rate (\u003cstrong\u003eFig. 8B\u003c/strong\u003e). These findings suggest that podocytes derived from the proband displayed reduced adhesive capacity while demonstrating greater motility compared to those from healthy individuals.\u003c/p\u003e\n\u003cp\u003eActin dynamics in podocytes are crucial for cellular morphology, motility, and intracellular transport [27-28]. We employed phalloidin staining to analyze actin structures in hiPSC-derived podocytes. Typically, podocyte cytoskeletons exhibit type A or B structures, while type C or D indicates abnormalities (\u003cstrong\u003eFig. 8C\u003c/strong\u003e). Untreated H-hiPSC-podocytes and P-hiPSC-podocytes preserved their cellular polarity through a well-organized cytoskeletal architecture. Quantitative analysis revealed that types A and B predominated in both groups (\u003cstrong\u003eFig. 8D\u003c/strong\u003e). After PAN treatment, both H-hiPSC-podocytes and P-hiPSC-podocytes predominantly exhibited types C and D of the cytoskeleton, accompanied by a clear disruption in stress fiber polarity (\u003cstrong\u003eFig. 8E\u003c/strong\u003e). Prior to PAN exposure, dexamethasone pretreatment led to a notable reduction in the proportions of type C and D within H-hiPSC-podocytes. Importantly, dexamethasone failed to restore aberrant skeletons in P-hiPSC-podocytes, underscoring hormone resistance. In H-hiPSC-podocytes, type A did not return to baseline levels observed without treatment, suggesting that dexamethasone was insufficient to fully restore damage inflicted on the podocyte cytoskeleton by PAN (\u003cstrong\u003eFig. 8F\u003c/strong\u003e). Podocytes from the proband exhibited clinical characteristics consistent with steroid-resistant nephropathy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is well-established that patients with BORS frequently present with renal malformations; however, there is a lack of studies investigating the characteristics and mechanisms of renal injury in these individuals. Moreover, despite being the primary pathogenic gene associated with BORS, reports on the impact of EYA1 on podocyte function are limited. In this study, we examined renal injury features in a family affected by BORS carrying an EYA1 mutation and explored potential cellular injuries in vitro using induced pluripotent stem cell-derived podocytes from the patient. Our findings provide novel insights into iPSC-derived podocytes from BORS patients harboring EYA1 mutations, which exhibit enhanced motility compared to those from healthy subjects. Furthermore, we observed significant glucocorticoid resistance in podocytes carrying EYA1 mutations. Collectively, our results demonstrate that the EYA1 mutation directly influences podocyte function by increasing cell motility and inducing cytoskeletal rearrangement unresponsive to glucocorticoid intervention. RNA-seq data revealed differential expression of several adhesion-related genes in podocytes derived from patients with EYA1 mutations. The findings elucidate potential effects and signaling targets of EYA1 on podocyte function, offering valuable directions for future research into the mechanistic underpinnings of kidney injury associated with EYA1.\u003c/p\u003e\n\u003cp\u003eCurrent reports indicate that the predominant clinical phenotypes in individuals with BORS and EYA1 gene mutations include deafness (98.5%), preauricular fovea (83.6%), branchial arch abnormalities (68.5%), renal abnormalities (38.2%), and external ear anomalies (31.5%) [29]. Investigations into EYA1\u0026apos;s role in kidney development have shown that conditional inactivation of Eya1 leads to loss of Six2 expression and premature epithelialization of renal progenitor cells, underscoring the essential coordination among Eya1, Six2, and Myc for nephron precursor proliferation and nephrogenesis [30]. Although infrequently reported, proteinuria has been observed to varying degrees in three recent BORS patients with EYA1 gene mutations [31]. This finding further underscores the necessity of enhancing proteinuria detection, including microalbuminuria assessment, for early identification of kidney damage among BORS patients.\u003c/p\u003e\n\u003cp\u003eIn this family, despite the proband\u0026rsquo;s mother and brother having deafness and preauricular fistula, no structural kidney abnormalities were identified. However, the proband exhibited nephrotic-level proteinuria and a solitary kidney. Whole exome sequencing revealed that the EYA1 gene mutation was inherited from the mother as a heterozygous splicing variant. These findings demonstrate significant heterogeneity in how mutations at the same site of EYA1 can influence renal phenotype within one family. Over 8 years of follow-up, both the patient\u0026apos;s mother and brother gradually developed proteinuria while their glomerular filtration rates progressively declined. Our results underscore the necessity of closely monitoring renal function among patients with BORS and conducting timely screenings for kidney damage.\u003c/p\u003e\n\u003cp\u003eCurrently, the underlying mechanism by which the EYA1 gene contributes to proteinuria and renal function decline remains unclear. The involvement of EYA1 in glomerular podocyte injury has not been previously reported. In this study, we aimed to induce in vitro differentiation of podocytes using established iPSCs derived from peripheral blood mononuclear cells obtained from the patient [20]. Induced podocytes at day 16, we observed the expression of key podocyte proteins including podocin, WT-1, and Synaptopodin. Additionally, the induced podocytes exhibited multiple protrusions with tight junctions between cells and aligned cytoskeletal fibers demonstrating significant polarity alignment, consistent with previous findings by Haynes JM et al. [32].\u003c/p\u003e\n\u003cp\u003eIt is widely recognized that the pathogenesis of podocyte injury primarily involves extensive fusion of foot processes. In vitro, this fusion leads to increased cell motility and cytoskeletal rearrangement. This study aimed to investigate whether patient-derived podocytes could recapitulate clinical manifestations in vitro. Our findings demonstrated that iPSC-derived podocytes from patients with EYA1 mutations exhibited typical morphological characteristics and specific protein expression associated with mature podocytes while also displaying enhanced cell motility and cytoskeletal rearrangement. These insights suggest that EYA1 mutations may impair the proper differentiation of nephron progenitors into functional podocytes and disrupt normal cellular functions. Notably, the observed functional abnormalities in cultured iPSC-derived podocytes corresponded well with the clinical phenotype of massive proteinuria, thereby providing an ideal cellular model for future investigations into the molecular mechanisms underlying podocyte injury. Furthermore, these findings underscore the potential utility of drug screening using renal organoid cultures as a promising avenue for therapeutic development.\u003c/p\u003e\n\u003cp\u003eUnfortunately, due to the presence of a solitary kidney in this patient, renal pathology data were unavailable. However, a renal biopsy on a child with EYA1 gene mutation BORS by Lin et al. revealed partial fusion of podocyte foot processes [31], consistent with our previously reported in vitro findings. Therefore, it is crucial to emphasize the importance of renal biopsies in children with proteinuria and EYA1 gene mutations to enhance our understanding of the pathological mechanisms underlying renal injury associated with this condition.\u003c/p\u003e\n\u003cp\u003eThe patient in this study exhibited clinical resistance to steroids, and we observed that the iPSC-derived podocytes from this patient demonstrated significant resistance to dexamethasone following puromycin aminonucleoside treatment. This finding is consistent with the clinical characteristics of steroid-resistant nephropathy. A critical question raised by this study is whether EYA1, a key transcriptional regulator, plays a role in hormone resistance mechanisms. However, our RNA-Seq data did not provide definitive evidence for regulatory relationships between EYA1 and known pathogenic genes associated with steroid-resistant nephropathy. Our results indicate that mutant podocytes exhibiting abnormal expression of adhesion and cytoskeletal signaling pathway genes persist even after glucocorticoid treatment. Future studies should focus on elucidating the potential role of EYA1 mutations in steroid-resistant patients to further clarify the underlying mechanisms.\u003c/p\u003e\n\u003cp\u003eIn summary, this study was directed towards probing into the potential ramifications and signaling targets of EYA1 gene mutations on podocyte injury. Our discoveries illuminate the underlying mechanism of proteinuria in patients afflicted with EYA1 gene mutations, furnishing indispensable data for clinical practice, especially for non-nephrologists, to optimize kidney damage screening in individuals with BORS. Furthermore, it is of paramount importance to explore the participation of the EYA1 gene in steroid-resistant nephropathy and its underlying mechanisms. This in vitro disease model employing mature hiPSC-derived podocytes holds promise for promoting the development and application of novel therapeutics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBORS\u0026nbsp;branchio-oto-renal syndrome\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBOS\u0026nbsp; \u0026nbsp;\u0026nbsp;Branchio-oto syndrome\u003c/p\u003e\n\u003cp\u003ehiPSC\u0026nbsp;\u0026nbsp;Human induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eH-hiPSC-podocytes\u0026nbsp; \u0026nbsp;\u0026nbsp;hiPSC-derived podocytes from the healthy individual\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP-hiPSC-podocytes \u0026nbsp; \u0026nbsp;\u0026nbsp;hiPSC-derived podocytes from the propositus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGSEA\u0026nbsp;Gene Set Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eGO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Gene Ontology Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eKEGG\u0026nbsp;Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003ePAN\u0026nbsp; \u0026nbsp;\u0026nbsp;puromycin aminonucleoside\u003c/p\u003e\n\u003cp\u003eDEX dexamethasone\u003c/p\u003e\n\u003cp\u003ePAN+DEX \u0026nbsp; \u0026nbsp; \u0026nbsp;pretreated with dexamethasone\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our sincere gratitude to the proband and her family for their invaluable support of the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGL, DL, LX, ML and XG designed and supervised research. GL, DL, LX, SZ JZ and LW performed the experiments. GL, DL, LX, LS and LW\u0026nbsp;analyzed data; XL and ZM supervised the project. GL and ML wrote the draft of the manuscript. GL and XG proofread the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis\u0026nbsp;research\u0026nbsp;was supported by grants from Science and Technology Program of Guangzhou (Grant No.\u0026nbsp;202102010222).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe source data and any additional information in this paper will be shared upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the submission and final publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Guangzhou Women and Children\u0026rsquo;s Medical Center (No.247A01). Informed consent in written form was acquired from the patients as well as family members.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMelnick M et al (1975) Autosomal dominant branchiootorenal dysplasia. Birth defects original article series, 11(5), 121\u0026ndash;128.\u003c/li\u003e\n\u003cli\u003eFraser FC, Sproule JR, Halal F (1980) Frequency of the branchio-oto-renal (BOR) syndrome in children with profound hearing loss. American journal of medical genetics, 7(3), 341\u0026ndash;349. https://doi.org/10.1002/ajmg.1320070316.\u003c/li\u003e\n\u003cli\u003eUnzaki A et al (2018) Clinically diverse phenotypes and genotypes of patients with branchio-oto-renal syndrome. 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BMC nephrology, 24(1), 248. https://doi.org/10.1186/s12882-023-03193-3.\u003c/li\u003e\n\u003cli\u003eHaynes JM et al (2018) Induced Pluripotent Stem Cell-Derived Podocyte-Like Cells as Models for Assessing Mechanisms Underlying Heritable Disease Phenotype: Initial Studies Using Two Alport Syndrome Patient Lines Indicate Impaired Potassium Channel Activity. The Journal of pharmacology and experimental therapeutics, 367(2), 335\u0026ndash;347. https://doi.org/10.1124/jpet.118.250142.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable1.\u003c/strong\u003e \u003cstrong\u003eThe 1050th nucleotide mutation in the coding region of the EYA1 gene retrieved from ClinVar is determined to be pathogenic\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"653\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 182px;\"\u003e\n \u003cp\u003eCondition(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eClinical significance\u003c/p\u003e\n \u003cp\u003e(Last reviewed)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eReview status\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eNM_000503.6(EYA1):\u003c/p\u003e\n \u003cp\u003ec.1051-2A\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 182px;\"\u003e\n \u003cp\u003eMelnick-Fraser syndrome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003cp\u003e(Apr 15, 2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003ecriteria provided,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003esingle submitter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eNM_000503.6(EYA1):\u003c/p\u003e\n \u003cp\u003ec.1050+1G\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 182px;\"\u003e\n \u003cp\u003eBranchiootic syndrome 1,\u003c/p\u003e\n \u003cp\u003eMelnick-Fraser syndrome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003cp\u003e(Aug 27, 2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003ecriteria provided,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003emultiple submitters,\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;no conflicts\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Clinical data of the pedigree.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"654\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eUrinary albumin/Urine creatinine (mg/\u0026mu;mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSerum creatinine\u003c/p\u003e\n \u003cp\u003e(\u0026mu;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBlood urea nitrogen\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eeGFR\u003c/p\u003e\n \u003cp\u003e(ml/min/1.73m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eII-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eII-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003emale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e94.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eI-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Branchio-oto-renal syndrome, Induced pluripotent stem cells, Podocytes","lastPublishedDoi":"10.21203/rs.3.rs-5591319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5591319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe primary cause of branchio-oto-renal syndrome (BORS) is mutations in the EYA1 gene. This study aimed to explore the impact and underlying mechanisms of EYA1 mutations on podocyte injury. We collected clinical and genetic data from a 4-year-old girl diagnosed with BORS and her family. Induced pluripotent stem cells (iPSC) were derived from peripheral blood mononuclear cells of both the patient and healthy individuals, which were differentiated into podocytes in vitro. RNA-seq was used to analyze differentially expressed genes in both groups. Here, the proband, along with his brother and mother, exhibited symptoms of BORS. WES analysis identified a heterozygous splicing variant at the EYA1 locus: c.1050\u0026thinsp;+\u0026thinsp;5G\u0026thinsp;\u0026gt;\u0026thinsp;A, inherited from his mother. The proband was initially glucocorticoid-resistant. After tacrolimus treatment, his urine protein/creatinine ratio significantly improved. Compared to healthy individuals, patient-derived podocytes displayed increased motility and pronounced cytoskeletal rearrangement. Dexamethasone was ineffective in ameliorating the pathological damage induced by puromycin aminonucleoside in patient-derived podocytes. RNA-Seq results indicated significant downregulation of cell adhesion molecule signaling pathway expression in patient-derived podocytes compared to healthy controls. In BORS patients with EYA1 mutations, podocytes exhibit cytoskeletal reorganization and enhanced motility in vitro while showing resistance to steroid treatment-indicating a unique damage response that warrants further investigation.\u003c/p\u003e","manuscriptTitle":"IPSC-induced podocytes from a BORS patient with EYA1 gene mutation showed glucocorticoid-resistant and cytoskeletal rearrangement","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-26 07:54:49","doi":"10.21203/rs.3.rs-5591319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-02-05T23:33:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-12-23T10:50:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-18T15:16:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-09T14:34:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-12-06T01:42:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"225d2208-ca0c-4733-a4fe-ebb6ddf79ded","owner":[],"postedDate":"December 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T20:33:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-26 07:54:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5591319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5591319","identity":"rs-5591319","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}