miR-135a-5p in Exosomes Promotes Childhood Nephrotic Syndrome via GSK-3β and TGF-β1 Crosstalk

miR-135a-5p in Exosomes Promotes Childhood Nephrotic Syndrome via GSK-3β and TGF-β1 Crosstalk | 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 miR-135a-5p in Exosomes Promotes Childhood Nephrotic Syndrome via GSK-3β and TGF-β1 Crosstalk Majid A Almeshary, Omali Y El-Khawaga, Dalia T Hussein, Mannar Refaat, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7328776/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nephrotic syndrome (NS) is an autoimmune kidney disorder that is characterized by severe protein loss in urine, which results in edema, hyperlipidemia, and hypoalbuminemia. Glucocorticoid therapy is the main treatment for NS patients; however, the unsuitable treatment of NS can lead to chronic glomerulonephritis and, in turn, end-stage renal disease (ESRD). Genetic and epigenetic variations between individuals lead to different responses to treatment. Therefore, this study aimed to investigate an early predictor for glucocorticoid response in NS patients. Therefore, this study aimed to investigate an early predictor for glucocorticoid response in NS patients. Methods This study was conducted on 70 NS patients enrolled from the Nephrology Unit of Mansoura University Children’s Hospital and 70 aberrantly healthy individuals as a control group. Peripheral blood samples were collected from all subjects for miR-135a-5p, GSK3β, and TGFB-1 expression. Results miR-135a-5p level was significantly decreased, while the GSK3β and TGFB-1 expression levels were increased in the NS group compared to the control group. Regarding the NS subtypes, the steroid-resistant nephrotic syndrome (SRNS) group exhibits a significant reduction in miR-135a-5p and a significant elevation in the levels of GSK3β and TGFB-1 compared to the steroid-sensitive nephrotic syndrome (SSNS) group. Conclusion These biomarkers demonstrated moderate diagnostic accuracy in distinguishing nephrotic syndrome (NS) from controls and effectively identified steroid-resistant NS (SRNS) cases. Their potential as early predictors of glucocorticoid response offers promising clinical utility for personalized therapeutic strategies in pediatric NS. Nephrotic syndrome Glucocorticoid miR-135a-5p GSK3β TGFB-1 children 1. Introduction Nephrotic syndrome (NS) is an immune-inflammatory kidney disorder that is one of the main causes of end-stage kidney disease (ESKD) [ 1 ]. This syndrome causes defects in the glomerular filtration barrier, leading to excessive loss of protein, fluid, and nutrition in the urine [ 2 ]. The main characteristics of NS are edema, proteinuria, hypoalbuminemia, and dyslipidemia [ 3 ]. NS can be divided into primary, secondary, and congenital NS. Primary NS approximately accounts for 90% of the total NS cases in children [ 4 ]. The incidence of NS varies across populations, ranging from two to seven per 100,000 children, with higher rates reported among individuals of African and South Asian ancestry [ 5 ]. The Middle East and North Africa region is expected to have a comparatively higher incidence of rare diseases than other parts of the world, largely due to the high prevalence of consanguinity [ 6 ]. In Egypt, the incidence of NS in children has increased over the years, which is accompanied by worsening kidney function [ 7 ]. Renal biopsy, the usual approach to determine disease nature and prognosis, is an invasive surgery with hidden risk and typically not practical for serial monitoring, especially in children. Improper tissue samples can lead to misdiagnosis [ 8 ]. Therefore, it is necessary to find new noninvasive diagnostic and prognostic biomarkers for the disease that are easy to assess, suited to accurately predicting long-term outcomes, and useful for both diagnosis and treatment. Glucocorticoid is the common treatment protocol for NS that acts as immunosuppressant and anti-inflammatory for several disorders [ 9 ]. The response to glucocorticoid treatment varies between patients. Depending on how well patients respond to glucocorticoid medication, NS is categorized into steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS). SRNS develops when glucocorticoid treatment fails to produce in complete remission within 4–6 weeks [ 10 ]. The etiology of SRNS is often attributed to structural and functional damage of the podocytes [ 11 ]. The response to treatment is distinct according to genetic variations between individuals. Early prediction of glucocorticoid response will allow for an alternative treatment protocol to avoid glucocorticoid side effects [ 12 ]. Micro-RNAs (miRNA), histone modifications, and DNA methylation (DNAm) are examples of epigenetic processes that modify gene expression without altering the underlying DNA sequence [ 13 ]. miRNA is a highly conserved, non-coding small RNA of 22 nucleotides that regulates gene expression at the post-transcriptional level [ 14 ]. They are involved in many valuable biological processes such as cell growth, differentiation, proliferation, and apoptosis [ 15 ]. miRNAs play a critical role in maintaining renal homeostasis under physiological and pathological conditions, as evidenced by the differential expression of specific miRNAs in diverse kidney diseases [ 16 ]. Previous studies demonstrated the role of miRNAs in the development and progression of glomerular diseases associated with podocyte dysfunction [ 17 ]. The alteration of miR-135a and miR-135b expression is a hallmark of podocyte injury, and this expression was observed in response to transforming growth factor beta (TGF-β). The binding of miR-135a and miR-135b to glycogen synthase kinase-3p (GSK3β) affects the GSK3β expression at the transcriptional and translational levels [ 18 ]. This study aimed to investigate the expression levels and functional role of circulating exosomal miR-135a-5p in regulating GSK-3β and TGF-β1 expression in patients with nephrotic syndrome (NS). Furthermore, it examined the correlation between these epigenetic and genetic markers and glucocorticoid responsiveness. 2. Patients and Methods 2.1 Patients and controls This study was performed in the Nephrology Unit of Mansoura University Children’s Hospital (MUCH). It includes 70 children with primary NS and 70 apparently healthy controls of matched age and sex. The NS patients were subdivided into three groups based on their initial response to steroid treatment: steroid sensitivity (SSNS), which includes 10 patients; steroid dependence with two consecutive relapses during therapy (SDNS), which includes 20 patients; and steroid resistance (SRNS), which includes 40 patients. Ethical approval The study was approved by the Mansoura Faculty of Medicine Institutional Research Board, code number: MDP.24.04.148. Informed consent was taken from parents of all subjects before the study. Inclusion criteria: children with primary NS and aged from 1 to 18 years of both sexes Exclusion criteria: patients with secondary or congenital NS, diabetes, leukemia, lupus nephritis, chronic inflammatory disease, and patients who have undergone kidney transplantation. 2.1.1. Sample collection From each NS patient and control, two peripheral blood samples were collected. The first was a gel tube to collect serum for isolation of exosomal miRNA, and the other was collected in an EDTA tube for extraction of total RNA. 2.2. Methods 2.2.1. Quantitative real-time PCR for Exosomal miRNA Exosomal miRNA was extracted from serum samples using miRNeasy Serum/Plasma Advanced Kit (cat. no. 217004, Qiagen, Germany), then converted to cDNA by miScript Reverse Transcription kit (cat. no. 218161, Qiagen, Germany). RT-PCR was performed using the Maximas SYBR Green/Fluorescein qPCR Master Mix (cat. no. K0243, Thermo Fisher Scientific, Waltham, MA, USA) for miR-135a-5p and U6 as a housekeeping gene following the manufacturer's instructions using Rotor-Gene Q (Qiagen, USA). The samples were performed in triplicate. The expression level of miRNA was calculated using the equation 2 −ΔΔCt . The primer sequences of the studied genes are listed in Table 1. 2.2.2. Quantitative real-time PCR mRNA Total RNA has been extracted from blood samples using TRIzols Reagent (cat. no. 15596026, Life Technologies, USA) following the manufacturer’s protocol. RNA samples were converted to complementary DNA (cDNA) with QuantiTect Reverse Transcription Kit (Cat no. 205311 Qiagen, USA). cDNA samples stored at -80°C. Quantitative RT-PCR was performed using Maximas SYBR Green/Fluorescein qPCR Master Mix (cat. no K0243, Thermo Fisher Scientific, Waltham, MA, USA). The expression of glycogen synthase kinase-3β (GSK-3β), and transforming growth factor beta-1 (TGFB-1) was quantified using Rotor-Gene Q (Qiagen, USA) using GAPDH as internal control. The samples were performed in triplicate. The expression level of mRNA was calculated using the equation 2 −ΔΔCt . The primer sequences of the studied genes are listed in Table 1. 2.2.3. Statistical analysis Statistical analysis was performed using the Statistical package for Social Science (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). The continuous data were expressed as mean ± standard deviation (SD) using the student's t-test to compare the statistical difference between two study groups, while the Kruskal-Wallis test was used to assess the statistical difference between more than two study groups. Categorical data, on the other hand, were compared using the Chi-square. The receiver operating characteristic (ROC) Curve was used to evaluate the sensitivity and specificity for quantitative diagnostic measures. Spearman's rho correlation coefficients ( sr ) were calculated for the variables. Logistic regression was used to predict risk factors when the dependent variable is categorical. p ≤ 0.05 was considered statistically significant. Conflict of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 3. Results Patients’ demographics and clinicopathological characteristics are shown in Table 2. Out of 70 NS patients, 54.3 % were males and 45.7 % were females; the mean ± SD age of all patients was 9.06 ± 3.75 years, while the mean ± SD age of onset was 5.70 ± 3.13 years. The disease duration averages 3.36 ± 2.88 years. Exosomal miR-135a-5p was significantly lower in NS patients compared to controls ( p < 0.001). Conversely, GSK-3β and TGFβ-1 gene expression levels were significantly higher in NS than in control groups ( p <0.001) (Table 3). Table 4 represents a relation between treatment response and gene expression. Regarding exosomal miR-153a-5p, the expression level showed significantly lower levels in SSNS, SDNS, and SRNS than the control group ( p <0.001). In comparing the SRNS and SSNS groups, the level was significantly lower in the SRNS group ( p =0.022), with no significant differences between SSNS versus SDNS and SRNS versus SDNS. The mRNA level of GSK-3β showed a significantly higher level in SDNS and SRNS than the control group ( p = 0.021, p <0.001, respectively), with a significant increase in the SRNS group compared to the SSNS group ( p <0.001), with no significant differences between the SSNS group versus the control or SDNS groups. Additionally, TGFβ-1 gene expression level showed significantly elevated levels in the SSNS, SDNS, and SRNS than the control group ( p <0.001), as well as in SRNS than SSNS and SDNS groups ( p = 0.008, p =0.016, respectively), with no significant differences between SSNS versus SDNS groups. The validity of the studied markers was evaluated in Table 5 for discriminating between NS patients and the control group using the ROC curve. The area under the curve (AUC) values indicate strong discriminatory power for exosomal miR-135a-5p, showing the highest AUC of 0.932, followed by TGFβ-1 at 0.917, then GSK-3β at 0.782 ( p = <0.001). The sensitivity and specificity values further support the utility of these markers. The cut-off values for each marker are provided, indicating the thresholds for optimal discrimination. As shown in Table 6, the AUC values indicate that GSK-3β has the highest predictive capabilities, with AUCs of 0.795 ( p <0.001), followed by TGFβ-1 (AUC= 0.764, p <0.001), then exosomal miR-135a-5p (AUC= 0.715, p = 0.002). The correlation between different markers in studied NS groups was presented in Table 7. Among total NS patients, exosomal miR-135a-5p gene expression showed a significant negative correlation with GSK-3β and TGFβ-1. In the SSNS group, GSK-3β gene expression showed a significant negative correlation with TGFβ-1. While among the SDNS and SRNS groups, no significant correlations were found between the studied markers. Table 8 presents a logistic regression analysis for predicting susceptibility to NS. The univariate analysis shows that lower exosomal miR-135a-5p and higher GSK-3β and TGFβ-1 are all significant predictors of NS susceptibility. While in the multivariate analysis, lower exosomal miR-135a-5p and higher TGFβ-1 remained significant predictors, suggesting that these markers could be valuable in assessing the risk of developing NS. Table 9 details a logistic regression analysis for predicting SRNS on top of the combination of SSNS and SDNS. The univariate analysis identifies several significant predictors, including the presence of consanguinity, lower exosomal miR-135a-5p, and higher GSK-3β and TGFβ-1, indicating their association with SRNS risk. In the multivariate analysis, higher GSK-3β and TGFβ-1 remained significant predictors, emphasizing their potential role in distinguishing SRNS from SSNS and SDNS. 4. Discussion In clinical nephrology, NS is the most prevalent disease in childhood. NS is typically a pathognomonic sign of glomerular disease; however, the underlying clinical etiology varies widely. Although renal function was normal in most NS patients at the beginning, prolonged NS will often result in progressive renal failure and a lifelong need for renal replacement therapy (dialysis or transplantation) [19]. More than 85% of NS cases in children under the age of 18 years are caused by focal segmental glomerulosclerosis (FSGS) and minimal change nephropathy (MCN). Conversely, prevalent causes in adult patients include amyloidosis, lupus nephritis, diabetic glomerulosclerosis, membranoproliferative glomerulonephritis, minimal change nephropathy, and focal glomerulosclerosis [20]. Previous studies in NS cleared that the main obstacle is in the accurate and early diagnosis, medical follow-up, and choosing the appropriate treatment for patients in terms of age, gender, race, and genetic differences. miRNAs play a crucial role in kidney dysfunction and structural damage, as well as maintaining normal regulatory systems. Previous research identified dysregulated miRNAs in the renal tissues of children with nephropathy, which was linked to the pathogenesis of nephrosis [21]. In this study, the quantification of exosomal miR-135a-5p was found to be significantly downregulated in NS patients compared to the control group. This significant downregulation shows that this miRNA may play an integrated or distinctive role in NS pathophysiology. Also, this exosomal miR-135a-5p was found to be significantly decreased in the SRNS group compared to either the healthy control group or the SSNS group. Previously, several studies reported that exosomal miRNA expression levels in urine have been found to be significantly altered in adults with kidney diseases such as chronic kidney disease (CKD), lupus nephritis, and IgA nephropathy [22]. This suggests that urinary exosomal miRNAs may be promising biomarkers of adult kidney disease. The clinical significance of the urine exosomal miRNA signature in pediatric idiopathic NS patients remains unknown [23]. Our results regarding miR-135a-5p were in agreement with the Iranian study, which stated that plasma samples from both the NS and membranous glomerulonephritis (MGN) groups showed significantly lower expression levels of miR-135a-5p ( p = 0.020 and p = 0.040, respectively) compared to the control group. The FSGS group had a significantly lower level of circulating miR-135a-5p ( p = 0.046) than the controls. There were no significant differences in miR-135a-5p expression levels between the FSGS and MGN groups in plasma samples ( p = 0.501) [24]. ROC analysis in our study showed that miR-135a-5p had an AUC of 0.932 ( p < 0.001), with reliable sensitivity and specificity in plasma, indicating high diagnostic power in distinguishing NS patients from healthy controls; also, miR-135a-5p showed a moderate diagnostic power at AUC = 0.715 ( p = 0.002) to differentiate the SRNS patients from the rest of the NS patients. This result is in contrast with the results of Ardalan et al., they stated that miR-135a-5p did not have reliable value in discriminating NS patients from controls, AUC = 0.605 in plasma samples and AUC = 0.510 in peripheral blood mononuclear cells (PBMCs) [24]. MiR-135 dysregulation has been observed in many non-neoplastic disorders, including NS, Alzheimer's disease, depression, Parkinson's disease, atherosclerosis, diabetes, pulmonary arterial hypertension, endometriosis, epilepsy, and allergies. Furthermore, a number of signalling pathways, including Wnt/β-catenin, TGF-β/SMAD, p38 MAPK, NF-κB, and IL-6/STAT3, have been shown to be modulated by miR-135. This broad method of action demonstrates the intricacy of the functional network impacted by miR-135 [25]. GSK-3β gene is one of the most potential target genes of the miR-135. The miR-135 family has two members: miR-135a and miR-135b, which play roles in many biological pathways [26]. GSK3β is a highly conserved serine/threonine kinase that was first identified as regulating the insulin signaling pathway and glycogen biosynthesis. Since then, it has been linked to numerous other pathophysiologic processes and conditions, such as organ damage and repair, cancer, neurodegenerative diseases, and more lately kidney diseases [27]. Referring to our molecular analysis comparison between NS patients and healthy control group regarding GSK-3b gene expression, there was a high significant elevation in NS patients compared to healthy control group ( p < 0.001). This elevation was observed in the SRNS group compared to the control and SSNS groups, while no statistically significant difference was observed between the control and SSNS groups. One of the results consistent with our results is a study by Ardalan et al., which stated that there were statistically significant increases in GSK-3β expression level in NS ( p = 0.001), MGN ( p = 0.002), and FSGS ( p = 0.015) groups compared to the control group [24]. The up-regulation of GSK-3β expression in tubular cells and glomeruli of the kidney indicates a detrimental role of GSK-3 in podocyte injury. Its over-activation is linked to various kidney diseases, including proteinuric glomerulopathies and CKD, as reported by Paeng et al. [28]. ROC diagram analysis in our study showed that GSK-3β with AUC = 0.782 ( p < 0.001) with reliable sensitivity and specificity in plasma had a significant moderate diagnostic ability in differentiating between NS patients and the healthy control group; also, GSK-3β showed a moderate diagnostic power at AUC = 0.795 ( p <0.001) to differentiate the SRNS patients from the rest of the NS patients. Our result is in agreement with Ardalan and colleagues, they approved that GSK-3β with AUC = 0.72 ( p = 0.002) and good sensitivity and specificity in PBMCs demonstrated diagnostic power in distinguishing NS patients from healthy controls [24]. Previous studies reported that high expression of GSK-3β in renal tubules has a destructive role [29], also GSK-3β has been associated with acute kidney injury (AKI) and progressive CKD, GSK-3β modulates renal tubular and interstitial damage [30]. GSK3β is predominantly expressed in the kidney's glomeruli and proximal renal tubules. Acute injuries increase GSK3β activity in glomeruli and renal tubules by suppressing inhibitory phosphorylation. GSK3β hyperactivity has been linked to kidney cell death and acute renal damage through various pathways, including sensitization of mitochondrial permeability transition, loss of cytoskeleton integrity, and potentiation of NF-κB-dependent inflammatory responses [31]. In a previous study by Zhang et al., stated that miR‑135a‑5p/SIRT1/Smad3 pathway was implicated in TGFβ1‑induced renal fibrosis [14]. TGF-β1 is a well-known profibrogenic cytokine that contributes significantly to the development of CKD. The fibrotic process results from the overproduction of pathological matrix that is produced in response to TGF-β1 stimulating matrix protein synthesis and decreasing its degradation [32]. In our study, the expression level of TGFβ-1 was significantly elevated in NS group compared to the healthy control group ( p <0.001). NS subtypes showed a significant increase in TGFβ-1 expression compared to the healthy control group, with a significant elevation in SRNS group compared to the SSNS and SDNS groups. ROC analysis in our study showed that TGFβ-1 has AUC = 0.917 ( p < 0.001) with strong reliable sensitivity and specificity in plasma and had high diagnostic power in separating NS patients from healthy controls; also, TGFβ-1 showed a moderate diagnostic power at AUC = 0.764 ( p <0.001) to differentiate the SRNS patients from the rest of the NS patients. Along with our results, previous studies have also demonstrated increased TGFβ1 levels in the NS group compared to the control group in rats [33]. Another study by Li et al. stated that the elevated TGFβ1 in serum samples in patients with tubulointerstitial damage compared to controls [34]. The underlined mechanism by which TGFβ-1 is involved in the fibrosis process is through the epithelial to mesenchymal transition (EMT). The tubular epithelial cells are transformed to mesenchymal fibroblasts and migrate to adjacent interstitial parenchyma along with local and circulating cells; this process includes proteinuria as well as hypoxia [35]. TGFβ-1 modulates biological reactions through interactions with Smads, MAPK, and the Jagged/Notch signaling pathways [36]. In conclusion, the expression of exosomal miR-135a-5p, GSK-3β, and TGFβ-1 has succeeded in distinguishing between healthy individuals and NS patients and can be used for predicting the response to glucocorticoid treatment. The importance of investigating molecular and epigenetic expression in children with NS and how they affect glucocorticoid treatment has been highlighted by this work. To overcome the limitations in this study, we recommend conducting further studies on a larger scale to demonstrate the role of glucocorticoids on the expression of these markers to strengthen the validity and applicability of the findings. Further investigations on different epigenetic markers that affect various signaling pathways could provide a more comprehensive understanding of the glucocorticoid treatment’s mechanisms and effects, leading to better clinical applications. Declarations Ethical approval The study was approved by the Mansoura Faculty of Medicine Institutional Research Board, code number: MDP.24.04.148. Informed consent was taken from parents of all subjects before the study. Conflict of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Politano SA, Colbert GB, Hamiduzzaman N (2020) Nephrotic Syndrome. Prim Care 47:597–613 Verma PR, Patil P (2024) Nephrotic Syndrome: A Review. Cureus 16:e53923 Lee J (2021) Children with Kidney Disease: An Overview of Pediatric Primary Nephrotic Syndrome. Pediatric nursing 47 Li FX, Hou YL, Zhou LS, Dong Y, Zhao JW, Li ZX (2021) The status and model of children primary nephrotic syndrome in continuing nursing. Ann Palliat Med 10:2398–2407 Alenazi SA, Alenazi SA (2024) Incidence and Pathological Patterns of Nephrotic Syndrome among Infants and Children: A Systematic Review. Cureus 16 Salman MA, Elgebaly A, Soliman NA (2024) Epidemiology and outcomes of pediatric autosomal recessive polycystic kidney disease in the Middle East and North Africa. Pediatr Nephrol 39:2569–2578 Eid R, Fathy AA, Hamdy N (2020) Health-related quality of life in Egyptian children with nephrotic syndrome. Qual Life Res 29:2185–2196 Huang Z, Zhang Y, Zhou J, Zhang Y (2017) Urinary exosomal miR-193a can be a potential biomarker for the diagnosis of primary focal segmental glomerulosclerosis in children. Biomed Res Int 2017:7298160 Zhao X, Hwang DY, Kao HY (2018) The Role of Glucocorticoid Receptors in Podocytes and Nephrotic Syndrome. Nucl Receptor Res 5:101323 Sachdeva S, Khan S, Davalos C, Avanthika C, Jhaveri S, Babu A, Patterson D, Yamani AJ (2021) Management of Steroid-Resistant Nephrotic Syndrome in Children. Cureus 13:e19363 Lee JM, Kronbichler A, Shin JI, Oh J (2021) Current understandings in treating children with steroid-resistant nephrotic syndrome. Pediatr Nephrol 36:747–761 Motavalli R, Majidi T, Pourlak T, Abediazar S, Shoja MM, Vahed SZ, Etemadi J (2021) The clinical significance of the glucocorticoid receptors: Genetics and epigenetics. J Steroid Biochem Mol Biology 213:105952 Berdasco M, Esteller M (2019) Clinical epigenetics: seizing opportunities for translation. Nat Rev Genet 20:109–127 Zhang J, Zhang L, Zha D, Wu X (2020) Inhibition of miRNA-135a-5p ameliorates TGF-β1-induced human renal fibrosis by targeting SIRT1 in diabetic nephropathy. Int J Mol Med 46:1063–1073 Chen L, Heikkinen L, Wang C, Yang Y, Sun H, Wong G (2019) Trends in the development of miRNA bioinformatics tools. Brief Bioinform 20:1836–1852 Schena FP, Serino G, Sallustio F (2013) MicroRNAs in kidney diseases: new promising biomarkers for diagnosis and monitoring. Nephrol Dialysis Transplantation 29:755–763 Liu F, Chen J, Luo C, Meng X (2022) Pathogenic Role of MicroRNA Dysregulation in Podocytopathies. Front Physiol 13:948094 Wang Y, Jarad G, Tripathi P, Pan M, Cunningham J, Martin DR, Liapis H, Miner JH, Chen F (2010) Activation of NFAT signaling in podocytes causes glomerulosclerosis. J Am Soc Nephrol 21:1657–1666 Szeto C-C (2014) Urine miRNA in nephrotic syndrome. Clin Chim Acta 436:308–313 Cameron JS (1996) Nephrotic syndrome in the elderly. Seminars in nephrology, pp 319–329 Lu M, Wang C, Yuan Y, Zhu Y, Yin Z, Xia Z, Zhang C (2015) Differentially expressed microRNAs in kidney biopsies from various subtypes of nephrotic children. Experimental Mol Pathol 99:590–595 Min QH, Chen XM, Zou YQ, Zhang J, Li J, Wang Y, Li SQ, Gao QF, Sun F, Liu J (2018) Differential expression of urinary exosomal micro RNA s in IgA nephropathy. J Clin Lab Anal 32:e22226 Chen T, Wang C, Yu H, Ding M, Zhang C, Lu X, Zhang CY, Zhang C (2019) Increased urinary exosomal microRNAs in children with idiopathic nephrotic syndrome. EBioMedicine 39:552–561 Ardalan M, Hejazian SM, Sharabiyani HF, Farnood F, Aghdam AG, Bastami M, Ahmadian E, Vahed SZ, Cucchiarini M (2020) Dysregulated levels of glycogen synthase kinase-3β (GSK-3β) and miR-135 in peripheral blood samples of cases with nephrotic syndrome. PeerJ 8:e10377 Kadkhoda S, Eslami S, Mahmud Hussen B, Ghafouri-Fard S (2022) A review on the importance of miRNA-135 in human diseases. Front Genet 13:973585 Yang X, Wang X, Nie F, Liu T, Yu X, Wang H, Li Q, Peng R, Mao Z, Zhou Q (2015) miR-135 family members mediate podocyte injury through the activation of Wnt/β-catenin signaling. Int J Mol Med 36:669–677 Patel P, Woodgett JR (2017) Glycogen synthase kinase 3: a kinase for all pathways? Curr Top Dev Biol 123:277–302 Paeng J, Chang JH, Lee SH, Nam BY, Kang H-Y, Kim S, Oh HJ, Park JT, Han SH, Yoo T-H (2014) Enhanced glycogen synthase kinase-3β activity mediates podocyte apoptosis under diabetic conditions. Apoptosis 19:1678–1690 Boini KM, Amann K, Kempe D, Alessi DR, Lang F (2009) Proteinuria in mice expressing PKB/SGK-resistant GSK3. Am J Physiol Ren Physiol 296:F153–159 Wang Z, Ge Y, Bao H, Dworkin L, Peng A, Gong R (2013) Redox-sensitive glycogen synthase kinase 3β-directed control of mitochondrial permeability transition: rheostatic regulation of acute kidney injury. Free Radical Biology Med 65:849–858 Chen B, Wang P, Liang X, Jiang C, Ge Y, Dworkin LD, Gong R (2021) Permissive effect of GSK3β on profibrogenic plasticity of renal tubular cells in progressive chronic kidney disease. Cell death disease 12:432 Ghobrial EE, Abdel-Aziz HA, Kaddah AM, Mubarak NA (2016) Urinary transforming growth factor β-1 as a marker of renal dysfunction in sickle cell disease. Pediatr Neonatology 57:174–180 Varghese R, Majumdar A (2024) All-trans-retinoic acid and valproic acid: A combinatorial approach for the treatment of nephrotic syndrome. Curr Res Biotechnol 7:100181 Li Y, Liu F-Y, Peng Y-M, Li J, Sun L, Chen X, Duan S-B, Ling G-H, Guo N, Liu Y-H (2010) The relationship between the TGF-β1 gene – 509C/T polymorphism and tubulointerstitial damage resulting from primary nephrotic syndrome. Ren Fail 32:420–427 Wang Y, Chang T, Wu T, Ye W, Wang Y, Dou G, Du H, Hui Y, Guo C (2021) Connective tissue growth factor promotes retinal pigment epithelium mesenchymal transition via the PI3K/AKT signaling pathway. Mol Med Rep 23:1–13 Gelat B, Malaviya P, Rathaur P, Trivedi K, Chaudhary P, Patel B, Johar K, Gelat R (2023) Regulation of epithelial-mesenchymal transition in retinal pigment epithelium and its associated cellular signaling cascades: an updated review. Beni-Suef Univ J Basic Appl Sci 12:94 Tables Table 1 to 9 are available in the Supplementary Files section. 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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-7328776","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502146649,"identity":"2b7f8053-4241-41be-8bd0-4112e62ad560","order_by":0,"name":"Majid A Almeshary","email":"","orcid":"","institution":"Mansoura University Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Majid","middleName":"A","lastName":"Almeshary","suffix":""},{"id":502146650,"identity":"714e494b-d0aa-4036-b557-02faca386e90","order_by":1,"name":"Omali Y El-Khawaga","email":"","orcid":"","institution":"Mansoura University Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Omali","middleName":"Y","lastName":"El-Khawaga","suffix":""},{"id":502146651,"identity":"2bb39999-3243-4e3c-a6bb-28fb3763fc95","order_by":2,"name":"Dalia T Hussein","email":"","orcid":"","institution":"Mansoura University Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"T","lastName":"Hussein","suffix":""},{"id":502146652,"identity":"f0b3be54-be69-4a7f-902a-de1f56e220d8","order_by":3,"name":"Mannar Refaat","email":"","orcid":"","institution":"Mansoura University Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Mannar","middleName":"","lastName":"Refaat","suffix":""},{"id":502146653,"identity":"8ea815c7-40de-4465-8916-0ecea69c74c4","order_by":4,"name":"Ahmed Mahmoud El-Refaey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYJCCD0Bc38/eAOUeIKyDcQaImNkDU0q0lg03EojUwj/78MGGHxWHmRluvk78XPCHQY7vRgLjwy94tEicS0ts7DlzmI1xdu5m6ZltDMaSNxKYjWXwWXOGx/wBb9thHmbp3A3SvA0MiUAXsklL4NEhf4b/Y+Pff4cl2CTPbv7N84ehHqiF/Tc+LQZneBibeRsOG/BI8G6T5mFjSDAA2sL4AY8WwzNshs0yx9ITJHhyt1nztkkYzjzzsFkan1fkzjA/bHxTY51gf/zs5ts8f2zk+Y4nH/z4A58eCGiGMUCeYGxg5iGspQ6Vy0iELaNgFIyCUTByAAAxYVK6PXB58gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1055-7131","institution":"Mansoura University","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"Mahmoud","lastName":"El-Refaey","suffix":""}],"badges":[],"createdAt":"2025-08-08 15:40:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7328776/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7328776/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91286010,"identity":"30b2aca5-1097-453a-af81-f53f8ea6f68c","added_by":"auto","created_at":"2025-09-14 18:18:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":483648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7328776/v1/c2f4545c-235f-4a2f-8d69-20789aaea995.pdf"},{"id":89919858,"identity":"ef1353f8-136f-4859-9190-44fb58a9d139","added_by":"auto","created_at":"2025-08-26 12:36:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36008,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7328776/v1/d4718e97406da75a433f18c3.docx"}],"financialInterests":"","formattedTitle":"miR-135a-5p in Exosomes Promotes Childhood Nephrotic Syndrome via GSK-3β and TGF-β1 Crosstalk","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNephrotic syndrome (NS) is an immune-inflammatory kidney disorder that is one of the main causes of end-stage kidney disease (ESKD) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This syndrome causes defects in the glomerular filtration barrier, leading to excessive loss of protein, fluid, and nutrition in the urine [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The main characteristics of NS are edema, proteinuria, hypoalbuminemia, and dyslipidemia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. NS can be divided into primary, secondary, and congenital NS. Primary NS approximately accounts for 90% of the total NS cases in children [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe incidence of NS varies across populations, ranging from two to seven per 100,000 children, with higher rates reported among individuals of African and South Asian ancestry [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The Middle East and North Africa region is expected to have a comparatively higher incidence of rare diseases than other parts of the world, largely due to the high prevalence of consanguinity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In Egypt, the incidence of NS in children has increased over the years, which is accompanied by worsening kidney function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRenal biopsy, the usual approach to determine disease nature and prognosis, is an invasive surgery with hidden risk and typically not practical for serial monitoring, especially in children. Improper tissue samples can lead to misdiagnosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, it is necessary to find new noninvasive diagnostic and prognostic biomarkers for the disease that are easy to assess, suited to accurately predicting long-term outcomes, and useful for both diagnosis and treatment.\u003c/p\u003e\u003cp\u003eGlucocorticoid is the common treatment protocol for NS that acts as immunosuppressant and anti-inflammatory for several disorders [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The response to glucocorticoid treatment varies between patients. Depending on how well patients respond to glucocorticoid medication, NS is categorized into steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS). SRNS develops when glucocorticoid treatment fails to produce in complete remission within 4\u0026ndash;6 weeks [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The etiology of SRNS is often attributed to structural and functional damage of the podocytes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The response to treatment is distinct according to genetic variations between individuals. Early prediction of glucocorticoid response will allow for an alternative treatment protocol to avoid glucocorticoid side effects [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicro-RNAs (miRNA), histone modifications, and DNA methylation (DNAm) are examples of epigenetic processes that modify gene expression without altering the underlying DNA sequence [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. miRNA is a highly conserved, non-coding small RNA of 22 nucleotides that regulates gene expression at the post-transcriptional level [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. They are involved in many valuable biological processes such as cell growth, differentiation, proliferation, and apoptosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. miRNAs play a critical role in maintaining renal homeostasis under physiological and pathological conditions, as evidenced by the differential expression of specific miRNAs in diverse kidney diseases [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous studies demonstrated the role of miRNAs in the development and progression of glomerular diseases associated with podocyte dysfunction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe alteration of miR-135a and miR-135b expression is a hallmark of podocyte injury, and this expression was observed in response to transforming growth factor beta (TGF-β). The binding of miR-135a and miR-135b to glycogen synthase kinase-3p (GSK3β) affects the GSK3β expression at the transcriptional and translational levels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the expression levels and functional role of circulating exosomal miR-135a-5p in regulating GSK-3β and TGF-β1 expression in patients with nephrotic syndrome (NS). Furthermore, it examined the correlation between these epigenetic and genetic markers and glucocorticoid responsiveness.\u003c/p\u003e"},{"header":"2. Patients and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Patients and controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in the Nephrology Unit of Mansoura University Children\u0026rsquo;s Hospital (MUCH). It includes 70 children with primary NS and 70 apparently healthy controls of matched age and sex. The NS patients were subdivided into three groups based on their initial response to steroid treatment: steroid sensitivity (SSNS), which includes 10 patients; steroid dependence with two consecutive relapses during therapy (SDNS), which includes 20 patients; and steroid resistance (SRNS), which includes 40 patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Mansoura Faculty of Medicine Institutional Research Board, code number: MDP.24.04.148. Informed consent was taken from parents of all subjects before the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion criteria:\u0026nbsp;\u003c/strong\u003echildren with primary NS and aged from 1 to 18 years of both sexes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria:\u003c/strong\u003e patients with secondary or congenital NS, diabetes, leukemia, lupus nephritis, chronic inflammatory disease, and patients who have undergone kidney transplantation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1. \u0026nbsp; Sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom each NS patient and control, two peripheral blood samples were collected. The first was a gel tube to collect serum for isolation of exosomal miRNA, and the other was collected in an EDTA tube for extraction of total RNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. \u0026nbsp;Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1. \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eQuantitative real-time PCR for Exosomal miRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExosomal miRNA was extracted from serum samples using miRNeasy Serum/Plasma Advanced Kit (cat. no. 217004, Qiagen, Germany), then converted to cDNA by miScript Reverse Transcription kit (cat. no. 218161, Qiagen, Germany). RT-PCR was performed using the Maximas SYBR Green/Fluorescein qPCR Master Mix (cat. no. K0243, Thermo Fisher Scientific, Waltham, MA, USA) for miR-135a-5p and U6 as a housekeeping gene following the manufacturer\u0026apos;s instructions using Rotor-Gene Q (Qiagen, USA). The samples were performed in triplicate. The expression level of miRNA was calculated using the equation 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e.\u0026nbsp;The primer sequences of the studied genes are listed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2. \u0026nbsp; \u0026nbsp;Quantitative real-time PCR mRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA has been extracted from blood samples using TRIzols Reagent (cat. no. 15596026, Life Technologies, USA) following the manufacturer\u0026rsquo;s protocol. RNA samples were converted to complementary DNA (cDNA) with QuantiTect Reverse Transcription Kit (Cat no. 205311 Qiagen, USA). cDNA samples stored at -80\u0026deg;C. Quantitative RT-PCR was performed using Maximas SYBR Green/Fluorescein qPCR Master Mix (cat. no K0243, Thermo Fisher Scientific, Waltham, MA, USA). The expression of glycogen synthase kinase-3\u0026beta; (GSK-3\u0026beta;), and transforming growth factor beta-1 (TGFB-1) was quantified using Rotor-Gene Q (Qiagen, USA) using GAPDH as internal control. The samples were performed in triplicate. The expression level of mRNA was calculated using the equation 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e. The primer sequences of the studied genes are listed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3. \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using the Statistical package for Social Science (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). The continuous data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) using the student\u0026apos;s t-test to compare the statistical difference between two study groups, while the Kruskal-Wallis test was used to assess the statistical difference between more than two study groups. Categorical data, on the other hand, were compared using the Chi-square. The receiver operating characteristic\u0026nbsp;(ROC) Curve was used to evaluate the sensitivity and specificity for quantitative diagnostic measures.\u0026nbsp;Spearman\u0026apos;s rho correlation coefficients (\u003cem\u003esr\u003c/em\u003e) were calculated for the variables. Logistic regression was used to predict risk factors when the dependent variable is categorical. \u003cem\u003ep\u003c/em\u003e \u0026le; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003ePatients\u0026rsquo; demographics and clinicopathological characteristics are shown in Table 2. Out of 70 NS patients, 54.3 % were males and 45.7 % were females; the mean \u0026plusmn; SD age of all patients was 9.06 \u0026plusmn; 3.75 years, while the mean \u0026plusmn; SD age of onset was 5.70 \u0026plusmn; 3.13\u0026nbsp;years. The disease duration averages\u0026nbsp;3.36 \u0026plusmn; 2.88\u0026nbsp;years.\u003c/p\u003e\n\u003cp\u003eExosomal miR-135a-5p was significantly lower in NS patients compared to controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Conversely, GSK-3\u0026beta; and TGF\u0026beta;-1 gene expression levels were significantly higher in NS than in control groups (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) (Table 3).\u003c/p\u003e\n\u003cp\u003eTable 4 represents a relation between treatment response and gene expression. Regarding exosomal miR-153a-5p, the expression level showed significantly lower levels in SSNS, SDNS, and SRNS than the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001). In comparing the SRNS and SSNS groups, the level was significantly lower in the SRNS group (\u003cem\u003ep\u003c/em\u003e=0.022), with no significant differences between SSNS versus SDNS and SRNS versus SDNS.\u003c/p\u003e\n\u003cp\u003eThe mRNA level of GSK-3\u0026beta; showed a significantly higher level in SDNS and SRNS than the control group (\u003cem\u003ep\u003c/em\u003e =\u0026nbsp;0.021,\u003cem\u003e\u0026nbsp;p\u003c/em\u003e\u0026lt;0.001, respectively), with a significant increase in the SRNS group compared to the SSNS group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001), with no significant differences between the SSNS group versus the control or SDNS groups.\u003c/p\u003e\n\u003cp\u003eAdditionally, TGF\u0026beta;-1 gene expression level showed significantly elevated levels in the SSNS, SDNS, and SRNS than the control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001), as well as in SRNS than SSNS and SDNS groups (\u003cem\u003ep\u003c/em\u003e = 0.008, \u003cem\u003ep\u003c/em\u003e =0.016, respectively), with no significant differences between SSNS versus SDNS groups.\u003c/p\u003e\n\u003cp\u003eThe validity of the studied markers was evaluated in Table 5 for discriminating between NS patients and the control group using the ROC curve. The area under the curve (AUC) values indicate strong discriminatory power for exosomal miR-135a-5p, showing the highest AUC of 0.932, followed by TGF\u0026beta;-1 at 0.917, then GSK-3\u0026beta; at\u0026nbsp;0.782\u0026nbsp;(\u003cem\u003ep\u0026nbsp;\u003c/em\u003e=\u0026nbsp;\u0026lt;0.001). The sensitivity and specificity values further support the utility of these markers. The cut-off values for each marker are provided, indicating the thresholds for optimal discrimination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Table 6, the AUC values indicate that GSK-3\u0026beta; has the highest predictive capabilities, with AUCs of 0.795 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001), followed by TGF\u0026beta;-1 (AUC=\u0026nbsp;0.764,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001), then exosomal miR-135a-5p (AUC=\u0026nbsp;0.715, \u003cem\u003ep\u003c/em\u003e =\u0026nbsp;0.002).\u003c/p\u003e\n\u003cp\u003eThe correlation between different markers in studied NS groups was presented in Table 7. Among total NS patients, exosomal miR-135a-5p gene expression showed a significant negative correlation with GSK-3\u0026beta; and TGF\u0026beta;-1. In the SSNS group, GSK-3\u0026beta; gene expression showed a significant negative correlation with TGF\u0026beta;-1. While among the SDNS and SRNS groups, no significant correlations were found between the studied markers.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;8 presents a logistic regression analysis for predicting susceptibility to NS. The univariate analysis shows that lower exosomal miR-135a-5p and higher GSK-3\u0026beta; and TGF\u0026beta;-1 are all significant predictors of NS susceptibility. While in the multivariate analysis, lower exosomal miR-135a-5p and higher TGF\u0026beta;-1 remained significant predictors, suggesting that these markers could be valuable in assessing the risk of developing NS.\u003c/p\u003e\n\u003cp\u003eTable 9 details a logistic regression analysis for predicting SRNS on top of the combination of SSNS and SDNS. The univariate analysis identifies several significant predictors, including the presence of consanguinity, lower exosomal miR-135a-5p, and higher GSK-3\u0026beta; and TGF\u0026beta;-1, indicating their association with SRNS risk. In the multivariate analysis, higher GSK-3\u0026beta; and TGF\u0026beta;-1 remained significant predictors, emphasizing their potential role in distinguishing SRNS from SSNS and SDNS.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn clinical nephrology, NS is the most prevalent disease in childhood. NS is typically a pathognomonic sign of glomerular disease; however, the underlying clinical etiology varies widely. Although renal function was normal in most NS patients at the beginning, prolonged NS will often result in progressive renal failure and a lifelong need for renal replacement therapy (dialysis or transplantation) [19].\u003c/p\u003e\n\u003cp\u003eMore than 85% of NS cases in children under the age of 18 years are caused by focal segmental glomerulosclerosis (FSGS) and minimal change nephropathy (MCN). Conversely, prevalent causes in adult patients include amyloidosis, lupus nephritis, diabetic glomerulosclerosis, membranoproliferative glomerulonephritis, minimal change nephropathy, and focal glomerulosclerosis [20].\u003c/p\u003e\n\u003cp\u003ePrevious studies in NS cleared that the main obstacle is in the accurate and early diagnosis, medical follow-up, and choosing the appropriate treatment for patients in terms of age, gender, race, and genetic differences.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;miRNAs play a crucial role in kidney dysfunction and structural damage, as well as maintaining normal regulatory systems. Previous research identified dysregulated miRNAs in the renal tissues of children with nephropathy, which was linked to the pathogenesis of nephrosis [21].\u003c/p\u003e\n\u003cp\u003eIn this study, the quantification of exosomal miR-135a-5p was found to be significantly downregulated in NS patients compared to the control group. This significant downregulation shows that this miRNA may play an integrated or distinctive role in NS pathophysiology. Also, this exosomal miR-135a-5p was found to be significantly decreased in the SRNS group compared to either the healthy control group or the SSNS group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePreviously, several studies reported that exosomal miRNA expression levels in urine have been found to be significantly altered in adults with kidney diseases such as chronic kidney disease (CKD), lupus nephritis, and IgA nephropathy [22]. This suggests that urinary exosomal miRNAs may be promising biomarkers of adult kidney disease. The clinical significance of the urine exosomal miRNA signature in pediatric idiopathic NS patients remains unknown [23].\u003c/p\u003e\n\u003cp\u003eOur results regarding miR-135a-5p were in agreement with the Iranian study, which stated that plasma samples from both the NS and membranous glomerulonephritis (MGN) groups showed significantly lower expression levels of miR-135a-5p (\u003cem\u003ep\u003c/em\u003e = 0.020 and \u003cem\u003ep\u003c/em\u003e = 0.040, respectively) compared to the control group. The FSGS group had a significantly lower level of circulating miR-135a-5p (\u003cem\u003ep\u003c/em\u003e = 0.046) than the controls. There were no significant differences in miR-135a-5p expression levels between the FSGS and MGN groups in plasma samples (\u003cem\u003ep\u003c/em\u003e = 0.501) [24].\u003c/p\u003e\n\u003cp\u003eROC analysis in our study showed that miR-135a-5p had an AUC of 0.932 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), with reliable sensitivity and specificity in plasma, indicating high diagnostic power in distinguishing NS patients from healthy controls; also, miR-135a-5p showed a moderate diagnostic power at AUC = 0.715 (\u003cem\u003ep\u003c/em\u003e = 0.002) to differentiate the SRNS patients from the rest of the NS patients. This result is in contrast with the results of Ardalan et al., they stated that miR-135a-5p did not have reliable value in discriminating NS patients from controls, AUC = 0.605 in plasma samples and AUC = 0.510 in peripheral blood mononuclear cells (PBMCs) [24].\u003c/p\u003e\n\u003cp\u003eMiR-135 dysregulation has been observed in many non-neoplastic disorders, including NS, Alzheimer\u0026apos;s disease, depression, Parkinson\u0026apos;s disease, atherosclerosis, diabetes, pulmonary arterial hypertension, endometriosis, epilepsy, and allergies. Furthermore, a number of signalling pathways, including Wnt/\u0026beta;-catenin, TGF-\u0026beta;/SMAD, p38 MAPK, NF-\u0026kappa;B, and IL-6/STAT3, have been shown to be modulated by miR-135. This broad method of action demonstrates the intricacy of the functional network impacted by miR-135 [25].\u003c/p\u003e\n\u003cp\u003eGSK-3\u0026beta; gene is one of the most potential target genes of the miR-135. The miR-135 family has two members: miR-135a and miR-135b, which play roles in many biological pathways [26]. GSK3\u0026beta; is a highly conserved serine/threonine kinase that was first identified as regulating the insulin signaling pathway and glycogen biosynthesis. Since then, it has been linked to numerous other pathophysiologic processes and conditions, such as organ damage and repair, cancer, neurodegenerative diseases, and more lately kidney diseases [27].\u003c/p\u003e\n\u003cp\u003eReferring to our molecular analysis comparison between NS patients and healthy control group regarding GSK-3b\u0026nbsp;gene expression, there was a high significant elevation in NS patients compared to healthy control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). This elevation was observed in the SRNS group compared to the control and SSNS groups, while no statistically significant difference was observed between the control and SSNS groups. One of the results consistent with our results is a study by Ardalan et al., which stated that there were statistically significant increases in GSK-3\u0026beta; expression level in NS (\u003cem\u003ep\u003c/em\u003e = 0.001), MGN (\u003cem\u003ep\u003c/em\u003e = 0.002), and FSGS (\u003cem\u003ep\u003c/em\u003e = 0.015) groups compared to the control group [24].\u003c/p\u003e\n\u003cp\u003eThe up-regulation of GSK-3\u0026beta; expression in tubular cells and glomeruli of the kidney indicates a detrimental role of GSK-3 in podocyte injury. Its over-activation is linked to various kidney diseases, including proteinuric glomerulopathies and CKD, as reported by Paeng et al. [28].\u003c/p\u003e\n\u003cp\u003eROC diagram analysis in our study showed that GSK-3\u0026beta; with AUC = 0.782 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) with reliable sensitivity and specificity in plasma had a significant moderate diagnostic ability in differentiating between NS patients and the healthy control group; also, GSK-3\u0026beta; showed a moderate diagnostic power at AUC = 0.795 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001) to differentiate the SRNS patients from the rest of the NS patients. Our result is in agreement with Ardalan and colleagues, they approved that GSK-3\u0026beta; with AUC = 0.72 (\u003cem\u003ep\u003c/em\u003e = 0.002) and good sensitivity and specificity in PBMCs demonstrated diagnostic power in distinguishing NS patients from healthy controls [24].\u003c/p\u003e\n\u003cp\u003ePrevious studies reported that high expression of GSK-3\u0026beta; in renal tubules has a destructive role [29], also GSK-3\u0026beta; has been associated with acute kidney injury (AKI) and progressive CKD, GSK-3\u0026beta; modulates renal tubular and interstitial damage [30].\u003c/p\u003e\n\u003cp\u003eGSK3\u0026beta; is predominantly expressed in the kidney\u0026apos;s glomeruli and proximal renal tubules. \u0026nbsp;Acute injuries increase GSK3\u0026beta; activity in glomeruli and renal tubules by suppressing inhibitory phosphorylation. GSK3\u0026beta; hyperactivity has been linked to kidney cell death and acute renal damage through various pathways, including sensitization of mitochondrial permeability transition, loss of cytoskeleton integrity, and potentiation of NF-\u0026kappa;B-dependent inflammatory responses [31].\u003c/p\u003e\n\u003cp\u003eIn a previous study by Zhang et al., stated that miR‑135a‑5p/SIRT1/Smad3 pathway was implicated in TGF\u0026beta;1‑induced renal fibrosis [14]. TGF-\u0026beta;1 is a well-known profibrogenic cytokine that contributes significantly to the development of CKD. The fibrotic process results from the overproduction of pathological matrix that is produced in response to TGF-\u0026beta;1 stimulating matrix protein synthesis and decreasing its degradation [32].\u003c/p\u003e\n\u003cp\u003eIn our study, the expression level of TGF\u0026beta;-1 was significantly elevated in NS group compared to the healthy control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). NS subtypes showed a significant increase in TGF\u0026beta;-1 expression compared to the healthy control group, with a significant elevation in SRNS group compared to the SSNS and SDNS groups. ROC analysis in our study showed that TGF\u0026beta;-1 has AUC = 0.917 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) with strong reliable sensitivity and specificity in plasma and had high diagnostic power in separating NS patients from healthy controls; also, TGF\u0026beta;-1 showed a moderate diagnostic power at AUC = 0.764 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001) to differentiate the SRNS patients from the rest of the NS patients. Along with our results, previous studies have also demonstrated increased TGF\u0026beta;1 levels in the NS group compared to the control group in rats [33]. Another study by Li et al. stated that the elevated TGF\u0026beta;1 in serum samples in patients with tubulointerstitial damage compared to controls [34].\u003c/p\u003e\n\u003cp\u003eThe underlined mechanism by which TGF\u0026beta;-1 is involved in the fibrosis process is through the epithelial to mesenchymal transition (EMT). The tubular epithelial cells are transformed to mesenchymal fibroblasts and migrate to adjacent interstitial parenchyma along with local and circulating cells; this process includes proteinuria as well as hypoxia [35]. TGF\u0026beta;-1 modulates biological reactions through interactions with Smads, MAPK, and the Jagged/Notch signaling pathways [36].\u003c/p\u003e\n\u003cp\u003eIn conclusion, the expression of exosomal miR-135a-5p, GSK-3\u0026beta;, and TGF\u0026beta;-1 has succeeded in distinguishing between healthy individuals and NS patients and can be used for predicting the response to glucocorticoid treatment. The importance of investigating molecular and epigenetic expression in children with NS and how they affect glucocorticoid treatment has been highlighted by this work. To overcome the limitations in this study, we recommend conducting further studies on a larger scale to demonstrate the role of glucocorticoids on the expression of these markers to strengthen the validity and applicability of the findings. Further investigations on different epigenetic markers that affect various signaling pathways could provide a more comprehensive understanding of the glucocorticoid treatment\u0026rsquo;s mechanisms and effects, leading to better clinical applications. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical approval\u003c/h2\u003e\u003cp\u003e The study was approved by the Mansoura Faculty of Medicine Institutional Research Board, code number: MDP.24.04.148. Informed consent was taken from parents of all subjects before the study.\u003c/p\u003e\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePolitano SA, Colbert GB, Hamiduzzaman N (2020) Nephrotic Syndrome. Prim Care 47:597\u0026ndash;613\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVerma PR, Patil P (2024) Nephrotic Syndrome: A Review. Cureus 16:e53923\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee J (2021) Children with Kidney Disease: An Overview of Pediatric Primary Nephrotic Syndrome. Pediatric nursing 47\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi FX, Hou YL, Zhou LS, Dong Y, Zhao JW, Li ZX (2021) The status and model of children primary nephrotic syndrome in continuing nursing. Ann Palliat Med 10:2398\u0026ndash;2407\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlenazi SA, Alenazi SA (2024) Incidence and Pathological Patterns of Nephrotic Syndrome among Infants and Children: A Systematic Review. Cureus 16\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalman MA, Elgebaly A, Soliman NA (2024) Epidemiology and outcomes of pediatric autosomal recessive polycystic kidney disease in the Middle East and North Africa. Pediatr Nephrol 39:2569\u0026ndash;2578\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEid R, Fathy AA, Hamdy N (2020) Health-related quality of life in Egyptian children with nephrotic syndrome. Qual Life Res 29:2185\u0026ndash;2196\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang Z, Zhang Y, Zhou J, Zhang Y (2017) Urinary exosomal miR-193a can be a potential biomarker for the diagnosis of primary focal segmental glomerulosclerosis in children. Biomed Res Int 2017:7298160\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao X, Hwang DY, Kao HY (2018) The Role of Glucocorticoid Receptors in Podocytes and Nephrotic Syndrome. Nucl Receptor Res 5:101323\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSachdeva S, Khan S, Davalos C, Avanthika C, Jhaveri S, Babu A, Patterson D, Yamani AJ (2021) Management of Steroid-Resistant Nephrotic Syndrome in Children. Cureus 13:e19363\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee JM, Kronbichler A, Shin JI, Oh J (2021) Current understandings in treating children with steroid-resistant nephrotic syndrome. Pediatr Nephrol 36:747\u0026ndash;761\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMotavalli R, Majidi T, Pourlak T, Abediazar S, Shoja MM, Vahed SZ, Etemadi J (2021) The clinical significance of the glucocorticoid receptors: Genetics and epigenetics. J Steroid Biochem Mol Biology 213:105952\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerdasco M, Esteller M (2019) Clinical epigenetics: seizing opportunities for translation. Nat Rev Genet 20:109\u0026ndash;127\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang J, Zhang L, Zha D, Wu X (2020) Inhibition of miRNA-135a-5p ameliorates TGF-β1-induced human renal fibrosis by targeting SIRT1 in diabetic nephropathy. Int J Mol Med 46:1063\u0026ndash;1073\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen L, Heikkinen L, Wang C, Yang Y, Sun H, Wong G (2019) Trends in the development of miRNA bioinformatics tools. Brief Bioinform 20:1836\u0026ndash;1852\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchena FP, Serino G, Sallustio F (2013) MicroRNAs in kidney diseases: new promising biomarkers for diagnosis and monitoring. Nephrol Dialysis Transplantation 29:755\u0026ndash;763\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu F, Chen J, Luo C, Meng X (2022) Pathogenic Role of MicroRNA Dysregulation in Podocytopathies. Front Physiol 13:948094\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Jarad G, Tripathi P, Pan M, Cunningham J, Martin DR, Liapis H, Miner JH, Chen F (2010) Activation of NFAT signaling in podocytes causes glomerulosclerosis. J Am Soc Nephrol 21:1657\u0026ndash;1666\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSzeto C-C (2014) Urine miRNA in nephrotic syndrome. Clin Chim Acta 436:308\u0026ndash;313\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCameron JS (1996) Nephrotic syndrome in the elderly. Seminars in nephrology, pp 319\u0026ndash;329\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu M, Wang C, Yuan Y, Zhu Y, Yin Z, Xia Z, Zhang C (2015) Differentially expressed microRNAs in kidney biopsies from various subtypes of nephrotic children. Experimental Mol Pathol 99:590\u0026ndash;595\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMin QH, Chen XM, Zou YQ, Zhang J, Li J, Wang Y, Li SQ, Gao QF, Sun F, Liu J (2018) Differential expression of urinary exosomal micro RNA s in IgA nephropathy. J Clin Lab Anal 32:e22226\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen T, Wang C, Yu H, Ding M, Zhang C, Lu X, Zhang CY, Zhang C (2019) Increased urinary exosomal microRNAs in children with idiopathic nephrotic syndrome. EBioMedicine 39:552\u0026ndash;561\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArdalan M, Hejazian SM, Sharabiyani HF, Farnood F, Aghdam AG, Bastami M, Ahmadian E, Vahed SZ, Cucchiarini M (2020) Dysregulated levels of glycogen synthase kinase-3β (GSK-3β) and miR-135 in peripheral blood samples of cases with nephrotic syndrome. PeerJ 8:e10377\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKadkhoda S, Eslami S, Mahmud Hussen B, Ghafouri-Fard S (2022) A review on the importance of miRNA-135 in human diseases. Front Genet 13:973585\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang X, Wang X, Nie F, Liu T, Yu X, Wang H, Li Q, Peng R, Mao Z, Zhou Q (2015) miR-135 family members mediate podocyte injury through the activation of Wnt/β-catenin signaling. Int J Mol Med 36:669\u0026ndash;677\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel P, Woodgett JR (2017) Glycogen synthase kinase 3: a kinase for all pathways? Curr Top Dev Biol 123:277\u0026ndash;302\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaeng J, Chang JH, Lee SH, Nam BY, Kang H-Y, Kim S, Oh HJ, Park JT, Han SH, Yoo T-H (2014) Enhanced glycogen synthase kinase-3β activity mediates podocyte apoptosis under diabetic conditions. Apoptosis 19:1678\u0026ndash;1690\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoini KM, Amann K, Kempe D, Alessi DR, Lang F (2009) Proteinuria in mice expressing PKB/SGK-resistant GSK3. Am J Physiol Ren Physiol 296:F153\u0026ndash;159\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Z, Ge Y, Bao H, Dworkin L, Peng A, Gong R (2013) Redox-sensitive glycogen synthase kinase 3β-directed control of mitochondrial permeability transition: rheostatic regulation of acute kidney injury. Free Radical Biology Med 65:849\u0026ndash;858\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen B, Wang P, Liang X, Jiang C, Ge Y, Dworkin LD, Gong R (2021) Permissive effect of GSK3β on profibrogenic plasticity of renal tubular cells in progressive chronic kidney disease. Cell death disease 12:432\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhobrial EE, Abdel-Aziz HA, Kaddah AM, Mubarak NA (2016) Urinary transforming growth factor β-1 as a marker of renal dysfunction in sickle cell disease. Pediatr Neonatology 57:174\u0026ndash;180\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVarghese R, Majumdar A (2024) All-trans-retinoic acid and valproic acid: A combinatorial approach for the treatment of nephrotic syndrome. Curr Res Biotechnol 7:100181\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Liu F-Y, Peng Y-M, Li J, Sun L, Chen X, Duan S-B, Ling G-H, Guo N, Liu Y-H (2010) The relationship between the TGF-β1 gene\u0026thinsp;\u0026ndash;\u0026thinsp;509C/T polymorphism and tubulointerstitial damage resulting from primary nephrotic syndrome. Ren Fail 32:420\u0026ndash;427\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Chang T, Wu T, Ye W, Wang Y, Dou G, Du H, Hui Y, Guo C (2021) Connective tissue growth factor promotes retinal pigment epithelium mesenchymal transition via the PI3K/AKT signaling pathway. Mol Med Rep 23:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGelat B, Malaviya P, Rathaur P, Trivedi K, Chaudhary P, Patel B, Johar K, Gelat R (2023) Regulation of epithelial-mesenchymal transition in retinal pigment epithelium and its associated cellular signaling cascades: an updated review. Beni-Suef Univ J Basic Appl Sci 12:94\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 9 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nephrotic syndrome, Glucocorticoid, miR-135a-5p, GSK3β, TGFB-1, children","lastPublishedDoi":"10.21203/rs.3.rs-7328776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7328776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNephrotic syndrome (NS) is an autoimmune kidney disorder that is characterized by severe protein loss in urine, which results in edema, hyperlipidemia, and hypoalbuminemia. Glucocorticoid therapy is the main treatment for NS patients; however, the unsuitable treatment of NS can lead to chronic glomerulonephritis and, in turn, end-stage renal disease (ESRD). Genetic and epigenetic variations between individuals lead to different responses to treatment. Therefore, this study aimed to investigate an early predictor for glucocorticoid response in NS patients. Therefore, this study aimed to investigate an early predictor for glucocorticoid response in NS patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis study was conducted on 70 NS patients enrolled from the Nephrology Unit of Mansoura University Children\u0026rsquo;s Hospital and 70 aberrantly healthy individuals as a control group. Peripheral blood samples were collected from all subjects for miR-135a-5p, GSK3β, and TGFB-1 expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003emiR-135a-5p level was significantly decreased, while the GSK3β and TGFB-1 expression levels were increased in the NS group compared to the control group. Regarding the NS subtypes, the steroid-resistant nephrotic syndrome (SRNS) group exhibits a significant reduction in miR-135a-5p and a significant elevation in the levels of GSK3β and TGFB-1 compared to the steroid-sensitive nephrotic syndrome (SSNS) group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese biomarkers demonstrated moderate diagnostic accuracy in distinguishing nephrotic syndrome (NS) from controls and effectively identified steroid-resistant NS (SRNS) cases. Their potential as early predictors of glucocorticoid response offers promising clinical utility for personalized therapeutic strategies in pediatric NS.\u003c/p\u003e","manuscriptTitle":"miR-135a-5p in Exosomes Promotes Childhood Nephrotic Syndrome via GSK-3β and TGF-β1 Crosstalk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-26 12:28:32","doi":"10.21203/rs.3.rs-7328776/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e05a949-02c4-41e8-8010-a8f68831c0f5","owner":[],"postedDate":"August 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-14T18:10:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-26 12:28:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7328776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7328776","identity":"rs-7328776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}