Expression and Significance of the GDF11 Protein in Ureteropelvic Junction Obstruction (UPJO)

Expression and Significance of the GDF11 Protein in Ureteropelvic Junction Obstruction (UPJO) | 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 Expression and Significance of the GDF11 Protein in Ureteropelvic Junction Obstruction (UPJO) Weihua Lao, Tong Shi, Qin Chen, Bao Qiao, Sai Ma, Cuiwei Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3873300/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 To explore the expression of growth differentiation factor 11 (GDF11), a member of the TGF-β superfamily, in pediatric ureteropelvic junction obstruction (UPJO) patients. Methods Five patients diagnosed with severe neonatal hydronephrosis underwent laparoscopic pyeloplasty between January 2021 and November 2022. Tissue samples from the constricted ureteropelvic junction and adjacent normal ureteral segments served as controls. The presence of the GDF11 protein in these tissues was examined using immunohistochemistry and Western blot techniques. Subsequently, a statistical analysis compared the protein expression levels in both groups. Results Immunohistochemical staining revealed that the frequency of high GDF11 protein expression in the narrowed ureter (60%) was significantly greater than that in the normal ureter (40%). Western blot analysis indicated that the expression of GDF11 was significantly greater in the narrowed ureter than in the normal ureter. Conclusions The increased expression of GDF11 in UPJO is noteworthy and deserves clinical attention. GDF11 UPJO Protein Figures Figure 1 Figure 2 1. Introduction Pediatric hydronephrosis represents a prevalent congenital anomaly, with 1%-5% of pregnant women encountering fetal hydronephrosis during prenatal ultrasound screenings. Among these cases, 19%-25% are attributed to ureteropelvic junction obstruction (UPJO). The advent of immunohistochemistry and advanced molecular biology techniques in recent years has enhanced the understanding of UPJO's pathogenesis. It has been established that the development of embryonic kidneys is influenced by a variety of genes. However, the precise mechanisms involved still necessitate further exploration. Consequently, elucidating the etiology of UPJO to diminish its incidence continues to be a primary focus in ongoing research. Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a secretory protein that belongs to the transforming growth factor β (TGF-β) superfamily. Molecules belonging to the TGF-β superfamily are extensively found across a range of organisms, from nematodes to mammals, and are crucial in regulating various cellular processes. These include cell proliferation, differentiation, adhesion, migration, apoptosis, and the induction of cartilage formation and differentiation. They are crucial in the development and postinjury repair of organisms and various organs and participate in numerous pathological processes. The maturation process of GDF11 closely resembles that of TGF-β1, TGF-β2, TGF-β3, and GDF8. These molecules have been demonstrated to be involved in kidney development and injury repair. As part of the TGF-β superfamily, GDF11 additionally plays a significant role in embryonic development. Recent studies have shown that GDF11 is involved in the development of metanephros and the regeneration of renal tubules after acute kidney injury, strongly indicating its potential value in treating UPJO. Therefore, this study explored the clinical significance of GDF11 expression in UPJO. 2. Materials and Methods 2.1 General information Between January 2021 and November 2022, five neonates with severe hydronephrosis underwent laparoscopic pyeloplasty at a selected hospital. Tissue samples were harvested from both the constricted ureteropelvic junction (UPJ) and the adjacent normal ureteral section of each patient for comparative analysis. These samples were immediately frozen for preservation and subsequently prepared for paraffin embedding to facilitate further analysis. The inclusion criteria for patients were as follows: ① Prenatal ultrasound detection and postnatal color Doppler ultrasound diagnosis of UPJO within 48 h after birth. Anteroposterior diameter (APD) of the renal pelvis ≥ 20 cm, classified as a Society for Fetal Urology (SFU) grade of 4; and ② Age at the time of surgery < 1 month. Exclusion criteria: Patients with other urinary tract anomalies, such as vesicoureteral reflux, stenosis at other locations of the ureter, or a duplicated kidney were excluded. Patients with severe liver and renal dysfunction, postoperative recurrence of UPJO, or whose parents refused surgery or were noncooperative during follow-up were also excluded. 2.2 Immunohistochemistry Paraffin sections were initially deparaffinized in water. Subsequently, these sections underwent a 5–10 min incubation with 3% H 2 O 2 at room temperature to suppress endogenous peroxidase activity. After rinsing with distilled water, the sections were immersed in phosphate buffer saline (PBS) for 5 min, a process repeated twice. If necessary, antigen retrieval could be conducted following this step. The sections were then blocked using 5–10% normal goat serum (diluted in PBS) at room temperature for 10 min, after which the serum was drained off without washing. The primary antibody solution was then applied, and the samples incubated at 37°C for 1–2 h or overnight at 4°C. This was followed by a PBS rinse for 5 min, repeated thrice. Subsequently, a biotinylated secondary antibody solution was added, and the sections were incubated at 37°C for 10–30 min. Another series of rinses with PBS for 5 min each, repeated thrice, was conducted. After rinsing, an adequate amount of horseradish peroxidase or alkaline phosphatase-conjugated streptavidin solution was applied, and the sections were incubated at 37°C for 10–30 min. This was succeeded by additional rinses with PBS for 5 min each, repeated thrice. The sections were then stained with a chromogen for 3–15 min, washed thoroughly with tap water, counterstained, dehydrated, cleared, and mounted. The GDF11 immunohistochemistry results underwent quantitative analysis by two pathologists using a two-tier scoring method. Cellular staining was scored as follows: 0 for no staining, 1 for light yellow, 2 for brownish yellow, and 3 for yellow-brown. The percentage of positive cells was determined (0–100%). The expression score was the product of the degree of cellular staining and the percentage of positive cells (0–3). A total score above 1 indicated high expression. 2.3 Western blot analysis Tissues underwent lysis and homogenization in RIPA buffer, followed by centrifugation at 13,000×g for the extraction of total proteins. The protein concentration was determined using the BCA method and normalized to 3000 ng/mL. The samples were then mixed with loading buffer and boiled for 10 min. Electrophoresis was subsequently conducted on a 10% polyacrylamide gel at 120 V, followed by protein transfer to a PVDF membrane at 200 mA. The membrane was blocked using 5% nonfat milk powder. Primary antibodies, including those against GDF11, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were incubated at 4°C overnight. This step was followed by incubation with corresponding secondary antibodies for specific binding. Protein visualization was achieved using an enhanced chemiluminescence (ECL) solution and a chemiluminescence imaging system. Band intensity was quantified using FlowJ software, and the relative expression levels of the target proteins were calculated, using GAPDH as an internal control protein. 2.4 Statistical methods The data were analyzed using SPSS 14.0 statistical software. Quantitative data are expressed as the mean ± standard deviation (x ± s). A t -test was used for comparisons, and a P value < 0.05 was used to indicate statistical significance. 3. Results 3.1 Immunohistochemistry results Immunohistochemical analysis indicated a high expression of GDF11 in UPJO tissues, exhibiting a significant expression rate of 60%. This rate is significantly greater than the 40% increase in expression observed in normal ureter tissues (Fig. 1). 3.2 Western blot analysis The expression of the GDF11 protein was significantly greater in UPJO tissues than in normal ureter tissues. A marked elevation in the grayscale intensity of GDF11 expression in UPJO tissues was observed, averaging at 55097.28 ± 351.67. This contrasted with the lower average grayscale intensity in normal ureter tissues, recorded at 32993.43 ± 487.95. The disparity in expression levels between these tissues was found to be statistically significant (P = 0.000) (Fig. 2). 4. Discussion Pediatric hydronephrosis is a common congenital anomaly, and prenatal ultrasound (US) reveals fetal hydronephrosis in approximately 1–5% of pregnant women. Among these instances, 19–25% are attributed to UPJO. This condition is more commonly observed in boys than in girls and is more prevalent on the left side compared to the right. Bilateral UPJO is noted in 10–40% of cases [ 1 ] [ 2 ]. The pathogenesis of UPJO remains incompletely understood and is currently in the early stages of exploration. However, recent advancements in immunohistochemistry and modern molecular biology have enhanced the understanding of UPJO's pathogenesis. Research indicates that the development of embryonic kidneys is regulated by various genes, yet the precise underlying mechanisms still demand further investigation. Thus, elucidating the etiology of UPJO to reduce its incidence is of significant clinical importance. Growth and differentiation factor 11 (GDF11), also known as BMP11, is a secretory protein and a member of the TGF-β superfamily [ 3 ]. GDF11 is pivotal in embryogenesis and development. It has been shown to inhibit the formation of skeletal muscle cells [ 4 ], stimulate ureteric bud formation, and contribute to kidney development [ 5 ]. GDF11 exhibits widespread expression in various embryonic tissues, including the tailbud, limbs, and nervous system [ 6 ]. Mice deficient in Gdf11 (Gdf11-/-) demonstrate perinatal lethality with abnormalities in the upper jaw and kidneys [ 7 ]. The relationship of GDF11 with tumorigenesis is notable, as its expression is altered in tumors. It can either enhance tumor growth in colon cancer or suppress cell proliferation in breast cancer, and it is being investigated as a potential biomarker for prognosis and aggressiveness in liver cancer, pancreatic cancer, melanoma, and OSCC [ 8 ]. While the role of GDF11 in inflammatory processes remains unclear, its influence on inflammation has been acknowledged [ 9 ]. This study explored the expression level of the GDF11 protein in UPJO tissues and revealed a significant increase in GDF11 expression compared to that in normal ureter tissues. Fibrosis in tissues and organs is a significant pathological alteration in the progression of numerous diseases, often resulting in functional impairment of these tissues and organs. Various members of the TGF-β superfamily play roles in the regulation of tissue fibrosis [ 10 ]. For example, TGF-β1 is known as a powerful pro-fibrotic cytokine, indicating that GDF11 might also contribute to fibrosis development. Research has suggested that GDF11 could act as an epithelial-mesenchymal transition (EMT) promoting factor, engaging in fibrosis and potentially exacerbating kidney diseases [ 11 , 12 ]. Histologically, in UPJO, the typical structure of a normal ureter comprises an organized arrangement of the mucosal layer, the inherent layer, and the muscular layer, all encased in adipose tissue. In UPJO cases, proliferation of fibrous tissue and alteration of smooth muscle in the ureteral wall can lead to thickening and rigidity of the UPJ wall, resulting in luminal narrowing and adversely affecting ureteral peristalsis. This can obstruct urine flow from the kidney, culminating in hydronephrosis [ 13 ]. Therefore, the expression of GDF11 in UPJ tissues holds considerable clinical significance. This retrospective study, which included limited sample size, underscores the need for further research with a larger sample size to validate these findings and to elucidate the specific molecular mechanisms of GDF11 in UPJO development and progression. Abbreviations growth differentiation factor (GDF11); ureteropelvic junction obstruction (UPJO); bone morphogenetic proteins 11 (BMP11); transforming growth factor β (TGF-β); Growth differentiation factor 8 (GDF8), Anteroposterior diameter (APD); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); phosphate buffer saline (PBS); enhanced chemiluminescence (ECL) Declarations Authors' contributions: TS: Conceptualization, Methodology, Software; WL: Data curation, Writing- Original draft preparation. QC: Visualization, Investigation; QB: Supervision; SM: Software, Validation; CL: Writing- Reviewing and Editing. All authors read and approved the final manuscript. Acknowledgements: Not applicable. Funding: This work was supported by the Medical Science and Technology Research Foundation of Guangdong Province (Grant number A20222241). Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interests: The authors declare that they have no competing interests. Ethics approval: The study was approved by the ethics review board of Guangdong Women and Children Hospital (No. 202101317) in accordance with the Declaration of Helsinki. Written informed consent have been obtained from a parent and/or legal guardian for study participation. Consent for publication: Not applicable. References Cost NG, Noh PH, Devarajan P, Ivancic V, Reddy PP, Minevich E, et al. Urinary NGAL levels correlate with differential renal function in patients with ureteropelvic junction obstruction undergoing pyeloplasty. J Urol. 2013;190(4 Suppl):1462–7. 10.1016/j.juro.2013.05.003 . Blanc T, Kohaut J, Elie C, Clermidi P, Pio L, Harte C, et al. Retroperitoneal approach for ureteropelvic junction obstruction: Encouraging preliminary results with robot-assisted laparoscopic repair. Front Pediatr. 2019;7:209. 10.3389/fped.2019.00209 . Walker RG, Poggioli T, Katsimpardi L, Buchanan SM, Oh J, Wattrus S, et al. Biochemistry and biology of GDF11 and myostatin: similarities, differences, and questions for future investigation. Circ Res. 2016;118(7):1125–41. 10.1161/circresaha.116.308391 . discussion 42. Gamer LW, Cox KA, Small C, Rosen V. Gdf11 is a negative regulator of chondrogenesis and myogenesis in the developing chick limb. Dev Biol. 2001;229(2):407–20. 10.1006/dbio.2000.9981 . Wu HH, Ivkovic S, Murray RC, Jaramillo S, Lyons KM, Johnson JE, et al. Autoregulation of neurogenesis by GDF11. Neuron. 2003;37(2):197–207. 10.1016/s0896-6273(02)01172-8 . Zhang Y, Wei Y, Liu D, Liu F, Li X, Pan L, et al. Role of growth differentiation factor 11 in development, physiology and disease. Oncotarget. 2017;8(46):81604–16. 10.18632/oncotarget.20258 . McPherron AC, Lawler AM, Lee SJ. Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11. Nat Genet. 1999;22(3):260–4. 10.1038/10320 . Król W, Machelak W, Zielińska M. GDF11 as a friend or an enemy in the cancer biology? Biochim Biophys Acta Rev Cancer. 2023;1878(5):188944. 10.1016/j.bbcan.2023.188944 . Wang L, Wang Y, Wang Z, Qi Y, Zong B, Liu M, et al. Growth differentiation factor 11 ameliorates experimental colitis by inhibiting NLRP3 inflammasome activation. Am J Physiol Gastrointest Liver Physiol. 2018;315(6):G909–g20. 10.1152/ajpgi.00159.2018 . Short KM, Smyth IM. The contribution of branching morphogenesis to kidney development and disease. Nat Rev Nephrol. 2016;12(12):754–67. 10.1038/nrneph.2016.157 . Li RX, Yiu WH, Tang SC. Role of bone morphogenetic protein-7 in renal fibrosis. Front Physiol. 2015;6:114. 10.3389/fphys.2015.00114 . Pons M, Koniaris LG, Moe SM, Gutierrez JC, Esquela-Kerscher A, Zimmers TA. GDF11 induces kidney fibrosis, renal cell epithelial-to-mesenchymal transition, and kidney dysfunction and failure. Surgery. 2018;164(2):262–73. 10.1016/j.surg.2018.03.008 . How GY, Chang KTE, Jacobsen AS, Yap TL, Ong CCP, Low Y, et al. Neuronal defects an etiological factor in congenital pelviureteric junction obstruction? J Pediatr Urol. 2018;14(1):51. 10.1016/j.jpurol.2017.07.014 . .e1-.e7 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3873300","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271721886,"identity":"0bdfd6e7-2e7b-4b8b-87ce-8e653cb96875","order_by":0,"name":"Weihua Lao","email":"","orcid":"","institution":"Guangdong Women and Children Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weihua","middleName":"","lastName":"Lao","suffix":""},{"id":271721887,"identity":"40389e68-0827-4d1b-9233-a19014862166","order_by":1,"name":"Tong Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIie3PoQrCUBTG8TMuXMsB6x0GX+EMQQwDH8Ry05Imq+C1mATrBF/CNzgyWBpYN2aYCCaDYh8KFtuuTfD+8/nB+QBcrh+sDYdHda/r+bJlLIm/4H4QS/bWyJaEEqYOvsgm1rafpax7iEdBxXmXwywcNQpvxXzaqoukMpoOII0mpokItTd0JYFUjvvKM0kzkd0zKNRCUZFZEoQUfOSE/BgtiYJMBhsT6Ta+tmibLUPORHU3oZatZJffZmEz+YyU/ub8Tb4VLpfL9R89Abd8RCki8ONXAAAAAElFTkSuQmCC","orcid":"","institution":"Guangdong Women and Children Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Shi","suffix":""},{"id":271721888,"identity":"27e42e0c-6eed-4b69-9998-ecf4925e8378","order_by":2,"name":"Qin Chen","email":"","orcid":"","institution":"Guangdong Women and Children Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Chen","suffix":""},{"id":271721889,"identity":"0576e1cc-d493-401b-84dc-9c171abf95bd","order_by":3,"name":"Bao Qiao","email":"","orcid":"","institution":"Guangdong Women and Children Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bao","middleName":"","lastName":"Qiao","suffix":""},{"id":271721890,"identity":"f1d1e4d6-bc12-419a-a169-0470346036e6","order_by":4,"name":"Sai Ma","email":"","orcid":"","institution":"Guangdong Women and Children Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sai","middleName":"","lastName":"Ma","suffix":""},{"id":271721891,"identity":"6c400822-1d84-4b04-904d-45ebf93a8522","order_by":5,"name":"Cuiwei Liu","email":"","orcid":"","institution":"Guangdong Women and Children Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cuiwei","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-17 15:48:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3873300/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3873300/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51017832,"identity":"02327030-beda-4545-8a1f-a6a9706d6d45","added_by":"auto","created_at":"2024-02-12 19:19:34","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":956275,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical expression of GDF11\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3873300/v1/f0af12e1f861c33ce3eefd0d.jpeg"},{"id":51017830,"identity":"305ab4a3-415e-496d-b900-f5f19128fd11","added_by":"auto","created_at":"2024-02-12 19:19:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12964,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-3873300/v1/6d881927105d1ff784e0ac9e.png"},{"id":63237664,"identity":"57809893-d66a-4131-ad73-a8f5b2d566a3","added_by":"auto","created_at":"2024-08-26 03:33:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1273126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3873300/v1/cd9a162a-06c5-445c-b983-2cb5a36bfeac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression and Significance of the GDF11 Protein in Ureteropelvic Junction Obstruction (UPJO)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePediatric hydronephrosis represents a prevalent congenital anomaly, with 1%-5% of pregnant women encountering fetal hydronephrosis during prenatal ultrasound screenings. Among these cases, 19%-25% are attributed to ureteropelvic junction obstruction (UPJO). The advent of immunohistochemistry and advanced molecular biology techniques in recent years has enhanced the understanding of UPJO's pathogenesis. It has been established that the development of embryonic kidneys is influenced by a variety of genes. However, the precise mechanisms involved still necessitate further exploration. Consequently, elucidating the etiology of UPJO to diminish its incidence continues to be a primary focus in ongoing research.\u003c/p\u003e \u003cp\u003eGrowth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a secretory protein that belongs to the transforming growth factor β (TGF-β) superfamily. Molecules belonging to the TGF-β superfamily are extensively found across a range of organisms, from nematodes to mammals, and are crucial in regulating various cellular processes. These include cell proliferation, differentiation, adhesion, migration, apoptosis, and the induction of cartilage formation and differentiation. They are crucial in the development and postinjury repair of organisms and various organs and participate in numerous pathological processes. The maturation process of GDF11 closely resembles that of TGF-β1, TGF-β2, TGF-β3, and GDF8. These molecules have been demonstrated to be involved in kidney development and injury repair. As part of the TGF-β superfamily, GDF11 additionally plays a significant role in embryonic development. Recent studies have shown that GDF11 is involved in the development of metanephros and the regeneration of renal tubules after acute kidney injury, strongly indicating its potential value in treating UPJO. Therefore, this study explored the clinical significance of GDF11 expression in UPJO.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 General information\u003c/h2\u003e \u003cp\u003eBetween January 2021 and November 2022, five neonates with severe hydronephrosis underwent laparoscopic pyeloplasty at a selected hospital. Tissue samples were harvested from both the constricted ureteropelvic junction (UPJ) and the adjacent normal ureteral section of each patient for comparative analysis. These samples were immediately frozen for preservation and subsequently prepared for paraffin embedding to facilitate further analysis.\u003c/p\u003e \u003cp\u003eThe inclusion criteria for patients were as follows: ① Prenatal ultrasound detection and postnatal color Doppler ultrasound diagnosis of UPJO within 48 h after birth. Anteroposterior diameter (APD) of the renal pelvis\u0026thinsp;\u0026ge;\u0026thinsp;20 cm, classified as a Society for Fetal Urology (SFU) grade of 4; and ② Age at the time of surgery\u0026thinsp;\u0026lt;\u0026thinsp;1 month.\u003c/p\u003e \u003cp\u003eExclusion criteria: Patients with other urinary tract anomalies, such as vesicoureteral reflux, stenosis at other locations of the ureter, or a duplicated kidney were excluded. Patients with severe liver and renal dysfunction, postoperative recurrence of UPJO, or whose parents refused surgery or were noncooperative during follow-up were also excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin sections were initially deparaffinized in water. Subsequently, these sections underwent a 5\u0026ndash;10 min incubation with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at room temperature to suppress endogenous peroxidase activity. After rinsing with distilled water, the sections were immersed in phosphate buffer saline (PBS) for 5 min, a process repeated twice. If necessary, antigen retrieval could be conducted following this step. The sections were then blocked using 5\u0026ndash;10% normal goat serum (diluted in PBS) at room temperature for 10 min, after which the serum was drained off without washing. The primary antibody solution was then applied, and the samples incubated at 37\u0026deg;C for 1\u0026ndash;2 h or overnight at 4\u0026deg;C. This was followed by a PBS rinse for 5 min, repeated thrice. Subsequently, a biotinylated secondary antibody solution was added, and the sections were incubated at 37\u0026deg;C for 10\u0026ndash;30 min. Another series of rinses with PBS for 5 min each, repeated thrice, was conducted. After rinsing, an adequate amount of horseradish peroxidase or alkaline phosphatase-conjugated streptavidin solution was applied, and the sections were incubated at 37\u0026deg;C for 10\u0026ndash;30 min. This was succeeded by additional rinses with PBS for 5 min each, repeated thrice. The sections were then stained with a chromogen for 3\u0026ndash;15 min, washed thoroughly with tap water, counterstained, dehydrated, cleared, and mounted. The GDF11 immunohistochemistry results underwent quantitative analysis by two pathologists using a two-tier scoring method. Cellular staining was scored as follows: 0 for no staining, 1 for light yellow, 2 for brownish yellow, and 3 for yellow-brown. The percentage of positive cells was determined (0\u0026ndash;100%). The expression score was the product of the degree of cellular staining and the percentage of positive cells (0\u0026ndash;3). A total score above 1 indicated high expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Western blot analysis\u003c/h2\u003e \u003cp\u003eTissues underwent lysis and homogenization in RIPA buffer, followed by centrifugation at 13,000\u0026times;g for the extraction of total proteins. The protein concentration was determined using the BCA method and normalized to 3000 ng/mL. The samples were then mixed with loading buffer and boiled for 10 min. Electrophoresis was subsequently conducted on a 10% polyacrylamide gel at 120 V, followed by protein transfer to a PVDF membrane at 200 mA. The membrane was blocked using 5% nonfat milk powder. Primary antibodies, including those against GDF11, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were incubated at 4\u0026deg;C overnight. This step was followed by incubation with corresponding secondary antibodies for specific binding. Protein visualization was achieved using an enhanced chemiluminescence (ECL) solution and a chemiluminescence imaging system. Band intensity was quantified using FlowJ software, and the relative expression levels of the target proteins were calculated, using GAPDH as an internal control protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical methods\u003c/h2\u003e \u003cp\u003eThe data were analyzed using SPSS 14.0 statistical software. Quantitative data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;s). A \u003cem\u003et\u003c/em\u003e-test was used for comparisons, and a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Immunohistochemistry results\u003c/h2\u003e\n \u003cp\u003eImmunohistochemical analysis indicated a high expression of GDF11 in UPJO tissues, exhibiting a significant expression rate of 60%. This rate is significantly greater than the 40% increase in expression observed in normal ureter tissues (Fig. 1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Western blot analysis\u003c/h2\u003e\n \u003cp\u003eThe expression of the GDF11 protein was significantly greater in UPJO tissues than in normal ureter tissues. A marked elevation in the grayscale intensity of GDF11 expression in UPJO tissues was observed, averaging at 55097.28\u0026thinsp;\u0026plusmn;\u0026thinsp;351.67. This contrasted with the lower average grayscale intensity in normal ureter tissues, recorded at 32993.43\u0026thinsp;\u0026plusmn;\u0026thinsp;487.95. The disparity in expression levels between these tissues was found to be statistically significant (P\u0026thinsp;=\u0026thinsp;0.000) (Fig. 2).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePediatric hydronephrosis is a common congenital anomaly, and prenatal ultrasound (US) reveals fetal hydronephrosis in approximately 1\u0026ndash;5% of pregnant women. Among these instances, 19\u0026ndash;25% are attributed to UPJO. This condition is more commonly observed in boys than in girls and is more prevalent on the left side compared to the right. Bilateral UPJO is noted in 10\u0026ndash;40% of cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The pathogenesis of UPJO remains incompletely understood and is currently in the early stages of exploration. However, recent advancements in immunohistochemistry and modern molecular biology have enhanced the understanding of UPJO's pathogenesis. Research indicates that the development of embryonic kidneys is regulated by various genes, yet the precise underlying mechanisms still demand further investigation. Thus, elucidating the etiology of UPJO to reduce its incidence is of significant clinical importance.\u003c/p\u003e \u003cp\u003eGrowth and differentiation factor 11 (GDF11), also known as BMP11, is a secretory protein and a member of the TGF-β superfamily [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. GDF11 is pivotal in embryogenesis and development. It has been shown to inhibit the formation of skeletal muscle cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], stimulate ureteric bud formation, and contribute to kidney development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. GDF11 exhibits widespread expression in various embryonic tissues, including the tailbud, limbs, and nervous system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mice deficient in Gdf11 (Gdf11-/-) demonstrate perinatal lethality with abnormalities in the upper jaw and kidneys [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The relationship of GDF11 with tumorigenesis is notable, as its expression is altered in tumors. It can either enhance tumor growth in colon cancer or suppress cell proliferation in breast cancer, and it is being investigated as a potential biomarker for prognosis and aggressiveness in liver cancer, pancreatic cancer, melanoma, and OSCC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While the role of GDF11 in inflammatory processes remains unclear, its influence on inflammation has been acknowledged [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This study explored the expression level of the GDF11 protein in UPJO tissues and revealed a significant increase in GDF11 expression compared to that in normal ureter tissues. Fibrosis in tissues and organs is a significant pathological alteration in the progression of numerous diseases, often resulting in functional impairment of these tissues and organs. Various members of the TGF-β superfamily play roles in the regulation of tissue fibrosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For example, TGF-β1 is known as a powerful pro-fibrotic cytokine, indicating that GDF11 might also contribute to fibrosis development. Research has suggested that GDF11 could act as an epithelial-mesenchymal transition (EMT) promoting factor, engaging in fibrosis and potentially exacerbating kidney diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Histologically, in UPJO, the typical structure of a normal ureter comprises an organized arrangement of the mucosal layer, the inherent layer, and the muscular layer, all encased in adipose tissue. In UPJO cases, proliferation of fibrous tissue and alteration of smooth muscle in the ureteral wall can lead to thickening and rigidity of the UPJ wall, resulting in luminal narrowing and adversely affecting ureteral peristalsis. This can obstruct urine flow from the kidney, culminating in hydronephrosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, the expression of GDF11 in UPJ tissues holds considerable clinical significance. This retrospective study, which included limited sample size, underscores the need for further research with a larger sample size to validate these findings and to elucidate the specific molecular mechanisms of GDF11 in UPJO development and progression.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003egrowth differentiation factor (GDF11); ureteropelvic junction obstruction (UPJO); bone morphogenetic proteins 11 (BMP11); transforming growth factor \u0026beta; (TGF-\u0026beta;); Growth differentiation factor 8 (GDF8), Anteroposterior diameter (APD); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); phosphate buffer saline (PBS); enhanced chemiluminescence (ECL)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e TS: Conceptualization, Methodology, Software; WL: Data curation, Writing- Original draft preparation. QC: Visualization, Investigation; QB: Supervision; SM: Software, Validation; CL: Writing- Reviewing and Editing. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the Medical Science and Technology Research Foundation of Guangdong Province (Grant number A20222241).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e The study was approved by the ethics review board of Guangdong Women and Children Hospital (No. 202101317) in accordance with the Declaration of Helsinki. Written informed consent have been obtained from a parent and/or legal guardian for study participation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCost NG, Noh PH, Devarajan P, Ivancic V, Reddy PP, Minevich E, et al. Urinary NGAL levels correlate with differential renal function in patients with ureteropelvic junction obstruction undergoing pyeloplasty. J Urol. 2013;190(4 Suppl):1462\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.juro.2013.05.003\u003c/span\u003e\u003cspan address=\"10.1016/j.juro.2013.05.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanc T, Kohaut J, Elie C, Clermidi P, Pio L, Harte C, et al. 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Neuronal defects an etiological factor in congenital pelviureteric junction obstruction? J Pediatr Urol. 2018;14(1):51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jpurol.2017.07.014\u003c/span\u003e\u003cspan address=\"10.1016/j.jpurol.2017.07.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.e1-.e7\u003c/span\u003e\u003cspan address=\"http://.e1-.e7\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"GDF11, UPJO, Protein","lastPublishedDoi":"10.21203/rs.3.rs-3873300/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3873300/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTo explore the expression of growth differentiation factor 11 (GDF11), a member of the TGF-β superfamily, in pediatric ureteropelvic junction obstruction (UPJO) patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFive patients diagnosed with severe neonatal hydronephrosis underwent laparoscopic pyeloplasty between January 2021 and November 2022. Tissue samples from the constricted ureteropelvic junction and adjacent normal ureteral segments served as controls. The presence of the GDF11 protein in these tissues was examined using immunohistochemistry and Western blot techniques. Subsequently, a statistical analysis compared the protein expression levels in both groups.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining revealed that the frequency of high GDF11 protein expression in the narrowed ureter (60%) was significantly greater than that in the normal ureter (40%). Western blot analysis indicated that the expression of GDF11 was significantly greater in the narrowed ureter than in the normal ureter.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe increased expression of GDF11 in UPJO is noteworthy and deserves clinical attention.\u003c/p\u003e","manuscriptTitle":"Expression and Significance of the GDF11 Protein in Ureteropelvic Junction Obstruction (UPJO)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 19:19:29","doi":"10.21203/rs.3.rs-3873300/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":"bd912a8a-8604-46f1-bde8-7e45c3a14697","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T03:25:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 19:19:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3873300","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3873300","identity":"rs-3873300","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}