The Effect of Placental Mesenchymal Stem Cells on The Protection of Chronic Renal Failure: The Role of Adiponectin

The Effect of Placental Mesenchymal Stem Cells on The Protection of Chronic Renal Failure: The Role of Adiponectin | 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 The Effect of Placental Mesenchymal Stem Cells on The Protection of Chronic Renal Failure: The Role of Adiponectin Ezgi Akan, Mehmet Sakıncı, Gultekin Suleymanlar, Emin Türkay Korgun, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6058870/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 Purpose Chronic glomerular and tubulointerstitial fibrosis is the leading cause of end-stage renal failure. Mesenchymal stem cell therapy shows great potential for kidney tissue regeneration. The use of measurable biomarkers is crucial for assessing the therapeutic effects of mesenchymal stem cells. Adiponectin is among the suggested biomarkers for monitoring the progression of chronic renal failure. Methods We extracted mesenchymal stem cells from the amnion membrane of term placentas. To establish the experimental groups, we partially ligated the left kidneys of male Wistar rats, fully removed the right kidney after two weeks, and observed them for an additional eight weeks. At the end of this period, the animals underwent a subtotal nephrectomy. After forming 5/6 nephrectomy, we transplanted stem cells via rat tail vein and waited for 15 and 30 days to form stem cell groups. We measured protein levels and mRNA expressions of Adiponectin, Adiponectin Receptor 1, Fibronectin and AMPK phosphorylation by western blot and Real-Time PCR methods respectively. Besides, urine and serum levels of adiponectin and urine levels of albumin measured by using a rat specific ELISA. Results Protein and mRNA expressions of Adiponectin, AdipoR1, Fibronectin, and AMPK phosphorylation were elevated in the nephrectomy groups compared to the controls; however, these increased gene and protein expressions declined following stem cell administration. Conclusion mesenchymal stem cells may have a therapeutic effect on chronic renal failure, and adiponectin may serve as a biomarker for monitoring disease progression. Mesenchymal stem cell placenta chronic renal failure adiponectin fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chronic kidney disease (CKD) is a progressive disorder affecting more than 800 million people worldwide, with a prevalence exceeding 10% of the global population. CKD is more common in older adults, women, racial minorities, and individuals with diabetes mellitus or hypertension [ 1 ]. Renal capillaries play a crucial role in kidney function, delivering oxygen and nutrients to the tubules, regulating vasoactivity, and maintaining angiogenic balance. Recent studies have identified capillary rarefaction and endothelial injury as key mechanisms contributing to renal fibrosis. These microvascular injuries exacerbate vasoconstriction, vascular permeability, complement system activation, oxidative stress, and inflammation, leading to endothelial apoptosis or necrosis [ 2 ]. Research on kidney regeneration has focused on pharmacological and genetic approaches, particularly in animal models. The subtotal (or 5/6) nephrectomy model is widely used for studying CKD, involving the removal of one kidney and partial resection of the remaining kidney [ 3 ]. Stem cells, particularly mesenchymal stem cells (MSCs), have emerged as promising therapeutic candidates for kidney disease due to their immunomodulatory and regenerative properties. MSCs differentiate into osteocytes, adipocytes, and chondrocytes and have been extensively investigated for their potential in kidney tissue repair [ 4 ]. Placental-derived MSCs present an ethical and minimally invasive alternative to other stem cell sources. Placental tissue, often discarded as medical waste, provides an abundant and easily accessible source of MSCs. Unlike adipose-derived MSCs, which require invasive collection procedures, placental MSCs can be obtained without ethical concerns associated with embryonic stem cells. Additionally, perinatal sources such as the umbilical cord, amniotic membrane, and chorionic plaque offer high cell purity and strong proliferative capacity [ 5 – 7 ]. Adiponectin, a key adipokine, plays a role in anti-inflammatory, insulin-sensitizing, and anti-atherogenic processes. It is the most abundant adipose-derived protein in human plasma and has been proposed as a potential biomarker for CKD [ 8 – 10 ]. Adiponectin signals through two primary receptors: AdipoR1 (highly expressed in skeletal muscle and kidneys) and AdipoR2 (primarily in the liver). In podocytes, AdipoR1 expression is predominant, whereas AdipoR2 expression is lower [ 11 , 12 ]. Studies on AdipoR1 and AdipoR2 deletions indicate that AdipoR1 activates 5’-adenosine monophosphate-activated protein kinase (AMPK), while AdipoR2 activates peroxisome proliferator-activated receptor (PPAR). AMPK functions as an energy sensor, balancing catabolic and anabolic pathways [ 13 – 15 ]. However, the role of adiponectin in CKD remains controversial. While increased plasma adiponectin levels have been linked to adiponectin receptor resistance and higher mortality in CKD patients, this paradox may be associated with inverse correlations between BMI and CKD-related mortality or uremia-related metabolic changes [ 16 ]. Podocytes, which are essential for glomerular filtration, have limited regenerative capacity. Their injury and apoptosis contribute to proteinuria and CKD progression. Adiponectin-knockout mice subjected to 5/6 nephrectomy demonstrate worsened renal function, whereas adiponectin infusion mitigates oxidative stress and albuminuria [ 17 – 19 ]. MSCs have been shown to reduce renal inflammation, apoptosis, and endoplasmic reticulum stress through paracrine immunomodulatory mechanisms. Studies suggest that MSC therapy improves kidney function, reduces oxidative stress, and attenuates fibrosis and microvascular remodeling [ 20 ]. However, limited data exist on adiponectin receptors and their signaling pathways in CKD. This study aims to evaluate the therapeutic potential of human placental-derived MSCs and investigate adiponectin as a potential biomarker for CKD progression. Materials and Methods Mesenchymal Stem Cell Isolation From Amniotic Membranes in Term Placentas All procedures followed ethical guidelines approved by the Ethical Committee of the Medical University of Akdeniz. Human term placentas from normal pregnancies (38–42 weeks, n = 6) were collected after spontaneous or cesarean deliveries with informed consent. Isolation of Mesenchymal Stem Cells from Amniotic Membranes of Term Placentas was performed according to the previously performed protocol [ 21 ]. Postpartum placentas were processed in a laminar flow cabinet in the cell culture laboratory. The amniotic membrane was segmented into roughly 3 x 3 cm pieces and incubated in PBS containing 2.4 U/mL dispase at 37°C for 9 minutes. Afterwards, these pieces were transferred into RPMI 1640 medium supplemented with FBS and 2.2 mM L-glutamine, where they remained at room temperature for 5–10 minutes. Next, the amniotic fragments were exposed to an enzyme solution composed of 0.75 mg/mL collagenase and 20 mg/mL DNA at 37°C for 2 hours. The resulting digest was then filtered through 100 µm cell strainers to collect the cells, which were subsequently centrifuged at 200 g for 10 minutes. Finally, the pellet obtained was resuspended in mesenchymal stem cell growth medium, seeded into culture flasks, and incubated in a 5% CO 2 atmosphere. Characterization of hAMSCs by Flow Cytometry The surface marker phenotype of hAMSCs has been previously analysed by flow cytometry [ 22 ]. The cells exhibited positive markers for CD44, CD90, CD73, and CD105, while lacking expression of CD34, CD45, CD14, CD19, and HLA-DR. Their characterization was carried out using a FACS Aria III Cell Sorter (BD Biosciences, New Jersey, USA). To evaluate their differentiation potential, hAMSCs were maintained in StemPro Osteogenic, StemPro Chondrogenic, and StemPro Adipogenic media (Life Technologies, Carlsbad, USA) supplemented appropriately. After a three-week period, the cells were fixed with 4% paraformaldehyde and stained to reveal the differentiation markers: Alizarin Red S and Kossa/Safranin O for osteogenic differentiation, toluidine blue and Heidenhayn Azan for chondrogenic differentiation, and Oil Red O for adipogenic differentiation. Animals All animal procedures received approval from Akdeniz University's Animal Care and Usage Committee and complied with the International Association for the Study of Pain guidelines. A total of 42 male Wistar rats, weighing between 250 and 300 g, were sourced from Akdeniz University's Experimental Animal Unit. These rats were kept at a controlled temperature of 22 ± 2°C under a 12-hour light/dark cycle and provided with standard pellet food and tap water throughout the duration of the experiment. To induce chronic kidney disease (CKD), a 5/6 nephrectomy model was implemented. Initially, two branches of the left kidney's renal artery were ligated, and two weeks later, the right kidney was completely removed. By the end of an 8-week period, the 5/6 nephrectomy procedure was fully established. Rats were randomly allocated into six groups: 1- Sham group, 2–5/6 nephrectomy (5/6 Nx) group, 3–5/6 Nx + 15 days group, 4–5/6 Nx + 30 days group, 5–5/6 Nx + hAMSC + 15 days group, 6 − 5/6 Nx + hAMSC + 30 days group. For the stem cell treatment groups, 10 6 hAMSCs suspended in 500 µL of DMEM medium (Lonza, Basel, Switzerland) were administered via tail vein injection. To mitigate graft rejection, cyclosporine A (Cell Signaling, Danvers, MA) was given at a dosage of 1 mg per day, beginning one day prior to the injection and continuing for seven days afterward. Urine samples were collected using metabolic cages, where rats were housed individually to separate urine and feces. Urine was collected over 24 hours. At the end of the experiment, rats were anesthetized with ether and sacrificed by cervical dislocation. Western Blot Kidney tissues were first homogenized and then centrifuged at 10,000 x g for 10 minutes to extract proteins. The protein concentrations were measured using the Bradford assay. For each sample, 20 µg of protein was separated via SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Hybond ECL blotting membrane, Amersham, UK) at 30 V overnight at 4°C. The membranes were then blocked with 5% BSA in TBS-T (Tris-buffered saline with Tween 20) and incubated overnight at 4°C with primary antibodies targeting adiponectin, AdipoR1, phosphorylated AMPK, AMPK, Fibronectin, and β-actin (Cell Signaling, Danvers, USA). This was followed by a 2-hour incubation at room temperature with HRP-conjugated secondary antibodies (BioRad). Protein bands were detected using chemiluminescence reagents (Thermo) and quantified by densitometry, with β-actin used as the loading control. Quantitative Real-Time PCR Total RNA was isolated from kidney tissues using Trizol reagent. Complementary DNA was then synthesized with the High Capacity cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR assays were performed employing the Power SYBR Green PCR Kit on a Quant Studio 3 system (Thermo Fisher Scientific). Expression levels of Adiponectin, AdipoR1, and Fibronectin mRNAs were normalized to β-actin and quantified using the 2 −ΔΔCt method. Primer sequences were: Adiponectin : forward 5'-TCTCTTCACCTACGACCAGT-3', reverse 5'-GGTAGAGAAGGAAGCCTGTA-3' AdipoR1 : forward 5'-GGAGAAGATGGAGGAGTTCG-3’, reverse 5'-TGGCCATGTAGCAGGTAGTC-3' Fibronectin : forward 5'-CGTCTATGCTCTCAAGGACA-3’, reverse 5'-CTGTCTTCGTTCTCCAGCTA-3' β-Actin : forward 5'-TATGCCAACACAGTGCTGTC-3', reverse 5'-GATAGAGCCACCAATCCACA-3' ELISA Analysis Adiponectin levels in serum and urine were measured using a rat-specific ELISA kit (Milipore, USA). Urine albumin levels were determined using a rat albumin ELISA kit (Elabscience, USA) in accordance with the manufacturer's protocol. Statistical Analysis All statistical analyses were carried out using GraphPad Prism software (version 5.0). Group comparisons were performed using Mann-Whitney U tests with Bonferroni correction, and differences were deemed statistically significant when the p-value was ≤ 0.05. Results Characterization and Differentiation of Isolated Mesenchymal Stem Cells Flow cytometry analysis revealed that mesenchymal stem cells were positive for CD44, CD90, CD73, and CD105, while negative for mesenchymal stem cells markers CD34, CD45, CD14, CD19, and HLA-DR. The proportion of positive cells was determined as follows: 82.4% for CD44, 87.8% for CD73, 98.4% for CD90, 85.5% for CD105, and negative for markers CD34, CD45, CD14, CD19, and HLA-DR (1.1%) ( Fig. 1 ) . Chondrocyte differentiation was confirmed by staining with Prussian Blue for cartilage proteoglycans and Sirius Red for cartilage collagen. Osteogenic differentiation was demonstrated using Alizarin Red S to detect calcium deposition. Adipogenic differentiation was verified through Oil Red O staining, which highlighted the presence of fat cells. The differentiation process showed successful transformation of MSCs into fat cells (stained with Oil Red O), bone cells (stained with Alizarin Red S), and cartilage cells (stained with Prussian Blue and Sirius Red) ( Fig. 2 a, b, c, d respectively) . Western Blot Analysis Western blot analysis showed a statistically significant decrease in adiponectin protein expression in the 5/6 Nx + hAMSC + 15 and 5/6 Nx + hAMSC + 30 groups compared to the 5/6 Nx group (p < 0.05). However, no significant difference was observed between the 5/6 Nx and 5/6 Nx + 30 groups ( Fig. 3 a ) . AdipoR1 protein expression was significantly lower in the 5/6 Nx + hAMSC + 15 group compared to the 5/6 Nx + hAMSC + 30 group (p < 0.005), whereas no statistically significant difference was observed between the 5/6 Nx and 5/6 Nx + 30 groups ( Fig. 3 b ) . For fibronectin protein expression, no significant difference was observed between the Sham group and the 5/6 Nx + 30 group. However, the 5/6 Nx + hAMSC + 30 group exhibited a statistically significant reduction in fibronectin levels compared to the 5/6 Nx + 30 group (p < 0.05) ( Fig. 3 c ). Regarding pAMPK and AMPK, a significant decrease in expression was detected between the 5/6 Nx + 30 group and the 5/6 Nx + hAMSC + 15 group (p < 0.05). Additionally, the decline was even more pronounced when comparing the 5/6 Nx + 30 group with the 5/6 Nx + hAMSC + 30 group, reaching statistical significance at p < 0.001 ( Fig. 3 d ). Quantitative Real-Time PCR Gene expression analysis revealed that adiponectin expression levels were significantly reduced following stem cell administration compared to the 5/6 Nx group (p < 0.005). A significant difference was also observed between the 5/6 Nx + hAMSC + 15 group and the 5/6 Nx + 30 group (p < 0.005). The most significant reduction was observed between the 5/6 Nx group and the 5/6 Nx + hAMSC + 30 group (p < 0.001) ( Fig. 4 a ) . AdipoR1 gene expression levels did not differ significantly between the Sham and 5/6 Nx groups. However, the 5/6 Nx + 30 group exhibited a statistically significant decrease in expression (p < 0.005) when compared to both the 5/6 Nx + hAMSC + 15 and 5/6 Nx + hAMSC + 30 groups ( Fig. 4 b ) . For fibronectin gene expression, no significant difference was detected between the Sham group and the nephrectomy groups. But, the 5/6 Nx + 30 group demonstrated a significant decrease (p < 0.001) in expression compared to both the 5/6 Nx + hAMSC + 15 and 5/6 Nx + hAMSC + 30 groups ( Fig. 4 c ) . Measurement of Serum and Urine Adiponectin Levels ELISA analysis indicated that serum adiponectin concentrations did not differ significantly between the Sham and nephrectomy groups. Nonetheless, the 5/6 Nx + hAMSC + 15 group exhibited a significant increase in serum adiponectin levels compared to the 5/6 Nx + 30 group ( Fig. 5 a ). In addition, urinary adiponectin levels, measured using a rat-specific ELISA kit, were significantly different (p < 0.005) between the Sham group and the 5/6 Nx + 30 group ( Fig. 5 b ) . Measurement of Urine Albumin Levels ELISA results demonstrated that urine albumin levels were significantly elevated in the 5/6 Nx group compared to the Sham group (p < 0.005). Additionally, a significant reduction in urine albumin was observed in the 5/6 Nx + hAMSC + 15 group relative to the 5/6 Nx group. Furthermore, both the 5/6 Nx + hAMSC + 15 and 5/6 Nx + hAMSC + 30 groups exhibited significantly lower urine albumin levels (p < 0.005) when compared to the 5/6 Nx + 30 group ( Fig. 6 ). Despite these differences, no significant variations were detected among the various nephrectomy groups. Discussion Chronic kidney failure is a worldwide epidemic marked by a persistent and gradual decline in the kidneys' metabolic functions as a result of decreased glomerular filtration. It affects 8–16% of the global population according to recent data [ 7 ]. While the kidney possesses a certain regenerative capacity following damage, this potential is limited under chronic conditions and is insufficient to prevent progressive glomerulosclerosis and tubulointerstitial fibrosis. These conditions constitute the primary factors contributing to end-stage renal failure, significantly impacting patients' quality of life [ 23 ]. Therefore, developing therapeutic strategies to prevent or slow the progression of kidney failure is crucial. Initially identified by Friedenstein as cells capable of differentiating from bone marrow fibroblasts [ 24 ], mesenchymal stem cells (MSCs) have garnered attention for their multipotency, immunomodulatory potential, and therapeutic applications in tissue regeneration. MSCs migrate to injury sites, modulate immune responses, secrete anti-inflammatory cytokines, and evade immune system detection [ 25 ]. Placenta, a temporary organ often discarded as medical waste, provides an ethical, cost-effective, and easily accessible source of MSCs. Unlike adipose tissue-derived MSCs, placental MSCs do not require invasive collection procedures and are free from the ethical concerns associated with embryonic stem cells [ 26 ]. Recent advancements in regenerative therapy have demonstrated the potential of MSCs in treating a range of conditions, including kidney diseases [ 27 ]. Experimental research has demonstrated that mesenchymal stem cells (MSCs) hold promise for enhancing kidney function across various chronic kidney disease models, such as diabetes, hypertension, and chronic nephropathy. Reliable biomarkers are essential for monitoring CKD progression and evaluating therapeutic interventions. Adiponectin, a prominent adipocytokine, is associated with kidney damage and has been shown to regulate endothelial function, oxidative stress, inflammation, and blood pressure [ 28 ]. In our investigation, we isolated mesenchymal stem cells from the amniotic membranes of term placentas, characterized them, and confirmed their multipotency by differentiating them into adipocytes, chondrocytes, and osteocytes. Protein levels of adiponectin, AdipoR1, Fibronectin, and AMPK phosphorylation were analyzed across all groups using Western blotting. Our findings revealed an increase in adiponectin and AdipoR1 protein levels in nephrectomized groups compared to sham groups, although this difference was not statistically significant. Following stem cell transplantation, significant decreases in adiponectin (p < 0.05) and AdipoR1 (p < 0.005) protein levels were observed in the Nx + 30 days group. Previous research has indicated that as kidney damage becomes more severe, there is a corresponding increase in adiponectin protein levels, alongside a gradual, time-dependent rise in receptor expression. Research using adiponectin-knockout mice demonstrated exacerbated proteinuria and renal fibrosis. Exogenous adiponectin reduced albuminuria, mesangial dilatation, and ROS production, thereby protecting against interstitial fibrosis [ 29 ]. These findings highlight adiponectin's protective role in reducing inflammation and oxidative stress, as well as its effect on podocytes in mitigating albuminuria [ 30 ]. Additionally, increased AdipoR1 and AdipoR2 expression in kidney tissues over time has been documented in CKD models [ 31 ]. Real-time PCR analysis revealed that adiponectin and AdipoR1 mRNA levels were significantly elevated in the Nx + 30 days group relative to the sham group (p < 0.005). Following stem cell therapy, significant decreases in gene expression were observed in the SC + 15 days and SC + 30 days groups compared to the Nx + 30 days group. These findings align with our Western blot results. Adiponectin exerts its protective effects on podocytes primarily through AMPK activation. AMPK, a key cellular energy sensor expressed in most eukaryotic cells, plays a central role in the effects of adiponectin by regulating intracellular ATP and AMP levels [ 32 ]. Our results showed increased AMPK phosphorylation in the Nx + 30 days group compared to the sham group. Stem cell therapy resulted in significant decreases in AMPK phosphorylation in the SC + 15 days (p < 0.05) and SC + 30 days (p < 0.001) groups. Our findings are in agreement with those of Maria et al. (2014), who documented elevated AdipoR1 levels and increased AMPK phosphorylation in patients with severe fibrosis compared to controls [ 33 ]. These results suggest that adiponectin mitigates albuminuria by activating AMPK via the AdipoR1 receptor pathway and reducing reactive oxygen species. Fibronectin, a matrix protein associated with fibrosis, increases in response to chronic injury or insufficient vascular supply. Our study showed increased fibronectin levels in groups with more severe kidney damage. Following stem cell therapy, fibronectin protein and mRNA levels significantly decreased in the SC + 30 days group compared to the Nx + 30 days group (p < 0.001). These results further support the antifibrotic effects of MSCs. Using a rat-specific ELISA kit, both urine and serum adiponectin levels were measured, revealing that serum adiponectin was significantly higher in the stem cell-treated groups compared to the Nx + 30 days groups. Conversely, urine adiponectin levels decreased significantly. These findings align with studies showing that reduced plasma adiponectin levels correlate with impaired renal function [ 34 ]. In studies on rodents, adiponectin exhibited renoprotective effects by reducing albuminuria [ 35 ]. In summary, our findings indicate that adiponectin not only exerts a protective effect against renal fibrosis but also holds potential as a biomarker for the progression of kidney disease. Additionally, MSC therapy significantly alleviates chronic renal failure, highlighting its therapeutic potential. The consistency of Western blot and real-time PCR findings underscores the reliability of adiponectin as a biomarker and the promise of MSCs in regenerative medicine. Declarations Competing Interests The authors declare that they have no financial or non-financial competing interests related to this study. Funding This study was supported by the Research Foundation of Akdeniz University, Turkey (Project Number: TYL-2017-2398). No external funding was received for this research. Author Contributions All authors have accepted responsibility for the entire content of this manuscript and approved its submission. All authors qualify for authorship by contributing significantly to this article. D.K.K. and E.A developed and drafted the original concept of this study. D.K.K and E.T.K. contributed to the methodology. G.S. and M.S. contributed to patients' diagnosis of and collection of tissues. E. A. performed animal modeling, western blotting, quantitative Real-Time PCR, ELISA methods. D.K.K. analyzed the results statistically, then created all figures. E.T.K. and D.K.K. assisted in both interpretation and evaluation of the data. All authors contributed to critical discussion, reviewed the final version of the article, and approved it for publication. Ethical Approval All procedures involving human participants were conducted in accordance with the ethical standards of the institutional and national research committee, as well as the 1964 Helsinki Declaration and its later amendments. The study was approved by the Ethical Committee of Akdeniz University Medical Faculty. All animal experiments complied with the ethical guidelines set by the Akdeniz University Animal Care and Usage Committee and adhered to the International Association for the Study of Pain guidelines. Informed Consent Informed consent was obtained from all human participants included in this study. Data Availability Statement The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Dr. Ezgi Akan (ORCID: 0000-0002-5589-8554): [email protected] Dr. Mehmet Sakıncı (ORCID:0000-0001-5074-0005): [email protected] Dr. Gultekin Suleymanlar (ORCID:0000-0001-7935-6402): [email protected] Dr. Emin Turkay Korgun (ORCID:0000-0003-4997-3869): [email protected] Dr. Dijle Kipmen-Korgun (ORCID:0000-0003-0692-7491): [email protected] References Kovesdy CP (2022) Epidemiology of chronic kidney disease: an update 2022. 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Best Pract Res Clin Endocrinol Metab 28(1), 71-9.https://doi.org/10.1016/j.beem.2013.08.002 Ross FA, MacKintosh C, Hardie DG (2016) AMP-activated protein kinase: a cellular energy sensor that comes in 12 flavours. Febs j 283(16), 2987-3001.https://doi.org/10.1111/febs.13698 Parker-Duffen JL, Nakamura K, Silver M, Zuriaga MA, MacLauchlan S, Aprahamian TR, et al. (2014) Divergent roles for adiponectin receptor 1 (AdipoR1) and AdipoR2 in mediating revascularization and metabolic dysfunction in vivo. J Biol Chem 289(23), 16200-13.https://doi.org/10.1074/jbc.M114.548115 Martinez Cantarin MP, Keith SW, Waldman SA, Falkner B (2014) Adiponectin receptor and adiponectin signaling in human tissue among patients with end-stage renal disease. Nephrol Dial Transplant 29(12), 2268-77.https://doi.org/10.1093/ndt/gfu249 Nakamaki S, Satoh H, Kudoh A, Hayashi Y, Hirai H, Watanabe T (2011) Adiponectin reduces proteinuria in streptozotocin-induced diabetic Wistar rats. Exp Biol Med (Maywood) 236(5), 614-20.https://doi.org/10.1258/ebm.2011.010218 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-6058870","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":418279696,"identity":"d0ec2ee5-f183-48c1-b292-684f46e31d4e","order_by":0,"name":"Ezgi Akan","email":"","orcid":"","institution":"Amasya University Medical Faculty","correspondingAuthor":false,"prefix":"","firstName":"Ezgi","middleName":"","lastName":"Akan","suffix":""},{"id":418279697,"identity":"381f9392-c337-4927-afe8-ab8c50e358e6","order_by":1,"name":"Mehmet Sakıncı","email":"","orcid":"","institution":"Akdeniz University Medical Faculty","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Sakıncı","suffix":""},{"id":418279699,"identity":"d4090cd8-ba29-4bf1-b4ce-4dbae300a55c","order_by":2,"name":"Gultekin Suleymanlar","email":"","orcid":"","institution":"Akdeniz University Medical Faculty","correspondingAuthor":false,"prefix":"","firstName":"Gultekin","middleName":"","lastName":"Suleymanlar","suffix":""},{"id":418279700,"identity":"73bf7995-7072-4e01-af67-9605a8581698","order_by":3,"name":"Emin Türkay Korgun","email":"","orcid":"","institution":"Akdeniz University Medical Faculty","correspondingAuthor":false,"prefix":"","firstName":"Emin","middleName":"Türkay","lastName":"Korgun","suffix":""},{"id":418279704,"identity":"faf88948-a759-4b3a-ac6b-99c247514c88","order_by":4,"name":"Dijle Kipmen-Korgun","email":"data:image/png;base64,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","orcid":"","institution":"Akdeniz University Medical Faculty","correspondingAuthor":true,"prefix":"","firstName":"Dijle","middleName":"","lastName":"Kipmen-Korgun","suffix":""}],"badges":[],"createdAt":"2025-02-18 20:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6058870/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6058870/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76821785,"identity":"5635d9ea-080b-49f9-8c41-62c685ae354c","added_by":"auto","created_at":"2025-02-21 07:03:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126437,"visible":true,"origin":"","legend":"\u003cp\u003eResults of FACS analysis of isolated mesenchymal stem cells. \u003cstrong\u003eA)\u003c/strong\u003e CD90, \u003cstrong\u003eB)\u003c/strong\u003e CD73, \u003cstrong\u003eC)\u003c/strong\u003e CD44, \u003cstrong\u003eD)\u003c/strong\u003e CD105 and \u003cstrong\u003eE)\u003c/strong\u003e negative control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/2e7d4017e49390d1975fb29e.png"},{"id":76821790,"identity":"270968fe-97a3-46c4-8147-fe1a58d3db14","added_by":"auto","created_at":"2025-02-21 07:03:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164695,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentiation staining of isolated mesenchymal stem cells. The differentiation of MSCs to cartilage cells after chondrogenesis (Prussian Blue \u003cstrong\u003e(2A)\u003c/strong\u003eand Sirius Red \u003cstrong\u003e(2B)\u003c/strong\u003e staining), differentiation to bone cells (Alizer's Red S \u003cstrong\u003e(2C)\u003c/strong\u003e) after osteogenesis and differentiation to fat cells after adipogenesis (Oil Red O) \u003cstrong\u003e(2D).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/6078fd799eb21c6c0c6befd8.png"},{"id":76825084,"identity":"e7e313dd-ce74-4b95-826f-6e60adad6ab9","added_by":"auto","created_at":"2025-02-21 07:27:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":165064,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot results of Adiponectin, AdipoR1, Fibronectin and phosporylated AMPK for all groups. \u003cstrong\u003ea)\u003c/strong\u003e The bands (A) and optical density (OD) chart graphics (B) of Adiponectin. \u003cstrong\u003eb)\u003c/strong\u003eThe bands (A) and optical density (OD) chart graphics (B) of AdipoR1. \u003cstrong\u003ec)\u003c/strong\u003e The bands (A) and optical density (OD) chart graphics (B) of Fibronectin. \u003cstrong\u003ed)\u003c/strong\u003eThe bands (A) and optical density (OD) chart graphics (B) of pAMPK and AMPK. The results for all groups were normalized according to β-actin. Data obtained by scanning the density of bands of western blot results are indicated as column charts (mean ± standard error, * p\u0026lt;0.05, ** p\u0026lt;0.005, *** p\u0026lt;0.001 and n= 6).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/9847081e91bc889adba003bd.png"},{"id":76823493,"identity":"bfb0e8ca-1ec6-40b2-a5a6-3951eb64bbee","added_by":"auto","created_at":"2025-02-21 07:11:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84208,"visible":true,"origin":"","legend":"\u003cp\u003eData of Quantitative Real Time-PCR results. \u003cstrong\u003ea)\u003c/strong\u003eAdiponectin mRNA expression charts for all groups. \u003cstrong\u003eb)\u003c/strong\u003e AdipoR1 mRNA expression charts for all groups. \u003cstrong\u003ec)\u003c/strong\u003e Fibronectin mRNA expression charts for all groups. The results for all groups were normalized according to housekeeping gene β-actin. Data of RT-PCR results are indicated as column charts (mean ± standard error, * p\u0026lt;0.05, ** p\u0026lt;0.005, *** p\u0026lt;0.001 and n= 6).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/d70baff36831f530a071758e.png"},{"id":76823699,"identity":"82f559eb-0cde-4aa7-89bd-54211e107c66","added_by":"auto","created_at":"2025-02-21 07:19:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110858,"visible":true,"origin":"","legend":"\u003cp\u003eELISA results for adiponectin. \u003cstrong\u003ea)\u003c/strong\u003e Serum adiponectin (ng/mL) levels. \u003cstrong\u003eb)\u003c/strong\u003eUrinary adiponectin (ng/mL) levels. All results are indicated as column charts (mean ± standard error, * p\u0026lt;0.05, ** p\u0026lt;0.005, *** p\u0026lt;0.001 and n= 6).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/e5ed6c5e37278ae362deee83.png"},{"id":76821791,"identity":"924391c0-c847-4679-831a-8e5a3818add0","added_by":"auto","created_at":"2025-02-21 07:03:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49741,"visible":true,"origin":"","legend":"\u003cp\u003eELISA result of urinary albumin (µg/mL) levels (mean ± standard error, * p \u0026lt;0.05 ** p\u0026lt;0.005 and n = 6).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/2c170f8bc49422687d6cb2fc.png"},{"id":77636393,"identity":"8e03ed34-d867-4add-9218-b9deec54d171","added_by":"auto","created_at":"2025-03-03 18:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1470387,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6058870/v1/a2227b1d-d1ca-4e0a-906f-78bcfd042e41.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effect of Placental Mesenchymal Stem Cells on The Protection of Chronic Renal Failure: The Role of Adiponectin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic kidney disease (CKD) is a progressive disorder affecting more than 800\u0026nbsp;million people worldwide, with a prevalence exceeding 10% of the global population. CKD is more common in older adults, women, racial minorities, and individuals with diabetes mellitus or hypertension [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Renal capillaries play a crucial role in kidney function, delivering oxygen and nutrients to the tubules, regulating vasoactivity, and maintaining angiogenic balance. Recent studies have identified capillary rarefaction and endothelial injury as key mechanisms contributing to renal fibrosis. These microvascular injuries exacerbate vasoconstriction, vascular permeability, complement system activation, oxidative stress, and inflammation, leading to endothelial apoptosis or necrosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Research on kidney regeneration has focused on pharmacological and genetic approaches, particularly in animal models. The subtotal (or 5/6) nephrectomy model is widely used for studying CKD, involving the removal of one kidney and partial resection of the remaining kidney [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStem cells, particularly mesenchymal stem cells (MSCs), have emerged as promising therapeutic candidates for kidney disease due to their immunomodulatory and regenerative properties. MSCs differentiate into osteocytes, adipocytes, and chondrocytes and have been extensively investigated for their potential in kidney tissue repair [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlacental-derived MSCs present an ethical and minimally invasive alternative to other stem cell sources. Placental tissue, often discarded as medical waste, provides an abundant and easily accessible source of MSCs. Unlike adipose-derived MSCs, which require invasive collection procedures, placental MSCs can be obtained without ethical concerns associated with embryonic stem cells. Additionally, perinatal sources such as the umbilical cord, amniotic membrane, and chorionic plaque offer high cell purity and strong proliferative capacity [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdiponectin, a key adipokine, plays a role in anti-inflammatory, insulin-sensitizing, and anti-atherogenic processes. It is the most abundant adipose-derived protein in human plasma and has been proposed as a potential biomarker for CKD [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Adiponectin signals through two primary receptors: AdipoR1 (highly expressed in skeletal muscle and kidneys) and AdipoR2 (primarily in the liver). In podocytes, AdipoR1 expression is predominant, whereas AdipoR2 expression is lower [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies on AdipoR1 and AdipoR2 deletions indicate that AdipoR1 activates 5\u0026rsquo;-adenosine monophosphate-activated protein kinase (AMPK), while AdipoR2 activates peroxisome proliferator-activated receptor (PPAR). AMPK functions as an energy sensor, balancing catabolic and anabolic pathways [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the role of adiponectin in CKD remains controversial. While increased plasma adiponectin levels have been linked to adiponectin receptor resistance and higher mortality in CKD patients, this paradox may be associated with inverse correlations between BMI and CKD-related mortality or uremia-related metabolic changes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePodocytes, which are essential for glomerular filtration, have limited regenerative capacity. Their injury and apoptosis contribute to proteinuria and CKD progression. Adiponectin-knockout mice subjected to 5/6 nephrectomy demonstrate worsened renal function, whereas adiponectin infusion mitigates oxidative stress and albuminuria [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMSCs have been shown to reduce renal inflammation, apoptosis, and endoplasmic reticulum stress through paracrine immunomodulatory mechanisms. Studies suggest that MSC therapy improves kidney function, reduces oxidative stress, and attenuates fibrosis and microvascular remodeling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, limited data exist on adiponectin receptors and their signaling pathways in CKD. This study aims to evaluate the therapeutic potential of human placental-derived MSCs and investigate adiponectin as a potential biomarker for CKD progression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMesenchymal Stem Cell Isolation From Amniotic Membranes in Term Placentas\u003c/h2\u003e \u003cp\u003eAll procedures followed ethical guidelines approved by the Ethical Committee of the Medical University of Akdeniz. Human term placentas from normal pregnancies (38\u0026ndash;42 weeks, n\u0026thinsp;=\u0026thinsp;6) were collected after spontaneous or cesarean deliveries with informed consent.\u003c/p\u003e \u003cp\u003eIsolation of Mesenchymal Stem Cells from Amniotic Membranes of Term Placentas was performed according to the previously performed protocol [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Postpartum placentas were processed in a laminar flow cabinet in the cell culture laboratory. The amniotic membrane was segmented into roughly 3 x 3 cm pieces and incubated in PBS containing 2.4 U/mL dispase at 37\u0026deg;C for 9 minutes. Afterwards, these pieces were transferred into RPMI 1640 medium supplemented with FBS and 2.2 mM L-glutamine, where they remained at room temperature for 5\u0026ndash;10 minutes. Next, the amniotic fragments were exposed to an enzyme solution composed of 0.75 mg/mL collagenase and 20 mg/mL DNA at 37\u0026deg;C for 2 hours. The resulting digest was then filtered through 100 \u0026micro;m cell strainers to collect the cells, which were subsequently centrifuged at 200 g for 10 minutes. Finally, the pellet obtained was resuspended in mesenchymal stem cell growth medium, seeded into culture flasks, and incubated in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of hAMSCs by Flow Cytometry\u003c/h3\u003e\n\u003cp\u003eThe surface marker phenotype of hAMSCs has been previously analysed by flow cytometry [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The cells exhibited positive markers for CD44, CD90, CD73, and CD105, while lacking expression of CD34, CD45, CD14, CD19, and HLA-DR. Their characterization was carried out using a FACS Aria III Cell Sorter (BD Biosciences, New Jersey, USA). To evaluate their differentiation potential, hAMSCs were maintained in StemPro Osteogenic, StemPro Chondrogenic, and StemPro Adipogenic media (Life Technologies, Carlsbad, USA) supplemented appropriately. After a three-week period, the cells were fixed with 4% paraformaldehyde and stained to reveal the differentiation markers: Alizarin Red S and Kossa/Safranin O for osteogenic differentiation, toluidine blue and Heidenhayn Azan for chondrogenic differentiation, and Oil Red O for adipogenic differentiation.\u003c/p\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003e All animal procedures received approval from Akdeniz University's Animal Care and Usage Committee and complied with the International Association for the Study of Pain guidelines. A total of 42 male Wistar rats, weighing between 250 and 300 g, were sourced from Akdeniz University's Experimental Animal Unit. These rats were kept at a controlled temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 12-hour light/dark cycle and provided with standard pellet food and tap water throughout the duration of the experiment.\u003c/p\u003e \u003cp\u003eTo induce chronic kidney disease (CKD), a 5/6 nephrectomy model was implemented. Initially, two branches of the left kidney's renal artery were ligated, and two weeks later, the right kidney was completely removed. By the end of an 8-week period, the 5/6 nephrectomy procedure was fully established.\u003c/p\u003e \u003cp\u003eRats were randomly allocated into six groups: 1- Sham group, 2\u0026ndash;5/6 nephrectomy (5/6 Nx) group, 3\u0026ndash;5/6 Nx\u0026thinsp;+\u0026thinsp;15 days group, 4\u0026ndash;5/6 Nx\u0026thinsp;+\u0026thinsp;30 days group, 5\u0026ndash;5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 days group, 6\u0026thinsp;\u0026minus;\u0026thinsp;5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 days group.\u003c/p\u003e \u003cp\u003eFor the stem cell treatment groups, 10\u003csup\u003e6\u003c/sup\u003e hAMSCs suspended in 500 \u0026micro;L of DMEM medium (Lonza, Basel, Switzerland) were administered via tail vein injection. To mitigate graft rejection, cyclosporine A (Cell Signaling, Danvers, MA) was given at a dosage of 1 mg per day, beginning one day prior to the injection and continuing for seven days afterward.\u003c/p\u003e \u003cp\u003eUrine samples were collected using metabolic cages, where rats were housed individually to separate urine and feces. Urine was collected over 24 hours. At the end of the experiment, rats were anesthetized with ether and sacrificed by cervical dislocation.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eKidney tissues were first homogenized and then centrifuged at 10,000 x g for 10 minutes to extract proteins. The protein concentrations were measured using the Bradford assay. For each sample, 20 \u0026micro;g of protein was separated via SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Hybond ECL blotting membrane, Amersham, UK) at 30 V overnight at 4\u0026deg;C. The membranes were then blocked with 5% BSA in TBS-T (Tris-buffered saline with Tween 20) and incubated overnight at 4\u0026deg;C with primary antibodies targeting adiponectin, AdipoR1, phosphorylated AMPK, AMPK, Fibronectin, and β-actin (Cell Signaling, Danvers, USA). This was followed by a 2-hour incubation at room temperature with HRP-conjugated secondary antibodies (BioRad). Protein bands were detected using chemiluminescence reagents (Thermo) and quantified by densitometry, with β-actin used as the loading control.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from kidney tissues using Trizol reagent. Complementary DNA was then synthesized with the High Capacity cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR assays were performed employing the Power SYBR Green PCR Kit on a Quant Studio 3 system (Thermo Fisher Scientific). Expression levels of Adiponectin, AdipoR1, and Fibronectin mRNAs were normalized to β-actin and quantified using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cp\u003ePrimer sequences were:\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAdiponectin\u003c/span\u003e: forward 5'-TCTCTTCACCTACGACCAGT-3', reverse 5'-GGTAGAGAAGGAAGCCTGTA-3'\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAdipoR1\u003c/span\u003e: forward 5'-GGAGAAGATGGAGGAGTTCG-3\u0026rsquo;, reverse 5'-TGGCCATGTAGCAGGTAGTC-3'\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFibronectin\u003c/span\u003e: forward 5'-CGTCTATGCTCTCAAGGACA-3\u0026rsquo;, reverse 5'-CTGTCTTCGTTCTCCAGCTA-3'\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eβ-Actin\u003c/span\u003e: forward 5'-TATGCCAACACAGTGCTGTC-3', reverse 5'-GATAGAGCCACCAATCCACA-3'\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISA Analysis\u003c/h2\u003e \u003cp\u003eAdiponectin levels in serum and urine were measured using a rat-specific ELISA kit (Milipore, USA). Urine albumin levels were determined using a rat albumin ELISA kit (Elabscience, USA) in accordance with the manufacturer's protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were carried out using GraphPad Prism software (version 5.0). Group comparisons were performed using Mann-Whitney U tests with Bonferroni correction, and differences were deemed statistically significant when the p-value was \u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization and Differentiation of Isolated Mesenchymal Stem Cells\u003c/h2\u003e \u003cp\u003eFlow cytometry analysis revealed that mesenchymal stem cells were positive for CD44, CD90, CD73, and CD105, while negative for mesenchymal stem cells markers CD34, CD45, CD14, CD19, and HLA-DR. The proportion of positive cells was determined as follows: 82.4% for CD44, 87.8% for CD73, 98.4% for CD90, 85.5% for CD105, and negative for markers CD34, CD45, CD14, CD19, and HLA-DR (1.1%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eChondrocyte differentiation was confirmed by staining with Prussian Blue for cartilage proteoglycans and Sirius Red for cartilage collagen. Osteogenic differentiation was demonstrated using Alizarin Red S to detect calcium deposition. Adipogenic differentiation was verified through Oil Red O staining, which highlighted the presence of fat cells. The differentiation process showed successful transformation of MSCs into fat cells (stained with Oil Red O), bone cells (stained with Alizarin Red S), and cartilage cells (stained with Prussian Blue and Sirius Red) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, c, d \u003cb\u003erespectively)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis showed a statistically significant decrease in adiponectin protein expression in the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 and 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 groups compared to the 5/6 Nx group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant difference was observed between the 5/6 Nx and 5/6 Nx\u0026thinsp;+\u0026thinsp;30 groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. AdipoR1 protein expression was significantly lower in the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 group compared to the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), whereas no statistically significant difference was observed between the 5/6 Nx and 5/6 Nx\u0026thinsp;+\u0026thinsp;30 groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. For fibronectin protein expression, no significant difference was observed between the Sham group and the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group. However, the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 group exhibited a statistically significant reduction in fibronectin levels compared to the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e Regarding pAMPK and AMPK, a significant decrease in expression was detected between the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group and the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the decline was even more pronounced when comparing the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group with the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 group, reaching statistical significance at p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003eGene expression analysis revealed that adiponectin expression levels were significantly reduced following stem cell administration compared to the 5/6 Nx group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). A significant difference was also observed between the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 group and the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). The most significant reduction was observed between the 5/6 Nx group and the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. AdipoR1 gene expression levels did not differ significantly between the Sham and 5/6 Nx groups. However, the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group exhibited a statistically significant decrease in expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) when compared to both the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 and 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. For fibronectin gene expression, no significant difference was detected between the Sham group and the nephrectomy groups. But, the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group demonstrated a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in expression compared to both the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 and 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Serum and Urine Adiponectin Levels\u003c/h2\u003e \u003cp\u003eELISA analysis indicated that serum adiponectin concentrations did not differ significantly between the Sham and nephrectomy groups. Nonetheless, the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 group exhibited a significant increase in serum adiponectin levels compared to the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e In addition, urinary adiponectin levels, measured using a rat-specific ELISA kit, were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) between the Sham group and the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Urine Albumin Levels\u003c/h2\u003e \u003cp\u003eELISA results demonstrated that urine albumin levels were significantly elevated in the 5/6 Nx group compared to the Sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). Additionally, a significant reduction in urine albumin was observed in the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 group relative to the 5/6 Nx group. Furthermore, both the 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;15 and 5/6 Nx\u0026thinsp;+\u0026thinsp;hAMSC\u0026thinsp;+\u0026thinsp;30 groups exhibited significantly lower urine albumin levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) when compared to the 5/6 Nx\u0026thinsp;+\u0026thinsp;30 group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Despite these differences, no significant variations were detected among the various nephrectomy groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChronic kidney failure is a worldwide epidemic marked by a persistent and gradual decline in the kidneys' metabolic functions as a result of decreased glomerular filtration. It affects 8\u0026ndash;16% of the global population according to recent data [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While the kidney possesses a certain regenerative capacity following damage, this potential is limited under chronic conditions and is insufficient to prevent progressive glomerulosclerosis and tubulointerstitial fibrosis. These conditions constitute the primary factors contributing to end-stage renal failure, significantly impacting patients' quality of life [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, developing therapeutic strategies to prevent or slow the progression of kidney failure is crucial.\u003c/p\u003e \u003cp\u003eInitially identified by Friedenstein as cells capable of differentiating from bone marrow fibroblasts [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], mesenchymal stem cells (MSCs) have garnered attention for their multipotency, immunomodulatory potential, and therapeutic applications in tissue regeneration. MSCs migrate to injury sites, modulate immune responses, secrete anti-inflammatory cytokines, and evade immune system detection [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlacenta, a temporary organ often discarded as medical waste, provides an ethical, cost-effective, and easily accessible source of MSCs. Unlike adipose tissue-derived MSCs, placental MSCs do not require invasive collection procedures and are free from the ethical concerns associated with embryonic stem cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Recent advancements in regenerative therapy have demonstrated the potential of MSCs in treating a range of conditions, including kidney diseases [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExperimental research has demonstrated that mesenchymal stem cells (MSCs) hold promise for enhancing kidney function across various chronic kidney disease models, such as diabetes, hypertension, and chronic nephropathy. Reliable biomarkers are essential for monitoring CKD progression and evaluating therapeutic interventions. Adiponectin, a prominent adipocytokine, is associated with kidney damage and has been shown to regulate endothelial function, oxidative stress, inflammation, and blood pressure [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our investigation, we isolated mesenchymal stem cells from the amniotic membranes of term placentas, characterized them, and confirmed their multipotency by differentiating them into adipocytes, chondrocytes, and osteocytes. Protein levels of adiponectin, AdipoR1, Fibronectin, and AMPK phosphorylation were analyzed across all groups using Western blotting.\u003c/p\u003e \u003cp\u003eOur findings revealed an increase in adiponectin and AdipoR1 protein levels in nephrectomized groups compared to sham groups, although this difference was not statistically significant. Following stem cell transplantation, significant decreases in adiponectin (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and AdipoR1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) protein levels were observed in the Nx\u0026thinsp;+\u0026thinsp;30 days group. Previous research has indicated that as kidney damage becomes more severe, there is a corresponding increase in adiponectin protein levels, alongside a gradual, time-dependent rise in receptor expression.\u003c/p\u003e \u003cp\u003eResearch using adiponectin-knockout mice demonstrated exacerbated proteinuria and renal fibrosis. Exogenous adiponectin reduced albuminuria, mesangial dilatation, and ROS production, thereby protecting against interstitial fibrosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These findings highlight adiponectin's protective role in reducing inflammation and oxidative stress, as well as its effect on podocytes in mitigating albuminuria [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Additionally, increased AdipoR1 and AdipoR2 expression in kidney tissues over time has been documented in CKD models [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReal-time PCR analysis revealed that adiponectin and AdipoR1 mRNA levels were significantly elevated in the Nx\u0026thinsp;+\u0026thinsp;30 days group relative to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). Following stem cell therapy, significant decreases in gene expression were observed in the SC\u0026thinsp;+\u0026thinsp;15 days and SC\u0026thinsp;+\u0026thinsp;30 days groups compared to the Nx\u0026thinsp;+\u0026thinsp;30 days group. These findings align with our Western blot results.\u003c/p\u003e \u003cp\u003eAdiponectin exerts its protective effects on podocytes primarily through AMPK activation. AMPK, a key cellular energy sensor expressed in most eukaryotic cells, plays a central role in the effects of adiponectin by regulating intracellular ATP and AMP levels [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our results showed increased AMPK phosphorylation in the Nx\u0026thinsp;+\u0026thinsp;30 days group compared to the sham group. Stem cell therapy resulted in significant decreases in AMPK phosphorylation in the SC\u0026thinsp;+\u0026thinsp;15 days (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and SC\u0026thinsp;+\u0026thinsp;30 days (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) groups.\u003c/p\u003e \u003cp\u003eOur findings are in agreement with those of Maria et al. (2014), who documented elevated AdipoR1 levels and increased AMPK phosphorylation in patients with severe fibrosis compared to controls [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These results suggest that adiponectin mitigates albuminuria by activating AMPK via the AdipoR1 receptor pathway and reducing reactive oxygen species.\u003c/p\u003e \u003cp\u003eFibronectin, a matrix protein associated with fibrosis, increases in response to chronic injury or insufficient vascular supply. Our study showed increased fibronectin levels in groups with more severe kidney damage. Following stem cell therapy, fibronectin protein and mRNA levels significantly decreased in the SC\u0026thinsp;+\u0026thinsp;30 days group compared to the Nx\u0026thinsp;+\u0026thinsp;30 days group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results further support the antifibrotic effects of MSCs.\u003c/p\u003e \u003cp\u003eUsing a rat-specific ELISA kit, both urine and serum adiponectin levels were measured, revealing that serum adiponectin was significantly higher in the stem cell-treated groups compared to the Nx\u0026thinsp;+\u0026thinsp;30 days groups. Conversely, urine adiponectin levels decreased significantly. These findings align with studies showing that reduced plasma adiponectin levels correlate with impaired renal function [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In studies on rodents, adiponectin exhibited renoprotective effects by reducing albuminuria [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, our findings indicate that adiponectin not only exerts a protective effect against renal fibrosis but also holds potential as a biomarker for the progression of kidney disease. Additionally, MSC therapy significantly alleviates chronic renal failure, highlighting its therapeutic potential. The consistency of Western blot and real-time PCR findings underscores the reliability of adiponectin as a biomarker and the promise of MSCs in regenerative medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or non-financial competing interests related to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Research Foundation of Akdeniz University, Turkey (Project Number: TYL-2017-2398). No external funding was received for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have accepted responsibility for the entire content of this manuscript and approved its submission. \u0026nbsp;All authors qualify for authorship by contributing significantly to this article. D.K.K. and E.A developed and drafted the original concept of this study. D.K.K and E.T.K. contributed to the methodology. G.S. and M.S. contributed to patients\u0026apos; diagnosis of and collection of tissues. E. A. performed animal modeling, western blotting, quantitative Real-Time PCR, ELISA methods. D.K.K. analyzed the results statistically, then created all figures. E.T.K. and D.K.K. assisted in both interpretation and evaluation of the data. All authors contributed to critical discussion, reviewed the final version of the article, and approved it for publication. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving human participants were conducted in accordance with the ethical standards of the institutional and national research committee, as well as the 1964 Helsinki Declaration and its later amendments.\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethical Committee of Akdeniz University Medical Faculty.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments complied with the ethical guidelines set by the Akdeniz University Animal Care and Usage Committee and adhered to the International Association for the Study of Pain guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all human participants included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDr. Ezgi Akan (ORCID: 0000-0002-5589-8554):\u003c/strong\u003e [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDr. Mehmet Sakıncı (ORCID:0000-0001-5074-0005):\u003c/strong\u003e [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDr. Gultekin Suleymanlar (ORCID:0000-0001-7935-6402):\u003c/strong\u003e\u0026nbsp; [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDr. Emin Turkay Korgun (ORCID:0000-0003-4997-3869):\u003c/strong\u003e\u0026nbsp; [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDr. Dijle Kipmen-Korgun (ORCID:0000-0003-0692-7491):\u003c/strong\u003e\u0026nbsp; [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKovesdy CP (2022) Epidemiology of chronic kidney disease: an update 2022. 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Mesenchymal stem cell therapy shows great potential for kidney tissue regeneration. The use of measurable biomarkers is crucial for assessing the therapeutic effects of mesenchymal stem cells. Adiponectin is among the suggested biomarkers for monitoring the progression of chronic renal failure.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe extracted mesenchymal stem cells from the amnion membrane of term placentas. To establish the experimental groups, we partially ligated the left kidneys of male Wistar rats, fully removed the right kidney after two weeks, and observed them for an additional eight weeks. At the end of this period, the animals underwent a subtotal nephrectomy. After forming 5/6 nephrectomy, we transplanted stem cells via rat tail vein and waited for 15 and 30 days to form stem cell groups. We measured protein levels and mRNA expressions of Adiponectin, Adiponectin Receptor 1, Fibronectin and AMPK phosphorylation by western blot and Real-Time PCR methods respectively. Besides, urine and serum levels of adiponectin and urine levels of albumin measured by using a rat specific ELISA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eProtein and mRNA expressions of Adiponectin, AdipoR1, Fibronectin, and AMPK phosphorylation were elevated in the nephrectomy groups compared to the controls; however, these increased gene and protein expressions declined following stem cell administration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003emesenchymal stem cells may have a therapeutic effect on chronic renal failure, and adiponectin may serve as a biomarker for monitoring disease progression.\u003c/p\u003e","manuscriptTitle":"The Effect of Placental Mesenchymal Stem Cells on The Protection of Chronic Renal Failure: The Role of Adiponectin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-21 07:03:51","doi":"10.21203/rs.3.rs-6058870/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":"66416b70-2fdb-4265-b60c-6db77213e760","owner":[],"postedDate":"February 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T08:23:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-21 07:03:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6058870","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6058870","identity":"rs-6058870","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}