Reactive oxygen species-evoked endoplasmic reticulum stress mediates albumin load-induced epithelial-mesenchymal transition in podocytes

Reactive oxygen species-evoked endoplasmic reticulum stress mediates albumin load-induced epithelial-mesenchymal transition in podocytes | 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 Reactive oxygen species-evoked endoplasmic reticulum stress mediates albumin load-induced epithelial-mesenchymal transition in podocytes Chien-An Chen, Jer-Ming Chang, Eddy-Essen Chang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8002591/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Feb, 2026 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Background Epithelial-mesenchymal transition (EMT) plays a significant role in cell migration and tissue fibrosis. Podocytes may undergo EMT after injury, leading to podocyte migration and detachment, which results ultimately in albuminuaria and renal fibrosis. The molecular mechanisms linking albuminuria to the progression of renal fibrosis is complex and not yet fully understood. Reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress play significant roles in renal fibrosis. In this study, we evaluate whether albumin load induces podocyte EMT through ROS/ER stress pathways. Methods Podocytes were exposed to medium alone or to high concentrations of delipidated, endotoxin-free human serum albumin (HSA, 10 mg/ml) (albumin load) with or without antioxidant (N-acetylcysteine, NAC) and ER stress inhibitors (4-phenylbutyrate (4-PBA) and salubrinal (Sal)). Intracellular ROS generation was measured using the fluorescent indicator 20, 70-dichlorofluorescin diacetate (DCF-DA). Both mRNA and protein levels of the EMT biomarker (α-smooth muscle actin, α-SMA) and the protein levels of ER stress biomarkers (GRP78 and CHOP) were assessed by real-time PCR and Western blotting. Results After albumin load, intracellular albumin was increased in podocytes. In addition, the elevated ROS generation and protein levels of both GRP78 and CHOP were caused under albumin load, both of which could be reversed by NAC. Also, 4-PBA attenuated ROS generation induced by albumin load. Both mRNA and protein levels of α-SMA were up-regulated under albumin load. NAC alleviated albumin load-induced alterations in both mRNA and protein levels of α-SMA. Also, both 4-PBA and Sal ameliorated albumin load –triggered both mRNA and protein levels of α-SMA. Conclusion Our findings suggested that albumin load promoted podocyte EMT mainly via ROS-mediated ER stress. The ROS-ER stress related pathways might be a potential intervention target for albuminuria-caused renal fibrosis. albuminuria epithelial-mesenchymal transition ROS ER stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The association between podocyte damage (podocytopathy) and albuminuria has been proven [ 1 ]. Mounting evidence has demonstrated that podocytes can undergo epithelial-mesenchymal transition (EMT) with expression of mesenchymal marker, α-smooth muscle actin (α-SMA), under triggered by various stimuli in the pathological states [ 2 , 3 ]. EMT may serve as a potential pathway leading to podocyte dysfunction, albuminuria and glomerulosclerosis [ 4 ]. Albuminuria is a key hallmark of renal disease, and its magnitude is a well-established adverse prognostic factor for the progression of chronic kidney disease regardless of the underlying pathologic cause [ 5 ]. In a study from the Stockholm CREAtinine Measurements (SCREAM) project, Carrero et al. found that a 4-fold increase in albuminuria was associated with a 3.08-fold (95% confidence interval 2.59 to 3.67) increase in risk of progression to end-stage renal disease [ 6 ]. However, the molecular mechanisms linking albuminuria to renal disease progression is complex and not yet fully understood. The endoplasmic reticulum (ER) plays significant roles in posttranslational modification, folding and trafficking of newly synthesized proteins [ 7 ]. Disturbance to ER homeostasis causes to ER stress response that leads to activation of tree ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6). The activated IRE1 regulates the transcription of the ER chaperones and enzymes. The activated ATF6 up-regulates the transcription the enzymes, which promote protein folding and maturation and increase the ER chaperones (including glucose-regulated protein 78, GRP78). The activated PERK causes phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which further activates activating transcription factor 4 (ATF4). The ATF6 and ATF4 pathways finally induce the expression of proapoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP), which induces apoptosis [ 7 ]. While the initial purpose of ER stress may be protective, prolonged or severe ER stress can eventually trigger cell damage. Several studies have demonstrated that ER stress and reactive oxygen species (ROS) play a vital role in the pathogenesis of albuminuria and nephrotic syndrome [ 8 – 10 ]. In this study, we hypothesized that albumin load could promote podocyte EMT through the ROS-ER stress pathways, thereby enhancing podocyte migration and contributing to further albuminuria and renal fibrosis. Materials and Methods Experimental design Cells were exposed to medium alone or to high concentrations of delipidated, endotoxin-free human serum albumin (HSA 10 mg/ml) (albumin load) (Sigma Chemical Co.) at 37°C for various time intervals. In some experiments, cells were pretreated for 1 h with ROS scavenger (50 uM N-acetylcysteine, NAC) (Sigma Chemical Co.), ER stress inhibitors (50 µM salubrinal, sal; an inhibitor for dephosphorylation of eIF2α) (Enzo Life Sciences) and 10 mM 4-phenylbutyrate, 4-PBA; an ER stress inhibitor, chemical chaperone that facilitates protein folding in the ER) (Sigma Chemical Co.)) prior to the addition of HSA. Following treatment, cells were processed for measuring intracellular albumin, intracellular ROS, ER stress biomarkers (GRP78 and CHOP), and EMT biomarker (α-SMA). Primary podocyte cell culture Primary podocyte culture was performed as described in our previous study [ 11 ]. All animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals, as approved by Kaohsiung Medical University. The research was carried out in compliance with the principles of the Declaration of Helsinki. Immunofluorescence confocal microscopy The endocytosis of HSA by podocytes was evaluated using a chicken polyclonal antibody to albumin (1:200; ab14225, Abcam, Cambridge Science Park, Cambridge, UK) in immunofluorescence confocal microscopy, as described in our previous study [ 12 ] Intracellular ROS assay Intracellular ROS generation was estimated using the fluorescent indicator 2',7'-dichlorofluorescin diacetate (DCF-DA) (Sigma Chemical Co.) as described in our previous study [ 13 ] Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) The α-SMA mRNA was detected by semiquantitative RT-PCR as described in previous research [ 14 ]. The primer pairs used were as follows: α-SMA, forward: 5’- TGCTCCAGCTATGTGTGAAGA-3’, reverse: 5’-AAGGTCGGATGCTCCTCTG-3’. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH), forward: 5’-TCACCATCTTCTAGGGAGA-3’, reverse: 5’-CTTCTGGGTGGCAGTGATG-3’ (337bp). The relative efficiency of RT-PCR amplification was quantified according to the relative intensity of GAPDH bands. Western blot analysis Cells were plated on collagen-coated plastic dishes in serum-free RPMI 1640 medium for 24 h, and then incubated with medium alone, HSA, NAC, 4-PBA, Sal, HSA + NAC, HSA + 4-PBA, or HSA + Sal, for different time intervals. Western blot analyses were then carried out as described previously [ 15 ]. The following primary antibodies were used: anti-megalin/LRP2 antibody (H-10) (1:1000; Santa Cruz Biotechnology, CA), 1A4 anti-α-SMA monoclonal antibody (1:1000; Sigma Chemical Co. St. Louis, Mo, USA), anti-GRP78 polyclonal antibody (1:1000; Santa Cruz Biotechnology, CA), and anti-CHOP antibody (1:500; Santa Cruz Biotechnology, CA). Equal loading was evaluated by testing for Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (1:1000) (Sigma Chemical Co.). Results are expressed as ratio (protein of interest/GAPDH) to correct for sample loading. Experiments were performed at least three times. Statistical analysis The GraphPad Prism 5.0 statistical package was used for analysis. Data are expressed as mean ± SE. Differences between groups were analyzed by ANOVA followed by Bonferroni post hoc testing, with p < 0.05 indicating statistical significance. Results Albumin uptake and megalin expression Megalin is the albumin receptor in podocyte [ 16 ]. We evaluated whether podocytes could up-take albumin and then, the intracellular albumin affects megalin expression. Albumin was found to be endocytosed at 1 h after HSA treatment (Fig. 1 A). The intracellular albumin was dominant at 1 h and decreased gradually at 24 and 48 h after HSA treatment (Fig. 1 B). Figure 1 C showed that megalin did not decrease at 12, 24, and 48 h after HSA treatment. Albumin load activated ER stress via ROS To evaluate whether the albumin load could mediate ROS generation, intracellular ROS was estimated using DCF-DA levels. Figure 2 A showed that DCF-DA levels increased at 30, 60, 90 and 120 min after HSA treatment, indicating that albumin load elicited ROS generation. To assess whether the albumin load could induce ER stress, ER stress biomarkers (GRP78 and CHOP) were examined at 12, 24 and 48 h after HSA treatment. As seen in Fig. 2 B, GRP78 and CHOP were up-regulated at 24 and 48 h after HSA treatment. Recent studies demonstrate that ROS mediates ER stress in hepatic cells after arsenite or cadmium exposure [ 17 , 18 ]. To evaluate whether the albumin load-induced ER stress was mediated through ROS, we used ROS scavenger (NAC) to ameliorate albumin load-induced ROS generation and ER stress-related biomarkers. NAC markedly reduced ROS generation at 5, 20, 40, 60, 90, 120, and 180 min after HSA treatment (Fig. 3 A). Moreover, NAC also reduced the protein levels of ER stress-related biomarkers (GRP78 and CHOP) at 48 h after HSA treatment (Fig. 3 B). These results suggested that albumin load activated ER stress through ROS in podocytes. Wang et al. found that inhibiting ER stress partially reversed the ROS generation induced by HDR1 deficiency in vascular smooth muscle cells [ 19 ]. In this study, we evaluated whether ER stress mediated ROS generation, therefore cells were pretreated with an ER stress inhibitor (4-PBA) and the production of ROS was examined at various time points after HSA treatment. Figure 3 C showed that 4-PBA partially reduced ROS generation at 5, 20, 40, 60, 90, 120 and 180 min after HSA treatment, indicating that ER stress also mediated ROS generation. Taken together, the above results revealed that albumin load induced ROS generation, contributing to activate ER stress, which in turn further up-regulated ROS generation, leading to a vicious cycle. Albumin load caused podocyte EMT For evaluating whether albumin load could mediate podocyte EMT, both mRNA and protein levels of α-SMA were examined after HSA treatment. The α-SMA mRNA levels were significantly up-regulated at 24 h after HSA treatment (Fig. 4 A). The α-SMA protein levels were also up-regulated at 48 h after HSA treatment (Fig. 4 B). These results showed that albumin load could induce podocyte EMT Inhibition of ER stress reversed albumin load-evoked podocyte EMT Accumulating evidence indicated that the significant association between ER stress and EMT in alveolar epithelial cells and pulmonary fibrosis [ 20 , 21 ]. We determined whether the albumin load-evoked podocyte EMT was mediated by ER stress. 4-PBA (an ER stress inhibitor, chemical chaperone that facilitates protein folding in the ER) and Sal (an inhibitor for dephosphorylation of eIF2α) have been shown to modulate ER stress pathways [ 22 ]. Both α-SMA mRNA and protein levels were examined after HSA treatment with or without 4-PBA. 4-PBA significantly alleviated albumin load-evoked α-SMA mRNA levels at 24 h after HSA treatment (Fig. 4 A). 4-PBA also significantly abolished albumin load-evoked α-SMA protein levels at 48 h after HSA treatment (Fig. 4 B). The studies showed that PERK-eIF2α-ATF4-CHOP pathway regulated apoptosis, autophage, EMT, fibrosis, and migration [ 22 – 24 ]. To evaluate the effect of the PERK-eIF2α axis on podocyte EMT, Sal was used to up-regulate activation of eIF2α (p-eIF2α). Figure 4 A showed that Sal abolished albumin load-evoked α-SMA mRNA levels at 24 h after HSA treatment. Sal also significantly abolished albumin load-evoked α-SMA protein levels at 48 h after HSA treatment (Fig. 4 B). Interestingly, ROS scavenger (NAC) reversed albumin-evoked both mRNA and protein levels of α-SMA at 24 h and 48 h respectively after HSA treatment (Fig. 4 A and 4 B). Altogether, these results verified that ROS-activated ER stress participated in albumin load-induced podocyte EMT. Discussion Albuminuria is associated with the progression of chronic kidney disease. The experimental data demonstrates that the abnormal modulation of podocyte differentiation or function, rather than podocyte loss, may be a trigger of albuminuria, because podocyte loss often occurs in late stage of chronic renal disease in which albuminuria is already distinct [ 4 , 25 ]. EMT has been demonstrated to play an important role in renal fibrogenesis [ 26 , 27 ]. Podocyte EMT is characterized by deficiency of podocyte-specific markers, including ZO-1, nephrin, and podocin, and de novo gains mesenchymal markers such as desmin, α-SMA, collagen type 1, and fibronectin [ 4 , 28 ]. Podocyte EMT has been found to be associated with the development of albuminuria [ 29 ]. This study found that podocyet EMT derived from albumin load was through ROS-ER stress signaling pathways, thereby contributing to further albuminuria. Megalin is expressed in podocytes and co-localize with podocin [ 16 ]. It can bind multiple ligands including albumin, lipoproteins, insulin, insulin-like growth factor, and drugs [ 30 ]. Several studies have shown that albumin endocytosis is megalin-dependent [ 16 , 31 ], and megalin-knockout mice exhibited increased albuminuria [ 32 ]. Urinary excretion of megalin is associated with the progression of albuminuria in type 2 diabetes mellitus [ 30 ]. This study found that albumin load did not reduce megalin protein levels in podocytes. From these results, we suggested that albuminuria did not modulate megalin to promote further albuminuria. ROS are important signaling messengers involved in regulating cytoskeleton remodeling, extracellular matrix remodeling, proliferation, apoptosis, autophagy, and migration [ 33 , 34 ]. This study showed that albumin load induced ROS generation. Several studies also showed that albumin load induced ROS generation in proximal tubule cells, renal collecting duct cells and podocytes [ 12 , 35 , 36 ]. Although the mechanisms by which albumin load induced ROS generation remains unclear, Imai et al. showed that albumin uptake activates PKC, which leaded to translocation of p47phox to the membrane and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in renal proximal tubule cells [ 37 ]. In the animal model of minimal change nephrotic syndrome, Kinugasa et al., found that an inhibitor of NADPH oxidase (apocynin) decreased ROS production in podocytes [ 38 ]. From these studies, we suggested that ROS generation induced by albumin load might be through NADPH oxidase in podocytes. ER performs multiple functions, including protein biosynthesis, rectifying abnormal protein folding/function and serving as a signaling platform [ 39 ]. ER stress refers to the accumulation of unfolded or misfolded proteins in the ER lumen, which disrupts ER functions. ER stress activates a series of integrative pathways called the unfolded protein response (UPR) to maintain cellular homeostasis and biological functions [ 39 ]. Lee et al. found that ROS act as upstream signals in the activation of ER stress [ 40 ]. This study showed that an antioxidant (NAC) attenuated intracellular albumin load-evoked ER stress and ER stress inhibitor (4-PBA) reduced ROS generation in podocytes. The ER membrane has three sensors for ER stress: protein kinase RNA (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol requiring enzyme 1α (IRE1α) [ 41 ]. Recent studies show that active X-box binding protein 1 (XBP1), a downstream target of IRE1α, is translated, leading to ROS generation through myo-inositol oxygenase (MIOX) and mitochondrial stress [ 41 , 42 ]. From these studies, we suggested that ROS induced ER stress, which in turn promoted more ROS generation. EMT is defined as the process where epithelial cells down-regulate epithelial markers and up-regulate mesenchymal markers such as α-SMA, while gaining the fibrotic capacity, motility and invasive characteristics of mesenchymal cells [ 40 , 43 ]. Recent studies have demonstrated that Podocyte EMT is through several molecular signaling pathways including advanced oxidative products, advanced glycation end products, TGF-β/Smad, Integrins/ILK, MAPKs, PI3K/AKT/mTOR, RTK/Ras/ERK, Wnt/β-catenin, Jagged/Notch, and NF-kB [ 44 ]. Moreover, crosstalk between these signaling pathways makes podocyte EMT molecular mechanism more complex. This study showed that albumin load induced podocyte EMT and an antioxidant (NAC) attenuated albumin load-evoked ROS, and EMT. Recent studies showed that ROS promoted the process of podocyte EMT, but the exact signaling pathways were unclear. [ 45 , 46 ]. ER stress plays a significant role in the pathogenesis of various renal diseases including focal segmental glomerusclerosis, membranous nephropathy, IgA nephropathy and diabetic kidney disease [ 47 , 48 ]. In this study, both mRNA and protein levels of α-SMA were up-regulated after albumin load, but down-regulated following an ER stress inhibitor (4-phenylbutyrate) co-treatment. In a study of hypoxia-induced EMT in alveolar epithelial cells (AECs), Delbrel et al. found that ER stress signaling pathways were involved in hypoxia-induced EMT both in vivo and in vitro [ 20 ]. These studies showed that ER stress promoted the process of podocyte EMT, but the exact signaling pathways were unclear. Meng et al. found that ER stress promoted EMT through the PERK signaling pathway in paraquat-induced pulmonary fibrosis [ 21 ]. Ni et al. showed that bavachin induced EMT of human proximal tubular epithelial cells was through GRP78/eIF2α/CHOP signaling pathway [ 23 ]. However in a study for evaluating ER stress and autophagy in the regulation of insulin resistance, Nguyen et al. found that salubrinal elicited eIF2α-ATF4-autophagy pathway leading to improve skeletal muscle insulin sensitivity [ 24 ]. This study showed that salubrinal significantly decreased both α-SMA mRNA and protein levels that were elevated by albumin load. From these results, we suggested that PERK-eIF2α-ATF4 activated autophagy leading to antagonize ER stress and provided a negative feedback mechanism to alleviate cell stress [ 41 ]. Recent studies showed that ER stress might indirectly regulate EMT. Yu et al. found that nickel chloride induced ER stress and EMT, and then an ER stress inhibitor (4-phenylbutyrate) alleviated nickel chloride-induced Smad2/3 activation, EMT, and migration in lung cancer cells (A549 cells) [ 49 ]. In a study of the relationship between ER stress and high-fat diet induced kidney damage, Mu et al. demonstrated that ER stress promoted renal fibrosis in high-fat diet fed mice through TGF-β/SMAD signaling pathway [ 50 ]. From these results, we suggested that ER stress directly and indirectly regulated EMT. In view of these results, we can conclude that albuminuria induces podocyte EMT through ROS-ER stress pathway (Fig. 5 ). The present findings further elucidate the molecular mechanism of albuminuria-induced glomerulosclerosis and renal fibrosis. ER stress was activated in podocytes after albumin load and was found to cross-talk with ROS. This interaction may lead to the secretion of pro-fibrotic cytokines, podocyte migration, and detachment, all of which promote the progression of albuminuria and glomerulosclerosis. Therefore, inhibiting ER stress-induced podocyte EMT may represent a key strategy for treating albuminuria and renal fibrosis in clinical settings. Declarations Funding This study is not funded. Conflict of interest The authors have declared that no conflict of interest exists Author contributions Chien-An Chen contributed to the conception, design, analysis and interpretation of the data, drafting of the article, critical revision and final approval. Jer-Ming Chang contributed to the design, analysis and interpretation of the data and final approval. Eddy-Essen Chang contributed to the analysis and interpretation of the data and final approval. Funding This study is not funded. Data availability No datasets were generated or analysed during the current study. Ethics declarations Conflict of interest The authors have declared that no conflict of interest exists Ethical approval All animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals, as approved by the Institutional Animal Care and Use Committee, Kaohsiung Medical University (approval number: IACUC-113108). The research was carried out in compliance with the principles of the Declaration of Helsinki. References Kopp JB, Anders H-J, Susztak K, Podestà MA, Remuzzi G, Hildebrandt F et al (2020) Podocytopathies Nat Rev Dise Primers 6(1):68 Chen CA, Hwang JC, Guh JY, Tsai JC, Chen HC (2006) TGF-beta1 and integrin synergistically facilitate the differentiation of rat podocytes by increasing alpha-smooth muscle actin expression. Transl Res 148(3):134–141 Dou Y, Shang Y, Shen Y, Qu J, Liu C, Cao J (2020) Baicalin alleviates adriamycin-induced focal segmental glomerulosclerosis and proteinuria by inhibiting the Notch1-Snail axis mediated podocyte EMT. Life Sci 257:118010 Li L, Feng Y, Zhang J, Zhang Q, Ren J, Sun C et al (2022) Microtubule associated protein 4 phosphorylation-induced epithelial-to-mesenchymal transition of podocyte leads to proteinuria in diabetic nephropathy. Cell Commun Signal 20(1):115 Jafar TH, Stark PC, Schmid CH, Landa M, Maschio G, Marcantoni C et al (2001) Proteinuria as a modifiable risk factor for the progression of non-diabetic renal disease. Kidney Int 60(3):1131–1140 Carrero JJ, Grams ME, Sang Y, Ärnlöv J, Gasparini A, Matsushita K et al (2017) Albuminuria changes are associated with subsequent risk of end-stage renal disease and mortality. Kidney Int 91(1):244–251 Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 115(10):2656–2664 Cybulsky AV, Papillon J, Guillemette J, Navarro-Betancourt JR, Chung CF, Iwawaki T et al (2024) Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice. Sci Rep 14(1):11718 Park S-J, Kim Y, Yang S-M, Henderson MJ, Yang W, Lindahl M et al (2019) Discovery of endoplasmic reticulum calcium stabilizers to rescue ER-stressed podocytes in nephrotic syndrome. Proc Natl Acad Sci 116(28):14154–14163 Fakhruddin S, Alanazi W, Jackson KE (2017) Diabetes-Induced Reactive Oxygen Species: Mechanism of Their Generation and Role in Renal Injury. J Diabetes Res 2017(1):8379327 Chen CA, Tsai JC, Su PW, Lai YH, Chen HC (2006) Signaling and regulatory mechanisms of integrinalpha3beta1 on the apoptosis of cultured rat podocytes. J Lab Clin Med 147(6):274–280 Cheng YC, Chen CA, Chang JM, Chen HC (2015) Albumin overload down-regulates integrin-beta1 through reactive oxygen species-endoplasmic reticulum stress pathway in podocytes. J Biochem 158(2):101–108 Chen CA, Chang JM, Chen HC, Chang EE (2022) Generation of endoplasmic reticulum stress-dependent reactive oxygen species mediates TGF-β1-induced podocyte migration. J Biochem 171(3):305–314 Chen CA, Hwang JC, Guh JY, Chang JM, Lai YH, Chen HC (2006) Reduced podocyte expression of alpha3beta1 integrins and podocyte depletion in patients with primary focal segmental glomerulosclerosis and chronic PAN-treated rats. J Lab Clin Med 147(2):74–82 Chen CA, Cheng YC, Hwang JC, Chang JM, Guh JY, Chen HC (2012) Cyclin D1 expression in podocytes: regulated by mitogens in collaboration with integrin-extracellular matrix interaction through extracellular signal-regulated kinase. Exp Biol Med (Maywood) 237(5):516–523 Schießl IM, Hammer A, Kattler V, Gess B, Theilig F, Witzgall R et al (2016) Intravital Imaging Reveals Angiotensin II-Induced Transcytosis of Albumin by Podocytes. J Am Soc Nephrol 27(3):731–744 Liu C, Zhang A (2020) ROS-mediated PERK-eIF2α-ATF4 pathway plays an important role in arsenite-induced L-02 cells apoptosis via regulating CHOP-DR5 signaling. EnviroToxicol 35(10):1100–1113 Zhaohui C, Cifei T, Di H, Weijia Z, Cairui H, Zecong L et al (2023) ROS-mediated PERK-CHOP pathway plays an important role in cadmium-induced HepG2 cells apoptosis. Environ Toxicol 38(9):2271–2280 Wang L, Wang M, Niu H, Zhi Y, Li S, He X et al (2024) Cholesterol-induced HRD1 reduction accelerates vascular smooth muscle cell senescence via stimulation of endoplasmic reticulum stress-induced reactive oxygen species. J Mol Cell Cardiol 187:51–64 Delbrel E, Uzunhan Y, Soumare A, Gille T, Marchant D, Planès C et al (2019) ER Stress is Involved in Epithelial-To-Mesenchymal Transition of Alveolar Epithelial Cells Exposed to a Hypoxic Microenvironment. Int J Mol Sci 20(6):1299 Meng X, Liu K, Xie H, Zhu Y, Jin W, Lu J et al (2021) Endoplasmic reticulum stress promotes epithelial–mesenchymal transition via the PERK signaling pathway in paraquat–induced pulmonary fibrosis. Mol Med Rep 24(1):525 Alsterda A, Asha K, Powrozek O, Repak M, Goswami S, Dunn AM et al (2021) Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway. Front Oncol 11:654940 Ni YH, Deng HF, Zhou L, Huang CS, Wang NN, Yue LX et al (2022) Ginsenoside Rb1 Ameliorated Bavachin-Induced Renal Fibrosis via Suppressing Bip/eIF2α/CHOP Signaling-Mediated EMT. Front Pharmacol 13:872474 Nguyen K, Tang J, Cho S, Ying F, Sung HK, Jahng JW et al (2024) Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance. Molr Metab 83:101921 Dai C, Stolz DB, Bastacky SI, St.-Arnaud R, Wu C, Dedhar S et al (2006) Essential Role of Integrin-Linked Kinase in Podocyte Biology: Bridging the Integrin and Slit Diaphragm Signaling. J Am Soc Nephrol 17(8):2164–2175 Hadpech S, Thongboonkerd V (2024) Epithelial–mesenchymal plasticity in kidney fibrosis. Genesis 62(1):e23529 Wang P, Chen W, li B, Yang S, Li W, Zhao S et al (2024) Exosomes on the development and progression of renal fibrosis. Cell Prolif 57(11):e13677 Chang YP, Sun B, Han Z, Han F, Hu SL, Li XY et al (2017) Saxagliptin Attenuates Albuminuria by Inhibiting Podocyte Epithelial- to-Mesenchymal Transition via SDF-1α in Diabetic Nephropathy. Front Pharmacoly 8:780 Hu S, Hang X, Wei Y, Wang H, Zhang L, Zhao L (2024) Crosstalk among podocytes, glomerular endothelial cells and mesangial cells in diabetic kidney disease: an updated review. Cell Commun Signal 22(1):136 Sun Y, Lu X, Danser AHJ (2020) Megalin: a Novel Determinant of Renin-Angiotensin System Activity in the Kidney? Curr Hypertens Rep 22(4):30 Elsakka EGE, Mokhtar MM, Hegazy M, Ismail A, Doghish AS (2022) Megalin, a multi-ligand endocytic receptor, and its participation in renal function and diseases: A review. Life Sci 308:120923 Weyer K, Storm T, Shan J, Vainio S, Kozyraki R, Verroust PJ et al (2011) Mouse model of proximal tubule endocytic dysfunction. Nephrol Dial Transpl 26(11):3446–3451 Jiang J, Wang K, Chen Y, Chen H, Nice EC, Huang C (2017) Redox regulation in tumor cell epithelial-mesenchymal transition: molecular basis and therapeutic strategy. Signal Transduct Target Ther 2:17036 Liu C, Liu Q, Mou Z (2024) Redox signaling and oxidative stress in systemic acquired resistance. J Exp Bot 75(15):4535–4548 Nazıroğlu M, Çiğ B, Yazğan Y, Schwaerzer GK, Theilig F, Pecze L (2019) Albumin evokes Ca(2+)-induced cell oxidative stress and apoptosis through TRPM2 channel in renal collecting duct cells reduced by curcumin. Sci Rep 9(1):12403 Eleftheriadis T, Pissas G, Golfinopoulos S, Efthymiadi M, Poulianiti C, Polyzou Konsta MA et al (2023) Routes of Albumin Overload Toxicity in Renal Tubular Epithelial Cells. Inte J Mol Sci 24(11):9640 Imai E, Nakajima H, Kaimori J-Y (2004) Albumin turns on a vicious spiral of oxidative stress in renal proximal tubules. Kidney Int 66(5):2085–2087 Kinugasa S, Tojo A, Sakai T, Tsumura H, Takahashi M, Hirata Y et al (2011) Selective albuminuria via podocyte albumin transport in puromycin nephrotic rats is attenuated by an inhibitor of NADPH oxidase. Kidney Int 80(12):1328–1338 Saito A, Imaizumi K (2018) Unfolded Protein Response-Dependent Communication and Contact among Endoplasmic Reticulum, Mitochondria, and Plasma Membrane. Int J Mol Sci. ;19(10) Lee JY, Chang JW, Yang WS, Kim SB, Park SK, Park JS et al (2011) Albumin-induced epithelial-mesenchymal transition and ER stress are regulated through a common ROS-c-Src kinase-mTOR pathway: effect of imatinib mesylate. Am J Physiol Ren Physiol 300(5):F1214–F1222 Wu D, Huang L-F, Chen X-C, Huang X-R, Li H-Y, An N et al (2023) Research progress on endoplasmic reticulum homeostasis in kidney diseases. Cell Death Dis 14(7):473 Zhang J, Zhao Y, Gong N (2023) XBP1 Modulates the Aging Cardiorenal System by Regulating Oxidative Stress. Antioxidants 12(11):1933 Manfioletti G, Fedele M (2023) Epithelial–Mesenchymal Transition (EMT). Int J Mol Sci 24(14):11386 Ying Q, Wu G (2017) Molecular mechanisms involved in podocyte EMT and concomitant diabetic kidney diseases: an update. Ren Fail 39(1):474–483 Zhou L, Chen X, Lu M, Wu Q, Yuan Q, Hu C et al (2019) Wnt/β-catenin links oxidative stress to podocyte injury and proteinuria. Kidney Int 95(4):830–845 Kang M-K, Kim S-I, Oh SY, Na W, Kang Y-H (2020) Tangeretin Ameliorates Glucose-Induced Podocyte Injury through Blocking Epithelial to Mesenchymal Transition Caused by Oxidative Stress and Hypoxia. Int J Mol Sci 21(22):8577 Jandeleit-Dahm KA, Kankanamalage HR, Dai A, Meister J, Lopez-Trevino S, Cooper ME et al (2024) Endothelial NOX5 obliterates the reno-protective effect of Nox4 deletion by promoting renal fibrosis via activation of EMT and ROS-sensitive pathways in diabetes. Antioxidants 13(4):396 Ricciardi CA, Gnudi L (2020) The endoplasmic reticulum stress and the unfolded protein response in kidney disease: Implications for vascular growth factors. J Cell Mol Med 24(22):12910–12919 Yu M, Chen F, Wang H, Fu Q, Yan L, Chen Z et al (2023) Endoplasmic reticulum stress mediates nickel chloride-induced epithelial–mesenchymal transition and migration of human lung cancer A549 cells through Smad2/3 and p38 MAPK activation. Ecotoxicol Environ Saf 249:114398 Mu Z, Li B, Chen M, Liang C, Gu W, Su J (2024) Endoplasmic reticulum stress induces renal fibrosis in high–fat diet mice via the TGF–β/SMAD pathway. Mol Med Reps 30(6):235 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 20 Feb, 2026 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 17 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor assigned by journal 02 Nov, 2025 Submission checks completed at journal 02 Nov, 2025 First submitted to journal 31 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8002591","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":540644623,"identity":"c9bda36b-db9e-4f7f-8673-e08c76a5b0d5","order_by":0,"name":"Chien-An Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACCTB5AIiZGx+AmAbMxGthbDaAawHxeYjQ0gZmG8D4uLTwz24+9oCh5o6cwbWDbVU3/tgxmLPzHnz8geGOnD0uS+4cSzdgOPbM2OB2Ytvt3LZkBstmvmSDAwzPjHHZYiCRYybB2HA4cQNYSwMzg8FhHjOJAwyHE3twasn/BtdSnPOnHqTF/AdQSz1uLTlscC3MOWyHwbYAvX84AZfDJG6kmUkkHDtsLHk7sVk6t+04D1CLscQZg8OGPQdwhNiM5GcSH2oOy/HdTj74OedPtZzB+TOGHyoqDsuzN+CwBgQSkNhQ5xjgUT4KRsEoGAWjgCAAALP+XEsgyYSbAAAAAElFTkSuQmCC","orcid":"","institution":"Tainan Sinlau Hospital","correspondingAuthor":true,"prefix":"","firstName":"Chien-An","middleName":"","lastName":"Chen","suffix":""},{"id":540644624,"identity":"e6df0989-f7a1-415e-9a13-b52cea0f4f64","order_by":1,"name":"Jer-Ming Chang","email":"","orcid":"","institution":"Kaohsiung Medical University 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10:37:13","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105647,"visible":true,"origin":"","legend":"","description":"","filename":"173849db7d7948d5831965f8c9450ebd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/3a5a4ae78ca586a1bf3026b9.xml"},{"id":95815902,"identity":"736fab7a-962a-49c6-8ffe-fc6eb8fe5b3c","added_by":"auto","created_at":"2025-11-13 09:40:14","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112184,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/723d5c8911094335ccf7e1e5.html"},{"id":95818977,"identity":"1c6bf181-bd99-472c-b629-4287d67dae37","added_by":"auto","created_at":"2025-11-13 10:36:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlbumin endocytosis and megalin expression in podocytes.\u003c/strong\u003e Cells were treated with 10 mg/ml HSA for different times, and intracellular albumin and megalin expression were then analyzed. (A) The endocytosed albumin was detected as green vesicles distributed in the cytosol at 1 h after HSA treatment. Bar, 10 μm. (B) The expression of megalin remained unchanged at 12, 24 and 48 h after HSA treatment.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/fbb77bd6b8847b85b6c4177a.jpg"},{"id":95819136,"identity":"60647c1b-b41c-4483-83b2-1b7f55d039ec","added_by":"auto","created_at":"2025-11-13 10:38:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlbumin load up-regulated ROS generation and ER stress.\u003c/strong\u003e Podocytes were incubated with 10 mg/ml HSA for different times, and intracellular ROS generation and ER stress biomarkers (GRP78 and CHOP) were then measured. (A) DCF-DA levels increased at 30, 60, 90 and 120 min after HSA treatment. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with at 0 min; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with at 30 min; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with at 60 min; \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with at 90 min. (B) The protein expressions of GRP78 and CHOP were up-regulated at 24 and 48 h after HSA treatment. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with at 0 h; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with at 24 h.\u003c/p\u003e","description":"","filename":"Figure2albuminROSERstresscrosstalk.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/21e13cb2da2d998a401b9989.jpg"},{"id":95815885,"identity":"325aa767-2b1c-41ec-af15-2b309e4fbbc5","added_by":"auto","created_at":"2025-11-13 09:40:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlbumin load led to cross-talk between ROS and ER stress.\u003c/strong\u003e Cells were pretreated with ROS scavenger (NAC, 50 uM) or ER stress inhibitor (4-PBA, 10 mM) for 1 h before the addition of HSA. Cells were then processed for measuring intracellular ROS and ER stress biomarkers (GRP78 and CHOP) at different times. (A) NAC attenuated intracellular ROS generation at 5, 20, 40, 60, 120 and 180 min after HSA treatment. (B) The protein expressions of GRP78 and CHOP were up-regulated at 48 h after HSA treatment. NAC attenuated the protein expressions of GRP78 and CHOP at 48 h after HSA treatment. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with normal control; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with HSA. (C) 4-PBA attenuated intracellular ROS generation at 5, 20, 40, 60, 120 and 180 min after HSA treatment.\u003c/p\u003e\n\u003cp\u003e4-PBA: 4-phenylbutyric acid.\u003c/p\u003e","description":"","filename":"newFig3HSANACERstresscrosstalk.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/9707b3b9ea56762a27dfbc1d.jpg"},{"id":95815883,"identity":"83077745-e4e3-4632-a61c-299a73069de8","added_by":"auto","created_at":"2025-11-13 09:40:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":111153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlbumin load regulated EMT expression through ROS and ER stress pathways.\u003c/strong\u003e Cells were pretreated with ROS scavenger (NAC, 50 uM), ER stress inhibitors (Sal, 50 μM; 4-PBA, 10 mM or 4-PBA, 10 mM) for 1 h before the addition of HSA. Cells were then processed for measuring α-SMA (an EMT biomarker) mRNA and protein expressions. (A) α-SMA mRNA expression was increased at 24 h after HSA treatment. NAC attenuated α-SMA mRNA expression at 24 h after HSA treatment. Both Sal and 4-PBA also attenuated α-SMA mRNA expression at 24 h after HSA treatment. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with normal control; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with HSA. (B) α-SMA protein expression was increased at 48 h after HSA treatment. NAC attenuated α-SMA protein expression at 48 h after HSA treatment. Both Sal and 4-PBA also attenuated α-SMA protein expression at 48 h after HSA treatment. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with normal control; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 compared with HSA.\u003c/p\u003e\n\u003cp\u003eSal: salubrinal; 4-PBA: 4-phenylbutyric acid.\u003c/p\u003e","description":"","filename":"Figure4albuminROSERstresscrosstalkinducedSMA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/00f87cd374587cf1cdef7e10.jpg"},{"id":95818923,"identity":"c0adebfe-6e0a-4549-bffa-4eec76ddbd9d","added_by":"auto","created_at":"2025-11-13 10:35:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of albumin load modulating podocyte EMT through ROS-ER stress pathway.\u003c/strong\u003e Albumin load initially activates ROS generation, which induces ER stress response. ER stress, in turn, promotes further ROS generation. Subsequently, ER stress up-regulates EMT. NAC inhibits albumin load-induced ROS generation and the ER stress response. The ER stress inhibitor 4-PBA also down-regulates albumin load-induced ROS generation. The ER stress inhibitors (4-PBA and Sal) down-regulate albumin load-induced α-SMA expression.\u003c/p\u003e\n\u003cp\u003eSal: salubrinal; 4-PBA: 4-phenylbutyric acid.\u003c/p\u003e","description":"","filename":"Fig5conclusion.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/b7804c82a4a3e67c7eaaea5d.jpg"},{"id":103251277,"identity":"b27d001a-a913-48d7-b685-5fdd18ed09ce","added_by":"auto","created_at":"2026-02-23 16:07:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1260649,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8002591/v1/f287368a-e848-47c5-b18b-bd4f6916cab3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reactive oxygen species-evoked endoplasmic reticulum stress mediates albumin load-induced epithelial-mesenchymal transition in podocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe association between podocyte damage (podocytopathy) and albuminuria has been proven [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mounting evidence has demonstrated that podocytes can undergo epithelial-mesenchymal transition (EMT) with expression of mesenchymal marker, α-smooth muscle actin (α-SMA), under triggered by various stimuli in the pathological states [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. EMT may serve as a potential pathway leading to podocyte dysfunction, albuminuria and glomerulosclerosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlbuminuria is a key hallmark of renal disease, and its magnitude is a well-established adverse prognostic factor for the progression of chronic kidney disease regardless of the underlying pathologic cause [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In a study from the Stockholm CREAtinine Measurements (SCREAM) project, Carrero et al. found that a 4-fold increase in albuminuria was associated with a 3.08-fold (95% confidence interval 2.59 to 3.67) increase in risk of progression to end-stage renal disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the molecular mechanisms linking albuminuria to renal disease progression is complex and not yet fully understood.\u003c/p\u003e\u003cp\u003eThe endoplasmic reticulum (ER) plays significant roles in posttranslational modification, folding and trafficking of newly synthesized proteins [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Disturbance to ER homeostasis causes to ER stress response that leads to activation of tree ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6). The activated IRE1 regulates the transcription of the ER chaperones and enzymes. The activated ATF6 up-regulates the transcription the enzymes, which promote protein folding and maturation and increase the ER chaperones (including glucose-regulated protein 78, GRP78). The activated PERK causes phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which further activates activating transcription factor 4 (ATF4). The ATF6 and ATF4 pathways finally induce the expression of proapoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP), which induces apoptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While the initial purpose of ER stress may be protective, prolonged or severe ER stress can eventually trigger cell damage. Several studies have demonstrated that ER stress and reactive oxygen species (ROS) play a vital role in the pathogenesis of albuminuria and nephrotic syndrome [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, we hypothesized that albumin load could promote podocyte EMT through the ROS-ER stress pathways, thereby enhancing podocyte migration and contributing to further albuminuria and renal fibrosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental design\u003c/h2\u003e\u003cp\u003eCells were exposed to medium alone or to high concentrations of delipidated, endotoxin-free human serum albumin (HSA 10 mg/ml) (albumin load) (Sigma Chemical Co.) at 37\u0026deg;C for various time intervals. In some experiments, cells were pretreated for 1 h with ROS scavenger (50 uM N-acetylcysteine, NAC) (Sigma Chemical Co.), ER stress inhibitors (50 \u0026micro;M salubrinal, sal; an inhibitor for dephosphorylation of eIF2α) (Enzo Life Sciences) and 10 mM 4-phenylbutyrate, 4-PBA; an ER stress inhibitor, chemical chaperone that facilitates protein folding in the ER) (Sigma Chemical Co.)) prior to the addition of HSA. Following treatment, cells were processed for measuring intracellular albumin, intracellular ROS, ER stress biomarkers (GRP78 and CHOP), and EMT biomarker (α-SMA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePrimary podocyte cell culture\u003c/h3\u003e\n\u003cp\u003ePrimary podocyte culture was performed as described in our previous study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. All animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals, as approved by Kaohsiung Medical University. The research was carried out in compliance with the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence confocal microscopy\u003c/h3\u003e\n\u003cp\u003eThe endocytosis of HSA by podocytes was evaluated using a chicken polyclonal antibody to albumin (1:200; ab14225, Abcam, Cambridge Science Park, Cambridge, UK) in immunofluorescence confocal microscopy, as described in our previous study [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003eIntracellular ROS assay\u003c/h3\u003e\n\u003cp\u003eIntracellular ROS generation was estimated using the fluorescent indicator 2',7'-dichlorofluorescin diacetate (DCF-DA) (Sigma Chemical Co.) as described in our previous study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003eSemiquantitative reverse transcriptase polymerase chain reaction (RT-PCR)\u003c/h3\u003e\n\u003cp\u003eThe α-SMA mRNA was detected by semiquantitative RT-PCR as described in previous research [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The primer pairs used were as follows: α-SMA, forward: 5\u0026rsquo;- TGCTCCAGCTATGTGTGAAGA-3\u0026rsquo;, reverse: 5\u0026rsquo;-AAGGTCGGATGCTCCTCTG-3\u0026rsquo;. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH), forward: 5\u0026rsquo;-TCACCATCTTCTAGGGAGA-3\u0026rsquo;, reverse: 5\u0026rsquo;-CTTCTGGGTGGCAGTGATG-3\u0026rsquo; (337bp). The relative efficiency of RT-PCR amplification was quantified according to the relative intensity of GAPDH bands.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eCells were plated on collagen-coated plastic dishes in serum-free RPMI 1640 medium for 24 h, and then incubated with medium alone, HSA, NAC, 4-PBA, Sal, HSA\u0026thinsp;+\u0026thinsp;NAC, HSA\u0026thinsp;+\u0026thinsp;4-PBA, or HSA\u0026thinsp;+\u0026thinsp;Sal, for different time intervals. Western blot analyses were then carried out as described previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The following primary antibodies were used: anti-megalin/LRP2 antibody (H-10) (1:1000; Santa Cruz Biotechnology, CA), 1A4 anti-α-SMA monoclonal antibody (1:1000; Sigma Chemical Co. St. Louis, Mo, USA), anti-GRP78 polyclonal antibody (1:1000; Santa Cruz Biotechnology, CA), and anti-CHOP antibody (1:500; Santa Cruz Biotechnology, CA). Equal loading was evaluated by testing for Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (1:1000) (Sigma Chemical Co.). Results are expressed as ratio (protein of interest/GAPDH) to correct for sample loading. Experiments were performed at least three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe GraphPad Prism 5.0 statistical package was used for analysis. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Differences between groups were analyzed by ANOVA followed by Bonferroni post hoc testing, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAlbumin uptake and megalin expression\u003c/h2\u003e\u003cp\u003eMegalin is the albumin receptor in podocyte [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We evaluated whether podocytes could up-take albumin and then, the intracellular albumin affects megalin expression. Albumin was found to be endocytosed at 1 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The intracellular albumin was dominant at 1 h and decreased gradually at 24 and 48 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC showed that megalin did not decrease at 12, 24, and 48 h after HSA treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAlbumin load activated ER stress via ROS\u003c/h2\u003e\u003cp\u003eTo evaluate whether the albumin load could mediate ROS generation, intracellular ROS was estimated using DCF-DA levels. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA showed that DCF-DA levels increased at 30, 60, 90 and 120 min after HSA treatment, indicating that albumin load elicited ROS generation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess whether the albumin load could induce ER stress, ER stress biomarkers (GRP78 and CHOP) were examined at 12, 24 and 48 h after HSA treatment. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, GRP78 and CHOP were up-regulated at 24 and 48 h after HSA treatment.\u003c/p\u003e\u003cp\u003eRecent studies demonstrate that ROS mediates ER stress in hepatic cells after arsenite or cadmium exposure [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To evaluate whether the albumin load-induced ER stress was mediated through ROS, we used ROS scavenger (NAC) to ameliorate albumin load-induced ROS generation and ER stress-related biomarkers. NAC markedly reduced ROS generation at 5, 20, 40, 60, 90, 120, and 180 min after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, NAC also reduced the protein levels of ER stress-related biomarkers (GRP78 and CHOP) at 48 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results suggested that albumin load activated ER stress through ROS in podocytes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWang et al. found that inhibiting ER stress partially reversed the ROS generation induced by HDR1 deficiency in vascular smooth muscle cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, we evaluated whether ER stress mediated ROS generation, therefore cells were pretreated with an ER stress inhibitor (4-PBA) and the production of ROS was examined at various time points after HSA treatment. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC showed that 4-PBA partially reduced ROS generation at 5, 20, 40, 60, 90, 120 and 180 min after HSA treatment, indicating that ER stress also mediated ROS generation.\u003c/p\u003e\u003cp\u003eTaken together, the above results revealed that albumin load induced ROS generation, contributing to activate ER stress, which in turn further up-regulated ROS generation, leading to a vicious cycle.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAlbumin load caused podocyte EMT\u003c/h2\u003e\u003cp\u003eFor evaluating whether albumin load could mediate podocyte EMT, both mRNA and protein levels of α-SMA were examined after HSA treatment. The α-SMA mRNA levels were significantly up-regulated at 24 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The α-SMA protein levels were also up-regulated at 48 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results showed that albumin load could induce podocyte EMT\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eInhibition of ER stress reversed albumin load-evoked podocyte EMT\u003c/h2\u003e\u003cp\u003eAccumulating evidence indicated that the significant association between ER stress and EMT in alveolar epithelial cells and pulmonary fibrosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We determined whether the albumin load-evoked podocyte EMT was mediated by ER stress. 4-PBA (an ER stress inhibitor, chemical chaperone that facilitates protein folding in the ER) and Sal (an inhibitor for dephosphorylation of eIF2α) have been shown to modulate ER stress pathways [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Both α-SMA mRNA and protein levels were examined after HSA treatment with or without 4-PBA. 4-PBA significantly alleviated albumin load-evoked α-SMA mRNA levels at 24 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). 4-PBA also significantly abolished albumin load-evoked α-SMA protein levels at 48 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThe studies showed that PERK-eIF2α-ATF4-CHOP pathway regulated apoptosis, autophage, EMT, fibrosis, and migration [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To evaluate the effect of the PERK-eIF2α axis on podocyte EMT, Sal was used to up-regulate activation of eIF2α (p-eIF2α). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA showed that Sal abolished albumin load-evoked α-SMA mRNA levels at 24 h after HSA treatment. Sal also significantly abolished albumin load-evoked α-SMA protein levels at 48 h after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eInterestingly, ROS scavenger (NAC) reversed albumin-evoked both mRNA and protein levels of α-SMA at 24 h and 48 h respectively after HSA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Altogether, these results verified that ROS-activated ER stress participated in albumin load-induced podocyte EMT.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlbuminuria is associated with the progression of chronic kidney disease. The experimental data demonstrates that the abnormal modulation of podocyte differentiation or function, rather than podocyte loss, may be a trigger of albuminuria, because podocyte loss often occurs in late stage of chronic renal disease in which albuminuria is already distinct [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. EMT has been demonstrated to play an important role in renal fibrogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Podocyte EMT is characterized by deficiency of podocyte-specific markers, including ZO-1, nephrin, and podocin, and de novo gains mesenchymal markers such as desmin, α-SMA, collagen type 1, and fibronectin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Podocyte EMT has been found to be associated with the development of albuminuria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This study found that podocyet EMT derived from albumin load was through ROS-ER stress signaling pathways, thereby contributing to further albuminuria.\u003c/p\u003e\u003cp\u003eMegalin is expressed in podocytes and co-localize with podocin [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It can bind multiple ligands including albumin, lipoproteins, insulin, insulin-like growth factor, and drugs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Several studies have shown that albumin endocytosis is megalin-dependent [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and megalin-knockout mice exhibited increased albuminuria [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Urinary excretion of megalin is associated with the progression of albuminuria in type 2 diabetes mellitus [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This study found that albumin load did not reduce megalin protein levels in podocytes. From these results, we suggested that albuminuria did not modulate megalin to promote further albuminuria.\u003c/p\u003e\u003cp\u003eROS are important signaling messengers involved in regulating cytoskeleton remodeling, extracellular matrix remodeling, proliferation, apoptosis, autophagy, and migration [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This study showed that albumin load induced ROS generation. Several studies also showed that albumin load induced ROS generation in proximal tubule cells, renal collecting duct cells and podocytes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Although the mechanisms by which albumin load induced ROS generation remains unclear, Imai et al. showed that albumin uptake activates PKC, which leaded to translocation of p47phox to the membrane and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in renal proximal tubule cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the animal model of minimal change nephrotic syndrome, Kinugasa et al., found that an inhibitor of NADPH oxidase (apocynin) decreased ROS production in podocytes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. From these studies, we suggested that ROS generation induced by albumin load might be through NADPH oxidase in podocytes.\u003c/p\u003e\u003cp\u003eER performs multiple functions, including protein biosynthesis, rectifying abnormal protein folding/function and serving as a signaling platform [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. ER stress refers to the accumulation of unfolded or misfolded proteins in the ER lumen, which disrupts ER functions. ER stress activates a series of integrative pathways called the unfolded protein response (UPR) to maintain cellular homeostasis and biological functions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Lee et al. found that ROS act as upstream signals in the activation of ER stress [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This study showed that an antioxidant (NAC) attenuated intracellular albumin load-evoked ER stress and ER stress inhibitor (4-PBA) reduced ROS generation in podocytes. The ER membrane has three sensors for ER stress: protein kinase RNA (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol requiring enzyme 1α (IRE1α) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recent studies show that active X-box binding protein 1 (XBP1), a downstream target of IRE1α, is translated, leading to ROS generation through myo-inositol oxygenase (MIOX) and mitochondrial stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. From these studies, we suggested that ROS induced ER stress, which in turn promoted more ROS generation.\u003c/p\u003e\u003cp\u003eEMT is defined as the process where epithelial cells down-regulate epithelial markers and up-regulate mesenchymal markers such as α-SMA, while gaining the fibrotic capacity, motility and invasive characteristics of mesenchymal cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Recent studies have demonstrated that Podocyte EMT is through several molecular signaling pathways including advanced oxidative products, advanced glycation end products, TGF-β/Smad, Integrins/ILK, MAPKs, PI3K/AKT/mTOR, RTK/Ras/ERK, Wnt/β-catenin, Jagged/Notch, and NF-kB [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, crosstalk between these signaling pathways makes podocyte EMT molecular mechanism more complex. This study showed that albumin load induced podocyte EMT and an antioxidant (NAC) attenuated albumin load-evoked ROS, and EMT. Recent studies showed that ROS promoted the process of podocyte EMT, but the exact signaling pathways were unclear. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eER stress plays a significant role in the pathogenesis of various renal diseases including focal segmental glomerusclerosis, membranous nephropathy, IgA nephropathy and diabetic kidney disease [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, both mRNA and protein levels of α-SMA were up-regulated after albumin load, but down-regulated following an ER stress inhibitor (4-phenylbutyrate) co-treatment. In a study of hypoxia-induced EMT in alveolar epithelial cells (AECs), Delbrel et al. found that ER stress signaling pathways were involved in hypoxia-induced EMT both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These studies showed that ER stress promoted the process of podocyte EMT, but the exact signaling pathways were unclear. Meng et al. found that ER stress promoted EMT through the PERK signaling pathway in paraquat-induced pulmonary fibrosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Ni et al. showed that bavachin induced EMT of human proximal tubular epithelial cells was through GRP78/eIF2α/CHOP signaling pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However in a study for evaluating ER stress and autophagy in the regulation of insulin resistance, Nguyen et al. found that salubrinal elicited eIF2α-ATF4-autophagy pathway leading to improve skeletal muscle insulin sensitivity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This study showed that salubrinal significantly decreased both α-SMA mRNA and protein levels that were elevated by albumin load. From these results, we suggested that PERK-eIF2α-ATF4 activated autophagy leading to antagonize ER stress and provided a negative feedback mechanism to alleviate cell stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recent studies showed that ER stress might indirectly regulate EMT. Yu et al. found that nickel chloride induced ER stress and EMT, and then an ER stress inhibitor (4-phenylbutyrate) alleviated nickel chloride-induced Smad2/3 activation, EMT, and migration in lung cancer cells (A549 cells) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In a study of the relationship between ER stress and high-fat diet induced kidney damage, Mu et al. demonstrated that ER stress promoted renal fibrosis in high-fat diet fed mice through TGF-β/SMAD signaling pathway [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. From these results, we suggested that ER stress directly and indirectly regulated EMT.\u003c/p\u003e\u003cp\u003eIn view of these results, we can conclude that albuminuria induces podocyte EMT through ROS-ER stress pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The present findings further elucidate the molecular mechanism of albuminuria-induced glomerulosclerosis and renal fibrosis. ER stress was activated in podocytes after albumin load and was found to cross-talk with ROS. This interaction may lead to the secretion of pro-fibrotic cytokines, podocyte migration, and detachment, all of which promote the progression of albuminuria and glomerulosclerosis. Therefore, inhibiting ER stress-induced podocyte EMT may represent a key strategy for treating albuminuria and renal fibrosis in clinical settings.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study is not funded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have declared that no conflict of interest exists\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eChien-An Chen contributed to the conception, design, analysis and interpretation of the data, drafting of the article, critical revision and final approval. Jer-Ming Chang contributed to the design, analysis and interpretation of the data and final approval. Eddy-Essen Chang contributed to the analysis and interpretation of the data and final approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study is not funded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have declared that no conflict of interest exists\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals, as approved by the Institutional Animal Care and Use Committee, Kaohsiung Medical University (approval number: IACUC-113108). The research was carried out in compliance with the principles of the Declaration of Helsinki.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKopp JB, Anders H-J, Susztak K, Podest\u0026agrave; MA, Remuzzi G, Hildebrandt F et al (2020) Podocytopathies Nat Rev Dise Primers 6(1):68\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CA, Hwang JC, Guh JY, Tsai JC, Chen HC (2006) TGF-beta1 and integrin synergistically facilitate the differentiation of rat podocytes by increasing alpha-smooth muscle actin expression. Transl Res 148(3):134\u0026ndash;141\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDou Y, Shang Y, Shen Y, Qu J, Liu C, Cao J (2020) Baicalin alleviates adriamycin-induced focal segmental glomerulosclerosis and proteinuria by inhibiting the Notch1-Snail axis mediated podocyte EMT. Life Sci 257:118010\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi L, Feng Y, Zhang J, Zhang Q, Ren J, Sun C et al (2022) Microtubule associated protein 4 phosphorylation-induced epithelial-to-mesenchymal transition of podocyte leads to proteinuria in diabetic nephropathy. Cell Commun Signal 20(1):115\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJafar TH, Stark PC, Schmid CH, Landa M, Maschio G, Marcantoni C et al (2001) Proteinuria as a modifiable risk factor for the progression of non-diabetic renal disease. Kidney Int 60(3):1131\u0026ndash;1140\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarrero JJ, Grams ME, Sang Y, \u0026Auml;rnl\u0026ouml;v J, Gasparini A, Matsushita K et al (2017) Albuminuria changes are associated with subsequent risk of end-stage renal disease and mortality. Kidney Int 91(1):244\u0026ndash;251\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 115(10):2656\u0026ndash;2664\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCybulsky AV, Papillon J, Guillemette J, Navarro-Betancourt JR, Chung CF, Iwawaki T et al (2024) Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice. Sci Rep 14(1):11718\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark S-J, Kim Y, Yang S-M, Henderson MJ, Yang W, Lindahl M et al (2019) Discovery of endoplasmic reticulum calcium stabilizers to rescue ER-stressed podocytes in nephrotic syndrome. Proc Natl Acad Sci 116(28):14154\u0026ndash;14163\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFakhruddin S, Alanazi W, Jackson KE (2017) Diabetes-Induced Reactive Oxygen Species: Mechanism of Their Generation and Role in Renal Injury. J Diabetes Res 2017(1):8379327\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CA, Tsai JC, Su PW, Lai YH, Chen HC (2006) Signaling and regulatory mechanisms of integrinalpha3beta1 on the apoptosis of cultured rat podocytes. J Lab Clin Med 147(6):274\u0026ndash;280\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng YC, Chen CA, Chang JM, Chen HC (2015) Albumin overload down-regulates integrin-beta1 through reactive oxygen species-endoplasmic reticulum stress pathway in podocytes. J Biochem 158(2):101\u0026ndash;108\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CA, Chang JM, Chen HC, Chang EE (2022) Generation of endoplasmic reticulum stress-dependent reactive oxygen species mediates TGF-β1-induced podocyte migration. J Biochem 171(3):305\u0026ndash;314\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CA, Hwang JC, Guh JY, Chang JM, Lai YH, Chen HC (2006) Reduced podocyte expression of alpha3beta1 integrins and podocyte depletion in patients with primary focal segmental glomerulosclerosis and chronic PAN-treated rats. J Lab Clin Med 147(2):74\u0026ndash;82\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CA, Cheng YC, Hwang JC, Chang JM, Guh JY, Chen HC (2012) Cyclin D1 expression in podocytes: regulated by mitogens in collaboration with integrin-extracellular matrix interaction through extracellular signal-regulated kinase. Exp Biol Med (Maywood) 237(5):516\u0026ndash;523\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchie\u0026szlig;l IM, Hammer A, Kattler V, Gess B, Theilig F, Witzgall R et al (2016) Intravital Imaging Reveals Angiotensin II-Induced Transcytosis of Albumin by Podocytes. J Am Soc Nephrol 27(3):731\u0026ndash;744\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu C, Zhang A (2020) ROS-mediated PERK-eIF2α-ATF4 pathway plays an important role in arsenite-induced L-02 cells apoptosis via regulating CHOP-DR5 signaling. EnviroToxicol 35(10):1100\u0026ndash;1113\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhaohui C, Cifei T, Di H, Weijia Z, Cairui H, Zecong L et al (2023) ROS-mediated PERK-CHOP pathway plays an important role in cadmium-induced HepG2 cells apoptosis. Environ Toxicol 38(9):2271\u0026ndash;2280\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang L, Wang M, Niu H, Zhi Y, Li S, He X et al (2024) Cholesterol-induced HRD1 reduction accelerates vascular smooth muscle cell senescence via stimulation of endoplasmic reticulum stress-induced reactive oxygen species. J Mol Cell Cardiol 187:51\u0026ndash;64\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDelbrel E, Uzunhan Y, Soumare A, Gille T, Marchant D, Plan\u0026egrave;s C et al (2019) ER Stress is Involved in Epithelial-To-Mesenchymal Transition of Alveolar Epithelial Cells Exposed to a Hypoxic Microenvironment. Int J Mol Sci 20(6):1299\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng X, Liu K, Xie H, Zhu Y, Jin W, Lu J et al (2021) Endoplasmic reticulum stress promotes epithelial\u0026ndash;mesenchymal transition via the PERK signaling pathway in paraquat\u0026ndash;induced pulmonary fibrosis. Mol Med Rep 24(1):525\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlsterda A, Asha K, Powrozek O, Repak M, Goswami S, Dunn AM et al (2021) Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway. Front Oncol 11:654940\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNi YH, Deng HF, Zhou L, Huang CS, Wang NN, Yue LX et al (2022) Ginsenoside Rb1 Ameliorated Bavachin-Induced Renal Fibrosis via Suppressing Bip/eIF2α/CHOP Signaling-Mediated EMT. Front Pharmacol 13:872474\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNguyen K, Tang J, Cho S, Ying F, Sung HK, Jahng JW et al (2024) Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance. Molr Metab 83:101921\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai C, Stolz DB, Bastacky SI, St.-Arnaud R, Wu C, Dedhar S et al (2006) Essential Role of Integrin-Linked Kinase in Podocyte Biology: Bridging the Integrin and Slit Diaphragm Signaling. J Am Soc Nephrol 17(8):2164\u0026ndash;2175\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHadpech S, Thongboonkerd V (2024) Epithelial\u0026ndash;mesenchymal plasticity in kidney fibrosis. Genesis 62(1):e23529\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang P, Chen W, li B, Yang S, Li W, Zhao S et al (2024) Exosomes on the development and progression of renal fibrosis. Cell Prolif 57(11):e13677\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang YP, Sun B, Han Z, Han F, Hu SL, Li XY et al (2017) Saxagliptin Attenuates Albuminuria by Inhibiting Podocyte Epithelial- to-Mesenchymal Transition via SDF-1α in Diabetic Nephropathy. Front Pharmacoly 8:780\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu S, Hang X, Wei Y, Wang H, Zhang L, Zhao L (2024) Crosstalk among podocytes, glomerular endothelial cells and mesangial cells in diabetic kidney disease: an updated review. Cell Commun Signal 22(1):136\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun Y, Lu X, Danser AHJ (2020) Megalin: a Novel Determinant of Renin-Angiotensin System Activity in the Kidney? Curr Hypertens Rep 22(4):30\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElsakka EGE, Mokhtar MM, Hegazy M, Ismail A, Doghish AS (2022) Megalin, a multi-ligand endocytic receptor, and its participation in renal function and diseases: A review. Life Sci 308:120923\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeyer K, Storm T, Shan J, Vainio S, Kozyraki R, Verroust PJ et al (2011) Mouse model of proximal tubule endocytic dysfunction. Nephrol Dial Transpl 26(11):3446\u0026ndash;3451\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang J, Wang K, Chen Y, Chen H, Nice EC, Huang C (2017) Redox regulation in tumor cell epithelial-mesenchymal transition: molecular basis and therapeutic strategy. Signal Transduct Target Ther 2:17036\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu C, Liu Q, Mou Z (2024) Redox signaling and oxidative stress in systemic acquired resistance. J Exp Bot 75(15):4535\u0026ndash;4548\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNazıroğlu M, \u0026Ccedil;iğ B, Yazğan Y, Schwaerzer GK, Theilig F, Pecze L (2019) Albumin evokes Ca(2+)-induced cell oxidative stress and apoptosis through TRPM2 channel in renal collecting duct cells reduced by curcumin. Sci Rep 9(1):12403\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEleftheriadis T, Pissas G, Golfinopoulos S, Efthymiadi M, Poulianiti C, Polyzou Konsta MA et al (2023) Routes of Albumin Overload Toxicity in Renal Tubular Epithelial Cells. Inte J Mol Sci 24(11):9640\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eImai E, Nakajima H, Kaimori J-Y (2004) Albumin turns on a vicious spiral of oxidative stress in renal proximal tubules. Kidney Int 66(5):2085\u0026ndash;2087\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKinugasa S, Tojo A, Sakai T, Tsumura H, Takahashi M, Hirata Y et al (2011) Selective albuminuria via podocyte albumin transport in puromycin nephrotic rats is attenuated by an inhibitor of NADPH oxidase. Kidney Int 80(12):1328\u0026ndash;1338\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaito A, Imaizumi K (2018) Unfolded Protein Response-Dependent Communication and Contact among Endoplasmic Reticulum, Mitochondria, and Plasma Membrane. Int J Mol Sci. ;19(10)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee JY, Chang JW, Yang WS, Kim SB, Park SK, Park JS et al (2011) Albumin-induced epithelial-mesenchymal transition and ER stress are regulated through a common ROS-c-Src kinase-mTOR pathway: effect of imatinib mesylate. Am J Physiol Ren Physiol 300(5):F1214\u0026ndash;F1222\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu D, Huang L-F, Chen X-C, Huang X-R, Li H-Y, An N et al (2023) Research progress on endoplasmic reticulum homeostasis in kidney diseases. Cell Death Dis 14(7):473\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang J, Zhao Y, Gong N (2023) XBP1 Modulates the Aging Cardiorenal System by Regulating Oxidative Stress. Antioxidants 12(11):1933\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManfioletti G, Fedele M (2023) Epithelial\u0026ndash;Mesenchymal Transition (EMT). Int J Mol Sci 24(14):11386\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYing Q, Wu G (2017) Molecular mechanisms involved in podocyte EMT and concomitant diabetic kidney diseases: an update. Ren Fail 39(1):474\u0026ndash;483\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou L, Chen X, Lu M, Wu Q, Yuan Q, Hu C et al (2019) Wnt/β-catenin links oxidative stress to podocyte injury and proteinuria. Kidney Int 95(4):830\u0026ndash;845\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKang M-K, Kim S-I, Oh SY, Na W, Kang Y-H (2020) Tangeretin Ameliorates Glucose-Induced Podocyte Injury through Blocking Epithelial to Mesenchymal Transition Caused by Oxidative Stress and Hypoxia. Int J Mol Sci 21(22):8577\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJandeleit-Dahm KA, Kankanamalage HR, Dai A, Meister J, Lopez-Trevino S, Cooper ME et al (2024) Endothelial NOX5 obliterates the reno-protective effect of Nox4 deletion by promoting renal fibrosis via activation of EMT and ROS-sensitive pathways in diabetes. Antioxidants 13(4):396\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRicciardi CA, Gnudi L (2020) The endoplasmic reticulum stress and the unfolded protein response in kidney disease: Implications for vascular growth factors. J Cell Mol Med 24(22):12910\u0026ndash;12919\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu M, Chen F, Wang H, Fu Q, Yan L, Chen Z et al (2023) Endoplasmic reticulum stress mediates nickel chloride-induced epithelial\u0026ndash;mesenchymal transition and migration of human lung cancer A549 cells through Smad2/3 and p38 MAPK activation. Ecotoxicol Environ Saf 249:114398\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMu Z, Li B, Chen M, Liang C, Gu W, Su J (2024) Endoplasmic reticulum stress induces renal fibrosis in high\u0026ndash;fat diet mice via the TGF\u0026ndash;β/SMAD pathway. Mol Med Reps 30(6):235\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"albuminuria, epithelial-mesenchymal transition, ROS, ER stress","lastPublishedDoi":"10.21203/rs.3.rs-8002591/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8002591/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eEpithelial-mesenchymal transition (EMT) plays a significant role in cell migration and tissue fibrosis. Podocytes may undergo EMT after injury, leading to podocyte migration and detachment, which results ultimately in albuminuaria and renal fibrosis. The molecular mechanisms linking albuminuria to the progression of renal fibrosis is complex and not yet fully understood. Reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress play significant roles in renal fibrosis. In this study, we evaluate whether albumin load induces podocyte EMT through ROS/ER stress pathways.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePodocytes were exposed to medium alone or to high concentrations of delipidated, endotoxin-free human serum albumin (HSA, 10 mg/ml) (albumin load) with or without antioxidant (N-acetylcysteine, NAC) and ER stress inhibitors (4-phenylbutyrate (4-PBA) and salubrinal (Sal)). Intracellular ROS generation was measured using the fluorescent indicator 20, 70-dichlorofluorescin diacetate (DCF-DA). Both mRNA and protein levels of the EMT biomarker (α-smooth muscle actin, α-SMA) and the protein levels of ER stress biomarkers (GRP78 and CHOP) were assessed by real-time PCR and Western blotting.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAfter albumin load, intracellular albumin was increased in podocytes. In addition, the elevated ROS generation and protein levels of both GRP78 and CHOP were caused under albumin load, both of which could be reversed by NAC. Also, 4-PBA attenuated ROS generation induced by albumin load. Both mRNA and protein levels of α-SMA were up-regulated under albumin load. NAC alleviated albumin load-induced alterations in both mRNA and protein levels of α-SMA. Also, both 4-PBA and Sal ameliorated albumin load \u0026ndash;triggered both mRNA and protein levels of α-SMA.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings suggested that albumin load promoted podocyte EMT mainly via ROS-mediated ER stress. The ROS-ER stress related pathways might be a potential intervention target for albuminuria-caused renal fibrosis.\u003c/p\u003e","manuscriptTitle":"Reactive oxygen species-evoked endoplasmic reticulum stress mediates albumin load-induced epithelial-mesenchymal transition in podocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 09:40:09","doi":"10.21203/rs.3.rs-8002591/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T15:09:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T04:15:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276241311991976432549877083153673626813","date":"2025-11-04T00:44:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-03T12:54:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T04:13:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T04:12:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-11-01T03:31:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"76fcb4be-3621-429a-99b2-04d9bc351b79","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:05:06+00:00","versionOfRecord":{"articleIdentity":"rs-8002591","link":"https://doi.org/10.1007/s11033-026-11582-8","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2026-02-20 15:59:18","publishedOnDateReadable":"February 20th, 2026"},"versionCreatedAt":"2025-11-13 09:40:09","video":"","vorDoi":"10.1007/s11033-026-11582-8","vorDoiUrl":"https://doi.org/10.1007/s11033-026-11582-8","workflowStages":[]},"version":"v1","identity":"rs-8002591","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8002591","identity":"rs-8002591","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}