β-Arrestin-2 enhances ER stress-induced glomerular endothelial cells injury through ATF6 in diabetic nephropathy

β-Arrestin-2 enhances ER stress-induced glomerular endothelial cells injury through ATF6 in diabetic nephropathy | 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 Short Report β-Arrestin-2 enhances ER stress-induced glomerular endothelial cells injury through ATF6 in diabetic nephropathy Jiang Liu, Xiaoyun Song, Xiuting Li, Mu Yang, Fang Wang, Ying Han, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3939362/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Glomerular endothelial cell (GENC) injury would be a characteristic of early stage diabetic nephropathy (DN) and the investigation of potential therapeutic targets for preventing GENC injury has clinical importance. Methods DN was induced in C57BL/6J mice by intraperitoneal injection of streptozotocin. GENC was transfected with plasmid containing siRNA-β-arrestin-2, shRNA-ATF6, pCDNA-β-arrestin-2 or pCDNA-ATF6. Additionally, we administrated adeno-associated virus (AAV) containing shRNA-β-arrestin-2 via tail vein injection in DN mice. Results The upregulation of β-arrestin-2 was observed in DN patients as well as in GENC from DN mice. Knockdown of β-arrestin-2 reduced endoplasmic reticulum stress (ER stress) and apoptosis in high glucose treated GENC which were reversed by overexpression of activating transcription factor 6 (ATF6). Moreover, overexpression of β-arrestin-2 led to the activation of ER stress and the apoptosis of GENC which could be mitigated by silencing of ATF6. Furthermore, knockdown of β-arrestin-2 by the administration of AAV-shRNA-β-arrestin-2 had alleviated renal injury in DN mice. Conclusions This study offer novel perspectives on the crucial involvement of β-arrestin-2 in GENC injury. Knockdown of β-arrestin-2 prevents GENC apoptosis by inhibiting ATF6-mediated ER stress in vivo and vitro. Consequently, β-arrestin-2 may represent a promising therapeutic target for the clinical management of patients with DN. Diabetic nephropathy Glomerular endothelial cell β-Arrestin-2 ATF6 ER stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Diabetic nephropathy (DN) is one of the microvascular complications of diabetes and about 30% diabetic patients will develop into DN, which is also the leading cause of end stage of renal failure in the whole world 1 . Lowering blood pressure and tight glycemic control are the common clinical treatments to the DN patients, but these strategies do not prevent the progression of DN 2 . As an early manifestation of DN, microalbuminuria is one of the main courses for the deterioration of DN, which also indicating the damage of glomerular filtration barrier (GFB). Podocyte is a member of the GFB, and previous studies have demonstrated that inhibition of podocyte damage can protect against DN. However, glomerular endothelial cell (GENC) is another member of the GFB, the study on the role of GENC in DN is sparse. Recently, more and more evidence explain that the damage of GENC is already present before podocyte injury 3 – 5 . In mice model, targeting the specific genes which induce endothelial damage enhance the development of DN 6 – 8 . GENC injury and apoptosis would be a characteristic of early stage DN but the mechanisms of GENC injury and apoptosis in diabetic nephropathy are still not very clear. Therefore, identifying the key molecules and mechanisms involve in GENC injury may provide clues to develop new therapeutic strategies for DN patients in the clinical practices. Arrestins are small molecular proteins with multiple functions, β-arrestin-1 and β-arrestin-2 are two subtypes widely expressed in various tissues in mammalian and they not only acted as negative regulators of G protein-coupled receptors (GPCRs) but also functioned as scaffold proteins to interact with different signaling molecules. β-Arrestins are closely related to the development of many diseases. In adriamycin induced nephropathy, β-arrestin-1 activated endothelin-A receptor signaling pathway to promote podocyte injury and apoptosis 9 . Our previous study discovered that high glucose induced upregulating of β-arrestin-1 and β-arrestin-2 which negatively regulated the conjugation of ATG5-ATG12 to suppress the autophagy in podocyte and then induced podocyte apoptosis in diabetic nephropathy 10 . However, the role of β-arrestin-1/2 in GENC in DN keeps unclear. In this study, we found that the level of β-arrestin-2 but not β-arrestin-1 was increased in GENC from DN mice. Silencing of β-arrestin-2 alleviated GENC injury and apoptosis under HG condition in vitro.In vivo, adeno-associated virus (AAV) contained shRNA-β-arrestin-2 was injected into the DN mice by tail vein and we detected the damage of kidney was improved significantly. Mechanistically, β-arrestin-2 activated ER stress through ATF6 signal pathway by increasing the expression of ATF6 and promoting ATF6 into nuclear. Therefore, our findings suggest β-arrestin-2 as a potential therapeutic target for the treatment of diabetic nephropathy in clinical. Materials and Methods Human renal biopsies. All the renal biopsy tissues were obtained from the department of pathology in The Second Hospital of Shandong University and the collection of clinical samples was described previously 11 (Supplementary Table 2). All patients had signed the informed consent and the investigations were approved by the ethics committee of the Second Hospital of Shandong University. Animal studies. Selected 8-week-old wide-type C57BL/6 male mice, constructed a diabetic nephropathy model by intraperitoneal injection of streptozotocin as described before 12 , 13 (The details of the methods were in the supplementary). The mice were sacrificed and blood, urine and kidney samples were collected for subsequent research. Ethics. The human renal biopsy collection was performed in accordance with the principles of the Declaration of Helsinki. All the experimental protocols for animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The investigations were approved by the ethics committee of the Second Hospital of Shandong University (Document No. KYLL-2020(LW)-072). Cell culture and treatments. Rat glomerular endothelial cells (GENCs) were kindly gifted from Professor Yifan in Shandong University and were cultured in RPMI 1640 medium containing 5.5mM of glucose and 10% fetal bovine serum (Gibco, USA). Chronic low-grade inflammation is key factor in the pathogenesis of DN. Diverse components of the immune system participate in the initiation and progression of DN including adhesion molecules, chemokines, and proinflammatory cytokines 14 – 16 . We then treated GENC under different stimuli. RNA interference and overexpression of β-arrestin-2 and ATF6. Small interference RNA (siRNA) to β-arrestin-2 (5′-CCUACAGGGUCAAGGUGAATT-3′) and negative control (5′-UUCUCCGCGUGUCACGUTT-3′) were synthesized by BioSune (Jinan, China). Small interference RNA to ATF6 was synthesized and constructed into pGPU6/GFP/Neo to get shRNA-ATF6 (5’-GAGTGAGCTGCAGGTGTATTA-3’) by Biomics Biotechnologies Co., Ltd. (Nantong, Jiangsu, China). In the experiments, all siRNA and shRNA were transfected by lipo 3000 (Invitrogen, USA) and operations followed the instruction. The overexpression plasmids contains β-arrestin-2 or ATF6 were purchased from BioSune (Jinan, China). GENC were also transfected with the pCDNA-β-arrestin-2 or pCDNA-ATF6 plasmids by lipo 3000 followed the instruction. Real-time RT-PCR. We extracted the total RNA from the cortex of the kidney and GENC by trizol reagent (Invitrogen) following the instruction. Then we detected the levels of β-arrestin-1/2 by real-time RT-PCR and the primers in this study are listed in Supplementary Table 4. Western blot analyses. Proteins extracted from the cortex of kidney and GENC, western blot analyses were performed as described previously 17 – 19 and the detailed procedure were in the supplementary. We also used nuclear protein extraction kit (Beyotime P0028) for protein extracted from nucleus. Antibodies used in this study were summarized in Supplementary Table 5. Morphologic, Immunohistochemical and Immunofluorescence staining. The fixed renal tissues were embedded in paraffin and slice to 4-µm, the kidney sections were stained with periodic acid-Schiff (PAS), immuno-histochemical (IHC) staining and terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) kits according to the manufacturer’s protocol 20 . Gene delivery by AAV into kidney. Recombinant adeno-associated virus (AAV) contained shRNA-β-arrestin-2 (5’-GGAACUCUGUGCGGCUUAUTT-3’) and negative control (AAV-Null: 5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from BioSune (Jinan, China). The details of intrarenal AAV delivery were in the supplementary 21 – 23 . Statistics. All data are showed as means ± SD. The significance of differences between multiple groups was detected by one-way ANOVA and P < 0.05 was considered statistically significant. Results Upregulation of β-arrestin-2 in DN patients and GENC from DN mice. We first assessed the expression pattern of β-arrestin-2 in the DN patients through renal biopsies. We found the upregulation of β-arrestin-2 in renal biopsies from DN patients compared with normal subjects by IHC staining (Fig. 1 a). We then constructed DN model by injection of STZ in mice, as shown in Supplementary Table 1, compared to the control mice, we found hyperglycemia and lower body weight in STZ-induced DN mice, the UACR and relative kidney weight in DN mice were higher than the control mice significantly, but the two groups had no difference in heart rate. Real-time RT-PCR and western blotting had shown that the expression of β-arrestin-2 was increased in renal cortex from the DN mice (Figs. 1 b, 1 c). We also demonstrated the same results in the paraffin sections of renal tissue by IHC (Fig. 1 d). Immunofluorescence double staining further confirmed that the expression of β-arrestin-2 (green) was significantly increased in GENC (red) from DN mice (Fig. 1 e). These results indicated that the upregulation of β-arrestin-2 in GENC from DN mice. We also detected the upregulation of β-arrestin-1 in renal biopsies from DN subjects (Supplementary Fig. 1a) and DN model mice (Supplementary Fig. 1b, 1c, 1d), but we did not find the expression of β-arrestin-1 increasing in GENC (Supplementary Fig. 1e). Expression of β-arrestin-2 was increased in GENC under various stimuli. Renin-angiotensin-aldosterone system (RAAS, Ang II), high glucose (HG) and advanced glycation end product (AGE) formation are important pathways to the development and progression of DN. Each pathway causes damage via multiple mediators or interacts with other pathways 24 . Though traditionally, DN has not been considered as an inflammatory disease, immune and inflammatory responses play an important role in the pathogenesis of DN. Inflammatory factors, such as IL-6, tumor necrosis factor (TNF-α), TGF-β1, and IL-18 are elevated in blood and have been shown to be involved in the development and progression of DN 25 . The expression of β-arrestin-2 was increased under 20mmol/L and 40mmol/L glucose concentration, but the expression of β-arrestin-2 was not changed under mannitol 40mmol/L treatment (Fig. 2 a). We also found that the expression of β-arrestin-2 was increased in GENC under common detrimental factors in DN such as AGE (Fig. 2 b), TNF-α (Fig. 2 c), AngII (Fig. 2 d) and TGF-β1 (Fig. 2 e) and the increased β-arrestin-2 expression in GENC in a concentration-dependent manner. Next experiments we used the concentration of glucose 40mmol/L as the HG stimuli to study the mechanism of GENC injury in vitro under HG treatment. Silencing of β-Arrestin-2 ameliorated HG induced GENC injury. To investigate the effect of β-arrestin-2 in GENC under HG stimulation, we knockdown the expression of β-arrestin-2 by siRNA in this study (Fig. 3 a). The membrane proteins zonula occludens-1 (ZO-1) and occludin are related to the permeability of endothelial tight junction, decreased ZO-1 and occludin levels are closely related to the progression of DN through disrupting the function of GENC 26 . Inhibited of eNOS reduced the production of NO and then led to endothelial injury, eNOS knockout mice developed to nodular diabetic glomerulosclerosis 27 , 28 .Western blotting (WB) showed that the expression of proteins ZO-1, occludin and eNOS were decreased in GENC under HG stimulation. Silencing of β-arrestin-2 recovered occludin, ZO-1 and eNOS in GENC under HG treatment (Fig. 3 b). We then detected the changes of apoptosis related proteins,such as bcl-2, bax and cleaved caspase 3. We found that HG induced upregulation of apoptosis related proteins was decreased by knockdown of β-arrestin-2 (Figs. 3 c and 3 d ), and flow cytometry further confirmed the results (Fig. 3 e and 3 f). Knockdown of β-arrestin-2 suppressed ER stress induced apoptosis in GENC through ATF6. ER stress plays vital role in the progression of diabetic nephropathy, bip and chop are marker proteins associated with ER stress. Our results showed that HG induced upregulation of bip and chop could be decreased by knockdown of β-arrestin-2 in GENC (Fig. 4 a). Indicating that silencing of β-arrestin-2 suppressed ER stress which was activated by HG in GENC. The role of ER stress in GENC was not clear, so we then detected the apoptosis of GENC by Annexin V/propidium iodide staining, the ER stress activators tunicamycin (TM), thapsigargin (TG) and ER stress inhibitor 4-phenyl butyric acid (4-PBA) were used in our study. Flow cytometry results confirmed that activated ER stress inducing GENC apoptosis which could be alleviated by ER stress inhibitor 4-PBA (Fig. 4 b). We also detected silencing of β-arrestin-2 could partially reduced apoptosis of GENC induced by ER stress activator TM (Fig. 4 c). Protein kinase R-like ER kinase (PERK), inositol requiring 1α (IRE1α) and activating transcription factor 6 (ATF6) are three signal pathways of ER stress. To determine the exact pathway by which β-arrestin-2 induced ER stress, we first detected the IRE1αand PERK signaling pathways. Immunoblot of the related proteins in the two pathway showed that HG dramatically enhanced expression of p-IRE1α, XBP1, p-eIF1α, p-PERK and ATF4, but knockdown of β-arrestin-2 did not change the upregulation of these proteins under HG treatment (Supplementary Figs. 2a, 2b). Then we detected the protein levels of ATF6 which is a transcription factor of ER stress. Western blotting results showed that HG increased the expression of ATF6 in GENC, knockdown of β-arrestin-2 significantly reduced the upregulation of ATF6 in GENC under HG treatment (Fig. 4 d). β-Arrestin-2 activated ER stress inducing GENC injury was blocked by knockdown of ATF6. We synthesized the overexpression plasmid of β-arrestin-2 (Supplementary Figure S3a) and shRNA of ATF6 (Supplementary Figure S3b). Western blotting results showed that overexpression of β-arrestin-2 would active ER stress in GENC through increasing expression of bip and CHOP which could be blocked by knockdown of ATF6 (Fig. 5 a). We also found that silencing of ATF6 could regain ZO-1 and occludin that were decreased by overexpression of β-arrestin-2 in GENC (Fig. 5 b). Western blotting and flow cytometry all confirmed that apoptosis of GENC induced by overexpression of β-arrestin-2 would be attenuated by knockdown of ATF6 (Fig. 5 c, 5 d). We also detected that overexpression of β-arrestin-2 could upregulate ATF6, but knockdown of ATF6 did not decrease the expression of β-arrestin-2 in GENC (Fig. 5 e). β-arrestin-2 increased expression of ATF6 and promoted ATF6 transport into nucleus. We then synthesized the overexpression plasmid of ATF6 (Supplementary Figure S3c). We found that overexpression of ATF6 increased protein level of bip and chop which were downregulated by knockdown of β-arrestin-2 in GENC with HG treatment (Fig. 6 a). Knockdown of β-arrestin-2 attenuated HG induced GENC injury was reversed by overexpression of ATF6 through the expression of protein ZO-1 and occludin (Fig. 6 b). Apoptosis of GENC was reduced by knockdown of β-arrestin-2 in GENC with HG treatment, but overexpression of ATF6 aggravated the apoptosis of GENC (Fig. 6 c, 6 d). We confirmed the results through the expression of cleaved caspase-3 (Supplementary Fig. 3d). Silencing of β-arrestin-2 reduced expression of ATF6 but overexpression of ATF6 did not change the expression of β-arrestin-2 (Fig. 6 d). In ATF6 signal pathway, ATF6 is cleaved and transferred to the nucleus to promote the transcription of downstream target genes. Immunoblots detected the expression of actived ATF6 in nucleus. The results showed that HG induced ATF6 transfferring into nucleus was blocked by silencing of β-arrestin-2 and overexpression of β-arrestin-2 promoted ATF6 transfferring into nucleaus which was inhibitted by knockdown of ATF6. We found the same tendency from the detection of mRNA levels of ATF6 target genes GRP78 and GRP94 (Fig. 6 e, Supplementary Fig. 3e). Silencing of β-arrestin-2 ameliorates kidney injury in vivo. To examine genetic therapeutic efficiency targeting to β-arrestin-2 in mice with diabetic nephropathy, we delivered adeno-associated virus (AAV) contained shRNA-β-arrestin-2 into the experimental mice by tail vein injection. Our results confirmed that mice received AAV-shRNA-β-arrestin-2 significantly decreased the mRNA and protein levels of β-arrestin-2 in the renal cortex (Fig. 7 a, b). Silencing of β-arrestin-2 reduced urinary albumin-to-creatinine ratio (Urinary-ACR, Fig. 7 c). PAS staining was used to evaluate renal histopathological changes. A widened mesangial area and an increased mesangial matrix were observed in DN mice, but in AAV-shRNA-β-arrestin-2 DN mice, mesangial area diminished and mesangial matrix decreased (Fig. 7 d). TUNEL assay was performed to detect cell apoptosis. Figure 7 e showed less apoptosis cells in AAV-shRNA-β-arrestin-2 DN mice compared to DN mice. And then, we also detected the expression of proteins related to the function of GENC. Western blotting results indicated that in AAV-shRNA-β-arrestin-2 transfected DN mice, the level of ZO-1, eNOS and occludin were significantly increased (Fig. 7 f). The ER stress was inhibited as evidenced by decreased protein level of bip, CHOP and ATF6 in diabetic nephropathy mice transfected with AAV-shRNA-β-arrestin-2 (Fig. 7 g). Discussion As a key component of the glomerular filtration barrier, GENC have been studied intensively in recent decades 29 . GENC is the first layer of glomerular filtration barrier,so GENC dysfunction arises early and plays a key role in the initiation and development of DN. Increasing data suggest that GENC injury plays an important role in the pathogenesis of DN 30 – 32 . Our previous study demonstrated that the mRNA level of β-arrestin-1 and β-arrestin-2 were negatively correlated with estimated glomerular filtration rate (eGFR) in all available subjects individually, but the functional role of β-arrestins in GENC remains to be elucidated. In this study, we found that the expression of β-arrestin-2 but not β-arrestin-1 was increased significantly in GENC from DN mice and we also detected upregulation of β-arrestin-2 in detrimental factors (such as HG, AGE, etc.) treated GENC in vitro. We then investigated the role of β-arrestin-2 in GENC. We found that HG reduced the expression of eNOS, occludin and ZO-1 in vitro, knockdown of β-arrestin-2 could recover the expression of proteins. We also found that knockdown of β-arrestin-2 could decrease HG induced GENC apoptosis. These data showed that knockdown of β-arrestin-2 alleviated HG induced GENC injury and apoptosis. Endoplasmic reticulum (ER) stress is one of the major cellular mechanisms involved in kidney injury in DN 33 . Numerous studies have demonstrated that the dysfunction of ER stress is associated with onset and progression of DN, ER stress inhibitors decreased ER stress and halted the progression of DN 34 , 35 . However, some studies demonstrated that activated ER stress had a protective role on DN which reflected the bidirectional control of ER stress in DN 36 . In this study, we found that activated ER stress induced apoptosis of GENC and the ER stress in GENC with HG treatment was activated. Additionally, silencing of β-arrestin-2 not only decreased the upregulation of ER stress related proteins bip and chop but also reduced ER stress induced apoptosis in GENC with HG treatment. There are three ER stress sensor pathways, including IRE1/sXBP1, PERK/EIf2α and ATF6 37 . It has reported that β-arrestin-2 interacted with eIF2α in intestinal epithelial cells which contributed to promote ERS/PUMA, thereby inducing mucosal apoptosis in colitis through the mitochondrial apoptotic pathway 38 . However, our present study found different outcomes in GENC under HG condition. Although p-PERK, p-eIF2α and ATF4 were increased in HG-treated GENC, knockdown of β-arrestin-2 had no effect on the expression of these proteins. The mixed results showed different contribution of β-arrestin-2 to ER stress in different diseases or different cell types. So how β-arrestin-2 regulates ER stress in GENC under HG condition is a question of fundamental importance. In this study, we provide potential mechanisms to answer this question. As we did not find the interaction between β-arrestin-2 and IRE1α pathway in GENC either, we focused on ATF6 sensor pathway. As a type II transmenbrane protein, ATF6 is an important molecule of ER stress pathway which participates in regulating cell apoptosis. In this study, we found that upregulation of ATF6 could be reduced by knockdown of β-arrestin-2 in GENC with HG treatment. Overexpression of β-arrestin-2 increasing the expression of ATF6 directly in GENC, but overexpression or knockdown of ATF6 did not change the expression of β-arrestin-2. We investigated that injury and apoptosis of GENC induced by overexpression of β-arrestin-2 could be alleviated by knockdown of ATF6, and silencing of β-arrestin-2 reducing injury and apoptosis of GENC would be reversed by overexpression of ATF6. The above results confirmed that β-arrestin-2 regulated the expression of ATF6 in GENC. When unfolded proteins accumulate, ATF6 is transported to the Golgi complex where it is proteolytically cleaved by S1P and S2P to release the NH2 terminal-domain 39 . The cleaved section of ATF6 translocated to the nucleus, where it activates gene transcription of target genes such as GRP78, GRP94 40, 41 . We then detected the expression of ATF6 in the nucleus, the results showed that β-arrestin-2 promoted ATF6 transported to the nucleus in GENC with HG treatment. The mRNA level of ATF6 target genes GPR78 and GPR94 also demonstrated the same results. But how β-arrestin-2 promoted ATF6 expression and transported to the nucleus needed to be investigated in the future. Gene therapy was used to treated many diseases recently. Adeno-associated virus vector, with low toxicity and antigenicity, is a promising vehicle for gene therapy, and commercial AAV gene therapy products have been approved by regulatory agencies and used in the clinic to treat spinal muscular atrophy, Leber congenital amaurosis and hemophilia A 42 – 44 . In the present study, we delivered AAV contained shRNA-β-arrestin-2 into the mice by tail vein injection. Our study demonstrated that AAV could stably transduce renal cells for 3 months and gene silencing of β-arrestin-2 could attenuate kidney damage in diabetic nephropathy mice. In addition, the GENC-specific β-arrestin-2 knockout mice would be an ideal model for investigating the role of β-arrestin-2 in GENC in diabetic nephropathy. So our findings need to be confirmed on the ideal model in the future studies. In conclusion, our study investigated that β-arrestin-2 aggravated GENC injury in diabetic nephropathy through ATF6 mediated ER stress by increasing the expression of ATF6 and promoting ATF6 transport to nucleus. This discovery provided us a new perspective for understanding the critical role of GENC in diabetic nephropathy and suggesting that β-arrestin-2 could be a new therapy target in clinical treatment for diabetic nephropathy. Declarations Author Contributions: Jiang Liu, Xiaoyun Song and Xiuting Li conducted in vivo and in vitro experiments, performed data analysis. Jiang Liu, Wen Zhang and Dongqi Tang helped write the manuscript. Writing-original draft, Jiang Liu; methodogy, Xiaoyun Song; formalanalysis, Xiuting Li, Ying Jiang and Yuxin Lei; software, Mu Yang; validation, Fang Wang and Miao Jiang; investigation, Ying Han; writing-review and editing, Wen Zhang and Dongqi Tang; funding acquisition, Jiang Liu, Wen Zhang and Dongqi Tang. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the grants of the Key Research and Development Program of Shandong Province (No. 2021CXGC011101) , the Special Fund for Taishan Scholars Project (tsqn202211324), the National Natural Science Foundation of China [81900669]; the Natural Science Foundation of Shandong Province, China [ZR2018PH007] and Multidisciplinary Innovation Center for Nephrology of the Second Hospital of Shandong University. Conflict of interest: The authors declare no conflicts of interest. References Mohamed Q, Gillies MC and Wong TY. Management of diabetic retinopathy: a systematic review. Jama. 2007; 298: 902–16. Al-Waili N, Al-Waili H, Al-Waili T and Salom K. Natural antioxidants in the treatment and prevention of diabetic nephropathy; a potential approach that warrants clinical trials. Redox report: communications in free radical research. 2017; 22: 99–118. Lassen E and Daehn IS. Molecular Mechanisms in Early Diabetic Kidney Disease: Glomerular Endothelial Cell Dysfunction. International journal of molecular sciences. 2020; 21. Li M, Deng L and Xu G. METTL14 promotes glomerular endothelial cell injury and diabetic nephropathy via m6A modification of alpha-klotho. Molecular medicine. 2021; 27: 106. Zhang Y, Ma KL, Gong YX, et al. Platelet Microparticles Mediate Glomerular Endothelial Injury in Early Diabetic Nephropathy. Journal of the American Society of Nephrology: JASN. 2018; 29: 2671–95. Maezawa Y, Takemoto M and Yokote K. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes. Journal of diabetes investigation. 2015; 6: 3–15. Natarajan M, Habib SL, Reddick RL, et al. Endothelial cell-specific overexpression of endothelial nitric oxide synthase in Ins2Akita mice exacerbates diabetic nephropathy. Journal of diabetes and its complications. 2019; 33: 23–32. Zheng X, Soroush F, Long J, et al. Murine glomerular transcriptome links endothelial cell-specific molecule-1 deficiency with susceptibility to diabetic nephropathy. PloS one. 2017; 12: e0185250. Buelli S, Rosano L, Gagliardini E, et al. beta-arrestin-1 drives endothelin-1-mediated podocyte activation and sustains renal injury. Journal of the American Society of Nephrology: JASN. 2014; 25: 523–33. Liu J, Li QX, Wang XJ, et al. beta-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy. Cell death & disease. 2016; 7: e2183. Liu M, Liang K, Zhen J, et al. Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling. Nature communications. 2017; 8: 413. Soler MJ, Riera M and Batlle D. New experimental models of diabetic nephropathy in mice models of type 2 diabetes: efforts to replicate human nephropathy. Experimental diabetes research . 2012; 2012: 616313. Liu J, Li QX, Wang XJ, et al. beta-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy. Cell death & disease. 2016; 7: e2183. Bruno G, Merletti F, Biggeri A, et al. Progression to overt nephropathy in type 2 diabetes: the Casale Monferrato Study. Diabetes Care. 2003; 26: 2150–5. Lim AK, Ma FY, Nikolic-Paterson DJ, Kitching AR, Thomas MC and Tesch GH. Lymphocytes promote albuminuria, but not renal dysfunction or histological damage in a mouse model of diabetic renal injury. Diabetologia. 2010; 53: 1772–82. Wada J and Makino H. Inflammation and the pathogenesis of diabetic nephropathy. Clinical science. 2013; 124: 139–52. Nanes BA. Slide Set: Reproducible image analysis and batch processing with ImageJ. BioTechniques. 2015; 59: 269–78. Sahar N, Bibi S, Masood N and Faryal R. Status of serine tyrosine kinase at germline and expressional levels in asthma patients. Molecular biology research communications. 2019; 8: 69–77. Gallo-Oller G, Ordonez R and Dotor J. A new background subtraction method for Western blot densitometry band quantification through image analysis software. Journal of immunological methods. 2018; 457: 1–5. Wang X, Liu J, Zhen J, et al. Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy. Kidney international. 2014; 86: 712–25. Rubin JD, Nguyen TV, Allen KL, Ayasoufi K and Barry MA. Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections. Human gene therapy. 2019; 30: 1559–71. Zincarelli C, Soltys S, Rengo G and Rabinowitz JE. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Molecular therapy: the journal of the American Society of Gene Therapy. 2008; 16: 1073–80. Ikeda Y, Sun Z, Ru X, Vandenberghe LH and Humphreys BD. Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector. Journal of the American Society of Nephrology: JASN. 2018; 29: 2287–97. Kopel J, Pena-Hernandez C and Nugent K. Evolving spectrum of diabetic nephropathy. World journal of diabetes. 2019; 10: 269–79. Shang J, Wang L, Zhang Y, et al. Chemerin/ChemR23 axis promotes inflammation of glomerular endothelial cells in diabetic nephropathy. Journal of cellular and molecular medicine. 2019; 23: 3417–28. Rincon-Choles H, Vasylyeva TL, Pergola PE, et al. ZO-1 expression and phosphorylation in diabetic nephropathy. Diabetes. 2006; 55: 894–900. Nakagawa T, Sato W, Glushakova O, et al. Diabetic endothelial nitric oxide synthase knockout mice develop advanced diabetic nephropathy. Journal of the American Society of Nephrology: JASN. 2007; 18: 539–50. Zhao HJ, Wang S, Cheng H, et al. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice. Journal of the American Society of Nephrology: JASN. 2006; 17: 2664–9. Song C, Wang S, Fu Z, et al. IGFBP5 promotes diabetic kidney disease progression by enhancing PFKFB3-mediated endothelial glycolysis. Cell death & disease. 2022; 13: 340. Zheng F, Ma L, Li X, et al. Neutrophil Extracellular Traps Induce Glomerular Endothelial Cell Dysfunction and Pyroptosis in Diabetic Kidney Disease. Diabetes. 2022; 71: 2739–50. Chen Z, Wang Z, Hu Y, et al. ELABELA/APJ Axis Prevents Diabetic Glomerular Endothelial Injury by Regulating AMPK/NLRP3 Pathway. Inflammation. 2023; 46: 2343–58. Shi Y and Vanhoutte PM. Macro- and microvascular endothelial dysfunction in diabetes. Journal of diabetes. 2017; 9: 434–49. Xie Y, E J, Cai H, et al. Reticulon-1A mediates diabetic kidney disease progression through endoplasmic reticulum-mitochondrial contacts in tubular epithelial cells. Kidney international. 2022; 102: 293–306. Su J, Peng J, Wang L, et al. Identification of endoplasmic reticulum stress-related biomarkers of diabetes nephropathy based on bioinformatics and machine learning. Frontiers in endocrinology. 2023; 14: 1206154. Sankrityayan H, Oza MJ, Kulkarni YA, Mulay SR and Gaikwad AB. ER stress response mediates diabetic microvascular complications. Drug discovery today. 2019; 24: 2247–57. Inoue T, Maekawa H and Inagi R. Organelle crosstalk in the kidney. Kidney international. 2019; 95: 1318–25. Di Conza G and Ho PC. ER Stress Responses: An Emerging Modulator for Innate Immunity. Cells. 2020; 9. Zeng LX, Tao J, Liu HL, et al. beta-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis. Mucosal immunology. 2015; 8: 683–95. Ye J, Rawson RB, Komuro R, et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Molecular cell. 2000; 6: 1355–64. Wang Y, Shen J, Arenzana N, Tirasophon W, Kaufman RJ and Prywes R. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J Biol Chem. 2000; 275: 27013–20. Read A and Schroder M. The Unfolded Protein Response: An Overview. Biology. 2021; 10. Li C and Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nature reviews Genetics. 2020; 21: 255–72. Leebeek FWG and Miesbach W. Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues. Blood. 2021; 138: 923–31. Wang D, Tai PWL and Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature reviews Drug discovery. 2019; 18: 358–78. Additional Declarations No competing interests reported. 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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-3939362","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":271745837,"identity":"da12a32f-04ca-4f85-aabd-31cdc6d3bf0e","order_by":0,"name":"Jiang Liu","email":"","orcid":"","institution":"Institute of Medical Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Liu","suffix":""},{"id":271745838,"identity":"9feb300a-5263-4997-8cfd-db07f2d572a6","order_by":1,"name":"Xiaoyun Song","email":"","orcid":"","institution":"Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyun","middleName":"","lastName":"Song","suffix":""},{"id":271745839,"identity":"0ac1801e-b677-44bd-8dcc-45e63f82d766","order_by":2,"name":"Xiuting Li","email":"","orcid":"","institution":"Shandong Institute of Medical Device and Pharmaceutical Packaging Inspection","correspondingAuthor":false,"prefix":"","firstName":"Xiuting","middleName":"","lastName":"Li","suffix":""},{"id":271745840,"identity":"b6f24546-8692-4d3a-84a0-68dcc9f28510","order_by":3,"name":"Mu Yang","email":"","orcid":"","institution":"Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Mu","middleName":"","lastName":"Yang","suffix":""},{"id":271745841,"identity":"8c3f674b-2666-435d-9146-1b176b346b8c","order_by":4,"name":"Fang Wang","email":"","orcid":"","institution":"Institute of Medical Sciences, The Second Hospital, 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University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Lei","suffix":""},{"id":271745845,"identity":"1b7a58c6-368e-462e-831c-bb394ec50a8f","order_by":8,"name":"Miao Jiang","email":"","orcid":"","institution":"Clinical Skill Training Centre, The Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Jiang","suffix":""},{"id":271745846,"identity":"707e9060-6c5e-4000-9efb-8c6411c693b0","order_by":9,"name":"Wen Zhang","email":"","orcid":"","institution":"Institute of Medical Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zhang","suffix":""},{"id":271745847,"identity":"39b7796a-8395-4061-9f2e-687d8c677618","order_by":10,"name":"Dongqi Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYLACHgYbBoYDYAZRqsFEGulaDpOgxZ797OEXb8rO2/PdSGB88LaNQd6coC08eWmWc87dTpx5I4HZcG4bg+HOBoIOyzEz5m27nWBwI4FNmreNIcHgACEt/G9AWs7ZA7Ww/yZOi0SO8WPetgOMG4C2MBOn5cYbM8Y555ITZ5552Cw555yE4QZCWtj7c4w/vCmzs+c7nnwQyLCRJ2gLELBJMLCBaMYGICFBWD0QMH+AaBkFo2AUjIJRgAMAAHUIP1HoFyZOAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Medical Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Dongqi","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2024-02-08 08:51:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3939362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3939362/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51018232,"identity":"957fc8db-ccb4-48c7-a1cb-dff5ebb426fb","added_by":"auto","created_at":"2024-02-12 19:21:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1025945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Arrestin-2 was increased significantly in renal biopsies from diabetic nephropathy patients and glomerular endothelial cell (GENC) from mice with diabetic nephropathy.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Representative images of IHC staining to detect the expression of β-arrestin-2 in paraffin sections of human renal from normal and diabetic nephropathy (DN) patients. (black bars=10μm, n=5) \u003cstrong\u003e(b)\u003c/strong\u003e Relative mRNA levels of β-arrestin-2 in the renal cortex from diabetic nephropathy mice (mean±SD, n=8). \u003cstrong\u003e(c) \u003c/strong\u003eThe expression of β-arrestin-2 in the renal cortex from diabetic nephropathy mice was analyzed by immunoblotting. (*P\u0026lt;0.05 vs. control, n=8) \u003cstrong\u003e(d)\u003c/strong\u003e Representative images of IHC staining to detect the expression of β-arrestin-2 in paraffin sections of kidney from control and DN mice. (red bars=20μm, n=8) \u003cstrong\u003e(e)\u003c/strong\u003e Detection of β-arrestin-2 expression in glomerular endothelial cell (GENC) in STZ-induced diabetic nephropathy mouse model by immunofluorescence double labeling: endothelin (red, mark protein in GENC), β-arrestin-2 (green). (white bars=20μm, n=8)\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/41751df14a10df08eae52315.png"},{"id":51018183,"identity":"86dce00b-7463-4bd2-8917-c73627158d5e","added_by":"auto","created_at":"2024-02-12 19:20:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":875505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Arrestin-2 was upregulated in GENC under HG and other stimuli in vitro.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Representative images and summarized data showing increased expression of β-arrestin-2 in GENC with high glucose (HG: with concentrations of 20 and 40 mmol/L, 40mmol/L mannitol was used as osmolarity control) stimulation for 24h by western blotting. (*P\u0026lt;0.05 vs. control, n=6) \u003cstrong\u003e(b)\u003c/strong\u003e Representative images and summarized data showing upregulation of β-arrestin-2 in GENC treated with advanced glycation end product (AGE:0, 50, 100, 200 μg/mL) for 24h by immunoblotting. (*P\u0026lt;0.05 vs. control, n=6) \u003cstrong\u003e(c)\u003c/strong\u003e Representative images and summarized data showing the increased expression of β-arrestin-2 in GENC treated with tumor necrosis factor α (TNF-α: 0, 20, 40, 80 ng/mL) for 24h by western blotting. (*P\u0026lt;0.05 vs. control, n=6) \u003cstrong\u003e(d)\u003c/strong\u003e Representative images and summarized data showing increased β-arrestin-2 expression in angiotensin II (Ang II: 0, 10\u003csup\u003e-7\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003e10\u003csup\u003e-6\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003e10\u003csup\u003e-5\u003c/sup\u003emol/L) treated GENC for 24h by immunoblotting. (*P\u0026lt;0.05 vs. control, n=6) \u003cstrong\u003e(e)\u003c/strong\u003e Images and summarized data reflecting upregulation of β-arrestin-2 in GENC treated with transforming growth factor-β1 (TGF-β1: 0, 2 , 4, 8 ng/mL) for 24h by western blotting. (*P\u0026lt;0.05 vs. control, n=6)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/f23e271eb85fcea08eac0f2d.png"},{"id":51018181,"identity":"7956cd2b-55b2-47be-aada-0a6240729356","added_by":"auto","created_at":"2024-02-12 19:20:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":490105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of β-arrestin-2 by siRNA ameliorated HG induced GENC injury and apoptosis.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003eImages showing the efficiency of silencing β-arrestin-2 with small interfering RNA (siRNA) by western blotting. (*P\u0026lt;0.05 vs. control, n=6) \u003cstrong\u003e(b)\u003c/strong\u003e Representative images showing the effect of β-arrestin-2 knockdown on the expression of occludin, ZO-1 and eNOS in HG-treated GENC by western blotting. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treatment, n=6) \u003cstrong\u003e(c)\u003c/strong\u003e Imunoblotting images showing the effect of β-arrestin-2 knockdown on the expression of apoptosis related proteins bcl-2 and bax in HG-treated GENC. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treatment, n=6) \u003cstrong\u003e(d)\u003c/strong\u003e Imunoblotting images showing the effect of β-arrestin-2 knockdown on the expression of cleave-caspase 3 and caspase 3 in GENC with HG treatment. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treatment, n=6) \u003cstrong\u003e(e)\u003c/strong\u003e Flow cytometric images showing decreased apoptosis of GENC with HG treatment by knockdown of β-arrestin-2. \u003cstrong\u003e(f)\u003c/strong\u003eSummarized flow cytometric data showing reduced apoptosis of GENC with HG treatment by silencing of β-arrestin-2. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treatment, n=6)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/f4f65fdb3d54300d6ee9a628.png"},{"id":51018187,"identity":"d0333500-945b-4000-ba9e-ca7eafb10157","added_by":"auto","created_at":"2024-02-12 19:21:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":945024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of β-arrestin-2 reduced ER stress and apoptosis of GENC with HG treatment by decreasing the expression of ATF6.\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images and summarize data showing knockdown of β-arrestin-2 inactivated ER stress by downregulating the expression of bip and chop in HG-treated GENC by western blotting. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treated group, n=6) \u003cstrong\u003e(b) \u003c/strong\u003eFlow cytometric analysis showing ER stress activators TM (tunicamycin) and TG (thapsigargin) induced GENC apoptosis and inhibitor 4-PBA (4-phenyl butyric acid) reduced GENC apoptosis.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;(*P\u0026lt;0.05 vs. control, \u0026amp;P\u0026lt;0.05 vs. TM treated group, n=6) \u003cstrong\u003e(c)\u003c/strong\u003e Summarized flow cytometric data showing the apoptosis of GENC under different stimuli. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treated group, \u0026amp;P\u0026lt;0.05 vs. scramble of TM treated group, n=6) \u003cstrong\u003e(d) \u003c/strong\u003eWestern blotting images showing the effects of β-arrestin-2 knockdown on the expression of ATF6 in GENC under HG treatment. (*P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. scramble of HG treated group, n=6).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/205ecf574a9c2119f9cda685.png"},{"id":51018184,"identity":"f32e2b39-2297-482f-aead-0f02fb0342d2","added_by":"auto","created_at":"2024-02-12 19:20:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":527379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of β-Arrestin-2 aggravated GENC injury and apoptosis by activating ER stress through the upregulation of ATF6. (a) \u003c/strong\u003eRepresentative images showing overexpression of β-arrestin-2 enhancing the expression of bip and chop which could be blocked by knockdown of ATF6 in GENC. (*P\u0026lt;0.05 vs. scramble control, $P\u0026lt;0.05 vs. pCDNA-β-arrestin-2 treated group, n=6) \u003cstrong\u003e(b) \u003c/strong\u003eRepresentative images showing overexpression of β-arrestin-2 reducing the expression of zo-1, occludin in GENC which would be recovered by knockdown of ATF6 in GENC. (*P\u0026lt;0.05 vs. scramble control, $P\u0026lt;0.05 vs. pCDNA-β-arrestin-2 treated group, n=6) \u003cstrong\u003e(c) \u003c/strong\u003eRepresentative images showing overexpression of β-arrestin-2 promoting apoptosis related proteins expression which were blocked by knockdown of ATF6 in GENC. (*P\u0026lt;0.05 vs. scramble control, $P\u0026lt;0.05 vs. pCDNA-β-arrestin-2 treated group, n=6) \u003cstrong\u003e(d) \u003c/strong\u003eSummarized flow cytometric data showing overexpression of β-arrestin-2 aggravating the apoptosis of GENC which was inhibited by knockdown of ATF6. (*P\u0026lt;0.05 vs. scramble control, $P\u0026lt;0.05 vs. pCDNA-β-arrestin-2 treated group, n=6) \u003cstrong\u003e(e)\u003c/strong\u003e Representative western blot images showing that overexpression of β-arrestin-2 upregulating the expression of ATF6, but knockdown of ATF6 did not affect the expression of β-arrestin-2.\u003cstrong\u003e \u003c/strong\u003e(*P\u0026lt;0.05 vs. scramble control, $P\u0026lt;0.05 vs. pCDNA-β-arrestin-2 treated group, n=6).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/f4b5864d23a078170e87bcb7.png"},{"id":51018186,"identity":"de6313ee-5296-4831-8489-c3b4181980b9","added_by":"auto","created_at":"2024-02-12 19:21:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":859400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Arrestin-2 enhanced the expression of ATF6 and promoted ATF6 transported into nucleus in GENC with HG treatment. (a) \u003c/strong\u003eRepresentative images and summarized data showing that knockdown of β-arrestin-2 reduced protein levels of bip and chop which would be reversed by overexpression of ATF6. (*P\u0026lt;0.05 vs. scramble control group, ΦP\u0026lt;0.05 vs. si-β-arrestin-2 and HG treated group) \u003cstrong\u003e(b) \u003c/strong\u003eRepresentative images and summarized data showing that knockdown of β-arrestin-2 recovered protein levels of zo-1 and occludin which would be reversed by overexpression of ATF6. (*P\u0026lt;0.05 vs. scramble control , ΦP\u0026lt;0.05 vs. si-β-arrestin-2 and HG treated group) \u003cstrong\u003e(c) \u003c/strong\u003eFlow cytometric analysis showing that knockdown of β-arrestin-2 reduced apoptosis of GENC with HG treatment was reversed by overexpression of ATF6. (*P\u0026lt;0.05 vs. scramble control , ΦP\u0026lt;0.05 vs. si-β-arrestin-2 and HG treated group) \u003cstrong\u003e(d) \u003c/strong\u003eRepresentative images and summarized data showing that knockdown of β-arrestin-2 reduced apoptosis related proteins expression which was reversed by overexpression of ATF6. And silencing of β-arrestin-2 decreased expression of ATF6, but overexpression of ATF6 did not change expression of β-arrestin-2. (*P\u0026lt;0.05 vs. scramble control group, ΦP\u0026lt;0.05 vs. si-β-arrestin-2 and HG treated group) \u003cstrong\u003e(e)\u003c/strong\u003e Representative western blot images and summarized data showing that the expresssion of cleaved ATF6 in nucleus under different stimuli.\u003cstrong\u003e \u003c/strong\u003e(*P\u0026lt;0.05 vs. scramble control, #P\u0026lt;0.05 vs. scramble of HG treated group, ΦP\u0026lt;0.05 vs. si-β-arrestin-2 and HG treated group) \u003cstrong\u003e(f)\u003c/strong\u003e Relative mRNA levels of GRP78 which is one of the target genes regulated by ATF6 in GENC with various stimuli (mean±SD). (*P\u0026lt;0.05 vs. scramble control, #P\u0026lt;0.05 vs. scramble of HG treated group, $P\u0026lt;0.05 vs. pCDNA-β-arrestin-2 treated group, ΦP\u0026lt;0.05 vs. si-β-arrestin-2 and HG treated group, \u0026amp;P\u0026lt;0.05 vs. TM treated group )\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/dfac58a646b38bd681026fa6.png"},{"id":51018182,"identity":"caf61bca-541f-44b1-a4fb-232659928176","added_by":"auto","created_at":"2024-02-12 19:20:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2064894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdeno-associated virus injection induced gene silencing of β-arrestin-2 ameliorated renal injury in streptozotocin (STZ)-induced diabetic nephropathy mice.\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eRelative mRNA levels of β-arrestin-2 in the renal cortex from the mice after AAV-Null (Scramble) and AAV-shRNA-β-arrestin-2 (shRNA-β-arrestin-2) tail injection. (mean±SD, *P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. STZ-induced diabetic mice, n=8). \u003cstrong\u003e(b) \u003c/strong\u003eWestern blotting images showing the protein level of β-arrestin-2 in the renal cortex from the mice after AAV-Null (Scramble) and AAV-shRNA-β-arrestin-2 (shRNA-β-arrestin-2) tail injection. (mean±SD, n=8). \u003cstrong\u003e(c)\u003c/strong\u003e Urinary ACR in different groups of mice. \u003cstrong\u003e(d) \u003c/strong\u003ePAS showing changes of glomerular structural in different groups of mice.(black bars=20μm, n=8) \u003cstrong\u003e(e)\u003c/strong\u003e TUNEL stainings showing the apoptosis of kidney in different groups of mice.(white bars=20μm, n=8) \u003cstrong\u003e(f) \u003c/strong\u003eRepresentative images showing the protein level of eNOS, ZO-1 and occludin in renal cortex from different groups of mice\u003cstrong\u003e \u003c/strong\u003e(CON means Control, *P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. STZ-induced diabetic mice, n=8)\u003cstrong\u003e. (g) \u003c/strong\u003eWestern blotting showing the expression of ER stress related proteins bip, chop and ATF6 in renal cortex from different groups of mice (CON means Control, *P\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. STZ-induced diabetic mice, n=8).\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/1e1a0b2ca4eec37c624d5683.png"},{"id":51027330,"identity":"ff319018-2edf-4df0-bd01-0ac5bc1b2c3a","added_by":"auto","created_at":"2024-02-12 23:13:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4168759,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/8aa41b8a-22bd-4d70-97ac-dc3ba82c3460.pdf"},{"id":51018185,"identity":"07bcc4c0-f381-420b-8f79-3e2befc080fe","added_by":"auto","created_at":"2024-02-12 19:21:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1769628,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-3939362/v1/5557000779e0ce3317740c57.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"β-Arrestin-2 enhances ER stress-induced glomerular endothelial cells injury through ATF6 in diabetic nephropathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic nephropathy (DN) is one of the microvascular complications of diabetes and about 30% diabetic patients will develop into DN, which is also the leading cause of end stage of renal failure in the whole world\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Lowering blood pressure and tight glycemic control are the common clinical treatments to the DN patients, but these strategies do not prevent the progression of DN\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As an early manifestation of DN, microalbuminuria is one of the main courses for the deterioration of DN, which also indicating the damage of glomerular filtration barrier (GFB). Podocyte is a member of the GFB, and previous studies have demonstrated that inhibition of podocyte damage can protect against DN. However, glomerular endothelial cell (GENC) is another member of the GFB, the study on the role of GENC in DN is sparse. Recently, more and more evidence explain that the damage of GENC is already present before podocyte injury\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In mice model, targeting the specific genes which induce endothelial damage enhance the development of DN\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. GENC injury and apoptosis would be a characteristic of early stage DN but the mechanisms of GENC injury and apoptosis in diabetic nephropathy are still not very clear. Therefore, identifying the key molecules and mechanisms involve in GENC injury may provide clues to develop new therapeutic strategies for DN patients in the clinical practices.\u003c/p\u003e \u003cp\u003eArrestins are small molecular proteins with multiple functions, β-arrestin-1 and β-arrestin-2 are two subtypes widely expressed in various tissues in mammalian and they not only acted as negative regulators of G protein-coupled receptors (GPCRs) but also functioned as scaffold proteins to interact with different signaling molecules. β-Arrestins are closely related to the development of many diseases. In adriamycin induced nephropathy, β-arrestin-1 activated endothelin-A receptor signaling pathway to promote podocyte injury and apoptosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Our previous study discovered that high glucose induced upregulating of β-arrestin-1 and β-arrestin-2 which negatively regulated the conjugation of ATG5-ATG12 to suppress the autophagy in podocyte and then induced podocyte apoptosis in diabetic nephropathy\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the role of β-arrestin-1/2 in GENC in DN keeps unclear. In this study, we found that the level of β-arrestin-2 but not β-arrestin-1 was increased in GENC from DN mice. Silencing of β-arrestin-2 alleviated GENC injury and apoptosis under HG condition in vitro.In vivo, adeno-associated virus (AAV) contained shRNA-β-arrestin-2 was injected into the DN mice by tail vein and we detected the damage of kidney was improved significantly. Mechanistically, β-arrestin-2 activated ER stress through ATF6 signal pathway by increasing the expression of ATF6 and promoting ATF6 into nuclear. Therefore, our findings suggest β-arrestin-2 as a potential therapeutic target for the treatment of diabetic nephropathy in clinical.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eHuman renal biopsies.\u003c/b\u003e All the renal biopsy tissues were obtained from the department of pathology in The Second Hospital of Shandong University and the collection of clinical samples was described previously\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (Supplementary Table\u0026nbsp;2). All patients had signed the informed consent and the investigations were approved by the ethics committee of the Second Hospital of Shandong University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimal studies.\u003c/b\u003e Selected 8-week-old wide-type C57BL/6 male mice, constructed a diabetic nephropathy model by intraperitoneal injection of streptozotocin as described before \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e(The details of the methods were in the supplementary). The mice were sacrificed and blood, urine and kidney samples were collected for subsequent research.\u003c/p\u003e \u003cp\u003e\u003cb\u003eEthics.\u003c/b\u003e The human renal biopsy collection was performed in accordance with the principles of the Declaration of Helsinki. All the experimental protocols for animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The investigations were approved by the ethics committee of the Second Hospital of Shandong University (Document No. KYLL-2020(LW)-072).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and treatments.\u003c/b\u003e Rat glomerular endothelial cells (GENCs) were kindly gifted from Professor Yifan in Shandong University and were cultured in RPMI 1640 medium containing 5.5mM of glucose and 10% fetal bovine serum (Gibco, USA). Chronic low-grade inflammation is key factor in the pathogenesis of DN. Diverse components of the immune system participate in the initiation and progression of DN including adhesion molecules, chemokines, and proinflammatory cytokines\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We then treated GENC under different stimuli.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA interference and overexpression of β-arrestin-2 and ATF6.\u003c/b\u003e Small interference RNA (siRNA) to β-arrestin-2 (5\u0026prime;-CCUACAGGGUCAAGGUGAATT-3\u0026prime;) and negative control (5\u0026prime;-UUCUCCGCGUGUCACGUTT-3\u0026prime;) were synthesized by BioSune (Jinan, China). Small interference RNA to ATF6 was synthesized and constructed into pGPU6/GFP/Neo to get shRNA-ATF6 (5\u0026rsquo;-GAGTGAGCTGCAGGTGTATTA-3\u0026rsquo;) by Biomics Biotechnologies Co., Ltd. (Nantong, Jiangsu, China). In the experiments, all siRNA and shRNA were transfected by lipo 3000 (Invitrogen, USA) and operations followed the instruction. The overexpression plasmids contains β-arrestin-2 or ATF6 were purchased from BioSune (Jinan, China). GENC were also transfected with the pCDNA-β-arrestin-2 or pCDNA-ATF6 plasmids by lipo 3000 followed the instruction.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReal-time RT-PCR.\u003c/b\u003e We extracted the total RNA from the cortex of the kidney and GENC by trizol reagent (Invitrogen) following the instruction. Then we detected the levels of β-arrestin-1/2 by real-time RT-PCR and the primers in this study are listed in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot analyses.\u003c/b\u003e Proteins extracted from the cortex of kidney and GENC, western blot analyses were performed as described previously\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and the detailed procedure were in the supplementary. We also used nuclear protein extraction kit (Beyotime P0028) for protein extracted from nucleus. Antibodies used in this study were summarized in Supplementary Table\u0026nbsp;5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorphologic, Immunohistochemical and Immunofluorescence staining.\u003c/b\u003e The fixed renal tissues were embedded in paraffin and slice to 4-\u0026micro;m, the kidney sections were stained with periodic acid-Schiff (PAS), immuno-histochemical (IHC) staining and terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) kits according to the manufacturer\u0026rsquo;s protocol \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene delivery by AAV into kidney.\u003c/b\u003e Recombinant adeno-associated virus (AAV) contained shRNA-β-arrestin-2 (5\u0026rsquo;-GGAACUCUGUGCGGCUUAUTT-3\u0026rsquo;) and negative control (AAV-Null: 5\u0026rsquo;-UUCUCCGAACGUGUCACGUTT-3\u0026rsquo;) were purchased from BioSune (Jinan, China). The details of intrarenal AAV delivery were in the supplementary\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics.\u003c/b\u003e All data are showed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The significance of differences between multiple groups was detected by one-way ANOVA and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eUpregulation of β-arrestin-2 in DN patients and GENC from DN mice.\u003c/b\u003e We first assessed the expression pattern of β-arrestin-2 in the DN patients through renal biopsies. We found the upregulation of β-arrestin-2 in renal biopsies from DN patients compared with normal subjects by IHC staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We then constructed DN model by injection of STZ in mice, as shown in Supplementary Table\u0026nbsp;1, compared to the control mice, we found hyperglycemia and lower body weight in STZ-induced DN mice, the UACR and relative kidney weight in DN mice were higher than the control mice significantly, but the two groups had no difference in heart rate. Real-time RT-PCR and western blotting had shown that the expression of β-arrestin-2 was increased in renal cortex from the DN mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We also demonstrated the same results in the paraffin sections of renal tissue by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Immunofluorescence double staining further confirmed that the expression of β-arrestin-2 (green) was significantly increased in GENC (red) from DN mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). These results indicated that the upregulation of β-arrestin-2 in GENC from DN mice. We also detected the upregulation of β-arrestin-1 in renal biopsies from DN subjects (Supplementary Fig.\u0026nbsp;1a) and DN model mice (Supplementary Fig.\u0026nbsp;1b, 1c, 1d), but we did not find the expression of β-arrestin-1 increasing in GENC (Supplementary Fig.\u0026nbsp;1e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of β-arrestin-2 was increased in GENC under various stimuli.\u003c/b\u003e Renin-angiotensin-aldosterone system (RAAS, Ang II), high glucose (HG) and advanced glycation end product (AGE) formation are important pathways to the development and progression of DN. Each pathway causes damage via multiple mediators or interacts with other pathways\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Though traditionally, DN has not been considered as an inflammatory disease, immune and inflammatory responses play an important role in the pathogenesis of DN. Inflammatory factors, such as IL-6, tumor necrosis factor (TNF-α), TGF-β1, and IL-18 are elevated in blood and have been shown to be involved in the development and progression of DN\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The expression of β-arrestin-2 was increased under 20mmol/L and 40mmol/L glucose concentration, but the expression of β-arrestin-2 was not changed under mannitol 40mmol/L treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We also found that the expression of β-arrestin-2 was increased in GENC under common detrimental factors in DN such as AGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), AngII (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) and TGF-β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) and the increased β-arrestin-2 expression in GENC in a concentration-dependent manner. Next experiments we used the concentration of glucose 40mmol/L as the HG stimuli to study the mechanism of GENC injury in vitro under HG treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing of β-Arrestin-2 ameliorated HG induced GENC injury.\u003c/b\u003e To investigate the effect of β-arrestin-2 in GENC under HG stimulation, we knockdown the expression of β-arrestin-2 by siRNA in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The membrane proteins zonula occludens-1 (ZO-1) and occludin are related to the permeability of endothelial tight junction, decreased ZO-1 and occludin levels are closely related to the progression of DN through disrupting the function of GENC\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Inhibited of eNOS reduced the production of NO and then led to endothelial injury, eNOS knockout mice developed to nodular diabetic glomerulosclerosis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.Western blotting (WB) showed that the expression of proteins ZO-1, occludin and eNOS were decreased in GENC under HG stimulation. Silencing of β-arrestin-2 recovered occludin, ZO-1 and eNOS in GENC under HG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We then detected the changes of apoptosis related proteins,such as bcl-2, bax and cleaved caspase 3. We found that HG induced upregulation of apoptosis related proteins was decreased by knockdown of β-arrestin-2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed ), and flow cytometry further confirmed the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of β-arrestin-2 suppressed ER stress induced apoptosis in GENC through ATF6.\u003c/b\u003e ER stress plays vital role in the progression of diabetic nephropathy, bip and chop are marker proteins associated with ER stress. Our results showed that HG induced upregulation of bip and chop could be decreased by knockdown of β-arrestin-2 in GENC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Indicating that silencing of β-arrestin-2 suppressed ER stress which was activated by HG in GENC. The role of ER stress in GENC was not clear, so we then detected the apoptosis of GENC by Annexin V/propidium iodide staining, the ER stress activators tunicamycin (TM), thapsigargin (TG) and ER stress inhibitor 4-phenyl butyric acid (4-PBA) were used in our study. Flow cytometry results confirmed that activated ER stress inducing GENC apoptosis which could be alleviated by ER stress inhibitor 4-PBA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). We also detected silencing of β-arrestin-2 could partially reduced apoptosis of GENC induced by ER stress activator TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtein kinase R-like ER kinase (PERK), inositol requiring 1α (IRE1α) and activating transcription factor 6 (ATF6) are three signal pathways of ER stress. To determine the exact pathway by which β-arrestin-2 induced ER stress, we first detected the IRE1αand PERK signaling pathways. Immunoblot of the related proteins in the two pathway showed that HG dramatically enhanced expression of p-IRE1α, XBP1, p-eIF1α, p-PERK and ATF4, but knockdown of β-arrestin-2 did not change the upregulation of these proteins under HG treatment (Supplementary Figs.\u0026nbsp;2a, 2b). Then we detected the protein levels of ATF6 which is a transcription factor of ER stress. Western blotting results showed that HG increased the expression of ATF6 in GENC, knockdown of β-arrestin-2 significantly reduced the upregulation of ATF6 in GENC under HG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003cb\u003eβ-Arrestin-2 activated ER stress inducing GENC injury was blocked by knockdown of ATF6.\u003c/b\u003e We synthesized the overexpression plasmid of β-arrestin-2 (Supplementary Figure S3a) and shRNA of ATF6 (Supplementary Figure S3b). Western blotting results showed that overexpression of β-arrestin-2 would active ER stress in GENC through increasing expression of bip and CHOP which could be blocked by knockdown of ATF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We also found that silencing of ATF6 could regain ZO-1 and occludin that were decreased by overexpression of β-arrestin-2 in GENC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Western blotting and flow cytometry all confirmed that apoptosis of GENC induced by overexpression of β-arrestin-2 would be attenuated by knockdown of ATF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). We also detected that overexpression of β-arrestin-2 could upregulate ATF6, but knockdown of ATF6 did not decrease the expression of β-arrestin-2 in GENC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eβ-arrestin-2 increased expression of ATF6 and promoted ATF6 transport into nucleus.\u003c/b\u003e We then synthesized the overexpression plasmid of ATF6 (Supplementary Figure S3c). We found that overexpression of ATF6 increased protein level of bip and chop which were downregulated by knockdown of β-arrestin-2 in GENC with HG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Knockdown of β-arrestin-2 attenuated HG induced GENC injury was reversed by overexpression of ATF6 through the expression of protein ZO-1 and occludin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Apoptosis of GENC was reduced by knockdown of β-arrestin-2 in GENC with HG treatment, but overexpression of ATF6 aggravated the apoptosis of GENC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). We confirmed the results through the expression of cleaved caspase-3 (Supplementary Fig.\u0026nbsp;3d). Silencing of β-arrestin-2 reduced expression of ATF6 but overexpression of ATF6 did not change the expression of β-arrestin-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). In ATF6 signal pathway, ATF6 is cleaved and transferred to the nucleus to promote the transcription of downstream target genes. Immunoblots detected the expression of actived ATF6 in nucleus. The results showed that HG induced ATF6 transfferring into nucleus was blocked by silencing of β-arrestin-2 and overexpression of β-arrestin-2 promoted ATF6 transfferring into nucleaus which was inhibitted by knockdown of ATF6. We found the same tendency from the detection of mRNA levels of ATF6 target genes GRP78 and GRP94 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, Supplementary Fig.\u0026nbsp;3e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing of β-arrestin-2 ameliorates kidney injury in vivo.\u003c/b\u003e To examine genetic therapeutic efficiency targeting to β-arrestin-2 in mice with diabetic nephropathy, we delivered adeno-associated virus (AAV) contained shRNA-β-arrestin-2 into the experimental mice by tail vein injection. Our results confirmed that mice received AAV-shRNA-β-arrestin-2 significantly decreased the mRNA and protein levels of β-arrestin-2 in the renal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). Silencing of β-arrestin-2 reduced urinary albumin-to-creatinine ratio (Urinary-ACR, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). PAS staining was used to evaluate renal histopathological changes. A widened mesangial area and an increased mesangial matrix were observed in DN mice, but in AAV-shRNA-β-arrestin-2 DN mice, mesangial area diminished and mesangial matrix decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). TUNEL assay was performed to detect cell apoptosis. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee showed less apoptosis cells in AAV-shRNA-β-arrestin-2 DN mice compared to DN mice. And then, we also detected the expression of proteins related to the function of GENC. Western blotting results indicated that in AAV-shRNA-β-arrestin-2 transfected DN mice, the level of ZO-1, eNOS and occludin were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). The ER stress was inhibited as evidenced by decreased protein level of bip, CHOP and ATF6 in diabetic nephropathy mice transfected with AAV-shRNA-β-arrestin-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a key component of the glomerular filtration barrier, GENC have been studied intensively in recent decades\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. GENC is the first layer of glomerular filtration barrier,so GENC dysfunction arises early and plays a key role in the initiation and development of DN. Increasing data suggest that GENC injury plays an important role in the pathogenesis of DN\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our previous study demonstrated that the mRNA level of β-arrestin-1 and β-arrestin-2 were negatively correlated with estimated glomerular filtration rate (eGFR) in all available subjects individually, but the functional role of β-arrestins in GENC remains to be elucidated. In this study, we found that the expression of β-arrestin-2 but not β-arrestin-1 was increased significantly in GENC from DN mice and we also detected upregulation of β-arrestin-2 in detrimental factors (such as HG, AGE, etc.) treated GENC in vitro. We then investigated the role of β-arrestin-2 in GENC. We found that HG reduced the expression of eNOS, occludin and ZO-1 in vitro, knockdown of β-arrestin-2 could recover the expression of proteins. We also found that knockdown of β-arrestin-2 could decrease HG induced GENC apoptosis. These data showed that knockdown of β-arrestin-2 alleviated HG induced GENC injury and apoptosis.\u003c/p\u003e \u003cp\u003eEndoplasmic reticulum (ER) stress is one of the major cellular mechanisms involved in kidney injury in DN\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Numerous studies have demonstrated that the dysfunction of ER stress is associated with onset and progression of DN, ER stress inhibitors decreased ER stress and halted the progression of DN\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, some studies demonstrated that activated ER stress had a protective role on DN which reflected the bidirectional control of ER stress in DN\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study, we found that activated ER stress induced apoptosis of GENC and the ER stress in GENC with HG treatment was activated. Additionally, silencing of β-arrestin-2 not only decreased the upregulation of ER stress related proteins bip and chop but also reduced ER stress induced apoptosis in GENC with HG treatment.\u003c/p\u003e \u003cp\u003eThere are three ER stress sensor pathways, including IRE1/sXBP1, PERK/EIf2α and ATF6\u003csup\u003e37\u003c/sup\u003e. It has reported that β-arrestin-2 interacted with eIF2α in intestinal epithelial cells which contributed to promote ERS/PUMA, thereby inducing mucosal apoptosis in colitis through the mitochondrial apoptotic pathway\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, our present study found different outcomes in GENC under HG condition. Although p-PERK, p-eIF2α and ATF4 were increased in HG-treated GENC, knockdown of β-arrestin-2 had no effect on the expression of these proteins. The mixed results showed different contribution of β-arrestin-2 to ER stress in different diseases or different cell types. So how β-arrestin-2 regulates ER stress in GENC under HG condition is a question of fundamental importance. In this study, we provide potential mechanisms to answer this question. As we did not find the interaction between β-arrestin-2 and IRE1α pathway in GENC either, we focused on ATF6 sensor pathway. As a type II transmenbrane protein, ATF6 is an important molecule of ER stress pathway which participates in regulating cell apoptosis. In this study, we found that upregulation of ATF6 could be reduced by knockdown of β-arrestin-2 in GENC with HG treatment. Overexpression of β-arrestin-2 increasing the expression of ATF6 directly in GENC, but overexpression or knockdown of ATF6 did not change the expression of β-arrestin-2. We investigated that injury and apoptosis of GENC induced by overexpression of β-arrestin-2 could be alleviated by knockdown of ATF6, and silencing of β-arrestin-2 reducing injury and apoptosis of GENC would be reversed by overexpression of ATF6. The above results confirmed that β-arrestin-2 regulated the expression of ATF6 in GENC. When unfolded proteins accumulate, ATF6 is transported to the Golgi complex where it is proteolytically cleaved by S1P and S2P to release the NH2 terminal-domain\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The cleaved section of ATF6 translocated to the nucleus, where it activates gene transcription of target genes such as GRP78, GRP94\u003csup\u003e40, 41\u003c/sup\u003e. We then detected the expression of ATF6 in the nucleus, the results showed that β-arrestin-2 promoted ATF6 transported to the nucleus in GENC with HG treatment. The mRNA level of ATF6 target genes GPR78 and GPR94 also demonstrated the same results. But how β-arrestin-2 promoted ATF6 expression and transported to the nucleus needed to be investigated in the future.\u003c/p\u003e \u003cp\u003eGene therapy was used to treated many diseases recently. Adeno-associated virus vector, with low toxicity and antigenicity, is a promising vehicle for gene therapy, and commercial AAV gene therapy products have been approved by regulatory agencies and used in the clinic to treat spinal muscular atrophy, Leber congenital amaurosis and hemophilia A\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In the present study, we delivered AAV contained shRNA-β-arrestin-2 into the mice by tail vein injection. Our study demonstrated that AAV could stably transduce renal cells for 3 months and gene silencing of β-arrestin-2 could attenuate kidney damage in diabetic nephropathy mice. In addition, the GENC-specific β-arrestin-2 knockout mice would be an ideal model for investigating the role of β-arrestin-2 in GENC in diabetic nephropathy. So our findings need to be confirmed on the ideal model in the future studies.\u003c/p\u003e \u003cp\u003eIn conclusion, our study investigated that β-arrestin-2 aggravated GENC injury in diabetic nephropathy through ATF6 mediated ER stress by increasing the expression of ATF6 and promoting ATF6 transport to nucleus. This discovery provided us a new perspective for understanding the critical role of GENC in diabetic nephropathy and suggesting that β-arrestin-2 could be a new therapy target in clinical treatment for diabetic nephropathy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Jiang Liu, Xiaoyun Song and Xiuting Li conducted in vivo and in vitro experiments, performed data analysis. Jiang Liu, Wen Zhang and Dongqi Tang helped write the manuscript. Writing-original draft, Jiang Liu; methodogy, Xiaoyun Song; formalanalysis, Xiuting Li, Ying Jiang and Yuxin Lei; software, Mu Yang; validation, Fang Wang and Miao Jiang; investigation, Ying Han; writing-review and editing, Wen Zhang and Dongqi Tang; funding acquisition, Jiang Liu, Wen Zhang and Dongqi Tang. \u0026nbsp; All authors have read and agreed to the published version of the manuscript. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the grants of the Key Research and Development Program of Shandong Province (No. 2021CXGC011101) , the Special Fund for Taishan Scholars Project (tsqn202211324), the National Natural Science Foundation of China [81900669]; the Natural Science Foundation of Shandong Province, China [ZR2018PH007] and Multidisciplinary Innovation Center for Nephrology of the Second Hospital of Shandong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMohamed Q, Gillies MC and Wong TY. Management of diabetic retinopathy: a systematic review. Jama. 2007; 298: 902\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Waili N, Al-Waili H, Al-Waili T and Salom K. Natural antioxidants in the treatment and prevention of diabetic nephropathy; a potential approach that warrants clinical trials. Redox report: communications in free radical research. 2017; 22: 99\u0026ndash;118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLassen E and Daehn IS. Molecular Mechanisms in Early Diabetic Kidney Disease: Glomerular Endothelial Cell Dysfunction. International journal of molecular sciences. 2020; 21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Deng L and Xu G. METTL14 promotes glomerular endothelial cell injury and diabetic nephropathy via m6A modification of alpha-klotho. Molecular medicine. 2021; 27: 106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Ma KL, Gong YX, et al. Platelet Microparticles Mediate Glomerular Endothelial Injury in Early Diabetic Nephropathy. Journal of the American Society of Nephrology: JASN. 2018; 29: 2671\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaezawa Y, Takemoto M and Yokote K. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes. Journal of diabetes investigation. 2015; 6: 3\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNatarajan M, Habib SL, Reddick RL, et al. Endothelial cell-specific overexpression of endothelial nitric oxide synthase in Ins2Akita mice exacerbates diabetic nephropathy. Journal of diabetes and its complications. 2019; 33: 23\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng X, Soroush F, Long J, et al. Murine glomerular transcriptome links endothelial cell-specific molecule-1 deficiency with susceptibility to diabetic nephropathy. PloS one. 2017; 12: e0185250.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuelli S, Rosano L, Gagliardini E, et al. beta-arrestin-1 drives endothelin-1-mediated podocyte activation and sustains renal injury. Journal of the American Society of Nephrology: JASN. 2014; 25: 523\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Li QX, Wang XJ, et al. beta-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy. Cell death \u0026amp; disease. 2016; 7: e2183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Liang K, Zhen J, et al. Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling. Nature communications. 2017; 8: 413.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoler MJ, Riera M and Batlle D. New experimental models of diabetic nephropathy in mice models of type 2 diabetes: efforts to replicate human nephropathy. \u003cem\u003eExperimental diabetes research\u003c/em\u003e. 2012; 2012: 616313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Li QX, Wang XJ, et al. beta-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy. Cell death \u0026amp; disease. 2016; 7: e2183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBruno G, Merletti F, Biggeri A, et al. Progression to overt nephropathy in type 2 diabetes: the Casale Monferrato Study. Diabetes Care. 2003; 26: 2150\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim AK, Ma FY, Nikolic-Paterson DJ, Kitching AR, Thomas MC and Tesch GH. Lymphocytes promote albuminuria, but not renal dysfunction or histological damage in a mouse model of diabetic renal injury. Diabetologia. 2010; 53: 1772\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWada J and Makino H. Inflammation and the pathogenesis of diabetic nephropathy. Clinical science. 2013; 124: 139\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNanes BA. Slide Set: Reproducible image analysis and batch processing with ImageJ. BioTechniques. 2015; 59: 269\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahar N, Bibi S, Masood N and Faryal R. Status of serine tyrosine kinase at germline and expressional levels in asthma patients. Molecular biology research communications. 2019; 8: 69\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallo-Oller G, Ordonez R and Dotor J. A new background subtraction method for Western blot densitometry band quantification through image analysis software. Journal of immunological methods. 2018; 457: 1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Liu J, Zhen J, et al. Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy. Kidney international. 2014; 86: 712\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRubin JD, Nguyen TV, Allen KL, Ayasoufi K and Barry MA. Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections. Human gene therapy. 2019; 30: 1559\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZincarelli C, Soltys S, Rengo G and Rabinowitz JE. Analysis of AAV serotypes 1\u0026ndash;9 mediated gene expression and tropism in mice after systemic injection. Molecular therapy: the journal of the American Society of Gene Therapy. 2008; 16: 1073\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkeda Y, Sun Z, Ru X, Vandenberghe LH and Humphreys BD. Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector. Journal of the American Society of Nephrology: JASN. 2018; 29: 2287\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKopel J, Pena-Hernandez C and Nugent K. Evolving spectrum of diabetic nephropathy. World journal of diabetes. 2019; 10: 269\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShang J, Wang L, Zhang Y, et al. Chemerin/ChemR23 axis promotes inflammation of glomerular endothelial cells in diabetic nephropathy. Journal of cellular and molecular medicine. 2019; 23: 3417\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRincon-Choles H, Vasylyeva TL, Pergola PE, et al. ZO-1 expression and phosphorylation in diabetic nephropathy. Diabetes. 2006; 55: 894\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakagawa T, Sato W, Glushakova O, et al. Diabetic endothelial nitric oxide synthase knockout mice develop advanced diabetic nephropathy. Journal of the American Society of Nephrology: JASN. 2007; 18: 539\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao HJ, Wang S, Cheng H, et al. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice. Journal of the American Society of Nephrology: JASN. 2006; 17: 2664\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong C, Wang S, Fu Z, et al. IGFBP5 promotes diabetic kidney disease progression by enhancing PFKFB3-mediated endothelial glycolysis. Cell death \u0026amp; disease. 2022; 13: 340.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng F, Ma L, Li X, et al. Neutrophil Extracellular Traps Induce Glomerular Endothelial Cell Dysfunction and Pyroptosis in Diabetic Kidney Disease. Diabetes. 2022; 71: 2739\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, Wang Z, Hu Y, et al. ELABELA/APJ Axis Prevents Diabetic Glomerular Endothelial Injury by Regulating AMPK/NLRP3 Pathway. Inflammation. 2023; 46: 2343\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Y and Vanhoutte PM. Macro- and microvascular endothelial dysfunction in diabetes. Journal of diabetes. 2017; 9: 434\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, E J, Cai H, et al. Reticulon-1A mediates diabetic kidney disease progression through endoplasmic reticulum-mitochondrial contacts in tubular epithelial cells. Kidney international. 2022; 102: 293\u0026ndash;306.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu J, Peng J, Wang L, et al. Identification of endoplasmic reticulum stress-related biomarkers of diabetes nephropathy based on bioinformatics and machine learning. Frontiers in endocrinology. 2023; 14: 1206154.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSankrityayan H, Oza MJ, Kulkarni YA, Mulay SR and Gaikwad AB. ER stress response mediates diabetic microvascular complications. Drug discovery today. 2019; 24: 2247\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue T, Maekawa H and Inagi R. Organelle crosstalk in the kidney. Kidney international. 2019; 95: 1318\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Conza G and Ho PC. ER Stress Responses: An Emerging Modulator for Innate Immunity. Cells. 2020; 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng LX, Tao J, Liu HL, et al. beta-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis. Mucosal immunology. 2015; 8: 683\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe J, Rawson RB, Komuro R, et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Molecular cell. 2000; 6: 1355\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Shen J, Arenzana N, Tirasophon W, Kaufman RJ and Prywes R. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J Biol Chem. 2000; 275: 27013\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRead A and Schroder M. The Unfolded Protein Response: An Overview. Biology. 2021; 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C and Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nature reviews Genetics. 2020; 21: 255\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeebeek FWG and Miesbach W. Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues. Blood. 2021; 138: 923\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Tai PWL and Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature reviews Drug discovery. 2019; 18: 358\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic nephropathy, Glomerular endothelial cell, β-Arrestin-2, ATF6, ER stress","lastPublishedDoi":"10.21203/rs.3.rs-3939362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3939362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGlomerular endothelial cell (GENC) injury would be a characteristic of early stage diabetic nephropathy (DN) and the investigation of potential therapeutic targets for preventing GENC injury has clinical importance.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDN was induced in C57BL/6J mice by intraperitoneal injection of streptozotocin. GENC was transfected with plasmid containing siRNA-β-arrestin-2, shRNA-ATF6, pCDNA-β-arrestin-2 or pCDNA-ATF6. Additionally, we administrated adeno-associated virus (AAV) containing shRNA-β-arrestin-2 via tail vein injection in DN mice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe upregulation of β-arrestin-2 was observed in DN patients as well as in GENC from DN mice. Knockdown of β-arrestin-2 reduced endoplasmic reticulum stress (ER stress) and apoptosis in high glucose treated GENC which were reversed by overexpression of activating transcription factor 6 (ATF6). Moreover, overexpression of β-arrestin-2 led to the activation of ER stress and the apoptosis of GENC which could be mitigated by silencing of ATF6. Furthermore, knockdown of β-arrestin-2 by the administration of AAV-shRNA-β-arrestin-2 had alleviated renal injury in DN mice.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study offer novel perspectives on the crucial involvement of β-arrestin-2 in GENC injury. Knockdown of β-arrestin-2 prevents GENC apoptosis by inhibiting ATF6-mediated ER stress in vivo and vitro. Consequently, β-arrestin-2 may represent a promising therapeutic target for the clinical management of patients with DN.\u003c/p\u003e","manuscriptTitle":"β-Arrestin-2 enhances ER stress-induced glomerular endothelial cells injury through ATF6 in diabetic nephropathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 19:20:13","doi":"10.21203/rs.3.rs-3939362/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":"d773daa6-6461-485b-9365-cb1c5da42a4b","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-12T23:05:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 19:20:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3939362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3939362","identity":"rs-3939362","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}