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Ets1 Associates with KMT5A to Participate in High Glucose

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Ets1 Associates with KMT5A to Participate in High Glucose Lu et al. Mol Med (2021) 27:74 https://doi.org/10.1186/s10020-021-00339-7 Molecular Medicine RESEARCH ARTICLE Open Access ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy Lihong Lu1†, Ziwen Zhong2,3†, Jiahui Gu2†, Ke Nan2*, Minmin Zhu1* and Changhong Miao2* Abstract Background: Diabetic nephropathy (DN) is currently the leading cause of end-stage renal disease globally. The endothelial-to-mesenchymal transition (EndMT) of glomerular endothelial cells has been reported to play a crucial role in DN. As a specifc form of epithelial-to-mesenchymal transition, EndMT and epithelial-to-mesenchymal transi- tion may exhibit mutual modulators. Proflin 2 (PFN2) has been reported to participate in epithelial-to-mesenchymal transition. Moreover, ETS proto-oncogene 1 (ets1) and lysine methyltransferase 5A (KMT5A) have been reported to contribute to high glucose-mediated endothelial injury and epithelial-to-mesenchymal transition. In this study, we hypothesize ets1 associates with KMT5A to modulate PFN2 transcription, thus participating in high glucose-mediated EndMT in glomerular endothelial cells. Methods: Immunohistochemistry (IHC) was performed to detect protein levels in the kidney tissues and/or aorta tis- sues of human subjects and rats. Western blot, qPCR and immunofuorescence were performed using human umbili- cal vein endothelial cells (HUVECs). Chromatin immunoprecipitation (ChIP) assays and dual luciferase assays were performed to assess transcriptional activity. The diference between the groups was compared by two-tailed unpaired t-tests or one-way ANOVAs. Results: Our data indicated that vimentin, αSMA, S100A4 and PFN2 levels were increased, and CD31 levels were reduced in glomerular endothelial cells of DN patients and rats. Our cell experiments showed that high glucose induced EndMT by augmenting PFN2 expression in HUVECs. Moreover, high glucose increased ets1 expression. si-ets1 suppressed high glucose-induced PFN2 levels and EndMT. ets1 overexpression-mediated EndMT was reversed by si- PFN2. Furthermore, ets1 was determined to associate with KMT5A. High glucose attenuated KMT5A levels and histone H4 lysine 20 methylation (H4K20me1), one of the downstream targets of KMT5A. KMT5A upregulation suppressed high glucose-induced PFN2 levels and EndMT. sh-KMT5A-mediated EndMT was counteracted by si-PFN2. Further- more, H4K20me1 and ets1 occupied the PFN2 promoter region. sh-KMT5A cooperated with ets1 overexpression *Correspondence: [email protected]; [email protected]; miao_ [email protected] †Lihong Lu, Ziwen Zhong and Jiahui Gu contributed equally to this work 1 Department of Anesthesiology, Department of Oncology, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai 200032, China 2 Department of Anesthesiology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. Lu et al. Mol Med (2021) 27:74 Page 2 of 19 to activate PFN2 promoter activity. Our in vivo study demonstrated that KMT5A was reduced, while ets1 was aug- mented, in glomerular endothelial cells of DN patients and rats. Conclusions: The present study indicated that ets1 cooperated with KMT5A to transcribe PFN2, thus contributing to hyperglycemia-induced EndMT in the glomerular endothelial cells of DN patients and rats. Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http:// www. chictr. org. cn/ showp roj. aspx? proj 48548 = Keywords: KMT5A, ets1, PFN2, EndMT, Diabetic nephropathy Background Lysine methyltransferase 5A (KMT5A) is the only Diabetic nephropathy (DN) has become one of the pri- known nucleosome-specifc methyltransferase that mary causes of mortality in diabetic patients and may modulates histone H4 lysine 20 through methylation result in end-stage renal disease (Packham et al. 2012; (H4K20me1) (Beck et al. 2012). Our previous studies Tomino and Gohda 2015). DN is characterized by a grad- have shown that KMT5A downregulation participates in ual elevation of urine albumin, a rise in blood pressure, high glucose-mediated vascular endothelial injury (Chen and a decline of the glomerular fltration rate (Boer et al. et al. 2018, 2020a, b; Qi et al. 2020; Wang et al. 2020; Shen 2011; Ng et al. 2016). However, current treatment strate- et al. 2020). Moreover, KMT5A was reported to mediate gies can only delay and cannot prevent DN progression. epithelial-to-mesenchymal transition (Hou et al. 2016). Once DN develops to end-stage renal disease, the cost However, whether KMT5A is involved in high glucose- of treatment and cardiovascular-related mortality both induced EndMT is still not well known. In the pre- increase (Alves and Lewis 2010; Xue et al. 2017). Tere- sent study, we hypothesize that ets1 may associate with fore, the search for the potential mechanisms underlying KMT5A to modulate PFN2 expression, thus performing DN is urgent. Te endothelial-to-mesenchymal transi- a vital role in high glucose-mediated EndMT in glomeru- tion (EndMT) of glomerular endothelial cells has been lar endothelium. reported to play a crucial role in the occurrence and pro- gression of DN (Li et al. 2010; Kanasaki et al. 2014). Methods EndMT is an intricate cellular diferentiation process Subjects whereby endothelial cells detach and migrate away from Twenty diagnosed DN patients (type II diabetes) and the endothelium, lose endothelial properties and acquire twenty control participants (renal cancer patients with mesenchymal features. EndMT is defned as endothe- normal renal function) were consecutively recruited. Tis lial cell loss of the endothelial phenotype, including research abided by the Declaration of Helsinki and was CD31, and acquisition of mesenchymal features, includ- authorized by the Ethics Committee of Huzhou Cen- ing vimentin (Liang et al. 2016). As EndMT is a specifc tral Hospital. Te license number of the present study form of epithelial-to-mesenchymal transition, it is possi- is 20191209-01. Written informed consent was acquired ble that epithelial-to-mesenchymal transition modulators from all the participants recruited. Te Chronic Kidney also play an important role in the modulation of EndMT Disease Epidemiology Collaboration equation (CKD- (Saito 2013). EPI) was used to calculate the glomerular fltration of Proflins are well known as actin-binding proteins human subjects. DN patients’ staging protocol was (Ding et al. 2012). Previous studies have indicated that defned according to the National Kidney Foundation’s proflin2 (PFN2) participates in epithelial-to-mesenchy- Kidney Disease Outcomes Quality Initiative classifcation mal transition in cancer cells (Tang et al. 2015; Zhang scheme. et al. 2018). However, whether PFN2 participates in hyperglycemia-mediated EndMT in endothelial cells has Rat model of DN not been reported. Male Sprague–Dawley rats weighing 180–200 g were ets1, a member of the ETS family of transcription fac- selected for the present study. Te animals were pur- tors, plays roles in kidney development and glomerular chased from Shanghai SLAC Laboratories. Tis study integrity (Razzaque et al. 2001). Moreover, ets1 serves abided by the Guide for the Care and Use of Labora- as a crucial transcription factor in the progression of tory Animals of Shanghai Medical College at Fudan DN (Geng et al. 2019; Liu et al. 2011). ets1 suppression University. Animals were housed in the care facilities as was reported to attenuate angiotensin II-induced car- described previously (Wang et al. 2020; Shen et al. 2020). diac fbrosis via the inhibition of EndMT (Xu et al. 2019). Twenty rats were randomly assigned to the control group EST1 may be a potential drug target for the treatment of (con, n = 10) and the DN group (DN, n = 10). In the con- diabetic nephropathy. trol group, the animals received a single intraperitoneal Lu et al. MolMed(2021)27:74 et al. Table 1 Characteristics of participants and rats in control (con) and Diabetic Nephropathy (DN) group Human Subjects Age BMI (kg/ SBP (mmHg) DBP HbA1C FBG (mmol/l) CREA ALB (g/L) CCr (ml/min) 24hUPQ TP (g/L) eGFR (ml/ (Year) m2) (mmHg) (%) (umol/l) (mg) min/) Normal control (n 20) = Male: 9/20 58.3 7.3 23.4 3.3 118.9 15.2 69.2 8.1 5.2 0.8 5.2 0.5 58.3 10.5 47.2 5.2 101.9 10.8 95.5 10.2 69.5 7.3 130.1 25.2 ± ± ± ± ± ± ± ± ± ± ± ± DN stage 2 (n 7) = Male: 3/7 57.4 4.7 22.5 2.3 133 10.2 75.3 8.3 6.8 1.2 6.4 0.8 100.2 10.3 35.4 3.2 80.9 7.6 1499.4 234.8 55.4 6.1 71.4 6.8 ± ± ± ± ± ± ± ± ± ± ± ± DN stage 3 (n 7) = Male: 4/7 60.2 4.4 24.6 2.5 142.8 12.1 78.3 8.2 7.2 2.0 7.2 2.3 155.9 12.4 32.2 2.9 65.5 7.1 4233.4 359.0 50.8 5.0 48.5 6.5 ± ± ± ± ± ± ± ± ± ± ± ± DN stage 4 (n 6) = Male: 3/6 59.1 5.9 21 4.3 147.3 6.2 85.1 6.9 8.9 2.5 9.2 3.1 265.8 20.9 27.5 3.6 39.8 4.6 6750.4 675.3 45.7 8.0 23.4 5.1 ± ± ± ± ± ± ± ± ± ± ± ± Rats Variables Con DM P-value CREA (umol/l) 21.0 2.2 39.7 9.7 < 0.0001 ± ± UREA (mmol/l) 2.3 0.2 6.4 1.1 < 0.0001 ± ± 24hUTP (mg) 31.7 4.5 357.9 70.3 < 0.0001 ± ± Data are presented as means SD.
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