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Research Article Protective Role of Tangshen Formula on the Progression of Renal Damage in Db/Db Mice by TRPC6/Talin1 Pathway in Podocytes

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Research Article Protective Role of Tangshen Formula on the Progression of Renal Damage in Db/Db Mice by TRPC6/Talin1 Pathway in Podocytes Hindawi Journal of Diabetes Research Volume 2020, Article ID 3634974, 19 pages https://doi.org/10.1155/2020/3634974 Research Article Protective Role of Tangshen Formula on the Progression of Renal Damage in db/db Mice by TRPC6/Talin1 Pathway in Podocytes Qian Wang,1,2 Xuefei Tian,3 Wei’e Zhou,2 Yan Wang,4 Hailing Zhao,2 Jialin Li,1,2 Xuefeng Zhou,1,2 Haojun Zhang,2 Tingting Zhao ,2 and Ping Li 2 1Beijing University of Chinese Medicine, Beijing 100029, China 2Beijing Key Laboratory for Immune-Mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China- Japan Friendship Hospital, Beijing 100029, China 3Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA 4Beijing Key Laboratory of Diabetes Research and Care, Center for Endocrine Metabolism and Immune Diseases, Lu He Hospital, Capital Medical University, Beijing 101149, China Correspondence should be addressed to Tingting Zhao; [email protected] and Ping Li; [email protected] Received 16 February 2020; Revised 11 August 2020; Accepted 29 August 2020; Published 17 September 2020 Academic Editor: Claudio De Lucia Copyright © 2020 Qian Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tangshen Formula (TSF) is a Chinese Medicine formula that has been reported to alleviate proteinuria and protect renal function in humans and animals with diabetic kidney disease (DKD). However, little is known about its mechanism in improving proteinuria. The dysregulation of podocyte cell-matrix adhesion has been demonstrated to play an important role in the pathogenesis and progression of proteinuric kidney diseases including DKD. In the present study, the underlying protective mechanism of TSF on podocytes was investigated using the murine model of type 2 DKD db/db mice in vivo and advanced glycation end products (AGEs)-stimulated primary mice podocytes in vitro. Results revealed that TSF treatment could significantly mitigate reduction of podocyte numbers and foot process effacement, reduce proteinuria, and protect renal function in db/db mice. There was a significant increase in expression of transient receptor potential canonical channel 6 (TRPC6) and a decrease in expression of talin1 in podocytes of db/db mice. The results of AGEs-stimulated primary mice podocytes showed increased cell migration and actin-cytoskeleton rearrangement. Moreover, primary mice podocytes stimulated by AGEs displayed an increase in TRPC6-dependent Ca2+ influx, a loss of talin1, and translocation of nuclear factor of activated T cell (NFATC) 2. These dysregulations in mice primary podocytes stimulated by AGEs could be significantly attenuated after TSF treatment. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC6 agonist, blocked the protective role of TSF on podocyte cell-matrix adherence. In conclusion, TSF could protect podocytes from injury and reduce proteinuria in DKD, which may be mediated by the regulation of the TRPC6/Talin1 pathway in podocytes. 1. Introduction number and/or structure of foot processes have been demon- strated to be the main cause leading to glomerular protein- Diabetic kidney disease (DKD) is one of the most common uria [3]. Under physiologic conditions, the podocyte actin and severe complications of diabetes mellitus and is charac- backbone is well organized to maintain their normal mor- terized by proteinuria and renal function impairment. DKD phology and function. Numerous studies on podocytes have is the major cause of end-stage renal disease (ESRD) world- shown that actin-cytoskeletal disorganization and related wide [1]. Accumulating evidence suggests that podocyte protein damage induced by multiple pathogenic factors lead injury is a typical manifestation and a core event in the pro- to podocyte injury [4, 5]. Cell-matrix adhesion core structure gression of DKD [2]. Podocytes are important components complexes, including integrins/talins/actin, are crucial for for glomerular filtration barrier integrity and maintenance maintaining the podocyte actin cytoskeleton. These adhesion of size selectivity in protein filtration. Changes in podocyte complexes allow podocytes to attach tightly to the glomerular 2 Journal of Diabetes Research basement membrane (GBM) to preserve the integrity of the loss and foot process effacement reduced proteinuria, and glomerular filtration barrier. Any deficit in the structural protected renal function in db/db mice, a classic genetic model components can disturb podocyte adhesion and lead to the of type 2 diabetes that has clinical and histologic features disorganization of the actin cytoskeleton [6]. of DKD resembling changes in humans [30]. Our results As a cell-matrix adhesion core structure complex, talin1 further showed that TSF might alleviate proteinuria mediated impairment can lead to integrin dysfunction, which is associ- by the regulation of the TRPC6/Talin1 pathway in podocytes, ated with a variety of pathologies, including thrombosis, following enhancement of the podocyte-matrix adhesion stroke, and cancer metastasis [7]. Tian and colleagues [8] ability. demonstrated that mice lacking talin1 specifically in podo- cytes displayed severe proteinuria, decreased podocyte 2. Materials and Methods adhesion, significant actin-cytoskeletal disorganization, foot process effacement, and progressive renal failure. Transient 2.1. Herbal Formulation and Reagents. TSF (Lot num- receptor potential canonical channel 6 (TRPC6) is a nonse- ber180408) was prepared and standardized by the Beijing lective Ca2+ channel protein, which has been reported to be Institute of Clinical Pharmacy, Beijing, China. TSF is com- closely related to the cleavage of talin1 [9]. Mutation of the posed of seven herbs: astragalus root (Astragalus membrana- TRPC6 gene in podocytes was first reported by Winn et al. ceus (Fisch) Bge.), burning bush twig (Euonymus alatus in patients with focal segmental glomerulosclerosis (FSGS), (Thunb.) Siebold.), rehmannia root (Rehmannia glutinosa suggesting the potential importance of TRPC6-mediated (Gaertn.) Li-bosch.), bitter orange fruit (Citrus aurantium Ca2+ dynamics for podocyte function [10]. More and more L.), cornus fruit (Cornus officinalis Sieb & Zucc.), rhubarb studies have shown that aberrant changes in TRPC6 in podo- root and rhizome (Rheum palmatum L.), and notoginseng cytes lead to proteinuria development and progression of root and rhizome (Panax Notoginseng (Burk.) F. H. Chen) in DKD, the mechanism by which may involve the rearrange- theratioof10:5:4:3.4:3:2:1 (w/w). TSF was prepared as ment of the podocyte actin-cytoskeleton [11, 12]. TRPC6- described in the Chinese Pharmacopeia, 2015. Six compo- dependent Ca2+ accumulation has been reported to lead to nents, including loganin, neohesperidin, naringenin, calyco- podocyte injury characterizing FSGS through reduced talin1 sin-7-O-β-D-glucoside, naringenine-7-rhamnosidoglucoside, expression [13]. Furthermore, knockdown of TRPC6 also and aloe-emodin, were identified in the chemical fingerprint results in decreased cleavage of talin1 [9]. However, it is of TSF for quality control as previously described [18]. Irbesar- unclear how the podocyte TRPC6/Talin1 signaling pathway tan (J20171089) was purchased from Sanofi Pharmaceutical affects the development and progression of DKD; thus, this (Hangzhou, ZJ, China). For the animal experiments, both mechanism needs to be investigated. TSF (0.36 g/mL) and irbesartan (2.25 mg/mL) were dissolved Tangshen Formula (TSF) is a Chinese Medicine for- in 0.5% carboxymethylcellulose sodium (CMC-Na). mula that is effective in treating DKD and is based on the Antibodies used in this study were as follows: TRPC6 empirical evidence of Chinese physicians [14]. TSF is com- (ab62461) (the specificity of the anti-TRPC6 antibody has prised of seven herbs: astragalus root, burning bush twig, been tested by MPC5 cells and Trpc6 siRNA in Figure S1), rehmannia root, bitter orange fruit, cornus fruit, rhubarb WT1 (ab89901), AGE-BSA (ab51995), fibronectin antibody root and rhizome, and notoginseng root and rhizome. (ab2413), collagen type I (ab34710), and collagen type IV Patients with stage III-IV DKD who were treated with (ab6586), all purchased from Abcam (Cambridge, MA, one component of TSF, astragalus root injection, experi- USA). siRNA against TRPC6 was purchased from Shanghai enced improved renal function and decreased proteinuria Generay Biotech (Shanghai, China). Nephrin antibody (GP- excretion compared with those in a control group [15]. A N2) was obtained from Progen (Heidelberg, Germany). decoction made from another TSF component, rehmannia Talin1 antibody (14168-1-AP), NFATC2 (nuclear factor of root, was found in vitro to dramatically suppress the pro- activated T cell) antibody (22023-1-AP), NFATC3 antibody duction of AGEs induced by inflammation [16]. Through (18222-1-AP), integrin β1 antibody (26918-1-AP), and liquid chromatography-mass spectrometry (LC-MS), we GAPDH antibody (60004-1-Ig) were purchased from have successfully detected 40 compounds in TSF and 12 Proteintech (Wuhan, HB, China). Paxillin antibody (sc- compounds in the serum of TSF-treated mice (supplemen- 365379) was purchased from Santa Cruz Biotechnology tary Tables S1 and S2). Six compounds, including loganin, (Dallas, TX, USA). N-2-hydroxyethylpiperazine-N-2-ethane neohesperidin, naringenin, calycosin-7-O-β-D-glucoside, sulfonic acid (HEPES) (15630080), RPMI 1640
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