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Extraneural CGRP Upregulates TGF-Β1 Through RAMP1 Signaling During Mechanical Stretch in Kidney Proximal Tubule Epithelial Cells

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Extraneural CGRP Upregulates TGF-Β1 Through RAMP1 Signaling During Mechanical Stretch in Kidney Proximal Tubule Epithelial Cells 해부 . 생물인류학 제 33 권 제 4 호 Anat Biol Anthropol Vol. 33, No. 4 (2020) pp. 173~180 https://doi.org/10.11637/aba.2020.33.4.173 Original Article Extraneural CGRP Upregulates TGF-β1 through RAMP1 Signaling during Mechanical Stretch in Kidney Proximal Tubule Epithelial Cells Daeun Moon 1, Babu J. Padanilam 2, Hee-Seong Jang 2, Jinu Kim 1,3 1Interdisciplinary Graduate Program in Advanced Convergence Technology & Science, Jeju National University 2Department of Cellular and Integrative Physiology, University of Nebraska Medical Center 3Department of Anatomy, Jeju National University School of Medicine Abstract : Calcitonin gene-related peptide (CGRP) derived from sensory neurons contributes to the development of renal tubulointerstitial fibrosis during ureteral obstruction through upregulation of profibrotic factors. However, the mechanism by which CGRP upregulates the profibrotic factors in the ureteral obstructive setting in the kidney tubule epithelial cells is not defined. In the human kidney proximal tubule epithelial cell line, HK-2, treatment with 1 nM CGRP significantly enhanced transforming growth factor-β1 (TGF-β1) production, its release, and protein kinase C (PKC) activity. Furthermore, mechanical stretch for 6 and 24 hours significantly increased expressions of receptor activity modifying protein 1 (RAMP1) mRNA and protein among CGRP receptor components in HK-2 cells. In addition, a combination treatment with CGRP and mechanical stretch synergistically increased TGF-β1 upregulation and PKC activation. However, RAMP1 deficiency, induced by RAMP1 double nickase plasmid transfection, abolished CGRP-induced TGF-β1 upregulation and PKC activation in HK-2 cells with or without mechanical stretch. Finally, pharmacological inhibition of PKC markedly reduced TGF-β1 production and release after treatment of HK-2 cells with CGRP. Taken together, these data suggest that extraneural CGRP upregulates TGF-β1 through RAMP1-PKC axis during mechanical stretch in kidney proximal tubule epithelial cells. Keywords : Fibrosis, Calcitonin gene-related peptide, RAMP1, TGF-β, Protein kinase C INTRODUCTION tubular epithelial cells occur mainly through fibroblast proliferation and extracellular matrix accumulation [1]. Fibrosis is responsible for sustained or repetitive pro- The injured kidney tubular epithelial cells have an active gressive tissue injury in various organs. During progression role in the development and progression of kidney fibrosis of acute kidney injury to chronic kidney disease (CKD), a through upregulation of profibrogenic factors [2]. Among persistent injury and/or an incomplete restoration in kidney various profibrogenic factors, transforming growth factor-β (TGF-β) is a well-established inducer of kidney fibrosis The author(s) agree to abide by the good publication practice guideline for and plays a key role in the pathogenesis of CKD [3-5]. medical journals. Upre gul ation of TGF-β in both tubular and interstitial cells The author(s) declare that there are no conflicts of interest. Received: December 4, 2020; Revised: December 14, 2020; is a universal finding in virtually every type of kidney fibro- Accepted: December 16, 2020 sis in human and rodent in vivo models [6,7]. In in vitro sys- Correspondence to: Jinu Kim (Department of Anatomy, Jeju National University School of Medicine 102 Jejudaehak-ro, Jeju 63243, Republic of Korea) tems, treatment with TGF-β stimulates interstitial fibroblast E-mail: [email protected] to undergo myofibroblast activation to produce extracel lular ⓒ 2020 Korean Association of Physical Anthropologists This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN 2671-566X (Online)·ISSN 2671-5651 (Print) 174 Daeun Moon, Babu J. Padanilam, Hee-Seong Jang, Jinu Kim matrix proteins including collagen and fibronectin [8]. TGF-β in kidney fibrosis. Here, we sought to determine whether signaling pathway in kidney tissue also integrates effects RAMP1 deficiency reduces CGRP-induced TGF-β upregu- of other profibrogenic factors including connective tissue lation during mechanical stretch in kidney proximal tubule growth factor [9]. Inhibition of TGF-β by multiple strate- epithelial cells. gies suppresses kidney fibrogenesis, resulting in the pre- vention of progressive kidney dysfunction [5]. Previously, our in vivo data have indicated that kidney denervation MATERIALS AND METHODS prevents the development of renal tubulointerstitial fibrosis induced by a unilateral ureteral obstruction (UUO), but Cell culture and mechanical stretch the local administration of calcitonin gene-related peptide The HK-2 human kidney proximal tubule epithelial cells (CGRP), which is a neuropathic peptide mostly secreted (American Type Culture Collection, Rockville, MD, USA) from sensory neurons among vertebrates [10], into dener- were grown in Roswell Park Memorial Institute (RPMI) vated and ureteral-obstructed kidneys revives the fibrotic 1640 (Welgene, Daegu, Korea) supplemented with 10% lesions observed in innervated and ureteral-obstructed kid- fetal bovine serum (FBS, Welgene) at 37℃ with 5% CO2, as neys [11]. Intriguingly, the CGRP neuropeptide is upregul- previously described [22-24]. When the cells grew until 70% ated in kidney proximal tubule epithelial cells during UUO confluence on Bioflex 6-well culture plates with flex ible [11]. Although our previous study has demonstrated that sheets coated with type I collagen (Flexcell International, exogenous CGRP upregulates TGF-β in kidney proximal McKeesport, PA, USA), mechanical stretch was carried tubule epithelial cells [12], it is still unclear whether and out in serum-free medium using a computer-controlled FX- how CGRP contributes to the upregulation of intracellular 5000 tension system (Flexcell International) with cycles of TGF-β under the ureteral obstructive condition. In order 5 second-stretch and 5 second-relaxation at a strain of 15%, to define the mechanism of an in vitro stress model, the as previously described [25]. The stretch was applied to the mechanical stretch was used to mimic conditions in UUO. cells for 0, 6 or 24 hours in CO2 incubator at 37℃ with 5% The mechanical stretch in kidney tubular epithelial cells is CO2. Phosphate-buffered saline (vehicle), CGRP (1 nM, the sole model that mimics the pathogenic effects of tubular R&D Systems, Minneapolis, MN, USA) or chelerythrine distension induced by UUO and can provide a mechanism chloride (1 to 100 μM, R&D Systems), a broad range PKC to upregulate fibrosis-related genes [13]. inhibitor, was added at the onset of stretch. CGRP receptor is a heterodimer consisting of receptor activity modifying protein 1 (RAMP1) and calcitonin recep- RAMP1 deficient cells tor-like receptor (CRLR) [14]. RAMP1 is required for the translocation of CRLR to form a functional CGRP receptor, Cells were transfected with either RAMP1 double nick- suggesting that RAMP1 is essential for CGRP receptor sig- ase plasmid (catalog no. sc-401396-NIC; Santa Cruz Bio- naling [10]. Expression of the CGRP receptor is detected in technology, Santa Cruz, CA, USA) for inducing RAMP1 the nervous system including trigeminal ganglia, brainstem, gene knockout (KO) or control double nickase plasmid cerebellum, and cerebral vasculature [15], as well as in kid- (Santa Cruz Biotechnology) for wild-type (WT) in 300 μL ney tubule epithelial cells [12,16]. The intracellular signal- of plasmid transfection medium (Santa Cruz Biotechnol- ing pathway of CGRP receptor can be involved in protein ogy) with 15 μL of UltraCruz transfection reagent (Santa kinase C (PKC) activation and translocation to the mem- Cruz Biotechnology) for 48 hours. After that, the transfect- brane fraction of cells [17,18]. The PKC family of serine/ ed cells were isolated using 10 μg/mL of puromycin (Sigma, threonine kinases is implicated in various fibrotic diseases St. Louis, MO, USA) for 4 days, as previously described and responses [19-21]. However, unlike an integral role of [26,27]. CGRP and its receptor signaling in the pathophysiology of migraine is established [10], the role of CGRP receptor Western blot in extra-neural sites is unclear. Thus, detailed knowledge Electrophoresis of protein extracts obtained from cell of RAMP1 downstream pathway and its regulation can be lysates using Tris-glycine buffer systems and subsequent essential to understanding the physiological role of CGRP blotting were performed as previously described [28-30]. CGRP/RAMP1 Axis-Induced TGF-β Upregulation during Mechanical Stretch 175 The membranes were incubated with antibodies against protocols. RAMP1 (1 : 2,500 dilution; catalog no. 10327-1-AP; Pro- teintech, Chicago, IL, USA), CRLR (1 : 2,500 dilution; cata- Statistical analysis log no. sc-18007; Santa Cruz Biotechnology), green fluores- All data are expressed as mean±standard error of the cent protein (GFP, Proteintech), and β-actin (Sigma). After mean (SEM). Analysis of variance was used to compare that, peroxidase-conjugated secondary antibodies (Vector data among groups using Systat SigmaPlot (Systat Software Laboratories, Burlingame, CA, USA) were incubated. To Inc., San Jose, CA, USA). Differences between the two detect the proteins, a chemiluminescence reagent (Perkin- groups were assessed using two-tailed unpaired Student’s Elmer, Boston, MA, USA) was used. Antibodies for GFP t-tests.
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