Cell Biol Toxicol DOI 10.1007/s10565-017-9399-4

ORIGINAL ARTICLE

Exogenous CGRP upregulates profibrogenic growth factors through PKC/JNK signaling pathway in kidney proximal tubular cells Sang Pil Yoon & Jinu Kim

Received: 23 February 2017 / Accepted: 16 May 2017 # Springer Science+Business Media Dordrecht 2017

Abstract Kidney denervation prevents the development of tubulointerstitial fibrosis, but the neuropeptide calcitonin gene-related peptide (CGRP) in the denervated kidneys restores the fibrotic feature through the upregulation of profibrogenic growth factors. CGRP is involved in aggravation of inflammation by increasing the number of circulating cells and chemotactic factors. However, it is not clear how CGRP contributes to the upregulation of profibrogenic factors during fibrogenesis. In both human and pig kidney proximal tubular cell lines, administration of 1 nM CGRP significantly increased the levels of transforming growth factor-β1 (TGF-β1) production and connective tissue growth factor (CTGF) expression at 6 and 24 h after the administration. Exogenous CGRP also increased the TGF-β1 and CTGF protein levels in the incubation media, indicating release of these proteins from the cells. Treatment with 100 nM CGRP receptor antagonist (CGRP8-37) for 24 h significantly inhibited the increase in intracellular levels and released levels of TGF-β1 and CTGF in CGRP-treated cells. Genetic inhibition of CGRP receptor using siRNA transfection also suppressed the increase in TGF-β1 production and release at 24 h after CGRP stimulation. Furthermore, treatment with a specific protein kinase C (PKC) inhibitor

: J. Kim

S. P. Yoon (*) Department of Anatomy, Jeju National University School of Medicine, 102 Jejudaehak-ro, Jeju 63243, Republic of Korea e-mail: [email protected] S. P. Yoon : J. Kim Department of Biomedicine and Drug Development, Jeju National University, Jeju 63243, Republic of Korea

chelerythrine (1 thru 10 μM) markedly reduced the upregulation and release of TGF-β1 and CTGF 6 h after CGRP administration. Finally, inhibition of c-Jun Nterminal protein kinase (JNK) phosphorylation using 1 μM SP600125 prevented the increase in TGF-β1 and CTGF upregulation and release 6 h after CGRP administration. Consistent with the in vitro data, exogenous CGRP in denervated UUO kidneys upregulated and secreted TGF-β1 and CTGF in dependence on PKC activation and JNK phosphorylation. In conclusion, these data suggest that exogenous CGRP induces the upregulation and secretion of profibrogenic TGF-β1 and CTGF proteins through the CGRP receptor/PKC/ JNK signaling pathway in kidney proximal tubular cells. Keywords Calcitonin gene-related peptide (CGRP) . Transforming growth factor-β1 (TGF-β1) . Connective tissue growth factor (CTGF) . Protein kinase C (PKC) . CJun N-terminal protein kinase (JNK) . Kidney proximal tubular cell

Introduction Fibrosis is responsible for chronic progressive tissue injury in various organs. During kidney fibrosis, incomplete tubular repair and persistent tubulointerstitial inflammation occur mainly through proliferation of fibroblasts and excessive deposition of extracellular matrix, a common characteristic of many types of kidney diseases and a primary determinant of progression to end-stage

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renal disease (Eddy 2014). Kidney tubular epithelial cells have been proposed to have an active role in the progression of fibrosis through upregulation of profibrogenic growth factors and/or epithelialmesenchymal transformation (EMT) (Liu 2010; Yokoi et al. 2001). However, there is little information available regarding the cellular mechanisms that facilitate the upregulation of profibrogenic growth factors. In unilateral ureteral obstruction (UUO) and ischemia reperfusion injury induced tubulointerstitial fibrosis models, our previous reports have shown that kidney denervation prevents fibrogenesis, whereas local infusion of calcitonin gene-related peptide (CGRP) into denervated kidneys mimics the fibrotic feature observed in innervated kidneys (Kim and Padanilam 2013, 2015). Furthermore, in kidney proximal tubular cells, CGRP increased the expression and release of profibrogenic growth factors such as transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) without EMT induction as represented by no expression of α-smooth muscle actin and phosphorylated SMAD3 (Kim and Padanilam 2013, 2015). However, currently, it is not clear how and to what extent CGRP contributes to the upregulation of profibrogenic growth factors. CGRP is a 37-amino acid neuropeptide, which is primarily localized to sensory fibers. These fibers display a broad innervation throughout the body, with extensive perivascular localization, and have a dual role in efferent and sensory function (Russell et al. 2014). CGRP is reduced in hypertensive patients, and its concentration in plasma is associated with nitric oxidemediated vasodilation (Calo et al. 2012). Although the mechanisms remain to be fully elucidated, it may include reduction in blood pressure, kidney efferent sympathetic nerve activity, and renin-angiotensin system (Clayton et al. 2011; DiBona 2005). CGRP receptor is composed of two subunits: calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) (Choksi et al. 2002). CGRP has been shown to activate the mitogen-activated protein kinases (MAPKs), which are phosphorylated following CGRP activation in gingival fibroblasts (Kawase et al. 1999), vascular smooth muscle cells (Schaeffer et al. 2003), and neuronal cells (Wang et al. 2010). It has also been shown that treatment with CGRP activates protein kinase C (PKC), as well as MAPKs, in bronchial epithelial cells (Zhou et al. 2013). We therefore speculated that CGRP-induced upregulation of profibrogenic growth factors might be implicated in PKC and MAPK

activations in kidney proximal tubular cells. To investigate this hypothesis, we sought to determine whether truncated CGRP peptide (CGRP8-37, a CGRP receptor antagonist) could reduce the upregulation of profibrogenic growth factors; and if so, whether the CGRP receptor antagonism could alter PKC and MAPK activations in CGRP-treated kidney proximal tubular cell lines and denervated UUO kidneys.

Materials and methods Cell culture and treatment The HK2 (human) and LLC-PK1 (pig) kidney proximal tubular cell lines (American Type Culture Collection, Rockville, MD) were cultured in RPMI 1640 and DMEM/high-glucose, respectively, supplemented with 10% FBS at 37 °C with 5% CO2, as previously described (Esposito et al. 2009; Song et al. 2016). The cells were grown until 70% confluence on 60-mm cell culture dishes and then changed to 3 ml of serum-free medium. After that, the culture was immediately treated with α-CGRP at a final concentration of 1 nM for 6 or 24 h. CGRP8-37 (100 nM) was added at 0, 1, 3, or 6 h after CGRP administration. Some cells were treated with either chelerythrine (1 thru 10 μM), SP600125 (1 μM), SB202190 (1 μM), FR180204 (1 μM), or vehicle (PBS or 1% DMSO) immediately after CGRP administration. All chemicals were purchased from R&D Systems (Minneapolis, MN, USA). After the above experiments, culture media was collected to obtain extracellular proteins. To obtain intracellular proteins, cultured cells were washed with phosphate buffered saline (PBS), harvested in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) including 1% protease inhibitor cocktail set III (Merck Millipore, Billerica, MA, USA), 0.5% phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO, USA), and 0.5% phosphatase inhibitor cocktail 3 (Sigma); and rocked for 10 min at 4 °C. The culture media and whole cell lysates were cleared by centrifugation at 17000×g for 10 min at 4 °C. RAMP1 knockdown To use a small interfering RNA (siRNA) targeting RAMP1, HK-2 cells were transfected with either RAMP1 siGENOME siRNA (siRAMP1; catalog no. M-003700-

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Fig. 1 Exogenous CGRP induces upregulation of profibrogenic growth factors in kidney proximal tubule epithelial cells. Pig kidney proximal tubule LLC-PK1 and human kidney proximal tubule HK-2 cells were cultured in DMEM/high-glucose and RPMI 1640, respectively, until the cells were 70% confluent. After change into serum-free medium, the cells were treated with CGRP at a final concentration of 1 nM for 6 or 24 h. a, b Intracellular levels of TGF-β1 production and CTGF expression after CGRP

administration using ELISA and Western blot analysis, respectively. c, d Released levels of TGF-β1 and CTGF in the supernatant of cells after CGRP administration using ELISA. e RAMP1 and CRLR expression in CGRP-treated HK-2 cells using Western blot analysis. Four experiments were performed to evaluate the protein expression and release. In each experiment, three samples per experimental condition were included. *P < 0.05 versus 0 h

02) or siGENOME nontargeting siRNA (siControl; catalog no. D-001206-13) purchased from Dharmacon (Lafayette, CA, USA) before treatment with 1 nM CGRP for 6 or 24 h. The cells were incubated with 200 pmol siRNA in 3 ml of culture medium with 30 μl of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol (Kim et al. 2009).

0 silk tie. Kidney denervation (DNx) was carried out 2 days prior to UUO, as previously described previously (Kim and Padanilam 2013, 2015). Briefly, the left kidney artery and vein were exposed through the abdominal incision and isolated from the surrounding connective tissue under anesthesia as described above. The kidney vessels were separated and painted for 2 min with 95% ethanol and for 2 min with PBS. CGRP (30 ng/kg body weight/day; R&D Systems) or control (0.9% saline) was continuously infused into the cortical region of the denervated kidney via an intrathecal catheter attached to a mini-osmotic pump (Alzet, Palo Alto, CA, USA) that was inserted at the same time as UUO. The catheter was anchored to the obstructed ureter, and the osmotic pump was placed toward the subcutaneous site. Some mice were given CGRP8-37 (R&D Systems, 120 μg/kg body weight/day), SP600125 (30 mg/kg body weight/day) (Nath et al. 2005), chelerythrine (5 mg/kg body weight/day) (Joo et al. 2007) or vehicle (0.9% saline or 10% DMSO in 0.9% saline) via intraperitoneal implantation of the mini-osmotic pump (Alzet) beginning 24 h prior to UUO.

Mouse preparation and surgery Male C57BL/6 mice aged 8 to 10 weeks were purchased from Orient Bio (Seongnam, Republic of Korea). All mice experiments were performed in accordance with the animal protocols approved by the Institutional Animal Care and Use Committee of Jeju National University. UUO was conducted as previously reported (Kim and Padanilam 2011, 2013). Briefly, the mice were anesthetized with an intraperitoneal injection of a cocktail containing ketamine (200 mg/kg body weight) and xylazine (16 mg/kg body weight). After exposing the left kidney through the left flank incision, the left ureter was ligated completely near the kidney pelvis using a 5–

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Enzyme-linked immunosorbent assay (ELISA) The levels of total TGF β1 production and release were measured in 20 μg protein in whole cell, 20 μg protein in tissue lysates and 100 μl of culture media, respectively, using human and porcine TGF-beta 1 Quantikine ELISA kits (R&D Systems, Minneapolis, MN). The level of CTGF release was measured in 100 μl of culture media, respectively, using human, porcine and mouse CTGF ELISA kits (MyBioSource, San Diego, CA, USA). PKC activity was measured in 20 μg protein in whole cell and tissue lysates using the PKC kinase activity kit (Enzo Life Sciences, Farmingdale, NY, USA). All ELISA experiments were rigorously performed according to the respective manufacturer’s protocols.

to detect proteins. The anti-β-actin antibody was used for loading control on stripped membranes. The bands were quantified using AzureSpot analysis software (Azure Biosystems, Dublin, CA, USA). Statistical analysis Results are expressed as mean ± SEM of at least three independent biological and technical replicates. Analysis of variance was used to compare data among groups using Systat SigmaPlot (Systat Software Inc., San Jose, CA, USA). Differences between two groups were assessed by two-tailed unpaired Student’s t tests. P values <0.05 were considered statistically significant.

Western blot

Results

Electrophoresis of 20 μg protein in cell lysates and 50 μg protein in tissue lysates on Any kD or 7.5% Mini-PROTEIN TGX gels (Bio-Rad, Hercules, CA, USA) using tris-glycine buffer systems and subsequent blotting onto PVDF membranes were performed as previously described (Kim 2016; Lee et al. 2015; Park et al. 2015; Song et al. 2016; Yoon and Kim 2015, 2016). Membranes were incubated with antibodies against CTGF (1:2000 dilution; catalog no. 23936-1AP; Proteintech, Chicago, IL, USA), RAMP1 (1:2000 dilution; catalog no. sc-11379, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CRLR (1:2000 dilution; catalog no. ab84467, Abcam, Cambridge, MA, USA), p-JNK (1:1000 dilution; catalog no. 4668; Cell Signaling, Beverly, MA, USA), JNK (1:1000 dilution; catalog no. 9252; Cell Signaling), p-p38 (1:1000 dilution; catalog no. 8690; Cell Signaling), p38 (1:1000 dilution; catalog no. 9212; Cell Signaling), p-ERK (1:1000 dilution; catalog no. 4370; Cell Signaling), ERK (1:1000 dilution; catalog no. 4695; Cell Signaling), and β-actin (1:5000 dilution; catalog no. A2228; Sigma, St. Louis, MO) overnight at 4 °C, respectively. After washing, peroxidase anti-rabbit IgG antibodies (1:5000 dilution; catalog no. WB-1000; Vector Laboratories, Burlingame, CA, USA) against CGRP, p-JNK, JNK, p-p38, p38, pERK and ERK antibodies, and peroxidase anti-mouse IgG antibodies (1:5000 dilution; catalog no. WB-2000; Vector Laboratories) against α-SMA and β-actin antibodies were applied for 1 h at room temperature. After that, Western Lighting chemiluminescence reagent (NEL101; PerkinElmer, Boston, MA, USA) was used

Exogenous CGRP induces upregulation and release of profibrogenic growth factors through its receptor in kidney proximal tubular cells To determine whether profibrogenic growth factors are upregulated after CGRP administration in kidney proximal tubular cells, we measured TGF-β1 production and CTGF expression at 6 and 24 h after CGRP administration in LLC-PK1 and HK-2 cells. Treatment with CGRP for 6 and 24 h significantly increased the levels of TGF-β1 production and CTGF expression (Fig. 1a, b). Next, we tested whether CGRP-treated kidney proximal tubular cells secrete TGF-β1 and CTGF proteins. The protein levels in the incubation media were elevated at 6 and 24 h after CGRP administration (Fig. 1c, d). These data indicate that exogenous CGRP induces upregulation and secretion of profibrogenic TGF-β1 and CTGF proteins in kidney proximal tubular cells. CGRP undergoes various intracellular signaling pathways through binding to its receptor composed of RAMP1 and CRLR (Russell et al. 2014) with no alteration of these expressions by CGRP (Fig. 1e). To investigate the role of the CGRP receptor in proximal tubular cells, we used the truncated CGRP peptide (CGRP8-37) as a competitive antagonist. The cells were treated with CGRP for 24 h, and the CGRP receptor antagonist was added at 0, 1, 3, or 6 h after CGRP administration. The addition of the CGRP receptor antagonist at 0 h significantly reduced the increased levels of TGF-β1 production and CTGF expression in the CGRP-treated cells (Fig. 2a, b). Release of these proteins from the CGRP-treated cells was also

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order to use genetic inhibition of RAMP1 in kidney proximal tubular cells, siRAMP1 was transfected into HK-2 cells. We confirmed that RAMP1 expression was lower in HK-2 cells transfected with siRAMP1 than in cells transfected with siControl (Fig. 2e). CGRP administration significantly increased TGF-β1 production and

reduced by the addition of the antagonist at 0 h after CGRP administration (Fig. 2c, d). However, treatment with the antagonist at 1, 3, and 6 h after CGRP administration did not significantly alter the levels of TGF-β1 production, CTGF expression, and their respective released levels in the CGRP-treated cells (Fig. 2a–d). In

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Fig. 2 CGRP receptor antagonist prevents CGRP-induced upregulation of profibrogenic growth factors in kidney proximal tubule epithelial cells. LLC-PK1 and HK-2 cells were grown until 70% confluence on culture plates and then changed to serum-free medium. After that, the cells were treated with CGRP at a final concentration of 1 nM for 24 h. Some cells were treated with 100 nM CGRP8-37, a peptidic CGRP receptor antagonist, at 0, 1, 3, or 6 h after CGRP administration. Some HK-2 cells were transfected with either 200 pmol siRAMP1 or 200 pmol siControl before treatment with CGRP for 24 h. a, b Intracellular levels of TGF-β1 production and CTGF expression at 24 h after CGRP

administration using ELISA and Western blot analysis, respectively. c, d Released levels of TGF-β1 and CTGF in the supernatant of CGRP-treated cells using ELISA. e Downregulation of RAMP1 protein by siRAMP1 transfection. f, g TGF-β1 production and release at 24 h after CGRP administration, respectively. Four experiments were performed to evaluate the protein expression and release. In each experiment, three samples per experimental condition were included. NT no treatment with CGRP8-37. * P < 0.05 versus NT, #P < 0.05 versus control, †P < 0.05 versus siControl

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Fig. 3 CGRP receptor antagonism prevents CGRP-induced PKC activity in kidney proximal tubule epithelial cells. LLC-PK1 and HK-2 cells in serum-free medium were treated with CGRP at a final concentration of 1 nM for 6 h. a Some cells were treated with either 100 nM CGRP8-37 or PBS (vehicle) immediately after CGRP administration. b Some HK-2 cells were transfected with either 200 pmol siRAMP1 or 200 pmol siControl before CGRP

administration. PKC activity was measured using the PKC kinase activity kit (Enzo Life Sciences, Farmingdale, NY). Four experiments were performed to evaluate the enzyme activity. In each experiment, three samples per experimental condition were included. *P < 0.05 versus control, #P < 0.05 versus vehicle, †P < 0.05 versus siControl

release in siControl-transfected HK-2 cells, but genetic inhibition of RAMP1 suppressed CGRP-induced increases of TGF-β1 production and release (Fig. 2f). Taken together, these data suggest that exogenous CGRP induces upregulation and secretion of profibrogenic growth factors through its receptor signaling pathway in kidney proximal tubular cells.

growth factors through PKC activation in kidney proximal tubular cells.

PKC activation is responsible for CGRP-induced profibrogenic growth factor upregulation in kidney proximal tubular cells To examine whether exogenous CGRP is involved in PKC activation in kidney proximal tubular cells, we assessed the intracellular PKC activity. As shown in Fig. 3a, PKC activity was remarkably increased in LLC-PK1 and HK-2 cells at 6 h after CGRP administration, but the CGRP receptor antagonist significantly prevented the increase in PKC activity. Genetic inhibition of RAMP1 in HK-2 cells also prevented CGRP-induced increase in PKC activity (Fig. 3b). These results indicate induction of PKC activation by the binding of CGRP to its receptor. To determine whether exogenous CGRP upregulates profibrogenic growth factors through PKC activation in kidney proximal tubular cells, we treated with a specific PKC inhibitor chelerythrine. High and low dose PKC inhibitor treatments markedly reduced TGF-β1 and CTGF upregulations in LLC-PK1 and HK-2 cells at 6 h after CGRP administration (Fig. 4a, b). The released level of TGF-β1 and CTGF proteins was also reduced by treatments with high and low doses of the PKC inhibitor (Fig. 4c, d). These data suggest that exogenous CGRP upregulates and secretes profibrogenic

CGRP-induced profibrogenic growth factor upregulation is implicated in JNK MAPK activation in kidney proximal tubular cells CGRP can activate MAPKs, leading to proliferation, drug tolerance, or apoptotic cell death in non-epithelial cells (Kawase et al. 1999; Schaeffer et al. 2003; Wang et al. 2010). To determine whether MAPK activation contributed to profibrogenic growth factor upregulation in CGRP-treated kidney proximal tubule epithelial cells, we assessed the phosphorylated forms of JNK, p38, and ERK MAPK in CGRP-treated HK-2 cells. As shown in Fig. 5a, the phosphorylation of JNK, p38 and ERK was markedly increased in these cells at 6 h after CGRP administration, compared with the non-administered control group. However, the addition of the CGRP receptor antagonist significantly prevented the increase in JNK, p38, and ERK phosphorylations. Treatment with the PKC inhibitor also significantly lessened increases in JNK, p38, and ERK phosphorylations in the CGRPtreated cells. In addition, HK-2 cells transfected with siControl showed significant increases in JNK, p38, and ERK phosphorylations at 6 h after CGRP administration, but downregulation of RAMP1 by siRNA prevented the increases of all MAPK phosphorylations (Fig. 5b). These data indicates that exogenous CGRP activates MAPKs through its receptor and PKC activation in kidney proximal tubular cells. To identify MAPKs that contributes to profibrogenic growth factor upregulation and release in CGRP-treated proximal tubule epithelial cells, we treated

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Fig. 4 PKC inhibition prevents CGRP-induced upregulation of profibrogenic growth factors in kidney proximal tubule epithelial cells. LLC-PK1 and HK-2 cells in serum-free medium were treated with CGRP at a final concentration of 1 nM for 6 h. Some cells were treated with 1 or 10 μM chelerythrine, a broad range PKC inhibitor, in PBS (vehicle, veh) immediately after CGRP administration. a, b Intracellular levels of TGF-β1 production and CTGF

expression at 6 h after CGRP administration using ELISA and Western blot analysis, respectively. c, d Released levels of TGFβ1 and CTGF in the supernatant of cells after CGRP administration using ELISA. Four experiments were performed to evaluate the protein expression and release. In each experiment, three samples per experimental condition were included. *P < 0.05 versus control, #P < 0.05 versus vehicle

the cells with respective selective inhibitors of MAPKs at 0 h after CGRP administration. Six hours after CGRP administration, the cells treated with the JNK inhibitor SP600125 showed a significant reduction in TGF-β1 production and CTGF expression; however, the levels of TGF-β1 production and CTGF expression were unaltered by respective treatments with the p38 inhibitor SB202190 and the ERK inhibitor FR180204 (Fig. 6a, b). Consistent with the patterns of TGF-β1 production and CTGF expression, the release of TGF-β1 and CTGF proteins from CGRP-treated cells was also prevented by treatment with the JNK inhibitor (Fig. 6c, d). However, respective

inhibitions of p38 and ERK did not alter the TGF-β1 and CTGF releases (Fig. 6c, d). Taken together, these data suggest that exogenous CGRP induces upregulation and secretion of profibrogenic growth factors through the CGRP receptor/PKC/JNK axis in kidney proximal tubular cells (Fig. 6e). Our previous report showed that kidney denervation abolished UUO-induced increase in TGF-β1 expression and collagen deposition, but treatment of denervated UUO kidneys with CGRP mimicked the fibrotic response observed in innervated obstructed kidneys (Kim and Padanilam 2013, 2015). Next, the in vitro data

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Fig. 5 CGRP receptor antagonism and PKC inhibition reduces CGRP-induced MAPK activation in kidney proximal tubule epithelial cells. LLC-PK1 and HK-2 cells in serum-free medium were treated with CGRP at a final concentration of 1 nM for 6 h. a Some cells were treated with 100 nM CGRP8-37, 10 μM chelerythrine or PBS (vehicle) immediately after CGRP administration. b Some HK-2 cells were transfected with either 200 pmol siRAMP1 or

200 pmol siControl before CGRP administration. The phosphorylations of JNK (p-JNK), p38 (p-p38), and ERK (p-ERK); and the respective total expressions were measured by Western blot analysis. Four experiments were performed to evaluate the MAPK activation. In each experiment, three samples per experimental condition were included. *P < 0.05 versus control, #P < 0.05 versus vehicle, †P < 0.05 versus siControl

on the role of CGRP in kidney proximal tubular cells were complemented by our studies in mouse denervated UUO kidneys. Ten days after UUO, the denervated kidneys treated with CGRP showed the increases of PKC activity, JNK phosphorylation, TGF-β1 production, and CTGF expression; however, treatments with CGRP receptor antagonist and PKC inhibitor significantly prevented these increases (Fig. 7a–d). Treatment of denervated UUO kidneys with JNK inhibitor significantly suppressed CGRP-induced increases of JNK phosphorylation, TGF-β1 production, and CTGF expression, but not PKC activity (Fig. 7a–d). These data indicates that, consistent with the in vitro data,

exogenous CGRP upregulates and secretes profibrogenic TGF-β1 and CTGF through the CGRP receptor/PKC/JNK axis in UUO kidneys.

Discussion Interstitial fibrosis is the hallmark of chronic kidney disease regardless of its cause (Zeisberg and Neilson 2010). During the past two decades, causative roles for fibroblast activation, inflammation, and tubular injury have been established in the tubulointerstitial fibrogenesis (Boor et al. 2010). Several molecules are implicated in the

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Tubulointerstitial fibrosis Fig. 6 JNK inhibition prevents CGRP-induced upregulation of profibrogenic growth factors in kidney proximal tubule epithelial cells. LLC-PK1 and HK-2 cells in serum-free medium were treated with CGRP at a final concentration of 1 nM for 6 h. Some cells were treated with 1 μM SP600125, a selective JNK inhibitor; 1 μM SB202190, a selective p38 inhibitor; 1 μM FR180204, a selective ERK inhibitor; or DMSO (vehicle) immediately after CGRP administration. a, b Intracellular levels of TGF-β1 production and CTGF expression after CGRP administration using

ELISA and Western blot analysis, respectively. c, d Released levels of TGF-β1 and CTGF in the supernatant of cells after CGRP administration using ELISA. e Scheme of CGRP-induced CGRP receptor/PKC/JNK signaling pathway during tubulointerstitial fibrosis. Four experiments were performed to evaluate the protein expression and release. In each experiment, three samples per experimental condition were included. *P < 0.05 versus control, #P < 0.05 versus vehicle

progression of kidney fibrosis. Among the profibrogenic growth factors, TGF-β1 is the principal factor that drives various fibrotic diseases, including glomerular and tubulointerstitial fibrosis, through promotion of myofibroblast activation and extracellular matrix deposition in multiple organs (Meng et al. 2016). CTGF also acts as an important fibrogenic growth factor in multiple organs, including the kidney, and it can be upregulated by both TGF-β1-dependent and -independent signaling (Gupta et al. 2000). Recently, our studies have shown that kidney denervation nearly completely prevented the development of tubulointerstitial fibrosis after kidney ischemia reperfusion injury and ureteral obstruction, while

administration of CGRP in the denervated kidneys restored tubulointerstitial fibrosis, suggesting that CGRP is a major trigger for kidney fibrosis (Kim and Padanilam 2013, 2015). However, to develop an effective treatment using CGRP inhibition, understanding of the underlying molecular mechanisms during the initiation and progression of fibrosis is required. Here, we found that in human and pig kidney proximal tubular cells, (1) exogenous CGRP induces upregulation and release of both TGF-β1 and CTGF through the CGRP receptor; (2) PKC activation is responsible for CGRP-induced upregulation and release of the factors; and (3) among MAPK proteins activated by CGRP, JNK mediates the upregulation and

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Fig. 7 CGRP upregulates TGF-β1 and CTGF through CGRP receptor, PKC and JNK in denervated UUO kidneys. Kidney denervation (DNx) in left kidneys of male C57/BL6 mice was carried out; 2 days after the onset, CGRP (30 ng/kg body weight per day) was continuously infused into kidneys via a mini-osmotic pump, and the left ureters were obstructed for 10 days. a PKC activity was measured using the PKC kinase activity kit (Enzo Life

Sciences). b The phosphorylation of JNK (p-JNK) and the expression of total JNK were measured by Western blot analysis. c Kidney tissue levels of TGF-β1 production and CTGF expression using ELISA and Western blot analysis, respectively. Six experiments were performed to evaluate the MAPK activation. In each experiment, three samples per experimental condition were included. *P < 0.05 versus control, #P < 0.05 versus vehicle

release of TGF-β1 and CTGF. Our present data suggest that CGRP derived from afferent fibers plays the role of a primary profibrogenic stimulator during the development of kidney fibrosis. Primary sensory nerve-derived CGRP is a key player in the regulation of peripheral inflammation and the immune response (Mignini et al. 2003). The intracellular signaling pathway of CGRP through binding to its receptor is involved in PKC activation and translocation to the membrane fraction of cells, as shown in previous reports (Wang et al. 2005; Zhou et al. 2013). The PKC family of serine/threonine kinases can exert its actions by phosphorylating a variety of cellular targets (Dekker and Parker 1994), and it is implicated in various fibrotic diseases and responses (Li et al. 1989; Mulsow et al. 2005). In kidney fibrosis, it has been reported that loss of PKCβ reduces fibrotic cytokine expression (Ohshiro et al. 2006). Consistent with this finding, our data

demonstrates that CGRP induces PKC activation through the CGRP receptor, and PKC inhibition prevents the upregulation of fibrotic cytokines including TGF-β1 and CTGF in CGRP-treated kidney proximal tubular cells. Furthermore, CGRP-induced release of these cytokines is blocked completely by PKC inhibition. CGRP has been shown to activate MAPKs, which are phosphorylated in various cells and tissues following CGRP administration, leading to either apoptotic cell death, cell proliferation, or drug tolerance (Kawase et al. 1999; Schaeffer et al. 2003; Wang et al. 2010). As reported previously, signaling events downstream of PKC activation also lead to phosphorylation of MAPKs in various cell types (Chien et al. 2015; Jin et al. 2013; Ye et al. 2017). Our data reveals that treatment with CGRP increases the phosphorylation of MAPKs through PKC activation in kidney proximal tubular

Cell Biol Toxicol

cells, but, intriguingly, among them, only JNK is implicated in TGF-β1 and CTGF upregulation and release induced by exogenous CGRP. This possibility is supported by previous reports demonstrating that JNK activation enhances TGF-β1 and CTGF gene expression and contributes to fibrosis, whereas inhibition of JNK activation protects against the development of fibrosis (Ma et al. 2009; Yang et al. 2010). It has been known that JNK enhances TGF-β1 and CTGF gene transcription and promote fibrosis (Ma et al. 2009). In conclusion, the present results demonstrate that kidney afferent nerve-derived CGRP induces upregulation of profibrogenic TGF-β1 and CTGF proteins through the CGRP receptor/PKC/JNK signaling pathway in kidney proximal tubular cells, and this pathway might be a cause that triggers the inflammation cascade and tubulointerstitial fibrosis. Although our present data have shown that CGRP-treated kidney tubular cells secretes both TGF-β1 and CTGF proteins as critical mediators in epithelial-mesenchymal transition, our previous data have observed that exogenous CGRP does not induce expression of α-smooth muscle actin as a marker for myofibroblasts and phosphorylation of SMAD3 as a TGF-β1 signaling mediator (Kim and Padanilam 2013). These data suggest that autocrine signaling through TGF-β1 and CTGF is not activated in kidney proximal tubular cells. How TGF-β1 and CTGF are released from kidney tubular cells, and further whether the released factors contribute to myofibroblast activation in tubulointerstitial fibrosis animal models remain to be explored. It is hoped that inhibiting the CGRP actions will represent a novel effective therapeutic strategy to prevent or limit kidney fibrogenesis in chronic kidney disease patients where it seems to be impaired. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1C1B2012080).

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