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Intermedin1–53 attenuates vascular calcification in rats with chronic kidney disease by see commentary on page 534 upregulation of a-Klotho Jin Rui Chang1,2,3,4,8, Jun Guo1,2,8, Yue Wang5, Yue Long Hou1,2,3, Wei Wei Lu1,2,6, Jin Sheng Zhang1,2,6, Yan Rong Yu6, Ming Jiang Xu3, Xiu Ying Liu7, Xiu Jie Wang7, You Fei Guan3, Yi Zhu3, Jie Du1,2, Chao Shu Tang1,2,3 and Yong Fen Qi1,2,3,6 1
The Key Laboratory of Remodeling-related Cardiovascular Diseases, Capital Medical University, Ministry of Education, Beijing, China; Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing Anzhen Hospital Affiliated with the Capital Medical University, Beijing, China; 3Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University Health Science Center, Beijing, China; 4Insititute of Basic Medicine Science, Xi’an Medical University, Xi’an, China; 5Renal Department, Peking University Third Hospital, Beijing, China; 6Department of Pathogen Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; and 7Key Laboratory of Genetic Network Biology, Collaborative Innovation Center of Genetics and Development, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 2
Deficiency in a-Klotho is involved in the pathogenesis of vascular calcification. Since intermedin (IMD)1–53 (a calcitonin/calcitonin gene-related peptide) protects against vascular calcification, we studied whether IMD1–53 inhibits vascular calcification by upregulating a-Klotho. A rat model of chronic kidney disease (CKD) with vascular calcification induced by the 5/6 nephrectomy plus vitamin D3 was used for study. The aortas of rats with CKD showed reduced IMD content but an increase of its receptor, calcitonin receptor-like receptor, and its receptor modifier, receptor activity-modifying protein 3. IMD1–53 treatment reduced vascular calcification. The expression of a-Klotho was greatly decreased in the aortas of rats with CKD but increased in the aortas of IMD1–53-treated rats with CKD. In vitro, IMD1–53 increased a-Klotho protein level in calcified vascular smooth muscle cells. a-Klotho knockdown blocked the inhibitory effect of IMD1–53 on vascular smooth muscle cell calcification and their transformation into osteoblastlike cells. The effect of IMD1–53 to upregulate a-Klotho and inhibit vascular smooth muscle cell calcification was abolished by knockdown of its receptor or its modifier protein, or treatment with the protein kinase A inhibitor H89. Thus, IMD1–53 may attenuate vascular calcification by upregulating a-Klotho via the calcitonin receptor/ modifying protein complex and protein kinase A signaling. Kidney International (2016) 89, 586–600; http://dx.doi.org/10.1016/ j.kint.2015.12.029 KEYWORDS: a-Klotho; calcitonin receptor-like receptor; chronic kidney disease; intermedin1-53; vascular calcification ª 2016 International Society of Nephrology
Correspondence: Yong Fen Qi, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing 100191, China. E-mail: yongfenqi@ 163.com 8
JRC and JG contributed equally to this work.
Received 4 March 2015; revised 18 November 2015; accepted 3 December 2015; published online 28 January 2016 586
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ascular calcification, a common feature of chronic kidney disease (CKD), diabetes, hypertension, and aging, is an independent risk factor for cardiovascular disease.1,2 It predicts mortality in patients, especially those with endstage renal disease.2 Vascular calcification has emerged as a highly regulated process that involves a phenotype transition of vascular smooth muscle cells (VSMCs) transforming into osteoblast-like cells.3 This transition is accompanied by a loss of VSMC contractile markers, including smoothelin, calponin, and smooth muscle 22a, and increased expression of osteogenic-profile genes, including alkaline phosphatase (ALP), osteopontin, osteocalcin, bone morphogenetic protein 2, core binding factor a 1, and osterix.4 Recently, loss of calcification inhibitors such as pyrophosphate, g-carboxyglutamic acid protein, and antiaging and anticalcification factor a-Klotho was found to greatly promote vascular calcification.1,2 The a-Klotho gene, a gene discovered in 19975 while coding for an antiaging protein, has high homology among humans, mice, and rats6,7 and is expressed primarily in renal tubules, the choroid plexus in the brain, and the parathyroid gland. The human a-Klotho gene spans about 50 kilobase pairs on chromosome 13q12 and consists of 5 exons;6 it encodes a single-pass transmembrane protein of 1012 amino acids acting as a coreceptor for fibroblast growth factor 23, the phosphaturic and vitamin D-regulating hormone.8 In addition to the membrane a-Klotho, secreted a-Klotho exists in circulation, generated from the a-Klotho gene via alternative RNA splicing and containing 3 exons or ectodomain shedding of membrane a-Klotho from the cell surface. This circulating a-Klotho acts as a humoral factor with a multitude of functions such as antiapoptosis and antisenescence effects.9 Mice lacking the a-Klotho gene develop a phenotype similar to premature human aging, including vascular calcification, endothelial dysfunction, and shortened lifespan.5 Several experimental studies showed that a-Klotho attenuates vascular calcification,10 improves endothelial dysfunction,9 and prevents cardiomyopathy.11 a-Klotho–haplo-insufficient mice with CKD show severe calcification, and transgenic mice overexpressing Kidney International (2016) 89, 586–600
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a-Klotho show less calcification,10 so a-Klotho may have a
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showed increased calcium content and ALP activity in aortas (Supplementary Figure S2A and B). Increased calciumphosphate salt deposition in CKD aortas was confirmed by von Kossa staining (Supplementary Figure S2C). The content of plasma IMD was decreased by 51.8% (P < 0.01) in CKD rats as compared with control rats (Figure 1a). The mRNA and protein levels of IMD were lower, by 51.5% and 45.8% (both P < 0.05), respectively, in calcified aortas of CKD rats than in control rats (Figure 1b–d), and the mRNA levels of CRLR and RAMP1, 2, and 3 were increased in calcified CKD aortas, by 5.88-fold (P < 0.05), 0.70-fold (P < 0.01), 1.11-fold (P < 0.05), and 7.90-fold (P < 0.05), respectively (Figure 1e). The protein levels of CRLR and RAMP3 were higher, by 4.70- and 2.21-fold (both P < 0.01) (Figure 1f and g), with no significant difference in protein levels of RAMP1 and 2 in CKD rats as compared with control rats.
pivotal role in vascular calcification. Recently, numerous studies, including our previous work,12 revealed that paracrine/autocrine factors are involved in vascular calcification. Most of these factors, vasoactive peptides such as adrenomedullin,12 ghrelin,13 parathyroid hormone-related peptide,14 and intermedin (IMD),15 are endogenous factors that inhibit vascular calcification. IMD, namely adrenomedullin 2, discovered in 2004, is a member of the calcitonin gene-related peptide (CGRP) family, which includes adrenomedullin, CGRP, calcitonin, and amylin.16,17 Human IMD gene encodes a prepropeptide of 148 amino acids with a signal peptide for secretion at the N terminus. IMD1-53 can be generated from prepro-IMD by proteolytic cleavage at Arg93-Arg94.18 The biological effects of IMD are mediated by calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs).16 RAMPs consist of 3 subtypes and interact with CRLR to modify its function: CRLR with RAMP1 is a CGRP receptor and with RAMP2 or 3 is an adrenomedullin or IMD receptor.19 IMD acts on CRLR/RAMP receptor complexes, which are rich in vasculature.20 The expression profile of IMD and its receptors is disturbed in different cardiovascular diseases.21,22 IMD is considered a potential endogenous protective peptide of the cardiovascular system by activating the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway.23 Several studies showed that IMD1–53 protects against myocardial ischemia-reperfusion, strengthens the contractility of the left ventricle, and inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes.18,24 Administration of IMD1–53 dilates coronary arteries and enhances coronary flow in isolated perfused rat hearts.18 We and others have reported that the formation of atherosclerotic lesions in apolipoprotein E/ mice25 and vascular calcification in rats could be inhibited by IMD1–53.15,22 Our previous studies showed downregulated levels of IMD in calcified aortas in vivo and in calcified VSMCs and that vascular calcification was alleviated with IMD1–53 treatment.22 However, the mechanism of IMD inhibiting vascular calcification is still unclear. CGRP, in the same family as IMD, was found to upregulate the expression of a-Klotho in angiotensin II-induced endothelial progenitor cells.26 In the present study, we investigated whether IMD1–53 attenuates vascular calcification by upregulating a-Klotho.
In rat CKD aortas, IMD1–53 treatment significantly reduced calcium content and ALP activity, by 75.7% (P < 0.01) and 60.1% (P < 0.01), respectively (Figure 2a and b). Hematoxylin and eosin staining revealed disordered elastic fibers in calcified CKD aortas; however, IMD1–53 treatment improved the vascular structure (Figure 2c). Alizarin-red S staining and von Kossa staining showed decreased calcium-phosphate salt deposition in IMD1–53-treated vascular calcification aortas (Figure 2c). As well, kidney damage and dysfunction were reduced in CKD rats with IMD1–53 treatment (Supplementary Figure S3). The phenotypic transition of VSMCs is an important feature during vascular calcification and involves decreased expression of SMC lineage markers and induced expression of osteoblast markers.4 We found reduced protein levels of the VSMC phenotype markers smoothelin and calponin in calcified rat CKD aortas (Figure 3a–c), which were increased by 2.52- and 6.17-fold (both P < 0.01) with IMD1–53 treatment (Figure 3a–c). The increased protein levels of the osteoblastlike cell markers osteopontin and osteocalcin in calcified CKD aortas were decreased significantly by 40.2% (P < 0.01) and 44.4% (P < 0.01), respectively, with IMD1–53 treatment (Figure 3a, d, and e). Furthermore, the upregulated mRNA levels of osteogenic markers core binding factor a 1, osterix, osteocalcin, and bone morphogenetic protein 2 in CKD aortas were downregulated with IMD1–53 treatment (Figure 3f–i).
RESULTS Level of IMD and its receptors in calcified CKD rat aortas
IMD1–53 inhibited VSMC calcification by increasing a-Klotho protein level
First, we assessed renal function and vascular calcification in rats. In rats with 5/6 nephrectomy plus vitamin D3 treatment (CKD group), compared with control rats, blood urea nitrogen and creatinine levels were significantly increased (Supplementary Figure S1A and B), as were plasma calcium and phosphorus levels (Supplementary Figure S1C–E). However, parathyroid hormone level was decreased (Supplementary Figure S1F), which was consistent with a previous report.27 Compared with control rats, CKD rats
whether IMD1–53 could inhibit vascular calcification by upregulating a-Klotho. First, we used RNA sequencing of normal and CKD rat aortas. Our reads were uniquely mapped to 14,072 genes; 3715 differentially expressed genes were obtained ($2-fold change in expression), including 1017 downregulated and 2698 upregulated genes in CKD rat aortas. The expression of a-Klotho was downregulated by
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IMD1–53 treatment attenuated vascular calcification in rat CKD aortas
a-Klotho deficiency results in vascular calcification,28 and a-Klotho can be regulated by CGRP.26 We investigated
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Figure 1 | Levels of intermedin (IMD) and its receptors in plasma and aortas of the chronic kidney disease (CKD) rat model. (a) Radioimmunoassay of plasma IMD in CKD rats (n ¼ 8). (b) Real-time polymerase chain reaction quantification of IMD mRNA level. (c) Western blot analysis of IMD (b-actin was a loading control) and (d) semiquantitative analysis. Data are mean SD (n ¼ 4). *P < 0.05; **P < 0.01 versus control (Con). (e) Real-time polymerase chain reaction quantification of mRNA levels of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP)1 to 3. (f) Western blot analysis of protein levels of CRLR, RAMP1 to 3 and (g) semiquantitative analysis. Data are mean SD (n ¼ 4). *P < 0.05, **P < 0.01 versus Con.
24.4-fold in CKD rat aortas (reads per kilobase per million: control group ¼ 0.1025, CKD group ¼ 0.0042). Then, we detected changes in a-Klotho expression in calcified CKD rat plasma and aortas with or without IMD1–53 treatment. a-Klotho protein level was decreased, by 19.5% (P < 0.01) and 56.3% (P < 0.01), in CKD plasma and aortas, respectively, as 588
compared with these levels in control rats (Figure 4a–c). In vitro, a-Klotho protein level was decreased in calcified VSMCs on day 3 and further reduced with calcification time (Figure 4d and e). IMD1–53 treatment significantly reversed the reduced a-Klotho protein level in calcified aortas (Figure 4f and g, Supplementary Figure S4A), kidney, and plasma in vivo Kidney International (2016) 89, 586–600
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Figure 2 | Intermedin (IMD)1–53 administration alleviated calcification in chronic kidney disease (CKD) rat aortas. Effect of IMD1–53 treatment on (a) calcium content (n ¼ 9) and (b) alkaline phosphatase (ALP) activity (n ¼ 7). Data are mean SD. **P < 0.01 versus control (Con); ##P < 0.01 versus CKD. (c) Hematoxylin and eosin (HE) staining of disordered elastic fibers in CKD rat aortas and with IMD1-53 treatment. Alizarin-red S and von Kossa staining of vascular calcium deposition. Bars ¼ 100 mm. Original magnification 200.
(Supplementary Figure S4B and C). The reduced a-Klotho protein expression in calcified VSMCs was restored by IMD1–53 (Figure 4h–k). To investigate the role of a-Klotho in IMD1–53 inhibiting calcification, we knocked down a-Klotho with its small, interfering RNA (siRNA). In cultured VSMCs, the mRNA and Kidney International (2016) 89, 586–600
protein levels of a-Klotho were greatly decreased with a-Klotho siRNA knockdown (Supplementary Figure S5). In calcified VSMCs, calcium content and ALP activity were significantly decreased with IMD1–53 treatment, which was eliminated with a-Klotho siRNA knockdown (Figure 5a and b). The observation was further verified by Alizarin-red 589
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JR Chang et al.: IMD1–53 inhibits VC by upregulating a-Klotho in CKD
Figure 3 | Intermedin (IMD)1–53 inhibited vascular smooth muscle cell phenotype transition in calcified chronic kidney disease (CKD) rat aortas. (a) Western blot analysis of effect of IMD1-53 on protein levels of smoothelin, calponin, osteopontin (OPN), and osteocalcin (OCN) and (b–e) semiquantitative analysis. (f–i) Quantitative real-time polymerase chain reaction analysis of the effect of IMD1-53 on mRNA levels of core binding factor a 1 (Cbfa-1), osterix, OCN, and bone morphogenetic protein 2 (BMP2). Data are mean SD (n ¼ 3). *P < 0.05, ** P < 0.01 versus control (Con); #P < 0.05, ##P < 0.01 versus CKD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
S staining, showing reduced calcification of VSMCs with IMD1–53 treatment, which was reversed with a-Klotho knockdown (Figure 5c). The levels of the contractile markers smoothelin, calponin, and smooth muscle 22a were increased with IMD1–53 treatment in scramble siRNA-treated calcified VSMCs but significantly decreased with a-Klotho siRNA knockdown (Figure 6a–d). The levels of osteoblast markers osteopontin and osteocalcin were decreased with IMD1–53 treatment in scramble siRNA-treated calcified VSMCs but significantly increased with a-Klotho siRNA knockdown (Figure 6a, e, and f). Thus, preventing reduced a-Klotho protein level is essential for IMD1–53 to inhibit VSMC calcification. IMD1–53 upregulated the protein level of a-Klotho and inhibited VSMC calcification via the CRLR/RAMP3 receptor complex and cAMP/PKA signaling
IMD exerts its biological action via CRLR/RAMP receptor complexes.16 Hence, we examined the expression of CRLR/RAMPs in calcified rat CKD aortas. The expression of the CRLR/RAMP3 receptor complex was significantly 590
increased in CKD aortas (Figure 1). To elucidate whether IMD1–53 increased a-Klotho level via CRLR/RAMP3, we used IMD17–47, an IMD receptor antagonist, or knocked-down CRLR or RAMP3 with siRNA in VSMCs. VSMCs were preincubated with IMD17–47 (106 mol/l) for 30 minutes, which reversed the inhibitory effects of IMD1–53 on calcificationdownregulated a-Klotho protein level (Figure 7a). Then, we knocked down CRLR or RMAP3 in cultured VSMCs, verified at both the mRNA and protein levels (Supplementary Figure S6). The upregulatory effect of IMD1–53 on a-Klotho protein level was abolished with CRLR or RAMP3 siRNA knockdown (Figure 7b and c), with levels decreased by 72.2% (P < 0.01) and 48.4% (P < 0.05), respectively. To investigate the signaling pathway by which IMD1–53 increases a-Klotho level in calcified VSMCs, we preincubated VSMCs with the PKA inhibitor H89, PI3K inhibitor LY294002, or extracellular signal–regulated kinase signaling inhibitor PD98059. Preincubation with H89 for 30 minutes inhibited the upregulatory effect of IMD1–53 on a-Klotho protein level (Figure 7d), but LY294002 or PD98059 had no effect (Supplementary Figure S7). Thus, the CRLR/RAMP3 Kidney International (2016) 89, 586–600
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Figure 4 | Level of a-Klotho in chronic kidney disease (CKD) rat aortas in vivo and calcified (Cal) vascular smooth muscle cells in vitro with or without intermedin (IMD)1–53 treatment. (a) Plasma levels of a-Klotho in normal and CKD rats (n ¼ 8). (b, d) Western blot analysis of protein level of a-Klotho and (c, e) semiquantitative analysis. b-actin was a loading control (Con). (f, h, and j) Western blot analysis and (g, i, and k) semiquantification of the effect of IMD1–53 on a-Klotho protein level in (f, g) calcified aortas in vivo or in (h-k) calcified vascular smooth muscle cells in vitro. IMD-7 and IMD-9 represent the concentration of the used IMD1–53 10-7 and 10-9 mol/l, respectively. Data are mean SD (n ¼ 4). *P < 0.05, **P < 0.01 versus Con; #P < 0.05, ##P < 0.01 versus CKD or Cal. d, days. Kidney International (2016) 89, 586–600
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Figure 5 | Deficiency of a-Klotho attenuated the inhibitory effect of intermedin (IMD)1–53 on vascular smooth muscle cell calcification (Cal). Cultured vascular smooth muscle cells transfected with a-Klotho small, interfering (siRNA) or scramble (sc) siRNA were treated with calcifying media plus IMD1-53 for 3 days (for alkaline phosphatase [ALP] activity assay) or 14 days (for calcium content assay and Alizarin-red S staining). a-Klotho knockdown blocked the (a) IMD1–53 inhibitory effect on calcium content (n ¼ 3) and (b) ALP activity (n ¼ 5) in calcified vascular smooth muscle cells. Data are mean SD. *P < 0.05, **P < 0.01 versus control (Con) in scramble; #P < 0.05, ##P < 0.01 versus Cal in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. (c) Alizarin-red S staining of vascular smooth muscle cell calcification. Bars ¼ 200 mm. Original magnification 100.
receptor complex and cAMP/PKA signaling were essential for IMD1–53 upregulating a-Klotho. To determine whether IMD1–53 inhibited vascular calcification via the CRLR/RAMP3 receptor complex and cAMP/ PKA signaling, we used siRNA knockdown of CRLR or RAMP3 or the PKA inhibitor H89. The inhibitory effect of IMD1–53 on VSMC calcification, as assessed by calcium content, ALP activity, and calcium deposition, was blocked 592
with CRLR or RAMP3 siRNA knockdown (Figure 8a–c and d–f). Preincubation with H89 for 30 minutes reversed the inhibitory effects of IMD1–53 on calcium content and ALP activity in calcified VSMCs (Figure 9a and b). This finding was further confirmed by Alizarin-red S staining, which showed significantly increased calcified nodules in VSMCs with H89 treatment (Figure 9c). Therefore, IMD1–53 attenuated VSMC calcification by upregulating a-Klotho via the Kidney International (2016) 89, 586–600
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Figure 6 | a-Klotho deficiency attenuated the effect of intermedin (IMD)1–53 inhibiting vascular smooth muscle cell phenotype transition in calcified vascular smooth muscle cells. (a) Western blot analysis of the effect of a-Klotho knockdown on protein levels of smoothelin, calponin, smooth muscle 22a (SM22a), osteopontin (OPN), and osteocalcin (OCN) in calcified vascular smooth muscle cells with IMD1–53 treatment for 6 days and (b–f) semiquantitative analysis. Data are mean SD (n ¼ 3). **P < 0.01 versus control (Con) in scramble (sc) small, interfering RNA (siRNA); ##P < 0.01 versus calcification (Cal) in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Kidney International (2016) 89, 586–600
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Figure 7 | Intermedin (IMD)1–53 augmented the protein level of a-Klotho in calcified vascular smooth muscle cells via the calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)3 receptor complex and cyclic adenosine monophosphate/ protein kinase A signaling pathway. (a) Western blot analysis and semiquantification of the effect of IMD17–47 on a-Klotho protein level in calcified (Cal) vascular smooth muscle cells with IMD1–53 treatment. IMD17–47 is the specific antagonist of the IMD receptor. Data are mean SD (n ¼ 4). **P < 0.01 versus control (Con); #P < 0.05 versus Cal; &P < 0.05 versus CalþIMD. (b and c) Western blot analysis of the effect of calcitonin receptor-like receptor or receptor activity-modifying proteins 3 knockdown on a-Klotho protein level in calcified vascular smooth muscle cells with IMD1–53 treatment and semiquantitative analysis. Data are mean SD (n ¼ 4). **P < 0.01 versus Con in scramble (sc) small, interfering RNA (siRNA); # P < 0.05, ##P < 0.01 versus Cal in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. (d) Western blot analysis of H89 (10 mmol/l, protein kinase A inhibitor) attenuating the effect of IMD1–53 on a-Klotho protein level in calcified vascular smooth muscle cells on day 6 and semiquantitative analysis. Data are mean SD (n ¼ 3). *P < 0.05 versus Con; #P < 0.05 versus Cal; &P < 0.05 versus CalþIMD.
CRLR/RAMP3 receptor complex and cAMP/PKA signaling pathway. DISCUSSION
In this study, rats with 5/6 nephrectomy plus vitamin D3 treatment to induce CKD showed typical vascular calcification along with increased plasma levels of blood urea nitrogen 594
and creatinine, as well as abnormal calcium and phosphorus levels. In addition, endogenous IMD level was decreased in calcified aortas, but the expression of the CRLR/RAMP3 receptor complex was upregulated. We identified significant transcript differences in a number of candidate genes with potential relevance in vascular calcification. An endogenous anticalcification factor, a-Klotho, was detected. a-Klotho Kidney International (2016) 89, 586–600
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Figure 8 | Calcitonin receptor-like receptor (CRLR) or receptor activity-modifying protein (RAMP)3 knockdown blocked the inhibitory effect of intermedin (IMD)1–53 on vascular smooth muscle cell calcification. (a, d) Calcium content and (b, e) alkaline phosphatase (ALP) activity in calcified (Cal) vascular smooth muscle cells treated with CRLR, RAMP3, or scramble (sc) small, interfering RNA (siRNA) with IMD1–53 for 14 days (calcium content) or 3 days (ALP activity), respectively. Data are mean SD (n ¼ 4). *P < 0.05, **P < 0.01 versus control (Con) in scramble; #P < 0.05, ##P < 0.01 versus Cal in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. (c, f) Alizarin-red S staining for vascular smooth muscle cell calcification on day 14. Bars ¼ 200 mm. Original magnification 100. Kidney International (2016) 89, 586–600
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Figure 9 | H89 (10 mmol/l) attenuated the inhibitory effect of intermedin (IMD)1–53 on vascular smooth muscle cell calcification. (a) Calcium content and (b) alkaline phosphatase (ALP) activity in calcified (Cal) vascular smooth muscle cells detected in vitro after incubation with calcifying media for 14 and 3 days, respectively. Data are mean SD (n ¼ 4). **P < 0.01 versus control (Con); #P < 0.05, ##P < 0.01 versus Cal; &P < 0.05, &&P < 0.01 versus CalþIMD. (c) Alizarin-red S staining of calcified vascular smooth muscle cells on day 14. Bars ¼ 200 mm. Original magnification 100.
expression was downregulated by 24.4-fold in CKD rat aortas, so it plays a key role during vascular calcification. Exogenous IMD1–53 administration increased a-Klotho protein level and inhibited vascular calcification in rat CKD aortas. The vasoprotective effects of IMD1–53 on vascular calcification were abolished with a-Klotho siRNA treatment in vitro. Use of CRLR or RAMP3 siRNA or the PKA inhibitor H89 reversed the upregulatory effects of IMD1–53 on a-Klotho protein level in vivo and on inhibiting VSMC 596
calcification in vitro. IMD1–53 inhibited vascular calcification by increasing a-Klotho protein level via the CRLR/ RAMP3 receptor complex and cAMP/PKA signaling (Figure 10). Rat vascular calcification in CKD in this experiment was performed according to a previous study29 with a minor modification. Rat CKD was induced by 5/6 nephrectomy then vitamin D3 injection (1 mg/kg) for 12 weeks. In the animal model of 5/6 nephrectomy, which has been used to study Kidney International (2016) 89, 586–600
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Figure 10 | Schematic representation of the effects of intermedin (IMD)1–53 attenuating vascular calcification by upregulating aLKlotho via calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)3 and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling in rats with chronic kidney disease and partly via improved kidney function. ALP, alkaline phosphatase; OCN, osteocalcin; OPN, osteopontin; SM22a, smooth muscle 22a.
vascular calcification outcomes in CKD,30 the model needs to include excessive vitamin D or dietary phosphorus.31,32 Without the concurrent use of vitamin D, 5/6 nephrectomy rats develop hyperparathyroidism, which is a feature of this model, whereas serum calcium level is minimally altered.33 In the 5/6 nephrectomy model, concurrent administration of vitamin D resulted in a mild increase in calcium level and substantial reduction in parathyroid hormone as compared to CKD animals not receiving concurrent vitamin D3.27,34 Vascular calcification can occur in the tunica intima, tunica media, or both. Intima calcification is seen mostly in the large arteriosclerotic plaques in the larger arteries. Medial calcification (also known as Monckeberg sclerosis) can occur in arteries of any size and is characterized by diffuse mineral deposition along elastic fibers.35 Medial calcification is often observed with CKD, age, and diabetes,1 whereas intimal calcification is observed in atherosclerotic lesions.36 We found vascular medial calcification in CKD, which is consistent with other reports. Coronary artery calcification is also accelerated in CKD, but this occurs as a result of 2 distinct pathologic processes that result in medial and intimal deposition.37 Calcification of the aorta and its major branches are associated with stiffness of the vascular tissue and hypertension, clinical conditions accompanied by increased risk of future cardiovascular events.38 Our previous investigation showed that levels of IMD were reduced in calcified aortic tissues and plasma of rats receiving vitamin D3 plus nicotine.22 In this work, we used the rat CKD model, which has several similarities to calcification in CKD patients, along with increased plasma levels of blood urea nitrogen and creatinine as well as abnormal calcium, phosphorus, and parathyroid hormone levels, which agree with previous results.39 Endogenous IMD level was reduced in our Kidney International (2016) 89, 586–600
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calcified rat CKD aortas, and IMD1–53 administration greatly ameliorated vascular calcification in vivo and in calcified VSMCs in vitro, which agreed with our previous studies.15,22 Kidney damage and dysfunction (plasma blood urea nitrogen and creatinine levels) were lessened in CKD rats with infusion of IMD1–53, so the IMD1–53 effects might have been due, at least in part, to improved kidney function. A number of studies have indicated that calcification inhibitors such as pyrophosphate, osteopontin, osteoprotegerin, and a-Klotho are necessary to prevent soft-tissue calcification.1 The level of a-Klotho, an endogenous anticalcification factor, was found decreased in early-stage CKD.40 a-Klotho is regulated by many factors: inflammation, oxidative stress, tumor necrosis factor, and angiotensin II decrease a-Klotho expression,41–43 whereas some bioactive peptides such as erythropoietin and fibroblast growth factor 2 promote the expression.44,45 CGRP, in the same family as IMD, can upregulate a-Klotho.26 Here, we found that exogenous IMD1–53 administration restored a-Klotho protein expression and retarded calcification in rat CKD aortas in vivo. IMD1–53 could not prevent VSMC calcification with a-Klotho siRNA knockdown, so the inhibitory effects of IMD1–53 on calcification are mediated at least in part by increased a-Klotho expression. a-Klotho functions as a cofactor of fibroblast growth factor 23 receptors and has been reported to cause fibroblast growth factor 23 action and specificity in the kidney. It was found that a-Klotho deficiency induced fibroblast growth factor 23 resistance.28,46 We found that with the upregulation of a-Klotho, plasma phosphorus levels were significantly decreased in CKD rats treatment with IMD1–53, probably due to the improved the fibroblast growth factor 23-a-Klotho axis. The role of fibroblast growth factor 23 in vascular calcification needs to be investigated in the future study. IMD exerts its biological effects by activating the CRLR/ RAMP system,20 and IMD shows selectivity for the CRLR/ RAMP3 receptor complex, with about 1 order of magnitude greater potency at that receptor than the CRLR/RAMP1 or CRLR/RAMP2 complexes have.23 Our previous study detected increased mRNA expression of RAMP2 and 3 in calcified VSMCs.47 Here, we found markedly increased mRNA and protein levels of CRLR and RAMP3 in calcified rat CKD aortas, probably because of higher reactivity. To confirm the specific receptor complex of IMD1–53 during VSMC calcification, we used CRLR and RAMP3 siRNA knockdown in cultured VSMCs. CRLR or RAMP3 knockdown could abolish the effects of IMD1–53 on a-Klotho upregulation and inhibition of VSMC calcification. Therefore, the CRLR/RAMP3 receptor complex is involved in these roles of IMD1–53. Fischer et al.48 reported that after binding to IMD, CRLR undergoes conformational alterations leading to Gs coupling and activation of adenylate cyclase and intracellular cAMP. 49 Our previous study demonstrated that IMD1–53 increased cAMP generation in cardiomyocytes, and H89 could block the effects of IMD on angiotensin II-induced hypertrophy in 597
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neonatal rat ventricular myocytes.24 In the current study, the PKA inhibitor H89 abolished the effect of IMD1–53 on a-Klotho upregulation and inhibition of VSMC calcification. Therefore, IMD1–53 consistently increased a-Klotho protein expression in calcified VSMCs by binding to the CRLR/ RAMP3 receptor complex, then activating PKA signaling to inhibit VSMC calcification. Both CGRP and a-Klotho mRNA expression were reduced in angiotensin II-induced senescent endothelial progenitor cells, whereas exogenous application of CGRP counteracted angiotensin II-induced endothelial progenitor cell senescence by increasing the production of a-Klotho and downregulating the expression of nicotinamide adenine dinucleotide phosphate oxidase and production of reactive oxygen species.26 We also observed that IMD1–53, a member of the CGRP family, upregulated a-Klotho protein level in VSMC calcification after binding to the CRLR/ RAMP3 receptor complex. Numerous studies, including our previous work, have shown that paracrine/autocrine factors are involved in vascular calcification and many agents could modify vascular calcification, including sodium pyrophosphate,50 lanthanum carbonate,51 angiotensin II receptor blocker losartan,52 and the bioactive peptide adrenomedullin.12 IMD has several benefits for inhibiting vascular calcification. First, as an endogenous bioactive peptide, it is extensively distributed throughout the body.23 Also, IMD is more potent than adrenomedullin, which shares the same family with IMD.53 Finally, within a wide range of concentration, IMD has various cardiovascular protective effects, including decreasing blood pressure,53 inhibiting calcification,22 enhancing cardiac contractile function,54 and protecting against schemia/reperfusion injury.21 In summary, we provide evidence that the paracrine/ endocrine factor IMD1–53 attenuates vascular calcification by upregulating a-Klotho level via the CRLR/RAMP3 receptor complex and activating cAMP/PKA signaling and partly by improving kidney function. The vasoactive peptide IMD1–53, an endogenous calcification inhibitor, may open new avenues for preventing vascular calcification in CKD. MATERIALS AND METHODS A detailed Method section and Supplementary Table S1 is available in the online-only supplementary material. Animals and materials Male Sprague-Dawley rats (200 10 g) were from the Animal Center, Peking University Health Science Center (Beijing, China). Vitamin D3, b-glycerophosphate, and Alizarin-red S were from Sigma (St. Louis, MO). Synthetic human IMD1–53 and primary IMD antibody were from Phoenix Pharmaceuticals (Belmont, CA). Primary antibodies for a-Klotho, smoothelin, calponin, SM22a, and osteocalcin were from Abcam (Cambridge, UK) and antibodies for osteopontin, CRLR, RAMP1-3, glyceraldehyde-3-phosphate dehydrogenase, and b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). The antimouse, antigoat, and antirabbit IgG secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). 598
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Rat CKD model All animal care and experimental protocols complied with the Guide for the Care and Use of Laboratory Animals55 and were approved by the Animal Care Committee of Peking University Health Science Center (Beijing, China). Rats were randomly assigned to 3 groups for treatment: control, CKD, and CKDþIMD. Rat CKD was induced by 5/6 nephrectomy via surgical removal of the right kidney and ligation of two-thirds of the arterial supply to the left kidney with silk ligatures; vitamin D3 was administered intramuscularly at 1 mg/kg body weight 3 times a week for 12 weeks as described29 with a minor modification. Antibiotics (penicillin 200,000 U/kg) were given postoperatively for 3 days. IMD1–53 was administered subcutaneously (100 ng/kg/h) in phosphate buffered saline during the last 4 weeks of CKD treatment via an Alzet Mini-osmotic Pump (Alzetw model 2004, DURECT Corp, Cupertino, CA). Evaluation of animal model Twelve weeks after 5/6 nephrectomy, rats were killed and blood was collected for determination of biochemical assay and radioimmunoassay. The aorta was immediately removed and subjected to calcification study or immunoblot analysis. And the detailed methods are shown in the supplementary methods. VSMC culture and VSMC calcification model VSMCs were isolated from the thoracic aortic arteries of SpragueDawley rats (150–180 g) and verified by anti-SM a-actin antibody staining.22 Briefly, after partial removal of external connective tissues, rat thoracic aortas were cut into small pieces (about 2–3 mm each), placed in Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin, and incubated at 37 oC in an incubator containing 95% air and 5% CO2. VSMCs migrating from explants were collected and maintained in growing medium (Dulbecco’s modified Eagle’s medium containing 10 mmol/l sodium pyruvate and 10% fetal bovine serum). VSMCs at passages 5 to 8 were used for all of the experiments. For calcification, confluent VSMCs were incubated in medium containing 2.5 mmol/l Ca2þ (0.7 mmol/l CaCl2 was added into Dulbecco’s modified Eagle’s medium containing 1.8 mmol/l CaCl2) and 5 mmol/l b-glycerophosphate. Each experiment was performed at least 3 times with triplicate cultures used in every experiment, unless stated otherwise. Expression analysis Expression levels of mRNA and protein were evaluated as described in the supplementary methods. Statistical analysis Statistical analyses involved the use of GraphPad Prism 5.00 for Windows (GraphPad Software Inc, San Diego, CA). Comparisons of 2 groups involved unpaired Student t test and more than 2 groups of 1-way analysis of variance followed by Student-Newman-Keuls test. Data are expressed as mean SD. P < 0.05 was considered statistically significant. DISCLOSURES
All the authors declare no competing interests. ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (nos. 91339203, 81270407, and 81170082 to Yong-Fen Qi; Kidney International (2016) 89, 586–600
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no. 31400984 to Jin-Rui Chang; no. 81470557 to Ming-Jiang Xu), Collaborative Innovation Center for Cardiovascular Disorders, the Ministry of Education Foundation for Doctoral Tutors (no. 20110001110012 to Yong-Fen Qi) and the Leading Academic Discipline Project of Beijing Education Bureau.
(n ¼ 3). *P < 0.05, **P < 0.01 versus control (Con); #P < 0.05, ##P < 0.01 versus Cal. Table S1. The forward and reverse PCR primers. Supplementary material is linked to the online version of the paper at www.kidney-international.org.
AUTHOR CONTRIBUTIONS
REFERENCES
JRC and JG designed, performed, and interpreted experiments; YW, YFG, YZ, and JD provided reagents and assisted to design experiments; YLH, WWL, MJX, and JSZ assisted in animal experiments and data analyses; YRY assisted with data analyses; XYL and XJW performed RNA sequence. CST interpreted data, and YFQ designed and interpreted the experimental work and wrote the manuscript.
1. Wu M, Rementer C, Giachelli CM. Vascular calcification: an update on mechanisms and challenges in treatment. Calcif Tissue Int. 2013;93: 365–373. 2. Shroff R, Long DA, Shanahan C. Mechanistic insights into vascular calcification in CKD. J Am Soc Nephrol. 2013;24:179–189. 3. Shanahan CM, Crouthamel MH, Kapustin A, Giachelli CM. Arterial calcification in chronic kidney disease: key roles for calcium and phosphate. Circ Res. 2011;109:697–711. 4. Shao JS, Cai J, Towler DA. Molecular mechanisms of vascular calcification: lessons learned from the aorta. Arterioscler Thromb Vasc Biol. 2006;26: 1423–1430. 5. Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390:45–51. 6. Matsumura Y, Aizawa H, Shiraki-Iida T, et al. Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem Biophys Res Commun. 1998;242:626–630. 7. Shiraki-Iida T, Aizawa H, Matsumura Y, et al. Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett. 1998;424:6–10. 8. Urakawa I, Yamazaki Y, Shimada T, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444:770–774. 9. Maekawa Y, Ohishi M, Ikushima M, et al. Klotho protein diminishes endothelial apoptosis and senescence via a mitogen-activated kinase pathway. Geriatr Gerontol Int. 2011;11:510–516. 10. Hu MC, Shi M, Zhang J, et al. Klotho deficiency causes vascular calcification in chronic kidney disease. J Am Soc Nephrol. 2011;22:124–136. 11. Xie J, Yoon J, An SW, et al. Soluble Klotho protects against uremic cardiomyopathy independently of fibroblast growth factor 23 and phosphate. J Am Soc Nephrol. 2015;26:1150–1160. 12. Zhou YB, Gao Q, Li P, et al. Adrenomedullin attenuates vascular calcification in fructose-induced insulin resistance rats. Acta Physiol (Oxf). 2013;207:437–446. 13. Liang QH, Jiang Y, Zhu X, et al. Ghrelin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the ERK pathway. PLoS One. 2012;7:e33126. 14. Huang Z, Li J, Jiang Z, et al. Effects of adrenomedullin, C-type natriuretic peptide, and parathyroid hormone-related peptide on calcification in cultured rat vascular smooth muscle cells. J Cardiovasc Pharmacol. 2003;42:89–97. 15. Chang JR, Duan XH, Zhang BH, et al. Intermedin1-53 attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase A pathway. Exp Biol Med (Maywood). 2013;238:1136–1146. 16. Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes. J Biol Chem. 2004;279:7264–7274. 17. Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator. FEBS Lett. 2004;556:53–58. 18. Yang JH, Jia YX, Pan CS, et al. Effects of intermedin (1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts. Biochem Biophys Res Commun. 2005;327:713–719. 19. McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature. 1998;393:333–339. 20. Bell D, McDermott BJ. Intermedin (adrenomedullin-2): a novel counterregulatory peptide in the cardiovascular and renal systems. Br J Pharmacol. 2008;153(suppl 1):S247–S262. 21. Teng X, Bian Y, Cai Y, et al. Downregulation of endogenous intermedin augmented myocardial injury in rats ischemic/reperfusion. Horm Metab Res. 2013;45:206–212. 22. Cai Y, Xu MJ, Teng X, et al. Intermedin inhibits vascular calcification by increasing the level of matrix gamma-carboxyglutamic acid protein. Cardiovasc Res. 2010;85:864–873.
SUPPLEMENTARY MATERIAL Detailed methods Figure S1. Plasma biochemistry of chronic kidney disease (CKD) induced in 8-week-old rats following a 2-step surgical procedure for partial renal ablation plus vitamin D3 injection for 12 weeks. (A–E) Assay of blood urea nitrogen (BUN), creatinine, calcium (Ca), and phosphate (Pi) levels in rats with CKD (n ¼ 8). (F) Level of intact parathyroid hormone (PTH) in rat plasma of control (Con) and CKD groups. Data are mean SD (n ¼ 6). **P < 0.01 versus Con. Figure S2. Vascular calcification in chronic kidney disease (CKD) rat aortas. (A) Calcium content and (B) alkaline phosphatase (ALP) activity measured in CKD rat aortas. Data are mean SD (n ¼ 8). * P < 0.05, **P < 0.01 versus control (Con) rats. (C) Von Kossa staining of CKD rat aortas. Bars ¼ 200 mm. Original magnification 100. Figure S3. The effects of intermedin (IMD)1–53 on kidney function in chronic kidney disease (CKD) rats. (A) Hematoxylin and eosin staining showed that kidney damage was lessened in CKD rats by infusion of IMD1–53. Bars in larger images ¼ 200 mm; bars in inset images ¼ 50 mm. Original magnification 100. (B–D) Assay of plasma blood urea nitrogen (BUN), creatinine, and phosphate (Pi) levels in CKD rats with use of IMD1–53. Data are mean SD (n ¼ 6). **P < 0.01 versus control (Con); ##P < 0.01 versus CKD. Figure S4. Intermedin (IMD)1–53 restored a-Klotho levels in aorta, kidney, and plasma in chronic kidney disease (CKD) rats. Immunohistochemistry staining of a-Klotho protein expression in (A) aorta and (B) kidney of CKD rats. Bars in larger images ¼ 200 mm; bars in inset images ¼ 50 mm. Original magnification 100. The arrows point to positive staining in (A) and (B). (C) Plasma levels of a-Klotho in CKD rats with infusion of IMD1-53. Data are mean SD (n ¼ 8). *P < 0.05 versus control (Con); ##P < 0.01 versus CKD. Figure S5. a-Klotho was knocked down by small, interfering RNA (siRNA). (A) Real-time polymerase chain reaction analysis of a-Klotho mRNA level with siRNA knockdown. (B) Western blot analysis of a-Klotho protein level with siRNA knockdown and (C) semiquantitative analysis. Data are mean SD (n ¼ 3). **P < 0.01 versus scramble siRNA. Figure S6. Calcitonin receptor-like receptor (CRLR) or receptor activity-modifying protein (RAMP)3 knockdown by small, interfering RNA (siRNA). Real-time polymerase chain reaction analysis of mRNA levels of (A) CRLR and (D) RAMP3 with siRNA knockdown. Western blot analysis of protein levels of (B) CRLR and (E) RAMP3 with siRNA knockdown and (C and F) semiquantitative analysis. Data are mean SD (n ¼ 3). **P < 0.01 versus scramble siRNA. Figure S7. Intermedin (IMD)1–53 upregulated a-Klotho protein expression in calcified (Cal) vascular smooth muscle cells not through PI3K/Akt or extracellular signal–regulated kinase signaling pathway. (A and B) Western blot analysis of a-Klotho protein level (b-actin was a loading control) and semiquantitative analysis. Data are mean SD Kidney International (2016) 89, 586–600
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23. Hong Y, Hay DL, Quirion R, Poyner DR. The pharmacology of adrenomedullin 2/intermedin. Br J Pharmacol. 2012;166:110–120. 24. Yang JH, Ma CG, Cai Y, et al. Effect of intermedin1-53 on angiotensin II-induced hypertrophy in neonatal rat ventricular myocytes. J Cardiovasc Pharmacol. 2010;56:45–52. 25. Dai XY, Cai Y, Mao DD, et al. Increased stability of phosphatase and tensin homolog by intermedin leading to scavenger receptor A inhibition of macrophages reduces atherosclerosis in apolipoprotein E-deficient mice. J Mol Cell Cardiol. 2012;53:509–520. 26. Zhou Z, Hu CP, Wang CJ, et al. Calcitonin gene-related peptide inhibits angiotensin II-induced endothelial progenitor cells senescence through up-regulation of klotho expression. Atherosclerosis. 2010;213:92–101. 27. Wu-Wong JR, Noonan W, Ma J, et al. Role of phosphorus and vitamin D analogs in the pathogenesis of vascular calcification. J Pharmacol Exp Ther. 2006;318:90–98. 28. Lim K, Lu TS, Molostvov G, et al. Vascular Klotho deficiency potentiates the development of human artery calcification and mediates resistance to fibroblast growth factor 23. Circulation. 2012;125:2243–2255. 29. Tamura K, Suzuki Y, Matsushita M, et al. Prevention of aortic calcification by etidronate in the renal failure rat model. Eur J Pharmacol. 2007;558: 159–166. 30. Shobeiri N, Adams MA, Holden RM. Vascular calcification in animal models of CKD: a review. Am J Nephrol. 2010;31:471–481. 31. Cardús A, Panizo S, Parisi E, et al. Differential effects of vitamin D analogs on vascular calcification. J Bone Miner Res. 2007;22:860–866. 32. Gauthier-Bastien A, Ung RV, Larivière R, et al. Vascular remodeling and media calcification increases arterial stiffness in chronic kidney disease. Clin Exp Hypertens. 2014;36:173–180. 33. Shalhoub V, Shatzen EM, Ward SC, et al. FGF23 neutralization improves chronic kidney disease-associated hyperparathyroidism yet increases mortality. J Clin Invest. 2012;122:2543–2553. 34. Mendoza FJ, Lopez I, Montes de Oca A, et al. Metabolic acidosis inhibits soft tissue calcification in uremic rats. Kidney Int. 2008;73:407–414. 35. Vervloet MG, Adema AY, Larsson TE, Massy ZA. The role of klotho on vascular calcification and endothelial function in chronic kidney disease. Semin Nephrol. 2014;34:578–585. 36. Callegari A, Coons ML, Ricks JL, et al. Bone marrow- or vessel wallderived osteoprotegerin is sufficient to reduce atherosclerotic lesion size and vascular calcification. Arterioscler Thromb Vasc Biol. 2013;33: 2491–2500. 37. Bashir A, Moody WE, Edwards NC, et al. Coronary artery calcium assessment in CKD: utility in cardiovascular disease risk assessment and treatment? Am J Kidney Dis. 2015;65:937–948. 38. Vlachopoulos C, Aznaouridis K, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol. 2010;55:1318–1327. 39. Krog M, Ejerblad S, Eriksson I, Johansson H. Arterial calcifications in uraemic rats treated with 1-alpha-hydroxycholecalciferol and parathyroidectomy. Scand J Urol Nephrol. 1984;18:227–239. 40. Shimamura Y, Hamada K, Inoue K, et al. Serum levels of soluble secreted a-Klotho are decreased in the early stages of chronic kidney disease, making it a probable novel biomarker for early diagnosis. Clin Exp Nephrol. 2012;16:722–729.
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41. Thurston RD, Larmonier CB, Majewski PM, et al. Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis. Gastroenterology. 2010;138:1384–1394, 1394.e1-2. 42. Mitobe M, Yoshida T, Sugiura H, et al. Oxidative stress decreases klotho expression in a mouse kidney cell line. Nephron Exp Nephrol. 2005;101: e67–e74. 43. Yoon HE, Ghee JY, Piao S, et al. Angiotensin II blockade upregulates the expression of Klotho the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy. Nephrol Dial Transplant. 2011;26: 800–813. 44. van Rijt WG, Nieuwenhuijs-Moeke GJ, van Goor H, et al. ARA290, a nonerythropoietic EPO derivative, attenuates renal ischemia/reperfusion injury. J Transl Med. 2013;11:9. 45. Xiao L, Naganawa T, Lorenzo J, et al. Nuclear isoforms of fibroblast growth factor 2 are novel inducers of hypophosphatemia via modulation of FGF23 and Klotho. J Biol Chem. 2010;285:2834–2846. 46. Galitzer H, Ben-Dov IZ, Silver J, Naveh-Many T. Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease. Kidney Int. 2010;77:211–218. 47. Pan CS, Qi YF, Wang SH, et al. Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells. Regul Pept. 2004;120:77–83. 48. Fischer JA, Muff R, Born W. Functional relevance of G-protein-coupledreceptor- associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs). Biochem Soc Trans. 2002;30:455–460. 49. Mittra S, Bourreau JP. Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes. Am J Physiol Heart Circ Physiol. 2006;290: H1842–H1847. 50. Riser BL, Barreto FC, Rezg R, et al. Daily peritoneal administration of sodium pyrophosphate in a dialysis solution prevents the development of vascular calcification in a mouse model of uraemia. Nephrol Dial Transplant. 2011;26:3349–3357. 51. Wang XH, Zhang X, Mu CJ, et al. Effects of lanthanum carbonate on vascular calcification in elderly maintenance hemodialysis patients. J Huazhong Univ Sci Technolog Med Sci. 2015;35:508–513. 52. Li M, Wu P, Shao J, et al. Losartan inhibits vascular calcification by suppressing the BMP2 and Runx2 expression in rats in vivo [e-pub ahead of print]. Cardiovasc Toxicol. 2015 Apr 21. Available at: http://www.ncbi. nlm.nih.gov/pubmed/?term¼Losartanþinhibitsþvascularþcalcification þbyþsuppressingþtheþBMP2þandþRunx2þexpressionþinþratsþ inþvivo. Accessed January 20, 2016. 53. Yuan Y, Wang X, Zeng Q, et al. Effects of continuous intermedin infusion on blood pressure and hemodynamic function in spontaneously hypertensive rats. J Geriatr Cardiol. 2012;9:17–27. 54. Dong F, Taylor MM, Samson WK, Ren J. Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes. J Appl Physiol (1985). 2006;101:778–784. 55. Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, Division on Earth and Life Sciences, National Research Council of the National Academies. Guide for the Care and Use of Laboratory Animals. 8th edition. Washington, D.C.: The National Academies Press; 2011.
Kidney International (2016) 89, 586–600
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Intermedin1–53 attenuates vascular calcification in rats with chronic kidney disease by see commentary on page 534 upregulation of a-Klotho Jin Rui Chang1,2,3,4,8, Jun Guo1,2,8, Yue Wang5, Yue Long Hou1,2,3, Wei Wei Lu1,2,6, Jin Sheng Zhang1,2,6, Yan Rong Yu6, Ming Jiang Xu3, Xiu Ying Liu7, Xiu Jie Wang7, You Fei Guan3, Yi Zhu3, Jie Du1,2, Chao Shu Tang1,2,3 and Yong Fen Qi1,2,3,6 1
The Key Laboratory of Remodeling-related Cardiovascular Diseases, Capital Medical University, Ministry of Education, Beijing, China; Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing Anzhen Hospital Affiliated with the Capital Medical University, Beijing, China; 3Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University Health Science Center, Beijing, China; 4Insititute of Basic Medicine Science, Xi’an Medical University, Xi’an, China; 5Renal Department, Peking University Third Hospital, Beijing, China; 6Department of Pathogen Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; and 7Key Laboratory of Genetic Network Biology, Collaborative Innovation Center of Genetics and Development, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 2
Deficiency in a-Klotho is involved in the pathogenesis of vascular calcification. Since intermedin (IMD)1–53 (a calcitonin/calcitonin gene-related peptide) protects against vascular calcification, we studied whether IMD1–53 inhibits vascular calcification by upregulating a-Klotho. A rat model of chronic kidney disease (CKD) with vascular calcification induced by the 5/6 nephrectomy plus vitamin D3 was used for study. The aortas of rats with CKD showed reduced IMD content but an increase of its receptor, calcitonin receptor-like receptor, and its receptor modifier, receptor activity-modifying protein 3. IMD1–53 treatment reduced vascular calcification. The expression of a-Klotho was greatly decreased in the aortas of rats with CKD but increased in the aortas of IMD1–53-treated rats with CKD. In vitro, IMD1–53 increased a-Klotho protein level in calcified vascular smooth muscle cells. a-Klotho knockdown blocked the inhibitory effect of IMD1–53 on vascular smooth muscle cell calcification and their transformation into osteoblastlike cells. The effect of IMD1–53 to upregulate a-Klotho and inhibit vascular smooth muscle cell calcification was abolished by knockdown of its receptor or its modifier protein, or treatment with the protein kinase A inhibitor H89. Thus, IMD1–53 may attenuate vascular calcification by upregulating a-Klotho via the calcitonin receptor/ modifying protein complex and protein kinase A signaling. Kidney International (2016) 89, 586–600; http://dx.doi.org/10.1016/ j.kint.2015.12.029 KEYWORDS: a-Klotho; calcitonin receptor-like receptor; chronic kidney disease; intermedin1-53; vascular calcification ª 2016 International Society of Nephrology
Correspondence: Yong Fen Qi, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing 100191, China. E-mail: yongfenqi@ 163.com 8
JRC and JG contributed equally to this work.
Received 4 March 2015; revised 18 November 2015; accepted 3 December 2015; published online 28 January 2016 586
V
ascular calcification, a common feature of chronic kidney disease (CKD), diabetes, hypertension, and aging, is an independent risk factor for cardiovascular disease.1,2 It predicts mortality in patients, especially those with endstage renal disease.2 Vascular calcification has emerged as a highly regulated process that involves a phenotype transition of vascular smooth muscle cells (VSMCs) transforming into osteoblast-like cells.3 This transition is accompanied by a loss of VSMC contractile markers, including smoothelin, calponin, and smooth muscle 22a, and increased expression of osteogenic-profile genes, including alkaline phosphatase (ALP), osteopontin, osteocalcin, bone morphogenetic protein 2, core binding factor a 1, and osterix.4 Recently, loss of calcification inhibitors such as pyrophosphate, g-carboxyglutamic acid protein, and antiaging and anticalcification factor a-Klotho was found to greatly promote vascular calcification.1,2 The a-Klotho gene, a gene discovered in 19975 while coding for an antiaging protein, has high homology among humans, mice, and rats6,7 and is expressed primarily in renal tubules, the choroid plexus in the brain, and the parathyroid gland. The human a-Klotho gene spans about 50 kilobase pairs on chromosome 13q12 and consists of 5 exons;6 it encodes a single-pass transmembrane protein of 1012 amino acids acting as a coreceptor for fibroblast growth factor 23, the phosphaturic and vitamin D-regulating hormone.8 In addition to the membrane a-Klotho, secreted a-Klotho exists in circulation, generated from the a-Klotho gene via alternative RNA splicing and containing 3 exons or ectodomain shedding of membrane a-Klotho from the cell surface. This circulating a-Klotho acts as a humoral factor with a multitude of functions such as antiapoptosis and antisenescence effects.9 Mice lacking the a-Klotho gene develop a phenotype similar to premature human aging, including vascular calcification, endothelial dysfunction, and shortened lifespan.5 Several experimental studies showed that a-Klotho attenuates vascular calcification,10 improves endothelial dysfunction,9 and prevents cardiomyopathy.11 a-Klotho–haplo-insufficient mice with CKD show severe calcification, and transgenic mice overexpressing Kidney International (2016) 89, 586–600
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a-Klotho show less calcification,10 so a-Klotho may have a
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showed increased calcium content and ALP activity in aortas (Supplementary Figure S2A and B). Increased calciumphosphate salt deposition in CKD aortas was confirmed by von Kossa staining (Supplementary Figure S2C). The content of plasma IMD was decreased by 51.8% (P < 0.01) in CKD rats as compared with control rats (Figure 1a). The mRNA and protein levels of IMD were lower, by 51.5% and 45.8% (both P < 0.05), respectively, in calcified aortas of CKD rats than in control rats (Figure 1b–d), and the mRNA levels of CRLR and RAMP1, 2, and 3 were increased in calcified CKD aortas, by 5.88-fold (P < 0.05), 0.70-fold (P < 0.01), 1.11-fold (P < 0.05), and 7.90-fold (P < 0.05), respectively (Figure 1e). The protein levels of CRLR and RAMP3 were higher, by 4.70- and 2.21-fold (both P < 0.01) (Figure 1f and g), with no significant difference in protein levels of RAMP1 and 2 in CKD rats as compared with control rats.
pivotal role in vascular calcification. Recently, numerous studies, including our previous work,12 revealed that paracrine/autocrine factors are involved in vascular calcification. Most of these factors, vasoactive peptides such as adrenomedullin,12 ghrelin,13 parathyroid hormone-related peptide,14 and intermedin (IMD),15 are endogenous factors that inhibit vascular calcification. IMD, namely adrenomedullin 2, discovered in 2004, is a member of the calcitonin gene-related peptide (CGRP) family, which includes adrenomedullin, CGRP, calcitonin, and amylin.16,17 Human IMD gene encodes a prepropeptide of 148 amino acids with a signal peptide for secretion at the N terminus. IMD1-53 can be generated from prepro-IMD by proteolytic cleavage at Arg93-Arg94.18 The biological effects of IMD are mediated by calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs).16 RAMPs consist of 3 subtypes and interact with CRLR to modify its function: CRLR with RAMP1 is a CGRP receptor and with RAMP2 or 3 is an adrenomedullin or IMD receptor.19 IMD acts on CRLR/RAMP receptor complexes, which are rich in vasculature.20 The expression profile of IMD and its receptors is disturbed in different cardiovascular diseases.21,22 IMD is considered a potential endogenous protective peptide of the cardiovascular system by activating the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway.23 Several studies showed that IMD1–53 protects against myocardial ischemia-reperfusion, strengthens the contractility of the left ventricle, and inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes.18,24 Administration of IMD1–53 dilates coronary arteries and enhances coronary flow in isolated perfused rat hearts.18 We and others have reported that the formation of atherosclerotic lesions in apolipoprotein E/ mice25 and vascular calcification in rats could be inhibited by IMD1–53.15,22 Our previous studies showed downregulated levels of IMD in calcified aortas in vivo and in calcified VSMCs and that vascular calcification was alleviated with IMD1–53 treatment.22 However, the mechanism of IMD inhibiting vascular calcification is still unclear. CGRP, in the same family as IMD, was found to upregulate the expression of a-Klotho in angiotensin II-induced endothelial progenitor cells.26 In the present study, we investigated whether IMD1–53 attenuates vascular calcification by upregulating a-Klotho.
In rat CKD aortas, IMD1–53 treatment significantly reduced calcium content and ALP activity, by 75.7% (P < 0.01) and 60.1% (P < 0.01), respectively (Figure 2a and b). Hematoxylin and eosin staining revealed disordered elastic fibers in calcified CKD aortas; however, IMD1–53 treatment improved the vascular structure (Figure 2c). Alizarin-red S staining and von Kossa staining showed decreased calcium-phosphate salt deposition in IMD1–53-treated vascular calcification aortas (Figure 2c). As well, kidney damage and dysfunction were reduced in CKD rats with IMD1–53 treatment (Supplementary Figure S3). The phenotypic transition of VSMCs is an important feature during vascular calcification and involves decreased expression of SMC lineage markers and induced expression of osteoblast markers.4 We found reduced protein levels of the VSMC phenotype markers smoothelin and calponin in calcified rat CKD aortas (Figure 3a–c), which were increased by 2.52- and 6.17-fold (both P < 0.01) with IMD1–53 treatment (Figure 3a–c). The increased protein levels of the osteoblastlike cell markers osteopontin and osteocalcin in calcified CKD aortas were decreased significantly by 40.2% (P < 0.01) and 44.4% (P < 0.01), respectively, with IMD1–53 treatment (Figure 3a, d, and e). Furthermore, the upregulated mRNA levels of osteogenic markers core binding factor a 1, osterix, osteocalcin, and bone morphogenetic protein 2 in CKD aortas were downregulated with IMD1–53 treatment (Figure 3f–i).
RESULTS Level of IMD and its receptors in calcified CKD rat aortas
IMD1–53 inhibited VSMC calcification by increasing a-Klotho protein level
First, we assessed renal function and vascular calcification in rats. In rats with 5/6 nephrectomy plus vitamin D3 treatment (CKD group), compared with control rats, blood urea nitrogen and creatinine levels were significantly increased (Supplementary Figure S1A and B), as were plasma calcium and phosphorus levels (Supplementary Figure S1C–E). However, parathyroid hormone level was decreased (Supplementary Figure S1F), which was consistent with a previous report.27 Compared with control rats, CKD rats
whether IMD1–53 could inhibit vascular calcification by upregulating a-Klotho. First, we used RNA sequencing of normal and CKD rat aortas. Our reads were uniquely mapped to 14,072 genes; 3715 differentially expressed genes were obtained ($2-fold change in expression), including 1017 downregulated and 2698 upregulated genes in CKD rat aortas. The expression of a-Klotho was downregulated by
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IMD1–53 treatment attenuated vascular calcification in rat CKD aortas
a-Klotho deficiency results in vascular calcification,28 and a-Klotho can be regulated by CGRP.26 We investigated
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Figure 1 | Levels of intermedin (IMD) and its receptors in plasma and aortas of the chronic kidney disease (CKD) rat model. (a) Radioimmunoassay of plasma IMD in CKD rats (n ¼ 8). (b) Real-time polymerase chain reaction quantification of IMD mRNA level. (c) Western blot analysis of IMD (b-actin was a loading control) and (d) semiquantitative analysis. Data are mean SD (n ¼ 4). *P < 0.05; **P < 0.01 versus control (Con). (e) Real-time polymerase chain reaction quantification of mRNA levels of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP)1 to 3. (f) Western blot analysis of protein levels of CRLR, RAMP1 to 3 and (g) semiquantitative analysis. Data are mean SD (n ¼ 4). *P < 0.05, **P < 0.01 versus Con.
24.4-fold in CKD rat aortas (reads per kilobase per million: control group ¼ 0.1025, CKD group ¼ 0.0042). Then, we detected changes in a-Klotho expression in calcified CKD rat plasma and aortas with or without IMD1–53 treatment. a-Klotho protein level was decreased, by 19.5% (P < 0.01) and 56.3% (P < 0.01), in CKD plasma and aortas, respectively, as 588
compared with these levels in control rats (Figure 4a–c). In vitro, a-Klotho protein level was decreased in calcified VSMCs on day 3 and further reduced with calcification time (Figure 4d and e). IMD1–53 treatment significantly reversed the reduced a-Klotho protein level in calcified aortas (Figure 4f and g, Supplementary Figure S4A), kidney, and plasma in vivo Kidney International (2016) 89, 586–600
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Figure 2 | Intermedin (IMD)1–53 administration alleviated calcification in chronic kidney disease (CKD) rat aortas. Effect of IMD1–53 treatment on (a) calcium content (n ¼ 9) and (b) alkaline phosphatase (ALP) activity (n ¼ 7). Data are mean SD. **P < 0.01 versus control (Con); ##P < 0.01 versus CKD. (c) Hematoxylin and eosin (HE) staining of disordered elastic fibers in CKD rat aortas and with IMD1-53 treatment. Alizarin-red S and von Kossa staining of vascular calcium deposition. Bars ¼ 100 mm. Original magnification 200.
(Supplementary Figure S4B and C). The reduced a-Klotho protein expression in calcified VSMCs was restored by IMD1–53 (Figure 4h–k). To investigate the role of a-Klotho in IMD1–53 inhibiting calcification, we knocked down a-Klotho with its small, interfering RNA (siRNA). In cultured VSMCs, the mRNA and Kidney International (2016) 89, 586–600
protein levels of a-Klotho were greatly decreased with a-Klotho siRNA knockdown (Supplementary Figure S5). In calcified VSMCs, calcium content and ALP activity were significantly decreased with IMD1–53 treatment, which was eliminated with a-Klotho siRNA knockdown (Figure 5a and b). The observation was further verified by Alizarin-red 589
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JR Chang et al.: IMD1–53 inhibits VC by upregulating a-Klotho in CKD
Figure 3 | Intermedin (IMD)1–53 inhibited vascular smooth muscle cell phenotype transition in calcified chronic kidney disease (CKD) rat aortas. (a) Western blot analysis of effect of IMD1-53 on protein levels of smoothelin, calponin, osteopontin (OPN), and osteocalcin (OCN) and (b–e) semiquantitative analysis. (f–i) Quantitative real-time polymerase chain reaction analysis of the effect of IMD1-53 on mRNA levels of core binding factor a 1 (Cbfa-1), osterix, OCN, and bone morphogenetic protein 2 (BMP2). Data are mean SD (n ¼ 3). *P < 0.05, ** P < 0.01 versus control (Con); #P < 0.05, ##P < 0.01 versus CKD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
S staining, showing reduced calcification of VSMCs with IMD1–53 treatment, which was reversed with a-Klotho knockdown (Figure 5c). The levels of the contractile markers smoothelin, calponin, and smooth muscle 22a were increased with IMD1–53 treatment in scramble siRNA-treated calcified VSMCs but significantly decreased with a-Klotho siRNA knockdown (Figure 6a–d). The levels of osteoblast markers osteopontin and osteocalcin were decreased with IMD1–53 treatment in scramble siRNA-treated calcified VSMCs but significantly increased with a-Klotho siRNA knockdown (Figure 6a, e, and f). Thus, preventing reduced a-Klotho protein level is essential for IMD1–53 to inhibit VSMC calcification. IMD1–53 upregulated the protein level of a-Klotho and inhibited VSMC calcification via the CRLR/RAMP3 receptor complex and cAMP/PKA signaling
IMD exerts its biological action via CRLR/RAMP receptor complexes.16 Hence, we examined the expression of CRLR/RAMPs in calcified rat CKD aortas. The expression of the CRLR/RAMP3 receptor complex was significantly 590
increased in CKD aortas (Figure 1). To elucidate whether IMD1–53 increased a-Klotho level via CRLR/RAMP3, we used IMD17–47, an IMD receptor antagonist, or knocked-down CRLR or RAMP3 with siRNA in VSMCs. VSMCs were preincubated with IMD17–47 (106 mol/l) for 30 minutes, which reversed the inhibitory effects of IMD1–53 on calcificationdownregulated a-Klotho protein level (Figure 7a). Then, we knocked down CRLR or RMAP3 in cultured VSMCs, verified at both the mRNA and protein levels (Supplementary Figure S6). The upregulatory effect of IMD1–53 on a-Klotho protein level was abolished with CRLR or RAMP3 siRNA knockdown (Figure 7b and c), with levels decreased by 72.2% (P < 0.01) and 48.4% (P < 0.05), respectively. To investigate the signaling pathway by which IMD1–53 increases a-Klotho level in calcified VSMCs, we preincubated VSMCs with the PKA inhibitor H89, PI3K inhibitor LY294002, or extracellular signal–regulated kinase signaling inhibitor PD98059. Preincubation with H89 for 30 minutes inhibited the upregulatory effect of IMD1–53 on a-Klotho protein level (Figure 7d), but LY294002 or PD98059 had no effect (Supplementary Figure S7). Thus, the CRLR/RAMP3 Kidney International (2016) 89, 586–600
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Figure 4 | Level of a-Klotho in chronic kidney disease (CKD) rat aortas in vivo and calcified (Cal) vascular smooth muscle cells in vitro with or without intermedin (IMD)1–53 treatment. (a) Plasma levels of a-Klotho in normal and CKD rats (n ¼ 8). (b, d) Western blot analysis of protein level of a-Klotho and (c, e) semiquantitative analysis. b-actin was a loading control (Con). (f, h, and j) Western blot analysis and (g, i, and k) semiquantification of the effect of IMD1–53 on a-Klotho protein level in (f, g) calcified aortas in vivo or in (h-k) calcified vascular smooth muscle cells in vitro. IMD-7 and IMD-9 represent the concentration of the used IMD1–53 10-7 and 10-9 mol/l, respectively. Data are mean SD (n ¼ 4). *P < 0.05, **P < 0.01 versus Con; #P < 0.05, ##P < 0.01 versus CKD or Cal. d, days. Kidney International (2016) 89, 586–600
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Figure 5 | Deficiency of a-Klotho attenuated the inhibitory effect of intermedin (IMD)1–53 on vascular smooth muscle cell calcification (Cal). Cultured vascular smooth muscle cells transfected with a-Klotho small, interfering (siRNA) or scramble (sc) siRNA were treated with calcifying media plus IMD1-53 for 3 days (for alkaline phosphatase [ALP] activity assay) or 14 days (for calcium content assay and Alizarin-red S staining). a-Klotho knockdown blocked the (a) IMD1–53 inhibitory effect on calcium content (n ¼ 3) and (b) ALP activity (n ¼ 5) in calcified vascular smooth muscle cells. Data are mean SD. *P < 0.05, **P < 0.01 versus control (Con) in scramble; #P < 0.05, ##P < 0.01 versus Cal in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. (c) Alizarin-red S staining of vascular smooth muscle cell calcification. Bars ¼ 200 mm. Original magnification 100.
receptor complex and cAMP/PKA signaling were essential for IMD1–53 upregulating a-Klotho. To determine whether IMD1–53 inhibited vascular calcification via the CRLR/RAMP3 receptor complex and cAMP/ PKA signaling, we used siRNA knockdown of CRLR or RAMP3 or the PKA inhibitor H89. The inhibitory effect of IMD1–53 on VSMC calcification, as assessed by calcium content, ALP activity, and calcium deposition, was blocked 592
with CRLR or RAMP3 siRNA knockdown (Figure 8a–c and d–f). Preincubation with H89 for 30 minutes reversed the inhibitory effects of IMD1–53 on calcium content and ALP activity in calcified VSMCs (Figure 9a and b). This finding was further confirmed by Alizarin-red S staining, which showed significantly increased calcified nodules in VSMCs with H89 treatment (Figure 9c). Therefore, IMD1–53 attenuated VSMC calcification by upregulating a-Klotho via the Kidney International (2016) 89, 586–600
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Figure 6 | a-Klotho deficiency attenuated the effect of intermedin (IMD)1–53 inhibiting vascular smooth muscle cell phenotype transition in calcified vascular smooth muscle cells. (a) Western blot analysis of the effect of a-Klotho knockdown on protein levels of smoothelin, calponin, smooth muscle 22a (SM22a), osteopontin (OPN), and osteocalcin (OCN) in calcified vascular smooth muscle cells with IMD1–53 treatment for 6 days and (b–f) semiquantitative analysis. Data are mean SD (n ¼ 3). **P < 0.01 versus control (Con) in scramble (sc) small, interfering RNA (siRNA); ##P < 0.01 versus calcification (Cal) in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Kidney International (2016) 89, 586–600
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Figure 7 | Intermedin (IMD)1–53 augmented the protein level of a-Klotho in calcified vascular smooth muscle cells via the calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)3 receptor complex and cyclic adenosine monophosphate/ protein kinase A signaling pathway. (a) Western blot analysis and semiquantification of the effect of IMD17–47 on a-Klotho protein level in calcified (Cal) vascular smooth muscle cells with IMD1–53 treatment. IMD17–47 is the specific antagonist of the IMD receptor. Data are mean SD (n ¼ 4). **P < 0.01 versus control (Con); #P < 0.05 versus Cal; &P < 0.05 versus CalþIMD. (b and c) Western blot analysis of the effect of calcitonin receptor-like receptor or receptor activity-modifying proteins 3 knockdown on a-Klotho protein level in calcified vascular smooth muscle cells with IMD1–53 treatment and semiquantitative analysis. Data are mean SD (n ¼ 4). **P < 0.01 versus Con in scramble (sc) small, interfering RNA (siRNA); # P < 0.05, ##P < 0.01 versus Cal in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. (d) Western blot analysis of H89 (10 mmol/l, protein kinase A inhibitor) attenuating the effect of IMD1–53 on a-Klotho protein level in calcified vascular smooth muscle cells on day 6 and semiquantitative analysis. Data are mean SD (n ¼ 3). *P < 0.05 versus Con; #P < 0.05 versus Cal; &P < 0.05 versus CalþIMD.
CRLR/RAMP3 receptor complex and cAMP/PKA signaling pathway. DISCUSSION
In this study, rats with 5/6 nephrectomy plus vitamin D3 treatment to induce CKD showed typical vascular calcification along with increased plasma levels of blood urea nitrogen 594
and creatinine, as well as abnormal calcium and phosphorus levels. In addition, endogenous IMD level was decreased in calcified aortas, but the expression of the CRLR/RAMP3 receptor complex was upregulated. We identified significant transcript differences in a number of candidate genes with potential relevance in vascular calcification. An endogenous anticalcification factor, a-Klotho, was detected. a-Klotho Kidney International (2016) 89, 586–600
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Figure 8 | Calcitonin receptor-like receptor (CRLR) or receptor activity-modifying protein (RAMP)3 knockdown blocked the inhibitory effect of intermedin (IMD)1–53 on vascular smooth muscle cell calcification. (a, d) Calcium content and (b, e) alkaline phosphatase (ALP) activity in calcified (Cal) vascular smooth muscle cells treated with CRLR, RAMP3, or scramble (sc) small, interfering RNA (siRNA) with IMD1–53 for 14 days (calcium content) or 3 days (ALP activity), respectively. Data are mean SD (n ¼ 4). *P < 0.05, **P < 0.01 versus control (Con) in scramble; #P < 0.05, ##P < 0.01 versus Cal in scramble; &P < 0.05, &&P < 0.01 versus CalþIMD in scramble. (c, f) Alizarin-red S staining for vascular smooth muscle cell calcification on day 14. Bars ¼ 200 mm. Original magnification 100. Kidney International (2016) 89, 586–600
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Figure 9 | H89 (10 mmol/l) attenuated the inhibitory effect of intermedin (IMD)1–53 on vascular smooth muscle cell calcification. (a) Calcium content and (b) alkaline phosphatase (ALP) activity in calcified (Cal) vascular smooth muscle cells detected in vitro after incubation with calcifying media for 14 and 3 days, respectively. Data are mean SD (n ¼ 4). **P < 0.01 versus control (Con); #P < 0.05, ##P < 0.01 versus Cal; &P < 0.05, &&P < 0.01 versus CalþIMD. (c) Alizarin-red S staining of calcified vascular smooth muscle cells on day 14. Bars ¼ 200 mm. Original magnification 100.
expression was downregulated by 24.4-fold in CKD rat aortas, so it plays a key role during vascular calcification. Exogenous IMD1–53 administration increased a-Klotho protein level and inhibited vascular calcification in rat CKD aortas. The vasoprotective effects of IMD1–53 on vascular calcification were abolished with a-Klotho siRNA treatment in vitro. Use of CRLR or RAMP3 siRNA or the PKA inhibitor H89 reversed the upregulatory effects of IMD1–53 on a-Klotho protein level in vivo and on inhibiting VSMC 596
calcification in vitro. IMD1–53 inhibited vascular calcification by increasing a-Klotho protein level via the CRLR/ RAMP3 receptor complex and cAMP/PKA signaling (Figure 10). Rat vascular calcification in CKD in this experiment was performed according to a previous study29 with a minor modification. Rat CKD was induced by 5/6 nephrectomy then vitamin D3 injection (1 mg/kg) for 12 weeks. In the animal model of 5/6 nephrectomy, which has been used to study Kidney International (2016) 89, 586–600
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Figure 10 | Schematic representation of the effects of intermedin (IMD)1–53 attenuating vascular calcification by upregulating aLKlotho via calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)3 and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling in rats with chronic kidney disease and partly via improved kidney function. ALP, alkaline phosphatase; OCN, osteocalcin; OPN, osteopontin; SM22a, smooth muscle 22a.
vascular calcification outcomes in CKD,30 the model needs to include excessive vitamin D or dietary phosphorus.31,32 Without the concurrent use of vitamin D, 5/6 nephrectomy rats develop hyperparathyroidism, which is a feature of this model, whereas serum calcium level is minimally altered.33 In the 5/6 nephrectomy model, concurrent administration of vitamin D resulted in a mild increase in calcium level and substantial reduction in parathyroid hormone as compared to CKD animals not receiving concurrent vitamin D3.27,34 Vascular calcification can occur in the tunica intima, tunica media, or both. Intima calcification is seen mostly in the large arteriosclerotic plaques in the larger arteries. Medial calcification (also known as Monckeberg sclerosis) can occur in arteries of any size and is characterized by diffuse mineral deposition along elastic fibers.35 Medial calcification is often observed with CKD, age, and diabetes,1 whereas intimal calcification is observed in atherosclerotic lesions.36 We found vascular medial calcification in CKD, which is consistent with other reports. Coronary artery calcification is also accelerated in CKD, but this occurs as a result of 2 distinct pathologic processes that result in medial and intimal deposition.37 Calcification of the aorta and its major branches are associated with stiffness of the vascular tissue and hypertension, clinical conditions accompanied by increased risk of future cardiovascular events.38 Our previous investigation showed that levels of IMD were reduced in calcified aortic tissues and plasma of rats receiving vitamin D3 plus nicotine.22 In this work, we used the rat CKD model, which has several similarities to calcification in CKD patients, along with increased plasma levels of blood urea nitrogen and creatinine as well as abnormal calcium, phosphorus, and parathyroid hormone levels, which agree with previous results.39 Endogenous IMD level was reduced in our Kidney International (2016) 89, 586–600
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calcified rat CKD aortas, and IMD1–53 administration greatly ameliorated vascular calcification in vivo and in calcified VSMCs in vitro, which agreed with our previous studies.15,22 Kidney damage and dysfunction (plasma blood urea nitrogen and creatinine levels) were lessened in CKD rats with infusion of IMD1–53, so the IMD1–53 effects might have been due, at least in part, to improved kidney function. A number of studies have indicated that calcification inhibitors such as pyrophosphate, osteopontin, osteoprotegerin, and a-Klotho are necessary to prevent soft-tissue calcification.1 The level of a-Klotho, an endogenous anticalcification factor, was found decreased in early-stage CKD.40 a-Klotho is regulated by many factors: inflammation, oxidative stress, tumor necrosis factor, and angiotensin II decrease a-Klotho expression,41–43 whereas some bioactive peptides such as erythropoietin and fibroblast growth factor 2 promote the expression.44,45 CGRP, in the same family as IMD, can upregulate a-Klotho.26 Here, we found that exogenous IMD1–53 administration restored a-Klotho protein expression and retarded calcification in rat CKD aortas in vivo. IMD1–53 could not prevent VSMC calcification with a-Klotho siRNA knockdown, so the inhibitory effects of IMD1–53 on calcification are mediated at least in part by increased a-Klotho expression. a-Klotho functions as a cofactor of fibroblast growth factor 23 receptors and has been reported to cause fibroblast growth factor 23 action and specificity in the kidney. It was found that a-Klotho deficiency induced fibroblast growth factor 23 resistance.28,46 We found that with the upregulation of a-Klotho, plasma phosphorus levels were significantly decreased in CKD rats treatment with IMD1–53, probably due to the improved the fibroblast growth factor 23-a-Klotho axis. The role of fibroblast growth factor 23 in vascular calcification needs to be investigated in the future study. IMD exerts its biological effects by activating the CRLR/ RAMP system,20 and IMD shows selectivity for the CRLR/ RAMP3 receptor complex, with about 1 order of magnitude greater potency at that receptor than the CRLR/RAMP1 or CRLR/RAMP2 complexes have.23 Our previous study detected increased mRNA expression of RAMP2 and 3 in calcified VSMCs.47 Here, we found markedly increased mRNA and protein levels of CRLR and RAMP3 in calcified rat CKD aortas, probably because of higher reactivity. To confirm the specific receptor complex of IMD1–53 during VSMC calcification, we used CRLR and RAMP3 siRNA knockdown in cultured VSMCs. CRLR or RAMP3 knockdown could abolish the effects of IMD1–53 on a-Klotho upregulation and inhibition of VSMC calcification. Therefore, the CRLR/RAMP3 receptor complex is involved in these roles of IMD1–53. Fischer et al.48 reported that after binding to IMD, CRLR undergoes conformational alterations leading to Gs coupling and activation of adenylate cyclase and intracellular cAMP. 49 Our previous study demonstrated that IMD1–53 increased cAMP generation in cardiomyocytes, and H89 could block the effects of IMD on angiotensin II-induced hypertrophy in 597
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neonatal rat ventricular myocytes.24 In the current study, the PKA inhibitor H89 abolished the effect of IMD1–53 on a-Klotho upregulation and inhibition of VSMC calcification. Therefore, IMD1–53 consistently increased a-Klotho protein expression in calcified VSMCs by binding to the CRLR/ RAMP3 receptor complex, then activating PKA signaling to inhibit VSMC calcification. Both CGRP and a-Klotho mRNA expression were reduced in angiotensin II-induced senescent endothelial progenitor cells, whereas exogenous application of CGRP counteracted angiotensin II-induced endothelial progenitor cell senescence by increasing the production of a-Klotho and downregulating the expression of nicotinamide adenine dinucleotide phosphate oxidase and production of reactive oxygen species.26 We also observed that IMD1–53, a member of the CGRP family, upregulated a-Klotho protein level in VSMC calcification after binding to the CRLR/ RAMP3 receptor complex. Numerous studies, including our previous work, have shown that paracrine/autocrine factors are involved in vascular calcification and many agents could modify vascular calcification, including sodium pyrophosphate,50 lanthanum carbonate,51 angiotensin II receptor blocker losartan,52 and the bioactive peptide adrenomedullin.12 IMD has several benefits for inhibiting vascular calcification. First, as an endogenous bioactive peptide, it is extensively distributed throughout the body.23 Also, IMD is more potent than adrenomedullin, which shares the same family with IMD.53 Finally, within a wide range of concentration, IMD has various cardiovascular protective effects, including decreasing blood pressure,53 inhibiting calcification,22 enhancing cardiac contractile function,54 and protecting against schemia/reperfusion injury.21 In summary, we provide evidence that the paracrine/ endocrine factor IMD1–53 attenuates vascular calcification by upregulating a-Klotho level via the CRLR/RAMP3 receptor complex and activating cAMP/PKA signaling and partly by improving kidney function. The vasoactive peptide IMD1–53, an endogenous calcification inhibitor, may open new avenues for preventing vascular calcification in CKD. MATERIALS AND METHODS A detailed Method section and Supplementary Table S1 is available in the online-only supplementary material. Animals and materials Male Sprague-Dawley rats (200 10 g) were from the Animal Center, Peking University Health Science Center (Beijing, China). Vitamin D3, b-glycerophosphate, and Alizarin-red S were from Sigma (St. Louis, MO). Synthetic human IMD1–53 and primary IMD antibody were from Phoenix Pharmaceuticals (Belmont, CA). Primary antibodies for a-Klotho, smoothelin, calponin, SM22a, and osteocalcin were from Abcam (Cambridge, UK) and antibodies for osteopontin, CRLR, RAMP1-3, glyceraldehyde-3-phosphate dehydrogenase, and b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). The antimouse, antigoat, and antirabbit IgG secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). 598
JR Chang et al.: IMD1–53 inhibits VC by upregulating a-Klotho in CKD
Rat CKD model All animal care and experimental protocols complied with the Guide for the Care and Use of Laboratory Animals55 and were approved by the Animal Care Committee of Peking University Health Science Center (Beijing, China). Rats were randomly assigned to 3 groups for treatment: control, CKD, and CKDþIMD. Rat CKD was induced by 5/6 nephrectomy via surgical removal of the right kidney and ligation of two-thirds of the arterial supply to the left kidney with silk ligatures; vitamin D3 was administered intramuscularly at 1 mg/kg body weight 3 times a week for 12 weeks as described29 with a minor modification. Antibiotics (penicillin 200,000 U/kg) were given postoperatively for 3 days. IMD1–53 was administered subcutaneously (100 ng/kg/h) in phosphate buffered saline during the last 4 weeks of CKD treatment via an Alzet Mini-osmotic Pump (Alzetw model 2004, DURECT Corp, Cupertino, CA). Evaluation of animal model Twelve weeks after 5/6 nephrectomy, rats were killed and blood was collected for determination of biochemical assay and radioimmunoassay. The aorta was immediately removed and subjected to calcification study or immunoblot analysis. And the detailed methods are shown in the supplementary methods. VSMC culture and VSMC calcification model VSMCs were isolated from the thoracic aortic arteries of SpragueDawley rats (150–180 g) and verified by anti-SM a-actin antibody staining.22 Briefly, after partial removal of external connective tissues, rat thoracic aortas were cut into small pieces (about 2–3 mm each), placed in Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin, and incubated at 37 oC in an incubator containing 95% air and 5% CO2. VSMCs migrating from explants were collected and maintained in growing medium (Dulbecco’s modified Eagle’s medium containing 10 mmol/l sodium pyruvate and 10% fetal bovine serum). VSMCs at passages 5 to 8 were used for all of the experiments. For calcification, confluent VSMCs were incubated in medium containing 2.5 mmol/l Ca2þ (0.7 mmol/l CaCl2 was added into Dulbecco’s modified Eagle’s medium containing 1.8 mmol/l CaCl2) and 5 mmol/l b-glycerophosphate. Each experiment was performed at least 3 times with triplicate cultures used in every experiment, unless stated otherwise. Expression analysis Expression levels of mRNA and protein were evaluated as described in the supplementary methods. Statistical analysis Statistical analyses involved the use of GraphPad Prism 5.00 for Windows (GraphPad Software Inc, San Diego, CA). Comparisons of 2 groups involved unpaired Student t test and more than 2 groups of 1-way analysis of variance followed by Student-Newman-Keuls test. Data are expressed as mean SD. P < 0.05 was considered statistically significant. DISCLOSURES
All the authors declare no competing interests. ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (nos. 91339203, 81270407, and 81170082 to Yong-Fen Qi; Kidney International (2016) 89, 586–600
JR Chang et al.: IMD1–53 inhibits VC by upregulating a-Klotho in CKD
basic research
no. 31400984 to Jin-Rui Chang; no. 81470557 to Ming-Jiang Xu), Collaborative Innovation Center for Cardiovascular Disorders, the Ministry of Education Foundation for Doctoral Tutors (no. 20110001110012 to Yong-Fen Qi) and the Leading Academic Discipline Project of Beijing Education Bureau.
(n ¼ 3). *P < 0.05, **P < 0.01 versus control (Con); #P < 0.05, ##P < 0.01 versus Cal. Table S1. The forward and reverse PCR primers. Supplementary material is linked to the online version of the paper at www.kidney-international.org.
AUTHOR CONTRIBUTIONS
REFERENCES
JRC and JG designed, performed, and interpreted experiments; YW, YFG, YZ, and JD provided reagents and assisted to design experiments; YLH, WWL, MJX, and JSZ assisted in animal experiments and data analyses; YRY assisted with data analyses; XYL and XJW performed RNA sequence. CST interpreted data, and YFQ designed and interpreted the experimental work and wrote the manuscript.
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SUPPLEMENTARY MATERIAL Detailed methods Figure S1. Plasma biochemistry of chronic kidney disease (CKD) induced in 8-week-old rats following a 2-step surgical procedure for partial renal ablation plus vitamin D3 injection for 12 weeks. (A–E) Assay of blood urea nitrogen (BUN), creatinine, calcium (Ca), and phosphate (Pi) levels in rats with CKD (n ¼ 8). (F) Level of intact parathyroid hormone (PTH) in rat plasma of control (Con) and CKD groups. Data are mean SD (n ¼ 6). **P < 0.01 versus Con. Figure S2. Vascular calcification in chronic kidney disease (CKD) rat aortas. (A) Calcium content and (B) alkaline phosphatase (ALP) activity measured in CKD rat aortas. Data are mean SD (n ¼ 8). * P < 0.05, **P < 0.01 versus control (Con) rats. (C) Von Kossa staining of CKD rat aortas. Bars ¼ 200 mm. Original magnification 100. Figure S3. The effects of intermedin (IMD)1–53 on kidney function in chronic kidney disease (CKD) rats. (A) Hematoxylin and eosin staining showed that kidney damage was lessened in CKD rats by infusion of IMD1–53. Bars in larger images ¼ 200 mm; bars in inset images ¼ 50 mm. Original magnification 100. (B–D) Assay of plasma blood urea nitrogen (BUN), creatinine, and phosphate (Pi) levels in CKD rats with use of IMD1–53. Data are mean SD (n ¼ 6). **P < 0.01 versus control (Con); ##P < 0.01 versus CKD. Figure S4. Intermedin (IMD)1–53 restored a-Klotho levels in aorta, kidney, and plasma in chronic kidney disease (CKD) rats. Immunohistochemistry staining of a-Klotho protein expression in (A) aorta and (B) kidney of CKD rats. Bars in larger images ¼ 200 mm; bars in inset images ¼ 50 mm. Original magnification 100. The arrows point to positive staining in (A) and (B). (C) Plasma levels of a-Klotho in CKD rats with infusion of IMD1-53. Data are mean SD (n ¼ 8). *P < 0.05 versus control (Con); ##P < 0.01 versus CKD. Figure S5. a-Klotho was knocked down by small, interfering RNA (siRNA). (A) Real-time polymerase chain reaction analysis of a-Klotho mRNA level with siRNA knockdown. (B) Western blot analysis of a-Klotho protein level with siRNA knockdown and (C) semiquantitative analysis. Data are mean SD (n ¼ 3). **P < 0.01 versus scramble siRNA. Figure S6. Calcitonin receptor-like receptor (CRLR) or receptor activity-modifying protein (RAMP)3 knockdown by small, interfering RNA (siRNA). Real-time polymerase chain reaction analysis of mRNA levels of (A) CRLR and (D) RAMP3 with siRNA knockdown. Western blot analysis of protein levels of (B) CRLR and (E) RAMP3 with siRNA knockdown and (C and F) semiquantitative analysis. Data are mean SD (n ¼ 3). **P < 0.01 versus scramble siRNA. Figure S7. Intermedin (IMD)1–53 upregulated a-Klotho protein expression in calcified (Cal) vascular smooth muscle cells not through PI3K/Akt or extracellular signal–regulated kinase signaling pathway. (A and B) Western blot analysis of a-Klotho protein level (b-actin was a loading control) and semiquantitative analysis. Data are mean SD Kidney International (2016) 89, 586–600
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