AT2 receptor stimulation inhibits phosphate-induced vascular calcification

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AT2 receptor stimulation inhibits phosphateinduced vascular calcification Masayoshi Kukida1,2, Masaki Mogi1, Harumi Kan-no1, Kana Tsukuda1, Hui-Yu Bai1, Bao-Shuai Shan1, Toshifumi Yamauchi1,3, Akinori Higaki1,2, Li-Juan Min1, Jun Iwanami1, Takafumi Okura2, Jitsuo Higaki2 and Masatsugu Horiuchi1 1

Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Tohon, Japan; Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University, Graduate School of Medicine, Tohon, Japan; and 3Department of Pediatrics, Ehime University, Graduate School of Medicine, Tohon, Japan

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Vascular calcification is a common finding in atherosclerosis and in patients with chronic kidney disease. The reninangiotensin system plays a role in the pathogenesis of cardiovascular remodeling. Here, we examined the hypothesis that angiotensin II type 2 receptor (AT2) stimulation has inhibitory effects on phosphate-induced vascular calcification. In vivo, calcification of the thoracic aorta induced by an adenine and high-phosphate diet was markedly attenuated in smooth muscle cell-specific AT2overexpressing mice (smAT2-Tg) compared with wild-type and AT2-knockout mice (AT2KO). Similarly, mRNA levels of relevant osteogenic and vascular smooth muscle cell marker genes were unchanged in smAT2-Tg mice, while their expression was significantly altered in wild-type mice in response to high dietary phosphate. Ex vivo, sections of thoracic aorta were cultured in media supplemented with inorganic phosphate. Aortic rings from smAT2-Tg mice showed less vascular calcification compared with those from wild-type mice. In vitro, calcium deposition induced by highphosphate media was markedly attenuated in primary vascular smooth muscle cells derived from smAT2-Tg mice compared with the two other mouse groups. To assess the underlying mechanism, we investigated the effect of PPAR-g, which we previously reported as one of the possible downstream effectors of AT2 stimulation. Treatment with a PPAR-g antagonist attenuated the inhibitory effects on vascular calcification observed in smAT2-Tg mice fed an adenine and high-phosphate diet. Our results suggest that AT2 activation represents an endogenous protective pathway against vascular calcification. Its stimulation may efficiently reduce adverse cardiovascular events in patients with chronic kidney disease. Kidney International (2018) j.kint.2018.07.028

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https://doi.org/10.1016/

KEYWORDS: phosphate; renin angiotensin system; vascular calcification Copyright ª 2018, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Correspondence: Masaki Mogi, Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. E-mail: [email protected] Received 18 November 2017; revised 9 July 2018; accepted 26 July 2018 Kidney International (2018) -, -–-

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n a recent report, about 2.6 million patients had undergone renal replacement therapy worldwide,1 and the number of the dialysis patients will further increase in the future. The risk of chronic kidney disease (CKD), which is the underlying condition of these patients, has attracted attention, and countermeasures for preventing and treating CKD are under study worldwide.2,3 There are 2 general problems associated with CKD. One is the high risk of subsequent end-stage renal disease.4 Another serious problem is the risk of developing cardiovascular disease.5,6 Unfortunately, it is assumed that many patients with CKD die of cardiovascular disease before progressing to end-stage renal disease.7 A pathological change strongly linked with this clinical outcome is vascular calcification. Vascular calcification is highly correlated with cardiovascular morbidity and mortality in CKD.8,9 Vascular calcification in CKD is mainly found in the media, but not intima, of blood vessels, and is strongly linked to systemic mineral imbalance such as hyperphosphatemia.10,11 This calcification mainly contributes to active change, which results in trans-differentiation of vascular smooth muscle cells (VSMCs) into osteo- and/or chondrogenic-like cells, but not passive change resulting in calcium (Ca) deposition in VSMCs.10,12 One of the stimuli for this trans-differentiation is flow of elevated serum inorganic phosphate (Pi) into VSMC through sodium-Pi cotransporters.13 As a result, there is loss of inhibitory factors for vascular calcification such as matrix Gla protein (MGP) and osteopontin (OPN), and upregulation of runt-related transcription factor 2 (Runx2), which is a key component of transformation in VSMCs, leading to transdifferentiation.11,14 It is assumed that vascular calcification involves apoptosis of VSMCs affected by hyperphosphatemia.15 However, vascular calcification is thought to involve many other factors, and the overall picture of the mechanisms leading to calcification has not been elucidated. As described in the national guidelines, the lowering of serum phosphorus with nutritional therapy such as a low-phosphate diet and administration of oral phosphate binders (and parathyroidectomy in some cases) are important therapies.16,17 However, a decrease in serum phosphorus alone cannot completely suppress vascular calcification. It is clinically important to consider how we can further suppress vascular calcification. 1

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In many CKD patients, the renin-angiotensin-aldosterone system (RAAS) is enhanced, leading to multiple organ damage. Much evidence suggests that activation of the angiotensin II type 1 receptor (AT1) plays a pivotal role in the pathogenesis and associated end-organ damage of hypertension.18,19 Moreover, it is recommended that hypertensive CKD patients are treated with an angiotensin-converting enzyme inhibitor or AT1 blocker (ARB). Clinically, elucidation of the relationship between the RAAS and calcification seems to be useful. In basic research, some recent reports suggest that the RAAS plays a role in the pathogenesis of vascular calcification.20,21 We previously reported that angiotensin II type 2 receptor (AT2) stimulation exerted beneficial counter-regulatory roles against AT1 action in cardiovascular remodeling.22 However, little has been reported about the relationship between AT2 stimulation and vascular calcification. Here, we investigated the possibility of whether AT2 stimulation is involved in inhibitory effects on phosphate-induced vascular calcification, using in vitro, ex vivo, and in vivo vascular calcification models. RESULTS Smooth muscle cell-specific AT2-overexpressing (smAT2-Tg) mice showed less vascular calcification compared with wildtype (WT) and AT2-knockout (AT2KO) mice with adenine and high-phosphate diet (in vivo)

An adenine and high-phosphate diet induced renal failure and hyperphosphatemia without an increase in Ca concentration (Supplementary Table S1A). There was no significant difference in serum biochemical measurements among the 3 adenine-diet groups. The adenine diet induced loss of body weight and tissue weight of the heart and kidney on the final indicated day (Supplementary Figure S1). The adenine diet had little effect on systolic blood pressure. There was no significant difference in levels of biochemical parameters among each adenine diet group. Histopathological analysis of the kidney of WT mice with an adenine diet showed deposition of symmetric crystalline structures in tubule lumens, micro-abscesses, dilated tubules, and dilated Bowman’s space (Supplementary Figure S2B) compared with mice fed a standard chow diet (Supplementary Figure S2A). Trichrome-Masson staining revealed mild interstitial fibrosis, and periodic acid–Schiff staining showed atrophic renal tubules with protein casts and tubular atrophy with thickening of the tubular basement membrane (Supplementary Figure S2B), indicating that an adenine diet induced tubulointerstitial nephropathy. Vascular calcification evaluated by Ca concentration in the aorta was significantly induced by an adenine diet in WT mice (Figure 1a). Vascular calcification in AT2KO mice was comparable with that in WT mice; however, smAT2-Tg mice showed less vascular Ca deposition compared with the other mouse groups. There was no significant difference in vascular Ca concentration among each group in mice fed standard chow (data not shown). Next, we examined the mRNA levels 2

M Kukida et al.: AT2 prevents vascular calcification

of osteogenic-related marker genes and a VSMC marker gene by real-time polymerase chain reaction (PCR) methods. Expression of Runx2 was increased, and expression of smooth muscle protein 22-a (SM22-a) and MGP was significantly decreased by an adenine diet compared with standard chow diet in WT mice (Figure 1b). In contrast, there was no significant difference in the expression of Runx2, SM-22a, and MGP in smAT2-Tg mice between the adenine diet and standard chow diet. SmAT2-Tg mice showed less aortic ring calcification with high-phosphate diet compared with WT and AT2KO mice (ex vivo)

Vascular calcification was induced by phosphate stimulation and increased time-dependently in WT mice (Figure 2a). Histological analysis showed significant vascular calcification in the aortic ring culture with this calcifying medium for 7 days (Figure 2b). Vascular calcification in AT2KO mice induced by a high-phosphate diet was comparable with that in WT mice, but smAT2-Tg mice showed less vascular calcification compared with other mice (Figure 2c). VSMC prepared from smAT2-Tg mice cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high phosphate showed less Ca deposition compared with WT and AT2KO mice (in vitro)

Next, we evaluated Ca deposition in primary mouse VSMCs isolated from the thoracic aorta of adult male mice induced by incubation in DMEM supplemented with high phosphate. To determine appropriate conditions, calcification of VSMC was induced by incubation in various calcifying media for 10 or 14 days with medium changes every 2 days (Supplementary Figure S3). As a result, in vitro study showed that Ca deposition was induced by phosphate stimulation, and increased dose- and time-dependently in VSMCs of WT mice. Interestingly, Ca deposition induced by incubation in DMEM supplemented with high phosphate plus 1% fetal bovine serum (FBS) was increased compared with DMEM plus 10% FBS, but the cell-free background level of Ca in DMEM plus 1% FBS was high compared with that in DMEM plus 10% FBS. On the basis of the prior study and a few other reports,23 we determined that vascular calcification was induced by culture in DMEM supplemented with 5% FBS and Pi at a final concentration of 2.8 mmol/l for 10 days. Ca deposition in AT2KO mice induced by high phosphate was comparable with that in WT mice, but smAT2-Tg mice showed less Ca deposition compared with that in other mice (Figure 3a and b). Expression of Runx2 was increased by high phosphate in WT mice, whereas there was no significant change in expression of Runx2 in smAT2-Tg mice (Figure 3c). Role of peroxisome proliferator-activated receptor-g (PPAR-g) in inhibitory effect on vascular calcification in smAT2-Tg mice

Next, we tried to elucidate the mechanism of the AT2 receptor-mediated inhibitory effects on VSMC calcification. Kidney International (2018) -, -–-

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Figure 1 | Comparison of vascular calcification in mice with adenine diet (in vivo study). (a) Calcium (Ca) concentration in the aorta was analyzed as described in Methods. Values are expressed as mean  SD of (n ¼ 5). *P < 0.01 versus wild-type (WT) mice fed standard chow. (b) Quantitative real-time reverse-transcription polymerase chain reaction analysis of aortic tissue in WT and smooth muscle cell–specific angiotensin II type 2 receptor–overexpressing (smAT2-Tg) mice. Values are expressed as mean  SD (n ¼ 3). AT2KO, angiotensin II type 2 receptor knockout; MGP, matrix Gla protein; Runx2, runt-related transcription factor 2; SM22a, smooth muscle protein 22a.

We previously reported that AT2 receptor stimulation by compound 21 (C21), a direct AT2 receptor agonist, is associated with PPARg activation, resulting in ameliorated insulin resistance in diabetic mice24 and inhibited vascular intimal proliferation.22 Therefore, we investigated the role of PPAR-g in the inhibitory effect on vascular calcification in smAT2-Tg mice. Dual-luciferase reporter assay showed that treatment of smAT2-Tg VSMCs with DMEM supplemented with 5% FBS and Pi more markedly increased PPARg activity than that in WT VSMCs (Figure 4a). Next, we assessed VSMCs of WT and smAT2-Tg mice incubated in this calcifying medium with GW9662. Interestingly, the inhibitory effect on Ca deposition in smAT2-Tg mice was attenuated by co-treatment with GW9662 (3mM) (Figure 4b and c). Kidney International (2018) -, -–-

Finally, we examined the effect of GW9662 on vascular calcification using WT and smAT2-Tg mice in vivo. Cotreatment with GW9662 canceled the reduction in vascular calcification observed in smAT2-Tg mice (Figure 4d). Effect of administration of C21 on vascular calcification in rats with adenine diet

Finally, we investigated the effect of AT2 receptor stimulation by C21 on vascular calcification. Mice fed an adenine diet containing 1.2% phosphate to induce vascular calcification were too weak to endure replacement of osmotic minipumps. Therefore, we used a rat model in direct AT2 receptor stimulation treatment. Wistar rats were randomly divided into standard diet and adenine diet groups with and without C21 (N ¼ 8 each). The distinct diets (chow vs. adenine) were 3

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Figure 2 | Comparison of aortic ring calcification with high phosphate (ex vivo study). (a) Time course analysis of vascular calcification induced by phosphate stimulation in wild-type (WT) mice. Values are expressed as mean  SD (n ¼ 5). *P < 0.01 versus day 1. (b) Histological analysis of aortic rings with Alizarin Red S and Von Kossa staining in WT mice. Bar ¼ 200 mm. (c) Calcium (Ca) concentration in aortic rings was analyzed as described in Methods. Values are expressed as mean  SD (n ¼ 5). AT2KO, angiotensin II type 2 receptor knockout; smAT2, smooth muscle cell–specific angiotensin II type 2 receptor. To optimize viewing of this image, please see the online version of this article at www. kidney-international.org.

started for 6 weeks following a 1-week chow diet for adaptation in all groups. The adenine diet induced vascular calcification with an elevated mRNA level of Runx2 in Wistar rats, as previously described.25,26 Interestingly, in vivo, vascular calcification in thoracic aortas induced by an adenine diet was markedly attenuated by C21 (Figure 5a and b). We examined the changes in mRNA levels of AT1 receptor and AT2 receptor in the thoracic aortas (Figure 5c). Expression of AT2 receptor, but not AT1 receptor, mRNA increased with an adenine diet. The adenine diet induced kidney injury, high phosphatemia, loss of body weight, and gain of kidney weight with or without C21 (Supplementary Figure S1E and F and Supplementary Table S1B). These results in rats did not deviate from those in mice. DISCUSSION

In the present study, we investigated the possibility of whether AT2 stimulation is involved in the inhibitory effects on 4

phosphate-induced vascular calcification in smAT2-Tg mice. Our results suggested that the presence of overexpressed AT2 ameliorated phosphate-induced vascular calcification partially due to PPARg activation. Here, we investigated the effect of AT2 on vascular calcification using gene-modified mice in in vivo, ex vivo, and in vitro studies by trial and error. In many previous reports, CKD and vascular calcification have been induced by a highfat diet and partial nephrectomy in apoE–/–KO and LDLR–/–KO mice in vivo.27 First, we tried to induce CKD and vascular calcification by a high-phosphate diet and 5/6 nephrectomy in WT mice, but this was not possible because of high mortality. In several recent reports,28,29 CKD has been induced by an adenine diet, which resulted in tubulointerstitial nephritis in rats. As described in these reports, we tried to generate a CKD-induced vascular calcification model induced by an adenine and high-phosphate diet in mice. First, we used a 0.45% adenine diet to induce CKD in mice, but Kidney International (2018) -, -–-

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Figure 3 | Comparison of calcium (Ca) deposition in vascular smooth muscle cells (VSMCs) cultured in Dulbecco’s modified Eagle’s medium containing high phosphate (Pi) (in vitro study). (a) Ca concentration in VSMCs was analyzed as described in Methods. Values are expressed as mean  SD (n ¼ 3). *P < 0.01 versus wild-type (WT) mice. (b) Ca deposition evaluated by Alizarin Red S staining. Left bar ¼ 1.2 cm; right bar ¼ 300 mm. (c) Quantitative real-time reverse-transcription polymerase chain reaction analysis of aortic tissue in WT and smooth muscle cell–specific angiotensin II type 2 receptor–overexpressing (smAT2-Tg) mice. Values are expressed as mean  SD (n ¼ 3). AT2KO, angiotensin II type 2 receptor knockout; Runx2, runt-related transcription factor 2; SM22a, smooth muscle protein 22a; smAT2, smooth muscle cell–specific angiotensin II type 2 receptor. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

almost all mice died within 4 weeks. In this study, the adenine diet-induced CKD mouse model was designed following an 8week program as described previously30,31 with partial modification. The mortality rate was approximately 10%. In this study, in order to simplify the stimulation for vascular calcification, the adenine diet only contained added phosphorous, and not Ca. As compared with previous reports,29,30 vascular calcification was induced similarly. Despite no significant difference in renal dysfunction and electrolyte imbalance, smAT2-Tg mice showed less vascular calcification compared with WT and AT2KO mice. Standard chow had little effect on vascular calcification in WT, AT2KO, and smAT2-Tg mice. In this study, despite CKD, there was no significant difference in systolic blood pressure measured by the tail-cuff method. In general, it is considered that hypertension associated with CKD is salt sensitive.32 This might be Kidney International (2018) -, -–-

the reason that mice did not undergo salt loading. Vascular calcification of aortic rings in WT mice was induced comparably to that described in previous reports.33 Moreover, we tried to generate a high-phosphorus–induced in vitro VSMC culture model for analyzing Ca deposition using mouse primary VSMCs. Most previous reports used rat primary VSMCs, human aortic smooth muscle cells (HASMCs), or mesenchymal stem cells (MSCs),34–36 but not primary mouse VSMCs. Therefore, WT VSMCs were incubated in various calcifying media for 10 or 14 days. Primary VSMCs showed an increase in Ca deposition in a dose- and timedependent manner with phosphate. Interestingly, a higher FBS concentration showed less vascular calcification. The reason may be preventive factors against vascular calcification such as MGP37 and Fetuin A, which makes calciprotein particles.38 Moreover, other reports have suggested that the 5

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Figure 4 | Role of proliferator-activated receptor-g (PPAR-g) in reduction of vascular calcification in smooth muscle cell–specific angiotensin II type 2 receptor–overexpressing (smAT2-Tg) mice. (a) Comparison of PPAR-g activity in vascular smooth muscle cells (VSMCs) between wild-type (WT) and smAT2-Tg mice. PPAR-g activity in VSMCs was analyzed as described in Methods. Values are expressed as mean  SD (n ¼ 3). P < 0.01 versus WT, 0.5% fetal bovine serum (FBS). †P < 0.05 versus smAT2-Tg, 0.5% FBS. Effect of GW9662 on calcium (Ca) deposition in VSMCs. (b) Comparison of Ca concentration in VSMC (in vitro study). Ca concentration in VSMCs was analyzed as described in Methods. Values are expressed as mean  SD (n ¼ 3). *P < 0.01 versus WT, GW9662 (–). †P < 0.05 versus smAT2, GW9662 (–), (c) Ca deposition evaluated by Alizarin Red S staining. Bar ¼ 1.2 cm. (d) Comparison of vascular calcification in mice with adenine diet (in vivo study). Ca concentration in the aorta was analyzed as described in Methods. Values are expressed as mean  SD (n ¼ 5). *P < 0.01 versus WT, standard chow. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

contractile phenotype of VSMC tends to occur more by transdifferentiation into osteogenic cells than does the synthetic phenotype of VSMC.39 Thus, a lower concentration of FBS may enhance Ca deposition in VSMC. The inhibitory effect on Ca deposition and vascular calcification in smAT2-Tg mice was attenuated by co-treatment with GW9662. Therefore, PPARg played a preventive role in vascular calcification, as shown in our previous report and other reports.22,40,41 However,

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GW9662 treatment did not completely cancel these inhibitory effects observed in smAT2-Tg mice. One of the reasons might be the smaller dose of GW9662 or the effect of other signaling than PPARg by AT2. Previous reports have suggested that angiotensin II stimulates Wnt/b-catenin signals through AT1,42,43 and AT2 stimulation also affects Wnt/b-catenin signals.44 Furthermore, AT2 stimulation has also been reported to enhance various phosphatase activity.45,46 Therefore, a possible mechanism via inhibition of

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Figure 5 | Effect of administration of compound 21 (C21) on vascular calcification in rats fed an adenine diet (in vivo study). (a) Calcium (Ca) concentration in the aorta was analyzed as described in Methods. Values are expressed as mean  SD (n ¼ 5). *P < 0.01 versus standard chow. (b,c) Quantitative real-time reverse-transcription polymerase chain reaction analysis of aortic tissue of mice fed standard chow and an adenine diet with and without C21. Values are expressed as mean  SD (n ¼ 3). *P < 0.01 versus standard chow. AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; Runx2, runt-related transcription factor 2.

Wnt/b-catenin activity and phosphorylation of ERK23 is involved. To investigate the role of Wnt/b-catenin signals in the inhibitory effect on vascular calcification in smAT2-Tg mice, the protein levels of Wnt3a, LRP-6, b-catenin, and active b-catenin were assessed by immunoblotting using VSMCs prepared from WT and smAT2-Tg mice in vitro and thoracoabdominal arteries of rats in vivo. However, we could not determine the effect of these signals on vascular calcification (data not shown). Angiotensin II type 2 receptor (AT2R) stimulation has been reported to alter the apoptosis rate, and this effect could be PPAR-g dependent.47 Therefore, we examined the mRNA levels of caspase 3, Bax, and Bcl2 by real-time PCR methods in the aorta of adenine-induced CKD rats. However, there was no significant difference in the expressions of apoptosisrelated genes between the adenine diet and standard chow diet (data not shown). Further investigation is needed to Kidney International (2018) -, -–-

reveal the detailed mechanism of the AT2-induced protective effect on vascular calcification. There was no significant difference in vascular calcification between VSMCs of AT2KO and WT mice in any model. Previous studies indicate that AT2R is usually expressed in fetal tissue or in some pathological conditions, but not in adult normal tissue. Moreover, detection of AT2R protein level by immunohistochemistry is much harder due to the lack of an appropriate antibody of AT2R. Therefore, we examined the mRNA level of AT2R in the aorta of adenine-induced CKD rats and confirmed the AT2R expression in them. Future studies are necessary to investigate AT2R expressions in human vascular samples of patients with CKD. We investigated the inhibitory effect of angiotensin (Ang) II on vascular calcification in smAT2-Tg VSMCs. However, the inhibitory effect on Ca deposition in smAT2Tg mice was not attenuated by co-treatment with Ang II 7

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(data not shown). This result might be due to Ang II-AT1 receptor signaling. We could not detect the beneficial effect of Ang II-AT2 receptor signaling because Ang II receptor expressions are different. Usually AT1 receptor is widely expressed in the vasculature, while AT2 receptor expression is not common in adult tissue and is upregulated in pathological conditions. Therefore, according to the vascular conditions, Ang II-AT1 receptor signaling may be strongly involved instead of Ang II-AT2 receptor signaling throughout Ang II administration. On the other hand, vascular calcification in rat thoracic aortas induced by an adenine diet was markedly attenuated by C21, indicating that specific AT2 receptor stimulation has a therapeutic benefit to prevent vascular calcification via avoiding AT1 receptor stimulation. Nakagami et al. have already demonstrated that administration of the ARB olmesartan decreases vascular calcification.20 ARB could prevent vascular calcification and relative AT2 receptor stimulation via unbound Ang II by ARB administration. It is for future investigation that we compared the beneficial effect between C21 and ARB treatment on vascular calcification. It has been suggested that the pathway between angiotensin-converting enzyme (ACE) 2, Ang-(1–7), and Mas exerts an antagonistic action in many physiological and pathophysiological processes in several systems and organs, including cardiovascular remodeling, via opposing the classical ACE/Ang II/AT1 receptor axis–mediated action. We have been investigating the possibility of whether the ACE2/Ang(1-7)/Mas pathway is involved in the inhibitory effects on vascular calcification induced via an adenine and highphosphate diet, using mice genetically modified for protective arms of the RAS. In summary, our results suggest that the presence of overexpressed AT2 ameliorates phosphate-induced vascular calcification partially due to PPARg activation. These results did not contradict the content of past reports.48 Regulation of AT2 signaling may decrease the risk of vascular calcification and subsequent adverse cardiovascular events in patients with CKD. MATERIALS AND METHODS This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. The experimental protocol was approved by the Animal Studies Committee of Ehime University. Animals Adult male C57BL/6J mice (WT), smAT2-Tg mice,49 and AT2 receptor-null mice (AT2KO)50 (10 weeks of age), all with a C57BL/6J genetic background, were used in this study. Wistar male rats (CLEA Japan; Tokyo, Japan) at 8 to 10 weeks of age were used for this study. The animals were housed in a room where lighting (12 hours on, 12 hours off) and room temperature (25C) were controlled. They were given free access to standard laboratory chow (CE2 rodent diet, CLEA Japan) and water.

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Animal model of CKD (in vivo) An adenine diet-induced CKD mouse model was designed following an 8-week program as described previously30,31,51 by varying the concentration of adenine as shown in Supplementary Figure S4A with partial modification. Casein (5%) was added to the adenine diet to conceal the taste and smell of adenine, and the same amount of casein was added to chow diet. Eight- to 10-week-old male mice were fed the adenine diet or casein-added chow (standard diet) diet. Phosphate (1.2%) was added to the adenine diet to accelerate the process of CKD and vascular calcification. WT, smAT2-Tg, and AT2KO mice were randomly divided into standard diet and adenine diet groups (N ¼ 5 each). The 8-week distinct diets (chow vs. adenine) were started following a 1-week chow diet for adaptation in all groups. Some mice were administered GW9662, a PPARg antagonist, at a dose of 0.35 mg/kg/d in drinking water. An adenine diet-induced CKD rat model was designed following a 6-week program as described previously25,26 by 0.75% adenine as shown in Supplementary Figure S4D. Eight- to 10-week-old male Wistar rats were fed the adenine diet or standard chow diet. Adenine-fed rats were treated with C21 (300 mg/kg/d) i.p. via an Alzet osmotic pump (models 2004 and 1002, Durect Corporation; Cupertino, CA) for 6 weeks. Wistar rats were randomly divided into standard diet and adenine diet groups with and without C21 (N ¼ 8 each). The distinct diets (chow vs. adenine) were started for 6 weeks following a 1-week chow diet for adaptation in all groups. The thoracoabdominal arteries of mice and rats were dissected to assay Ca deposition. Plasma levels of blood urea nitrogen (BUN), creatinine (Cre), Ca, and phosphate were measured. Systolic blood pressure was measured by the tail-cuff method (BP-98A, Softron Co., Ltd.; Tokyo, Japan). Aortic ring calcification (ex vivo) Thoracic aortas were dissected from WT, smAT2-Tg, and AT2KO mice at 14 to 16 weeks of age. After removing the adventitia, we cut the vessels into 2- to 3-mm rings and cultured them in DMEM (Life Technologies, Inc.; Gaithersburg, MD) supplemented with 15% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in a humidified atmosphere containing 5% CO2. The culture medium was changed every 2 days. To induce calcification, Pi (Na2HPO4/ NaH2PO4, pH 7.4) was added to supplement DMEM at a final concentration of 2.8 mmol/l. After the indicated incubation periods (1, 3, 5, 7, and 10 days), samples were taken and analyzed. The study protocol is described in Supplementary Figure S4B. To remove endothelial cells, a 5-cm thread (Daiso-Sangyo Inc.; Hiroshima, Japan) was passed once through the lumen to excoriate the endothelium. This aortic ring culture method has been reported previously.33,51 Cell culture and treatment (in vitro) Primary mouse VSMCs were isolated from the thoracic aorta of adult male WT, smAT2-Tg, and AT2KO mice by the explant method52 and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in a humidified atmosphere containing 5% CO2. Cells at passage 4 to 8 were used for the experiments. Calcification of VSMC was induced by incubation in various calcifying media (DMEM supplemented with 1 or 10% FBS, and NaH2PO4/Na2HPO4 [pH 7.4] at a final concentration of 2.8 or 3.8 mmol/l) for 10 or 12 days with medium changes every 2 days (Supplementary Figure S4C).23,34

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M Kukida et al.: AT2 prevents vascular calcification

Quantification of VSMC and aortic calcification (Ca assay) To evaluate vascular calcification, dissolved Ca content was measured. In in vivo and ex vivo studies, after the indicated periods, the treated aortas were weighed and then incubated with 1 N hydrogen chloride overnight at 4 C. In in vitro studies, after the indicated incubation periods with calcifying media, the treated VSMCs were incubated with 0.6 N hydrogen chloride overnight at 4 C. After the hydrogen chloride supernatant was removed, the remaining cell or tissue pellets were dissolved in 0.1 N sodium hydroxide and 0.1% sodium dodecylsulfate for protein concentration analysis. The protein lysate was collected in 0.1 N sodium hydroxide and 0.1% sodium dodecylsulfate. Protein concentration was determined by bicinchoninic acid assay. Ca concentration in the hydrogen chloride supernatant was determined by the O-cresolphthalein method using a Ca Reagent Set (Teco Diagnostics; Anaheim, CA). Ca data were normalized to tissue weight or overall protein data. Histological analysis In ex vivo studies, to evaluate vascular calcification, Von Kossa and Alizarin Red S staining were performed. Digital photographs of the stained culture plates were obtained using a microscope. After the incubation periods, the treated aortas were fixed with 10% neutralbuffered formalin for 24 hours. Paraffin-embedded cross-sections were prepared. Each sample was sliced at 5-mm thickness, and deparaffinized before staining. For Von Kossa staining, samples were incubated with 5% silver nitrate under ultraviolet light for 1 hour, and then un-reacted sliver was removed by incubation in 5% sodium thiosulfate for 5 minutes. Nuclei were counterstained with hematoxylin. For Alizarin Red S staining, samples were incubated with Alizarin Red solution (pH 4.1–4.3 with 10% ammonium hydroxide) for 5 minutes. In in vitro studies, to characterize calcific nodules, Alizarin Red S staining was performed. After the indicated incubation periods (10 days), VSMCs in 6-cm dishes were washed 3 times with ice-cold phosphate-buffered saline, fixed with 10% (v/v) formaldehyde for 1 h, washed 3 times with phosphate-buffered saline, exposed to 2% Alizarin Red S for 15 min, and washed with ice-cold phosphatebuffered saline. Positively stained cells displayed a reddish or purple color. Quantitative real-time PCR In in vitro studies, total RNA was extracted from VSMCs after 6 days of incubation in calcifying medium (DMEM supplemented with 5% FBS, Pi at final concentration of 2.8 mmol/l). In in vivo studies, total RNA was extracted from rat thoracic aorta after a 6-week program. Total mRNA was purified with an RNeasy Mini Kit (Qiagen; Hilden, Germany) following the manufacturer’s protocol. Real-time quantitative reverse-transcription PCR was performed with a Premix Ex Taq (Takara Bio Inc.; Shiga, Japan). The mRNA levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase. The sequences of PCR primers are provided in Supplementary Table S2A and B. Luciferase activity assay PPARg activity was analyzed by luciferase activity assay. VSMCs seeded in 12-well plates were transfected with 500 ng PPARgluciferase reporter vector (Qiagen) using Lipofectamine PLUS (Invitrogen Corp.; Carlsbad, CA) according to the manufacturer’s instructions. After 4 hours, the transfection medium was replaced with DMEM medium containing 0.5% FBS and 1% antibiotics as well as 5% FBS and phosphorus at a final concentration of Kidney International (2018) -, -–-

2.8 mmol/l. At 24 hours after transfection, stimulated cells were lysed and subjected to luciferase activity assay, using a DualLuciferase Reporter Assay System (Promega; Madison, WI) on a luminometer (AB-2200, ATTO Corp.; Tokyo, Japan). Data were expressed as fold change after normalization to the activity of Renilla luciferase. Statistical analysis All values in the text and figures are expressed as mean  SD. Data were evaluated by analysis of variance. If a statistically significant effect was found, post hoc analysis (Bonferroni method or Student ttest) was performed to detect the difference between the groups. Values of P < 0.05 were considered statistically significant. DISCLOSURE

All the authors declared no competing interests.

FUNDING SUPPORT

This study was supported by JSPS KAKENHI (grant nos. 25293310 to MH, 25462220 to MM, 15K19974 to JI, and 26860567 to LM), and research grants from Astellas Pharma Inc., Daiichi-Sankyo Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., and Takeda Pharmaceutical Co. Ltd. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. SUPPLEMENTARY MATERIAL Table S1. (A) Plasma levels of markers of renal function in 8-week proof-of-concept study of adenine-induced renal failure in mice. (B) Plasma levels of markers of renal function in 6-week proof-of-concept study of adenine-induced renal failure in rats. Table S2. (A) Sequences of polymerase chain reaction (PCR) primers in mice. (B) Sequences of PCR primers in rats. Figure S1. Body weight, tissue weights of heart and kidney, and systolic blood pressure. (A–D) clinical parameters in 8-week proof-ofconcept study of adenine-induced renal failure in mice. Values are expressed as mean  SD (n ¼ 5). *P < 0.01 versus wild-type (WT) mice fed standard chow. (E,F) Clinical parameters in 6-week proof-ofconcept study of adenine-induced renal failure in rats. Values are expressed as mean  SD (n ¼ 8). *P < 0.01 versus rats fed standard chow. Figure S2. Adenine-induced tubulointerstitial nephropathy in wildtype (WT) mice. Representative histopathological findings in kidney tissues of mice fed a (A) standard chow diet and (B) adenine diet. Histological examination shows deposition of symmetric crystalline structures in a tubular lumen (green arrow), micro-abscesses (white arrow), dilated tubules (blue arrowheads), and dilated Bowman’s space (yellow arrowheads). Trichrome-Masson staining reveals mild interstitial fibrosis. Periodic acid–Schiff staining shows atrophic renal tubules with protein casts and tubular atrophy with thickening of the tubular basement membrane. Figure S3. Time-course of calcium deposition in primary vascular smooth muscle cells (VSMCs) prepared from wild-type (WT) mice cultured in Dulbecco’s modified Eagle’s medium containing various concentrations of fetal bovine serum (FBS) and phosphate (Pi). Values are expressed as mean  SD (n ¼ 3). *P < 0.01 versus 0.9 mM Pi, 10 days, 10% FBS. yP < 0.01 versus 0.9 mM Pi, 10 days, 1% FBS. Figure S4. Experimental procedure. (A) Schematic view of 8-week proof-of-concept study of adenine-induced renal failure in mice. (B) Schematic view of ex vivo study of aortic ring culture. (C) Schematic 9

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