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Dietary L-Lysine Prevents Arterial Calcification inAdenine-Induced Uremic Rats

Akihiro Shimomura,* Isao Matsui,* Takayuki Hamano,† Takuya Ishimoto,‡ Yumiko Katou,§

Kenji Takehana,| Kazunori Inoue,* Yasuo Kusunoki,* Daisuke Mori,* Chikako Nakano,*Yoshitsugu Obi,* Naohiko Fujii,¶ Yoshitsugu Takabatake,* Takayoshi Nakano,‡

Yoshiharu Tsubakihara,† Yoshitaka Isaka,* and Hiromi Rakugi*

Departments of *Geriatric Medicine and Nephrology and †Comprehensive Kidney Disease Research, OsakaUniversity Graduate School of Medicine, Suita, Osaka, Japan; ‡Division of Materials and Manufacturing Science,Graduate School of Engineering, Osaka University, Suita, Osaka, Japan §Applied Analytical Group, FundamentalTechnology Laboratories, Institute for Innovation, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki, Kanagawa, Japan;|Pharmacology Research Laboratory, Research Institute, Ajinomoto Pharmaceutical Co., Ltd., Kawasaki-ku, Kawasaki,Kanagawa, Japan; and ¶Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia,Pennsylvania

ABSTRACTVascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes ashortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects ofdietary L-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13weeksweredivided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (groupAde),LPdiet+adenine+glycine (groupGly) as a control amino acid group, and LPdiet+adenine+L-lysinezHCl (groupLys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severeVC. L-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creati-nine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calciumin group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary L-lysine stronglysuppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis.The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects ofL-lysine. Dietary L-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Anal-yses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth musclecells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of L-lysine ameliorated VC by modifying keypathways that exacerbate VC.

J Am Soc Nephrol 25: 1954–1965, 2014. doi: 10.1681/ASN.2013090967

Medial vascular calcification is common in aging,diabetes, and CKD.1–4 Because the presence of vas-cular calcification is strongly associated with in-creased cardiovascular morbidity and mortality,several studies in both animals and humans havesought ways to reduce the extent of vascular calci-fication.5–10 However, satisfactory therapies havenot yet been established.11

Adenine-induced renal failure is one of thecommonly used animal models for studyingthe development of vascular calcification, but theprevalence of vascular calcification in this model is

not very high. Indeed, Price et al. reported that vas-cular calcification was detected in only 30% of rats

Received September 13, 2013. Accepted January 7, 2014.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Yoshitaka Isaka, Department of GeriatricMedicine and Nephrology, Osaka University Graduate School ofMedicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Email:[email protected]

Copyright © 2014 by the American Society of Nephrology

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with adenine-induced chronic renal failure (adenine rats)fed a normal-protein diet.5 These authors speculated that con-sistent vascular calcification might require a longer period ofadenine feeding. On the basis of this idea, they designed a low-protein (LP) diet in an attempt to reduce the nitrogen load andthus enable the rats to thrive on the adenine diet for longerperiods. As a result of this attempt, Price et al. unexpectedlyfound that adenine rats fed a LP diet had extensive vascularcalcification without a longer feeding period.5 All 13 adeninerats fed the LP diet had uniform alizarin red staining of theaorta, whereas only 3 of the 11 adenine rats fed a normal-protein diet had partial calcification.5 These findings indi-cated that dietary protein deficiency correlates with the extent ofvascular calcification.

Proteins are usually made from 20 kinds of amino acids. Onthe basis of nutritional requirements, these amino acids can bedivided into two groups: essential amino acids (EAAs) andnon-EAAs. Because restriction of dietary protein results in ashortage of EAAs, the level of dietary EAAs may be relevant tothe extent of vascular calcification. Among nine EAAs, thisstudy focused on L-lysine (L-Lys) based on the following threereasons. First, L-Lys is the first-limiting amino acid in mostcereal grains.12 Second, the safety of L-Lys supplementationhas been verified in the area of animal husbandry. L-Lys haslong been added to feed grains in order to improve the utilityof feed proteins.13 Third, several studies have demonstratedthat dietary supplementation with L-Lys protects bones fromosteoporosis, a pathologic condition that often coexists withvascular calcification.14,15 These points prompted us to hy-pothesize that supplementation with L-Lys would amelioratevascular calcification. Therefore, in this study, we tested thishypothesis using adenine rats.

RESULTS

L-Lys Ameliorated Arterial Calcification in Adenine RatsTo assess the effects of L-Lys on vascular calcification, we usedLP diet–based adenine rats. The rats at age 13 weeks were di-vided randomly into four groups: LP diet (group LP), LP diet+0.75% adenine (group Ade), LP diet+0.75% adenine+2.5%glycine (group Gly), and LP diet+0.75% adenine+2.5%L-LyszHCl (group Lys) (Figure 1). Glycine served as an aminoacid control in this study because glycine is the amino acidwith the simplest structure.

VonKossa stainingof the thoracic aorta revealed that the ratsin group LP had no calcification (Figure 2Aa), whereas the ratsin groups Ade and Gly had medial calcification (Figure 2, Aband Ac). Calcification was not observed in adenine rats sup-plemented with L-Lys (Figure 2Ad). Alizarin red staining of theaorta showed similar results (Figure 2B). We extracted min-erals from the aortic tissues and quantified the levels of cal-cium, magnesium, and phosphate. In accordance with theresults of von Kossa and alizarin red staining, the levels ofthese minerals were elevated in groups Ade and Gly, but not

in group Lys (Figure 2C). Analyses of aortic root tissues alsoconfirmed that L-Lys inhibited vascular calcification in ade-nine rats (Figure 3).The inhibitory effect of L-Lys on ectopiccalcification was not limited to vascular calcification. We alsofound that L-Lys ameliorated nephrocalcinosis in parathyroidhormone (PTH)–treated rats (Supplemental Figure 1).

Because apoptosis of vascular smoothmuscle cells (VSMCs)is one of the major promoters of vascular calcification, weexamined the effects of dietary L-Lys on apoptosis (Figure 4).Both terminal deoxynucleotidyl transferase–mediated digox-igenin-deoxyuridine nick-end labeling (TUNEL) staining andWestern blot analysis demonstrated that uremia induced ap-optosis of the thoracic aorta in groups Ade and Gly, but not ingroup Lys (Figure 4).

L-Lys Ameliorated Arterial Calcification withoutAffecting Renal FunctionWe measured several parameters to investigate the underlyingmechanism of how dietary L-Lys ameliorated arterial calcifi-cation in adenine rats (Tables 1–4). Compared with group LP,three adenine-loaded groups (groups Ade, Gly, and Lys) hadlow levels of food intake, low body weight, and high levels ofwater intake (Table 1). These parameters were not differentamong the three adenine-loaded groups (Table 1). Serum cre-atinine, urea nitrogen, magnesium, and phosphate equallyincreased, whereas serum pH, 25-hydroxyvitamin D, and1,25-dihydroxyvitamin D equally decreased in the threeadenine-loaded groups (Table 2). All four groups had similarlevels of serum albumin (Table 2). Serum calcium levels ingroups Ade and Gly were lower than in group LP. The rats ingroup Lys had slightly but significantly higher serum calciumlevels than in group Ade (Table 2). Reflecting the high serumcalcium level, a high urinary calcium/creatinine ratio was ob-served in group Lys (Table 3). The urinary volume and urinaryphosphate/creatinine ratio were equally elevated in the threeadenine-loaded groups (Table 3). All four groups had comparable

Figure 1. Experimental design of adenine-induced uremic rats.Twelve-week-old male Sprague-Dawley (SD) rats are maintainedunder a normal MF diet for one week. At age 13 weeks, theanimals are divided randomly into four groups: LP diet (group LP),LP diet+0.75% adenine (group Ade), LP diet+0.75% adenine+2.5% glycine (group Gly), and LP diet+0.75% adenine+2.5%L-LyszHCl (group Lys). At age 18 weeks, the rats are anesthetizedand processed.

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levels of the urinary magnesium/creatinineratio (Table 3). These results indicate thatwe need to explore other mechanisms to un-derstand the effects of dietary L-Lys.

L-Ly Protected the Femora fromOsteoporotic Changes in AdenineRatsWe found that the rats in group Lys hadstrongly suppressed plasma intact PTH(iPTH) (Figure 5A). The strong suppres-sion seemed to depend on the elevated se-rum calcium in group Lys (Table 2) becausethe reduction of maximum cross-sectionalarea of the parathyroid glands in group Lyswas minor (Figure 5, B and C).

Although hyperparathyroidism oftencorrelates with the presence of vascularcalcification, PTH itself cannot directlyinduce calcification in cultured VSMCs orin cultured aortic rings.16 The causal rela-tionship between hyperparathyroidismand vascular calcification depends, at leastin part, on the bone-vascular axis.17 There-fore, we examined the effects of L-Lys onthe femora. Both transverse sections of thefemoral diaphyses (Figure 6A) and three-dimensional reconstructed images of thedistal femora (Figure 6B) demonstratedthat the rats in group LP had intact femora,whereas the rats in groups Ade and Gly hadosteoporotic femora. Dietary supplementa-tionwith L-Lys suppressed these osteoporoticchanges (Figure 6). Three vital predictors ofbone strength—bone mineral density (Fig-ure 6C), porosity (Figure 6D), and the ori-entation of biologic apatite (Figure 6E)—ingroup Lys were identical to those in groupLP, representing the bone-protective effectsof dietary L-Lys.18–21

Dietary Supplementation with L-Lys inAdenine Rats Did Not Elevate PlasmaLys LevelUsing liquid chromatography–electrosprayionization–tandem mass spectrometry, weassessed plasma amino acid levels (Table4).22 All four groups had comparable levelsof plasma Gly, Val, Ile, Leu, Tyr, Met, Cys2,Asp, and a-aminobutyric acid. Bothb-aminoisobutyric acid and g-aminobutyricacid were almost undetectable in all groups.The three adenine-loaded groups hadequally elevated plasma Lys, Phe, andequally suppressed Ser, Trp, and Glu.

Figure 2. L-Lys ameliorates vascular calcification in adenine-induced uremic rats.(A) Calcification of the thoracic aorta is visualized by von Kossa staining. Repre-sentative micrographs from group LP (a), group Ade (b), group Gly (c), and groupLys (d) are shown. Group LP has no calcification, whereas groups Ade and Gly showextensive medial calcifications (black area). Group Lys has no von Kossa–positivearea. (B) Macroscopically, alizarin red staining shows no vascular calcification ingroup LP. The majority of group Ade (six of eight animals) and all rats in group Glyhave extensive calcification (red area) throughout the vasculature. In group Ade,the calcification of the thoracic aorta is relatively weak in two of eight animals. Themajority (six of eight animals) of group Lys has no calcification. Only two of eightanimals in group Lys develop partial vascular calcification. (C) In contrast with ar-teries from group LP, those from groups Ade and Gly contain high levels of cal-cium, magnesium, and phosphate. Compared with group Ade, arteries fromgroup Lys have significantly low amounts of these minerals. All results are pre-sented as means6SD (n=6–8 in each group). ***P,0.001 (Dunnett’s test). Scalebar, 100 mm.

1956 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1954–1965, 2014


Plasma Thr and the His levels did not correlate with the pres-ence of arterial calcification. Elevated plasma a-aminoadipicacid and homoarginine, two metabolites of L-Lys, in group Lysindicated that the rats in group Lys had increased intestinalabsorption of L-Lys.23–25 However, the elevation of plasmaLys in group Lys was not statistically significant. As far as weexamined, plasma Ala, Pro, Arg, Gln, Asn, homoarginine, anda-aminoadipic acid in group Lys seemed to play some roles inthe suppression of vascular calcification. Dietary L-Lys–dependentelevation of plasma Ala, Pro, Arg, Gln, Asn, homoarginine,and a-aminoadipic acid was not limited to the condition

of CKD. Compared with the rats in groupLP (Table 4), rats fed a LP diet containing2.5% L-LyszHCl without 0.75% adeninehad elevated plasma Ala, Pro, Arg, Gln,Asn, homoarginine, and a-aminoadipicacid (Supplemental Table 1).

L-Ala and L-Pro Inhibited Apoptosis ofCultured Human VSMCsUsing the hydrophilic amino acids amongAla, Pro, Arg, Gln, Asn, homoarginine, anda-aminoadipic acid (i.e., Ala, Pro, Arg, andhomoarginine), we further analyzed theunderlying mechanism. Both passive andactive pathways play roles in the pathogen-esis of vascular calcification.26,27 Amongseveral active pathways, apoptosis ofVSMCs is one of the key phenomena thatpromote vascular calcification.28–30 There-fore, using cultured human VSMCs(hVSMCs), we examined the effects ofL-Ala, L-Pro, L-Arg, and L-homoarginine onapoptosis. As shown in Figure 7, calcium/phosphate-boosted DMEM induced cell ap-optosis. Both TUNEL staining (Figure 7, Aand B) and Western blotting (Figure 7C) re-vealed that L-Ala and L-Pro, but not L-Lys,Gly, L-Arg, or L-homoarginine, inhibitedthe apoptosis. These results indicated thatelevated plasma Ala and Pro in group Lys(Table 4) contributed, at least in part, tothe inhibition of aortic apoptosis (Figure 4).

L-Lys, L-Arg, and L-HomoarginineInhibited the Precipitation of Mineralsin a Solution of SupersaturatedCalcium/PhosphateFinally, we examined the effects of aminoacids on the passive pathway of vascularcalcification. We used HEPES-bufferedamino acid solutions (pH 7.4) supersatu-rated with calcium/phosphate for the anal-yses of mineral precipitations. Because thesolubility product of hydroxyapatite is ex-

tremely low, the mixed calcium/phosphate solutions imme-diately developed mineral precipitates (data not shown). Thequantification of calcium and phosphate in the resultantprecipitates showed that L-Lys, L-Arg, and L-homoarginine,but not Gly, L-Ala, or L-Pro, attenuated the mineral precipita-tions (Figure 8A). These findings indicated that L-Arg andL-homoarginine, two amino acids elevated in the plasma ofgroup Lys (Table 4), inhibited the passive pathway of vascularcalcification. The importance of L-Arg in the suppression ofarterial calcification was confirmed in adenine rats supple-mented with dietary 2.5% L-ArgzHCl (Supplemental Figure 2).

Figure 3. L-Lys ameliorates valvular calcification in adenine-induced uremic rats. (A)Calcified areas of aortic root tissues from group LP (a), group Ade (b), group Gly (c),and group Lys (d) are visualized by von Kossa staining. (B) Calcified area fractions (vonKossa–positive area/cross-sectional area of aortic root) are quantitatively analyzed. Allresults are presented as means6SD (n=6–7 in each group). *P,0.05 (Dunnett’s test).Scale bar, 500 mm.

Figure 4. L-Lys suppresses apoptosis of VSMCs in adenine rats. Apoptosis of the aortais assessed by TUNEL staining (A) and Western blotting (B). (A) Thoracic aortae areTUNEL stained in brown. Representative micrographs from group LP (a), group Ade(b), group Gly (c), and group Lys (d) are shown (n=6–7 in each group). (B) Using anti-cleaved caspase-3 antibody, thoracic aortic tissues are subjected to Western blotanalysis. Three independent experiments show similar results. Scale bar, 100 mm.





We also assessed the effects of these six amino acids on thedissolution of hydroxyapatite (Figure 8B). HEPES-bufferedhydroxyapatite mixtures were incubated in the presence orabsence of amino acids for 12 hours. The resultant superna-tants were subjected to calcium/phosphate quantification.Figure 8B shows that Gly, L-Lys, L-Ala, L-Pro, and L-Arg didnot dissolve hydroxyapatite. Although L-homoarginine in-creased the supernatant calcium levels in a dose-dependentmanner, it seemed that calcium contamination in purchasedL-homoarginine caused the elevation (Figure 8Bc). Indeed,calcium quantification of purchased L-homoarginine byboth the methylxylenol blue method and atomic absorptionspectroscopy indicated that the reagent had calcium contam-ination (data not shown). The constant phosphate levels in the

supernatant confirmed that L-homoarginine did not dissolvehydroxyapatite (Figure 8Bd). L-Arg and L-homoarginine in-hibited the precipitation of minerals, but they did not dissolvehydroxyapatite.

DISCUSSION

In this study, we investigated the effects of dietary L-Lys onvascular calcification. Our results clearly demonstrated thatdietary L-Lys ameliorates vascular calcification by modifyingseveral key pathways.

Three observations in our study suggested that dietaryL-Lys increased intestinal absorption of calcium. First, threeadenine-loaded groups had similar levels of food intake (Table 1).Second, group Lys had an increased urinary calcium/creatinineratio (Table 3), in addition to elevated serum calcium (Table 2).Third, L-Lys protected the bones from adenine-induced oste-oporotic changes (Figure 6). Indeed, using orally adminis-tered radioactive calcium as a tracer, Comar et al. reportedthat dietary supplementation of L-Lys facilitates intestinalabsorption of calcium.31,32 Because intraperitoneally injectedL-Lys does not facilitate calcium absorption,32 it is likely thatdietary L-Lys works within the gastrointestinal tract. Ourresults in Figure 8A showed that L-Lys elevated the solubilityof calcium. This phenomenon seems to explain the reason, atleast in part, why dietary L-Lys facilitates intestinal absorptionof calcium.

Osteoporosis is one of the serious pathologic conditionsassociated with vascular calcification. In addition to morpho-logic changes, this study revealed that the orientation ofbiologic apatite in the femora was degraded in groups Ade andGly (Figure 6E). Dietary L-Lys almost completely preventedthese changes (Figure 6E). Because the orientation of biologicapatite is the key determinant of the bone intrinsic materialproperty, a bone possessing a more elevated degree of apatiteorientation shows a higher Young’s modulus in the orienteddirection.19 Therefore, all of our data in Figure 6 confirm the

bone-protective effects of L-Lys. DietaryL-Lys plays a vital role in bone microstruc-ture and mechanical function.

In accordance with some previous re-ports, we demonstrated that both L-Ala andL-Pro inhibit Ca/P-induced apoptosis incultured hVSMCs (Figure 7).33,34 There-fore, elevated plasma Ala and Pro in groupLys seemed to be responsible, at least inpart, for the protection of VSMCs from ap-optotic death (Figure 4, Table 4). Severalpreceding studies revealed the underlyingmechanisms of how L-Ala and L-Pro inhibitapoptosis. Grosser et al. demonstrated thatL-Ala protects cultured human endothelialcells from hydrogen peroxide–mediatedcytotoxicity by inducing heme oxygenase

Table 1. Physical parameters of the adenine rats

ParameterAdenine Administration Period

0 wk 2 wk 4 wk 5 wk

Food intake (g/d)Group LPa 18.762.4 27.063.0 29.067.9 33.067.6Group Adeb 19.761.4 18.363.9 11.763.5 11.561.0Group Gly 21.162.5 15.467.3 15.168.9 10.065.5Group Lys 22.363.4 15.367.2 17.069.5 14.868.1

Water intake(ml/d)

Group LPc 33.063.3 21.761.5 18.363.9 18.364.5Group Adeb 36.666.3 52.964.9 41.466.4 38.5614.4Group Gly 35.765.6 46.666.7 44.666.7 36.364.8Group Lys 35.363.7 57.065.6 48.764.8 44.4615.2

Body weight (g)Group LPa 396.068.4 400.069.7 389.0618.9 388.3616.7Group Adeb 400.366.9 332.9610.3 289.7618.5 255.5619.8Group Gly 400.0614.8 324.3616.1 283.4611.9 240.3610.5Group Lys 400.765.8 315.0611.8 279.7614.2 242.8618.0

Physical parameters are summarized. All results are presented asmeans6SD.Statistical significance was evaluated by multivariate ANOVA (n=4–7 in eachgroup).aP,0.001.bReference group.cP,0.05.

Table 2. Serum parameters of the adenine rats at 5 weeks

Parameter Group LP Group Adea Group Gly Group Lys

Creatinine (mg/100 ml) 0.5160.13b 6.3260.95 5.7660.92 5.3761.53Urea nitrogen (mg/100 ml) 6.6463.66b 180.9649.1 220.8664.4 129.7671.6pH 7.6560.04b 7.2360.12 7.2260.14 7.1960.12Albumin (g/100 ml) 3.1460.31 3.0360.21 3.0060.28 3.3560.44Calcium (mg/100 ml) 9.6560.35b 8.6060.40 8.2660.56 10.0860.34b

Magnesium (mg/100 ml) 2.4260.40b 4.3160.81 3.9960.41 4.1260.34Phosphate (mg/100 ml) 6.8963.04b 32.1964.35 36.6368.02 26.3366.2025-hydroxyvitamin D (ng/ml) 37.8168.73b 23.1964.87 22.1363.04 20.2663.011,25-dihydroxyvitamin D (pg/ml) 103.97633.79b 5.0360.90 5.0260.73 5.1360.98

Serum parameters are summarized. All results are presented asmeans6SD. Statistical significance wasevaluated by Dunnett’s test. n=10 in each group for creatinine, urea nitrogen, pH, albumin, calcium,magnesium, and phosphate. n=9 in each group for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.aReference group.bP,0.001.



and ferritin proteins.35 Furthermore, Krishnan et al. demon-strated that L-Pro modulates the intracellular redox status andprotects cells from H2O2 stress.36 These molecular mecha-nisms might also be responsible for the protection of VSMCsunder Ca/P stimuli (Figure 7) and adenine-induced uremia(Figure 4).

Hydroxyapatite is one of the main components of calcifiedaortic tissues.37 Because of its hexagonal crystallographicstructure, hydroxyapatite has two crystal planes: a-planesand c-planes.18,38,39 The a-planes are rich in calcium ionsand are positively charged, whereas the c-planes are rich inphosphate ions and are negatively charged.40,41 This propertyof hydroxyapatite affects the growth of hydroxyapatite itself.Using self-assembled monolayers that have CH3, PO4H2,COOH, CONH2, OH, or NH2 terminal functional groups,Tanahashi et al. revealed that the negatively charged groupsstrongly promote hydroxyapatite formation.42 The results ofTanahashi et al. suggest that the electrical adsorption of cal-cium ions on the negatively charged surface, but not the ad-sorption of phosphate ions on the positively charged surface,promotes hydroxyapatite formation.42 Indeed, Tanase et al.demonstrated that the growth of hydroxyapatite on thec-plane of crystallographically aligned hydroxyapatite is fasterthan that on the a-plane.43 In this study, we revealed that L-Lys,L-Arg, and L-homoarginine, but not Gly, L-Ala, or L-Pro, at-tenuated mineral precipitations in supersaturated calcium/phosphate solutions (Figure 8A). As shown in SupplementalFigure 3, the precipitate-inhibitory amino acids have two

amino groups (2NH2) and are positivelycharged under physiologic pH. Positivelycharged amino acids might interfere withthe electrical interaction between calciumand a negatively charged surface, althoughthis is not experimentally confirmed in thisstudy.

Homoarginine has caught researchers’attention in recent years because results oftwo clinical studies revealed that low serumhomoarginine, a metabolite of L-Lys, is in-dependently associatedwith cardiovascularmortality and fatal strokes.23,44 The causalrelationship between low homoarginineand an increased incidence of cardiovascu-lar events has been mainly attributed tosuppressed nitric oxide (NO), because ho-moarginine induces NO by two pathways:(1) homoarginine itself serves as a substratefor NO synthase; and (2) homoarginine in-hibits arginase and thereby increasesarginine, a major substrate for NOsynthase.23 In addition to the property ofhomoarginine as an inducer of NO, our cur-rent study revealed that homoarginineworksas an inhibitor of mineral precipitations(Figure 8A). The property of homoarginine

as an inhibitor of arginase seems to explain the reason, at least inpart, why plasma Arg, another precipitate-inhibitory aminoacid, was elevated in group Lys (Figure 8A, Table 4).23 There-fore, homoarginine is likely to have both direct and indirectprecipitate-inhibitory effects in vivo. Because vascular calcifi-cation has been revealed to be associated with an increasedincidence of cardiovascular events,1–4 the property of homo-arginine as a precipitate-inhibitor seems to play importantroles.

Dietary supplementation of L-Lys in adenine rats elevatedplasma Ala, Pro, Arg, and homoarginine, but not Lys itself(Table 4). The putative underlying metabolic pathways thatcaused these changes in group Lys are summarized in Supple-mental Figure 4. First, the statistically unchanged plasma Lyslevel in group Lysmight be caused by an induction of catabolicenzymes for L-Lys due to prolonged dietary supplementationof L-Lys. Indeed, the elevation of plasma a-aminoadipic acidand homoarginine, two metabolites of L-Lys, in group Lys in-dicated that the rats in group Lys more rapidly catabolizedL-Lys than the rats in group Ade. In contrast with the rats ingroup Lys (Table 4), however, the rats fed the LP diet contain-ing 2.5% L-LyszHCl without 0.75% adenine (SupplementalTable 1) had elevated plasma Lys. Because the adenine ratsate a lower amount of the diet (Table 1), a balance betweenintestinal absorption and the catabolism of L-Lys seems to de-termine plasma Lys levels. Second, an induction of amino acidtransporters, such as b0,+-AT and y+LAT-1 systems, in the in-testine might increase intestinal absorption of Arg and His in

Table 3. Urinary parameters of the adenine rats

ParameterAdenine Administration Period

0 wk 2 wk 4 wk 5 wk

Urinary volume (ml/6 h)Group LPa 5.8361.79 2.3761.44 1.9561.20 2.3162.12Group Adeb 5.5962.49 7.6563.16 6.7162.28 4.5064.80Group Gly 3.2761.53 5.9961.90 5.0462.21 3.3762.82Group Lys 4.0961.99 5.8562.68 6.4262.59 3.0162.95

Calcium/creatinineGroup LP 0.14160.090 0.04160.021 0.09860.067 0.08560.033Group Adeb 0.14660.070 0.01860.012 0.12360.073 0.22460.037Group Gly 0.16460.060 0.01260.011 0.13860.055 0.30660.111Group Lysc 0.17660.078 0.06460.043 0.27460.084 0.49060.155

Magnesium/creatinineGroup LP 0.53460.251 0.12360.042 0.13060.064 0.21260.125Group Adeb 0.53360.225 0.39660.099 0.35160.108 0.45360.135Group Gly 0.44860.115 0.26060.065 0.39960.093 0.20360.170Group Lys 0.36960.172 0.28260.082 0.36360.060 0.15260.137

Phosphate/creatinineGroup LPa 0.09860.072 2.04461.112 2.50360.686 2.33861.076Group Adeb 0.39960.486 2.11760.340 2.93060.526 4.47460.868Group Gly 0.40560.403 1.88960.459 2.76060.306 4.45060.958Group Lys 0.33460.383 2.18560.415 2.77560.531 4.14260.548

Urinary parameters are summarized. All results are presented asmeans6SD. Statistical significancewasevaluated by multivariate ANOVA (n=4–7 in each group).aP,0.05.bReference group.cP,0.01.









group Lys. He et al. recently reported thatdietary supplementation of L-Lys upregu-lates jejunal b0,+-AT and y+LAT-1 in pig-lets.45 Absorbed L-Arg and L-His can beconverted to L-Pro, L-Gln, and/or L-Alathrough the pathway shown in Supplemen-tal Figure 4. The presence of elevatedplasma ornithine, a metabolite of L-Arg,in group Lys supports the idea that themet-abolic flow from L-Arg to L-Pro, L-Gln,and/or L-Ala was very high in group Lys(Supplemental Table 2). Third, skeletalmuscle of group Lys might serve as a sourceof plasma Gln and Ala. L-Gln and L-Ala aretwo major amino acids supplied from skel-etal muscles.46 Although we did not di-rectly evaluate the effect of dietary L-Lyson skeletalmuscle in adenine rats, theobservedsuppression of plasma 3-methylhistidine, anindex of the muscular breakdown rate,in group Lys suggested that the rats ingroup Lys were protected from musculardegradation (Supplemental Table 2).47

Our findings agreed with those of a studyof Ishida et al., in which they demonstratedthat dietary L-Lys downregulates E3 ubiquitinligase atrogin-1 (also known as muscleatrophy F-box), a key enzyme for muscledegradation.48 Fourth, the similar plasmaGlu levels among the three adenine-loadedgroups might be attributed to proximal tu-bular damage by adenine. The proximaltubule is the major expression site ofglutaminase, a key enzyme that generatesGlu+NH4

+ from Gln.49 Because the proxi-mal tubules were severely damaged, the ratsin group Lys might have been unable toconvert Gln to Glu+NH4

+, even under thehigh Gln condition. The rats fed the LP dietcontaining L-Lys without adenine showedan elevation in Glu, which is in sharp con-trast with the rats with adenine. This obser-vation supports the idea, at least in part, thatproximal tubular glutaminase contributedto the elevation of plasma Glu (Supplemen-tal Table 1).

Although we have demonstrated in thisstudy that dietary L-Lys prevents arteriesfrom calcification in adenine-induced ure-mic rats, the therapeutic potential of die-tary L-Lys for treating preexisting vascularcalcification remains obscure. Furtherstudies are required to address this issue.

In conclusion, dietary supplementationwith L-Lysprevented arteries fromcalcification

Table 4. Plasma amino acid levels of the adenine rats at 5 weeks

Amino Acid (nmol/ml) Group LP Group Adea Group Gly Group Lys

Glycine (Gly) 249.0674.8 265.6683.1 400.46216.5 416.06193.9Alanine (Ala) 561.9668.4b 346.56100.1 304.6681.9 473.76126.7c

Serine (Ser) 339.1649.7b 118.6630.0 167.0676.9 163.7652.1Threonine (Thr) 31.566.0b 417.66120.1 203.9680.5b 268.1696.9d

Valine (Val) 78.4610.4 86.3626.5 110.3653.0 142.16155.7Isoleucine (Ile) 37.965.3 40.6611.3 52.0622.7 57.6656.1Leucine (Leu) 77.166.8 72.4622.1 92.0642.6 111.66114.5Lysine (Lys) 210.0635.9d 432.36110.0 405.66164.1 498.66242.9Arginine (Arg) 89.2618.0 91.3618.9 94.2624.2 125.6628.0d

Histidine (His) 53.2610.2d 86.0612.7 92.5629.7 121.8628.5d

Tyrosine (Tyr) 29.968.5 29.564.3 36.8614.3 36.2617.2Phenylalanine (Phe) 28.663.4c 42.366.5 53.1619.2 45.7611.0Tryptophan (Trp) 47.067.9b 12.561.8 13.664.6 12.562.9Methionine (Met) 23.264.0 25.263.7 28.569.9 28.966.4Cystin (Cys2) 17.664.3 25.3610.2 24.968.8 26.865.7Proline (Pro) 192.3629.1b 120.6629.8 107.8621.9 173.4643.1d

Glutamine (Gln) 574.0681.6 520.76107.8 522.96106.3 759.16169.2d

Glutamic acid (Glu) 78.7619.3d 45.4612.8 45.4614.8 49.7616.3Asparagine (Asn) 34.265.6 38.666.3 36.668.2 53.768.7b

Asparatic acid (Asp) 6.961.9 7.361.8 7.662.7 6.162.1a-Aminoadipic acid 1.260.5 2.360.4 2.260.5 4.162.5c

Homoarginine (Homo-Arg) 3.260.5 4.460.7 3.861.0 6.563.0c

a-aminobutyric acid 3.060.5 3.561.4 2.560.7 4.762.3b-aminoisobutyric acid Not detectable Not detectable Not detectable 0.160.3g-aminobutyric acid Not detectable Not detectable Not detectable Not detectable

Plasma amino acid levels are summarized. All results are presented as means6SD. Statistical signifi-cance was evaluated by Dunnett’s test (n=10 in each group).aReference group.bP,0.001.cP,0.05.dP,0.01.

Figure 5. Dietary L-Lys suppresses plasma iPTH level in adenine rats. (A) The plasmaiPTH level is determined by ELISA. Compared with group LP, groups Ade and Glyhave elevated plasma iPTH. L-Lys supplementation strongly suppresses plasma iPTH.Results are presented as means6SD (n=10 in each group). (B) Maximum cross-sectionalarea of the parathyroid glands is assessed using hematoxylin and eosin–stained sec-tions. Results are presented as means6SD (n=5–13 in each group). (C) Representativehematoxylin and eosin–stained micrographs of parathyroid glands from group LP (a),group Ade (b), group Gly (c), and group Lys (d) are shown. *P,0.05; ***P,0.001(Dunnett’s test). Scale bar, 100 mm.









by modifying several key pathways via the following mechanisms:(1) dietary L-Lys strongly suppresses plasma iPTH; (2) dietary L-Lyssupports a proper bone-vascular axis; (3) dietary L-Lys elevatesplasma alanine and proline, thereby inhibiting apoptosis ofVSMCs; and (4) dietary L-Lys elevates plasma arginine and homo-arginine, thereby inhibiting mineral precipitation. Our findingsprovide a novel preventive approach for managing vascular calci-fication.

CONCISE METHODS

AnimalsTwelve-week-old male Sprague-Dawley rats were purchased from

Japan SLC (Hamamatsu, Japan) and were fed a normal MF diet

(Oriental Yeast, Tokyo, Japan) for 1week. At age 13weeks, the animals

were divided randomly into four groups (Figure 1). Glycine served as

an amino acid control in this study because it is the amino acid that

has the simplest structure. Adenine rats fed a LP diet containing 2.5%

L-ArgzHCl were prepared in a similar way (Supplemental Figure 2).

Another group of rats were fed a LP diet with 2.5% L-LyszHCl but

without 0.75% adenine; this group was prepared for the purpose of

testing whether the changes in plasma amino acid levels (Table 4)

were unique to CKD (Supplemental Table 1). Based on the compo-

sition of the TD05030 diet (Harlan Teklad, Madison, WI), a LP diet

containing 2.5% protein, 1.062% calcium, and 0.923% phosphorus

was prepared by CLEA Japan (Tokyo, Japan). Adenine, glycine,

L-LyszHCl, and L-ArgzHCl were purchased fromWako Pure Chemical

Industries (Osaka, Japan).

Nephrocalcinosis in 6-week-old male Wistar rats was induced by

continuous injection of rat PTH 1-34 (Bachem, Bubendorf, Switzer-

land) at a dosage of 40 mg/kg per day via an osmotic mini-pump

(Alzet, Cupertino, CA) for 50 hours.50 L-LyszHCl or glycine at a

dose of 20 mmol/kg was administered via a gastric tube at 2 hours,

14 hours, 26 hours, and 38 hours after the implantation of the os-

motic pump.

At the indicated periods, the rats were processed in similar ways, as

previously described.5,50,51 All rats were handled in a humanemanner

in accordance with the guidelines of the Animal Committee of Osaka

University.

Figure 6. L-Lys protects the femora from osteoporotic changes. Bone morphology is assessed by micro x-ray computed tomography.Transverse sections of the femoral diaphyses (A) and three-dimensional reconstructed images of the distal femora (B) from group LP (a),group Ade (b), group Gly (c), and group Lys (d) are shown. In addition to these morphologic changes, bone mineral density (C), porosity(D), and orientation of the biologic apatite (E) demonstrate the bone-protective effects of L-Lys (n=5 in each group in A and B and n=6–7 in each group in C–E). **P,0.01; *** P,0.001 (Dunnett’s test).





Biochemical ParametersSerum and urinary biochemical parameters, urea nitrogen, albumin,

calcium, inorganic phosphate, and magnesium were determined by

clinical diagnostic reagents (Wako Pure Chemical Industries; and

Kainos Laboratories, Inc., Tokyo, Japan). Serum creatinine and

plasma iPTH were measured by Kainos CRE reagent and the Rat

BioActive Intact PTH ELISA Kit (Immutopics, Inc., San Clemente,

CA), respectively. Serum 1,25(OH)2D levels were

determined by RIA (SRL, Inc., Tokyo, Japan).

Serum samples were sent to Kyowa Medex

Inc. (Shizuoka, Japan) for the serum 25(OH)D

assay using the DiaSorin LIAISON 25-hydroxy

OH vitamin D TOTAL Assay (DiaSorin, Inc.,

Stillwater, MN).52 Plasma amino acid levels

were evaluated by liquid chromatography–elec-

trospray ionization–tandem mass spectrome-

try.22 To investigate mineral accumulation levels

in the aorta, the appropriate alizarin red–stained

tissues were minced and freeze-dried. After

weighing the dried aorta, the minerals were ex-

tracted with 150 mM HCl overnight at room

temperature.

Histologic AnalysesThe thoracic aorta, abdominal aorta, and fem-

oral arteries from each rat were dissected as a

unit, stainedwithAlizarinRedS (Sigma-Aldrich,

St. Louis, MO), and photographed. Histologic

analyses by von Kossa staining and TUNEL

staining (In SituApoptosis DetectionKit; Takara

Bio, Shiga, Japan) were performed using 4%

paraformaldehyde-fixed aortic valve and tho-

racic aorta sections.

Bone AnalysesMorphometric analyses of isolated femoralmid-

shafts were performed using micro x-ray com-

puted tomography (R_mCT2; Rigaku, Tokyo,

Japan). The settings of the R_mCT2 were as

follows: The unit operated with x-ray power at

90 kV and 160 mA with an exposure time of 3

minutes. Three-dimensional microstructural

images were visualized using TRI/3D-BON soft-

ware (RATOC, Tokyo, Japan).

Bone mineral density of the femoral mid-

shafts was measured using peripheral quantita-

tive computed tomography (XCTResearch SA+;

Stratec Medizintechnik, Birkenfeld, Germany)

with a resolution of 803803460mm. Bone tissue

was judged over a threshold value of 690mg/cm3.

The bone mineral density in the femoral mid-

shaft cross-sections was averaged.

The crystallographic orientation of the bi-

ologic apatite c axis was analyzed by conducting

microbeam x-ray diffraction (R-Axis BQ; Ri-

gaku) with a transmission optical system. The apatite orientation

is a valuable index for the analysis of bone microstructure (which

largely depends on the bone type, such as long bones, skull, and

mandible), prediction of bone mechanical function, evaluation of re-

generative and pathologic conditions, and validation of administration

of antiosteoporotic agents.18–20,53,54 Molybdenum-Ka radiation was

generated at 50 kV and 90 mA. The whole femur was vertically

Figure 7. L-Ala and L-Pro suppress apoptosis of cultured hVSMCs. Apoptosis ofhVSMCs is analyzed. Apoptosis is induced by calcium/phosphate (Ca/P)-boostedmedium in the absence or presence of amino acid supplementation. (A) TUNEL-positive nuclei are stained in red. All nuclei are counterstained in blue. Representativemicrographs are shown as follows: (a) negative control [Ca/P-boost (2), amino acidsupplementation (2)], (b) positive control [Ca/P-boost (+), amino acid supplementa-tion (2)], and (c–h) Ca/P-stimulated hVSMCs treated with L-Lys (c), Gly (d), L-Arg (e),L-homoarginine (L-Homo-Arg) (f), L-Ala (g), or L-Pro (h). (B) TUNEL-positive nuclei arequantified using the In Cell Analyzer 6000. (C) Western blot analysis for cleavedcaspase-3 is shown. L-Ala and L-Pro, but not L-Lys, Gly, L-Arg, or L-Homo-Arg inhibitcell apoptosis. All results are presented as means6SD (n=8 in each group).***P,0.001 (Dunnett’s test). Scale bar, 100 mm.



mounted on the specimen holder, and the inci-

dent beam was collimated into an 800-mm cir-

cular spot by a double-pinhole metal collimator.

The incident x-ray radiated vertically onto the

specimen in the anteroposterior axis, and dif-

fracted x-rays were collected by an imaging plate

(FujiFilm, Tokyo, Japan) placed behind the speci-

men for 300 seconds. From the Debye ring ob-

tained, the degree of biologic apatite orientation

was quantitatively analyzed as the intensity ratio of

(002) diffraction peak to (310) peak along the fe-

mur long axis.21 The (002) crystal plane is the rep-

resentative plane of the biologic apatite c axis, and

the (310) plane is orthogonal to the (002) plane.

Calcium-Phosphate PrecipitationAssaysThe formation of calcium-phosphate precipi-

tates in vitro was assessed in HEPES-buffered-

glycine, -L-Lys, -L-Arg, -L-homoarginine, -L-Ala,

and -L-Pro solution. All amino acids were pur-

chased from Wako Pure Chemical Industries.

Amino acid solutions at desired concentrations

were obtained by adding amino acid stock solu-

tions (pH 7.4) to 500 mM HEPES buffer (pH

7.4). To increase the concentrations of calcium

and phosphate, 0.4 M CaCl2 stock solution was

added to an aliquot of HEPES-buffered amino

acid solution to achieve twice the desired final

levels, and 0.4 M sodium phosphate (pH 7.4)

stock solution was added to a second aliquot

of HEPES-buffered amino acid solution to

achieve twice the desired final levels. Fifty micro-

liters of calcium-boosted amino acid solution

was then expelled rapidly into 50 ml of phos-

phate-boosted amino acid solution, and vor-

texed. After 10 minutes of incubation at room

temperature, the mixed solutions were centri-

fuged at 18903g for 10 seconds. The resultant

pellets were dissolved in 150 mM HCl for the

analyses of calcium and phosphate.

Hydroxyapatite Dissolution AssaysThe solubility of hydroxyapatite (Sigma-Aldrich)

was assessed in HEPES-buffered amino acid

solutions. Amino acid solutions at desired con-

centrations were obtained by adding amino acid

stock solutions (pH 7.4) to 500 mM HEPES

buffer (pH 7.4). We added hydroxyapatite to an

aliquot of HEPES-buffered amino acid solution

to achieve the desired final levels. After 12 hours

of rotation at 37°C, the incubated mixtures were

centrifuged at 18903g for 10 seconds. The re-

sultant supernatants were subjected to calcium/

phosphate quantification.

Figure 8. L-Lys, L-Arg, and L-homoarginine (L-Homo-Arg) inhibit the precipitation ofminerals in a solution of supersaturated calcium/phosphate. (A) HEPES-buffered cal-cium/phosphate solution (pH 7.4) is incubated at room temperature for 10 minutes inthe absence or presence of amino acids. All mixtures contain 10 mM calcium and 10mM phosphate. After incubation, the precipitates are collected by centrifugation at18903g for 10 seconds. Calcium (a and c) and phosphate (b and d) levels in the re-sultant pellet are examined. L-Lys, L-Arg, and L-Homo-Arg inhibit mineral precipitationin a dose-dependent manner (n=5 in each point). (B) HEPES-buffered hydroxyapatitemixture (2.0 mM) is incubated at 37°C for 12 hours in the absence or presence ofamino acids. After incubation, the mixtures are centrifuged at 18903g for 10 seconds.The resultant supernatants are subjected to calcium (a and c) or phosphate (b and d)quantification. (c) Contamination with calcium in purchased L-Homo-Arg causes cal-cium elevation in the supernatant. (a–d) All amino acids do not dissolve hydroxyapatite(n=5 in each point). *P,0.05; **P,0.01; ***P,0.001 (Dunnett’s test).



Cell CulturehVSMCs were maintained in DMEM supplemented with 10% FCS.

For the evaluation of cell apoptosis, hVSMCs at 90% confluence were

stimulated with calcium/phosphate-boosted (final calcium 2.7 mM

and phosphate 2.0 mM) DMEM supplemented with 0.5% BSA.

Aminoacid stock solution (pH7.4)was added to the calcium-boosted

medium to elevate each amino acid concentration by 2.0 mM. Af-

ter incubation for 24 hours, samples were subjected to Western

blot and TUNEL analyses. TUNEL-stained cells were analyzed using

the In Cell Analyzer 6000 (GE Healthcare Bio-Sciences, Piscataway,

NJ).

Western Blot AnalysesAntibodies against specific molecules were obtained as follows:

cleaved caspase-3 (no. 9664; Cell Signaling Technology, Danvers,

MA) and b-actin (A5441; Sigma-Aldrich). The dilution rates of the

primary antibodies were 1:10,000 for b-actin and 1:1000 for cleaved

caspase-3. Western blot analyses were carried out in similar ways, as

previously described.55

Statistical AnalysesStatistical significance among multiple experimental values was

evaluated by Dunnett’s test. To analyze the time course of serum

and urine parameters with respect to treatment assignment, changes

in these parameters over time were analyzed with linear mixedmod-

els for repeated measures with random intercept and slope. This

method was applied to take into account the correlation between

repeated measurements within the same rat. The multivariate mod-

els contained the treatment group as well as the number of measure-

ments (time) as independent variables. The group difference was

investigated by adding interaction terms between the exposures

(groups) and time. All results are presented as means6SD. Statisti-

cal significance was defined as P,0.05. All data were statistically

analyzed using JMP Pro 10.0.2 for Windows (SAS Institute, Cary,

NC) or STATA/SE 11.0 for Windows (StataCorp LP, College Station,

TX).

ACKNOWLEDGMENTS

The authors thank Naoko Horimoto for her technical assistance. The

authors also thank the Center of Medical Research and Education,

OsakaUniversity Graduate School ofMedicine, for technical support.

This researchwas supportedbyagrant-in-aid for scientific research

fromthe JapaneseMinistryofEducation,Culture, Sports, Science, and

Technology (24790844 to I.M.), and grants from the Osaka Kidney

Foundation (OKF12-0001 to I.M.) and the Kidney Foundation, Japan

(JKFB11-8 to I.M.).

DISCLOSURESK.T. is an employee of Ajinomoto Pharmaceuticals Co. Ltd. Y.K. is an

employee of Ajinomoto Co. Inc. T.H. and Y.Ts. are members of a department

that has received donations from Chugai Pharmaceutical Co. Ltd.

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