* Division of Nephrology; Division of Cardiovascular Medicine, Department of Medicine; Department of Pathology, Kurume University, School of Medicine, Kurume, Japan; and Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan
Address correspondence to: Dr. Seiji Ueda, Division of Nephrology, Department of Medicine, Kurume University, School of Medicine, 67 Asahi-machi, Kurume, 830-0011, Japan. Phone: +81-942-31-7002; Fax: +81-942-31-7763; E-mail: ueda{at}med.kurume-u.ac.jp
Received for publication December 29, 2005.
Accepted for publication May 17, 2006.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitorof nitric oxide synthase. ADMA is generated by protein methyltransferase(PRMT) and is metabolized mainly by dimethylarginine dimethylaminohydrolase(DDAH). ADMA levels are reported to increase in patients withchronic kidney disease (CKD), thereby playing a role in thepathogenesis of accelerated atherosclerosis in this population.However, the precise mechanism underlying ADMA accumulationin these patients is not fully understood. This study investigatedthe molecular mechanism for the elevation of ADMA levels inCKD, using a rat remnant kidney model that represents progressiveCKD. After male Sprague-Dawley rats underwent baseline measurementof BP and renal function, 5/6 subtotal nephrectomy (5/6Nx) and4/6 nephrectomy were performed. Plasma and urinary levels ofADMA and symmetric dimethylarginine, an inert isomer of ADMA,were measured by HPLC. Expression levels of PRMT genes and DDAHproteins were analyzed by semiquantitative reverse transcription–PCRand Western blotting, respectively. Plasma ADMA levels wereelevated in the Nx groups in proportion to the degree of nephrectomydespite marked increases in renal clearance of ADMA. In contrast,renal clearance of symmetric dimethylarginine was decreasedand its plasma levels were increased in the Nx groups. Furthermore,both liver and kidney gene expression of PRMT was increased,whereas DDAH protein expression was decreased in the 5/6Nx group.Plasma ADMA levels were correlated with systolic BP levels.Moreover, adenovirus-mediated DDAH gene transfer into the 5/6Nxrats prevented the elevation of BP levels, which was associatedwith the reduction of plasma and urinary ADMA levels. The resultspresented here suggest that decreased DDAH levels as well asincreased PRMT gene expression could cause the elevation ofplasma ADMA levels, thereby eliciting hypertension in CKD. Substitutionof DDAH protein or enhancement of its activity may become anovel therapeutic strategy for the treatment of hypertension-relatedvascular injury in CKD.
Cardiovascular disease is a major cause of death in patientswith ESRD (1). Endothelial dysfunction as a result of reducedbioavailability of nitric oxide (NO) is an initial step of atherosclerosisin patients with chronic kidney disease (CKD) (2,3). NO is synthesizedby stereospecific oxidation of the terminal guanidine nitrogenof l-arginine by the action of the NO synthase (NOS). The synthesisof NO can be blocked by inhibition of the NOS active site withguanidino-substituted analogues of l-arginine, such as asymmetricdimethylarginine (ADMA) (2,3). This modified amino acid is deliveredfrom proteins that have been posttranslationally methylatedand subsequently hydrolyzed (4). ADMA is formed by protein methyltransferase(PRMT) (5) and metabolized mainly by NG,NG-dimethylargininedimethylaminohydrolase (DDAH) (6–8).
We, along with others, have demonstrated that high levels ofADMA are observed in hypertension (9), hypercholesterolemia(10), diabetes (11), and CKD (3). Furthermore, we previouslyshowed that there was a close association between plasma ADMAlevels and carotid intima-media thickness, one of the surrogatemarkers of atherosclerosis in the general population (12). Inaddition, ADMA is a strong prognostic marker of cardiovascularevents in patients with coronary artery disease (13), diabetes(14), and ESRD (15). These observations suggest that the elevationof ADMA could contribute to accelerated atherosclerosis by inactivatingNOS in CKD. However, the precise molecular mechanism underlyingADMA elevation in this population is not fully understood. Therefore,in this study, we investigated the kinetics of methylated arginines,ADMA and symmetric dimethylarginine (SDMA), an inert isomerof ADMA, and expression levels of PRMT genes and DDAH proteins,using a rat remnant kidney model, an experimental model of progressiveCKD.
Experimental Protocol
After male Sprague-Dawley rats (200 to 250 g) underwent baselinemeasurement of BP and renal function, 4/6 nephrectomy (4/6Nx;right nephrectomy with surgical resection of the lower thirdof left kidney) or 5/6 nephrectomy (5/6Nx; right nephrectomywith surgical resection of the lower and upper thirds of leftkidney) were performed as described previously (16). Sham-operatedrats (sham) underwent the same procedure without the surgicalreduction of the kidney. Twelve weeks after the operation, BPwas measured using a tail-cuff sphygmomanometer and an automatedsystem with a photoelectric sensor (BP-98A; Softron, Tokyo,Japan). After measurements of BP, the rats were transferredto metabolic cages for 2 d for urinalysis and then killed. Allexperimental procedures were conducted in accord with the NationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals and were approved by the ethical committee of our institution.
Chemical Analysis
Plasma and urinary levels of SDMA, ADMA, and nitrate and nitrite(NOx) were measured by HPLC as described previously (8). Creatinine(Cr) and blood urea nitrogen were determined with commercialkits (DENKA SEIKEN Co., Tokyo, Japan, and Alfresa Pharma Co.,Osaka, Japan, respectively). Cr clearance (Ccr; ml/min) wasdetermined by the following formula: [urine Cr (mg/dl)] x [urinevolume (ml/d)]/[plasma Cr (mg/dl)]/[1440 (min)]. Renal clearanceof dimethylarginines (ADMA and SDMA) was calculated by the followingformula [urine dimethylarginine (µM)] x [urine volume(ml/d)]/[plasma dimethylarginine (µM)]/[1440 (min)].
Antibodies against DDAH-I and DDAH-II
A mAb against DDAH-I was prepared as described previously (8).A rabbit polyclonal antibody against DDAH-II–specificpeptide (LHRGGGDLPNSQE [amino acids 235 to 247]) were preparedby Sigma Genosys (Ishikari, Japan). We confirmed that the antibodyagainst DDAH-II peptide actually bounds to a recombinant DDAH-IIprotein (data not shown).
Western Blot Analysis
The kidney cortex or liver tissues were homogenized (Polytron;Brinkmann Instruments, Westbury, NY) and lysed with 500 µlof 25 mmol/L Tris-HCl (pH 7.4) that contained 1% Triton X-100,0.1% SDS, 2 mmol/L EDTA, and 1% protease inhibitor cocktail(Nakarai tesque, Kyoto, Japan). Then the supernatant was collectedafter centrifugation at 14,000 rpm for 30 min at 4°C. Westernblot analysis was performed as described previously (17). Forconfirmation that equal amounts of the proteins were appliedfor each lane, the membranes were reprobed with an antibodyfor -actin (Sigma, St. Louis, MO). Densitometric analysis wasperformed with National Institutes of Health image software,and the relative ratio to the -actin expression was calculatedin each sample.
Primers and Probes
Primer sequences that were used in semiquantitative reversetranscription–PCR (RT-PCR) were 5'-AACTGAAGCTCGCACTCTCG-3'and 5'-TCAGCACAGATCTCCTTGGC-3' for PRMT-1, 5'-TCGGACAACCAAGTACCACA-3'and 5'-CCACCTTCTGCTGAAACACA-3' for PRMT-2, and 5'-CCTCAGTGTTCTCCAGCCATAGCC-3'and 5'-GGCTATGGCTGGAGAACACTGAGG-3' for PRMT-3. Sequences ofthe upstream and downstream primers that were used in semiquantitativeRT-PCR for detecting rat glyceraldehyde-3-phosphate dehydrogenasemRNA were the same as described previously (18).
Semiquantitative RT-PCR
Poly(A)+RNA were isolated from kidney cortex or liver tissuesand analyzed by RT-PCR as described previously (18). The amountsof poly(A)+RNA templates (30 ng) and cycle numbers (28 cyclesfor PRMT-1 gene, 33 cycles for PRMT-2 gene, 32 cycles for PRMT-3gene, and 22 cycles for glyceraldehyde-3-phosphate dehydrogenasegene) for amplification were chosen in quantitative ranges,where reactions proceeded linearly, which had been determinedby plotting signal intensities as functions of the templateamounts and cycle numbers (18).
Recombinant Adenoviruses
The plasmid, including the entire coding region of rat DDAH-IcDNA, was cloned as described previously (8). For producingAdv-DDAH, an adenovirus vector that expresses DDAH protein underthe control of the cytomegalovirus promoter, the entire codingregion of DDAH was inserted into an E1, E3-deleted, human adenovirusserotype 5 mutant, dl7001, with homologous recombination in293 cells (American Tissue Culture Collection, Bethesda, MD)as described previously (8). Four weeks after 5/6Nx, BP wasmeasured and blood and urine samples were collected. Then therats were randomly divided into two groups; one was injectedwith 1.5 x 1010 plaque-forming units of Adv-DDAH through thetail vein, the other with Adv-LacZ, which encodes bacterial-galactosidase. Determination of infection efficiencies by insitu X-Gal staining of the liver revealed that at least 80%of cells were positive for -galactosidase 14 d after the infectionwith Adv-LacZ (data not shown). DDAH infection was found actuallyto increase its level and enzymatic activity in the liver byapproximately 2.5-fold compared with that of Adv-LZ–infectedrats (data not shown).
Statistical Analyses
All data were expressed as mean ± SE. Experimental groupswere compared by ANOVA and, when appropriate, with Scheffe testfor multiple comparisons. Linear regression analysis was performedbetween plasma levels of ADMA and SDMA, Ccr, urinary proteinexcretion, and total cholesterol levels and BP. A level of P< 0.05 was accepted as statistically significant.
Characteristics of the Experimental Model
As shown in Table 1, body weight was significantly lower inthe 5/6Nx group compared with the sham and the 4/6Nx groups.Resection of the kidney increased blood urea nitrogen (sham18.6 ± 0.6; 4/6Nx 41.0 ± 7.9; 5/6Nx 109 ±30 mg/dl) and Cr levels (sham 0.34 ± 0.04; 4/6Nx 0.85± 0.2; 5/6Nx 2.2 ± 0.5 mg/dl) and decreased Ccrvalues (50% decrease in 4/6Nx, 90% decrease in 5/6Nx). Subtotalnephrectomy (4/6Nx and 5/6Nx) also increased systolic BP levelsand urinary protein excretions (Table 1).
Plasma and Urine Levels of NOx, lArginine, and Methylated Arginines
As shown in Table 2, plasma ADMA and SDMA were increased inproportion to the degree of nephrectomy. Renal clearance ofADMA was markedly increased in the Nx groups, whereas that ofSDMA was decreased. Furthermore, urinary excretion of NOx wassignificantly decreased in proportion to the degree of nephrectomy(sham 11.6 ± 2.1; 4/6Nx 6.4 ± 2.7; 5/6Nx 2.4 ±0.9 µmol/d; P < 0.01), whereas the plasma levels ofl-arginine, a NO substrate, were similar among the three groups(sham 192 ± 44; 4/6Nx 188 ± 24; 5/6Nx 165 ±7.2 µmol/L; NS). Plasma levels of ADMA were negativelycorrelated to urinary excretion levels of NOx (r2 = 0.2, P <0.05).
Table 2. Plasma and urinary excretion levels of methylated arginines and their renal clearancea
ADMA-Related Enzyme Expression in Both Kidney and Liver
Because impaired renal clearance of ADMA was not a cause ofADMA accumulation in this model, we next examined expressionlevels of PRMT genes and DDAH proteins. As shown in Figure 1,A and B, renal and liver gene expression levels of type I PRMT(PRMT-1 and PRMT-3), which catalyze the asymmetric dimethylationand monomethylation of arginine residues and resultantly producesADMA and NG-monomethyl-larginine (5), were significantly increasedin the 5/6Nx group. The level of type II PRMT (PRMT-2), whichproduces SDMA and NG-monomethyl-larginine but not ADMA (5),had a tendency to increase in the 5/6Nx group (Figure 1A).
Figure 1. Protein methyltransferase (PRMT) gene and dimethylarginine dimethylaminohydrolase (DDAH) protein levels 12 wk after nephrectomy. (Top) Representative reverse transcription–PCR (RT-PCR) bands of PRMT in kidney (A) and liver (B) and Western blots of DDAH proteins in kidney (C) and liver (D). (Bottom) Quantitative representation of PRMT genes and DDAH proteins. Data were normalized by the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or -actin protein and were expressed as mean ± SE (n = 6). *P < 0.05 compared with the value of the sham-operated rats.
It has been reported that two isoforms of DDAH (DDAH-I and DDAH-II)are expressed in both kidney and liver (19). Therefore, we nextinvestigated the expression levels of DDAH in our model. Asshown in Figure 1, C and D, the renal and liver expression levelsof DDAH-I and DDAH-II were significantly decreased in the 5/6Nxgroup. As shown in Figure 2, the changes in DDAH and PRMT levelswere observed at an early phase of nephrectomy. In parallelto these alterations, urinary ADMA excretion was found to beincreased.
Figure 2. Time course of changes in PRMT gene and DDAH protein expression after nephrectomy. (A) Levels of urinary asymmetric dimethylarginine (ADMA) excretion were measured on the indicated days (n = 5). Data were normalized by the concentration of urinary creatinine. *P < 0.01 compared with the value of the rats before nephrectomy. (B, top) Representative RT-PCR bands of PRMT in kidney. (B, bottom) Quantitative representation of PRMT genes. (C, top) Western blots of DDAH proteins in kidney. (C, bottom) Quantitative representation of DDAH proteins. Data were normalized by the intensity of GAPDH gene or -actin protein and were expressed as mean ± SE (n = 5). *P < 0.05 compared with the value of the rats before nephrectomy.
ADMA and BP
To assess the pathologic role for ADMA in CKD, we next investigatedthe relationship between ADMA and BP. By linear regression analysis,plasma levels of ADMA (Figure 3A) and SDMA (Figure 3B), Ccr(r2 = 0.61, P < 0.0001), urinary protein excretion (r2 =0.55, P = 0.0003), and total cholesterol levels (r2 = 0.55,P = 0.0002) were found to correlate to BP. Multiple stepwiseregression analysis revealed that Ccr was a sole determinantfor BP (data not shown). Ccr was more strongly and negativelycorrelated with plasma levels of SDMA (r2 = 0.66, P < 0.01)than those of ADMA (r2 = 0.33, P < 0.01). Therefore, to investigatethe direct involvement of ADMA in BP, we examined the effectof DDAH overexpression on BP in our model. DDAH infection significantlyreduced plasma and urinary ADMA levels (plasma levels of ADMAbefore infection 0.64 ± 0.05 µM and 14 d afterinfection 0.56 ± 0.03 µM [P < 0.05]; urinarylevels of ADMA before infection 7.6 ± 1.6 µmol/gCr and 14 d after infection 2.2 ± 0.9 µmol/g Cr[P < 0.01]) and subsequently prevented the elevation of BPin the 5/6Nx rats (Figure 4). It has been reported that micethat overexpress DDAH have a higher heart rate (20), whereasinfusion of ADMA into humans lowers the heart rate (21). However,the heart rate was not changed during the experimental periodsin this study (before infection 385 ± 13 bpm; 14 d afterinfection 379 ± 10 bpm).
Figure 3. Correlations between systolic BP and plasma levels of ADMA (A) and symmetric dimethylarginine (SDMA; B). sham, sham-operated rats (; n = 9), 4/6Nx; 4/6 nephrectomy rats (•; n = 7), 5/6Nx; 5/6 nephrectomy rats (; n = 6).
Figure 4. Effect of DDAH overexpression on BP in the remnant kidney model. Four weeks after the 5/6Nx, adenovirus vector encoding DDAH (Adv-DDAH; ; n = 18) or encoding -galactosidase (Adv-LZ; •; n = 12) were injected into the rats. Then BP was measured on the indicated days. *P < 0.05 compared with the value of the Adv-LZ.
The salient findings of this study were that (1) renal excretionof ADMA was increased rather than decreased; (2) PRMT, whichis an enzyme to synthesize ADMA, was increased and DDAH, whichis a key-limiting enzyme to metabolize ADMA but not SDMA, wasdecreased; (3) plasma levels of ADMA were correlated with BPlevels; and (4) DDAH overexpression decreased plasma levelsof ADMA and subsequently prevented the elevation of BP levelsin the animal model of CKD.
There is a growing body of evidence to show that ADMA is implicatedin the pathogenesis of atherosclerosis (2,12). Indeed, plasmaADMA levels are elevated in patients with CKD (3,22–24)and are a strong predictor for cardiovascular disease and mortalityin these patients (15,25). Until now, it was assumed that impairedurinary clearance could account, at least in a part, for theelevation of ADMA level in CKD. However, in this study, we demonstratedthat urinary clearance of ADMA was increased rather than decreasedin the Nx groups than in the sham-operated group. In contrast,renal clearance of SDMA was disturbed in the Nx groups. Theseobservations suggest that impaired renal clearance of SDMA couldbe a main cause of the elevation of this methylated arginine,whereas that of ADMA cannot explain its elevation in our CKDmodel. It has been reported that when dimethylarginines areinjected intravenously, 66% of SDMA is recovered in the urine,whereas only 5% of ADMA is recovered in the urine (26). Theseobservations suggest that SDMA could be almost entirely excretedby kidney, whereas ADMA could be metabolized extensively ratherthan excreted in urine. Similar findings were reported by Banchaabouchiet al. (27). In their study, ADMA levels in 80% nephrectomizedrats increased not only in the plasma but also in the urine;urinary ADMA levels increased up to eight-fold, but they alsoshowed that patients with CKD and nephrectomized mice had adecreased renal clearance of ADMA, suggesting marked differencesof urinary clearance of ADMA between species.
To investigate further the molecular mechanism for the elevationof ADMA, we next examined expression levels of PRMT genes andDDAH proteins. In this study, we found that expression levelsof PRMT-1 and PRMT-3 were increased in the 5/6Nx group. Thesefindings suggest that enhancement of ADMA production could beone possible mechanism for the elevation of ADMA in our CKDmodel. In support of our findings, Okubo et al. (28) demonstratedthat inhibition of PRMT by adenosine dialdehyde decreased plasmaADMA levels in an experimental model of ESRD. Furthermore, wedemonstrated here that DDAH-I and DDAH-II protein expressionswere significantly decreased in the 5/6Nx group. These observationssuggest that impaired metabolism of ADMA as a result of reducedDDAH levels also may be a causal factor for the elevation ofADMA in this model. More than 90% of ADMA is eliminated by theaction of DDAH in rats (29). In addition, the impaired abilityof DDAH was associated with ADMA accumulation in diabetic ratsor hypercholesterolemic rabbits without renal dysfunction (11,30).These findings strongly support that decreased levels or activityof DDAH also could contribute to the elevation of ADMA levelsin CKD.
In this study, we cannot clarify the molecular mechanisms ofupregulation of PRMT and downregulation of DDAH in our CKD model.However, we speculate that oxidative stress could be involvedin the dysregulation of PRMT and DDAH by the following evidence:(1) Gene expressions of type I PRMT were increased by oxidizedLDL in cultured endothelial cells (EC) through a redox-regulatedmechanism (5); (2) DDAH activities in EC and smooth muscle cellswere reduced under high glucose conditions, which were preventedby an antioxidant, polyethylene glycol–conjugated superoxidedismutase (11); and (3) it is widely known that oxidative stressgeneration is increased in patients with CKD (31,32). Uremia-relatedoxidative stress as well as uremic toxins such as homocysteineand advanced glycation end products, which decrease DDAH activity(33,34), may have contributed to dysregulation of these enzymes.Furthermore, recently, coupling factor 6, an endogenous inhibitorof prostacyclin synthesis, was found to be elevated in patientswith ESRD and positively associated with plasma levels of ADMA(35). In addition, coupling factor 6 increased EC gene expressionof PRMT, whereas it decreased that of DDAH (36). Taken together,oxidative stress and/or coupling factor 6 could be involvedin dysregulation of PRMT and DDAH in our models. Moreover, inthis study, the time-course experiments (Figure 2) revealedthat the alterations of DDAH and PRMT levels were observed atearly phase of nephrectomy, whose changes were parallel to theincrease in urinary ADMA excretion. These results suggest thatearly hemodynamic changes after nephrectomy also may affectthe levels of DDAH and PRMT. We also do not know the mechanismsby which renal resection increased urinary ADMA excretion inthis study. However, it is possible that the increased urinaryexcretion was due to the systemic accumulation of ADMA by overproductionand decreased degradation of ADMA. In this study, DDAH overexpressionreduced urinary excretion of ADMA, also suggesting decreasedextrarenal clearance of ADMA.
Hypertension is the most common complication in patients withCKD and is not only a predictor of mortality of cardiovascularcomplications but also a determinant for progression of renaldisease (37). In this study, plasma levels of ADMA were correlatedto BP levels. Furthermore, DDAH overexpression decreased plasmalevels of ADMA and subsequently prevented the elevation of BPlevels. Moreover, Dayoub and colleagues (20,38) recently reportedthat BP is significantly lower in DDAH-transgenic mice thanin wild-type mice. These observations suggest the pathologicrole for the increase of plasma ADMA levels in the elevationof BP in CKD. However, several papers showed no correlationbetween ADMA and BP (39,40). Other factors than ADMA have beenknown to participate in the BP elevation after subtotal nephrectomy,such as angiotensin II (41). Furthermore, several factors, suchas age, insulin resistance, and dyslipidemia, also could affectthe plasma levels of ADMA (5,10,12,30,42). These confoundingfactors and/or cardiac medications might attenuate the positivecorrelation between plasma ADMA levels and BP, which could explainthe negative clinical studies.
Decreased DDAH as well as increased PRMT expression but notimpaired renal clearance of ADMA could cause the elevation ofplasma ADMA levels, thereby eliciting BP elevation in CKD. Substitutionof DDAH or enhancement of its activity may become a novel therapeuticstrategy for the treatment of hypertension-associated vascularinjury in CKD.
Acknowledgments
This work was supported in part by a grant from Grant-in-Aidfor Scientific Research from the Ministry of Education, Scienceand Culture, Tokyo; a grant from Japan Foundation of CardiovascularResearch, Tokyo; and a grant from the Ishibashi Foundation forthe Promotion of Science, Kurume.
Parts of this work were presented at the 38th annual meetingof American Society of Nephrology, November 8 to 13, 2005; Philadelphia,PA; and at the 3rd World Congress of Nephrology, Singapore,June 26 to 30, 2005.
We thank F. Imamura for excellent technical support.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-actin (Sigma, St. Louis, MO). Densitometric analysis was performed with National Institutes of Health image software, and the relative ratio to the 
; n = 9), 4/6Nx; 4/6 nephrectomy rats (•; n = 7), 5/6Nx; 5/6 nephrectomy rats (
; n = 6).