Degrading Pathways in Coronary Endothelial Dysfunction in Chronic Kidney Disease

Role of Nitric Oxide–Producing and –Degrading Pathways in Coronary Endothelial Dysfunction in Chronic Kidney Disease

Satoru Tatematsu,* Shu Wakino,* Takeshi Kanda,* Koichiro Homma,* Kyoko Yoshioka,* Kazuhiro Hasegawa,* Naoki Sugano,* Masumi Kimoto,† Takao Saruta,* and Koichi Hayashi* *Department of Internal Medicine, School of Medicine, Keio University, Tokyo, and †Department of Nutritional Science, Okayama Prefectural University, Okayama, Japan

Cardiovascular events are accelerated in chronic kidney disease (CKD). Although deranged nitric oxide (NO) pathways and asymmetric dimethylarginine (ADMA) cause endothelial dysfunction, no direct evidence for coronary artery endothelial dysfunction in CKD has been documented. CKD was induced in male dogs by heminephrectomy (1/2Nx) or five-sixths nephrectomy (5/6Nx). After 4 wk, renal ablation reduced GFR (control 76 [54 to 85]; 1/2Nx 38 [29 to 47]; 5/6Nx 15 [12 to 46] ml/min) and elevated plasma ADMA (control 1.88 [1.68 to 2.54]; 1/2Nx 2.51 [2.11 to 3.55]; 5/6Nx 3.84 [2.16 to 3.95] ␮mol/L). Coronary circulatory responses to acetylcholine revealed marked increases in coronary blood flow in control group (83 ؎ 17% increment) but blunted responses in 1/2Nx (34 ؎ 8% increment) and 5/6Nx (20 ؎ 4% increment). The acetylcholine-induced changes in epicardial arteriolar diameter, using needle-lens probe charge-coupled device videomicroscopy, showed similar results. The responsiveness to sodium nitroprusside did not differ among three groups. Plasma nitrite/nitrate levels decreased in 1/2Nx and 5/6Nx, and the mRNA expressions of dimethylarginine dimethylaminohydrolase-II (DDAH-II), ADMA-degrading enzyme, and endothelial NO synthase (eNOS) in coronary arteries were downregulated in 1/2Nx and 5/6Nx. Finally, 4-wk treatment with all-trans retinoic acid restored the impaired endothelium-dependent vasodilation and reversed the expression of eNOS but not DDAH-II. Coronary endothelial function is impaired in the early stage of CKD. The dysfunction is attributed to the downregulation of eNOS and/or DDAH-II in coronary arteries. Furthermore, the manipulation of NO pathways may constitute a therapeutic strategy for the prevention of coronary dysfunction in CKD. J Am Soc Nephrol 18: 741–749, 2007. doi: 10.1681/ASN.2006040367

here is a growing body of evidence that several cardio- pendent vasodilation is impaired in several diseases that are vascular risk factors reside in chronic kidney disease associated with atherosclerosis, which may contribute to the T (CKD) (1,2), and the incidence of cardiovascular events progression of atherosclerosis (9). Although the mechanisms of increases even in the stage of apparent macro- (3) or microalbu- endothelial dysfunction are multifactorial, one contributing ab- minuria (4). Notably, endothelial dysfunction is established as normality is decreased expression and/or activity of eNOS in a predictor for cardiovascular outcomes (5,6), and the impaired the vascular tissue (10,11). Recent studies have demonstrated endothelium-dependent vasodilation is observed in patients that the increased level of asymmetric dimethylarginine who have CKD and are on maintenance hemodialysis (7). Al- (ADMA), an endogenous NOS inhibitor, contributes to the though endothelial injury/dysfunction constitutes a critical de- development of endothelial dysfunction in various diseases terminant of atherosclerosis through multiple processes, in- (12). Because its levels are increased in patients with CKD, it is cluding lipid accumulation, smooth muscle proliferation, and surmised that plasma ADMA levels are predictive of future thrombosis (8), whether the progression of CKD impairs the cardiovascular events and overall mortality in CKD (13). In- endothelial function in the coronary artery has not been eluci- deed, in patients with CKD stages 1 through 4, ADMA is well dated. correlated with cardiovascular events and survival (14,15). Fi- One of the most important functions of the endothelium is a nally, the short-term administration of ADMA causes de- source of various vasoactive substances. Nitric oxide (NO) is a creased cardiac output and elevated systemic vascular resis- potent vasodilator that is produced by endothelial NO synthase tance (16,17). Of note, ADMA is metabolized by the enzyme (eNOS) and is deemed an antiatherogenic substance. NO-de- dimethylarginine dimethylaminohydrolase (DDAH), which was shown recently to have two isoforms, DDAH-I and DDAH-II, and different tissue distribution and regulation for Received April 19, 2006. Accepted December 29, 2006. each isoform (18). Furthermore, whereas symmetric dimethyl- Published online ahead of print. Publication date available at www.jasn.org. arginine (SDMA), an enantiomer of ADMA, is eliminated al- Address correspondence to: Dr. Koichi Hayashi, Department of Internal Medi- most exclusively by urinary excretion, ADMA is partly elimi- cine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Phone: ϩ81-3-5363-3796; Fax: ϩ81-3-3359-2745; E-mail: nated by urinary excretion and extensively metabolized by [email protected] DDAH (16,19). It is possible, therefore, that DDAH constitutes

Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1803-0741 742 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 741–749, 2007 a crucial determinant of plasma ADMA levels and contributes New York, NY) placed at the proximal portion of the left circumflex to the pathogenesis of endothelial dysfunction in CKD. coronary artery (20). The changes in CBF after 5 min of the commence- In this study, we investigated the role of NO and ADMA in ment of each drug administration were evaluated. mediating the endothelial dysfunction of the coronary artery in End-diastolic diameters of epicardial coronary arterioles were measured with intravital needle-lens charge-coupled device videomicros- dogs with various degrees of CKD, using an intravital needle- copy (Nihon Kohden, Tokyo, Japan) (20). The needle-lens probe was lens charge-coupled device videomicroscopy (20). Further- enclosed in a sheath with a doughnut-shaped balloon on the tip to more, we examined whether the expressions of eNOS and avoid direct compression of the arteriole by the needle tip, and the tip DDAH in the coronary artery were altered. We demonstrated of the probe was placed gently on the epicardial surface. A green filter that coronary endothelial dysfunction developed even in the was applied to contrast the image of the vessel with the surrounding early stage of CKD. Furthermore, the expression levels of both tissue. Images were converted into black and white video signals and eNOS and DDAH-II in the coronary vessels were downregu- were analyzed by a computer (840AV; Apple Computer, Cupertino, lated in CKD. These studies endorse the important role of the CA) with a freeze-frame modality (20). deranged regulation of eNOS and DDAH/ADMA pathways in the initiation of cardiovascular abnormalities in CKD. Finally, Biochemical Analyses the manipulation of the NO pathway and ADMA could con- Plasma triglyceride, total cholesterol, HDL cholesterol, LDL choles- stitute a therapeutic strategy for the prevention of cardiovas- terol, and glucose were measured with the enzymatic colorimetric cular events in CKD. assay. Insulin levels were measured with the immunoradiometric assay method (Abbot Japan, Tokyo, Japan). Plasma C-reactive protein, total Materials and Methods plasminogen activator inhibitor-1, and von Willebrand factor were Animal Preparation determined by ELISA. Plasma concentrations of homocysteine, l-arginine, ADMA, and SDMA were determined by HPLC (23). Plasma Adult male hybrid dogs (American foxhounds, Labrador retrievers, nitrite/nitrate (NOx) concentrations were evaluated using the Griess and beagles, 16 to 20 kg) were fed a standard diet (80 mmol/d sodium, reaction (24). 20 g/d nitrogen; Oriental Yeast Co., Tokyo, Japan) and had free access to tap water at all times. A Tygon tube was inserted into the abdominal aorta through the right femoral artery for measurement of mean arterial mRNA Isolation, cDNA Synthesis, and Real-Time PCR pressure and heart rate under the conscious condition (21). CKD was Total RNA was isolated from the dog coronary artery with Trizol ϭ induced in dogs by heminephrectomy (1/2Nx group; n 8) or by the Reagent (Invitrogen, Carlsbad, CA). Total RNA (50 ng) was reverse- ligation of two or three branches of the left renal artery and the transcribed for cDNA synthesis with the SuperScript First-Strand Syn- subsequent contralateral nephrectomy (five-sixths nephrectomy thesis System (Invitrogen) for the quantification of mRNA expression. ϭ [5/6Nx] group; n 10). Four weeks after the operation, each dog was Real-time PCR was performed using the ABI PRISM-7700 sequence used for experiment. Sham-operated dogs were used for normal control detector (PE Applied Biosystems, Foster City, CA) with SYBER Green I ϭ (n 8). In additional experiments, all-trans retinoic acid (atRA; 2 Dye (PE Applied Biosystems) for the detection of PCR reaction. The mg/kg per d orally), which was recently reported to increase NO sequences that were used for PCR were as follows: DDAH-I 5Ј-catg- synthesis in endothelial cells (22), was administered to 5/6Nx dogs for gctgggcctaacctaat-3Ј for forward primer and 5Ј-tgagtttgtcatagcggtg- 4 wk, and the same experimental procedures were conducted (control gtc-3Ј for reverse primer; DDAH-II 5Ј-cagctgctgactgcctctttc-3Ј for for- ϭ ϭ ϩ ϭ n 7; 5/6Nx n 6; 5/6Nx atRA n 7). ward primer and 5Ј-aggtaccagggtgacatcagaga-3Ј for reverse primer; For the evaluation of the coronary vascular responsiveness and car- eNOS 5Ј-cagcacaagagttataagatccgc-3Ј for forward primer and 5Ј-actg- diac function, dogs were anesthetized with sodium pentobarbital (25 gactccttcctcttccg-3Ј for reverse primer; and 18S rRNA 5Ј-ggttgatcctgc- mg/kg) and were under the control of a ventilator. A thoracotomy was cagtagcatatg-3Ј for forward primer and 5Ј-ggccgtgcgtacttagacatg-3Ј for performed in the left fifth intercostal space, and the heart was sus- reverse primer. pended in a pericardial cradle. A 6-Fr pigtail catheter (Goodtec, Gifu, Japan) was inserted into the left ventricular (LV) cavity through the right carotid artery to measure LV pressure (LVP). The positive first Immunohistochemistry derivative of LVP was obtained to assess the cardiac contractility. The protein expressions of eNOS, DDAH-I, and DDAH-II in the For measurement of renal plasma flow (RPF) and GFR, 7-Fr balloon coronary artery were examined by immunohistochemistry. Tissue sec- catheters (Create Medic, Yokohama, Japan) were temporarily inserted tions were incubated with primary antibodies against eNOS (Transduc- in the bladder through the urethra for urine collection. Bolus injection tion Laboratories, Lexington, KY) and DDAH-I and II (Abcam, Cam- of 20% p-aminohippurate (200 mg) and 25% inulin (1200 mg) was bridge, UK) at dilution 1:200 in 5% nonfat milk in PBS for 2 h at 37°C followed by a continuous infusion of p-aminohippurate (3 mg/min) for primary antibodies. After washing with PBS, tissue sections were and inulin (12 mg/min) at a rate of 1.5 ml/min. The infusion was incubated with secondary anti-goat IgG antibodies conjugated with started 90 min before the experiments to obtain stable blood levels of FITC (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:50 dilution. For each agent. All experimental procedures were conducted according to negative control, the same procedure was performed except for the the guideline of the Animal Care Committee of Keio University. addition of primary antibodies. Immunolabeled sections were examined with confocal laser scanning microscopy (LSM-510; Carl Zeiss, Oberkochen, Germany) at an excitation wavelength of 488 nm. Experimental Protocols After a 30-min stabilization period, acetylcholine (ACh; 0.001 to 0.1 ␮g/kg per min; Sigma, St. Louis, MO) or sodium nitroprusside (SNP; Statistical Analyses 0.01 to 1 ␮g/kg per min; Maruishi Seiyaku, Tokyo, Japan) was infused Data are expressed as median (range) or mean Ϯ SEM. Results are directly into the left circumflex coronary artery. Coronary blood flow analyzed by one-way/two-way ANOVA, as appropriate, followed by (CBF) was measured with an ultrasonic flowmeter (Transonic Systems, Fisher post hoc test. P Ͻ 0.05 was considered statistically significant. J Am Soc Nephrol 18: 741–749, 2007 Coronary Endothelial Function in CKD 743

Results microscopy (Figure 2). The vasodilator response of coronary Baseline Data of Dogs in CKD arterioles to ACh (0.1 ␮g/kg per min) was blunted in a renal As shown in Table 1, 1/2Nx and 5/6Nx reduced both GFR function–dependent manner (28.3 Ϯ 7.7, 14.6 Ϯ 3.1, and 5.5 Ϯ and RPF in a renal mass reduction–dependent manner. Al- 2.3% increment, for control, 1/2Nx, and 5/6Nx, respectively; though body weight was decreased in dogs with CKD (1/2Nx P Ͻ 0.05; each n ϭ 5; Figure 2E). In contrast, no difference in and 5/6Nx), neither mean arterial pressure nor heart rate was arteriolar response to SNP was observed at any dosage exam- altered. Food consumption was not different among these ined among control, 1/2Nx, and 5/6Nx (each n ϭ 5; Figure 2F). groups. No significant difference in baseline CBF, LV end- These results coincide with those of CBF in response to ACh diastolic pressure, or positive first derivative of LVP was noted and SNP. among control, 1/2Nx, and 5/6Nx. Blood glucose, insulin, and lipid levels were not different Plasma NOx Levels and Expression Levels of eNOS and among the three groups (Table 2). Neither C-reactive protein DDAH-I/II in Coronary Artery nor the markers that are associated with endothelial dysfunc- Plasma NOx levels were decreased in both 1/2Nx and 5/6Nx tion (fibrinogen, total plasminogen activator inhibitor-1, and as compared with those in control, although no significant von Willebrand factor) differed among these groups. Plasma difference was observed between 1/2Nx and 5/6Nx (control homocysteine concentrations increased by reducing renal mass, 22.1 Ϯ 2.8 ␮mol/L [n ϭ 5]; 1/2Nx 13.4 Ϯ 0.9 ␮mol/L [n ϭ 8; although the differences did not attain statistical significance. P Ͻ 0.01]; 5/6Nx 10.6 Ϯ 2.4 ␮mol/L [n ϭ 6; P Ͻ 0.001]; Figure Plasma ADMA concentrations were elevated in 1/2Nx and 3A). The eNOS mRNA level in 1/2Nx did not differ from that 5/6Nx, compared with control group. The l-arginine/ADMA in the control group, but marked downregulation was observed ratio, a surrogate marker of NO production capacity, was sig- in coronary arteries from 5/6Nx (Figure 3B). Whereas no dif- nificantly decreased in 1/2Nx and 5/6Nx. Plasma SDMA levels ference in DDAH-I mRNA expression levels was noted among were elevated as renal mass was reduced. Of note, GFR was three groups (n ϭ 5; Figure 3C), the DDAH-II mRNA expres- significantly correlated with plasma SDMA levels (r ϭϪ0.851, sion was pronouncedly downregulated in both 1/2Nx and P Ͻ 0.0001), as well as serum creatinine (r ϭϪ0.749, P ϭ 5/6Nx (n ϭ 5; Figure 3D). 0.0013). Figure 4 illustrates the immunohistochemical analysis on the coronary artery showing the protein expression levels of eNOS Effects of ACh and SNP on Coronary Circulation and DDAH-I/II. In the control group, eNOS, DDAH-I, and The ACh-induced changes in CBF were markedly reduced in DDAH-II antibody produced intense labeling primarily along 1/2Nx and 5/6Nx, compared with those in normal control the endothelial layer (Figure 4). In the 1/2Nx and 5/6Nx (32.8 Ϯ 7.9, 10.2 Ϯ 5.5, and 0.8 Ϯ 2.4% increments at 0.01 ␮g/kg groups, there was a loss of immunoreactivity of each molecule, per min; 83.0 Ϯ 16.7, 33.5 Ϯ 7.9, and 19.6 Ϯ 3.6% increments at the degree of which seemed to be greater in eNOS and 0.1 ␮g/kg per min, for control, 1/2Nx, and 5/6Nx, respectively; DDAH-II than in DDAH-I. each n ϭ 5; Figure 1A). In contrast, SNP elicited dosage-dependent vasodilation, similar in magnitude, in control, 1/2Nx, and Effect of atRA on CBF and Its Related Enzymes 5/6Nx at any dosage examined (each n ϭ 5; Figure 1B). Four-week treatment with atRA did not alter GFR (5/6Nx 17 Next, we directly evaluated whether endothelium-dependent [12 to 31]) ml/min [n ϭ 5]; 5/6NxϩatRA 22 [12 to 25] ml/min vasodilation of the coronary microcirculation was impaired in [n ϭ 6]), BP (5/6Nx 105 [86 to 114] mmHg [n ϭ 5]; CKD, using intravital needle-lens charge-coupled device video- 5/6NxϩatRA 102 [96 to 114] mmHg [n ϭ 5]), or LV function

Table 1. Baseline hemodynamic parameters in dogs with normal kidneys and CKDa

Control 1/2Nx 5/6Nx Parameter (n ϭ 5) (n ϭ 5) (n ϭ 5)

GFR (ml/min) 76 (54 to 85) 38 (29 to 47)b 15 (12 to 46)b,d RPF (ml/min) 175 (147 to 296) 155 (144 to 179)b 86 (61 to 113)b,d Body weight (kg) 18.0 (17.5 to 19.5) 17.0 (16.2 to 18.2)c 16.7 (16.0 to 18.6)b MAP, conscious (mmHg) 105 (93 to 119) 100 (83 to 107) 111 (92 to 114) HR, conscious (beats/min) 100 (84 to 115) 99 (87 to 106) 103 (93 to 122) CBF (ml/min) 40 (29 to 65) 47 (32 to 56) 43 (27 to 58) LVEDP (mmHg) 5.0 (3.9 to 8.4) 5.7 (4.1 to 6.7) 4.4 (4.1 to 5.3) LVdP/dt (mmHg/s) 3230 (2674 to 4599) 2633 (1450 to 4023) 3996 (1785 to 4458)

aData are median (range). 1/2Nx, heminephrectomy; 5/6Nx, five-sixths nephrectomy; CKD, chronic kidney disease; RPF, renal plasma flow; MAP, mean arterial pressure; HR, heart rate; CBF, coronary blood flow; LVEDP, left ventricular end- diastolic pressure; LVdP/dt, peak positive derivative of LV pressure. bP Ͻ 0.01, cP Ͻ 0.05 versus control. dP Ͻ 0.05 versus 1/2Nx. 744 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 741–749, 2007

Table 2. Baseline characteristics in dogs with normal kidneys and CKDa

Control 1/2Nx 5/6Nx Characteristic (n ϭ 5) (n ϭ 5) (n ϭ 5)

Blood glucose (mmol/L) 4.1 (3.6 to 5.2) 3.9 (3.2 to 4.2) 4.4 (3.9 to 4.8) IRI (␮U/ml) 5.0 (5.0 to 12.0) 4.7 (1.3 to 23.0) 5.0 (4.0 to 17.0) Triglyceride (mmol/L) 0.29 (0.23 to 0.47) 0.34 (0.28 to 0.67) 0.36 (0.16 to 0.58) Total cholesterol (mmol/L) 3.85 (3.03 to 4.50) 3.96 (3.67 to 4.91) 4.37 (3.78 to 4.73) HDL cholesterol (mmol/L) 2.87 (2.35 to 3.28) 2.90 (2.48 to 3.21) 3.39 (2.33 to 3.49) LDL cholesterol (mmol/L) 0.10 (0.03 to 0.13) 0.21 (0.08 to 0.52) 0.13 (0.05 to 0.28) C-reactive protein (mg/dl) 0.1 (0.0 to 0.1) 0 (0.0 to 0.1) 0 (0.0 to 0.1) Fibrinogen (␮mol/L) 8.0 (6.7 to 10.7) 10.4 (9.6 to 14.4) 7.7 (5.5 to 12.9) Total PAI-1 (ng/ml) 10 (9 to 11) 10 (10 to 11) 10 (9 to 13) von Willebrand factor (%) 12 (10 to 45) 10 (9 to 62) 32 (12 to 35) Homocysteine (␮mol/L) 6.3 (4.5 to 9.2) 7.7 (4.7 to 10.4) 7.8 (6.8 to 12.2) l-arginine (␮mol/L) 127.3 (90.6 to 144.0) 90.4 (81.0 to 121.7) 106.7 (97.4 to 112.9) ADMA (␮mol/L) 1.88 (1.68 to 2.54) 2.51 (2.11 to 3.55)b 3.84 (2.16 to 3.95)c l-arginine/ADMA 56.5 (54.6 to 63.4) 33.7 (22.9 to 57.7)b 28.2 (25.4 to 51.7)c SDMA (␮mol/L) 0.29 (0.22 to 0.36) 0.49 (0.47 to 0.54)c 0.81 (0.49 to 0.85)d,e

aData are median (range). ADMA, asymmetric dimethylarginine; IRI, immunoreactive insulin; PAI-1, plasminogen activator inhibitor-1; SDMA, symmetric dimethylarginine. bP Ͻ 0.05, cP Ͻ 0.01, dP Ͻ 0.001 versus controls. eP Ͻ 0.01 versus 1/2Nx.

7; Figure 6C). In contrast, atRA failed to alter the expression of DDAH-I (Figure 6D) or DDAH-II (Figure 6E). In immunohistochemical analysis, the diminished eNOS staining was restored by the 4-wk treatment with atRA (Figure 7A), although atRA had no effect on the staining of DDAH-I (Figure 7B) or DDAH-II (Figure 7C).

Discussion In this study, we demonstrated that the coronary artery Figure 1. Effects of acetylcholine (ACh) or sodium nitroprusside manifests blunted responsiveness to ACh at the stage of mild to (SNP) on coronary blood flow (CBF) in chronic kidney disease moderate CKD (Figure 1). In contrast, the vasodilator response (CKD) and correlation between plasma asymmetric dimethyl- to SNP is preserved even in moderate CKD. These observations arginine (ADMA) levels and endothelium-dependent vasodila- indicate that the endothelium-dependent vasodilation of the tion. (A) ACh-induced increase in CBF was markedly impaired coronary artery is impaired not only in advanced (i.e., moder- in dogs with heminephrectomy (1/2Nx; Ⅺ) and five-sixths ate) CKD but also in early-stage (i.e., mild) CKD. Several recent nephrectomy (5/6Nx; ‚) compared with that in control dogs studies have shown impaired endothelial function in various (E). (B) SNP-induced increase in CBF did not differ among the three groups. Percentage change from basal levels of CBF is vascular beds, including isolated mesenteric (25), carotid (26), expressed as mean Ϯ SEM. **P Ͻ 0.01, ***P Ͻ 0.001 versus and subcutaneous resistance arteries (27). Our findings there- control; ##P Ͻ 0.01 versus baseline. fore provide direct in vivo evidence for endothelial dysfunction of the coronary artery. Of note, our experimental model did not manifest elevation in BP (Table 1), a finding that is in accor- (data not shown). Similarly, atRA had no effect on plasma dance with previous studies that showed that dogs with rem- homocysteine or ADMA levels. Treatment with atRA, however, nant kidneys are resistant to the induction of hypertension markedly restored the ACh-induced changes in CBF in 5/6Nx (28,29). Furthermore, cardiac dysfunction is not apparent in our (Figure 5A) without affecting the SNP-induced vasodilation model. It is most likely that impaired renal function per se (Figure 5B). constitutes a critical determinant of the coronary endothelial Whether the ameliorated vasodilator response to ACh was function in CKD, which may be associated with increased risks mediated by the alterations in eNOS or DDAH was examined for cardiovascular events in CKD. (Figure 6). atRA increased plasma NOx levels but had no effect A growing body of evidence suggests that ADMA is respon- on plasma ADMA levels in 5/6Nx. Concomitantly, atRA re- sible for the endothelial dysfunction in various disorders. Pre- stored the suppressed eNOS expression in 5/6Nx to the level of vious studies demonstrated the relationship between plasma normal control (control n ϭ 5; 5/6Nx n ϭ 6; 5/6NxϩatRA n ϭ ADMA levels and the degree of endothelial dysfunction of J Am Soc Nephrol 18: 741–749, 2007 Coronary Endothelial Function in CKD 745

Figure 3. Plasma nitrite/nitrate (NOx) levels and mRNA expression of endothelial nitric oxide synthase (eNOS), dimethylargi- Figure 2. Effects of ACh or SNP on coronary arteriole diameters nine dimethylaminohydrolase-I (DDAH-I), and DDAH-II in the in CKD. (A through D) Representative images of epicardial coronary artery of CKD. (A) Plasma NOx levels were decreased coronary arterioles are shown. (A) Baseline in control dogs. (B) as renal function was impaired. The mRNA expressions of ACh 0.1 ␮g/kg per min in control dogs. (C) Baseline in 5/6Nx eNOS (B; control n ϭ 5; 1/2Nx n ϭ 8; 5/6Nx n ϭ 6), DDAH-I dogs. (D) ACh 0.1 ␮g/kg per min in 5/6Nx dogs. Diameters of (C; control n ϭ 6; 1/2Nx n ϭ 6; 5/6Nx n ϭ 7), and DDAH-II (D; arterioles were measured by marking the vessels on the camera control n ϭ 5; 1/2Nx n ϭ 6; 5/6Nx n ϭ 6) in the coronary artery screen. (E) ACh-induced coronary vasodilation was impaired in were analyzed by real-time PCR. The mRNA expression levels dogs with 1/2Nx (Ⅺ) and 5/6Nx (‚) compared with that in of each molecule were normalized by that of 18S rRNA. The control dogs (E). (F) SNP elicited the coronary vasodilation, mRNA expression of eNOS and DDAH-II was markedly down- similar in magnitude in control, 1/2Nx, and 5/6Nx dogs. Per- regulated in the 1/2Nx and 5/6Nx groups. The expression of centage dilation from basal levels of diameter of coronary ar- DDAH-I was also reduced, but the change did not attain sta- teriole is expressed as mean Ϯ SEM. *P Ͻ 0.05, **P Ͻ 0.01 versus tistical significance. Results are expressed as means Ϯ SEM. control. **P Ͻ 0.01, ***P Ͻ 0.001 versus control; †P Ͻ 0.05 versus 1/2Nx. forearm vascular beds in patients who were undergoing maintenance hemodialysis (30) and in hypertensive patients (31). Although our study indicates significant increases in sys- Furthermore, coronary endothelial dysfunction is indepen- temic ADMA levels in CKD, the regulatory mechanisms of dently associated with the plasma ADMA concentration in men ADMA have not been elucidated. Because the kidney provides with early coronary atherosclerosis (32). Although reduced re- one route for clearance of ADMA, it is not surprising that nal function is associated with attenuated coronary vasodilator plasma ADMA levels are elevated in patients with CKD (35). capacity in patients with nonobstructive coronary artery dis- However, we have demonstrated that renal function is not a ease (33), no investigation has examined the endothelial func- determinant of the plasma ADMA level because the ADMA tion of the coronary artery in CKD. We demonstrate that cor- level is not correlated with GFR, a finding that is in accordance onary endothelial dysfunction is already apparent in mild to with previous reports (36). Furthermore, induction of total moderate CKD, which endorses the recent findings that the risk nephrectomy was recently demonstrated to decrease, rather for cardiovascular events is already high in patients with kid- than increase, plasma ADMA levels in rats (37). Alternatively, ney disease and microalbuminuria (4). In this regard, CKD is our study demonstrated that the mRNA level of DDAH-II, a characterized by hyperhomocysteinemia in humans (34). Be- degrading enzyme for ADMA, is decreased in the tissues of the cause our study failed to show a significant elevation in homo- coronary artery at the early stage of CKD (Figure 3D). In this cysteine levels, our canine model may not completely reflect the regard, we previously reported that the renal mRNA level of situation in humans with CKD. In addition, a potential limita- DDAH-II was decreased and suggested that this alteration was tion of using dogs as an experimental animal is the low number responsible for the endothelial dysfunction of renal arterioles in each study group. (38). Therefore, the downregulation of DDAH-II in various 746 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 741–749, 2007

Figure 4. Tissue expressions of eNOS, DDAH-I, and DDAH-II in Figure 6. Effects of atRA on plasma NOx and ADMA and coronary arteries of CKD. Representative fluorescence immu- mRNA expression of eNOS, DDAH-I, and DDAH-II in coro- nohistochemical images of coronary artery for eNOS (A), nary artery. Decreased plasma NOx levels were restored by DDAH-I (B), and DDAH-II (C) in control, 1/2Nx, and 5/6Nx atRA in 5/6Nx (A), whereas plasma ADMA levels were not dogs are shown. Immunoreactivities of eNOS and each isoform altered (B). The treatment with atRA restored the suppressed of DDAH were detected along the endothelial layer. Fluores- eNOS (C) expression in 5/6Nx to the level of normal control. In contrast, atRA failed to alter the expression of DDAH-I (D)/ cence intensities of eNOS and DDAH-II seemed to be decreased Ϯ Ͻ in the 1/2Nx and 5/6Nx groups as compared with the control DDAH-II (E). Results are expressed as means SEM. *P 0.05, Ͻ ‡ Ͻ group. **P 0.01 versus control; P 0.05 versus 5/6Nx.

mocysteine [40], hyperglycemia [41]) reduce the activity of DDAH without the alteration of its expression. Furthermore, oxidative stress downregulates the expressions of DDAH-II (42). Whereas we show distinct regulation of DDAH-I and DDAH-II in dogs with CKD (Figure 3), Matsuguma et al. (43) demonstrated the downregulation of both DDAH-I and DDAH-II in subtotally nephrectomized rats, which manifest elevated BP. Although the reason for these divergent effects of CKD is unknown, different experimental settings such as animal species and BP might affect these differences. Figure 5. Effects of all-trans retinoic acid (atRA) on endotheli- Of interest, our study shows the intimate correlation between um-dependent and -independent vasodilation of coronary ar- GFR (estimated by inulin clearance) and SDMA (r ϭϪ0.851). tery. (A) Four-week treatment with atRA markedly restored the This intriguing finding is in accordance with the results of a decreased ACh-induced changes in CBF in 5/6Nx. (B) The recent meta-analysis (44) and therefore lends support to the treatment with atRA did not affect the SNP-induced vasodila- premise that SDMA constitutes a novel marker of renal function. E, control; Ⅺ, 5/6Nx; ‚, 5/6NxϩatRA. **P Ͻ 0.01, ***P Ͻ tion. Further investigations would establish the role of SDMA 0.001 versus control; ‡‡P Ͻ 0.01 versus 5/6Nx; ##P Ͻ 0.01 versus baseline. as a reliable index of renal function. Besides ADMA as an endogenous substance for coronary endothelial dysfunction, our study has demonstrated that the tissues, including the kidney and coronary vessels, would con- mRNA expression of eNOS is downregulated in coronary ar- tribute to the elevation of circulating ADMA levels. It was terial tissues in moderate (i.e., 5/6Nx) CKD (Figure 3B). Fur- demonstrated that several factors (e.g., oxidized LDL [39], ho- thermore, our findings that atRA ameliorates endothelium- J Am Soc Nephrol 18: 741–749, 2007 Coronary Endothelial Function in CKD 747

Therapeutic strategy for cardiovascular protection in CKD merits comment. We recently demonstrated that the treatment with a peroxisome proliferator–activated receptor-␥ ligand, pioglitazone, enhances renal DDAH-II expression and urinary NOx excretion, as well as reduces plasma ADMA levels, in hypertensive rat models (49). Furthermore, atRA is reported to increase DDAH-II expression and reduce ADMA in cultured endothelial cells without affecting eNOS expression (22). Al- though our study failed to show upregulation of DDAH-II by the 4-wk treatment with atRA, it markedly upregulates the eNOS expression and reverses the endothelium-dependent vasodilation of the coronary artery in vivo (Figures 6 and 7). These observations suggest that the in vivo effects of atRA on eNOS and DDAH-II expression in CKD differ from those in the in vitro condition. Nevertheless, it is tempting to speculate that these agents alter the course of CKD by manipulating NO and ADMA levels and ameliorating endothelial dysfunction. Of course, further investigations are required to clarify this important issue.

Conclusion This study demonstrates impaired coronary endothelial function in dogs with CKD, which is apparent at the mild to moderate stage of CKD. The derangement is presumably attributed to ADMA-mediated NO suppression and/or the de- Figure 7. Effects of atRA on tissue expression of eNOS, DDAH-I, creased expression of eNOS. These data underscore CKD as a and DDAH-II in coronary artery. Representative fluorescence immunohistochemical images of coronary artery for eNOS (A), potential cardiovascular risk factor and warrant early therapeu- DDAH-I (B), and DDAH-II (C) in control group, 5/6Nx, and tic intervention in CKD for the prevention of cardiovascular 5/6NxϩatRA are shown. The diminished DDAH-II staining events. was reversed by the 4-wk treatment with atRA. Acknowledgments This study was supported by a Grant-in-Aid for Young Scientists (B; dependent vasodilation (Figure 5) and upregulates eNOS 14770557) from the Ministry of Education, Culture, Sports, Science and expression (Figure 6C) suggest that the suppressed eNOS ac- Technology of Japan. tivity accounts for the impaired endothelium-dependent vasodilation of the coronary artery at least in part in moderate CKD. Disclosures The downregulation of eNOS in CKD was previously reported None. in the aortic tissue (10) and gastric mucosa (11) of rat model with five-sixths nephrectomy, and parathyroid hormone and References the change in intracellular calcium concentration were pro- 1. Rostand SG, Brunzell JD, Cannon RO 3rd, Victor RG: Car- posed as a potential mechanism (10). Alternatively, CKD and diovascular complications in renal failure. J Am Soc Nephrol its metabolic consequence, including increased oxidative stress, 2: 1053–1062, 1991 are supposed to play substantial roles in eNOS downregulation 2. Shulman NB, Ford CE, Hall WD, Blaufox MD, Simon D, (45). Paradoxic, previous studies showed elevated plasma NOx Langford HG, Schneider KA: Prognostic value of serum levels in patients with CKD, which was probably due to de- creatinine and effect of treatment of hypertension on renal creased excretion of NOx (46,47). To the extent that pulsatile function. Results from the hypertension detection and fol- stretch and distension of the arterial wall favor NO release in low-up program. The Hypertension Detection and Fol- hypertension (48), it is speculated that lack of hypertension in low-up Program Cooperative Group. Hypertension 13: I80– our canine model and the subsequent reduction in systemic NO I93, 1989 production may result in no increases in plasma NOx levels. Of 3. Samuelsson O: Hypertension in middle-aged men. Man- agement, morbidity and prognostic factors during long- note, in mild (i.e., 1/2Nx) CKD, the eNOS expression was term hypertensive care. Acta Med Scand Suppl 702: 1–79, relatively preserved, whereas the DDAH-II expression was 1985 markedly downregulated (Figure 3). These findings suggest 4. Agrawal B, Berger A, Wolf K, Luft FC: Microalbuminuria that different mechanisms affect the regulation of eNOS and screening by reagent strip predicts cardiovascular risk in DDAH-II expression, probably because these molecules are hypertension. J Hypertens 14: 223–228, 1996 transcribed from different genes and regulated by different 5. Schachinger V, Britten MB, Zeiher AM: Prognostic impact promoters of each gene. of coronary vasodilator dysfunction on adverse long-term 748 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 741–749, 2007

outcome of coronary heart disease. Circulation 101: 1899– cardial arterioles and venules during prolonged diastole in 1906, 2000 canine left ventricles. Circ Res 75: 393–397, 1994 6. Halcox JP, Schenke WH, Zalos G, Mincemoyer R, Prasad 21. Murakami M, Suzuki H, Naitoh M, Matsumoto A, A, Waclawiw MA, Nour KR, Quyyumi AA: Prognostic Kageyama Y, Tsujimoto G, Saruta T: Blockade of the renin- value of coronary vascular endothelial dysfunction. Circu- angiotensin system in heart failure in conscious dogs. J Hy- lation 106: 653–658, 2002 pertens 13: 1405–1412, 1995 7. van Guldener C, Janssen MJ, Lambert J, ter Wee PM, 22. Achan V, Tran CT, Arrigoni F, Whitley GS, Leiper JM, Jakobs C, Donker AJ, Stehouwer CD: No change in im- Vallance P: All-trans-retinoic acid increases nitric oxide paired endothelial function after long-term folic acid ther- synthesis by endothelial cells: A role for the induction of apy of hyperhomocysteinaemia in haemodialysis patients. dimethylarginine dimethylaminohydrolase. Circ Res 90: Nephrol Dial Transplant 13: 106–112, 1998 764–769, 2002 8. Ross R: The pathogenesis of atherosclerosis: A perspective 23. Vester B, Rasmussen K: High performance liquid chroma- for the 1990s. Nature 362: 801–809, 1993 tography method for rapid and accurate determination of 9. Cooke JP, Dzau VJ: Derangements of the nitric oxide syn- homocysteine in plasma and serum. Eur J Clin Chem Clin thase pathway, L-arginine, and cardiovascular diseases. Biochem 29: 549–554, 1991 Circulation 96: 379–382, 1997 24. Ohta K, Araki N, Shibata M, Hamada J, Komatsumoto S, 10. Vaziri ND, Ni Z, Wang XQ, Oveisi F, Zhou XJ: Downregu- Shimazu K, Fukuuchi Y: A novel in vivo assay system for lation of nitric oxide synthase in chronic renal insuffi- consecutive measurement of brain nitric oxide production ciency: Role of excess PTH. Am J Physiol 274: F642–F649, combined with the microdialysis technique. Neurosci Lett 1998 176: 165–168, 1994 11. Tomikawa M, Ohta M, Vaziri ND, Kaunitz JD, Itani R, Ni 25. Hasdan G, Benchetrit S, Rashid G, Green J, Bernheim J, Z, Tarnawski AS: Decreased endothelial nitric oxide syn- Rathaus M: Endothelial dysfunction and hypertension in thase in gastric mucosa of rats with chronic renal failure. 5/6 nephrectomized rats are mediated by vascular super- Am J Physiol 274: F1102–F1108, 1998 oxide. Kidney Int 61: 586–590, 2002 12. Vallance P, Leiper J: Cardiovascular biology of the asym- 26. Kohler R, Eichler I, Schonfelder H, Grgic I, Heinau P, Si H, metric dimethylarginine:dimethylarginine dimethylamin- Hoyer J: Impaired EDHF-mediated vasodilation and func- ohydrolase pathway. Arterioscler Thromb Vasc Biol 24: 1023– tion of endothelial Ca-activated K channels in uremic rats. 1030, 2004 Kidney Int 67: 2280–2287, 2005 13. Vallance P, Leone A, Calver A, Collier J, Moncada S: Ac- 27. Morris ST, McMurray JJ, Spiers A, Jardine AG: Impaired cumulation of an endogenous inhibitor of nitric oxide syn- endothelial function in isolated human uremic resistance thesis in chronic renal failure. Lancet 339: 572–575, 1992 arteries. Kidney Int 60: 1077–1082, 2001 14. Fliser D, Kronenberg F, Kielstein JT, Morath C, Bode-Boger 28. Brown SA, Finco DR, Navar LG: Impaired renal autoreg- SM, Haller H, Ritz E: Asymmetric dimethylarginine and ulatory ability in dogs with reduced renal mass. JAmSoc progression of chronic kidney disease: The mild to mod- Nephrol 5: 1768–1774, 1995 erate kidney disease study. J Am Soc Nephrol 16: 2456–2461, 29. Michell AR, Bodey AR, Gleadhill A: Absence of hyperten- 2005 sion in dogs with renal insufficiency. Ren Fail 19: 61–68, 15. Ravani P, Tripepi G, Malberti F, Testa S, Mallamaci F, 1997 Zoccali C: Asymmetrical dimethylarginine predicts pro- 30. Cross JM, Donald A, Vallance PJ, Deanfield JE, Woolfson gression to dialysis and death in patients with chronic RG, MacAllister RJ: Dialysis improves endothelial function kidney disease: A competing risks modeling approach. in humans. Nephrol Dial Transplant 16: 1823–1829, 2001 J Am Soc Nephrol 16: 2449–2455, 2005 31. Perticone F, Sciacqua A, Maio R, Perticone M, Maas R, 16. Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, Boger RH, Tripepi G, Sesti G, Zoccali C: Asymmetric dim- MacAllister R, Vallance P: Asymmetric dimethylarginine ethylarginine, L-arginine, and endothelial dysfunction in causes hypertension and cardiac dysfunction in humans essential hypertension. J Am Coll Cardiol 46: 518–523, 2005 and is actively metabolized by dimethylarginine dimeth- 32. Elesber AA, Solomon H, Lennon RJ, Mathew V, Prasad A, ylaminohydrolase. Arterioscler Thromb Vasc Biol 23: 1455– Pumper G, Nelson RE, McConnell JP, Lerman LO, Lerman 1459, 2003 A: Coronary endothelial dysfunction is associated with 17. Kielstein JT, Impraim B, Simmel S, Bode-Boger SM, Tsikas erectile dysfunction and elevated asymmetric dimethyl- D, Frolich JC, Hoeper MM, Haller H, Fliser D: Cardiovas- arginine in patients with early atherosclerosis. Eur Heart J cular effects of systemic nitric oxide synthase inhibition 27: 824–831, 2006 with asymmetrical dimethylarginine in humans. Circula- 33. Chade AR, Brosh D, Higano ST, Lennon RJ, Lerman LO, tion 109: 172–177, 2004 Lerman A: Mild renal insufficiency is associated with re- 18. Leiper JM, Santa Maria J, Chubb A, MacAllister RJ, Charles duced coronary flow in patients with non-obstructive cor- IG, Whitley GS, Vallance P: Identification of two human onary artery disease. Kidney Int 69: 266–271, 2006 dimethylarginine dimethylaminohydrolases with distinct 34. van Guldener C: Why is homocysteine elevated in renal tissue distributions and homology with microbial arginine failure and what can be expected from homocysteine-low- deiminases. Biochem J 343: 209–214, 1999 ering? Nephrol Dial Transplant 21: 1161–1166, 2006 19. Ogawa T, Kimoto M, Watanabe H, Sasaoka K: Metabolism 35. MacAllister RJ, Rambausek MH, Vallance P, Williams D, of NG, NG-and NG, NЈG-dimethylarginine in rats. Arch Hoffmann KH, Ritz E: Concentration of dimethyl-L-argi- Biochem Biophys 252: 526–537, 1987 nine in the plasma of patients with end-stage renal failure. 20. Hiramatsu O, Goto M, Yada T, Kimura A, Tachibana H, Nephrol Dial Transplant 11: 2449–2452, 1996 Ogasawara Y, Tsujioka K, Kajiya F: Diameters of subendo- 36. Kielstein JT, Boger RH, Bode-Boger SM, Frolich JC, Haller J Am Soc Nephrol 18: 741–749, 2007 Coronary Endothelial Function in CKD 749

H, Ritz E, Fliser D: Marked increase of asymmetric dim- 43. Matsuguma K, Ueda S, Yamagishi S, Matsumoto Y, ethylarginine in patients with incipient primary chronic Kaneyuki U, Shibata R, Fujimura T, Matsuoka H, Kimoto renal disease. J Am Soc Nephrol 13: 170–176, 2002 M, Kato S, Imaizumi T, Okuda S: Molecular mechanism for 37. Carello KA, Whitesall SE, Lloyd MC, Billecke SS, D’Alecy elevation of asymmetric dimethylarginine and its role for LG: Asymmetrical dimethylarginine plasma clearance per- hypertension in chronic kidney disease. J Am Soc Nephrol sists after acute total nephrectomy in rats. Am J Physiol 17: 2176–2183, 2006 Heart Circ Physiol 290: H209–H216, 2006 44. Kielstein JT, Salpeter SR, Bode-Boeger SM, Cooke JP, Fliser 38. Okubo K, Hayashi K, Wakino S, Matsuda H, Kubota E, D: Symmetric dimethylarginine (SDMA) as endogenous Honda M, Tokuyama H, Yamamoto T, Kajiya F, Saruta T: marker of renal function: A meta-analysis. Nephrol Dial Role of asymmetrical dimethylarginine in renal microvas- Transplant 21: 2446–2451, 2006 cular endothelial dysfunction in chronic renal failure with 45. Bojunga J, Dresar-Mayert B, Usadel KH, Kusterer K, hypertension. Hypertens Res 28: 181–189, 2005 Zeuzem S: Antioxidative treatment reverses imbalances of 39. Boger RH, Sydow K, Borlak J, Thum T, Lenzen H, Schubert nitric oxide synthase isoform expression and attenuates B, Tsikas D, Bode-Boger SM: LDL cholesterol upregulates tissue-cGMP activation in diabetic rats. Biochem Biophys Res synthesis of asymmetrical dimethylarginine in human en- Commun 316: 771–780, 2004 dothelial cells: Involvement of S-adenosylmethionine-de- 46. Schmidt RJ, Yokota S, Tracy TS, Sorkin MI, Baylis C: Nitric pendent methyltransferases. Circ Res 87: 99–105, 2000 oxide production is low in end-stage renal disease patients 40. Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, on peritoneal dialysis. Am J Physiol 276: F794–F797, 1999 Cooke JP: Homocysteine impairs the nitric oxide synthase 47. Schmidt RJ, Baylis C: Total nitric oxide production is low in pathway: Role of asymmetric dimethylarginine. Circulation patients with chronic renal disease. Kidney Int 58: 1261– 104: 2569–2575, 2001 1266, 2000 41. Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto 48. Aiello S, Noris M, Todeschini M, Zappella S, Foglieni C, M, Tsuji H, Reaven GM, Cooke JP: Impaired nitric oxide Benigni A, Corna D, Zoja C, Cavallotti D, Remuzzi G: synthase pathway in diabetes mellitus: Role of asymmetric Renal and systemic nitric oxide synthesis in rats with renal dimethylarginine and dimethylarginine dimethylamin- mass reduction. Kidney Int 52: 171–181, 1997 ohydrolase. Circulation 106: 987–992, 2002 49. Wakino S, Hayashi K, Tatematsu S, Hasegawa K, Taka- 42. Jia SJ, Jiang DJ, Hu CP, Zhang XH, Deng HW, Li YJ: matsu I, Kanda T, Homma K, Yoshioka K, Sugano N, Lysophosphatidylcholine-induced elevation of asymmetric Saruta T: Pioglitazone lowers systemic asymmetric dim- dimethylarginine level by the NADPH oxidase pathway in ethylarginine by inducing dimethylarginine dimethylami- endothelial cells. Vascul Pharmacol 44: 143–148, 2006 nohydrolase in rats. Hypertens Res 28: 255–262, 2005