Objectives: Peripheral arterial diseases associated with an increased risk of death in kidney transplant patients. Natriuretic peptide has anti-atherosclerotic effects. We sought to evaluate the relation between ankle-brachial index and fasting serum long-acting natriuretic peptide concentrations in kidney transplant patients.

Materials and Methods: Fasting blood samples were obtained from 69 kidney transplant patients. Serum long-acting natriuretic peptide concentrations were measured using a commercially available enzyme immunoassay kit. Left or right ankle-brachial index values that were < 0.9 were included in the low ankle-brachial index group.

Results: Fifteen patients (21.7%) were enrolled in the low ankle-brachial index group. Increased waist circumference (P = .013), higher serum total cholesterol levels (P = .019), higher triglyceride levels (P = .002), and decreased serum long-acting natriuretic peptide concentrations (P = .006) were noted in the low ankle-brachial index group. Univariate linear regression analysis indicated that the left/right ankle-brachial index values of the subjects were negatively correlated with serum triglycerides (P = .008 or P < .001) and fasting glucose levels (P = .034 or P = .012), but were positively correlated with long-acting natriuretic peptide concentrations (P = .011 or P = .011). Multivariate forward stepwise linear regression analyses of the significant variables revealed that serum triglycerides and long-acting natriuretic peptide levels were independent predictors of the left/right ankle-brachial index values of kidney transplant patients.

Conclusions: Serum long-acting natriuretic peptide concentrations correlate positively with ankle-brachial index values among the kidney transplant patients.

Key words : Long-acting natriuretic peptide, Ankle-brachial index, Kidney transplant, Peripheral arterial disease

Introduction

Cardiac natriuretic peptides consist of a family of peptide hormones synthesized by 3 different genes, which are then stored as 3 different prohormones (namely, 126-amino acid atrial natriuretic peptide [ANP], 108-amino acid B-type natriuretic peptide, and 103-amino acid C-type natriuretic peptide prohormones).¹ Within the 126-amino ANP prohormone are 4 peptide hormones; these are long-acting natriuretic peptide ([LANP]; N-terminal pro-ANP 1-30), vessel dilator (N-terminal pro-ANP 31-67), kaliuretic peptide (N-terminal pro-ANP 79-98), and ANP (α-ANP; N-terminal pro-ANP 99-126), and these peptide hormones have significant diuretic, natriuretic, and blood-pressure–lowering properties in animals and humans.² The biological effects of these 4 peptide hormones include vasodilatation mediated via enhancing guanylate cyclase activity with a resultant increase in intracellular messenger cyclic guanosine monophosphate.¹ 126-amino acid atrial natriuretic peptide represses the fibrogenic activation of vascular smooth muscle cells, inhibits pressure-induced cardiac remodeling and fibrosis, and has a vascular relaxation effect.³ 126-amino acid atrial natriuretic peptide also decreases atherosclerosis in cholesterol-fed anesthetized rabbits.⁴

Peripheral artery disease (PAD) is a general term used to describe progressive atherosclerotic narrowing of the peripheral arteries, and is most often used to refer to the arteries of the lower extremities.⁵ Ankle-brachial index (ABI), which is the ratio of ankle-to-brachial systolic blood pressure, provides a simple, noninvasive, useful method for assessing subclinical PAD.^5,6 Epidemiologic studies have demonstrated that lower ABI values are independent predictors of cardiovascular events and mortality.^7,8 An ABI of less than 0.9 can predict mortality in both advanced chronic kidney disease and in hemodialysis patients.^9,10 Moreover, PAD is associated with an increased risk of death among patients with and without diabetes among kidney transplant patients.¹¹

Until now, no study has investigated serum LANP values in relation to ABI among renal transplant recipients. We sought to determine the relation between serum LANP concentrations and ABI among renal transplant recipients.

Materials and Methods

Patients
Sixty-nine renal transplant recipients (43 men, 26 women; age range, 31-73 y) were studied in April 2010 at a medical center in Hualien, Taiwan. The study was approved by the Protection of Human Subjects Institutional Review Board of Tzu-Chi University and Hospital. Patients were excluded if they had any acute infection, malignancy, acute rejection, acute myocardial infarction, pulmonary edema, heart failure, were using cilostazol or pentoxifylline, or using any antiplatelet drug (aspirin or clopidogrel) at the time of blood sampling, or if they refused to provide informed consent for the study. All protocols were approved by the ethics committee of the institution before the study began, and the protocols conformed with the ethical guidelines of the 1975 Helsinki Declaration, and written, informed consent was obtained from all patients.

Anthropometric analysis
Body weight was measured to the nearest half-kilogram with the patient in light clothing and without shoes. Height was measured to the nearest half-centimeter. Waist circumference was measured to the nearest half-centimeter at the shortest point below the lower rib margin and the iliac crest. Body mass index was calculated as the weight (kilograms) divided by height squared (meters).^12,13

Biochemical determinations
Fasting blood samples of approximately 0.5 mL were immediately centrifuged at 3000g for 10 minutes after collection. Serum samples were stored at 4°C and used for biochemical analyses within 1 hour of collection. Serum levels of blood urea nitrogen, creatinine, fasting glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total calcium, and phosphorus were measured using an autoanalyzer (COBAS Integra 800, Roche Diagnostics, Basel, Switzerland). Serum LANP levels (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) were measured using a commercially available enzyme immunoassay kit. The limit of detection calculated as the concentration of human LANP corresponding to the blank average minus 3 standard deviations was 0.1 ng/mL. The interassay and intraassay coefficients of variation for LANP and were 6.2% and 5.4%.^12,13

Ankle-brachial index measurements
The ankle-brachial index (ABI) values were measured using an ABI-form device (VaSera VS-1000, Fukuda Denshi Co, Ltd, Tokyo, Japan) that automatically and simultaneously measures blood pressure in both arms and ankles using an oscillometric method. Ankle-brachial index was calculated as the ratio of the ankle systolic blood pressure divided by the arm systolic blood pressure, and the lower value of the ankle systolic blood pressure was used for the calculation. Occlusion and monitoring cuffs were placed tightly around 4 extremities and the electrocardiogram and heart sounds during the measurement in the supine position at rest were measured for at least 10 minutes. We repeatedly measured these parameters for both legs of each patient and expressed those as the means. The ranges of these markers in control subjects are ABIs from 0.9 to 1.2.^9,10,14 Peripheral artery disease can easily be diagnosed based on an ABI of < 0.90.^5,6 In this study, left or right ABI values of < 0.9 were used to define the low ABI group.

Statistical analyses
Data are expressed as means ± standard deviation (SD) and were tested for normal distribution by Kolmogorov-Smirnov test. Comparisons between patients were done using an independent t test (2-tailed) for normally distributed data or the Mann-Whitney U test for parameters that presented with non-normal distribution (fasting glucose, blood urea nitrogen, LANP). Data expressed as the number of patients were analyzed by the chi-square test. Clinical variables that correlated with ABI in renal transplant recipients were evaluated by univariate linear regression analyses. Variables that were significantly associated with ABI in renal transplant recipients were tested for independence by multivariate forward stepwise regression analysis. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 13.0, IBM Corporation, Armonk, NY, USA). A P value < .05 was considered statistically significant.

Results

The demographic, biochemical, and clinical characteristics of the 69 renal transplant recipients are presented in Table 1. Comorbid conditions included diabetes (n=12; 17.4%), and hypertension (n=37; 53.6%). Prescribed therapeutic agents included tacrolimus (n=53; 76.8%), mycophenolate mofetil or mycophenolic acid (n=65; 94.2%), steroids (n=67; 97.1%), rapamycin (n=3; 4.3%), and cyclosporine (n=14; 20.3%).

The clinical characteristics in both the normal and low ABI groups among the renal transplant recipients are presented in Table 2. Fifteen patients (21.7%) were enrolled in low ABI group among the renal transplant recipients. Increased waist circumference (P = .013), a higher serum total cholesterol level (P = .019), a higher triglyceride level (P = .002), and a decreased serum LANP level (P = .006) were noted in low ABI group among the renal transplant recipients.

Clinical characteristics and ABI values are presented in Table 3. Left or right ABI values did not differ statistically with the presence of diabetes, hypertension, transplant model, and the use of a tacrolimus, mycophenolate mofetil or mycophenolic acid, steroids, rapamycin, or cyclosporine.

The univariate linear analysis of the clinical variables associated with left/right ABI values presented in Table 4. Serum triglyceride (r=-0.317; P = .008) and fasting glucose (r=-0.255; P = .034) were negatively correlated, while serum LANP (r=0.304; P = .011) was positively correlated with the left ABI values among the renal transplant recipients. Similar results also were noted for right ABI values of the renal transplant recipients, namely that serum triglycerides (r=-0.433; P < .001) and fasting glucose (r=-0.300; P = .012) were negatively correlated, while serum LANP (r=0.305; P = .011) was positively correlated.

Multivariate forward stepwise linear regression analysis of the variables significantly associated with left ABI values revealed that serum trigly­­cerides (r²change = 0.100; P = .008) and LANP (r²change = 0.071; P = .021) levels were independent predictors of left ABI values among the renal transplant recipients (Table 5). Similar results also noted for serum triglyceride (r²change = 0.188; P < .001) and LANP (r²change = 0.063; P = .022) levels with respect to the right ABI values in the renal transplant recipients.

Discussion

The results of this study show that there was significant correlation between increased waist circumference, higher serum total cholesterol level, and higher triglyceride level, with a decreased serum LANP level among the low ABI group of renal transplant recipients. Triglyceride levels and LANP levels were found to be independent predictors of the ABI values among renal transplant recipients.

Peripheral artery disease results from progressive narrowing of the arteries secondary to atherosclerosis and is defined as an ABI of < 0.9.^5,6 Peripheral artery disease is associated with an increased risk of death among patients with and without diabetes after kidney transplant.¹¹ The incidence of PAD seems to vary and has been reported to range from 6.8% to 15% after a kidney transplant.^15,16 The cumulative incidence of PAD has been found to be 20% and 5% in patients with and without diabetes at 3 years after transplant.¹¹ The prevalence of low ABI in this study was about 21.7%. Peripheral artery disease is highly prevalent among the elderly and subjects with atherosclerotic risk factors such as diabetes mellitus, hypertension, and hyperlipidemia.⁶

In our study, older age, diabetes, or hypertension were increased in the low ABI group, although there were no statistically significant differences between the 2 groups for these factors. It also was found that fasting glucose was negatively was correlated with ABI value among the renal transplant recipients. Peripheral artery disease patients also had elevated serum levels of triglyceride compared with those without PAD.^17-19 A higher serum triglyceride level has been shown to be associated with a low ABI value in asymptomatic patients between 50 and 70 years of age.20 Our results also identified higher serum total cholesterol and higher triglyceride levels in the low ABI group, and the serum triglyceride level was negatively correlated with the ABI value for renal transplant recipients. Patients with aortoiliac PAD also have a higher waist circumference and increased waist circumference is an independent risk factor for PAD.^{21, 22} Our results also found that increased waist circumference was significantly correlated with being a member of the low ABI group among renal transplant recipients.

Peripheral artery disease is similar to atherosclerosis elsewhere in the body; the initiating insult is endothelial injury, followed by inflammation and progressive atherosclerotic narrowing of the vessel.⁵ Development of atherosclerosis is regulated by multiple complex mechanisms that include endothelial dysfunction with impaired nitric oxide bioavailability, oxidative stress, inflammation, hemostasis, and activation of the renin–angiotensin–aldosterone system.^23-25 126-amino acid atrial natriuretic peptide has an antifibrosis and vascular relaxation effect.^3,26 Inhibition of RhoA through phosphorylation at Ser188, which is the site targeted by the natriuretic peptide effect or cyclic guanosine monophosphate-dependent protein kinase I, is critical to being able to fully exert the antifibrotic potential relative to vascular fibrosis.²⁷ 126-amino acid atrial natriuretic peptide elicits its anti-inflammatory effect by reducing production of 2 proinflammatory cytokines (TNF-α and IL-12) while enhancing production of IL-10.^28,29 126-amino acid atrial natriuretic peptide regulates blood pressure, and this occurs in part through an inhibition of the renin-angiotensin-aldosterone system.³ In an animal study, ANP decreases aorta stiffness and atherosclerosis in cholesterol-fed anesthetized rabbits.⁴ In a human study, NT-pro–C-type natriuretic peptide exhibited significant negative associations with carotid-femoral pulse wave velocity and carotid intima-media thickness as well as a positive association with flow-mediated dilatation of the brachial artery.³⁰ Our results noted a lower LANP level in the low ABI group and LANP was positively correlated with the ABI values of renal transplant recipients. Further studies are required to elucidate the relation between ABI value and LANP level among renal transplant recipients.

Certain immunosuppressive drugs may influence cardiovascular and metabolic profiles in renal transplant recipients. Corticosteroids and cyclosporine are the agents with the most negative effect on weight gain, blood pressure, and lipids. Target of rapamycin inhibitors increase the risk of new-onset diabetes mellitus and have the greatest effect on risk factors for posttransplant hyperlipidemia.³¹ However, some studies noted tacrolimus has been responsible for significant changes in plasma lipid concentrations only for the first 6 months, but not for the remaining time of observation, and target of rapamycin inhibitors have emerging antiatherosclerotic effect.^32,33 In the present study, no relations between tacrolimus, mycophenolate mofetil or mycophenolic acid, steroids, rapamycin, or cyclosporine therapy and ABI values were observed.

Our study has some limitations. First, the number of patients enrolled was small. Second, this study has a cross-sectional design. Third, most studies have focused on the effects of ANP on atherosclerosis, and there is a paucity of studies on the effects of LANP. Therefore, our findings support the need for continued long-term prospective studies to confirm a causal relation between serum LANP and ABI values among renal transplant recipients. In conclusion, in this study, serum fasting LANP level was shown to be positively associated with ABI values among renal transplant recipients.

References:

1. Vesely DL. Atrial natriuretic peptides: anticancer agents. J Investig Med. 2005;53(7):360-365.
   CrossRef - PubMed
2. Vesely DL. Which of the cardiac natriuretic peptides is most effective for the treatment of congestive heart failure, renal failure and cancer? Clin Exp Pharmacol Physiol. 2006;33(3):169-176.
   CrossRef - PubMed
3. Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev. 2006;27(1):47-72.
   CrossRef - PubMed
4. Kallaras K, Babas G, Stergiou-Michailidou V, Karamouzis M, Zaraboukas T. Atrial natriuretic peptide decreases aorta stiffness in cholesterol-fed anesthetized rabbits. Pharmacol Res. 2009;60(4):324-331. doi: 10.1016/j.phrs.2009.04.014.
   CrossRef - PubMed
5. Shanmugasundaram M, Ram VK, Luft UC, Szerlip M, Alpert JS. Peripheral arterial disease--what do we need to know? Clin Cardiol. 2011;34(8):478-482. doi: 10.1002/clc.20925.
   CrossRef - PubMed
6. Ferreira AC, Macedo FY. A review of simple, non-invasive means of assessing peripheral arterial disease and implications for medical management. Ann Med. 2010;42(2):139-150. doi: 10.3109/07853890903521070.
   CrossRef - PubMed
7. Ostergren J, Sleight P, Dagenais G, et al. Impact of ramipril in patients with evidence of clinical or subclinical peripheral arterial disease. Eur Heart J. 2004;25(1):17-24.
   CrossRef - PubMed
8. Perlstein TS, Creager MA. The ankle-brachial index as a biomarker of cardiovascular risk: it's not just about the legs. Circulation. 2009;120(21):2033-2035. doi: 10.1161/CIRCULATIONAHA.109.907238.
   CrossRef - PubMed
9. Kato A, Takita T, Furuhashi M, Kumagai H, Hishida A. A small reduction in the ankle-brachial index is associated with increased mortality in patients on chronic hemodialysis. Nephron Clin Pract. 2010;114(1):c29-c37. doi: 10.1159/000245067.
   CrossRef - PubMed
10. Chen SC, Chang JM, Hwang SJ, et al. Ankle brachial index as a predictor for mortality in patients with chronic kidney disease and undergoing haemodialysis. Nephrology (Carlton). 2010;15(3):294-299. doi: 10.1111/j.1440-1797.2010.01187.x.
    CrossRef - PubMed
11. Snyder JJ, Kasiske BL, Maclean R. Peripheral arterial disease and renal transplantation. J Am Soc Nephrol. 2006;17(7):2056-2068.
    CrossRef - PubMed
12. Hsu BG, Shih MH, Yang YC, Ho GJ, Lee MC. Fasting long-acting natriuretic peptide correlates inversely with metabolic syndrome in kidney transplant patients. Transplant Proc. 2012;44(3):646-650. doi: 10.1016/j.transproceed.2011.11.008.
    CrossRef - PubMed
13. Hsu BG, Ho GJ, Lee CJ, et al. Inverse association of serum long-acting natriuretic peptide and bone mineral density in renal transplant recipients. Clin Transplant. 2012;26(2):E105-E110. doi: 10.1111/j.1399-0012.2011.01575.x.
    CrossRef - PubMed
14. Kato A, Ishida J, Endo Y, et al. Association of abdominal visceral adiposity and thigh sarcopenia with changes of arteriosclerosis in haemodialysis patients. Nephrol Dial Transplant. 2011;26(6):1967-1976. doi: 10.1093/ndt/gfq652.
    CrossRef - PubMed
15. Ott U, Busch M, Steiner T, Wolf G. Presence of cardiovascular disease in patients on a waiting list for renal transplantation and in patients after kidney transplantation in a single center. Transplant Proc. 2010;42(9):3450-3454. doi: 10.1016/j.transproceed.2010.08.047.
    CrossRef - PubMed
16. Barrionuevo JD, Vargas-Machuca MF, Pulido FG, Sacaluga LG, Govantes MA, Martínez Martínez A. Prevalence of cardiovascular disease in kidney transplant candidates: outpatient cardiac evaluation. Transplant Proc. 2010;42(8):3126-3127. doi: 10.1016/j.transproceed.2010.05.077.
    CrossRef - PubMed
17. Gimeno SG, Hirai AT, Harima HA, et al. Fat and fiber consumption are associated with peripheral arterial disease in a cross-sectional study of a Japanese-Brazilian population. Circ J. 2008;72(1):44-50.
    CrossRef - PubMed
18. Blanes JI, Cairols MA, Marrugat J; ESTIME. Prevalence of peripheral artery disease and its associated risk factors in Spain: The ESTIME Study. Int Angiol. 2009;28(1):20-25.
    PubMed
19. Berger JS, Ballantyne CM, Davidson MH, et al. Peripheral artery disease, biomarkers, and darapladib. Am Heart J. 2011;161(5):972-978. doi: 10.1016/j.ahj.2011.01.017.
    CrossRef - PubMed
20. Kravos A, Bubnic-Sotosek K. Ankle-brachial index screening for peripheral artery disease in asymptomatic patients between 50 and 70 years of age. J Int Med Res. 2009;37(5):1611-1619.
    CrossRef - PubMed
21. Jakovljević B, Stojanov V, Lović D, Paunović K, Radosavljević V, Tutić I. Obesity and fat distribution as predictors of aortoiliac peripheral arterial disease in middle-aged men. Eur J Intern Med. 2011;22(1):84-88. doi: 10.1016/j.ejim.2010.07.019.
    CrossRef - PubMed
22. Brouwer BG, Visseren FL, Stolk RP, van der Graaf Y; SMART Study Group. Abdominal fat and risk of coronary heart disease in patients with peripheral arterial disease. Obesity (Silver Spring). 2007;15(6):1623-1630.
    CrossRef - PubMed
23. Higashi Y, Noma K, Yoshizumi M, Kihara Y. Endothelial function and oxidative stress in cardiovascular diseases. Circ J. 2009;73(3):411-418.
    CrossRef - PubMed
24. Montecucco F, Pende A, Mach F. The renin-angiotensin system modulates inflammatory processes in atherosclerosis: evidence from basic research and clinical studies. Mediators Inflamm. 2009;2009:752406. doi: 10.1155/2009/752406.
    CrossRef - PubMed
25. Koh KK, Han SH, Oh PC, Shin EK, Quon MJ. Combination therapy for treatment or prevention of atherosclerosis: focus on the lipid-RAAS interaction. Atherosclerosis. 2010;209(2):307-313. doi: 10.1016/j.atherosclerosis.2009.09.007.
    CrossRef - PubMed
26. Nishikimi T, Maeda N, Matsuoka H. The role of natriuretic peptides in cardioprotection. Cardiovasc Res. 2006;69(2):318-328.
    CrossRef - PubMed
27. Sawada N, Itoh H, Miyashita K, et al. Cyclic GMP kinase and RhoA Ser188 phosphorylation integrate pro- and antifibrotic signals in blood vessels. Mol Cell Biol. 2009;29(22):6018-6032. doi: 10.1128/MCB.00225-09.
    CrossRef - PubMed
28. Kiemer AK, Hartung T, Vollmar AM. cGMP-mediated inhibition of TNF-alpha production by the atrial natriuretic peptide in murine macrophages. J Immunol. 2000;165(1):175-181.
    PubMed
29. Morita R, Ukyo N, Furuya M, Uchiyama T, Hori T. Atrial natriuretic peptide polarizes human dendritic cells toward a Th2-promoting phenotype through its receptor guanylyl cyclase-coupled receptor A. J Immunol. 2003;170(12):5869-5875.
    PubMed
30. Vlachopoulos C, Ioakeimidis N, Terentes-Printzios D, et al. Amino-terminal pro-C-type natriuretic peptide is associated with arterial stiffness, endothelial function and early atherosclerosis. Atherosclerosis. 2010;211(2):649-655. doi: 10.1016/j.atherosclerosis.2010.03.020.
    CrossRef - PubMed
31. Marcén R. Immunosuppressive drugs in kidney transplantation: impact on patient survival, and incidence of cardiovascular disease, malignancy and infection. Drugs. 2009;69(16):2227-2243. doi: 10.2165/11319260-000000000-00000.
    CrossRef - PubMed
32. Tarantino G, Palmiero G, Polichetti G, et al. Long-term assessment of plasma lipids in transplant recipients treated with tacrolimus in relation to fatty liver. Int J Immunopathol Pharmacol. 2010;23(4):1303-1308.
    PubMed
33. Pieri M, Miraglia N, Polichetti G, Tarantino G, Acampora A, Capone D. Analytical and pharmacological aspects of therapeutic drug monitoring of mTOR inhibitors. Curr Drug Metab. 2011;12(3):253-267.
    CrossRef - PubMed