Regular Article Influence of Klotho genotypes on plasma NO x levels in South Indian population Vijaya Majumdar, Dania Jose, Rita Christopher ⁎ Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore 560029, India abstract article info Article history: Received 30 November 2010 Received in revised form 31 March 2011 Accepted 2 April 2011 Available online 4 May 2011 Keywords: Klotho KL-VS Klotho C1818T Nitric oxide Aging Endothelial dysfunction South Indians Introduction: Despite experimental evidences of the influence of the aging suppressor gene Klotho, on the modulation of endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production, the contribution of its variants to the phenotypic variance of plasma nitrite and nitrate (NO x ) has not been addressed to date. In the present study, we aimed to determine the influence of two exonic variants, KL-VS and C1818T of Klotho, on circulating NO x levels in South Indian population. Materials and Methods: We genotyped the two Klotho KL-VS and C1818T variants in 429 healthy South Indians and measured their plasma NO x concentrations by the Griess method. Results: Genotype frequencies were compared in subjects with low and high NO x levels. An age-specific association of the Klotho C1818T variant was found with plasma NO x levels in subjects aged N 40 years (p = 0.027); the CC homozygotes were more prevalent in the low compared to the high plasma NO x group. However, the variant was not associated with plasma NO x levels in subjects aged ≤ 40 years (p = 0.799). The KL-VS variant did not have any influence on plasma NO x status (p = 0.260). Conclusions: Our results suggest that the effect of Klotho C1818T variant on levels of plasma NO x becomes pronounced with age probably implying the adaptive capability of Klotho alleles to meet the age-related increasing physiological load. © 2011 Elsevier Ltd. All rights reserved. Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular homeostasis [1,2], maintains basal vasodilator tone, inhibits platelet aggregation, attenuates leukocyte adhesion to the endothelium, modulates smooth muscle proliferation and is largely responsible for the maintenance of normal endothelial function [3]. Endothelial dysfunction with reduced bioactivity of NO predisposes to major adverse cardiovascular events like coronary artery disease (CAD), diabetes, peripheral arterial occlusive disease, and arterial hypertension. Thus, elucidating the role of genes and their variants in the formation and degradation of NO is of great physiological relevance. Deficiency of Klotho (MIM 604824) in mice leads to the manifestation of pleiotropic phenotypes resembling human aging; endothelial dysfunction and extensive medial calcification of aorta, being few of the salient features related to vasculature [4,5]. There is a significant attenuation of relaxation of aortic rings in response to acetylcholine in Klotho-deficient mice, which suggests severe impair- ment of vascular endothelial function [5]. Further, in-vivo Klotho gene delivery has been found to ameliorate vascular endothelial dysfunc- tion in a rat model with multiple atherogenic risk factors [6]. An interplay between generation of reactive oxygen species and antioxidants, with reduced production of NO has been suggested to underlie the mechanism of endothelial dysfunction in Klotho-deficient mice [7]. The in-vivo reduction in the endogenous NO production of Klotho-deficient mice is reflected by their reduced urinary excretion of NO metabolites and cGMP (an indicator of NO synthesis) [5,8]. Further, in-vitro experimental findings lend support for Klotho- induced enhancement in NO production in human endothelial cells via modulation of the activity of endothelial nitric oxide synthase (eNOS), the enzyme responsible for generation of NO [7,9]. A number of studies have been carried out to examine whether polymorphisms in eNOS gene are associated with plasma NO levels [10–16]. However, the results are controversial. Based on the reported findings of Klotho's role in modulation of eNOS activity and regulation of NO production and modulation, we aimed to study the effect of genetic variants of Klotho on plasma NO x levels. For this purpose we chose two of the most widely studied variants of the gene to test their association with plasma NO x levels. The functional KL-VS variant is composed of six sequence variants in perfect linkage disequilibrium (LD), two of which result in amino acid substitutions F352V and C370S [17]. Due to the presence of LD across single nucleotide polymorphisms (SNPs), the single variant, F352V, has been used to tag the KL-VS haplotype. The presence of phenylalanine at position 352 in the human Klotho gene is highly conserved and its substitution to valine has been demonstrated to alter the in-vitro secretion and Thrombosis Research 128 (2011) 251–255 Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; CAD, coronary artery disease; cAMP, cyclic adenosine 3',5'-monophosphate; PKA, protein kinase A; VNTR, variable number of tandem repeats; SNPs, single nucleotide polymorphisms; TG, triglyceride; TC, total cholesterol; NADPH, nicotinamide adenine dinucleotide phosphate; TNF, tumor necrosis factor. ⁎ Corresponding author. Tel.: + 91 80 26995162; fax: + 91 80 26564830. E-mail addresses: rita@nimhans.kar.nic.in, rita.nimhans@yahoo.com (R. Christopher). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.04.002 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres