The Influence of a Single Nucleotide Polymorphism within <em>CNDP1</em> on Susceptibility to Diabetic Nephropathy in Japanese Women with Type 2 Diabetes

<div><h3>Background</h3><p>Several linkage analyses have mapped a susceptibility locus for diabetic nephropathy to chromosome 18q22–23, and polymorphisms within the carnosine dipeptidase 1 gene (<em>CNDP1)</em>, located on 18q22.3, have been shown to be associated with diabetic nephropathy in European subjects with type 2 diabetes. However, the association of this locus with diabetic nephropathy has not been evaluated in the Japanese population. In this study, we examined the association of polymorphisms within the <em>CNDP1/CNDP 2</em> locus with diabetic nephropathy in Japanese subjects with type 2 diabetes.</p> <h3>Methodology/Principal Findings</h3><p>We genotyped a leucine repeat polymorphism (D18S880) that is within <em>CNDP1</em> along with 29 single nucleotide polymorphisms (SNPs) in the <em>CNDP1</em>/<em>CNDP2</em> locus for 2,740 Japanese subjects with type 2 diabetes (1,205 nephropathy cases with overt nephropathy or with end-stage renal disease [ESRD], and 1,535 controls with normoalbuminuria). The association of each polymorphism with diabetic nephropathy was analysed by performing logistic regression analysis. We did not observe any association between D18S880 and diabetic nephropathy in Japanese subjects with type 2 diabetes. None of the 29 SNPs within the <em>CNDP1/CNDP2</em> locus were associated with diabetic nephropathy, but a subsequent sex-stratified analysis revealed that 1 SNP in <em>CNDP1</em> was nominally associated with diabetic nephropathy in women (rs12604675-A; <em>p</em> = 0.005, odds ratio [OR] = 1.76, 95% confidence interval [CI], 1.19−2.61). Rs12604675 was associated with overt proteinuria (<em>p</em> = 0.002, OR = 2.18, 95% CI, 1.32−3.60), but not with ESRD in Japanese women with type 2 diabetes.</p> <h3>Conclusions/Significance</h3><p>Rs12604675-A in <em>CNDP1</em> may confer susceptibility to overt proteinuria in Japanese women with type 2 diabetes.</p> </div

Predictive Properties of Plasma Amino Acid Profile for Cardiovascular Disease in Patients with Type 2 Diabetes

<div><p>Prevention of cardiovascular disease (CVD) is an important therapeutic object of diabetes care. This study assessed whether an index based on plasma free amino acid (PFAA) profiles could predict the onset of CVD in diabetic patients. The baseline concentrations of 31 PFAAs were measured with high-performance liquid chromatography-electrospray ionization-mass spectrometry in 385 Japanese patients with type 2 diabetes registered in 2001 for our prospective observational follow-up study. During 10 years of follow-up, 63 patients developed cardiovascular composite endpoints (myocardial infarction, angina pectoris, worsening of heart failure and stroke). Using the PFAA profiles and clinical information, an index (CVD-AI) consisting of six amino acids to predict the onset of any endpoints was retrospectively constructed. CVD-AI levels were significantly higher in patients who did than did not develop CVD. The area under the receiver-operator characteristic curve of CVD-AI (0.72 [95% confidence interval (CI): 0.64–0.79]) showed equal or slightly better discriminatory capacity than urinary albumin excretion rate (0.69 [95% CI: 0.62–0.77]) on predicting endpoints. A multivariate Cox proportional hazards regression analysis showed that the high level of CVD-AI was identified as an independent risk factor for CVD (adjusted hazard ratio: 2.86 [95% CI: 1.57–5.19]). This predictive effect of CVD-AI was observed even in patients with normoalbuminuria, as well as those with albuminuria. In conclusion, these results suggest that CVD-AI based on PFAA profiles is useful for identifying diabetic patients at risk for CVD regardless of the degree of albuminuria, or for improving the discriminative capability by combining it with albuminuria.</p></div

Crude and multivariate-adjusted hazard ratios for the cardiovascular composite endpoint in patient subgroups stratified according to urinary albumin excretion rate and the CVD-AI.

<p>Subjects were categorized as being above or below a UAER of 20 µg/min and above or below the CVD-AI cut-off value of −1.662. Crude (unadjusted) and adjusted hazard ratios were calculated using Cox proportional hazards regression models.</p>a<p>Estimates were adjusted for the conventional risk factors of cardiovascular disease, including age, sex, HbA1c, total cholesterol, triglyceride, high density lipoprotein cholesterol, estimated glomerular filtration rate, body mass index and hypertension.</p><p><i>Abbreviations:</i> CVD-AI, cardiovascular disease-amino acid based index; UAER, urinary albumin excretion rate.</p

Absolute levels of 31 plasma amino acids in patients who did (cases) and did not (controls) experience cardiovascular events during follow-up.

<p><i>Abbreviations:</i> HMDB ID: Human Metabolome Database ID.</p

Hazard ratios for the cardiovascular composite endpoint.

<p>The variables listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101219#pone-0101219-t001" target="_blank">Table 1</a> and CVD-AI were firstly assessed in the univariate analysis of the Cox proportional hazards regression model. Only variables shown to be statistically significant in the univariate model are shown in this table.</p>a<p>Each estimate was adjusted for all variables shown in this table.</p><p><i>Abbreviations:</i> BP, blood pressure; CI, confidence interval, CVD-AI, cardiovascular disease-amino acid based index; HDL, high density lipoprotein; UAER, urinary albumin excretion rate; eGFR, estimated glomerular filtration rate; baPWV, brachial-ankle pulse wave velocity.</p

Association of rs12604675-A with diabetic nephropathy.

<p>OR represents the odds ratio per copy of risk allele (A). P-values were calculated using a logistic regression analysis with additive model (adjusted for sex, age, log-transformed body mass index and duration of diabetes).</p

Clinical characteristics of the participants.

<p>Abbreviations: BMI, body mass index; HbA1c, hemoglobin A1c; SBP, systolic blood pressure; DBP, diastolic blood pressure.</p>a<p>4 unknown,</p>b<p>14 unknown.</p>#<p>Mean ± SD, HbA1c; NGSP.</p

Association of single nucleotide polymorphisms (SNPs) within <i>CNDP1/CNDP2</i> locus with diabetic nephropathy.

<p>Results of association studies are shown using: A) men and women, B) men only, or C) women only. The x-axis represents the position in chromosome 18, and the y-axis shows the absolute values of log₁₀-transformed association <i>p</i> values. Open squares represent unadjusted values, and black squares represent values adjusted for age, log-transformed body mass index, and duration of diabetes. Thresholds for nominal (<i>p</i> = 0.05) or statistical (<i>p</i> = 0.0004) significance are shown as broken lines.</p

Baseline characteristics of patients who did (cases) and did not (controls) experience cardiovascular events during follow-up.

<p>Data are expressed as mean ± SD for normally distributed continuous variables or median (interquartile range) for skewed continuous variables.</p><p><i>Abbreviations:</i> GFR, glomerular filtration rate; HDL, high density lipoprotein; baPWV, brachial-ankle pulse wave velocity.</p

Knockdown of PPARδ gene expression does not affect GW501516-mediated inhibition of <i>MCP-1</i> expression in mouse proximal tubular (mProx) cells treated with palmitate or TNFα.

<p>(A) Representative immunoblot showing protein expression of PPARδ in siRNA-mediated PPARδ-knockdown and control cells. (B) mRNA expression of PPARδ-target genes (<i>PDK4</i> and <i>ADRP</i>) in PPARδ-knockdown and control cells treated with or without GW501516. Results are expressed as fold change relative to the mRNA from the control group. <i>PDK4</i>: Pyruvate dehydrogenase kinase 4; <i>ADRP</i>: Adipocyte differentiation-related protein. The effect of PPARδ knockdown on GW501516-mediated inhibition of palmitate- (C) and TNFα- (D) induced <i>MCP-1</i> expression. Results are expressed as fold change relative to the mRNA from the control group. Data are shown as means ± SEM of three independent experiments. MCP-1: monocyte chemoattractant protein-1; TNFα: tumor necrotic factor α.</p