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AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure
Accepted Manuscript AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure
Xiao-Lei Hu, Wen-Jing Zeng, Mu-Peng Li, Yong-Long Yang, DaBin Kuang, He Li, Yan-Jiao Zhang, Chun Jiang, Li-Ming Peng, Hong Qi, Ke Zhang, Xiao-Ping Chen PII: DOI: Reference:
S0378-1119(17)30757-6 doi:10.1016/j.gene.2017.09.038 GENE 42187
To appear in:
Gene
Received date: Revised date: Accepted date:
12 June 2017 5 August 2017 19 September 2017
Please cite this article as: Xiao-Lei Hu, Wen-Jing Zeng, Mu-Peng Li, Yong-Long Yang, Da-Bin Kuang, He Li, Yan-Jiao Zhang, Chun Jiang, Li-Ming Peng, Hong Qi, Ke Zhang, Xiao-Ping Chen , AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/ j.gene.2017.09.038
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ACCEPTED MANUSCRIPT AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure Xiao-Lei Hu a, Wen-Jing Zeng a, Mu-Peng Li a, Yong-Long Yang b, Da-Bin Kuang a, He Li a,
Department of Clinical pharmacology, Xiangya Hospital, Central south university, Changsha,
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a
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Yan-Jiao Zhang a, Chun Jiang a, Li-Ming Peng c, Hong Qi c, Ke Zhang d* and Xiao-Ping Chen a*
b
Haikou People's Hospital and Affiliated Haikou Hospital of Xiangya Medical School,
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Central South University, Haikou, 570311, China;
Department of Cardiovascular Medicine, Xiangya Hospital, Central south university,
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c
Changsha, 410008, China.
Department of Nephrology, The Third Xiangya Hospital of Central south university,
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Changsha, 410013, China.
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d
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410008, China;
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Abbreviations: CHF, chronic heart failure; BUN, blood urea nitrogen; AGXT2, alanine-glyoxylate aminotransferase 2; ADMA, Asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; WRF, worsening renal function. * Corresponding author at: Department of Clinical pharmacology, Xiangya Hospital,
Central south university, No.110 Xiangya road, Changsha, Hunan 410008, China; E-mail address: [email protected] (Xiaoping Chen); [email protected] (Ke Zhang)
ACCEPTED MANUSCRIPT Abstract Patients with chronic heart failure (CHF) are often accompanied with varying degrees of renal diseases. The purpose of this study was to identify rs37369 polymorphism of AGXT2 specific to the renal function of CHF patients. A total of 1012 southern
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Chinese participants, including 487 CHF patients without renal diseases and 525
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healthy volunteers, were recruited for this study. Polymerase chain reaction-restriction
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fragment length polymorphism (PCR-RFLP) was used to determine the genotypes of AGXT2 rs37369 polymorphism. Levels of blood urea nitrogen (BUN) and serum
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creatinine (SCr) were detected to indicate the renal function of the participants. BUN
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level was significantly higher in CHF patients without renal diseases compared with healthy volunteers (p=0.000). And the similar result was also obtained in SCr
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(p=0.000). Besides, our results indicated that the level of BUN correlated significantly
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with SCr in CHF patients without renal diseases (r = 0.4533, p<0.0001) and volunteers (r = 0.2489, p<0.0001). Furthermore, we found AGXT2 rs37369
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polymorphism could significantly affect the level of BUN in CHF patients without
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renal diseases (p=0.036, AA+AG vs GG). Patients with rs37369 GG genotype showed a significantly reduced level of BUN compared to AA genotype (p=0.024), and the significant difference was still observed in the smokers of CHF patients without renal diseases (p=0.023). In conclusion, we found CHF might induce the impairment of kidney and cause deterioration of renal function. AGXT2 rs37369 polymorphism might affect the renal function of CHF patients free from renal diseases, especially in patients with cigarette smoking.
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Keywords: BUN, renal function, CHF, AGXT2, polymorphism
ACCEPTED MANUSCRIPT 1. Introduction Heart failure (HF) may often threaten human life and has now become a global public health issue worldwide. Report of Framingham Heart Study (FHS) indicates the incidence of HF approaches 1% in population above 65 years old, which will be
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an important contributor to both the burden and cost of healthcare expenditures
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(Heidenreich et al., 2013; Lloyd-Jones et al., 2002; Mozaffarian et al., 2016). Chronic
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heart failure (CHF) represents a relatively stable state of HF and CHF patients are often accompanied with varying degrees of renal diseases. A novel study found in
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heart failure patients that the association between estimated glomerular filtration rate
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(eGFR) and the higher risk of all-cause mortality might be largely dependent on concomitant elevation of blood urea nitrogen (BUN) (Kajimoto et al., 2016). Renal
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failure is reported to be one of the causes of HF (Heusch et al., 2014), and worsening
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renal function (WRF) has also been proved to be associated with reduced survival in patients with HF over the past two decades (Damman et al., 2014; Damman et al.,
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2015). Therefore, HF and renal disease might interact with each other as a reciprocal
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causation relation. A large number of studies indicated that CHF patients (including the patients accompanied with renal diseases) showed poor renal function, however, few reports demonstrated the phenomenon in CHF patients free from renal diseases, especially in Chinese. Besides, although much has been learned on cardiorenal interaction in HF, more dedicated mechanistic and controlled trials in HF patients should be provided eagerly in the coming decade. Nitric oxide (NO) plays a pivotal role in the homeostasis of cardiovascular system
ACCEPTED MANUSCRIPT and participates in the control of contractility and heart rate, contributes to the protection of cardiac function and limits the deleterious effects of cardiac remodeling after myocardial infarction (Naseem, 2005; Rastaldo et al., 2007). Asymmetric dimethylarginine (ADMA) could competitively bond with nitric oxide synthase (NOS)
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and obstruct the synthesis of nitric oxide (NO). Increasing evidences indicate that
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ADMA is a crucial and independent cardiovascular risk factor (Boger, 2004; Boger et
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al., 2005; Boger et al., 2009; De Gennaro et al., 2009; Duckelmann et al., 2007; Perticone et al., 2005; Willeit et al., 2015; Wolf et al., 2012). In CHF patients, the
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plasma level of ADMA is significantly elevated and positively associated with the
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progress of CHF (Hsu et al., 2012; Usui et al., 1998). Besides, increased circulating ADMA was also reported to be a powerful predictor of progressive chronic kidney
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disease (CKD), independent of eGFR and other traditional risk factors (Fliser et al.,
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2005; Zoccali et al., 2001). Symmetric dimethylarginine (SDMA), the structural isomer of ADMA, is transported efficiently by hCAT-2B and exchanged against
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intracellular L-arginine, resulting in a suppression of NO synthesis indirectly. A
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clinical-related research reported that SDMA might be an important parameter for severity of coronary artery disease and renal function (Bode-Boger et al., 2006; Tang et al., 2008). The major route of ADMA elimination is the hydrolytic degradation whereas SDMA is mostly eliminated by renal excretion (McDermott, 1976; Ogawa et al., 1987). There are two known metabolic pathways for the elimination of ADMA in humans, the first pathway is the hydrolysis of ADMA to citrulline and dimethylamine in the cytoplasm by dimethylarginine dimethylaminohydrolases (DDAH) and the
ACCEPTED MANUSCRIPT second is the transamination of ADMA to α-keto-δ-(NG, NG-dimethylguanidino) valeric acid (DMGV) via alanine-glyoxylate aminotransferase 2 (AGXT2) (Rodionov et al., 2010). AGXT2 is a promiscuous aminotransferase and a fraction of SDMA may as well be degraded into α-keto-δ-(NG, N’G-dimethylguanidino) valeric acid (DM’GV)
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by AGXT2 (Caplin et al., 2012; Kittel et al., 2013; Ogawa et al., 1987).
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There are two isoforms of AGXT observed in human: AGXT1 and AGXT2, both
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of which are pyridoxal phosphate (PLP)-dependent and catalyze the transfer of an amino group from alanine to glyoxylate (Baker et al., 2004; Noguchi et al., 1978).
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AGXT1 is primarily distributed in peroxisomes while AGXT2 is mainly located in
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mitochondria, and it is AGXT1 that plays a major role in the clearance of glyoxylate. Besides, two isoenzymes of human AGXT show different organ distribution in the
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human body. AGXT1 is mainly found in the liver while AGXT2 is primarily
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expressed in the kidney (Figure 1) (Kamoda et al., 1980; Luneburg et al., 2014). AGXT1 has been extensively studied owing to its crucial role in the detoxification of
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glyoxylate and the pathogenesis of primary hyperoxaluria type I (PH1) (Danpure et al.,
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1989; Mesa-Torres et al., 2013; Oppici et al., 2013). Whereas, few researchers showed their interests in AGXT2 until Roman and his colleagues ‘rediscovered’ the potential pathophysiological roles of AGXT2 and demonstrated AGXT2 could protect endothelial cells from ADMA-induced inhibition of nitric oxide production (Rodionov et al., 2010). Data from a genome-wide association study (GWAS) for metabolic traits indicated that there was a strong association between AGXT2 rs37369 (V140I) polymorphism
ACCEPTED MANUSCRIPT and human urinary β-aminoisobutyrate (BAIB) (Suhre et al., 2011). This polymorphism was also reported to be associated with diastolic blood pressure (Suhre et al., 2011). Besides, another GWAS revealed that the rs37369 polymorphism was significantly associated with heart rate variability which was indicated to be an
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important risk factor of CHF long before (Ponikowski et al., 1997), and the authors
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also indicated a significant correlation between rs37369 polymorphism and
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circulating SDMA levels (Seppala et al., 2014). Coincidentally, we had previously demonstrated that rs37369 polymorphism was associated with increased risk of CHF
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and coronary heart disease (CHD) in Chinese population (Hu et al., 2016; Zhou et al.,
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2014). As there is every indication that AGXT2 rs37369 polymorphism might imply a potential significance in cardiorenal interaction system, we assessed the potential
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association between the polymorphism and renal function of CHF patients in the
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present study.
ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Study Participants From December 2011 to March 2015, a total of 487 consecutive patients with CHF and 525 healthy volunteers were screened from XiangYa Hospital, Central South
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University (Changsha, China). All patients collected in our study were free from renal
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diseases and diagnosed as New York Heart Association (NYHA) functional class
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(II/Ⅲ/IV). Patients with congenital heart disease, severe liver disease and any other confirmation of life-threatening disease were excluded from this study. All the healthy
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volunteers were free from CHD, hypertension, diabetes mellitus, ischemic heart
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disease and any other CVDs. The levels of BUN and SCr were detected to indicate the renal function of participants. And all patients' blood was collected for the detection
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of renal function when the patients were hospitalized and had not taken drugs.
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Detection results of BUN and SCr were provided by the hospital's laboratory. Smokers in our study were moderate or severe smokers and defined with cigarette
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smoking equal or greater than 27 packages a year. The study protocol was in
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accordance with the principles of Declaration of Helsinki and approved by ethics committee of Pharmaceutical College, Central South University, Changsha. The registration number in Chinese Clinical Trial Registry (http://www.chictr.org.cn/) was ChiCTR-RCC-12002817.
2.2 Genotyping of rs37369 polymorphism The venous blood samples of all participants were collected into EDTA
ACCEPTED MANUSCRIPT anticoagulant tubes and stored at -20 °C for extraction of DNA. Method of phenol-chloroform was used to isolate the genomic DNA of participants. AGXT2 rs37369 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as we previously described (Hu et al.,
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2016). The target fragment of 520 bp was amplified using primers 5 ´
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-tagggacgctcccctagaat-3´(forward) and 5´-tctaagcccaaacttcctcct-3´ (reverse). Two
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microliters of PCR products were digested with 3 U BbsI restriction enzyme (Thermo Fisher Scientific, USA) at 37 ℃ overnight. Then the digested PCR products were
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analyzed by electrophoresis on an agarose gel followed by GelRed (Biotium, USA)
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staining. In addition, a random selection of 5% of the samples was also genotyped by
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2.3 Statistical analysis
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Sanger sequencing and the genotyping were confirmed in 100%.
Data of continuous variables were showed as mean ± SD, and categorical variables
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were presented as numbers and percentages. Chi-square test was used for comparison
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of the categorical variables and independent sample t test for continuous variables. Spearman correlation analysis was used to perform the correlation analysis between variables. Chi-square test was also used to assess the deviation from Hardy-Weinberg equilibrium. Differences in the level of BUN and SCr between CHF patients and healthy volunteers were compared by independent sample t-test. One-way ANOVA or independent sample t-test was used to analyze the effect of rs37369 polymorphism on the level of BUN (or SCr) and the results were adjusted for gender, age, history of
ACCEPTED MANUSCRIPT smoking, history of alcohol consumption, hypertension, diabetes mellitus and coronary heart disease by univariate general linear model. All of the above analyses were performed in SPSS software version 18.0 (IBM corporation, Armonk, NY, USA)
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and a two-tailed p<0.05 was considered to be statistically significant.
ACCEPTED MANUSCRIPT 3. Results 3.1 Characteristics of Participants General information of participants were briefly summarized in Table 1. CHF patients and healthy controls were matched in gender, but the age of CHF patients was
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slightly but significantly older than that of healthy controls. Compared to the control,
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patients with CHF exhibited significantly higher systolic blood pressure (SBP),
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diastolic blood pressure (DBP), and lower level of high-density lipoprotein cholesterol (HDL). The prevalence of smoking was greater in patients with CHF in
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comparison with that observed in healthy controls.
3.2 CHF itself is associated with renal impairment
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We determined the levels of BUN and SCr in 487 CHF patients without renal
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diseases and 525 healthy controls. Compared to the controls, the level of BUN was significantly elevated in the CHF patients free from renal diseases after adjusting for
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age and gender (6.30±2.53 vs 4.75±1.28 mmo/L, p=0.000, Figure 2A). And the
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similar result was also obtained when we analyzed the level of SCr in cases and controls (93.6±29.3 vs 61.8±14.5 μmol/L, p=0.000, Figure 2B).
3.3 The association between BUN and SCr It is widely acknowledged that BUN and SCr are two main indicators of renal function. Here, we verified the correlationship between BUN and SCr in our present data. As expected, the level of BUN was significantly correlated with the level of SCr
ACCEPTED MANUSCRIPT in healthy objects (r = 0.2489, p<0.0001, Figure 2C) and in CHF patients without renal diseases (r = 0.4533, p<0.0001, Figure 2D).
3.4 AGXT2 rs37369 polymorphism could influence the BUN level in patients
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To find the effect of AGXT2 rs37369 polymorphism on the function of kidney, the
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levels of BUN and SCr were determined in 487 CHF cases and 525 healthy volunteers.
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Genotype distribution of the polymorphism investigated in our study was in accordance with Hardy–Weinberg equilibrium in both case and control samples. For
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the patients without renal diseases, significant difference in BUN level among the
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genotypes was observed and carriers of GG genotype showed significantly decreased BUN level compared to that of AA genotype carriers (adjusted p=0.024, Table 2,
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Figure 3A). Despite a downward trend of the level of SCr was observed in patients
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carrying the GG genotype, no significant difference was observed (Table 2, Figure 3B). Besides, we also could not find significant differences of the BUN/SCr level in
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the healthy volunteers among three different genotypes (Table 3).
3.5 AGXT2 rs37369 polymorphism could influence the BUN level in smoking patients
Considering that smoking was not only a very common risk factor for cardiovascular disease but also an important factor of CKD, we had performed a stratification analysis by smoking status to evaluate the effect of smoking on renal function in CHF patients without renal diseases. We found carriers of GG genotype
ACCEPTED MANUSCRIPT showed significantly reduced level of BUN in smokers of patients without renal diseases compared to the patients with AA genotype (adjusted p=0.023, Table 2, Figure 3C). A similar trend (without significant) was also observed in the non-smokers of CHF patients free from renal diseases (Table 2, Figure 3D). Besides,
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smoking could also marginally increased the BUN level in CHF patients with AA and
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AG genotypes and inversely reduced the BUN level in CHF patients with GG
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genotypes, although both of them were out of significant (Figure 3E).
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4. Discussion
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In this study, we verified that CHF was associated with renal impairment and bring about worsening renal function. Furthermore, we investigated the association between
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AGXT2 rs37369 polymorphism and renal function in CHF patients without renal
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diseases or in healthy volunteers for the first time. Our results demonstrated that CHF patients (free from renal diseases) carrying rs37369 GG genotype showed a decreased
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BUN level.
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The clinical relevance of HF with renal diseases has attracted much interest in recent years. Cardiac and renal diseases were regulated by some common factors, such as inflammation, cellular immunity, metabolic and nutritional changes, and they interact each other in a complex bidirectional and interdependent manner (Schefold et al., 2016). The clinical related evidence is urgent to verify the mechanism underlying the interaction. BUN and SCr are nitrogenous end products of protein metabolism and both of them could be filtered at the glomerulus freely owing to their relatively small
ACCEPTED MANUSCRIPT molecules. The level of BUN was correlated with the level of Scr and the result was verified in our study both in CHF patients and in healthy donors. In fact, it is universally recognized that both of BUN and SCr are confirmed as critical markers of renal function, and increasing evidences indicate that BUN and SCr are associated
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with clinical outcomes in patients with HF (Damman et al., 2014; Matsue et al., 2016;
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van Veldhuisen et al., 2016). In this study, we found the levels of BUN and SCr were
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significantly elevated in the CHF patients free from renal diseases which indicated that CHF itself might make contributions to the impairment of kidney and
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deterioration of renal function. The result might be beneficial to clarifying the
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mechanism between CHF and renal function to a certain extent. In addition, we are the first to report the association between AGXT2 rs37369
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polymorphism and renal function in CHF patients or in healthy controls. In our study,
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we found the level of BUN was significantly elevated in the CHF patients (free from renal diseases) with rs37369 AA genotype, especially in smoking patients. An
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analogous trend (without significant) was also observed in the level of SCr. ADMA is
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an endogenous inhibitor of NOS which can competitively obstruct NO synthesis, and there is no doubting that elevated plasma levels of ADMA are associated with poor outcomes. However, James and his colleagues recently found therapeutic ADMA reduction might even be deleterious to renal function, and they put forward that reduced renal ADMA metabolism might protect against progressive kidney damage (Tomlinson et al., 2015). In our previous study, we had found the enzyme AGXT2 (rs37369 A allele) showed significantly higher ADMA metabolic activity in vitro (Hu
ACCEPTED MANUSCRIPT et al., 2016) which might indicate therapeutic ADMA reduction in patients with rs37369 AA genotype. It might be the reason why we found the polymorphism might influence the renal function in CHF patients. Since we did not detect the level of renal tubular ADMA in different rs37369 genotypes, further investigation are deserved to
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clear the specific reasons causing this difference urgently. Besides, plasma SDMA
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concentration was reported to be highly significantly correlated with both glomerular
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filtration rate (GFR, r=-0.837, P<0.01) and SCr (r=0.894, P<0.01) (Fliser et al., 2005) and might be an important parameter for predicting the early stages of renal diseases
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(Bode-Boger et al., 2006). SDMA is mostly eliminated by renal excretion in the body.
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However, there was still a small fraction of SDMA metabolized by AGXT2 despite of renal excretion (Caplin et al., 2012). Data from the Leeds Stroke Study demonstrated
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that patients carrying AGXT2 rs37369 AA genotype showed significantly enhanced
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SDMA plasma concentration which is also consistent with our results (Luneburg et al., 2014).
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It was interesting that we did not find significant effect of rs37369 polymorphism
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on the renal function of healthy volunteers. We suspected that it might be caused by the lower level of circulating dimethylarginine in healthy population. Strong evidence indicated that ADMA was dramatically elevated in the patients with CHF (Usui et al., 1998), and it might be precisely because of the high concentration of ADMA in CHF patients that highlighted the role of AGXT2 in dimethylarginine metabolism. Meanwhile, we must acknowledge that there are several limitations of this study. Our results were obtained just from one population and with limited samples,
ACCEPTED MANUSCRIPT therefore, multicenter studies are eagerly needed to establish the clinical significance of AGXT2 rs37369 polymorphism in renal function of CHF patients. Moreover, we recognize that we failed to detect the level of ADMA and SDMA in patients and healthy volunteers owing to the limited volume of blood samples collected in our
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study, which might not directly and perfectly support our findings. Nevertheless, we
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hope our findings can arouse interest for further investigations into mechanism
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underlying the association between AGXT2 and renal function of CHF. In summary, we are the first study to reveal the association between AGXT2
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rs37369 polymorphism and renal function in CHF patients and demonstrate that
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rs37369 polymorphism might predict the renal function of patients with CHF, especially in the smokers of CHF patients. Meanwhile, we also verified CHF itself
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might lead to worsening renal function and the impairment of kidney. Further
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investigations in other races and larger cohorts may be required to verify our findings. More comprehensive surveys and functional experiments are also needed to
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illuminate the exact mechanism underlying the association.
Conflict of interest The authors have no conflict of interest to declare.
ACCEPTED MANUSCRIPT Acknowledgements This work was supported by National Natural Science Foundation of China (No.81170091, No.81373489, No.81403018 and No.81422052), National Science and Technology Major Project (2013ZX09509107), and the Fundamental Research Funds
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for the Central Universities of Central South University (2017zzts223).
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Angelantonio E. and Chowdhury R. 2015. Asymmetric dimethylarginine and
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cardiovascular risk: systematic review and meta-analysis of 22 prospective studies. J Am Heart Assoc. 4, e1833.
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Zhou J.P., Bai Y.P., Hu X.L., Kuang D.B., Shi R.Z., Xiong Y., Zhang W., Xia J., Chen B.L., Yang T.L. and Chen X.P. 2014. Association of the AGXT2 V140I polymorphism with risk for coronary heart disease in a Chinese population. J Atheroscler Thromb. 21, 1022-1030. Zoccali C., Bode-Boger S., Mallamaci F., Benedetto F., Tripepi G., Malatino L., Cataliotti A., Bellanuova I., Fermo I., Frolich J. and Boger R. 2001. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet. 358, 2113-2117.
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Figure legends
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Figure 1. Organ distribution of AGXT in the human body. The data were
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obtained from the human protein atlas (http://www.proteinatlas.org/).
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Figure 2. BUN and SCr in volunteers and CHF patients without renal diseases. (A) and (B): Comparison of the levels of BUN and SCr in healthy volunteers and
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CHF patients without renal diseases. (C) and (D): The correlation between BUN and SCr in healthy controls and CHF patients without renal diseases. CHFa: CHF patients
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who were free from renal diseases. *p<0.05 **p<0.01 ***p<0.001.
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Figure 3. AGXT2 rs37369 polymorphism and renal function in CHF patients
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without renal diseases. (A and B): Association between AGXT2 rs37369 polymorphism and BUN and SCr in CHF patients without renal diseases. (C, D, and E): Influence of AGXT2 rs37369 polymorphism on the BUN level in smoking patients. CHFa: CHF patients who were free from renal diseases. *p<0.05.
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Fig. 2
Fig. 3
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Table 1. General characteristics of the study population. Characteristics
T P
CHF without renal diseases
Healthy volunteers
N
487
525
Male (%)
290 (59.5)
Age (years)
59±13
SBP (mmHg)
130±23
DBP (mmHg)
78±13
I R
SC
p value
284 (54.1)
0.080
56±10
<0.001
118±15
<0.001
75±10
<0.001
1.15±0.34
1.27±0.30
<0.001
2.33±0.89
2.23±0.60
0.070
204 (41.9)
135 (25.7)
<0.001
Positive history of alcohol consumption (%)
149 (30.6)
130 (24.8)
0.118
Coronary heart disease (%)
365 (74.9)
0 (0)
<0.001
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HDL (mmol/L)
E C
LDL (mmol/L)
C A
Positive history of smoking (%)
A M
U N
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Hypertension (%)
284 (58.3)
0 (0)
<0.001
Diabetes mellitus (%)
104 (36.6)
0 (0)
<0.001
I R
T P
Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density
C S U
lipoprotein.
N A
D E
T P E
A
C C
M
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Table 2. Influence of rs37369 polymorphism on the level of BUN in CHF patients without renal diseases BUN(mmo/L)
SCr(μmol/L)
T P
I R
rs37369 genotypes
N
Mean
p
pa
—
—
152
94.0±30.2
—
—
0.496
0.264
248
93.9±29.9
0.634
0.770
Mean
p
AA
152
6.64±3.00
AG
248
6.28±2.31
Entire cohort
Dominant
Recessive
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T P E
GG
C S U pa
N
N A
M
87
5.77±2.12
0.029
0.024
87
92.1±26.0
0.611
0.646
152
6.64±3.00
—
—
152
94.0±30.2
—
—
AG+GG
335
6.15±2.27
0.072
0.075
335
93.4±28.9
0.830
0.978
AG+AA
400
6.42±2.60
—
—
400
93.9±30.0
—
—
GG
87
5.77±2.12
0.031
0.036
87
92.1±26.0
0.607
0.428
AA
C C
A
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Dominant
Recessive
AA
67
6.91±3.07
—
—
67
AG
96
6.39±2.33
0.217
0.269
96
GG
41
5.69±2.44
0.020
AA
67
6.91±3.07
—
AG+GG
137
6.18±2.38
0.065
AG+AA
163
6.61±2.66
D E
T P E
GG
—
—
97.9±29.3
0.945
0.973
T P
I R
C S U
97.6±28.0
0.023
41
91.2±23.3
0.249
0.086
—
67
97.6±28.0
—
—
0.079
137
95.9±27.7
0.683
0.513
—
—
163
97.8±28.7
—
—
N A
M
41
5.69±2.44
0.047
0.046
41
91.2±23.3
0.177
0.089
85
6.43±2.94
—
—
85
91.2±31.7
—
—
AG
152
6.21±2.31
0.505
0.625
152
91.3±30.1
0.982
0.623
GG
46
5.85±1.83
0.191
0.262
46
93.0±28.5
0.755
0.592
Non-Smokers
C C
AA
A
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Recessive
a
AA
85
6.43±2.94
—
—
85
91.2±31.7
—
—
AG+GG
198
6.13±2.21
0.335
0.427
198
91.7±29.7
0.560
AG+AA
237
6.29±2.55
—
—
237
T P
0.905
91.3±30.6
—
—
GG
46
5.85±1.83
0.260
93.0±28.5
0.732
0.687
C S U
I R
0.298
46
Adjusted for gender, age, history of smoking, history of alcohol consumption, hypertension, diabetes mellitus and coronary heart disease
N A
by univariate general linear model.
D E
T P E
A
C C
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Table 3. The rs37369 polymorphism did not affect the level of BUN and Scr in healthy volunteers. BUN(mmo/L)
SCr(μmol/L)
T P
I R
rs37369 genotypes
N
Mean
p
pa
—
—
184
61.1±14.9
—
—
0.262
0.383
269
62.3±14.6
0.386
0.808
Mean
p
AA
184
4.67±1.26
AG
269
4.80±1.29
GG
Entire cohort
Dominant
Recessive
D E
C S U pa
N
N A
M
72
4.84±1.30
T P E
0.328
0.221
72
61.4±13.0
0.863
0.848
184
4.67±1.26
—
—
184
61.1±14.9
—
—
AG+GG
341
4.81±1.29
0.216
0.289
341
62.1±14.3
0.435
0.758
AG+AA
453
4.75±1.28
—
—
453
61.8±14.7
—
—
GG
72
4.84±1.30
0.574
0.433
72
61.4±13.0
0.836
0.881
AA
A
C C
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Smokers
Dominant
Recessive
AA
52
4.75±1.26
—
—
52
AG
65
4.57±1.09
0.427
0.293
65
GG
18
5.15±1.08
0.215
0.306
AA
52
4.75±1.26
—
AG+GG
83
4.70±1.11
0.813
AG+AA
117
4.66±1.17
GG
69.4±12.6
0.522
0.541
18
69.4±10.0
0.740
0.708
—
52
67.0±11.7
—
—
0.620
83
69.4±12.1
0.252
0.547
—
—
117
68.3±12.2
—
—
C S U
N A
M
I R
T P
T P E
0.097
0.105
18
69.4±10.0
0.724
0.969
132
4.63±1.26
—
—
132
58.8±15.4
—
—
204
4.87±1.35
0.101
0.110
204
60.1±14.5
0.434
0.841
54
4.74±1.37
0.624
0.448
54
58.8±12.9
0.997
0.960
C C
A
—
5.15±1.08
Non-Smokers
AG
—
18
GG
AA
D E
67.0±11.7
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Dominant
Recessive
a
AA
132
4.63±1.26
—
—
132
58.8±15.4
—
—
AG+GG
258
4.85±1.35
0.131
0.137
258
59.8±14.2
0.517
0.912
AG+AA
336
4.78±1.32
—
—
336
59.6±14.9
—
—
GG
54
4.74±1.37
0.826
0.990
58.8±12.9
0.720
0.867
C S U
I R
T P
54
Adjusted for gender, age, history of smoking and history of alcohol consumption by univariate general linear model.
N A
D E
T P E
A
C C
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AGXT2 rs37369 polymorphism predicts the renal function in patients
Highlights:
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(1) CHF itself might lead to worsening renal function.
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with chronic heart failure
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(2) AGXT2 rs37369 polymorphism could significantly affect the level of BUN in CHF patients without renal diseases.
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(3) AGXT2 rs37369 polymorphism could also significantly affect the level of
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BUN in smokers of CHF patients without renal diseases.
Xiao-Lei Hu, Wen-Jing Zeng, Mu-Peng Li, Yong-Long Yang, DaBin Kuang, He Li, Yan-Jiao Zhang, Chun Jiang, Li-Ming Peng, Hong Qi, Ke Zhang, Xiao-Ping Chen PII: DOI: Reference:
S0378-1119(17)30757-6 doi:10.1016/j.gene.2017.09.038 GENE 42187
To appear in:
Gene
Received date: Revised date: Accepted date:
12 June 2017 5 August 2017 19 September 2017
Please cite this article as: Xiao-Lei Hu, Wen-Jing Zeng, Mu-Peng Li, Yong-Long Yang, Da-Bin Kuang, He Li, Yan-Jiao Zhang, Chun Jiang, Li-Ming Peng, Hong Qi, Ke Zhang, Xiao-Ping Chen , AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/ j.gene.2017.09.038
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ACCEPTED MANUSCRIPT AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure Xiao-Lei Hu a, Wen-Jing Zeng a, Mu-Peng Li a, Yong-Long Yang b, Da-Bin Kuang a, He Li a,
Department of Clinical pharmacology, Xiangya Hospital, Central south university, Changsha,
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a
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Yan-Jiao Zhang a, Chun Jiang a, Li-Ming Peng c, Hong Qi c, Ke Zhang d* and Xiao-Ping Chen a*
b
Haikou People's Hospital and Affiliated Haikou Hospital of Xiangya Medical School,
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Central South University, Haikou, 570311, China;
Department of Cardiovascular Medicine, Xiangya Hospital, Central south university,
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c
Changsha, 410008, China.
Department of Nephrology, The Third Xiangya Hospital of Central south university,
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Changsha, 410013, China.
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d
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410008, China;
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Abbreviations: CHF, chronic heart failure; BUN, blood urea nitrogen; AGXT2, alanine-glyoxylate aminotransferase 2; ADMA, Asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; WRF, worsening renal function. * Corresponding author at: Department of Clinical pharmacology, Xiangya Hospital,
Central south university, No.110 Xiangya road, Changsha, Hunan 410008, China; E-mail address: [email protected] (Xiaoping Chen); [email protected] (Ke Zhang)
ACCEPTED MANUSCRIPT Abstract Patients with chronic heart failure (CHF) are often accompanied with varying degrees of renal diseases. The purpose of this study was to identify rs37369 polymorphism of AGXT2 specific to the renal function of CHF patients. A total of 1012 southern
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Chinese participants, including 487 CHF patients without renal diseases and 525
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healthy volunteers, were recruited for this study. Polymerase chain reaction-restriction
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fragment length polymorphism (PCR-RFLP) was used to determine the genotypes of AGXT2 rs37369 polymorphism. Levels of blood urea nitrogen (BUN) and serum
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creatinine (SCr) were detected to indicate the renal function of the participants. BUN
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level was significantly higher in CHF patients without renal diseases compared with healthy volunteers (p=0.000). And the similar result was also obtained in SCr
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(p=0.000). Besides, our results indicated that the level of BUN correlated significantly
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with SCr in CHF patients without renal diseases (r = 0.4533, p<0.0001) and volunteers (r = 0.2489, p<0.0001). Furthermore, we found AGXT2 rs37369
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polymorphism could significantly affect the level of BUN in CHF patients without
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renal diseases (p=0.036, AA+AG vs GG). Patients with rs37369 GG genotype showed a significantly reduced level of BUN compared to AA genotype (p=0.024), and the significant difference was still observed in the smokers of CHF patients without renal diseases (p=0.023). In conclusion, we found CHF might induce the impairment of kidney and cause deterioration of renal function. AGXT2 rs37369 polymorphism might affect the renal function of CHF patients free from renal diseases, especially in patients with cigarette smoking.
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Keywords: BUN, renal function, CHF, AGXT2, polymorphism
ACCEPTED MANUSCRIPT 1. Introduction Heart failure (HF) may often threaten human life and has now become a global public health issue worldwide. Report of Framingham Heart Study (FHS) indicates the incidence of HF approaches 1% in population above 65 years old, which will be
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an important contributor to both the burden and cost of healthcare expenditures
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(Heidenreich et al., 2013; Lloyd-Jones et al., 2002; Mozaffarian et al., 2016). Chronic
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heart failure (CHF) represents a relatively stable state of HF and CHF patients are often accompanied with varying degrees of renal diseases. A novel study found in
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heart failure patients that the association between estimated glomerular filtration rate
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(eGFR) and the higher risk of all-cause mortality might be largely dependent on concomitant elevation of blood urea nitrogen (BUN) (Kajimoto et al., 2016). Renal
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failure is reported to be one of the causes of HF (Heusch et al., 2014), and worsening
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renal function (WRF) has also been proved to be associated with reduced survival in patients with HF over the past two decades (Damman et al., 2014; Damman et al.,
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2015). Therefore, HF and renal disease might interact with each other as a reciprocal
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causation relation. A large number of studies indicated that CHF patients (including the patients accompanied with renal diseases) showed poor renal function, however, few reports demonstrated the phenomenon in CHF patients free from renal diseases, especially in Chinese. Besides, although much has been learned on cardiorenal interaction in HF, more dedicated mechanistic and controlled trials in HF patients should be provided eagerly in the coming decade. Nitric oxide (NO) plays a pivotal role in the homeostasis of cardiovascular system
ACCEPTED MANUSCRIPT and participates in the control of contractility and heart rate, contributes to the protection of cardiac function and limits the deleterious effects of cardiac remodeling after myocardial infarction (Naseem, 2005; Rastaldo et al., 2007). Asymmetric dimethylarginine (ADMA) could competitively bond with nitric oxide synthase (NOS)
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and obstruct the synthesis of nitric oxide (NO). Increasing evidences indicate that
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ADMA is a crucial and independent cardiovascular risk factor (Boger, 2004; Boger et
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al., 2005; Boger et al., 2009; De Gennaro et al., 2009; Duckelmann et al., 2007; Perticone et al., 2005; Willeit et al., 2015; Wolf et al., 2012). In CHF patients, the
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plasma level of ADMA is significantly elevated and positively associated with the
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progress of CHF (Hsu et al., 2012; Usui et al., 1998). Besides, increased circulating ADMA was also reported to be a powerful predictor of progressive chronic kidney
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disease (CKD), independent of eGFR and other traditional risk factors (Fliser et al.,
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2005; Zoccali et al., 2001). Symmetric dimethylarginine (SDMA), the structural isomer of ADMA, is transported efficiently by hCAT-2B and exchanged against
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intracellular L-arginine, resulting in a suppression of NO synthesis indirectly. A
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clinical-related research reported that SDMA might be an important parameter for severity of coronary artery disease and renal function (Bode-Boger et al., 2006; Tang et al., 2008). The major route of ADMA elimination is the hydrolytic degradation whereas SDMA is mostly eliminated by renal excretion (McDermott, 1976; Ogawa et al., 1987). There are two known metabolic pathways for the elimination of ADMA in humans, the first pathway is the hydrolysis of ADMA to citrulline and dimethylamine in the cytoplasm by dimethylarginine dimethylaminohydrolases (DDAH) and the
ACCEPTED MANUSCRIPT second is the transamination of ADMA to α-keto-δ-(NG, NG-dimethylguanidino) valeric acid (DMGV) via alanine-glyoxylate aminotransferase 2 (AGXT2) (Rodionov et al., 2010). AGXT2 is a promiscuous aminotransferase and a fraction of SDMA may as well be degraded into α-keto-δ-(NG, N’G-dimethylguanidino) valeric acid (DM’GV)
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by AGXT2 (Caplin et al., 2012; Kittel et al., 2013; Ogawa et al., 1987).
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There are two isoforms of AGXT observed in human: AGXT1 and AGXT2, both
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of which are pyridoxal phosphate (PLP)-dependent and catalyze the transfer of an amino group from alanine to glyoxylate (Baker et al., 2004; Noguchi et al., 1978).
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AGXT1 is primarily distributed in peroxisomes while AGXT2 is mainly located in
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mitochondria, and it is AGXT1 that plays a major role in the clearance of glyoxylate. Besides, two isoenzymes of human AGXT show different organ distribution in the
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human body. AGXT1 is mainly found in the liver while AGXT2 is primarily
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expressed in the kidney (Figure 1) (Kamoda et al., 1980; Luneburg et al., 2014). AGXT1 has been extensively studied owing to its crucial role in the detoxification of
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glyoxylate and the pathogenesis of primary hyperoxaluria type I (PH1) (Danpure et al.,
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1989; Mesa-Torres et al., 2013; Oppici et al., 2013). Whereas, few researchers showed their interests in AGXT2 until Roman and his colleagues ‘rediscovered’ the potential pathophysiological roles of AGXT2 and demonstrated AGXT2 could protect endothelial cells from ADMA-induced inhibition of nitric oxide production (Rodionov et al., 2010). Data from a genome-wide association study (GWAS) for metabolic traits indicated that there was a strong association between AGXT2 rs37369 (V140I) polymorphism
ACCEPTED MANUSCRIPT and human urinary β-aminoisobutyrate (BAIB) (Suhre et al., 2011). This polymorphism was also reported to be associated with diastolic blood pressure (Suhre et al., 2011). Besides, another GWAS revealed that the rs37369 polymorphism was significantly associated with heart rate variability which was indicated to be an
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important risk factor of CHF long before (Ponikowski et al., 1997), and the authors
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also indicated a significant correlation between rs37369 polymorphism and
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circulating SDMA levels (Seppala et al., 2014). Coincidentally, we had previously demonstrated that rs37369 polymorphism was associated with increased risk of CHF
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and coronary heart disease (CHD) in Chinese population (Hu et al., 2016; Zhou et al.,
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2014). As there is every indication that AGXT2 rs37369 polymorphism might imply a potential significance in cardiorenal interaction system, we assessed the potential
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association between the polymorphism and renal function of CHF patients in the
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present study.
ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Study Participants From December 2011 to March 2015, a total of 487 consecutive patients with CHF and 525 healthy volunteers were screened from XiangYa Hospital, Central South
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University (Changsha, China). All patients collected in our study were free from renal
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diseases and diagnosed as New York Heart Association (NYHA) functional class
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(II/Ⅲ/IV). Patients with congenital heart disease, severe liver disease and any other confirmation of life-threatening disease were excluded from this study. All the healthy
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volunteers were free from CHD, hypertension, diabetes mellitus, ischemic heart
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disease and any other CVDs. The levels of BUN and SCr were detected to indicate the renal function of participants. And all patients' blood was collected for the detection
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of renal function when the patients were hospitalized and had not taken drugs.
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Detection results of BUN and SCr were provided by the hospital's laboratory. Smokers in our study were moderate or severe smokers and defined with cigarette
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smoking equal or greater than 27 packages a year. The study protocol was in
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accordance with the principles of Declaration of Helsinki and approved by ethics committee of Pharmaceutical College, Central South University, Changsha. The registration number in Chinese Clinical Trial Registry (http://www.chictr.org.cn/) was ChiCTR-RCC-12002817.
2.2 Genotyping of rs37369 polymorphism The venous blood samples of all participants were collected into EDTA
ACCEPTED MANUSCRIPT anticoagulant tubes and stored at -20 °C for extraction of DNA. Method of phenol-chloroform was used to isolate the genomic DNA of participants. AGXT2 rs37369 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as we previously described (Hu et al.,
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2016). The target fragment of 520 bp was amplified using primers 5 ´
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-tagggacgctcccctagaat-3´(forward) and 5´-tctaagcccaaacttcctcct-3´ (reverse). Two
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microliters of PCR products were digested with 3 U BbsI restriction enzyme (Thermo Fisher Scientific, USA) at 37 ℃ overnight. Then the digested PCR products were
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analyzed by electrophoresis on an agarose gel followed by GelRed (Biotium, USA)
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staining. In addition, a random selection of 5% of the samples was also genotyped by
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2.3 Statistical analysis
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Sanger sequencing and the genotyping were confirmed in 100%.
Data of continuous variables were showed as mean ± SD, and categorical variables
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were presented as numbers and percentages. Chi-square test was used for comparison
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of the categorical variables and independent sample t test for continuous variables. Spearman correlation analysis was used to perform the correlation analysis between variables. Chi-square test was also used to assess the deviation from Hardy-Weinberg equilibrium. Differences in the level of BUN and SCr between CHF patients and healthy volunteers were compared by independent sample t-test. One-way ANOVA or independent sample t-test was used to analyze the effect of rs37369 polymorphism on the level of BUN (or SCr) and the results were adjusted for gender, age, history of
ACCEPTED MANUSCRIPT smoking, history of alcohol consumption, hypertension, diabetes mellitus and coronary heart disease by univariate general linear model. All of the above analyses were performed in SPSS software version 18.0 (IBM corporation, Armonk, NY, USA)
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and a two-tailed p<0.05 was considered to be statistically significant.
ACCEPTED MANUSCRIPT 3. Results 3.1 Characteristics of Participants General information of participants were briefly summarized in Table 1. CHF patients and healthy controls were matched in gender, but the age of CHF patients was
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slightly but significantly older than that of healthy controls. Compared to the control,
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patients with CHF exhibited significantly higher systolic blood pressure (SBP),
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diastolic blood pressure (DBP), and lower level of high-density lipoprotein cholesterol (HDL). The prevalence of smoking was greater in patients with CHF in
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comparison with that observed in healthy controls.
3.2 CHF itself is associated with renal impairment
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We determined the levels of BUN and SCr in 487 CHF patients without renal
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diseases and 525 healthy controls. Compared to the controls, the level of BUN was significantly elevated in the CHF patients free from renal diseases after adjusting for
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age and gender (6.30±2.53 vs 4.75±1.28 mmo/L, p=0.000, Figure 2A). And the
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similar result was also obtained when we analyzed the level of SCr in cases and controls (93.6±29.3 vs 61.8±14.5 μmol/L, p=0.000, Figure 2B).
3.3 The association between BUN and SCr It is widely acknowledged that BUN and SCr are two main indicators of renal function. Here, we verified the correlationship between BUN and SCr in our present data. As expected, the level of BUN was significantly correlated with the level of SCr
ACCEPTED MANUSCRIPT in healthy objects (r = 0.2489, p<0.0001, Figure 2C) and in CHF patients without renal diseases (r = 0.4533, p<0.0001, Figure 2D).
3.4 AGXT2 rs37369 polymorphism could influence the BUN level in patients
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To find the effect of AGXT2 rs37369 polymorphism on the function of kidney, the
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levels of BUN and SCr were determined in 487 CHF cases and 525 healthy volunteers.
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Genotype distribution of the polymorphism investigated in our study was in accordance with Hardy–Weinberg equilibrium in both case and control samples. For
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the patients without renal diseases, significant difference in BUN level among the
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genotypes was observed and carriers of GG genotype showed significantly decreased BUN level compared to that of AA genotype carriers (adjusted p=0.024, Table 2,
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Figure 3A). Despite a downward trend of the level of SCr was observed in patients
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carrying the GG genotype, no significant difference was observed (Table 2, Figure 3B). Besides, we also could not find significant differences of the BUN/SCr level in
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the healthy volunteers among three different genotypes (Table 3).
3.5 AGXT2 rs37369 polymorphism could influence the BUN level in smoking patients
Considering that smoking was not only a very common risk factor for cardiovascular disease but also an important factor of CKD, we had performed a stratification analysis by smoking status to evaluate the effect of smoking on renal function in CHF patients without renal diseases. We found carriers of GG genotype
ACCEPTED MANUSCRIPT showed significantly reduced level of BUN in smokers of patients without renal diseases compared to the patients with AA genotype (adjusted p=0.023, Table 2, Figure 3C). A similar trend (without significant) was also observed in the non-smokers of CHF patients free from renal diseases (Table 2, Figure 3D). Besides,
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smoking could also marginally increased the BUN level in CHF patients with AA and
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AG genotypes and inversely reduced the BUN level in CHF patients with GG
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genotypes, although both of them were out of significant (Figure 3E).
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4. Discussion
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In this study, we verified that CHF was associated with renal impairment and bring about worsening renal function. Furthermore, we investigated the association between
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AGXT2 rs37369 polymorphism and renal function in CHF patients without renal
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diseases or in healthy volunteers for the first time. Our results demonstrated that CHF patients (free from renal diseases) carrying rs37369 GG genotype showed a decreased
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BUN level.
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The clinical relevance of HF with renal diseases has attracted much interest in recent years. Cardiac and renal diseases were regulated by some common factors, such as inflammation, cellular immunity, metabolic and nutritional changes, and they interact each other in a complex bidirectional and interdependent manner (Schefold et al., 2016). The clinical related evidence is urgent to verify the mechanism underlying the interaction. BUN and SCr are nitrogenous end products of protein metabolism and both of them could be filtered at the glomerulus freely owing to their relatively small
ACCEPTED MANUSCRIPT molecules. The level of BUN was correlated with the level of Scr and the result was verified in our study both in CHF patients and in healthy donors. In fact, it is universally recognized that both of BUN and SCr are confirmed as critical markers of renal function, and increasing evidences indicate that BUN and SCr are associated
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with clinical outcomes in patients with HF (Damman et al., 2014; Matsue et al., 2016;
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van Veldhuisen et al., 2016). In this study, we found the levels of BUN and SCr were
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significantly elevated in the CHF patients free from renal diseases which indicated that CHF itself might make contributions to the impairment of kidney and
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deterioration of renal function. The result might be beneficial to clarifying the
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mechanism between CHF and renal function to a certain extent. In addition, we are the first to report the association between AGXT2 rs37369
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polymorphism and renal function in CHF patients or in healthy controls. In our study,
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we found the level of BUN was significantly elevated in the CHF patients (free from renal diseases) with rs37369 AA genotype, especially in smoking patients. An
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analogous trend (without significant) was also observed in the level of SCr. ADMA is
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an endogenous inhibitor of NOS which can competitively obstruct NO synthesis, and there is no doubting that elevated plasma levels of ADMA are associated with poor outcomes. However, James and his colleagues recently found therapeutic ADMA reduction might even be deleterious to renal function, and they put forward that reduced renal ADMA metabolism might protect against progressive kidney damage (Tomlinson et al., 2015). In our previous study, we had found the enzyme AGXT2 (rs37369 A allele) showed significantly higher ADMA metabolic activity in vitro (Hu
ACCEPTED MANUSCRIPT et al., 2016) which might indicate therapeutic ADMA reduction in patients with rs37369 AA genotype. It might be the reason why we found the polymorphism might influence the renal function in CHF patients. Since we did not detect the level of renal tubular ADMA in different rs37369 genotypes, further investigation are deserved to
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clear the specific reasons causing this difference urgently. Besides, plasma SDMA
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concentration was reported to be highly significantly correlated with both glomerular
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filtration rate (GFR, r=-0.837, P<0.01) and SCr (r=0.894, P<0.01) (Fliser et al., 2005) and might be an important parameter for predicting the early stages of renal diseases
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(Bode-Boger et al., 2006). SDMA is mostly eliminated by renal excretion in the body.
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However, there was still a small fraction of SDMA metabolized by AGXT2 despite of renal excretion (Caplin et al., 2012). Data from the Leeds Stroke Study demonstrated
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that patients carrying AGXT2 rs37369 AA genotype showed significantly enhanced
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SDMA plasma concentration which is also consistent with our results (Luneburg et al., 2014).
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It was interesting that we did not find significant effect of rs37369 polymorphism
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on the renal function of healthy volunteers. We suspected that it might be caused by the lower level of circulating dimethylarginine in healthy population. Strong evidence indicated that ADMA was dramatically elevated in the patients with CHF (Usui et al., 1998), and it might be precisely because of the high concentration of ADMA in CHF patients that highlighted the role of AGXT2 in dimethylarginine metabolism. Meanwhile, we must acknowledge that there are several limitations of this study. Our results were obtained just from one population and with limited samples,
ACCEPTED MANUSCRIPT therefore, multicenter studies are eagerly needed to establish the clinical significance of AGXT2 rs37369 polymorphism in renal function of CHF patients. Moreover, we recognize that we failed to detect the level of ADMA and SDMA in patients and healthy volunteers owing to the limited volume of blood samples collected in our
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study, which might not directly and perfectly support our findings. Nevertheless, we
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hope our findings can arouse interest for further investigations into mechanism
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underlying the association between AGXT2 and renal function of CHF. In summary, we are the first study to reveal the association between AGXT2
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rs37369 polymorphism and renal function in CHF patients and demonstrate that
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rs37369 polymorphism might predict the renal function of patients with CHF, especially in the smokers of CHF patients. Meanwhile, we also verified CHF itself
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might lead to worsening renal function and the impairment of kidney. Further
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investigations in other races and larger cohorts may be required to verify our findings. More comprehensive surveys and functional experiments are also needed to
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illuminate the exact mechanism underlying the association.
Conflict of interest The authors have no conflict of interest to declare.
ACCEPTED MANUSCRIPT Acknowledgements This work was supported by National Natural Science Foundation of China (No.81170091, No.81373489, No.81403018 and No.81422052), National Science and Technology Major Project (2013ZX09509107), and the Fundamental Research Funds
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for the Central Universities of Central South University (2017zzts223).
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Figure legends
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Figure 1. Organ distribution of AGXT in the human body. The data were
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obtained from the human protein atlas (http://www.proteinatlas.org/).
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Figure 2. BUN and SCr in volunteers and CHF patients without renal diseases. (A) and (B): Comparison of the levels of BUN and SCr in healthy volunteers and
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CHF patients without renal diseases. (C) and (D): The correlation between BUN and SCr in healthy controls and CHF patients without renal diseases. CHFa: CHF patients
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who were free from renal diseases. *p<0.05 **p<0.01 ***p<0.001.
CE
Figure 3. AGXT2 rs37369 polymorphism and renal function in CHF patients
AC
without renal diseases. (A and B): Association between AGXT2 rs37369 polymorphism and BUN and SCr in CHF patients without renal diseases. (C, D, and E): Influence of AGXT2 rs37369 polymorphism on the BUN level in smoking patients. CHFa: CHF patients who were free from renal diseases. *p<0.05.
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
Fig. 1
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
Fig. 2
Fig. 3
AC
CE
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Table 1. General characteristics of the study population. Characteristics
T P
CHF without renal diseases
Healthy volunteers
N
487
525
Male (%)
290 (59.5)
Age (years)
59±13
SBP (mmHg)
130±23
DBP (mmHg)
78±13
I R
SC
p value
284 (54.1)
0.080
56±10
<0.001
118±15
<0.001
75±10
<0.001
1.15±0.34
1.27±0.30
<0.001
2.33±0.89
2.23±0.60
0.070
204 (41.9)
135 (25.7)
<0.001
Positive history of alcohol consumption (%)
149 (30.6)
130 (24.8)
0.118
Coronary heart disease (%)
365 (74.9)
0 (0)
<0.001
D E
PT
HDL (mmol/L)
E C
LDL (mmol/L)
C A
Positive history of smoking (%)
A M
U N
ACCEPTED MANUSCRIPT
Hypertension (%)
284 (58.3)
0 (0)
<0.001
Diabetes mellitus (%)
104 (36.6)
0 (0)
<0.001
I R
T P
Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density
C S U
lipoprotein.
N A
D E
T P E
A
C C
M
ACCEPTED MANUSCRIPT
Table 2. Influence of rs37369 polymorphism on the level of BUN in CHF patients without renal diseases BUN(mmo/L)
SCr(μmol/L)
T P
I R
rs37369 genotypes
N
Mean
p
pa
—
—
152
94.0±30.2
—
—
0.496
0.264
248
93.9±29.9
0.634
0.770
Mean
p
AA
152
6.64±3.00
AG
248
6.28±2.31
Entire cohort
Dominant
Recessive
D E
T P E
GG
C S U pa
N
N A
M
87
5.77±2.12
0.029
0.024
87
92.1±26.0
0.611
0.646
152
6.64±3.00
—
—
152
94.0±30.2
—
—
AG+GG
335
6.15±2.27
0.072
0.075
335
93.4±28.9
0.830
0.978
AG+AA
400
6.42±2.60
—
—
400
93.9±30.0
—
—
GG
87
5.77±2.12
0.031
0.036
87
92.1±26.0
0.607
0.428
AA
C C
A
ACCEPTED MANUSCRIPT
Smokers
Dominant
Recessive
AA
67
6.91±3.07
—
—
67
AG
96
6.39±2.33
0.217
0.269
96
GG
41
5.69±2.44
0.020
AA
67
6.91±3.07
—
AG+GG
137
6.18±2.38
0.065
AG+AA
163
6.61±2.66
D E
T P E
GG
—
—
97.9±29.3
0.945
0.973
T P
I R
C S U
97.6±28.0
0.023
41
91.2±23.3
0.249
0.086
—
67
97.6±28.0
—
—
0.079
137
95.9±27.7
0.683
0.513
—
—
163
97.8±28.7
—
—
N A
M
41
5.69±2.44
0.047
0.046
41
91.2±23.3
0.177
0.089
85
6.43±2.94
—
—
85
91.2±31.7
—
—
AG
152
6.21±2.31
0.505
0.625
152
91.3±30.1
0.982
0.623
GG
46
5.85±1.83
0.191
0.262
46
93.0±28.5
0.755
0.592
Non-Smokers
C C
AA
A
ACCEPTED MANUSCRIPT
Dominant
Recessive
a
AA
85
6.43±2.94
—
—
85
91.2±31.7
—
—
AG+GG
198
6.13±2.21
0.335
0.427
198
91.7±29.7
0.560
AG+AA
237
6.29±2.55
—
—
237
T P
0.905
91.3±30.6
—
—
GG
46
5.85±1.83
0.260
93.0±28.5
0.732
0.687
C S U
I R
0.298
46
Adjusted for gender, age, history of smoking, history of alcohol consumption, hypertension, diabetes mellitus and coronary heart disease
N A
by univariate general linear model.
D E
T P E
A
C C
M
ACCEPTED MANUSCRIPT
Table 3. The rs37369 polymorphism did not affect the level of BUN and Scr in healthy volunteers. BUN(mmo/L)
SCr(μmol/L)
T P
I R
rs37369 genotypes
N
Mean
p
pa
—
—
184
61.1±14.9
—
—
0.262
0.383
269
62.3±14.6
0.386
0.808
Mean
p
AA
184
4.67±1.26
AG
269
4.80±1.29
GG
Entire cohort
Dominant
Recessive
D E
C S U pa
N
N A
M
72
4.84±1.30
T P E
0.328
0.221
72
61.4±13.0
0.863
0.848
184
4.67±1.26
—
—
184
61.1±14.9
—
—
AG+GG
341
4.81±1.29
0.216
0.289
341
62.1±14.3
0.435
0.758
AG+AA
453
4.75±1.28
—
—
453
61.8±14.7
—
—
GG
72
4.84±1.30
0.574
0.433
72
61.4±13.0
0.836
0.881
AA
A
C C
ACCEPTED MANUSCRIPT
Smokers
Dominant
Recessive
AA
52
4.75±1.26
—
—
52
AG
65
4.57±1.09
0.427
0.293
65
GG
18
5.15±1.08
0.215
0.306
AA
52
4.75±1.26
—
AG+GG
83
4.70±1.11
0.813
AG+AA
117
4.66±1.17
GG
69.4±12.6
0.522
0.541
18
69.4±10.0
0.740
0.708
—
52
67.0±11.7
—
—
0.620
83
69.4±12.1
0.252
0.547
—
—
117
68.3±12.2
—
—
C S U
N A
M
I R
T P
T P E
0.097
0.105
18
69.4±10.0
0.724
0.969
132
4.63±1.26
—
—
132
58.8±15.4
—
—
204
4.87±1.35
0.101
0.110
204
60.1±14.5
0.434
0.841
54
4.74±1.37
0.624
0.448
54
58.8±12.9
0.997
0.960
C C
A
—
5.15±1.08
Non-Smokers
AG
—
18
GG
AA
D E
67.0±11.7
ACCEPTED MANUSCRIPT
Dominant
Recessive
a
AA
132
4.63±1.26
—
—
132
58.8±15.4
—
—
AG+GG
258
4.85±1.35
0.131
0.137
258
59.8±14.2
0.517
0.912
AG+AA
336
4.78±1.32
—
—
336
59.6±14.9
—
—
GG
54
4.74±1.37
0.826
0.990
58.8±12.9
0.720
0.867
C S U
I R
T P
54
Adjusted for gender, age, history of smoking and history of alcohol consumption by univariate general linear model.
N A
D E
T P E
A
C C
M
ACCEPTED MANUSCRIPT
AGXT2 rs37369 polymorphism predicts the renal function in patients
Highlights:
RI
(1) CHF itself might lead to worsening renal function.
PT
with chronic heart failure
SC
(2) AGXT2 rs37369 polymorphism could significantly affect the level of BUN in CHF patients without renal diseases.
NU
(3) AGXT2 rs37369 polymorphism could also significantly affect the level of
AC
CE
PT E
D
MA
BUN in smokers of CHF patients without renal diseases.