Part One Genetic Polymorphisms Of MnSOD Modify The Impacts Of Environmental Melamine On Oxidative Stress And Early Kidney Injury in Calcium Urolithiasis Patients

Abstract

Environmental melamine exposure increases the risks of oxidative stress and early kidney injury. Manganese superoxide dismutase (MnSOD), glutathione peroxidase, and catalase can protect the kidneys against oxidative stress and maintain normal function. We evaluated whether their single-nucleotide polymorphisms (SNPs) could modify melamine’s effects. A total of 302 patients diagnosed with calcium urolithiasis were enrolled. All patients were provided one-spot overnight urine samples to measure their melamine levels, urinary biomarkers of oxidative stress, and renal tubular injury. Median values were used to dichotomize levels into high and low. Subjects carrying the T allele of rs4880 and high melamine levels had a 3.60 times greater risk of high malondialdehyde levels than those carrying the C allele of rs4880 and low melamine levels after adjustment. Subjects carrying the G allele of rs5746136 and high melamine levels had a 1.73 times greater risk of high N-Acetyl-β-D-glucosaminidase levels than those carrying the A allele of rs5746136 and low melamine levels. In conclusion, the SNPs of MnSOD, rs4880, and rs5746136, influence the risk of oxidative stress and renal tubular injury, respectively, in calcium urolithiasis patients. In the context of high urinary melamine levels, their effects on oxidative stress and renal tubular injury were further increased.

Keywords

manganese superoxide dismutase; genetic polymorphism; melamine; kidney injury; oxidative stress; calcium urolithiasis.

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Introduction

Melamine is a synthetic chemical used in manufacturing a variety of commercial daily life products including housewares, countertops, fabrics, glues, and flame retardants [1,2]. Because it has a high nitrogen content, it has been misused in animal feed and milk to deceptively elevate the protein content [1,2]. The adverse effects of melamine exposure drew worldwide attention after its scandalous addition to animal feeds, leading to the deaths of thousands of pet animals in the US in 2007, as well as to infant formula, resulting in urolithiasis in more than 50,000 children and six deaths in China in 2008 [1,2]. To date, melamine remains ubiquitously present in our environment. Several studies have detected melamine in water, soil, crops, daily food products, and animal tissues [3–5], and others detected it in most urine samples of several general populations from different countries [6–9].

After melamine intake, ninety percent of its original form will be excreted in urine within 24 h, so the kidneys may be more susceptible to melamine [1]. In addition to the effects of high doses of melamine on acute nephrotoxicity in children in 2008, long-term low-dose exposure to melamine has been linked to the risk of kidney complications, including stone formation and deterioration of renal function in adults [10–12]. One probable mechanism that chronic low-dose melamine exposure could lead to early kidney injury and stone formation is its adverse effect on renal tubules, as was found by two of our human studies in melamine tableware workers [13] and adult patients with calcium urolithiasis [14]. We also conducted an in vitro study by using human renal proximal tubular HK-2 cells and found that melamine could induce renal tubular damage by increasing oxidative stress [15]. In two recent human studies, we also found that exposure to melamine increased the urinary biomarkers of oxidative stress, malondialdehyde (MDA), and 8-oxo-20 -deoxyguanosine (8-OHdG), with MDA mediating 36–53% of the total effect of melamine on a biomarker of renal tubular injury, N-Acetyl-β-D-glucosaminidase (NAG) [16]. Those findings suggest that exposure to low-dose environmental melamine might increase oxidative stress and further the risk of early kidney injury in humans.

Oxidative stress occurs when the generation of pro-oxidants or reactive oxygen species (ROS) exceeds the endogenous antioxidant capacity. Our kidneys are particularly sensitive to oxidative stress, which is thought to be an important factor in the initiation, development, and progression of most kidney diseases [17,18]. The cause of oxidative stress in kidneys may be associated with the interactions between medical diseases and environmental exposure to chemicals such as melamine [16]. Endogenous antioxidant systems including manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPX1), and catalase (CAT) have been found to protect the kidneys against oxidative stress and subsequently help maintain normal function [18]. MnSOD can decompose toxic ROS, and superoxide anions, to hydrogen peroxide, which is then converted to non-toxic water and oxygen by GPX1 and CAT in the mitochondria [18]. Several single-nucleotide polymorphisms (SNPs) of those antioxidant enzyme genes have been associated with various diseases [19], including kidney diseases [20–22].

Since environmental toxicant-associated kidney damage might be influenced by genetic factors [23], it is possible that the SNPs of antioxidant enzyme genes (e.g., MnSOD, GPX1, CAT) could modify the effects of environmental melamine exposure on the risk of oxidative stress and renal tubular injury in humans. To find out, we selected five candidate SNPs of antioxidant enzyme genes (MnSOD: rs4880 and rs5746136, GPX1: rs1800668, CAT: rs1001179 and rs769217), most of which have been linked to kidney diseases [20–22], to study each of their effects when combined with environmental melamine exposure on 8-OHdG and MDA, two biomarkers of oxidative stress, and NAG, a biomarker of renal tubular injury, in urine.

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Materials and Methods

1. Subjects

In total, 309 patients diagnosed with upper urinary tract calcium urolithiasis were enrolled between November 2010 and January 2015. The detailed study designs and protocols for their inclusion were described previously [14,16]. In brief, all patients were from Kaohsiung Medical University-affiliated hospitals in southwestern Taiwan. The eligible patients were individuals aged ≥ 20 years who had been diagnosed with urolithiasis in the upper urinary tract by radiography or ultrasonography, and who had provided stone specimens confirmed to have calcium components by infrared spectroscopy analysis (Spectrum RX I Fourier Transform-Infrared System, PerkinElmer, Waltham, MA, USA). None of the participants was found by X-ray to have radiolucent stones or by clinical evaluation to have uric acid or cystine stones.

Patient subjects were excluded if they had a history of chronic urinary tract infection, chronic diarrhea, gout, hyperparathyroidism, renal tubular acidosis, renal failure, or cancer. Any subjects who had regularly taken vitamin D, calcium supplements, diuretics, or potassium citrate more than once per week within six months before the diagnosis of urolithiasis or interview were also excluded. The study protocol was approved by the Institutional Review Board of KMUH and all eligible patients provided signed informed written consent forms. This study followed the guidelines of STREGA [24] (see Supplementary Materials Table S1).

2. Collection of Clinical Data and Biological Samples

Upon admission, all participants were provided blood and one-spot first-void morning urine samples after overnight fasting and before any treatment of urolithiasis for biochemical and genetic analyses. They also answered a structured questionnaire to collect their demographic data, medical history, and substance use (cigarette, alcohol, and betel quid) [14,16]. Participants were defined as cigarette smokers, alcohol drinkers, or betel quid chewers if they had regularly smoked ≥ 10 cigarettes per week, had consumed any alcoholic beverage ≥ 1 time per week, or had chewed ≥ 7 betel quids per week, respectively, for at least six months. Current users were those still practicing the above habits within one year before the diagnosis of urolithiasis or the interview [11,25].

Clinical information, including stone location, stone number, stone diameter, and stone episodes, was also collected by questionnaire and further reviewed using the patients’ medical charts by one urologist (C.-C.L.) who was unaware of the exposure of interest, including melamine and biomarkers of oxidative stress (8-OHdG and MDA) and renal tubular injury (NAG) in urine and genotyping of antioxidant enzyme genes.

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3. Analyses of Melamine, Biomarkers of Oxidative Stress (MDA and 8-OHdG), and Renal Tubular Injury (NAG) in Urine

Urinary melamine was measured using an isotopic liquid chromatography-tandem mass spectrometry method (LC-MS/MS) (API4000Q, Applied Biosystems/MDS SCIEX, Concord, Vaughan, ON, Canada) [13]. Urinary MDA was measured using high-performance liquid chromatography with fluorescence (HPLC-FL) detection in a reversed-phase column (Luna C18, 250 × 4.6 nm) [26]. Urinary 8-OHdG was measured using a validated method for online solid-phase extraction (SPE) LC-MS/MS [27]. The detailed methods were described previously [14,16]. The limits of detection (LOD) for urinary melamine and biomarkers of oxidative stress were 0.4 ng/ml for melamine, 0.02 µmol/L for MDA, and 0.01 ng/mL for 8-OHdG. All urinary measurements of MDA and 8-OHdG were detectable. In contrast, 31 (10.0%) of the 309 urinary melamine measurements were below the LOD and were substituted as LOD/√ 2.

Urinary NAG was measured using a NAG assay kit (Diazyme Laboratory, Poway, CA, USA) [13,14]. All urinary NAG measurements were detectable. Urinary creatinine was analyzed by spectrophotometry (U-2000; Hitachi, Tokyo, Japan) set at a wavelength of 520 nm to measure the creatinine–picrate reaction [14]. The measurement of all biochemical parameters above was performed by two different laboratory technicians blinded to each other’s findings, study design, and participant information.

4. Genotyping of Five SNPs

DNA was extracted from peripheral whole blood using a Puregene DNA Isolation Kit (Gentra Systems Inc., Minneapolis, MN, USA). The five SNPs (MnSOD-rs4880, MnSODrs5746136, GPX1-rs1800668, CAT-rs1001179, and CAT-rs769217) were analyzed using an assay-on-demand SNP genotyping kit for a TaqMan 5’ allelic discrimination assay (Applied Biosystems, Foster City, CA, USA). Briefly, SNP amplification assays including 10 ng of sample DNA in 25 µL of reaction solution contained 12.5 µL of the 2× TaqMan®Universal PCR Mix (Applied Biosystems), while 1.25 µL of pre-developed assay reagent from the SNP genotyping product (Applied Biosystems) contained two primers. Two MCB-Taqman probes were performed following the instructions of the manufacturer. Polymerase chain reactions (PCRs) were performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems) [28,29]. These genotypes were confirmed by direct sequencing after full-scale genotyping. Randomly, ~10% of the study samples (30 cases) were repeated for quality control, with the results showing 100% accuracy.

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5. Statistical Analyses

Quantitative data were expressed as means ± standard deviations (SD) or medians with interquartile ranges (IQR), and categorical data were presented as numbers (n) and percentages. The Hardy–Weinberg equilibrium for the distribution of genotypes was checked using the chi-square test.

Urinary melamine levels and urinary biomarkers (MDA, 8-OHdG, NAG) were corrected by urinary creatinine values before further analyses. After correction, urinary biomarkers of oxidative stress (MDA and 8-OHdG) and renal tubular injury (NAG) were dichotomized into high or low using their median values [14,16]. Simple logistic regression models were first used to evaluate the associations between genotypes of antioxidant enzyme genes and urinary biomarkers of oxidative stress and renal tubular injury. If a significant relationship was noted in the initial analysis, the combined effects between genotypes of antioxidant enzyme genes and urinary melamine levels dichotomized by median values were further tested by multiple logistic regression analyses after adjusting for covariates, such as age, sex, BMI, educational level, personal habits, stone number, stone size, stone location, and comorbidities. All statistical analyses were performed using the SAS statistical package. All p-values were two-sided and considered significant if <0.05.

6. Sensitivity Analysis

To examine the robustness of our findings, we performed sensitivity analyses to compare the results when urinary melamine levels were divided into tertiles. Furthermore, we also compare the findings using a new method of covariate-adjusted standardization plus creatinine adjustment to correct urine concentration and complicated confounding structures [14,30].

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Chia-Chu Liu 1,2,3,4 , Chia-Fang Wu 1,5, Yung-Chin Lee 2,3,6, Tsung-Yi Huang 2 , Shih-Ting Huang 1,7 , Hsun-Shuan Wang 6 , Jhen-Hao Jhan 6 , Shu-Pin Huang 2,3, Ching-Chia Li 2,3, Yung-Shun Juan 2,3 , Tusty-Jiuan Hsieh 1,7 , Yi-Chun Tsai 1,8,9, Chu-Chih Chen 1,10 and Ming-Tsang Wu 1,11,12,13,

1 Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan; ccliu0204@gmail.com (C.-C.L.); cfwu27@nuu.edu.tw (C.-F.W.); u107800006@kmu.edu.tw (S.-T.H.); hsiehjun@kmu.edu.tw (T.-J.H.); 920254@kmuh.org.tw (Y.-C.T.); ccchen@nhri.edu.tw (C.-C.C.)

2 Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung City 807, Taiwan; 890197@kmuh.org.tw (Y.-C.L.); 970417@kmuh.org.tw (T.-Y.H.); shpihu@kmu.edu.tw (S.-P.H.); 850144@kmuh.org.tw (C.-C.L.); 840066@kmuh.org.tw (Y.-S.J.)

3 Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan

4 Department of Urology, Pingtung Hospital, Ministry of Health and Welfare, Pingtung City 900, Taiwan

5 International Master Program of Translational Medicine, National United University, Miaoli 360, Taiwan

6 Department of Urology, Kaohsiung Municipal Siaogang Hospital, Kaohsiung City 812, Taiwan; 940199@kmuh.org.tw (H.-S.W.); 1030398@kmuh.org.tw (J.-H.J.)

7 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan

8 Department of Internal Medicine, Divisions of Nephrology and General Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung City 807, Taiwan

9 Department of Internal Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan

10 Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes, Miaoli 350, Taiwan

11 Environmental and Occupational Medicine and Graduate Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan

12 Department of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung City 807, Taiwan

13 Department of Public Health, College of Health Sciences, Kaohsiung Medical University, Kaohsiung City 807, Taiwan