Nucleic acid oxidation plays an important role in the pathophysiology progress of a variety of diseases. 8-oxo-7,8-dihydro-2
Reactive oxygen species (ROS) are persistently generated by living cells under normal physiological conditions as a consequence of cellular metabolism and external environmental factors, such as smoke and ultraviolet radiation. Previous studies have suggested that ROS can damage nucleic acids, lipids, and proteins. DNA and RNA precursor nucleotides are also subjected to oxidative damage. Among the various types of oxidized purine and pyrimidine bases thus produced, guanine has the lowest oxidation potential; it is most readily oxidized to form 8-oxo-7,8-dihydro-2
CKD, defined by the presence of kidney damage (resulting in proteinuria) or reduced kidney function (estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2) [
In our research, we applied an accurate method based on isotope dilution ultra high-performance liquid chromatography triple-quadrupole mass spectrometry (UHPLC-MS/MS) to simultaneously assay the concentrations of 8-oxo-dGsn and 8-oxo-Gsn in the plasma and urine of 146 patients with CKD. Our data revealed the relevance of nucleic acid oxidative products in CKD diagnosis and could present a novel evaluation index for ESRD.
The 8-oxo-Gsn (>98% purity), 8-oxo-dGsn (>98% purity), and heavy-isotope-labeled 8-oxo-dGsn were obtained from Cambridge Isotope Laboratories (USA). The 8-oxo-[15N213C1]Gsn was purchased from Toronto Research Chemicals (Canada), HPLC-grade ammonium acetate was purchased from Fisher Scientific (USA), and HPLC-grade solvents were obtained from Merck (Germany). Water used for the analysis was of Milli-Q quality (18.2 M
All subjects were enrolled in the department of Nephrology of Beijing Hospital, and all participants were adults (age ≥ 18 years) with a confirmed diagnosis of CKD. The 146 patients with CKD were divided into five disease stages according to the national kidney foundation guidelines, with approximately 30 participants in each stage. The diseases included in this study encompassed a variety of kidney diseases, except for diabetic nephropathy. Thus, the diseases included in the study were primary renal disease (IgA nephropathy, membrane nephropathy, minimal change disease, etc.); secondary renal disease (hypertensive nephropathy, gout nephropathy, lupus nephritis, etc.); renal tubular interstitial disease; and genetic diseases. All patients fasted before sample collection. Whole blood samples were collected in vacuum tubes pretreated with the anticoagulant Na2 EDTA and immediately centrifuged at 2500 rpm for 10 min. Plasma specimens (1 mL) were then isolated. Fresh urine specimens were obtained in the morning from midstream urine. Plasma and urine samples were frozen at −80°C before analysis.
The frozen plasma specimens were thawed on ice. After vortexing for approximately 1 min, samples were centrifuged at 14000
The frozen urine was thawed and heated at 37°C for 5 min and then centrifuged at 7500
All samples were analyzed by an Agilent 1290 Infinity UHPLC instrument equipped with an Agilent triple-quadrupole mass spectrometer with a Jet Stream ESI source and iFunnel (Agilent, 6490, USA). UHPLC separation was achieved on an Agilent C18 (3
Mass spectrometric data acquisition was in the positive ion detection mode. Optimum nitrogen pressure for the nebulizer was 30 psi and ESI needle voltage was adjusted to 2000 V. The temperatures of dry gas and sheath gas were set at 200°C and 400°C, respectively, and the flow rates of dry gas and sheath gas were16 L/min and 12 L/min, respectively. Multiple reaction mode (MRM) was monitored for quantitative analysis. The high-pressure RF and low-pressure RF were 120 V and 50 V, respectively. UHPLC conditions and optimized parameters are presented in supplementary Tables
Data are presented as the ratio of the concentrations of nucleosides (8-oxo-dGsn and 8-oxo-Gsn) and creatinine in urine. The continuous parameters are expressed as mean ± standard derivation (SD) and analyzed by SPSS Version 22.0. The values of
In this study, 47.95% of the patients were male (
Demographic and clinical characteristics of the study population.
CKD 1 |
CKD 2 |
CKD 3 |
CKD 4 |
CKD 5 |
|
---|---|---|---|---|---|
|
|||||
Age (years) | 41.63 ± 14.38 |
53.13 ± 17.77 |
61.47 ± 11.05 | 63.23 ± 16.38 | 63.96 ± 12.28 |
Male sex (%) | 13 (43.3) | 17 (56.7) | 14 (45.2) | 18 (60) | 8 (32) |
|
|||||
PRD | 26 | 25 | 15 | 18 | 18 |
SRD | 4 | 3 | 12 | 10 | 9 |
TIN | 1 | 1 | 3 | 2 | 3 |
Others | 0 | 1 | 1 | 0 | 0 |
The data are expressed as the mean ± SD;
The levels of creatinine (Cr), 8-oxo-dGsn and 8-oxo-Gsn in fasting plasma and morning urine were measured in all stable patients to measure the level of nucleic acid oxidation. The results were showed in Table
Creatinine and products derived from nucleic acid oxidation.
CKD 1 | CKD 2 | CKD 3 | CKD 4 | CKD 5 | |
---|---|---|---|---|---|
Plasma creatinine ( |
61.60 ± 14.21a | 90.33 ± 26.26ab | 142.87 ± 63.91b | 202.03 ± 53.21b | 590.40 ± 277.87bc |
Urinary creatinine ( |
12,494 ± 6926.21a | 10270.07 ± 6238.45a | 7359.17 ± 3882.42b | 6447.93 ± 4354.35bc | 4268.64 ± 5782.24bc |
eGFR | 132.53 ± 33.21a | 76.98 ± 9.1b | 45.81 ± 8.5c | 24.73 ± 4.56d | 7.84 ± 2.9e |
Urinary 8-oxo-dGsn ( |
6.56 ± 5.11a | 5.14 ± 3.54a | 3.97 ± 2.58ab | 2.03 ± 1.46b | 1.92 ± 2.04b |
Urinary 8-oxo-Gsn ( |
10.87 ± 6.15a | 9.49 ± 4.74ab | 8.39 ± 5.52ab | 6.17 ± 3.78bc | 4.29 ± 2.62bc |
Urinary 8-oxo-dGsn/Cr ( |
1.87 ± 0.87 | 1.92 ± 1.01 | 1.84 ± 0.81 | 1.47 ± 0.89 | 1.60 ± 1.59 |
Urinary 8-oxo-Gsn/Cr ( |
3.07 ± 1.07a | 3.42 ± 1.34a | 3.72 ± 1.47a | 3.90 ± 1.93b | 3.75 ± 2.26a |
Plasma 8-oxo-Gsn ( |
0.17 ± 0.12a | 0.24 ± 0.18ab | 0.37 ± 0.20b | 0.49 ± 0.22bc | 1.10 ± 0.57bcd |
Plasma/urinary 8-oxo-Gsn | 0.02 ± 0.02a | 0.03 ± 0.02a | 0.06 ± 0.04a | 0.10 ± 0.05b | 0.34 ± 0.03bc |
The data are expressed as the mean ± SD; letters indicate statistical significance (
Figure
(a) Urinary 8-oxo-dGsn/Cr and 8-oxo-Gsn/Cr levels in CKD patients. There were significant differences in the levels of 8-oxo-Gsn between CKD1 and CKD4 (
The changes in concentration of urinary 8-oxo-dGsn, 8-oxo-Gsn, and creatinine in all stages of patients are shown in Figure
The concentration of urinary 8-oxo-dGsn, 8-oxo-Gsn, and creatinine in CKD patients. CKD: chronic kidney disease; 8-oxo-dGsn: 8-oxo-7,8-dihydro-2
Spearman’s correlation analysis between urinary biomarkers of oxidative damage and creatinine.
8-oxo-dGsn | 8-oxo-Gsn | |
---|---|---|
8-oxo-Gsn | 0.744 |
|
Creatinine | 0.564 |
0.630 |
The Spearman’s coefficient (
The ratio of 8-oxo-Gsn in urine to plasma was consistent with the eGFR, as shown in Figure
The eGFR and the ratio of 8-oxo-Gsn in urine and plasma in CKD patients. Spearman’s correlation analysis was performed and the coefficient (
The ratio of creatinine and 8-oxo-Gsn in plasma and urine in CKD patients. Spearman’s correlation analysis was performed and the coefficient (
8-oxo-dGsn and 8-oxo-Gsn levels can be influenced by age. In fact, they have been historically used as indicators of aging. Considering this problem, we analyzed the influence of age and the renal function on plasma 8-oxo-Gsn and the ratio of 8-oxo-Gsn in plasma to urine with multiple regression analyses. Plasma 8-oxo-Gsn = serum creatinine × 0.656 − 0.233 (
The largest cause of mortality in CKD patients is cardiovascular disease, especially in patients with ESRD. In the past decade, many efforts have been made to determine the causative or associated factors that contribute to high mortality from cardiovascular disease. Traditional risk factors, such as hypertension and hypercholesterolemia, were unable to account for the high-mortality rate from cardiovascular disease [
To our knowledge, the changes in nucleic acid oxidative damage in renal disease have not been investigated. A study of DNA oxidation in dialysis patients has been reported, and patients in the predialysis group showed higher values for most of the oxidized molecules. The peritoneal dialysis group showed a better oxidation-antioxidation balance, with no significant differences in levels of mitochondrial 8-oxo-dGsn when compared to the control group [
However, the unexpected result was that urinary 8-oxo-Gsn/Cr did not increase but, in fact, declined in stage 5 CKD patients. We theorized that this could be related to the lower excretion ability of the kidneys. Severe damage of renal function may affect the excretion of 8-oxo-Gsn, a phenomenon evident in proteinuria. We found that proteinuria decreased in ESRD patients. The glomerular filtration membrane has a three-layer structure, which (from the inside out) consists of the following: endothelial cells, the basement membrane, and epithelial cells of the capsule (podocytes). There are pores between the endothelial cells, which are about ~500 to 1000 Å. Small solutes and water can easily pass through these holes. The basement membrane is a continuous dense structure with no holes and a thickness of ~3200 to 3400 Å. The membrane surface is coated with a negatively charged binding substance, the main component of which is a mucopolysaccharide. Long thin gaps exist between the podocytes, and the gap widths are about ~100 to 400 Å, with lengths of about ~200 to 900 Å. Only molecules equal to or less than the size of albumin (approximately 68,000 Da) are able to pass through the glomerular filtration membrane pores. The molecular weights of the oxidation products are very small. The weight of 8-oxo-dGsn is approximately 283 Da, and 8-oxo-Gsn is approximately 299 Da, and they are freely able to pass through the kidney like creatinine, whose weight is approximately 113 Da. However, no research has reported the exact metabolism pathway until now. The oxidation products 8-oxo-dGsn and 8-oxo-Gsn can be produced by all kinds of cells, tissues, and organs. The results of our previous report suggested that larger amounts of 8-oxo-dGsn and 8-oxo-Gsn are detected in urine [
RNA may have higher levels of oxidative lesions compared with DNA. Reasons for this may include the following: RNA is a single-stranded nucleic acid without the protection of histone proteins, RNA polymerase lacks the correction function, and RNA is widely distributed and highly expressed in cells [
The plots of urinary nucleic acid oxidation products and creatinine concentration were consistent, as shown in Figure
8-oxo-Gsn was first studied as an index of aging. In our research, patients with stage 1 and stage 2 CKD were younger. This was a clinical characteristic of the disease that we could not control. We analyzed the influence of age with regression analyses and found that 8-oxo-Gsn was only related to renal function.
In conclusion, (1) RNA oxidative damage is present in patients with renal disease and increases with deterioration of the disease. (2) The level of 8-oxo-Gsn in plasma and urine is a novel evaluation index of ESRD.
There are still many limitations in our research, for example, the excretion of 8-oxo-Gsn via the kidneys is still speculation, and more experimental evidence is needed. In addition, the sample size was small, and it was a single-center study in a Chinese population. Therefore, additional studies are required to confirm the results presented here.
The authors take responsibility that this study has been reported honestly, accurately, and transparently, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned have been explained.
The authors declare that they have no conflicts of interest.
Jian-Ping Cai and Sadayoshi Ito provided the research idea and designed the study. Yong-Hui Mao, Leng-Nan Xu, Qing-Hua Weng, Xiang-Yu Li, Ban Zhao, Jing-Jing Nie, Ji-Hong Hu, Li-Qun Zhang, and Ming-Zhang Zuo acquired the data. Jian-Ping Cai, Yong-Hui Mao, Leng-Nan Xu, Qing-Hua Weng, Xiang-Yu Li, Ban Zhao, Jing-Jing Nie, Ji-Hong Hu, Li-Qun Zhang, Zhe Chen, and Ming-Zhang Zuo analyzed and interpreted the data. Statistical analysis was performed by Leng-Nan Xu, Qing-Hua Weng, and Xiang-Yu Li. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. Yong-Hui Mao and Leng-Nan Xu contributed equally to this work.
The authors are grateful to the members of the Institute of Geriatrics of the Ministry of Health for their assistance and advice. This work was supported by the National Natural Science Foundation of China (no. 81171028 and no. 81571058).
Table S1: LC conditions for urine. Table S2: conditions for different compounds in three samples. Table S3: conditions for different samples. Figure S1: the levels of plasma and urinary creatinine in CKD patients. CKD: chronic kidney disease.