Correlation of SOD-Mn gene polymorphism with renal function and
oxidative injury in patients with diabetic nephropathy
Wei-Kai Zhang
1, Ling Zhang
21. Nephrology and Endocrinology Department, Yingshan County People’s Hospital in Hubei Province, Huanggang, Hubei Province, 438700 2. Nephrology Department, Huanggang Central Hospital in Hubei Province, Huanggang, Hubei Province, 438000
Journal of Hainan Medical University
http://www.hnykdxxb.com
ARTICLE INFO ABSTRACT
Article history: Received 2 Jul 2018
Received in revised form 9 Jul 2018 Accepted 20 Jul 2018
Available online 28 Jul 2018
Keywords:
Diabetic nephropathy SOD-Mn
Gene polymorphism Oxidative stress response
Corresponding author: Ling Zhang, Nephrology Department, Huanggang Central
Hospital in Hubei Province, Huanggang, Hubei Province, 438000.
Fund Project: Huanggang Clinical Medical College, Yangtze University No: WJ2016-YZ-10.
1. Introduction
Diabetic nephropathy is a common microvascular complication in diabetic patients and also the most common cause of end-stage renal disease in clinic[1,2]. The mass generation of reactive oxygen species and the over-activation of oxidative stress response are the important pathological changes in the development of diabetic nephropathy[3]. SOD-Mn is an important antioxidant enzyme in the body, which can scavenge the reactive oxygen species in the mitochondria and reduce the oxidative stress injury induced by reactive oxygen species. When the expression of SOD-Mn changes or its activity decreases,
the antioxidant capacity of the body is weakened, the reactive oxygen species cannot be removed in time, and the risk of oxidative stress injury of multiple organs in the whole body is increased. The kidney is a target organ prone to oxidative stress damage, and the increase of ROS production in patients with diabetes can cause oxidative stress damage in the kidney, and thus cause the occurrence of diabetic nephropathy. At present, the regulating mechanism of oxidative stress response in diabetic nephropathy is still unclear. Gene polymorphism is an important factor that influences the activity of SOD-Mn, and the SOD-Mn gene rs4880 locus mutation can affect the transfer of coding product from the cytoplasm to mitochondria, and then influence the antioxidant function of the molecule[4]. In the following studies, we specifically analyzed the correlation of SOD-Mn gene polymorphism with renal function and oxidative injury in patients with diabetic nephropathy.
Objective: To study the correlation of SOD-Mn gene polymorphism with renal function and oxidative injury in patients with diabetic nephropathy. Methods: Patients who were diagnosed with diabetic nephropathy in Yingshan County People’s Hospital between March 2014 and March 2017 were chosen as DN group, and healthy volunteers who received physical examination during the same period were selected as the control group. The SOD-Mn gene rs4880 locus polymorphism and oxidative stress molecule expression in peripheral blood as well as the contents of renal function indexes and oxidative stress indexes in serum were determined. Results: The constituent ratio of SOD-Mn gene CC genotype in DN group was lower than that in control group whereas the constituent ratio of CT+TT genotype was higher than that in control group; serum T-AOC contents as well as peripheral blood Nrf2 and SIRT1 expression intensity of DN group of patients with CC genotype and CT+TT genotype were significantly lower than those of control group whereas serum Scr, BUN, CysC, β 2-MG, MDA, AOPP, 8-OHdG and 8-Iso-PGF2α contents as well as peripheral blood NOX4, NOX5, p38MAPK and NF-κB expression intensity were significantly higher than those of control group, and serum T-AOC content as well as peripheral blood Nrf2 and SIRT1 expression intensity of patients with CT+TT genotype was significantly lower than those of patients with CC genotype whereas serum Scr, BUN, CysC, β2-MG, MDA, AOPP, 8-OHdG and 8-Iso-PGF2α contents as well as peripheral blood NOX4, NOX5, p38MAPK and
2. Case information and research methods
2.1 General case information
Patients who were diagnosed with diabetic nephropathy in Yingshan County People’s Hospital between March 2014 and March 2017 were selected as the DN group of the study, all patients had the history of type 2 diabetes mellitus and were with eGFR＜60 mL/ min/1.73 m2 or male UAER＞2.5 mg/mmoL and female UAER＞ 3.5 mg/mmoL, and the renal dysfunction caused by other primary kidney diseases were ruled out. Healthy volunteers who received physical examination during the same period were selected as the control group of the study, and they were confirmed to be healthy by physical examination and had normal renal function. There were 103 cases in the DN group, including 59 males and 44 females who were 41-64 years old; there were 60 cases in the control group, including 34 males and 26 females who were 39-62 years old. There was no significant difference in the general data between the two groups (P>0.05).
2.2 Gene polymorphism detection
Peripheral blood specimens were taken, the genomic DNA extraction kit was used to separate the DNA from peripheral blood, SOD-Mn gene rs4880 locus primers were designed for PCR reaction, the reaction products underwent enzyme digestion reaction with Pid I restriction enzyme and then underwent electrophoresis in agarose gel, the development was done under ultraviolet light to judge the polymorphism, that with development in 107 bp was TT genotype, that with development in 89 bp was CC genotype and that with development in both 107 bp and 89 bp was CT genotype.
2.3 Serum index detection
The serum samples were taken and the steps of automatic biochemical analyzer were followed to determine the contents of Scr and BUN; the instructions of Elisa kit were referred to determine the contents of CysC and β2-MG; the contents of T-AOC, MDA, AOPP, 8-OHdG and 8-Iso-PGF2α were detected according to the instructions of radioimmunoprecipitation kit.
2.4 Peripheral blood signal molecule expression detection
The peripheral blood specimens were taken, used to incubate the fluorescent antibody of NOX4, NOX5, Nrf2, SIRT1, p38MAPK and NF-κB for 20 min, washed with PBS and centrifuged, and then flow cytometer was used to determine the fluorescence intensity of corresponding molecules.
2.5 Statistical methods
Software SPSS 20.0 was used to input data, the count data were analyzed by chi-square test, the measurement data were by variance analysis and P<0.05 meant statistical significance in the differences.
3. Results
3.1 Peripheral blood SOD-Mn genotype distribution
38 cases in DN group were with SOD-Mn gene CC genotype, 42 cases were with CT genotype and 23 cases were with TT genotype; 24 cases in control group were with SOD-Mn gene CC genotype, 20 cases were with CT genotype and 16 cases were with TT genotype. After chi-square test, the constituent ratio of SOD-Mn gene CC genotype in DN group was lower than that in control group whereas the constituent ratio of CT+TT genotype was higher than that in control group.
3.2 Serum renal function index contents
Analysis of serum renal function indexes SCr (μmol/L), BUN (mmol/L), Cys-C (μg/mL) and β2-MG (μg/mL) among the research subjects was as follows: serum Scr, BUN, CysC and β2-MG contents of DN group of patients with CC genotype and CT+TT genotype were significantly higher than those of control group (P<0.05), and serum Scr, BUN, CysC and β2-MG contents of patients with CT+TT genotype were significantly higher than those of patients with CC genotype (P<0.05).
Table 1.
Comparison of serum renal function indexes.
Groups n Scr BUN CysC β2-MG
DN group CT+TT genotype 38 147.5±22.1*△ 15.48±1.76*△ 3.26±0.42*△ 5.26±0.72*△ CC genotype 38 121.2±14.6* 9.93±1.06* 2.47±0.29* 3.48±0.55*
Control group 65 68.6±8.4 5.86±0.77 1.26±0.16 2.01±0.32
3.3 Serum oxidative stress index contents
Analysis of serum oxidative stress indexes T-AOC (U/mL), MDA (nmol/mL), AOPP (nmol/mL), 8-OHdG (ng/mL) and 8-iso-PGF2 (pg/mL) among the research subjects was as follows: serum T-AOC contents of DN group of patients with CC genotype and CT+TT genotype were significantly lower than that of control group whereas MDA, AOPP, 8-OHdG and 8-Iso-PGF2α contents were significantly higher than those of control group (P<0.05), and serum T-AOC content of patients with CT+TT genotype was significantly lower than that of patients with CC genotype whereas MDA, AOPP, 8-OHdG and 8-Iso-PGF2α contents were significantly higher than those of patients with CC genotype (P<0.05).
3.4 Peripheral blood oxidative stress molecule contents
Analysis of peripheral blood oxidative stress molecules NOX4, NOX5, Nrf2, SIRT1, p38MAPK and NF-κB among the research subjects was as follows: peripheral blood NOX4, NOX5, p38MAPK and NF-κB expression intensity of DN group of patients with CC genotype and CT+TT genotype were significantly higher than those of control group whereas Nrf2 and SIRT1 expression intensity were significantly lower than those of control group (P<0.05), and peripheral blood NOX4, NOX5, p38MAPK and NF-κB expression intensity of patients with CT+TT genotype were significantly higher than those of patients with CC genotype whereas Nrf2 and SIRT1 expression intensity was significantly lower than those of patients with CC genotype (P<0.05).
4. Discussion
The excessive activation of oxidative stress is an important pathological change in patients with diabetic nephropathy. The substandard blood glucose control and the increased formation
of advanced glycation end products in the course of diabetes will stimulate increased formation of reactive oxygen species. When the abnormally increased reactive oxygen species exceed the body's antioxidant capacity, they will accumulate in the tissue and cause oxidative stress injury to it[5,6]. SOD-Mn is an important antioxidant enzyme in mitochondria, which has the ability of catalyzing reduction reaction and scavenging oxygen free radicals. However, when the gene encodes SOD-Mn mutates, its antioxidant activity will be significantly weakened. The rs4880 site is located in the promoter region of SOD-Mn gene, and the mutation of the allele of this site from C to T will affect the transfer of the encoding products to the mitochondria, and then weaken the antioxidant capacity[7,8]. In the study, the analysis of SOD-Mn gene rs4880 locus polymorphism in patients with diabetic nephropathy showed that the constituent ratio of SOD-Mn gene CC genotype in DN group was lower than that in control group whereas the constituent ratio of CT+TT genotype was higher than that in control group. This indicates that the mutation of SOD-Mn gene rs4880 locus allele from C to T is related to the occurrence of diabetic nephropathy; after the above SOD-Mn gene mutation, the antioxidant activity of the encoding products is weakened, and they are unable to effectively remove reactive oxygen species, and increase the oxidative stress reaction to cause renal damage in patients with diabetes and eventually result in the occurrence of diabetic nephropathy.
Scr and BUN are the common biochemical indexes for clinical evaluation of renal function, and both are products of protein metabolism and excreted by the kidneys; when renal function is impaired, Scr and BUN excretion are blocked and they accumulate in the blood circulation, which is manifested as increased serum contents. CysC and β2-MG are newly discovered indexes to evaluate renal function in recent years, both are micromolecular proteins, and they can freely pass through the glomeruli and be reabsorbed and degraded in the kidney tubules; the damage of renal function can affect the excretion as well as reabsorption and Table 2.
Comparison of serum oxidative stress indexes.
Groups n T-AOC MDA AOPP 8-OHdG 8-Iso-PGF2α
DN group CT+TT genotype 38 20.12±3.25*△ 15.23±1.84*△ 39.62±5.52*△ 237.5±33.6*△ 351.2±42.7*△ CC genotype 65 32.64±4.46* 10.56±1.42* 24.51±3.26* 147.6±20.3* 203.6±26.8* Control group 60 48.59±6.47 5.48±0.72 10.93±1.36 73.8±8.9 137.5±17.8
*: compared with control group, P＜ 0.05; △: compared with CC genotype, P＜ 0.05.
Table 3.
Comparison of peripheral blood oxidative stress molecule.
Groups n NOX4 NOX5 Nrf2 SIRT1 p38MAPK NF-κB
DN group CT+TT genotype 38 2.64±0.36*△ 2.81±0.42*△ 0.39±0.06*△ 0.41±0.07*△ 2.33±0.36*△ 3.06±0.52*△ CC genotype 65 1.76±0.25* 1.82±0.26* 0.65±0.08* 0.78±0.09* 1.56±0.25* 1.85±0.26* Control group 60 1.02±0.15 0.99±0.12 1.04±0.11 1.05±0.15 0.97±0.13 1.03±0.15
degradation of CysC and β2-MG, and then increase the contents of corresponding molecules in the serum[9,10]. In the study, analysis of the changes of the above renal function indexes in serum showed that serum Scr, BUN, CysC and β2-MG contents of DN group were significantly higher than those of control group. This indicates that in the occurrence and development of diabetic nephropathy, various molecules abnormally accumulate in the body and the serum contents of corresponding molecules significantly increase. Further analysis of the effect of SOD-Mn on renal function indexes in patients with diabetic nephropathy showed that serum Scr, BUN, CysC and β2-MG contents of DN group of patients with CT+TT genotype were significantly higher than those of patients with CC genotype. This indicates that the mutation of SOD-Mn gene rs4880 locus allele from C to T can aggravate the renal function damage in patients with diabetic nephropathy.
In the progression of diabetic nephropathy, when SOD-Mn gene rs4880 locus mutates from C to T, the coding products can't enter into the mitochondria from the cytoplasm to exert antioxidant effect, so the body’s antioxidant capacity is weakened, which is characterized by the decline of T-AOC content[11]. Meanwhile, the weakening of antioxidant capacity will also lead to the accumulation of reactive oxygen species and cause the oxidation reaction of various elements in local tissue through the strong oxidizing property of reactive oxygen species[12], MDA, AOPP and 8-OHdG are the oxidation reaction products of lipid, protein and nucleic acid in local tissue respectively, and 8-Iso-PGF2α is the oxidation reaction product of lipid metabolite prostaglandin[13,14]. Analysis of the changes in above oxidative stress indexes in the study showed that serum T-AOC content of DN group was significantly lower than that of control group whereas MDA, AOPP, 8-OHdG and 8-Iso-PGF2α contents were significantly higher than those of control group. This indicates that there is excessive activation of oxidative stress response in patients with diabetic nephropathy, the generation of multiple oxidation products increases and the antioxidant capacity is significantly weakened. Further analysis of the influence of SOD-Mn on oxidative stress indexes in patients with diabetic nephropathy showed that serum T-AOC content of patients with CT+TT genotype was significantly lower than that of patients with CC genotype whereas MDA, AOPP, 8-OHdG and 8-Iso-PGF2α contents were significantly higher than those of patients with CC genotype. It shows that the mutation of SOD-Mn gene rs4880 locus allele from C to T will weaken the antioxidant capacity and increase the formation of oxidation products to increase the extent of oxidative stress. The mass generation of reactive oxygen species in patients with diabetic nephropathy is closely related to the biological activities of NOX4, NOX5 and p38MAPK, NOX4 and NOX5 are the NOX family members closely associated with diabetic nephropathy, and they can catalyze the electron transfer to oxygen and generate
reactive oxygen species[15,16]; P38MAPK is a member of the MAPK family, which activates NF-κB by signaling cascade transduction to mediate the generation of ROS[17]. Nrf2 and SIRT1 are molecules with antioxidant activity, the former can catalyze the expression of antioxidant enzymes to exert antioxidant effect, and the latter inhibit the activation of NF-κB to exert antioxidant effect[18]. In the study, in order to further clarify the change of oxidative stress response in patients with diabetic nephropathy and the effect of SOD-Mn gene polymorphism on the oxidative stress reaction, the expression levels of oxidative stress signal molecules in peripheral blood were analyzed in the study, and the result showed that peripheral blood NOX4, NOX5, p38MAPK and NF-κB expression intensity of DN group were significantly higher than those of control group whereas Nrf2 and SIRT1 expression intensity were significantly lower than those of control group, and peripheral blood NOX4, NOX5, p38MAPK and NF-κB expression intensity of patients with CT+TT genotype were significantly higher than those of patients with CC genotype whereas Nrf2 and SIRT1 expression intensity was significantly lower than those of patients with CC genotype. It indicates that the high expression of pro-oxidative stress molecules and the low expression of antioxidant molecules are closely related to the occurrence of diabetic nephropathy, and the mutation of SOD-Mn gene rs4880 locus allele from C to T will further increase the expression of pro-oxidative stress molecules and further decrease the expression of antioxidant molecules to increase the activation extent of oxidative stress reaction.
The above study results indicate that there is SOD-Mn gene polymorphism in patients with diabetic nephropathy, which is specifically characterized by increased mutation of rs4880 locus allele from C to T; the above change of polymorphism can aggravate the renal function damage and oxidative stress response in patients with diabetic nephropathy.
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