The Role Of DNA Oxidation In Renal Injury Of Type 2 Diabetes Mellitus

Diabetes mellitus is a lifetime metabolic disease with the main characteristic of the increased level of glucose in blood. Nearly 95% of the patients with diabetes are suffered from type 2 diabetes mellitus. The continuing high blood glucose attacks the systemic micro vascular and macrovascular relentlessly, and lead to all kinds of complications. According to statistics, about 5%～10% T2DM patients in China eventually developed to DN. Diabetic nephropathy (DN) is one of the most common microvascular complications. With the extension of course and the depth of lesion, DN eventually develops to end-stage renal disease (ESRD). The patients have to rely on replacement therapy such as dialysis to sustain life. Therefore, it is very important to study the pathogenesis of DN and looking for the effective prevention and treatment measures.Docosahaexaenoic acid (DHA) is one of the n-3 polyunsaturated fatty acids (n-3 PUFAs). It contains six carbon-carbon double bonds and has high instauration. In vivo, DHA can be oxidized easily by other oxides and generate a series of secondary metabolites. Numerous studies have demonstrated that DHA and its metabolic derivatives have many important biological functions, such as anti-inflammatory, anti-oxidative stress, participating in the composition of cell membrane, increasing the fluidity of cell membrane, regulating the ion-channel and gene expression.Up to now, the mechanisms of T2DM and DN remain uncertain. In the present study, Firstly, a new method named MEKC-UV was established for determination of the oxidative DNA damage marker 8-OHdG in urine. This method was then applied to analyze the content of urinary 8-OHdG in T2DM and DN patients. Secondly, the animal experiments were carried out to investigate the effects of DHA on renal oxidative stress in type 2 diabetes mellitus rats. It provides experimental and theoretical basis for the application of DHA on prevention and treatment of DN. Part I Evaluation of oxidative DNA damage biomarker 8-OHdG by MEKC-UVObjective:To lay the methodology foundation for the the research on oxidative DNA level in patients with T2DM and DN.Methods:1 Evaluation of the Oxidative DNA Damage Biomarker 8-OHdG in Urine by SPE-MEKC-UV.2 The urinary creatinine was analyzed by a new CZE-UV method.Results:We developed a SPE-MEKC-UV method for the analysis of urinary 8-OHdG. The sensitivity of MEKC-UV was improved using reasonable UV system, injection mode, and SPE. The parameters affecting MEKC and SPE were also optimized. The calibration curve was linear within the range from 1 to 500 μg L-1. The limits of detection and limits of quantification were 0.27 μg L-1 and 0.82 μg L-1, respectively. Interday and intraday precision were both< 5.6%. The recovery of 8-OHdG in urine ranged from 99.8% to 103.2%.Summary:The MEKC-UV method proposed in this study was proven to be a convenient, sensitive, rapid, and selective way of quantifying urinary 8-OHdG. This method can be used in daily clinical practice to assess oxidative stress in patients.Part II The relationship between oxidative DNA damage and renal injury in T2DMObjective:To investigate the relationship between oxidative DNA damage and renal injury in T2DM.Methods:1 Evaluation of the Oxidative DNA Damage Biomarker 8-OHdG in Urine by SPE-MEKC-UV.2 The urinary creatinine was analyzed by CZE-UV method.3 Correlations between 8-OHdG/creatinine ratio and other clinical indicators were determined with the person correlation test.Results:The dates of urinary 8-OHdG were adjusted by urinary creatinine, and the mean±SD of urinary 8-OHdG was 16.37±4.97 ug g-1 in T2DM group. And T2DM group showed higher urinary 8-OHdG levels than CON group (11.72±2.38μg g-1), the difference was significant (P<0.05). The urinary 8-OHdG levels of DN group (45.14±12.68μg g-1) were significantly higher than those of T2DM and CON group (P<0.05).The index of urinary 8-OHdG has no significant correlation with uric acid (UA). There is a significant positive correlation between urinary 8-OHdG level and glycosylated hemoglobin HbAlc (%), and There is also a significant positive correlation between urinary 8-OHdG level and 24h urinary protein.Summary:There is a correlation between oxidative DNA damage and renal injury in T2DM.Part III DHA alleviated renal injury in type 2 diabetic rats via reducing the level of oxidative DNAObjective:To investigate whether DHA could alleviate the renal damage in T2DM via reducing the level of oxidative DNA and oxidative stress.Methods:1 T2DM rats were induced by the combination of a HFD and a ow-dose STZ, and some of the T2DM rats were fed with a HFD plus DHA supplementation.2 The physiological and biochemical indexes of renal "unction were analyzed by HE staining and Elisa kits.3 The ROS levels in kidney of different groups were detected by using DHE staining and spectrophotometer.4 Lipid peroxide in kidney tissue of rats were detected by immunofluorescence method and Elisa kits.5 DNA damage was analyzed by capillary electrophoresis and immunofluorescence method.6 The contents of important antioxidants were detected by Elisa kits.7 Effects of DHA on the three antioxidant system in kidney were detected at mRNA level by Real time-PCR.8 Effects of DHA on the subunits of NADPH oxidase in kidney were test by Western blot.Results: 1 DHA reduced the 24-hour urine protein excretion rate of T2DM rats.The 24-hour urine protein excretion rate was significantly lower in the CON and DHA group than in the DM group (P< 0.05). It was also 3 up (P<0.05). significantly lower in the CON group than in the DHA grou2 DHA improved the kidney structure of T2DM rats.HE staining showed:There were no pathological changes in the kidney of CON group. The glomeruli of DM group distributed irregularly, and the nucleus arranged in disorder, the renal tubular epithelial cells appeared edema, with diffuse matrix increased in DM group.The mean glomerular area was significantly higher in the DM group (21.59±5.01 103μm2) than in the CON group (11.05±3.52 103μm2) (P<0.05) The mean ± SD of mean glomerular area was 17.15±4.66 103 μm2 in DHA group, and it was found in the between of DM group and CON group. 3 DHA reduced the DNA damage in kidney tissue of T2DM rats. 3.1 DHA reduced the level of 8-OHdG in kidney tubules of T2DM rats.Immunofluorescent staining showed:The contents of 8-OHdG in the glomeruli were no significant differences among the three groups. But the level of 8-OHdG in the renal tubules of DM group was significantly higher than that in CON and DHA group (P<0.05). And there was no significant change in the renal tubules between the CON and DHA group (P>0.05). 3.2 DHA alleviated the apoptosis of renal cells induced by T2DM in rats.The result of TUNEL showed:the level of apoptosis in the renal tissue of DM group was significantly higher than that in CON and DHA group (P< 0.05). And there was significant higher in the DHA group than in the CON group (P<0.05).4 DHA reduced the levels of oxidative stress in the kidney of T2DM rats.4.1 DHA reduced the ROS level in the kidney of T2DM rats.At the end of the experiment (32 weeks), the ROS level was significantly lower in the DHA group than in the DM group (P< 0.05). It was also significantly lower in the CON group than in the DM group (P<0.05), but not than in the DHA group (P>0.05).4.2 DHA reduced the gene expression of NADPH oxidase subunit in renal tissue of T2DM rats both on mRNA level and on protein level.NADPH oxidase can induce ROS under high glucose conditions. The esults of real time PCR showed:DHA can significantly down regulate the mRNA expression of Nox2, Nox4 and P22 phox. And the results of western blot showed:DHA can significantly down regulate the protein expression of Nox2, Nox4, P22 phox and p-P47 phox. 5 The content of lipid peroxide in kidney tissue of different groups. 5.1 DHA reduced the MDA level in the kidney of T2DM rats.The content of MDA in kidney of DM group (1.20±0.18 nmol/mg prot) was higher than that in CON group (0.86±0.13 nmol/mg prot) and that in DHA group (1.01±0.19 nmol/mg prot) (P< 0.05). The content of MDA in DHA group was also higher than CON group (P<0.05).5.2 DHA reduced the 4-HNE level in the renal tubules of T2DM rats.The level of 4-HNE in the renal tubules was higher in DM group than that in CON and DHA group (P<0.05). There was no significant change in the renal tubules between the CON and DHA group (P>0.05). The content of 4-HNE in the glomeruli of CON group was lower than that in DM and DHA group (P<0.05). There was no significant change in the glomeruli between the DM and DHA group (P>0.05).6 DHA improved the antioxidant capacity in the kidney of T2DM rats. 6.1 DHA increased the T-AOC level in the kidney of T2DM rats.The T-AOC level was significantly lower in the DM group than that in the CON and DHA group (P<0.05). It was no significant deference between CON group and DHA group (P>0.05). 6.2 DHA increased the activity of antioxidant enzymes, including GR, CAT and Txnrd in the kidney of T2DM rats. 6.3 DHA increased the GSH level in the kidney of T2DM rats. 6.4 DHA increased the 4-HHE level both in the renal tubules and in the glomeruli of DHA group. 7 DHA increased the mRNA expression of SOD1, Gp×1 and Gpx4-1. Summary:1 DHA improved the renal function and renal injury in T2DM rats.2 DHA reduced the oxidative DNA damage and oxidative stress in renal tissue of T2DM rats via self oxidation and increasing the activity of antioxidant enzymes.Conclusions:1 There is a correlation between oxidative DNA damage and renal injury in T2DM.2 DHA improves the renal function and renal injury in T2DM rats. DHA reduces the DNA oxidative damage and oxidative stress in renal tissue of T2DM rats via self oxidation and increasing the activity of antioxidant enzymes.