Study On The Relationship Between Soluble Epoxide Hydrolytic Enzyme And Diabetic Nephropathy And The Intervention Effect

Section oneBackground:Diabetic nephropathy (DN) is the most common microvascular complication of diabetes, which causes large amount of health expenditure especially when renal replacement therapy is needed in its end-stage renal disease. Pathogenesis of diabetic nephropathy has not been fully elucidated. Current drug therapies for diabetic nephropathy are not entirely satisfactory. In recent years, soluble epoxide hydrolase (sEH) received much attention due to its potential effects in the pathogenesis of diabetic nephropathy.Objectives:This study aimed to verify whether sEH expression is upregulated in DN, through examining the protein level of sEH in the kidney of DN patients. The current study compared the functions of two different rat EPHX2 (the gene coding sEH) phenotypes in NF-κB inflammatory signaling pathway, TNF-a induced apoptosis pathway and cholesterol biosynthesis, to explore if the EPHX2 gene in OLETF spontaneous diabetic rat plays a role in the pathogenesis of DN. Moreover, this study also evaluated the effect of Tangshen Formula in regulating human EPHX2 promoter activities, in order to demonstrate whether the treatment effect of Tangshen Formula in diabetic nephropathy is at least partially due to its EPHX2 regulating function.Methods:1. Study of sEH protein expression in the kidney of DN patients:This study includes patients who registered in Shanxi Provincial Hospital of Traditional Chinese Medicine and went through renal biopsy during April 2011 to July 2014. The study compared renal sEH protein expression between DN patients and IgA nephropathy. Para-carcinoma tissue separated from renal carcinoma patients is used as normal control. All cases should meet the clinical diagnostic criteria, and be confirmed by renal biopsy. Paraffin embedding kidney samples are cut into 6μm section by automatic slicing machine. After dewaxing, immunohistochemical staining of sEH is performed in each sample slices. Image-Pro Plus 6.0 software was used for mean optical density semi-quantitative analysis. Relationship of sEH expression and 24 hour urinary protein level were analyzed by Pearson correlations.2. Study of the function difference of EPHX2 gene phenotypes between OLETF spontaneous diabetic rat and LETO non-diabetic rat, in NF-kB inflammatory signaling pathway, TNF-a induced apoptosis pathway and cholesterol biosynthesis:(1) Construction of EPHX2 gene over expressed cell model:Four different EPHX2 mRNA fragments including the full length EPHX2 mRNA of LETO and OLETF rat, and a shorter fragment EPHX2-X1 which is lack the first 198 nucleotide of 5’end of LETO and OLETF rat, were amplified, using cDNA extracted from renal cortex of OLETF spontaneous diabetic rat and LETO non-diabetic rat as templates. The 5’end of forward primer contained EcoR V restriction enzyme cutting site, and the 5’end of reverse primer contained Xbal I restriction enzyme cutting site. The amplified cDNA were connected to p-UCT vector using a p-UCT DNA Ligation Kit. Then the DNA fragment were subcloned to eukaryotic vector pcDNA3.1/V5-His-A. The plasmid DNA were finally went through enzyme identification and DNA sequencing to confirm the plasmid DNA contained the right insertion element. The 4 recombinant plasmid DNA and pcDNA3.1/V5-His-A empty vector were transient transfected into rat renal tubular epithelial cell line NRK-52E, by Lipofectamine2000 transfection reagents. GFP was also transfected in the same way to observe the transfection efficiency. Fusion protein expression was observed by Western Blot using anti-V5 antibody. The mRNA expression of transfected plasmid DNA was detected by reverse transcription-PCR.(2) The effect of sEH over expression on inflammation and apoptosis:Recombinant plasmid DNA transfected NRK-52E cells were stimulated with or without 11,12-EET or sEH inhibitor TPPU added to the cell culture medium.11,12-DHET concentration in the culture medium of each group was tested by an ELISA kit to observe the hydrolysis activity of different plasmid DNA. Inflammation was induced by stimulation with inflammatory factor TNF-a in transfected NRK-52E cells. Real time-PCR and Western blot were applied to detect the inflammatory markers p65, IκBα MCP-1、IL-1β、PPARα、PPARγ and apoptosis markers TNFR1、caspase-3、Bcl-2.(3) The effect of sEH over expression on cholesterol biosynthesis:four recombinant plasmid DNA were transfected into NRK-52E cells, and empty vector was used as negative control. Another group was treated with TPPU after transfection. Total cholesterol in the culture medium was evaluated by an ELISA kit. Real time-PCR and Western blot were applied to detect the expression of HMGCR in each group.3. The effect of Chinese herbal formula Tangshen formula in regulating human EPHX2 promoter activities:Human EPHX2 promoter recombinant plasmid hEPHX2/pGL3 was transient transfected into HEK293T cell. The cells then received different concentrations of TSF treatment for 24 hours. Dual fluorescein enzyme report gene method was used to compare the promoter activities between TSF treatment groups and negative control group. Human EPHX2 promoters with 3 different length were transfected into HEK293T cell, then each group received 400μM,600uM and 800μM H2O2 stimulation for 4 hours. Promoter activities after H2O2 stimulation were detected with dual fluorescein enzyme report gene method.Results:1. A total of 8 DN,6 IgA nephropathy and 3 renal carcinoma cases were included in this study. sEH was identically expressed at the cytoplasm of renal tubular epithelial cells, mostly in proximal tubular. No specific staining was observed at the small vessels nor glomeruli. Mean optical density of sEH was statistically elevated in the diabetic nephropathy group, compared with normal control group (P< 0.05). Mean optical density in the diabetic nephropathy group was higher than the IgA nephropathy group but without statistically significant difference. Mean optical density of sEH was positively correlated with proteinuria level in patients with both diabetic nephropathy and IgA nephropathy.2. The LETO type and OLETF type of recombinant plasmid DNA EPHX2/pcDNA3.1/V5-His-A and EPHX2/pcDNA3.1/V5-His-A were successfully constructed, and the sequences were confirmed by restriction enzyme cutting and DNA sequencing test. However, when using Western blot method to detect the fusion protein levels, the 2 full length EPHX2 recombinant plasmids displayed overexpression, but the 2 EPHX2-X1 recombinant plasmids could not overexpress at the protein level. Further study in the mRNA level expression test showed that the 4 recombinant plasmids expressed equal levels of mRNA after transfection, which indicated the low expression of EPHX2-X1 was due to post translational regulation. Therefore, the 2 EPHX2-X1 recombinant plasmids was not suitable for the following study, and only the 2 full length EPHX2 recombinant plasmids were used.After adding 11,12-EET into the medium,11,12-DHET levels in the transfected cell groups were remarkably elevated, compared with empty vector transfection group. No difference was observed between LETO type and OLETF type. Protein levels of NF-κB p65, p-p65 were elevated in the two transfected group compared with empty vector group. After TNF-α stimulation, p-p56 in transfected group went through a further rise, and IκBα was compatibly decreased in each group.11,12-EET administration could decrease p-p56 and increase IκBα expression. IL-1β protein was upregulated by TNF-α stimulation and downregulated by 11,12-EET in transfected groups. No difference was observed between LETO type and OLETF type. As for the downstream gene expression of NF-κB signaling, MCP-1 and IL-1β was upregulated by TNF-a stimulation and downregulated by 11,12-EET in transfected groups. PPARa and PPARy were downregulated after transfection, further decreased by TNF-a stimulation and upregulated by 11,12-EET in transfected groups. No difference of these gene expression was observed between LETO type and OLETF type.The rate-limiting enzyme of cholesterol synthesis HMGCR was upregulated by recombinant plasmid transfection, and was further increased by TPPU. OLETF type plasmid displayed a greater degree of HMGCR increase compared with LETO type but without statistically significant difference. This result indicated that the N terminal of sEH functions to increase cholesterol synthesis.3. The 4.8kb human EPHX2 promoter plasmid DNA showed notably activity after transfected into HEK293T cell line, compared with pGL3 empty vector group. Different concentration of Tangshen formula stimulation ranging from 100 μ.g/ml to 750 μg/ml, did not have an effect on the promoter activity. When stimulated by different concentration of H2O2, all three human EPHX2 promoter fragments were dose dependent activated.Conclusion:sEH expression is upregulated in the kidney of diabetic nephropathy patients. The functions of EPHX2 gene phenotypes between OLETF spontaneous diabetic rat and LETO non-diabetic rat are not differ in NF-κB inflammatory signaling pathway, TNF-a induced apoptosis pathway and cholesterol biosynthesis. The current study does not show that Tangshen formula has an effect on the 4.8kb human EPHX2 promoter activity. Human EPHX2 promoter displays a positive response to stimulation, which could be a potential breakthrough point for future study of sEH.Section twoYi Qi Qing Re Gao (YQQRG) formula is a traditional Chinese herbal medicine used to treat chronic nephritis. This study was designed to evaluate the underlying mechanism in the use of YQQRG formula to treat nephrosis induced by puromycin aminonucleoside (PAN).Methods:Thirty-six male Wistar rats were randomly divided into three groups of 12 rats:a sham group, a vehicle-treated PAN model group (PAN) and a group treated with YQQRG (PAN+ YQQRG). The PAN model was established by a single intravenous injection of PAN at a dose of 40 mg/kg body weight; the rats in the sham group received the same volume of saline. Twenty-four hour urinary protein were measured 0,3,5,10, and 15 days after the injection. The rats were sacrificed at day 10 and day 15 and the serum lipid profile examined. The renal cortex of each rat was stained with periodic acid-Schiff reagents and the pathological alterations and ultrastructural changes were examined by transmission electron microscopy. In situ cell apoptosis was detected by a terminal deoxynucleotidyl transferase-mediated uridine 5’-triphosphate-biotin nick end-labeling (TUNEL) assay. Transcriptive levels of inflammatory markers and molecules associated with apoptosis were detected by a real-time polymerase chain reaction and the expression of proteins was examined by either immunohistochemistry or Western blot analysis.Results:YQQRG remarkably decreased the levels of urinary protein, increased serum albumin and lowered serum lipid levels. YQQRG also attenuated histological lesions in the rat kidneys. The activation of inflammatory markers was largely restored by the administration of YQQRG. The TUNEL assay showed that YQQRG decreased the number of apoptotic cells. Both the mRNA and protein levels of caspase-3 were significantly reduced in the group treated with YQQRG, whereas the expression of the Bcl-2 protein increased in the YQQRG group.Conclusions:YQQRG alleviated kidney injuries of PAN-treated rats, possibly through anti-inflammatory anti-apoptosis effects.