The Aberrant Expression Of Histone Deacetylase5in NOD Mice Spleen CD4~+T Cells And Its Role In The Pathogenesis Of Diabetic Nephropathy

Diabetic nephropathy (Diabetic nephropathy, DN) is a common and serious diabetes complication, which is caused by micro-vascular lesion and the main reason developing to the end-stage renal disease (ESRD).There is an upward trend in the incidence of our country. Its etiology and pathological mechanism is still unclear. And most of the researches showed that the etiology of DN was closely related to the genetic predisposition, poor blood sugar control, aging, gender factor and duration of diabetes. Recent studies have found that excessive activation of T cells and inflammatory infiltration is the main immune pathological mechanism of diabetic kidney injury. While epigenetic regulation play an important role in gene expression, differentiation and activation of T cells.Epigenetics is emerging discipline from the1980s, which means on the premise DNA sequence of gene encoding is unchanged, gene expression or cell phenotype is heritable changed through some mechanism. The main mechanisms includes DNA methylation, histone modification and small RNA interference and etc. Histone acetylation is one of the most important modification and the main driving force of gene expression. Histone acetyltransferase(HATs) and histone deacetyltransferase (HDACs) co-participates in maintaining dynamic balance of acetylation of histone modifications and relates to gene transcription activation. In the transcription-on region of core histone, the level of acetylation is higher, while lower in the transcription-off region. Core protein can be acetylated by HATs, making the chromatin structure loose in the related area and transcription activated; HDACs can remove acetylation decorate of the core protein, making chromatin superhelix in relevant region and inhibiting DNA in combination with RNA polymerase,related transcription factors, then close the transcription. Contrary to the function of the HATs, HDACs can close the transcription by getting rid of the histone acetylation. It has been found18types of HDACs in humans, according to the homology of three kinds of HDACs from saccharomyces cerevisiae (apd3, Hdal and Sir2), HDACs can be divided into three types:category1has similar structure and homology with apd3of yeast, including HDAC1, HDAC2, HDAC3, HDAC8, HDAC11; category2has similar catalytic structure with yeast Hdal and can be divided into sub-A and sub-B. Class Ⅱ-A is the transcription Corepressor, including HDAC4, HDAC5, HDAC7, HDAC9,while class Ⅱ-B includes HDAC6, HDAC10. Category1and category2have the similar catalytic activity, but of them the structure and function are obviously different; Category3, homologous with Sir2 of yeast, has been identified to seven kinds, SirTl-7respectively.With the development of research, we already know clearly about a large number of molecular mechanisms involved in the pathogenesis of diabetes complications, especially the DN. Protein kinase C(PKC) and reactive oxygen species(ROS) are now recognized as important signaling molecules leading to diabetes renal injury. In DN patients, the expression level of most pro-inflammatory factors and fibrosis factors were increased, such as transforming growth factor-b1(TGF-b1), connective tissue growth factors(CTGF), vascular endothelial growth factor(VEGF), a-smooth muscle actin(a-SMA) and extracellular matrix(ECM) protein, etc, while the expression of endogenous anti-fibrosis cytokines BMP7(morphogenetic protein-7) and E-calcium mucin were decreased. The abnormal expression of a variety of promoting or anti-fibrosis related genes in renal cell from DN patients suggests that may be there is some sort of imbalance in upstream molecular mechanisms which could influence the expression of related genes. HDACs can gene transcription by altering histone acetylation level. Studies have shown that HDAC inhibitor can effectively protect the kidney from DN patients and obviously relieve the clinical symptoms. It indicates us that abnormal expression of HDACs may be involved in the pathological process of DN. By comparing the renal pathology from non-obese diabetic mice(NOD) before and after onset, we found infiltration of glomerular T, B and CD11c+dendritic cells, accompanied with deposition of IgG and C3in the incident NOD mice. In addition, anti-glomerular autoantibodies can be detected in serum from the NOD mice.And these phenomena in onset of diabetes in incident NOD mice, which was not observed in undiseased NOD mice Therefore, the occurrence of diabetic nephropathy is closely related to the dysfunction of immune cells. This previous study found that, compared with normal controls, histone H3, H4acetylation level were decreased obviously in CD4+T cells from Type1diabetes patients, HDAC5expression level was increased significantly, and H3acetylation level significantly negatively correlated with blood sugar levels of patients. It implicates us that the globle histone hypo-acetylation and abnormal hyper-expression of HDAC5may lead to the dysfunction of CD4+T cells, which participates in the pathogenesis of diabetes and diabetic nephropathy. In this study, we choose diabetic NOD mice model as the research object, through testing the globle H3, H4acetylation level and the expression of HDACs, correlation analysis with related indicators, further clarify the relationship between abnormal histone acetylation modification and DN development in CD4+T cells. Part Ⅰ The relationship between histone acetylation modification and the pathogenesis of diabetic nephropathy in spleenCD4+T cells from NOD mice Section Ⅰ Globle H3, H4acetylation level and modification enzyme spectrum in spleen CD4+T cells from different weeks of NOD micePurpose:To test the globle H3, H4acetylation level and modification enzyme spectrum in spleen CD4+T cells from different weeks of NOD mice.Methods:NOD mice were randomly divided into4groups(diet, drinking), isolate CD4+T cells from spleen in12,18,24and30weeks NOD mice, then extract histone, total RNA and protein. Using ELISA kit ordered from American Epigentek company to test globle H3,H4acetylation level in CD4+T cells from NOD mice. Real-time quantitative PCR to detect the related mRNA level of histone acetylation modification enzyme in CD4+T cells. Western Blot to further verify the abnormal expression genes picked from the results of real-time PCR, and makes correlation analysis between the abnormal expression of modification enzyme and globle H3,H4acetylation level. Clatify the H3,H4acetylation modification status in CD4+T cells from NOD mice and explore its relationship with DN.Results:We found that compared with12weeks of mice, globle H3,H4acetylation level in spleen CD4+T cells from24and30weeks old NOD mice was obviously lower, so was the18weeks old NOD mice. Through Real-Time PCR detection,we found that P300, PCAF, HDAC2mRNA level declined obviously while HDAC5mRNA level was significantly increases in24weeks and30weeks NOD mice,but there was no difference of CREBBP expression level. Compared with12weeks of mice, HDAC5mRNA level was only slightly increases in18weeks of NOD mice, there was no significant difference in the rest of mRNA. Western Blot results shew that compared with12weeks of mice, HDAC5protein expression level is significantly lower than24and30weeks of NOD mice in spleen CD4+T cells.Conclusion:With the progress of the NOD mice disease, its globle H3, H4acetylation level significantly decreased and HDAC5expression level was increased significantly in spleen CD4+T cell,and there were significantly negative correlation. Section Ⅱ The relationship between abnormal histone acetylation modification and index of diabetic nephropathy in CD4+T cells from NOD micePurpose:To test random blood glucose and trace urinary albumin excretion in different weeks of NOD mice, then correlate globle H3/H4acetylation level with HDAC5expression level in spleen CD4+T cells. Clarify the relationship between HDAC5and DN in spleen CD4+T cells.Method:Using blood glucose meter and test paper to detected in random blood glucose from12,18,24and30weeks of mice. Using mice tail reflection method to collect urine of mice from the4group.Using ELISA kit ordered from RB companies in the United States to detect urinary albumin and creatinine, respectively, calculating the urinary albumin/creatinine ratio to get urinary albumin excretion rate.Results:Compared with12weeks of mice, there was a significant rise in blood glucose levels and urinary albumin/creatinine ratio of24and30weeks of mice, but thers was a slightly raise of18weeks of NOD mice in blood sugar, while urinary albumin/creatinine ratio had no obvious change. H3, H4acetylation level significantly negatively correlated with urinary albumin/creatinine ratio and HDAC5level positively correlated with urinary albumin/creatinine ratio in24and30weeks of mice.Conclusion:Abnormal HDAC5expression is closely related to the NOD mice kidney damage in spleen CD4+T cells. Part Ⅱ Using siRNA plasmid to interfere the expression of HDAC5Purpose:Using siRNA interference technology to construct HDAC5plasmid, screening effective targets preparing for late intervention in spleen CD4+T cells from NOD mice.Methods:According to HDAC5mRNA sequence from genebank, using online design tools BLOCK-iTTM RNAi Designer provided by ambion to find the interference targets. Chosing the target sequence to clone into pLKD. CMV. G&PR. U6. ShRNA plasmids, then transfecting the succesful cloning plasmid to CD4+T cells from NOD mice,using real-time PCR and western blot to test interference efficiency, finally screening effective targets.Results:Using BLOCK-iTTM RNAi Designer to find ouy four highest score of HDAC5interfering targets, and successfully cloned them into interference carrier. Using real-time PCR and Western Blot to detect the best interference effect of siRNA-HDAC5.Conclusion:SiRNA technology can successfully intervent the expression of HDAC5in CD4+T cells from NOD mice. Part Ⅲ The intervention and therapeutic effect of siRNA-HDAC5on abnormal histone modification in NOD micePurpose:Through intervening the expression of HDAC5in spleen CD4+T cells of NOD mice, detect related indicators of renal function and assess therapeutic effect of siRNA-HDAC5on NOD mice, further clear the relationship between HDAC5and DN.Method:Mices were randomly divided into three groups:siRNA-HDAC5group, siRNA-Control group and normal saline group. Then collected samples from the mice and tested related indexes at18,24and30weeks old. Using Real-time PCR to detect CD11a, CCR5and CX3CR1transcription level in spleen CD4+T cells, ELISA to test serum IL-1, IL-6, IL-18and TNF alpha level,glucose meter to test blood sugar, ELISA to detect the urinary albumin excretion rate, finally pathological section to detect the kidney damage of the three groups.Results:Compared with the control group which was not intervened, CD11a, CCR5and CX3CR1were significantly decreased in spleen CD4+T cells in mice of HDAC5intervention group, so were serum cytokine of IL-1, IL-6, IL-18, TNF alpha level, the blood sugar and urine albumin excretion rate. Compared with control group, renal pathology results shew that there was no obvious changes in glomerular pathological damage of the24weeks old mice in intervention group, while there was only mild hyperplasia of mesangial cells, slightly increasing of matrix, less neutrophil infiltration, no thickening of basement membrane, a small amount of inflammatory cell infiltration in interstitia, obvious fibrosis of renal tubular cells in30weeks old mice..Conclusion:Blocking the expression HDAC5in CD4+T cells could effectively therapy the kidney damage causing by diabetes.