| Objective:1.To identify and analyse urine proteins between normal stage when the models have been made or not and the early stage of kidney disease when the models have been made in diabetic rats separately with"shotgun"which is a study strategy of proteomics. By doing this we can provide fundamental experimental basis to search for new urinary protein markers and investigate the pathogenesis of diabetic nephropathy.2.To investigate the role of mitochondria in the occurrence and development of diabetic nephropathy through comparing the related indicators of renal cortical oxidative stress and mitochondrial dysfunction and the change in PGC-lαmRNA expression between normal and the early stages of diabetic nephropathy rats.3.To investigate the pathogenesis of diabetic nephropathy at the sub-cellular proteins level by analyzing and identifying differential proteins of renal cortical mitochondria in normal rats and diabetic nephropathy rats through two-dimensional fluorescence difference gel electrophoresi(s2D-DIGE)as well as MALDI-TOF-MS.Methods:The research objects are diabetic Wistar rat models which are induced by streptozotocin (STZ).1.Collect urine specimen of normal control period when the models have been made or not and the early stage of kidney disease when the models have been made separately from 7 rats. After ultrafilter concentration, depletion of high abundant proteins by chromatographic column and removing impurity we preliminarily separated the protein samples by 1-DE(SDS-PAGE). Then enzymatic hydrolysis of proteins in the glue, capillary high performance liquid chromatographic separation, mass spectrum analysis by LTQ-Velos and searching protein databases have been done to identify proteins. Use bioinformatics technology to study the cellular localization, functional groups, biological process and signaling pathways of proteins which have been identified. In order to search for new urinary protein markers and investigate the pathogenesis of diabetic nephropathy we search the data base further to analyze specific proteins which we are interested in among rats of the early stage of kidney disease.2.Take renal cortical separately from normal controls(n=6) and the early diabetic nephropathy group(n=7) of rats to compare indexes such as superoxide dismutase (SOD) activity which is an oxidative stress index and can be detected by chemical colorimetric method, the content of malondialdehyde (MDA) ,the activity of amber acid dehydrogenase (SDH) which is a mitochondrial function index and the expression of PGC-lαmRNA which can be tested by Real-time fluorescence quantitative RT - PCR detection.3.Extract high purity and crude extract of renal cortical mitochondria and renal cortical tissue proteins by velocity centrifugation method from three normal rats which are chosen by random. Compare the concentration and integrity between high purity and crude extract of mitochondria and identify purity of mitochondria preliminarily by electron microscope. Compare GAPDH which is a biomarker protein of cytoplasm among high purity and crude extract of mitochondria and histones. Identify the purity of the mitochondrial protein. Extract high purity of renal cortical mitochondria from normal controls and the early diabetic nephropathy group of rats. Lyse mitochondria to extract proteins and meanwhile remove impurities. Then identify different proteins by two-dimensional fluorescence difference gel electrophoresis(2D-DIGE), software analysis, glue preparation, cutting glue, enzymolysis, MALDI-TOF-MS technique and searching in proteins database.Results:1. There are 274 proteins which are identified by shotgun analysis and database search among urine proteins during the two stages, 121 of them are proteins during the normal stage and 153 of them are proteins during the early stage of diabetic nephropathy. There are 56 urine proteins which are the same during the two stages. There are 65 urine proteins uniquely during the normal stage and 97 urine proteins uniquely during the early stage of diabetic nephropathy. Analyze cellular localization, functional groups, biological process, signaling pathways and so on by bioinformatics approach. Analyze biological pathway of 97 proteins during the early stage of diabetic nephropathy and then select one proteins which called Alpha-actinin-4.This proteins have been proved to be related to focal adhesion kinase(FAK ) signal transduction pathway which is induced by extra cellular matrix(ECM)and growth factor(GF), phosphatidyl-inositide 3 kinase/AKT (PI3K/AKT) signal transduction pathway , protein kinase signal transduction pathway which is activated by mitogen and RhoA-ROCK signal transduction pathway.2.Compared to normal stage, the levels of malondialdehyde (MDA) is higher while activity of superoxide dismutase (SOD) and succinate dehydrogenase (SDH)is lower in the renal cortex of rats during the early stage of diabetic nephropathy. The real-time fluorescence quantitative RT - PCR detection has proved the expression of PGC-lαmRNA in the renal cortex of rats during the early stage of diabetic nephropathy is lower than normal.3.Being observed under electron microscope, both high purity and crude extract of mitochondria have intact membranes and the concentration and purity is higher in high purity of mitochondria. Compared with organization extraction protein, crude extract of mitochondria shows extremely few GAPDH biomarker proteins of intracellular compositions while high purity of mitochondria shows none of it by Western blot.4. 357±32 spots have been isolated successfully from the high purity of renal cortical mitochondria samples by two-dimensional fluorescence difference gel electrophoresis(2D-DIGE).There are 13 spots where differences between the two groups are greater than three times. We have successfully identified 8 proteins by MALDI-TOF-MS.,which are lower during the early stage of diabetic nephropathy ,The proteins link to oxidative phosphorylation and amino acid and fatty acid metabolism in the mitochondria suggest there may be metabolic disorders of three macronutrients during the early stage of diabetic nephropathy by bioinformatics methods.Conclusion:1.Shotgun is an efficient, rapid, good detection limit and high flux proteomic technique, which is suitable for protein analysis of complex specimens which are rich for salinity and impurity such as urine. Proteomic technique may help researchers to analyze urine proteins panoramically, which is helpful for find out diabetic nephropathy relevant biomarker proteins more quickly from urine.2.Following and analyzing the proteome changes of urine by shotgun technology during the different stages of disease in diabetic rats has been seldom reported both at home and abroad. In this study, we found that Alpha-actinin-4 appear on the early stage of diabetic nephropathy and do with several important signal transduction pathways link to diabetic nephropathy. These findings suggest that this proteins can not only be candidate markers of early diagnosis but also be biomarker proteins for the study on the mechanisms of diabetic nephropathy.3. In this study, SOD is less active and MDA levels rise, which confirm the perspective that diabetic nephropathy is closely connected to oxidative stress again. At the same time, SDH is less active and PGC-lαmRNA levels drop, which suggest that diabetic nephropathy is related to the dysfunction and synthesis obstacles of mitochondria. Relations between diabetic nephropathy and PGC-lαhas been seldom reported both at home and abroad, which need to do more research.4.Two-dimensional fluorescence difference gel electrophoresis(2D-DIGE)has been used to analyse different proteins among the early stage of diabetic nephropathy and normal stage of rats in the study, which identify 8 different proteins that substantially decrease. These proteins are related to mitochondrial oxidative phosphorylation, acid metabolism and fatty acid metabolism, which suggest that diabetic nephropathy is connected to mitochondria and the mechanism requires further researching.
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