Transcription Factor Activity Profile Of Systemic Lupus Erythematosus And Acute Rejection After Renal Transplantation

Systemic lupus erythematosus (SLE) is an unpredictable autoimmune disease involving multiple systems. Lupus nephritis is most common and most serious complication of it. At clinical statistics, SLE patient with renal damage is up to 95%, while the biopsy results are even more close to 100%. Since the glucocorticoid hormones and immunosuppressive agents have been used to treat SLE, the patient's prognosis has been significantly improved, but the disease is still not optimistic about the treatment status. About 15% of the patients die and appear renal failure in five years. Meanwhile, Relapse during treatment is also facing a serious problem.Renal transplantation is the most ideal and oecumenical treatment of choice for end-stage renal disease (ESRD) patients. But transplantation rejection is still a strong risk factor for recipients of renal grafts. At present, the diagnosis of renal transplantation rejection can only be made by renal biopsy, which is costly, inconvenient and carries risks of complication.Clinical management of SLE and renal transplant patients would be improved if new rapid and reliable methods for detecting biomarkers of SLE and rejection were available. Despite a lot of work have been done, current clinical treatment of SLE commonly used glucocorticoid and/ or immunosuppressive drugs to suppress the body's immune function. However, it is no target-oriented and lack targeting with facing immune functional disorder of SLE. At the same time, many patients are forced to withdrawal due to side effects of these drugs, leading to treatment interruption. Now investigators have not been able to use the results of rejection studies for early biomarkers form urine and peripheral blood of the patients in clinically.Transcription factor (TF) is a DNA-binding protein and also known as trans-acting factors. It can take place specificity interact with cis-acting element of eukaryotic gene promoter region-specific. Through them and with other relevant interactions between proteins, it is activating or inhibiting transcription. A transcription factor can control hundreds of genes' expression, while the expression of a gene can also be regulated by a number of transcription factors. The structure and function of transcription factor defects are correlation to a number of human diseases (such as cancer and inflammation). Many transcription factors are still the role of drug targets.Novel biomarkers are readily, accessible and predict SLE and the fate of the transplant early and could help to further illustrate the mechanism of SLE and rejection. Our study Objective was to compare the levels of TFs expression in SLE and renal biopsies of acute rejection after renal transplantation between the normal samples and tried to reveal the mechanism of SLE and acute rejection after renal transplantation.Materials and Methods1. Fifteen cases of peripheral blood mononuclear cell of patients with SLE as experimental group, ten cases of peripheral blood mononuclear cell of healthy volunteers as normal control group; collected three biopsies of patients with acute rejection after renal transplantation as acute rejection group, three renal cortexes of tissue eumorphism patients as normal control group.2. Nuclear extracts were prepared using Nuclear Extract Kit according to the manufacturer's instructions. Measure the protein concentration of each sample using a protein quantitation assay with Bradford method. Then it was quantitately subpackaged and stored in-80℃. Total RNA was extracted using trizol according to the manufacturer's instructions and assessed RNA yield and quality by UV absorbance and denaturing agarose gel electrophoresis.3. The total TFs of nuclear extracts samples were labeling and concentrating and hybridization with TF array using TranSignalTM Protein/DNA Array microarray kit.4. Through X-film exposure, scan the slide using the Genepix 4000B.The images were saved as gray scale TIF files. Analyzed the data in ScanAlyze and saved the results as EXCEL files.5. Validated the results of TF array through EMSA.Results1. The quality of nuclear extracts was satisfied the demand of TF microarray experiment through the quantitative detection.2. In SLE, there were 92 TFs significant differential expression of which 78 up-regulated and 14 down-regulated.3. In acute rejection after kidney transplantation, there were 99 TFs significant differential expression of which 95 up-regulated and 4 down-regulated. 4. AP-1, Pbx1 and MEF-2 with a significant differential expressionin in SLE and acute rejection were selected for EMSA. It showed that the results were conformable to the result of array.Conclusions1. Microarray chip is the effective approach for the high-throughput analysis of TFs expression profile in SLE and acute rejection of renal transplantation.2. There were 92 TFs significant differential expression in SLE and 99 TFs significant differential expression in acute rejection after renal transplantation by Microarray chip. TFs were significant differential expression in SLE and acute rejection of renal transplantation.3.3 TFs were selected for EMSA. All results were conformable to the result of array which confirmed the reliability of Microarray chip results. EMSA was an effective approach for detecting expression level of the single TF.TFs may be the new biomarkers for SLE and acute rejection of renal transplantation.The study of differentially expressed TFs could help to further investigate the mechanism underlying the development of SLE and acute rejection of renal transplantation.