Study On The Urinary Proteomics Of Chronic Kideny Disease And Mechanisms Of Action Of Shengqingjiangzhuo Capsule

ObjectiveChronic kidney disease (CKD) is becoming a wordwide public health problem. The prevalence of CKD is increasing significantly in recent years and CKD becomes a serious threat to human health and life. Identification of patients at an early disease stage and early diagnosis of the specific renal disease enable us to improve therapeutic treatment and alleviate the financial burden to patients and society. Therefore, the identification of a specific set of early biomarkers for CKD is extremely important to clarifying pathogenesis, improving diagnosis and treatment or identifying therapeutic targets for the development of new drugs.The etiology of CKD include:glomerular diseases, tubulointerstitial diseases, renal vascular diseases, the renal damage of metabolic diseases or connective tissue diseases, the renal damage of infectious diseases, congenital or hereditary kidney disease, etc. In China, epidemiological survey showed that the prevalence of CKD was between9%and14%, and the most popular etiology is primary chronic glomerulonephritis. With CKD developing to the mid or late stage (stage3-5CKD), there is a gradual decline in renal function, especially the function of excretion, secretion and regulation. And the clinical abnormalities are showed as the disorder of organism in excreting metabolic waste and the disturbing of the water-electrolyte and sour-alkali balances. In the category of TCM, stage3-5CKD belongs to the range of consumptive disease, edema, uroschesis, obstruction and rejection. According to Chinese medicine theory, TCM pathogenesis of stage3-5CKD is due to deficiency of spleen and kidney, as well as obstruction of turbid toxins and blood stasis. Shengqingjiangzhuo Capsule, which was developed by a professor named Hong Qinguo who comes from the first affiliated hospital of guangzhou university of TCM, according to TCM pathogenesis of stage3-5CKD. And the Capsule has been obtaining satisfactory results in clinical. In this study, we treated patients with stage3-5CKD who developed from primary chronic glomerulonephritis with Shengqingjiangzhuo Capsule, and explore the effect and mechanisms of Shengqingjiangzhuo Capsule by observing the TCM syndrome integral, index of renal function, and other laboratory parameters.In this study, we use urinary proteomics techniques to detect the specific protein markers of CKD, aiming at investigating the pathogenesis. Moreover, in order to clarify the mechanism of action of ShengQingJiangZhuo capsule, we further screen the differential proteins in urine of the patients with stage3-5CKD before and after treatment.Methods1. The clinical study selected79cases with stage3-5CKD in non-dialysis phase,41with stage3CKD,19with stage4CKD, and20with stage5CKD. In addition to the basic treatment of feeding with low albumen food, controlling blood pressure, maintaining water-electrolyte and acid-base balance, all patients received the treatment with Shengqingjiangzhuo Capsule. After12weeks of treatment, before and after treatment, the TCM syndrome integral, laboratory parameters such as serum creatinine(Scr), blood urea nitrogen (BUN), glomerular filtration rate(GFR), albumin(ALB), cholesterol(CHOL), triglycerides(TG), hemoglobin(HGB), serum calcium(Ca), serum phosphorus (P), calcium-phosphorus product and uric acid(UA) were assayed.2.40patients stage1-5CKD in non-dialysis phase and10healthy volunteers (group A) were selected in the experimental study.40patients were divided into four group:group B (stage1CKD, n=10), group C (stage2CKD, n=10), group D (stage3CKD, n=10) and group E (stage4-5CKD, n=10). The group D and group E were randomly selected from the clinical study participants, all of them had received12weeks of treatment with Shengqingjiangzhuo Capsule. Mid-stream clean catch urine were collected from five groups, and urine were collected again from group D and group E after treatment with Shengqingjiangzhuo Capsule. Equal amount of10urine samples intra-group were mixed together. The urinary proteins were extracted with cold acetone and the high-abundance proteins were removed. Then the proteins were purified by2D-clean up Kit. Bradford method which was used to measure the concentration of urinary proteins, and SDS-PAGE was applied to separating those proteins. After SDS-PAGE, proteins were digested by trypsin, and then we labeld proteins with iTRAQ isotopic reagents, followed by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) to screen the differential proteins. With the mass spectrum files as original ones, we used the Mascot software to search petide information in the IPI database. The differential expressed proteins were defined as the relative abundance of iTRAQ labeling proteins. At last, the functions of differential proteins were analysed.Results1. Clinical studies(1)General data analysis:The patients with stage3CKD (23males and17females), aged29years to65years, mean age was43.93±9.20years. The patients with stage4CKD (10males and9females), aged24years to68years, mean age was48.95±14.68years. The patients with stage5CKD (7males and13females), aged28years to70years, mean age was55.95±13.40years. The mean age of patients among three groups had significative difference (P<0.01), with the progression of CKD, the mean age increased gradually. The mean age of the patients with stage5CKD was obviously older than the patients with stage3CKD(P<0.01). There was no statistical significance in sex distribution of the three groups of patients(P>0.05).(2)TCM syndrome integral:In all groups, TCM syndrome integral was different from before(P<0.01). For the TCM syndrome efficiency after treatment,10cases were obviously efficient,30efficient and0inefficient, the total effective rate was100%in stage3CKD group;5cases were obviously efficient,14efficient and0inefficient, the total effective rate was100%in stage4CKD group;4cases were obviously efficient,14efficient and2inefficient, the total effective rate was90%in stage5CKD group. There was no obvious difference among three groups(P>0.05).(3)Laboratory parameters:After treatment, the levels of Scr were significant lower in all groups (P<0.05or P<0.01), while the stage4CKD group and stage5CKD group decreased more significantly (P<0.05) and. After treatment, the levels of eGFR were remarkable higher in all groups (P<0.05or P<0.01), while the effect on the increase of eGFR was insignificant among three groups. After treatment, the levels of BUN, ALB, CHOL and TG had no significant changes in all groups (P>0.05). After treatment, the levels of HGB was significant higher in stage5CKD group, while, there was no significant change in stage3CKD group and stage4CKD group. After treatment, the levels of Ca increased in all groups, while the levels of P, calcium-phosphorus product and UA decreased in all groups, showed no significant change.2. Experimental Study(1) Total of1576proteins were identified in this study, and there were144differentially expressed proteins among healthy group and four CKD groups, which may be relative to the pathogenesis of CKD. Among these differentially expressed proteins, there were123down-regulated proteins and21up-regulated ones in four CKD groups. Protein function analysis revealed that the molecular function of differentially expressed proteins mainly included binding, catalytic activity and receptor activity. Biological processes which they were mainly involved in were single-organism process, cellular process, biological regulation, regulation biological process, and the cellular component distributed mainly in cell, membrane, extracellular region.(2) We have selected8proteins from these144differentially expressed proteins, which can be the early biomarkers for CKD. In these differential expressed Proteins, one had higher expression in four CKD groups than healthy group, which may be related closely to the mechanisms of CKD. The others had low expression compared among four CKD groups and healthy group A, which may be the protective factors for CKD. Protein function analysis revealed that the molecular function of these proteins mainly included binding and catalytic activity. They were mainly involved in regulation biological process and signaling pathway. And the cellular component localized in extracellular region.(3) We have selected17proteins from all144differentially expressed proteins, which may related to progression of CKD. With the progress of CKD from stage1to5,2proteins expression were up-regulated in turn, but15proteins expression were down-regulated in turn. These differential expressed proteins may be used as indicators to monitor activity and prognosis of CKD, or to predict and monitor response to treatment. Protein function analysis revealed that the molecular function of these proteins mainly included binding, enzyme activity and receptor activity. Biological processes which they were mainly involved in were metabolic processes, regulation biological process, cellular process and signaling pathway, and the cellular component localized in cytomembrane, organelle and extracellular region.(4) We have selected6proteins from all these144differentially expressed proteins, which may become drug targets for Shengqingjiangzhuo Capsule to treat CKD. Among these proteins, the expression intensity of5down-regulated proteins and1up-regulated protein decreased in stage3CKD group and stage4-5CKD group by Shengqingjiangzhuo Capsule treatment. Protein function analysis revealed that the molecular function of these proteins mainly included binding, enzyme activity and cytokine activity. Meanwhile, they were mainly involved in metabolic processes and regulation biological process, and the cellular component mainly localized in organelle and extracellular region.Conelusion1.Clinical studies Our results showed that ShengQingJiangZhuo capsule-can reduce serum creatinine and increase the level of e-GFR in patients with stage3-5CKD in non-dialysis. In addition, it can obviously improve the clinical symptoms of TCM and decrease the scores of TCM syndrome, and it can significantly improve the effect of TCM syndrome (total effective rate in the90-100%). So, our results proved that ShengQingJiangZhuo capsule can relieve clinical symptoms of patients with stage3-5CKD in non-dialysis, protect the residual function of renal and delay the progress of CKD.2. Experimental Study(1)We have established urinary proteome profiling of CKD in different clinical stages identified by iTRAQ coupled with2D LC-MS/MS, and we also have established urinary proteome profiling of stage3CKD and stage4-5CKD after treatment with Shengqingjiangzhuo Capsule.(2) The study discoverd a lot of differential proteins between all stage of CKD and normal subjects. Some proteins were selected from the differentially expressed proteins, which may become early biomarkers for CKD, or be used as indicators to monitor activity and prognosis of CKD, even can be used to predict and monitor response to treatment. Making further investigation of the function of these proteins, may develop a new way to clarify the pathogenesis and progression of CKD, then help to provide a new basis for the diagnosis and treatment of CKD at an early stage.(3) Basis on the clinical studies which showed a effective treatment in stage3-5CKD with Shengqingjiangzhuo Capsule, We have successfully screened some proteins which may become drug targets for Shengqingjiangzhuo Capsule to treat CKD. Through analyzing the function of these proteins, it develops a new way to clarify the pathogenesis and drug targets of Shengqingjiangzhuo Capsule, and it is helpful for us to clarify the essentials of TCM pathogenesis of CKD by urinary proteomics, then to develop the modernization of Traditional Chinese Medicine.