| ObjectsBiomarkers are the measurable changes associated with a physiological or pathophysiological process. Urine is the place that bears and accumulates different changes to a high degree. Therefore, it is possible to magnify and detect specific biomarkers associated with a physiological or pathophysiological process in urine. Urine has the potential to become a better biological source for disease biomarker discovery, particularly for renal diseases, because this fluid accumulates changes in the body and is the direct excreta of the kidney. Urinary proteins are affected; therefore, identifying the specific variables associated with a particular pathophysiological condition in clinical samples remains challenging. To circumvent these issues, simpler systems such as animal models should be used to establish a direct relationship between disease progression and corresponding changes in urine.Renal fibrosis is regarded as the final common pathway for most forms of progressive renal disease and involves glomerular sclerosis and/or interstitial fibrosis. Most renal disorders lead to renal fibrosis; therefore, there is great interest in identifying biomarkers for early diagnosis or therapeutic monitoring. In obstructive nephropathy, interstitial fibrosis is caused by tubular injury and the proliferation of interstitial fibroblasts, and this process is considered the most serious pathological change in end-stage renal disease. Many pre-clinical studies have provided extensive insights into the common pathways of fibrosis; however, few studies have focused on identifying urinary biomarkers involved in this pathological process. The unilateral ureteral obstruction (UUO) model was developed to identify useful biomarkers in different stages of obstructive nephropathy. UUO animal models have been considered the classic models of obstructive nephropathy because UUO can be easily manipulated by changing the time, severity and duration of the experimental setup. In this study, urine samples were collected from the residuary ureter linked to the kidney following UUO, providing samples similar to the specimens obtained from patients with obstructive nephropathy.Starvation can lead to different disorder of the body. During the process of starvation, there show a series of energy metabolism changes which leads to the decrease of body weight, the change of the body component and the metabolic gene expression. Short time starvation can cause the acceleration of protein metabolism and the increase of nitrogen excrement, intra leucine flux and oxidation. All of above illustrate that the effect of starvation on the body may possibly reflect by urinary protein changes. Posttranslational modification is the step of attaching the biochemical functional groups on one or more animo acids of polypeptide chains before they become the mature protein product.Proteins undergoing posttranslational modification play an important role in cell signaling and modulatory of enzyme activity. The type and extent of posttranslational modificantion can change under different circumstances and effects. Therefore, the study on the urinary proteome changes after starvation should focus not only on the difference of quantity and constituent of urinary protein, but also on the difference of the type and location of protein posttranslational modification. This may be important for the understanding of different changes caused by starvation. In this study, urinary protein changes after starvation 0,4 and 7 days were ovserved by LC-MS/MS to distinguish the possible effects of starvation.Methods1. This study was designed to identify urinary biomarkers related to renal tubular injury and interstitial fibrosis in the early (1 week) and late (3 week) stages of a rat UUO model using urinary proteomic profiling.After the UUO model was established, the laboratory chemical analysis was carried out. The renal histology was observed by HE and Masson staining. The urinary proteomic changes were tested by LC-MS/MS and were validated by western blot and immunohistochemistry.2. This study was designed to investigate the different changes in urine proteins after the rats were starved for 4 and 7 days. The body weight and laboratory chemicals were measured and analyzed. The urinary proteomic changes were tested by LC-MS/MS and were measured by Scaffold and Peaks analysis.Results1. Five hundred proteins were identified and quantified by LC-MS/MS, out of which 7 and 18 significantly changed in the UUO 1- and 3-week groups, respectively, compared with the sham-operation group. Validation by western blot and immunohistochemistry showed increased levels of Alpha-actinin-1 and Moesin in the UUO 1-week group, and significantly increased levels of Vimentin, Annexin A1 and Clusterin in the UUO 3-week group.2. Six hundred and sixty four proteins were identified by LC-MS/MS, our of which 5 and 13 significantly changed in Starvation 4- and 7-day groups, respectively, compared with the Starvation 0 day group. The posttranslational modification of urinary proteins changed after starvation for 4 and 7 days.Conclusions1. Significantly changed proteins increased after 1 week of UUO may be used as potential biomarkers of tubular injury, whereas significantly changed proteins increased after 3 weeks of UUO may be the candidate biomarkers of renal fibrosis.2. Starvation can lead to a variety changes in urinary proteins. The significantly changed proteins during the process of starvation can be exclued as disruptor in the discovery of disease candidate biomarker. It would be beneficial for the discovery of specific urine biomarkers in certain kind of disease. In addition, the significantly changed PTMs after starvation were Formylation and Phosphorylation, and these may provide clues for the exploration of the meaning why proteins exist in urine.
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