Using Proteomics To Study Renal Protein Handling Function

Background:With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form "reference profiles" of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology. Methodology/Principal Findings:After removing secreted proteins downstream of the kidney,2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched.Conclusion/Significance:The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma) and output (urine), it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It can also be anticipated that there will be more applications for proteomics in organ function research Urinary proteome includes proteins from plasma and proteins generated by the kidney. And the latter part has important significance on understanding kidney functions. Therefore, it is necessary to differentiate proteins generated by kidney with proteins from plasma in the urine. We believe that the kidney has a great contribution to the urinary proteome, however there is no direct evidence to confirm it, which is usually speculated based on the comparing of plasma and urine proteome data. Studying the contribution of kidney to urine proteome needs to exclude the interference of plasma, which is not simple. Isolated perfusion kidney technology is a traditional technique, which is widely used in drug metabolism and toxicology studies. It can preserve an intact structure and functions of the kidney in certain period of time. We applied proteomic techniques and isolated perfusion kidney techniques into the study of kidney functions by analyzing the urine proteome generated from the perfused kidney.We established the isolated kidney perfusion techniques and applied high performance liquid chromatography with mass spectrometry technology to analyze the urinary proteome generated from the perfused kidney. Two isolated perfusion kidney strategies were used in this study, supplimented with 02 or not.527 urinary proteins were identified with a false positive rate around 1%. Based on the enrichment analysis, we found that 103 proteins were enriched in both perfusate proteome under the oxygenated state comparing to urine proteome. This indicated that these proteins come from kidney. Comparing perfusate proteome with O2 suppliment with that without O2, we found 22 changed proteins. The expression of these proteins increased as perfusion goes on, suggesting that these proteins may be kidney damage related proteins and might be used as biomarkers of renal injury. In this study, the proteome of perfusion fluid in the blood vessels is also analyzed, and 210 proteins were identified. These proteins are mainly derived from plasma proteins, suggesting the existence of residual plasma before the start of perfuse. However, we found that the proteins identification between contaminated plasma proteins and normal plasma were not the same. We found that 11 middle and high abundance proteins in normal plasma proteins were not identified in perfusion fluid in the blood vessels. This indicated that the residual plasma proteins were very different from proteins in normal plasma. We feel that these proteins adhere to the renal vascular endothelial more tightly.In summary, we applied proteomics technology and classic isolated kidney perfusion techniques to the kidney physiological studies and obtained useful information.