LAUSR

The physiological and related body fluids, urine, plasma and hemodialysis (HD) fluid spent dialysate (a mixture of technical HD fluid and patient´s ultrafiltrate) are rich biological sources of information especially in the context of kidney diseases. Of particular relevance are proteins and peptides, as they reflect essentially every biological process. Urine is produced as a result of glomerular plasma filtration and subsequent tubular reabsorption in the kidney. In analogy, HD spent dialysate is a result of ultrafiltration by an artificial kidney (dialysis membrane), but lacking tubular reabsorption and thereby accumulating in huge amounts of > 100 L per session comparable to primary urine. A recent study suggested that tubular reabsorption may be selective for specific proteins, and this process may represent a major difference between “natural” and artificial kidney. In this study, we aim at gaining insight into the composition of the peptidome/proteome in these body fluids of renal significance in a comprehensive analysis, in order to shed light on the relevant pathophysiological processes that take place in kidney, which may help developing better strategy in advanced-stage chronic kidney disease and detoxification in renal replacement therapies (RRT). We collected 15 plasma, 15 urine and 13 HD filtrate (spent dialysate during the first 30min of RRT) from age and sex matched subjects with stage 4 or above chronic kidney disease. Peptide identification and quantification were performed with capillary-electrophoresis coupled mass spectrometry and tandem mass spectrometry (CE-MS). The abundance, overlap and differences of the peptidome/proteome of the three body fluids were evaluated. We were able to detect 6281 urinary peptides, 1750 plasma peptides and 1728 peptides from spent dialysate. Of these we could identify 1764, 452 and 374 sequenced peptides, respectively Among them, there are 318 peptides detectable in both urine and spent dialysate, 307 detectable in both plasma and urine and only 191 detectable in both plasma and spent dialysate. Among the most abundant peptides in plasma were Thymosine-ß4 and fragments from hemoglobin, fibrinogen and collagen. In dialysate, the most abundant proteins detected in these patients were ß-2-microglobuline, Thymosine-ß4, and fragments from fibrinogen, while in urine fragments derived from collagen, albumin, and from alpha-1-antitrypsin were of the highest abundance. When investigating proteins/peptides found in at least 2 body fluids, a strong correlation in peptide abundance between urine and spent dialysate (P=4.2e-27, Rho=0.56), and a moderately strong correlation between HD fluid and plasma (P=3.7e-5, Rho=0.29) were detected. However, there is no significant correlation between peptide abundance in urine and plasma (P=0.22, Rho=0.07). Collagen peptides are the most abundant peptides in all three body fluids. This dataset will serve as a valid basis to define the protein and peptide contents in blood, urine, and spent dialysate. The data show, as expected, substantial similarity between plasma and spent dialysate, but, surprisingly, very little similarity between urine and plasma. This further supports the hypothesis that tubular reabsorption may be selective for specific proteins and peptides, possibly excluding collagen-derived peptides. A more detailed investigation into the process and relevance of tubular reabsorption appears warranted in the light of the data. The fact that the samples investigated in this study are derived from advanced stage CKD patients must be taken into account, significant differences exist in comparison to urine produced by healthy kidney. The identification of molecular differences between urine and dialysate spent may present an opportunity to design future “biologically adapted” removal devices.