I. V. Dobrokhotov

Detection of Renal Tissue and Urinary Tract Proteins in the Human Urine after Space Flight

The urine protein composition samples of ten Russian cosmonauts (male, aged of 35 up to 51) performed long flight missions and varied from 169 up to 199 days on the International Space Station (ISS) were analyzed. As a control group, urine samples of six back-up cosmonauts were analyzed. We used proteomic techniques to obtain data and contemporary bioinformatics approaches to perform the analysis. From the total number of identified proteins (238) in our data set, 129 were associated with a known tissue origin. Preflight samples contained 92 tissue-specific proteins, samples obtained on Day 1 after landing had 90 such proteins, while Day 7 samples offered 95 tissue-specific proteins. Analysis showed that consistently present proteins in urine (under physiological conditions and after space flight) are cubilin, epidermal growth factor, kallikrein-1, kininogen-1, megalin, osteopontin, vitamin K-dependent protein Z, uromodulin. Variably present proteins consists of: Na(+)/K(+) ATPase subunit gamma, β-defensin-1, dipeptidyl peptidase 4, maltasa-glucoamilasa, cadherin-like protein, neutral endopeptidase and vascular cell adhesion protein 1. And only three renal proteins were related to the space flight factors. They were not found in the pre-flight samples and in the back-up cosmonaut urine, but were found in the urine samples after space flight: AFAM (afamin), AMPE (aminopeptidase A) and AQP2 (aquaporin-2). This data related with physiological readaptation of water-salt balance. The proteomic analysis of urine samples in different phases of space missions with bioinformation approach to protein identification provides new data relative to biomechemical mechanism of kidney functioning after space flight.

Permanent proteins in the urine of healthy humans during the Mars-500 experiment

Urinary proteins serve as indicators of various conditions in human normal physiology and disease pathology. Using mass spectrometry proteome analysis, the permanent constituent of the urine was examined in the Mars-500 experiment (520 days isolation of healthy volunteers in a terrestrial complex with an autonomous life support system). Seven permanent proteins with predominant distribution in the liver and blood plasma as well as extracellular localization were identified. Analysis of the overrepresentation of the molecular functions and biological processes based on Gene Ontology revealed that the functional association among these proteins was low. The results showed that the identified proteins may be independent markers of the various conditions and processes in healthy humans and that they can be used as standards in determination of the concentration of other proteins in the urine.

Variability of urine proteome in healthy humans during a 105-day isolation in a pressurized compartment

The protein composition (proteome) of the body fluids is rather flexible; it can change, responding to various factors of the external environment and changes in the internal environment. In order to study the variability of the proteome profile in healthy humans under the conditions of total control of vital rhythm, physical activity, and diet, urine samples were collected from subjects who had been selected according to special criteria and qualified as healthy by a special physical evaluation board. The subjects took part in an experiment with a 105-day-long isolation in a pressurized compartment, carried out by using an autonomous life-support system at the Institute of Biomedical Problems, Russian Academy of Sciences. The purification and concentration of proteins from the urine samples were carried out using a MB-HIC C8 magnetic bead set (Bruker Daltonics). The mass spectra have been obtained using an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics) in the positive lineal mode. One hundred and seventeen peaks were obtained for each urine sample; technological errors of the method have been studied. The high variability of the urine proteome profile (36 protein MC peaks on average) was shown in healthy humans under the conditions of isolation and controlled vital activity.

Reconstruction of associative protein networks connected with processes of sodium exchange regulation and sodium deposition in healthy volunteers based on urine proteome analysis

The study was conducted during the experiment with a 105-day isolation in an experimental complex. Urine samples were collected from six healthy volunteers. The physical activity, diurnal rhythm, temperature parameters, and levels of oxygen and carbon dioxide were controlled during the experiment. According to the program, food intake (electrolysis, water, calories, fat, carbohydrates, protein, vitamins, etc.) at each stage of the experiment was normalized to the body weight of each subject. All samples were analyzed using an LTQ FT MS ionic cyclotron resonance mass spectrometer with Fourier transform (Thermo) on the basis of the accurate mass and time (AMT) tag approach. Among more than 20 000 peptides, 690 proteotypical proteins were found. A total of 600 urine proteins were identified to be included in the database of healthy human urine proteins. For physiological interpretation of the proteome data, computer ANDCell and AND-Viso systems were used. Clustering of proteins and the application of these systems revealed proteins that were most closely associated with the regime of sodium intake and allowed building the network of their interactions.

Study of the urine proteome in healthy humans

A new branch of molecular biology, proteomics, has been developed recently due to a success in genomics and informatics. Proteomics is currently solving problems of the full proteome mapping of various biological substances, e.g., body fluids, cells, and tissues in the normal state and pathology; and also search for biomarkers of pathologies, including tumors. Data on the urine proteome have been analyzed in this review. Analysis of the methods used in proteomics, including sample preparation, study strategy, as well as published data on urine proteome over the past five years are presented.

Characteristics of age-dependent changes in urine proteome in healthy men

This paper studies the age dynamics of the proteomic profile of urine in healthy volunteers. The proteome composition was determined by chromatography-mass-spectrometry based on a nanostream highly efficient liquid chromatograph (Agilent 1100), and mass-spectra were obtained with a LTQ-FT hybrid mass-spectrometer. The urine samples obtained from 52 healthy men aged 19–54 years were found to contain 259 various proteins. According to the TiGER database, the tissue origin was established for 141 of them, and 715 biological processes in which they participate were identified. A significant positive correlation of the number (R = 0.566, p-value = 1.24E–05) and weight of proteins (R = 0.45; p-value = 8.17E–04) with age was found. We identified 23 proteins that are significantly more frequent in urine with increasing age of the subjects (p < 0.05) and only one protein, RGSL1, that is a regulator of signal transmission through receptors connected with G-protein (MW 125.69), which becomes less frequent with increasing age.

Constant urinary proteins in healthy humans in a 520-day isolation experiment

The aim of this study was the search of permanent proteins of the urinary proteome during a 520-day isolation experiment at the Institute of Biomedical Problems (IBMP) Ground-Based Test Facility in controlled conditions, using an autonomous life support system. The object of the study was urine sampled from 6 normal male subjects aged 25 to 37. The biological material samples (the second morning urine fractions) were collected for proteomic investigations against the background, on the 50th, 93rd, 124th, 153rd, 180th, 251st, 274th, 303rd, 330th, 371st, 400th, and 427th days of isolation, and on the 7th day after its completion. The samples were analyzed using chromatography–mass spectrometry, while the obtained results were analyzed using bioinformatics resources. The following seven permanent proteins were identified and observed during the entire period of urine investigations: epidermal growth factor, polymeric immunoglobulin receptor, plasma serine protease inhibitor, Alpha 1 microglobulin/bikunin precursor (AMBP), keratin (type II cytoskeletal 1), collagen alpha-1 (VI) chain, and serum albumin.

Direct proteomic profiling of human urine and blood serum in an experiment with five-day dry immersion

Changes in the proteome of urine and blood serum obtained from 14 healthy humans (aged 21–29 years), medically approved for the experiment with dry immersion, have been studied. Urine and serum samples were fractionated and concentrated using MB–WCX and MB–HIC magnetic particles, respectively. Direct mass spectrometry profiling by MSLDI–TOF was carried out using a ClinProt robotic device (Bruker Daltonics). As a result, on average, 143 proteins peaks in urine samples have been identified. The most plastic fraction of the urine proteome has been identified by a high variation coefficient (double of technical variation) in 23.7% of protein peaks. In blood serum, 175 peaks per sample have been identified, on average. Comparison of immersion mass spectra of the blood proteome and baseline revealed significant differences. It is concluded that the identified increase in peak areas for several protein fragments, including fragments of the C3 and C4 serum complement components and high-molecular-weight kinogen and fibrinogen, can be ascribed to human body adaptation to the experimental conditions.

Identification of proteins of cardiovascular system in healthy subjects’ urine during “dry” immersion

We analyzed the urine proteome in 14 healthy volunteers who were subjected to 5-day dry immersion using proteomic analysis methods and bioinformatics approach. We identified nine proteins related to the cardiovascular system. It was shown that 5-day dry immersion modifies the urine proteomic profile, indicating renal, endocrine, circulatory, and metabolic changes. Most of these changes are characterized by both a very rapid development and very rapid restoration within return to normal conditions.

Functioning of the kidneys and human body fluids during five-day immersion

Renal function and body composition, including fluids, were studied during space flight conditions imitation with five-day dry immersion in 14 apparently healthy men. Noninvasive research of water spaces of the body was performed using methods of bioimpedance analysis (BIA). It was shown that renal excretion of fluids increased and negative aqueous balance developed during dry immersion. Bioimpedance research revealed decreased volume of the total body and the extracellular fluids, and the amount of circulating plasma also decreased. Thus, under the conditions of immersion, hypohydration developed in the body. In the recovery period, these changes in the hydration level rapidly returned to the initial values. The lean body mass during immersion was reduced insignificantly and the amount of fat, in contrast, exceeded the baseline levels. Furthermore, the experiment showed technical impossibility to obtain reliable impedancemetry data under the dry immersion conditions.