Effects of anesthetics pentobarbital sodium and chloral hydrate on urine proteome

Experiment animals

Rats were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science & Peking Union Medical College. The experiment was approved by Institute of Basic Medical Sciences Animal Ethics Committee, Peking Union Medical College (Animal Welfare Assurance Number: ACUC-A02-2013-015). All animals were kept with standard laboratory diet under controlled indoor temperature (22 ± 1 °C) and humidity (65–70%). The study was performed according to guidelines developed by Institutional Animal Care and Use Committee of Peking Union Medical College.

Rat models

Twelve male Sprague-Dawley rats (weight = 200 g) were divided into two groups. One group was anesthetized by intraperitoneal injection of pentobarbital sodium (n = 6, 50 mg/kg), and the other group was by chloral hydrate (n = 6, 300 mg/kg). Urine samples before anesthesia were collected as control (about 2 mL). Anesthesia affected urine was collected for three hours during anesthesia (about 2 mL). Anesthesia was supported for four hours and the activities of the anesthetics were detected by measuring muscle relaxation. The urinary protein and creatinine concentration were measured at the Peking Union Medical College Hospital. The self-controlled experiment was conducted in two phases: for the discovery phase, differential protein identification was performed in three independent rats each group; for the validation phase, samples were obtained from the three remaining rats.

Sample preparation

Urine was centrifuged at 2,000 g for 30 min immediately after collection. Three volumes of acetone were added after removing the pellets and precipitated at 4 °C. Then, lysis buffer (8 M urea, 2 M thiourea, 25 mM dithiothreitol and 50 mM Tris) was used to re-dissolve the pellets. Proteins were digested by trypsin (Trypsin Gold, Mass Spec Grade, Promega, Fitchburg, Wisconsin, USA) using filter-aided sample preparation methods (Wisniewski et al., 2009). Briefly, after proteins were loaded on the filter unit (Pall, Port Washington, New York, USA), UA buffer (8 M urea in 0.1 M Tris–HCl, pH 8.5) and 50 mM NH₄HCO₃ was added. Proteins were denatured at 50 °C for 1 h by the addition of 20 mM dithiothreitol and alkylated in the dark for 40 min by the addition of 50 mM iodoacetamide. Proteins were digested by trypsin (1:50) at 37 °C overnight. The digested peptides were desalted using Oasis HLB cartridges (Waters, Milford, Massachusetts, USA).

LC-MS/MS analysis

The digested peptides were dissolved in 0.1% formic acid and loaded on a Michrom Peptide Captrap column (MW 0.5–50 kD, 0.5 × 2 mm; Michrom Bioresources, Auburn, California, USA). The eluent was transferred to a reversed-phase microcapillary column (0.1 × 150 mm, packed with Magic C18, 3 µm, 200 Å; Michrom Bioresources, Auburn, California, USA) by an Agilent 1200 HPLC system. Peptides were analyzed by a LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The LTQ-OrbitrapVelos was operated in data-dependent acquisition mode. Survey MS scans were acquired in the Orbitrap using a 300–2,000 m/z range with the resolution set to 60,000. The 20 most intense ions per survey scan were selected for collision-induced dissociation fragmentation, and the resulting fragments were analyzed in the LTQ. Dynamic exclusion was employed with a 60 s window to prevent the repetitive selection of the same peptide.

Data analysis

All MS/MS spectra were analyzed using the Mascot search engine (version 2.4.1, Matrix Science, London, UK), and proteins were identified by searching against the Swissprot_ 2013_ 07 database (taxonomy: Rattus; containing 9,354 sequences). The parameters were set as follows: carbamidomethylation of cysteines was set as a fixed modification, and oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Trypsin was set as the digestion enzyme, and two missed trypsin cleavage sites were allowed. The precursor mass tolerance was set to 10 ppm, and the fragment mass tolerance was set to 0.5 Da. Peptide and protein identifications were validated by Scaffold (version 4.0.1, Proteome Software Inc., Portland, Oregon, USA). Peptide identifications were accepted if they could be detected with ≥95.0% probability by the Scaffold local false discovery rate algorithm, and protein identifications were accepted if they could be detected with ≥99.0% probability and contained at least 2 identified peptides (Nesvizhskii et al., 2003). The acquired raw files were loaded to Progenesis LC-MS/MS software (version 4.1, Nonlinear, Newcastle upon Tyne, UK), and label-free quantification was conducted as previously described (Hauck et al., 2010). For quantification, all peptides (with Mascot score >30 and p < 0.01) of an identified protein were included.

Western blot analysis

Urine proteins were prepared as described in Materials and Methods; 20 µg of each sample were separated by 10% SDS-PAGE and transferred to PVDF membranes (Whatman, Maidstone, UK) in transfer buffer (10% methanol, 25 mM Tris base, 192 mM glycine, PH 8.0). Membranes were incubated overnight at 4 °C with primary antibody against alpha-1-antiproteinase (Species reactivity: rat; dilution 1:1000; ab106582, Abcam, Cambridge, UK) or transferrin (Species reactivity: rat; dilution 1:10000; ab82411, Abcam, Cambridge, UK). The membranes were then washed and incubated with peroxidase-conjugated anti-chicken and anti-rabbit IgG (1:10000; zSgb-Bio, Beijing, China) at room temperature for 2 h and proteins were visualized using enhanced chemiluminescence (ECL) reagents. Intensity of each protein band was quantified using Image J analysis software (National Institutes of Health, Bethesda, Maryland, USA).

Urine protein-to-creatinine ratios were increased with either pentobarbital sodium or chloral hydrate anesthesia

The urinary creatinine concentrations were first reduced with anesthesia than in normal condition (in pentobarbital sodium group, creatinine decreased to 0.9-fold and in chloral hydrate group, also decreased to about 0.9-fold; Table S1). When compared with changes of creatinine concentration, changes of urinary proteins concentrations were much more significant. As a result, the urine protein-to-creatinine values with anesthesia increased 2.4-fold (in pentobarbital sodium group, 107.1 ± 21.1 versus 259.1 ± 81.1 mg/mmol, n = 6, P value <0.05) and 2.1-fold (in chloral hydrate group, 107.5 ± 16.5 versus 220.8 ± 79.0 mg/mmol, n = 6, P value <0.05). With pentobarbital sodium and chloral hydrate anesthesia, the urine protein-to-creatinine ratio of all rats were significantly increased in both groups, which were consistent with the values that have been reported in previous studies (Mercatello et al., 1991; Vaden et al., 2010). Figure 1 showed the different effects of each anesthetic on rat urine protein concentration.

Urinary proteome changes with anesthesia

Twelve urine samples before and after anesthesia from 6 rats (n = 3 in each group) in the pentobarbital sodium and chloral hydrate group were profiled by LC-MS/MS. In the pentobarbital sodium and chloral hydrate group, label-free quantitation data of proteins identified were listed in Table S2.

In the pentobarbital sodium group, the relative abundance of 22 proteins changed according to the following criteria: fold change >2 for each rat and p value <0.05 (data were analyzed by t test); 6 proteins had increased relative abundance and 16 proteins had decreased relative abundance. In the chloral hydrate group, the relative abundance of 23 proteins changed: 9 proteins had increased relative abundance and 14 proteins had decreased relative abundance. Among the proteins with altered relative abundance, 7 had the same trends in all six rats that were anesthetized with either pentobarbital sodium or chloral hydrate; one protein increased relative abundance and six proteins had decreased relative abundance (Table 1).

Verification of affected proteins by Western blot

Two changed proteins were selected to be validated in six more rats for the following reasons: (1) were identified previously in biomarker discovery; (2) were at relatively high abundance and easier to be detected in western blot; (3) had commercially available antibodies. In the pentobarbital sodium group, the levels of transferrin were analyzed and in the chloral hydrate group, the levels of alpha-1-antiproteinase were analyzed. With anesthesia, transferrin and alpha-1-antiproteinase expression levels were upregulated in three more rats (Fig. 2), consistent with the MS quantification data.

Comparison with previous studies

In the pentobarbital sodium anesthesia group, the relative abundance of 22 proteins were changed. Compared with the Urinary Protein Biomarkers Database (Shao et al., 2011), 11 out of 22 proteins were considered as candidate biomarkers, such as uromodulin and serotransferrin. Among these proteins, some exhibited the opposite trend. For example, the relative abundance of aminopeptidase N was increased in septic rats with acute renal failure (Wang et al., 2008), whereas their relative abundance decreased with pentobarbital sodium anesthesia. In the chloral hydrate anesthesia group, the relative abundance of 23 proteins changed and chloral hydrate had a relatively different impact on the urine proteome. Compared with the Urinary Protein Biomarkers Database, 8 out of 23 proteins were considered as candidate biomarkers, such as uromodulin and parvalbumin alpha. However, the relative abundance of clusterin was increased under conditions of gentamicin administration (Takahashi, 1995), but it decreased with chloral hydrate anesthesia.

Rat proteins were converted to their human orthologs using the Ensembl homolog database as reported (Jia et al., 2013). Stable proteins in healthy human urine are more likely to become candidate biomarkers when changed (Sun et al., 2009). In this study, differently expressed proteins with anesthesia were compared with the human core urinary proteome, which were considered stable and relatively high in abundance. Data from the “stable urinary proteome,” which represented the common and most easily identifiable proteins from urine, were determined by Mann (Nagaraj & Mann, 2011). The dataset contains 587 proteins that were identified in each of the 7 participant’s urinary proteomes on three consecutive days. The changes of high abundant proteins are likely to be real, as it is unlikely to be caused by data dependent sampling of low abundant peptides by MS. 6 out of 22 proteins (Uromodulin, Kallikrein-1, Serotransferrin, Serum albumin, Gamma-glutamyl hydrolase, Neutral and basic amino acid transport protein rBAT) affected by pentobarbital sodium had stable relative abundance in healthy human urine. Twelve out of 23 proteins (Uromodulin, Kallikrein-1, Superoxide dismutase (Cu–Zn), Putative uncharacterized protein, Parvalbumin alpha, Corticosteroid-binding globulin, E-cadherin, Alpha/beta hydrolase domain-containing protein 14B, Retinoid-inducible serine carboxypeptidase, Apolipoprotein E, Na(+)/H(+) exchange regulatory cofactor NHE-RF3, Nucleobindin-1) affected by chloral hydrate were stable. Two proteins (Uromodulin, Kallikrein-1) were shared by both groups (Table 2 listed the changed proteins which exist in human core urinary proteins).