Approval of the method of electrophoretic separation and identification of some urine proteins in rats with toxic nephropathy

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Abstract

The aim of the article. The aim was to test the method for determining certain protein markers in rat urine by electrophoretic separation followed by mass spectrometric identification for early diagnostic of nephrotoxicity. The objectives of the study included assessing the reproducibility of the protein separation method, comparing rats urine protein profiles normal and after gentamicin sulfate administration, as well as a biochemical and morphological study of kidney biopsy specimens.

Materials and methods. Gentamicin sulfate (GS) was administered intramuscularly to rats of both sexes at a dose of 60 mg/kg b.w. for consequence 5 days. Daily urine was collected in the background, on the 3rd, 7th and 14th day from the start of the administration of GS in the metabolic cages. We measured the concentrations of total protein and creatinine in urine samples, the level of lipids in the homogenates of the kidneys. Electrophoretic separation of urine proteins was performed in the Mini-PROTEAN Tetra Vertical Electrophoresis. The gels were stained, and the stained zones were excised and enzymatic hydrolysis of the proteins was performed. Spectra of tryptic peptides were recorded on an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker, Germany). Proteins were identified in the Biotools program by accessing the Mascot database (matrixscience.com).

Results. Assessment of reproducibility of the results of electrophoretic separation of rat urine proteins showed an acceptable value of the coefficient of variation (on average 8.03%) and the error of analysis (Δanalysis = 14.53%). It that normally in rats of males the alpha-2-uroglobulin precursor dominates in urine and in females immunoglobulin G light chains dominate was show. Against the background of the administration of GS, leukocytes in pathological concentrations appear in the urine in rats of both sexes; in males, the proteomic profile has a strong intraspecific dispersion, including due to sperm contamination; in females, well identified zones of albumin, alpha-1-antitrypsin, aminopeptidase M, beta-2-microglobulin, and transferrin was appear. A biochemical study of kidney homogenates an increase in total cholesterol (males) and triacylglycerides (females) revealed. Pathomorphological changes were similar in male and female rats in the form of fatty degeneration of the proximal tubule nephrothelium and leukocyte infiltration of interstitium and confirmed changes in the spectrum of urine proteins.

Conclusion. Based on the analysis of the experimental results it was found that the use of the technology of electrophoretic separation of proteins in PAGE under denaturing conditions followed by MALDI mass spectrometric identification (PAGE-MALDI-TOF/TOF) and densitometric determination of the percentage of protein fractions is applicable for the detection of nephrotoxicity. Due to gender differences in the protein spectrum it is preferable to examine the urine of female rats. Pathomorphological changes did not have gender differences.

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About the authors

Konstantin V. Sivak

Smorodintsev Research Institute of Influenza

Author for correspondence.
Email: kvsivak@gmail.com
ORCID iD: 0000-0003-4064-5033
SPIN-code: 7426-8322
Scopus Author ID: 35269910300

PhD in Biology, Head of the Department of Preclinical Trials

Russian Federation, St. Petersburg

Yana A. Zabrodskaya

Smorodintsev Research Institute of Influenza; Petersburg Nuclear Physics Institute named by B.P. Konstantinov of NRC “Kurchatov Institute”; Peter the Great Saint Petersburg Polytechnic University

Email: zabryaka@yandex.ru
ORCID iD: 0000-0003-2012-9461

PhD in Physic, Researcher of the Department of Molecular Biology of Viruses

Russian Federation, St. Petersburg

Olga A. Dobrovolskaya

Smorodintsev Research Institute of Influenza

Email: dobrovolskaya.od@gmail.com
ORCID iD: 0000-0002-0857-4733

Researcher of the Laboratory of Genetic Engineering and Recombinant Protein Expression

Russian Federation, St. Petersburg

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2. Fig. 1. The determined zones of the PAGE-electrophoregrams of rat urine proteins (M — males and F — females) and their molecular weights (kDa). MM is a molecular weight marker (Bio-Rad). * the alpha-2u-globulin precursor or major urinary protein characteristic of males is indicated; # — number of sample

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3. Fig. 2. Fragment of the mass spectrum of tryptic peptides (top), which reliably identified the protein alpha-2u globulin precursor (Rattus norvegicus), Score = 135 (threshold value 92). Ions and the corresponding numbers of amino acid residues are noted. The estimated sequences of the peptides, as well as their mass and error are presented below

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4. Fig. 3. Fragment of the mass spectrum of tryptic peptides by which the Ig kappa chain (Rattus norvegicus) protein was reliably identified, Score = 78 (threshold value 72). Ions and the corresponding numbers of amino acid residues are noted (а). The table shows the expected sequences of the peptides, as well as their mass and error. The fragmentation spectrum of ion 1004.52, reliably identified as Ig kappa chain (Rattus norvegicus), Score = 46 (threshold 42), sequence WKIDGTER (b)

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5. Fig. 4. Dynamics of leukocyturia in rats treated with gentamicin sulfate (a — males, b — female rats)

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6. Fig. 5. Pathomorphological changes in the kidney in male (а) and female (b) rats after exposure to gentamicin. * fatty degeneration of the nephroepithelium; # leukocyte infiltration of interstitium. Paraffin sections, hematoxylin and eosin staining, x400 magnification

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Copyright (c) 2019 Sivak K.V., Zabrodskaya Y.A., Dobrovolskaya O.A.

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