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. 2013 Jun 15;6:232.
doi: 10.1186/1756-0500-6-232.

Changes in Male Rat Urinary Protein Profile During Puberty: A Pilot Study

Free PMC article

Changes in Male Rat Urinary Protein Profile During Puberty: A Pilot Study

Ariane Vettorazzi et al. BMC Res Notes. .
Free PMC article

Abstract

Background: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication between adults, especially in the dark where reliance is on smell and taste. Crystal structure of the first-characterised lipocalin of male rats, α2u-globulin, has been determined and peptide sequences for others are available, but no study of occurrence during early puberty has been made. We have followed temporal occurrence in urine of juveniles (n = 3) for non-invasive pilot study by high resolution gradient mini-gel electrophoresis, tryptic digest of excised protein bands, and LC-MS/MS of digest to identify peptide fragments and assign to specific lipocalins. Study objective refers directly to external availability for social communication but also indirectly to indicate kinetics of circulating lipocalins to which some xenobiotics may bind and constitute determinants of renal disease.

Results: Mini-gels revealed greater lipocalin complexity than hitherto recognised, possibly reflecting post-translational modifications. Earliest patterns comprised rat urinary protein 1, already evident in Sprague-Dawley and Wistar strains at 36 and 52 days, respectively. By 44 and 57 days major rat protein (α2u-globulin) occurred as the progressively more dominant protein, though as two forms with different electrophoretic mobility, characterised by seven peptide sequences. No significant change in urinary testosterone had occurred in Wistars when major rat protein became evident, but testosterone surged by 107 days concomitant with the marked abundance of excreted lipocalins.

Conclusions: Qualitative temporal changes in the composition of excreted lipocalins early in puberty, and apparent increase in major urinary protein as two resolvable forms, should catalyse systematic non-invasive study of urinary lipocalin and testosterone dynamics from early age, to illuminate this aspect of laboratory rodent social physiology. It could also define the potential temporal onset of nephrotoxic ligand risk, applicable to young animals used as toxicological models.

Figures

Figure 1
Figure 1
Growth of groups of young male rats. The Wistar group (n = 3) started with weight-matched individuals which were fed the maintenance diet. Sprague-Dawley (SD) groups (similar littermates, n = 3 or 4) were fed either a maintenance diet (14% protein) or a high protein (21%) formulation. Error bars for the SD high protein diet group have been omitted to avoid visual confusion in the graphic; the high protein diet appeared to promote a lower growth rate, evident after about 60 days of age (~250 g).
Figure 2
Figure 2
A, Gel of Sprague-Dawley urines from increasing age (right to left) showing, in the 14-20 kDa region, transition from an initial pair of bands to a group of four in which two attributed to α2u-globulin become dominant (see also Figure 3). A band attributed to an albumin, by analogy with that in Figure 4B indicated (arrow), in the 45-66 kDa region apparently increased with age. B, Gel of Wistar urines from increasing age (right to left) showing a pattern analogous to that in Sprague-Dawley rats (Figure 4A). A band indicated by the arrow was identified from tryptic digest data as an albumin.
Figure 3
Figure 3
Coomassie-stained SDS-PAGE (Phastgel gradient 10-15%) of urinary proteins of three different Sprague-Dawley male rats fed with maintenance diet. Gel replicates of the same rats are shown (not for rat 1). The body weights of each animal at bands 1 and 3 first appearance have been highlighted with a square. The mean weight (± SD) of the animals when bands 1 and 3 appeared is also indicated. MW: molecular weight marker. α2u: α2u-globulin standard.
Figure 4
Figure 4
Coomassie-stained SDS-PAGE (Phastgel gradient 10-15%) of urinary proteins of two independent experiments performed with Sprague-Dawley male rats fed with high protein diet (n = 3 rats per experiment). The body weights of each animal at bands 1 and 3 first appearance have been highlighted with a square. The mean (± SD) animal weight at bands 1 and 3 first appearance is also indicated per experiment. MW: molecular weight marker. α2u: α2u globulin standard.
Figure 5
Figure 5
Coomassie-stained SDS-PAGE (Phastgel gradient 10-15%) of urinary proteins of three different Wistar male rats fed with maintenance diet. Gel replicates of the same rats are shown. The body weights of each animal at bands 1 and 3 first appearance have been highlighted with a square. The mean weight (± SD) of the animals when bands 1 and 3 appeared is also indicated. MW: molecular weight marker. α2u: α2u-globulin standard.
Figure 6
Figure 6
Coded illustration of changes in composition of ~ 18 kDa urinary proteins during early puberty in Sprague-Dawley male rats on high protein diet, represented in Figure 2A. identified proteins in this gel. Red, Major urinary protein; Dark blue, Rat urinary protein 1; Light blue, Rat urinary protein 2. Blank spaces indicate either no obvious band, or that no identification from tryptic digest analysis was obtained. Numbers refer to MASCOT score.
Figure 7
Figure 7
Amino acid sequence of Rat urinary proteins 1 and 2 with matching highlighted sequences recognised in tryptic digest peptide sequences [37][38].
Figure 8
Figure 8
Product ion spectrum of the triply protonated peptide of m/z 626.3 corresponding to the sequence VFMQHIDVLENSLGFK from MUP_RAT.
Figure 9
Figure 9
Amino acid sequence of rat major urinary protein (α2u-globulin) with matching highlighted sequences recognised in tryptic digest peptide sequences. Colouring of peptide sequences does not correlate with that in Figure 6.
Figure 10
Figure 10
Coded location of lipocalins in Wistar rat urine shown in Figure 2B in the right hand four tracks showing temporal transition from rat urinary protein 1 to dominant α2u-globulin (matching the standard in track 4). Numbers relate to MASCOT score.
Figure 11
Figure 11
Coomassie-stained SDS-PAGE (Phastgel gradient 10-15%) of urinary proteins of six different F 344 male adult rats (15 weeks old) fed with normal diet. α2u: α2u-globulin standard.

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