Changes in male rat urinary protein profile during puberty: a pilot study

BMC Research Notes

Peter Mantle³  Jose Ignacio Monreal⁴  Judit Nagy⁵  Robin Wait²  Ariane Vettorazzi¹ 

[1] Current address: Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain;Current address: Kennedy Institute of Rheumatology, Nuffield Department of Rheumatology and Musculoskeletal Sciences, University of Oxford, London, UK;Centre for Environmental Policy, Imperial College London, London SW7 2AZ, UK;Clinical Chemistry Department, University Clinic of Navarra, University of Navarra, Pamplona, Spain;Imperial College London, Institute of Biomedical Engineering, London SW7 2AZ, UK

关键词: Androgen-dependent α2u-globulin;    Rat renal carcinogenesis;    Ochratoxin A;    Protein droplet nephropathy;    Lipocalins;    PhastGel electrophoresis;    Proteomics;   

Others  :  1142420
DOI  :  10.1186/1756-0500-6-232

 received in 2013-03-12, accepted in 2013-06-03,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Vettorazzi et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

【 参考文献 】

• [1]Beynon RJ, Hurst JL: Urinary proteins and the modulation of chemical scents in mice and rats. Peptides 2004, 25(9):1553-1563.
• [2]Cavaggioni A, Mucignat-Caretta C: Major urinary proteins, alpha(2U)-globulins and aphrodisin. Biochim Biophys Acta 2000, 1482(1–2):218-228.
• [3]Roberts SA, Simpson DM, Armstrong SD, Davidson AJ, Robertson DH, McLean L, Beynon RJ, Hurst JL: Darcin: a male pheromone that stimulates female memory and sexual attraction to an individual male’s odour. BMC Biol 2010, 8:75. BioMed Central Full Text
• [4]Cohen SM, Meek ME, Klaunig JE, Patton DE, Fenner-Crisp PA: The human relevance of information on carcinogenic modes of action: overview. Crit Rev Toxicol 2003, 33(6):581-589.
• [5]Roy AK, Neuhaus OW: Proof of the hepatic synthesis of a sex-dependent protein in the rat. Biochim Biophys Acta 1966, 127(1):82-87.
• [6]Borghoff SJ, Lehman-McKeeman LD, Short BG, Hard GC, Swenberg JA: Critique of R. Melnick's "An alternative hypothesis on the role of chemically induced protein droplet (alpha 2u-globulin) nephropathy in renal carcinogenesis". Regul Toxicol Pharmacol 1993, 18(2):357-364.
• [7]Melnick RL: Mechanistic data in scientific public health decisions. Regul Toxicol Pharmacol 1992, 16(2):109-110.
• [8]Melnick RL: An alternative hypothesis on the role of chemically induced protein droplet (alpha 2u-globulin) nephropathy in renal carcinogenesis. Regul Toxicol Pharmacol 1992, 16(2):111-125.
• [9]Melnick RL: Critique does not validate assumptions in the model on alpha 2u-globulin and renal carcinogenesis. Regul Toxicol Pharmacol 1993, 18(2):365-368.
• [10]Doi AM, Hill G, Seely J, Hailey JR, Kissling G, Bucher JR: alpha 2u-globulin nephropathy and renal tumors in national toxicology program studies. Toxicol Pathol 2007, 35(4):533-540.
• [11]Roy AK, Nath TS, Motwani NM, Chatterjee B: Age-dependent regulation of the polymorphic forms of alpha 2u-globulin. J Biol Chem 1983, 258(16):10123-10127.
• [12]Pahler A, Blumbach K, Herbst J, Dekant W: Quantitation of alpha2u-globulin in rat kidney cytosol by capillary electrophoresis. Anal Biochem 1999, 267(1):203-211.
• [13]Chatterjee B, Motwani NM, Roy AK: Synthesis and processing of the dimorphic forms of rat alpha 2u-globulin. Biochim Biophys Acta 1982, 698(1):22-28.
• [14]Laperche Y, Lynch KR, Dolan KP, Feigelson P: Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in the submaxillary gland. Cell 1983, 32(2):453-460.
• [15]Gubits RM, Lynch KR, Kulkarni AB, Dolan KP, Gresik EW, Hollander P, Feigelson P: Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland. J Biol Chem 1984, 259(20):12803-12809.
• [16]Rasonyi T, Schlatter J, Dietrich DR: The role of alpha2u-globulin in ochratoxin A induced renal toxicity and tumors in F344 rats. Toxicol Lett 1999, 104(1–2):83-92.
• [17]Mantle PG, Nagy JM: Binding of ochratoxin A to a urinary globulin: a new concept to account for gender difference in rat nephrocarcinogenic responses. Int J Mol Sci 2008, 9(5):719-735.
• [18]Chaudhuri BN, Kleywegt GJ, Bjorkman J, Lehman-McKeeman LD, Oliver JD, Jones TA: The structures of alpha 2u-globulin and its complex with a hyaline droplet inducer. Acta Crystallogr D: Biol Crystallogr 1999, 55(Pt 4):753-762.
• [19]Vicente-Vicente L, Ferreira L, Gonzalez-Buitrago JM, Lopez-Hernandez FJ, Lopez-Novoa JM, Morales AI: Increased urinary excretion of albumin, hemopexin, transferrin and VDBP correlates with chronic sensitization to gentamicin nephrotoxicity in rats. Toxicology 2013, 304:83-91.
• [20]Thongboonkerd V, Klein JB, Arthur JM: Proteomic identification of a large complement of rat urinary proteins. Nephron Exp Nephrol 2003, 95(2):e69-e78.
• [21]Kulkarni AB, Gubits RM, Feigelson P: Developmental and hormonal regulation of alpha 2u-globulin gene transcription. Proc Natl Acad Sci USA 1985, 82(9):2579-2582.
• [22]Elliott BM, Ramasamy R, Stonard MD, Spragg SP: Electrophoretic variants of alpha 2u-globulin in the livers of adult male rats: a possible polymorphism. Biochim Biophys Acta 1986, 870(1):135-140.
• [23]Wilkinson JM, Halley S, Towers PA: Comparison of male reproductive parameters in three rat strains: Dark Agouti. Sprague-Dawley and Wistar. Lab Anim 2000, 34(1):70-75.
• [24]Kostanski JW, Jiang G, Dani BA, Murty SB, Qiu W, Schrier B, Thanoo BC, DeLuca PP: Return to fertility after extended chemical castration with a GnRH antagonist. BMC Cancer 2001, 1:18. BioMed Central Full Text
• [25]Takeyoshi M, Mitoma H, Yamasaki K: Changes in serum alpha2u-globulin levels in castrated male rats treated with testosterone propionate in a Hershberger assay. J Appl Toxicol 2005, 25(2):176-178.
• [26]Mantle PG, Dobrota M, Gillett CE, Odell EW, Pinder SE: Oncological outcomes in rats given nephrocarcinogenic exposure to dietary ochratoxin a, followed by the tumour promoter sodium barbital for life: a pilot study. Toxins (Basel) 2010, 2(4):552-571.
• [27]Lehman-McKeeman LD, Caudill D: d-Limonene induced hyaline droplet nephropathy in alpha 2u-globulin transgenic mice. Fundam Appl Toxicol 1994, 23(4):562-568.
• [28]Boorman GA: Toxicology and Carcinogenesis studies of Ochratoxin A (CAS No. 303-47-9) in F344/N Rats (Gavage Studies). NC, USA: Research Triangle Park: Research Triangle region; 1989. [Technical Report 358]
• [29]Mantle PG, Kulinskaya E, Nestler S: Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A. Food Addit Contam 2005, 22(Supplement 1):58-64.
• [30]Wilson MJ, McMillin JM, Seal US, Ahmed K: Circadian variation of serum testosterone in the adult male rat with a late morning acrophase. Experientia 1976, 32(7):944-945.
• [31]Gatenbeck L, Eneroth P, Johansson B, Stromberg L: Plasma testosterone concentrations in male rats during short and long-term stress stimulation. Scand J Urol Nephrol 1987, 21(2):139-142.
• [32]Heywood LH: Testosterone levels in the male laboratory rat: variation under experimental conditions. Int J Androl 1980, 3(5):519-529.
• [33]Vettorazzi A, Gonzalez-Penas E, Troconiz IF, Arbillaga L, Corcuera LA, Gil AG, de Cerain AL: A different kinetic profile of ochratoxin A in mature male rats. Food Chem Toxicol 2009, 47(8):1921-1927.
• [34]Wait R, Miller I, Eberini I, Cairoli F, Veronesi C, Battocchio M, Gemeiner M, Gianazza E: Strategies for proteomics with incompletely characterized genomes: the proteome of Bos taurus serum. Electrophoresis 2002, 23(19):3418-3427.
• [35]Keller A, Nesvizhskii AI, Kolker E, Aebersold R: Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 2002, 74(20):5383-5392.
• [36]Nesvizhskii AI, Keller A, Kolker E, Aebersold R: A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 2003, 75(17):4646-4658.
• [37]Southan C, Cutler P, Birrell H, Connell J, Fantom KG, Sims M, Shaikh N, Schneider K: The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data. Proteomics 2002, 2(2):187-196.
• [38]Wait R, Gianazza E, Eberini I, Sironi L, Dunn MJ, Gemeiner M, Miller I: Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison. Electrophoresis 2001, 22(14):3043-3052.