Vettorazzi Ariane, Wait Robin, Nagy Judit, Monreal Jose Ignacio, Mantle Peter
BMC Res Notes. 2013 Jun 15;6:232. doi: 10.1186/1756-0500-6-232.
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.
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.
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.
雄激素依赖性蛋白（脂质运载蛋白）在雄性大鼠和小鼠的血液中循环，因其分子量小（约18 kDa），可自由进入肾小球滤液。部分蛋白在近端肾单位被重吸收，但部分会随尿液排出。几种有机分子可与这些蛋白结合，在蛋白被重吸收的情况下引发肾病，包括肾皮质恶性肿瘤。在尿液中，游离脂质运载蛋白及其配体在成年个体的社交交流中发挥着越来越重要的生物学作用，尤其是在依赖嗅觉和味觉的黑暗环境中。已确定雄性大鼠首个被表征的脂质运载蛋白α2u球蛋白的晶体结构，其他脂质运载蛋白的肽序列也已获得，但尚未有关于青春期早期出现情况的研究。我们通过高分辨率梯度迷你凝胶电泳、对切下的蛋白条带进行胰蛋白酶消化以及对消化产物进行液相色谱 - 串联质谱分析，追踪了幼年大鼠（n = 3）尿液中脂质运载蛋白的时间出现情况，以鉴定肽片段并确定其所属的特定脂质运载蛋白，进行非侵入性初步研究。研究目的直接指向社交交流中的外部可利用性，也间接表明了循环脂质运载蛋白的动力学，一些外源性物质可能与之结合并构成肾病的决定因素。
迷你凝胶显示脂质运载蛋白的复杂性比以往认识的更高，这可能反映了翻译后修饰。最早的模式包括大鼠尿蛋白1，在斯普拉格 - 道利和Wistar品系中分别在36天和52天时已很明显。到44天和57天时，主要大鼠蛋白（α2u球蛋白）出现并逐渐成为更主要的蛋白，不过以两种具有不同电泳迁移率的形式存在，由七个肽序列表征。当主要大鼠蛋白明显出现时，Wistar大鼠尿液中的睾酮没有显著变化，但睾酮在107天时激增，同时排泄的脂质运载蛋白显著增多。
青春期早期排泄的脂质运载蛋白组成的定性时间变化，以及主要尿蛋白以两种可分辨形式的明显增加，应促使从幼年开始对尿脂质运载蛋白和睾酮动态进行系统的非侵入性研究，以阐明实验啮齿动物社交生理学的这一方面。这也可以确定肾毒性配体风险的潜在时间起始，适用于用作毒理学模型的幼年动物。
