Hadpech Sudarat, Peerapen Paleerath, Chaiyarit Sakdithep, Sritippayawan Suchai, Thongboonkerd Visith
Medical Proteomics Unit, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Division of Nephrology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
J Adv Res. 2025 May 23. doi: 10.1016/j.jare.2025.05.040.
Various urinary parameters are used for determining kidney stone risk. However, almost all of the widely used lithogenic indices rely on urinary concentrations of small molecules/ions and pH.
To address whether urinary macromolecules (especially oxidatively modified proteins) also play a critical role in determining the stone risk.
Complexed urinary proteins (proteome) were purified from healthy individuals and calcium oxalate (CaOx) stone formers and performed various crystal assays and quantitative proteomics to compare them. Bioinformatic analyses were performed to gain additional insights, and the obtained data were verified by ELISA.
While the normal urinary proteome inhibited CaOx stone-forming mechanisms (i.e., crystallization, growth and aggregation), the stone formers' urinary proteome promoted all these CaOx crystal parameters. Descriptive proteomics by nanoLC-ESI-LTQ-Orbitrap-MS/MS analysis identified 203 and 381 proteins in the urine of healthy individuals and stone formers, respectively. Analyses of physicochemical properties revealed only molecular mass and isoelectric point that slightly increased in the stone formers' urine, whereas instability index, grand average of hydrophathicity (GRAVY) and amino acid composition were comparable. Interestingly, proportion of oxidatively modified proteins (particularly those with methionine oxidation, methionine dioxidation and cysteine trioxidation) markedly increased (∼2.5-fold) in the stone formers' urine. Quantitative proteomics revealed 89 increased and 56 decreased proteins in the stone formers' urine. The oxidized proteins had a greater proportion (>3-fold) in the increased proteins (77 %) compared with the decreased ones (23 %), whereas the non-oxidized proteins showed comparable proportions (54 % and 46 %, respectively). Functional enrichment analyses revealed a correlation between the increased proteins and oxidative stress biological processes and molecular functions. Finally, ELISA confirmed the significantly increased levels of oxidized proteins in the stone formers' urine compared with that of healthy individuals.
These data implicate that oxidatively modified proteome serves as a key pathogenic factor or risk for CaOx kidney stone formation.
多种尿液参数用于确定肾结石风险。然而，几乎所有广泛使用的结石形成指数都依赖于小分子/离子的尿液浓度和pH值。
探讨尿液大分子（尤其是氧化修饰蛋白）在确定结石风险中是否也起关键作用。
从健康个体和草酸钙（CaOx）结石患者中纯化复合尿液蛋白（蛋白质组），并进行各种晶体分析和定量蛋白质组学以进行比较。进行生物信息学分析以获得更多见解，并通过酶联免疫吸附测定（ELISA）验证所得数据。
正常尿液蛋白质组抑制CaOx结石形成机制（即结晶、生长和聚集），而结石患者的尿液蛋白质组促进所有这些CaOx晶体参数。通过纳升液相色谱-电喷雾电离-线性离子阱-轨道阱串联质谱（nanoLC-ESI-LTQ-Orbitrap-MS/MS）分析进行的描述性蛋白质组学分别在健康个体和结石患者的尿液中鉴定出203种和381种蛋白质。物理化学性质分析显示，仅结石患者尿液中的分子量和等电点略有增加，而不稳定指数、亲水性总平均值（GRAVY）和氨基酸组成相当。有趣的是，氧化修饰蛋白（特别是那些甲硫氨酸氧化、甲硫氨酸二氧化和半胱氨酸三氧化的蛋白）在结石患者尿液中的比例显著增加（约2.5倍）。定量蛋白质组学显示结石患者尿液中有89种蛋白质增加，56种蛋白质减少。与减少的蛋白质（23%）相比，氧化蛋白质在增加的蛋白质中所占比例更大（>3倍）（77%），而非氧化蛋白质的比例相当（分别为54%和46%）。功能富集分析揭示增加的蛋白质与氧化应激生物学过程和分子功能之间存在相关性。最后，ELISA证实结石患者尿液中氧化蛋白质的水平与健康个体相比显著增加。
这些数据表明氧化修饰的蛋白质组是CaOx肾结石形成的关键致病因素或风险因素。
