Caffeine causes cell cycle arrest at G0/G1 and increases of ubiquitinated proteins, ATP and mitochondrial membrane potential in renal cells

Caffeine is a well-known purine alkaloid commonly found in coffee. Several lines of previous and recent evidence have shown that habitual coffee drinking is associated with lower risks for chronic kidney disease (CKD) and nephrolithiasis. However, cellular and molecular mechanisms underlying its renoprotective effects remain largely unknown due to a lack of knowledge on cellular adaptive response to caffeine. This study investigated cellular adaptive response of renal tubular cells to caffeine at the protein level. Cellular proteome of MDCK cells treated with caffeine at a physiologic concentration (100 μM) for 24 h was analyzed comparing with that of untreated cells by label-free quantitative proteomics. From a total of 936 proteins identified, comparative analysis revealed significant changes in levels of 148 proteins induced by caffeine. These significantly altered proteins were involved mainly in proteasome, ribosome, tricarboxylic acid (TCA) (or Krebs) cycle, DNA replication, spliceosome, biosynthesis of amino acid, carbon metabolism, nucleocytoplasmic transport, cell cycle, cytoplasmic translation, translation initiation, and mRNA metabolic process. Functional validation by various assays confirmed that caffeine decreased cell population at G2/M, increased cell population at G0/G1, increased level of ubiquitinated proteins, increased intracellular ATP and enhanced mitochondrial membrane potential in MDCK cells. These data may help unravelling molecular mechanisms underlying the biological effects of caffeine on renal tubular cells at cellular and protein levels.

咖啡因是一种公认的嘌呤生物碱，广泛存在于咖啡中。多项既往及近期研究证据表明，习惯性饮用咖啡与慢性肾脏病（CKD）和肾结石病的发病风险降低相关。但由于对咖啡因诱导的细胞适应性应答机制认知不足，其肾保护作用的细胞与分子基础仍不甚明确。本研究从蛋白质组层面探究了肾小管细胞对咖啡因的适应性应答反应：采用无标记定量蛋白质组学（label-free quantitative proteomics）技术，对经生理浓度（100 μM）咖啡因处理24小时的MDCK细胞与未处理对照组细胞的细胞蛋白质组进行了对比分析。在总共鉴定出的936种蛋白质中，对比分析显示咖啡因处理后有148种蛋白质的表达水平发生了显著变化。这些差异表达蛋白质主要富集于蛋白酶体、核糖体、三羧酸（TCA）循环（或称Krebs循环）、DNA复制、剪接体、氨基酸生物合成、碳代谢、核质运输、细胞周期、胞质翻译、翻译起始以及mRNA代谢过程等通路。通过多种实验手段开展的功能验证证实，咖啡因可使MDCK细胞的G2/M期细胞群占比降低，G0/G1期细胞群占比升高；同时可提升细胞内泛素化蛋白质水平、增加细胞内ATP含量并增强线粒体膜电位。本研究数据有助于从细胞及蛋白质组层面阐明咖啡因对肾小管细胞产生生物学效应的分子机制。