Inter-Laboratory Variability in the Madin-Darby Canine Kidney Cell ProteomeInter-Laboratory Variability in the Madin-Darby Canine Kidney Cell Proteome

Madin-Darby Canine Kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate trans-epithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits and as such laboratory-to-laboratory variability in drug permeability assessments are observed. Consequently, In Vitro-In Vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of eleven filter-grown parental or mock-transfected MDCK monolayers from eight different pharmaceutical laboratories using the Total Protein Approach (TPA) is provided. TPA enabled estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cell due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundance SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that Claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 proteins copy number and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

马-达二氏犬肾（Madin-Darby Canine Kidney, MDCK）细胞是研究上皮细胞功能的经典模型，应用范围广泛。因其内源性药物转运蛋白表达水平较低，该细胞系在转染后成为探究上皮跨膜渗透及药物转运蛋白活性的理想实验系统。MDCK细胞具有多样的表型特征，因此不同实验室间的药物渗透性评估结果存在显著差异。据此，采用渗透性及/或转运蛋白活性数据的体外-体内外推（In Vitro-In Vivo Extrapolation, IVIVE）方法需进行校准。本数据集提供了来自8家不同制药实验室的11株滤膜培养的亲本或空白转染MDCK单层细胞的蛋白质组定量数据，分析采用总蛋白质组学方法（Total Protein Approach, TPA）完成。TPA可用于估算单层细胞密度、体积等关键形态计量参数。整体而言，由于所需酶类表达水平较低，MDCK细胞对外源化合物的代谢易感性较为有限。其中，与外源性化合物活性相关的丰度最高的溶质转运蛋白为溶质载体家族16成员1（SLC16A1，即MCT1），而丰度最高的ATP结合盒转运蛋白则为ATP结合盒转运蛋白家族C成员4（ABCC4，即MRP4）。本研究数据佐证了已有研究结论：紧密连接蛋白-2（Claudin-2）的表达水平可能与紧密连接调控相关，进而影响上皮跨膜电阻。该独特数据库涵盖了超过8000种蛋白质的拷贝数与浓度数据，可用于深入评估各实验室所使用的对照单层细胞的特征。