基于单细胞测序及蛋白质组数据分析尿酸转运蛋白在肾脏的精细表达定位

Analysis of precision expression localization of uric acid transporters in kidney tissues based on single cell RNA sequencing and proteome data

目的利用单细胞测序、微量蛋白质组学及免疫组化三部分数据深入分析肾脏尿酸转运蛋白的细胞分布及表达丰度。方法利用2个成人健康肾脏的单细胞测序数据集(GSE131685)分析12个肾脏尿酸转运基因的细胞定位及表达丰度,进一步利用激光微切割联合质谱检测大鼠肾小管14个区段蛋白质数据集12个蛋白的定位和丰度,最后利用ProteinAtlas数据库检索上述蛋白在肾脏中的免疫组化染色结果。结果单细胞测序数据集GSE131685中,12个尿酸转运蛋白集中表达于近端小管细胞,而在KIT数据集中,除了ABCG2、SLC22A12和SLC22A13外,其余都集中表达于近端肾小管S1,S2和S3段细胞。KEAT蛋白数据集的分析结果与KIT数据结果类似:ABCG2,SLC17A3,SLC22A12,SLC22A6和SLC22A8在近端小管S1,S2和S3段有显著的表达,尤其是SLC22A6仅富集在S2段肾小管,而ABCG2在S3段有较强的富集,其它的蛋白表达丰度相对较低。ProteinAtlas的免疫组化数据显示出与单细胞测序及蛋白质组的结果有较大的差异:ABCC2、SLC17A4、SLC22A6、SLC22A11、SLC22A12、SLC22A13及SLC2A9显示在近端小管比较特异的表达定位,其中ABCC2、SLC22A6、SLC22A11和SLC22A13信号较强。ABCC4、ABCG2和SLC16A9的表达丰度相对较低,特异性较差。结论尿酸转运蛋白在肾脏主要集中表达于小管的S1,S2和S3段细胞,蛋白质组数据和单细胞测序数据比较接近。单细胞测序及微切割的微量蛋白组学是研究蛋白精细定位的良好工具。本研究为今后研究尿酸转运蛋白的功能及研发降尿酸药物提供基础依据。

Objective To analyze the cell distribution and expression abundance of uric acid transporters in kidney by single cell sequencing,microproteomics,and immunohistochemical data.MethodsThe cell localization and expression abundances of 12 uric acid transporter genes were analyzed by single cell sequencing data sets(GSE131685)of 2 healthy adult kidneys.Furtherly,laser microdissection combined with mass spectrometry was used to detect the localization and abundance of 12 proteins in the protein data set of 14 segments of rat renal tubules.Finally,the immunohistochemical staining results of the above proteins in the kidney were retrieved in the ProteinAtlas database.Results In the single-cell sequencing dataset GSE131685,12 uric acid transporters were concentratedly expressed in proximal tubule cells.In the KIT dataset,except for uric acid transporters of ABCG2、SLC22A12,and SLC22A13,the rest uric acid transporters were concentratedly expressed in proximal tubular cells of S1,S2,and S3 segments.The analysis results of the KEAT protein dataset were similar to those of the KIT dataset:uric acid transporters of ABCG2,SLC17A3,SLC22A12,SLC22A6,and SLC22A8 were significantly expressed in proximal tubule cells of S1,S2,and S3 segments,while uric acid transporter of SLC22A6 was especially enriched in the renal tubule cells of S2 segment,and uric acid transporter of ABCG2 was strongly enriched in the proximal tubule cells of S3 segment.The other proteins showed relatively lower abundance and specificity.Immunohistochemical data of the ProteinAtlas showed results significantly different from those of the single-cell sequencing and proteome:uric acid transporters of ABCC2,SLC17A4,SLC22A6,SLC22A11,SLC22A12,SLC22A13,and SLC2A9 showed a specific localization in proximal tubules,among which the uric acid transporters of ABCC2,SLC22A6,SLC22A11,and SLC22A13 has stronger abundance,while ABCC4,ABCG2,and SLC16A9 showed relatively lower expression abundance and specificity.ConclusionUric acid transporters were mainly expressed in the renal tubule cells of S1,S2,and S3 segments.The results of proteome data and single-cell sequencing data were similar.The microproteomics of single-cell sequencing and laser capture microdissection is an effective tool to study precise localization of proteins.This study provided a basis for future research on the function of uric acid transporters and the development of uric acid-lowering drugs.