Introduction: The kidney is a complicated organ constituted with nephrons. The nephron has several parts, which have different functions and are damaged by various injuries. To know the whole kidney damages, kidney biopsy tissues are examined histologically. We aimed to select urine biomarkers for the kidney nephron part damages by proteomics and immunoassay.

Methods: Proteins were extracted from urine and nephron tissues, which were laser-micro-dissected from formalin-fixed paraffin-embedded kidney. The extracted proteins were digested into peptides with trypsin and identified using LC-MS in a Data Dependent Acquisition (DDA) manner. Amounts of the proteins selected as kidney nephron/site-unique ones were measured in urine samples obtained from healthy volunteers and patients with various kidney diseases (IgA nephropathy, diabetic kidney diseases, nephrosclerosis, and other chronic kidney disease) by surface plasmon resonance (SPR) using antibodies. Then, quantitative a Data Independent Acquisition (DIA) proteomics was also applied for quantification.

Results: By LC-MS, ten proteins were selected as nephron/site-unique ones in the human kidney. SPR assay using antibodies against these proteins demonstrated their up-regulation in urine from kidney disease patients, indicating that they were candidates of urine biomarkers for kidney-site specific injuries.

Discussion: We performed DDA analysis of urine proteins from kidney disease patients using LC-MS, and succeeded to select candidate proteins as biomarkers for kidney-site specific injuries. However, DIA quantitative proteomics with recently advanced MS technology was useful to quantify many urinary proteins accurately for biomarker discovery and validation.