URINARY PROTEOMIC PROFILING FOR POTENTIAL BIOMARKERS OF EARLY NON-DIABETIC CHRONIC KIDNEY DISEASE

Proteinuria is established as a key indicator of chronic kidney disease (CKD) progression and severity. The kidneys excrete hundreds of different proteins. There is a paucity of data relating to significant urinary proteomic biomarkers in the specific context of early non-diabetic CKD. The non-diabetic population living with early stage CKD is a prevalent cohort at risk of adverse outcomes, and early disease identification followed by treatment initiation is important for disease optimization. In this pilot study, we conducted urinary proteomic profiling analyses to identify potential biomarkers of early CKD in non-diabetic individuals.

Our group conducted a cross-sectional study performing urinary proteomic profiling for 20 adult male non-diabetic patients who underwent kidney biopsy. This included a cohort of individuals with no known CKD (undergoing a nephrectomy) compared to a cohort undergoing kidney biopsy for investigation of potential CKD (Table 1). CKD status was determined by interstitial fibrosis and tubular atrophy (IFTA) grading upon evaluation of biopsy tissue. There were 10 patients with IFTA 0-10% (no CKD) and 10 patients with IFTA 10-25% (early CKD). The two groups were statistically similar in age and estimated glomerular filtration rate. In the early CKD group, there were 7 patients with micro or macroalbuminuria. Urine samples were collected immediately prior to kidney biopsy and prepared for data independent acquisition (DIA) liquid chromatography-mass spectrometry analysis. Database searches for DIA data and statistical differential abundances analysis of the searched data by RStudio (v4.4.2) were completed, followed by data filtering and normalization.

759 proteins were quantified through label-free proteomic methodologies, in which analysis of the human ortholog-mapped dataset through proteomic profiling detected 237 proteins with regulated expression where log₂ fold change is between 1 and –1 and false discovery rate is below 0.1. A volcano plot representing the findings from this analysis is shown, noting the most significantly upregulated and downregulated urinary proteins in early non-diabetic CKD (Fig 1A). A heatmap of the 20 most upregulated and 20 most downregulated proteins is presented (Fig 1B). Functional enrichment analysis validated the biological significance of these observed protein changes. Disruptions in lysosomal organisation, extracellular matrix–receptor interaction, PI3K-Akt signaling pathways, and epithelial remodeling processes were the most significantly regulated pathways in early non-diabetic CKD. Kinase radar mapping analysis indicated altered activity in tyrosine and serine/threonine kinases, which points to the disrupted cellular signaling patterns typically seen during CKD development. 

This study demonstrated that various urinary proteins differed between the early non-diabetic CKD and no CKD groups. The validity of utilizing urinary proteomics as a biomarker tool in early non-diabetic CKD is confirmed by reflecting similar molecular processes relating to the development of non-diabetic CKD as per findings from previously published studies. Further research is required to confirm the regulation of identified proteins in the kidneys of patients with early non-diabetic CKD and to assess the utility of urinary proteomics in monitoring treatment response.