EXPLORING THE PATHOGENESIS OF DIABETIC KIDNEY DISEASE PROGRESSION BY URINARY SINGLE-CELL RNA SEQUENCING

Identifying the risk of progression to end-stage kidney disease (ESKD) from early diabetic kidney disease (DKD) is important for optimization of therapy. The availability of single-cell RNA sequencing (scRNAseq) technology allows for measurement of gene expression at a single-cell level and provides detailed insights on subpopulations of cells in kidney disease & their roles in disease progression. There is potential in utilizing scRNAseq as a prognostic biomarker tool in early DKD. Urinary exfoliated kidney cells have been used as non-invasive biomarkers for DKD prognostication. Whilst the pathogenesis of DKD was previously explored using serum scRNAseq, few studies have done so based on urinary scRNAseq. There were no published studies which explored the utility of urinary scRNAseq in determining the pathogenesis of DKD progression over time. Our pilot study aimed to address this.

Eight adults with early DKD at study baseline (i.e. eGFR 60-90ml/min/1.73m2) were recruited. 4 individuals were classified in the ‘progressive’ DKD group and 4 in the ‘non-progressive’ DKD group (‘progression’ & ‘non-progression’ defined as per the KDIGO 2024 guideline definition). There were 2 males and 2 females in each group. 800-1000ml of urine were collected from each individual at baseline. Urinary exfoliated kidney cells in each sample were washed, collected, labelled with barcoding antibodies (BD Rhapsody Enhanced Cartridge Reagent Kit), and stained with viability dyes (Calcein AM & Draq7). The same number of viable cells from each sample were pooled and sorted by an INCYTO hemocytometer, after which the pooled samples were subjected to the BD Rhapsody Single Cell Analysis System followed by standard bioinformatics analyses.

Numerous kidney, hematopoietic and epithelial cell types were identified in significant amounts from urine of the 8 DKD patients (Fig 1). Proximal tubule cells (PTCs) were the predominant cell type amongst the urinary exfoliated kidney cells, accounting for >20% of the cell population on average. Individuals in the ‘progressive’ DKD group exfoliated significantly more PTCs compared to those in the ‘non-progressive’ DKD group (Fig 2). scRNAseq analysis of the urinary cells elucidated genes which were specifically expressed in the different cell types, as well as genes which were significantly regulated in these cells between the ‘progressive’ versus ‘non-progressive’ DKD groups. The top 10 genes expressed (Fig 3a) and significantly regulated between the ‘progressive’ versus ‘non-progressive’ DKD groups (Fig 3b) in urinary podocytes, Loop of Henle and PTCs are shown. Signaling pathways that are significantly regulated between the 2 groups were identified (Fig 4). These included mitochondrial & oxidative stress pathways, fibrosis pathways (TGFβ & SMAD signaling pathways), inflammation pathways (IFNγ, IL-6, Activator Protein-1 which modulate NF-κB activity), and metabolic pathways (PPAR signaling & toll-like receptors).

Our data strongly suggest that scRNAseq analysis of urinary exfoliated cells provides a non-invasive, unprecedented insight into the cellular processes which underly DKD progression. Further study is required to correlate the changes in molecular signatures over time in urinary exfoliated cells to treatment response and select individuals likely to benefit from subsequent therapeutic intervention.