Flow-cytometric quantification of urine kidney epithelial cells specifically reflects tubular damage in acute kidney diseases
Despite tubular injury being one of the main mechanisms driving acute kidney injury (AKI), clinicians still have a limited diagnostic repertoire to precisely monitor damage to tubular epithelial cells (TEC). In our previous work we used single cell sequencing to identify TEC subsets as main component of the urine signature in AKI. The aim of this study was to establish TEC as a clinical marker for tubular damage. In total 243  patients were analyzed. For sequencing, we collected eight urine samples of  patients with AKI and glomerular disease. By aligning urinary single-cell transcriptomes and TEC-surface proteins using Cellular Indexing of Transcriptome and Epitope Sequencing (CITE-Seq), we developed a protocol for flow cytometric quantification of CD10/CD13+ proximal and CD227/CD326+ distal TEC in urine. The marker combinations were confirmed in kidney biopsies. We validated our approach across four cohorts totaling 235  patients, consisting of patients with AKI (n=63), SARS-CoV-2 infection (n=47), ANCA-associated vasculitis (AAV) with active disease and stable remission (n=110) and healthy controls (n=15). Our findings demonstrate that CD10/CD13 and CD227/CD326 adequately identify proximal and distal urinary TEC, respectively. Distal urinary TEC counts correlate with the severity of AKI based on KDIGO stage and acute  eGFR loss in two separate cohorts and can successfully discriminate AKI from healthy controls as well as glomerular disease. We propose urinary CD227/CD326+ TEC counts as a specific, non-invasive marker for tubular injury in AKI. Our protocol provides the basis for a deeper phenotypic analysis of urinary TECs Overall design: Single-cell RNAseq of flow cytometry sorted viable cells from 8  human urine specimen of patients with acute kidney injury (n=6), membranous nephropathy  (n=1) and active lupus nephritis class IV (n=1)