An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles.

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.

Urinary Proteomics as a Tool to Identify Kidney Responders to Dipeptidyl Peptidase-4 Inhibition: A Hypothesis-Generating Analysis from the MARLINA-T2D Trial.

PURPOSE: Chronic kidney disease (CKD) is a serious complication of hyperglycemia and treatment options to slow its progression are scarce. Dipeptidyl peptidase-4 (DPP-4) inhibitors are common glucose-lowering drugs in type 2 diabetes (T2D). Among these, linagliptin has been suggested to exert kidney protective effects. It is investigated whether an effect of linagliptin on kidney function could be unmasked by characterizing the urinary proteome profile (UPP) in albuminuric T2D individuals. EXPERIMENTAL DESIGN: Participants of the MARLINA-T2D trial (NCT01792518) are randomized 1:1 to receive either linagliptin 5 mg or placebo for 24 weeks. A previously developed proteome-based classifier, CKD273, is assessed. RESULTS: Results confirm a significant correlation between CKD273 and clinical kidney parameters as well as with eGFR decline. Patient stratification using CKD273 at baseline, show a trend toward attenuation of renal function loss in high CKD-risk patients treated with linagliptin. Moreover, characterized are linagliptin affected peptides of which the majority contained a DPP-4 target sequence. CONCLUSIONS AND CLINICAL RELEVANCE: CKD273 is a promising tool for identifying patients at high risk for CKD progression and may unmask a potential of linagliptin to slow progressive kidney function loss in high CKD-risk patients. UPP characterization reveals a significant impact of linagliptin on urinary peptides.