The Authors Reply

We thank Gleadle et al. for their interest in our article and comments. We agree that the isolation method for urine exosomes is a very important issue. In our study, we chose differential centrifugation and ultracentrifugation, which have been the most frequently used approach to isolate urinary extracellular vesicles.1,2 Size distribution measurements, such as nanoparticle tracking analysis were recommended by the International Society for Extracellular Vesicle as the minimal requirements for characterization of extracellular vesicles.3 Thus, nanoparticle tracking analysis was applied in our study to measure urinary exosomes, and it revealed increased urinary particles in patients with IgA nephropathy. To confirm the finding and to exclude the influence of other nonextracellular vesicle particles, we also measured the exosome markers Alix, CD9, and CD63 by Western blotting analysis. Importantly, we confirmed the results by additional experiments with the EXOCET Exosome Quantitation Kit (System Biosciences, CA), which measured the esterase activity known to be within exosomes. The finding of Gleadle et al. that albumin can form exosome-like particles is important and interesting,4 and confirms the importance of combining techniques for characterizing urinary extracellular vesicles, rather than a single approach like nanoparticle tracking analysis. The choice of a specific isolation technique for exosomes should probably also depend on the aim of the study. The focus of our study was to explore the functional cargo of exosome CCL2 mRNA in mediating the crosstalk between tubular cells and macrophages, and abundant protein did not seem to influence RNA profiling of this cargo of extracellular vesicles.5 Nevertheless, we agree that when evaluating exosomes in proteinuric urine, the potential influence of protein on the exosome purification should be considered.