Circulating extracellular vesicles of steroid sensitive nephrotic syndrome patients have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes

Am J Physiol Renal Physiol. 2021 Sep 27. doi: 10.1152/ajprenal.00097.2021. Online ahead of print.

Abstract

Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (SSNS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with SSNS in relapse and remission, 10 healthy controls and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry and western blotting. The major proteins from the plasma EVs were identified via mass spectrometry. A Gene Ontology classification analysis and integuinity pathway analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and the particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively express proteins which involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared to remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho p38 (p-p38) and decreased levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the nephrotic syndrome phenotype in podocytes in vitro.

Keywords: Nephrotic syndrome; RAC1; children; extracellular vesicles; phospho-p38.