Extracellular vesicles of podocytes impact intraglomerular signaling and parietal epithelial cell activation
Background
Extracellular vesicles (EVs) have the ability to impact basic pathological processes such as malignant, metabolic and autoimmune diseases through intercellular signaling. However, we lack a concise knowledge about their role in kidney health and disease. Our study aims to characterize the intraglomerular signalling propagated by medium-sized (mEVs) and small EVs (sEVs) shed by podocytes.
Methods
Using differential (ultra-)centrifugation we separated mEVs and sEVs from cell culture supernatants, kidney tissue and urine samples. Using Western Blot, immunofluorescence microscopy employing antibody stainings and pKH membrane dyes as well as image flow cytometry and cryo-electron microscopy (cryo-EM) we investigated the release dynamics of podocyte-specific EVs in different models of murine podocyte damage in vitro and in vivo. Potential signaling factors contained in EVs were analysed through proteomics. Live microscopy and cross-culture experiments were used to determine the effect of podocyte specific EVs on parietal epithelial cells (PECs).
Results
Upon podocyte damage, we detected a unified response in the form of an increase in EV release with a size-shift towards larger vesicles revealed in Cryo-EM. Depending on EV size and the initial insult, podocyte-specific EVs exerted different effects on the migratory behavior and proliferation of PECs while proteomics revealed limited differences in the EV proteome in different stress conditions. We shortlisted the first candidate proteins potentially propagating the effect on PECs. In vivo, decreased EV release by podocytes resulted in reduced PEC activation and limited recruitment of macrophages in a model of crescentic glomerulonephritis.
Conclusion
We present essential insights on podocyte-specific release of different sizes of EVs, their protein contents and functional implications in health and upon podocyte damage. Ongoing experiments focus on further elucidating the impact of podocyte-specific release in vivo and the impact of knocking-out identified EV candidate proteins.
Keywords
Extracellular Vesicles, Kidney Disease, Podocytes
Authors
Pamella Marie Sendon1,2, Alexander Pausch1,2, Annika Gathmann1,2, Kilian Teicher1,2, Inka Homeyer1,2, Moritz Lassé1,2, Milagros N. Wong1,2,3,4, Anja Obser1,2, Kelly Dryden6, Hetty Wong6, Nicola Wanner1,2, Maja T. Lindenmeyer1,2, Lawrence B. Holzman6, Markus Rinschen1,2, Franz L. Ricklefs7, Uta Erdbrügger8, Victor G. Puelles1,2,3,4, Tobias B. Huber1,2, Fabian Braun1,2,9 (Corresponding Author)