Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells

Barros, Eric; Carotti, Valentina; Vries, Christine R S de; Witsel, Femke; Arntz, Onno J.; Loo, Fons A. J. van de; Carvajal, Cristián A.; Bindels, René J. M.; Hoenderop, Joost G. J.; Rigalli, Juan Pablo

doi:10.1096/fj.202002559r

Cited by 10 publications

(11 citation statements)

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“…EVs from the culture medium of wild type and Pkd1 -/- mDCT15 and mIMCD3 cells were isolated by ultracentrifugation as described previously ( 20 , 32 ). Briefly, cells were plated in 8 T-175 flasks (1.2x10 6 cells/flask for mDCT15 cells and 1.4x10 6 cells/flask for mIMCD3 cells).…”

Section: Methodsmentioning

confidence: 99%

“…Extracellular vesicles (EVs) represent a heterogenous family of lipid vesicles carrying several cellular components (e.g., lipids, nucleic acids, proteins), which play an essential role in cell-to-cell communication. In the kidney, EVs have been shown to mediate proximal-to-distal communication and, in this way, regulate electrolyte transport ( 20 ). EVs comprise microvesicles and exosomes, which arise from the outward budding of the plasma membrane as well as from the endosomal compartment, respectively ( 21 ).…”

Section: Introductionmentioning

confidence: 99%

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Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells

et al. 2022

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Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells

et al. 2022

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“…Finally, given the role of EVs in intercellular communication, EVs may mediate regulation between tubular segments (Figure 4). In vitro studies have demonstrated that proximal tubular EVs modify the activity of the epithelial sodium channel (ENaC) in the distal nephron [95], [96]. Similarly, EVs isolated from engineered proximal tubule cells were shown to improve the functional maturation of "tubuloids" (organoids showing a predominant tubular phenotype) including enhanced transport capacity of organic anion transporter 1 (OAT1) [97].…”

Section: Evs In Tubular Function and Diseasementioning

confidence: 99%

Extracellular Vesicles in Kidney Diseases: Moving Forward

Erdbrügger

Hoorn

et al. 2022

Kidney360

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Extracellular vesicles (EVs) are evolving as novel cell mediators, biomarkers and therapeutic targets in kidney health and disease. They are naturally deriving from cells within but also outside the kidney and carry cargo which mirrors the state of the parent cell. Thus, they are potentially more sensitive and disease specific as biomarkers and messengers in various kidney diseases. Beside their role as novel communicators within the nephron they likely communicate between different organs affected by various kidney diseases. Study of urinary EVs can help to fill current knowledge gaps in kidney diseases. However, separation and characterization are challenged by their heterogeneity in size, shape and cargo. Fortunately, more sensitive and direct EV measuring tools are in development. Many clinical syndromes in nephrology from acute to chronic kidney and glomerular to tubular diseases have been studied. Yet validation of biomarkers in larger cohorts is warranted and simpler tools are needed. Translation from in vitro to in vivo studies is also urgently needed. The therapeutic role of urinary EVs in kidney diseases has been studied extensively in rodent models of AKI. Based on the current exponential growth of EV research the field of EV diagnostics and therapeutics is moving forward.

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“…The copyright holder for this this version posted February 9, 2022. ; https://doi.org/10.1101/2022.02.08.479621 doi: bioRxiv preprint strategy in regulating kidney regeneration [24,25]. These vesicles are nanosized lipid bilayer structures that mediate cellular communication through the transfer of bioactive molecules, including proteins, nucleic acids and lipids [26]. Based on biogenesis, size and content, EVs can be classified as exosomes (30 -150 nm), microvesicles (100 nm -1 μm) and apoptotic bodies (50 nm -5 μm), presenting different biological functions based on their cargo [27].…”

Section: Introductionmentioning

confidence: 99%

“…For instance, urinary EVs isolated from rats and from conditioned medium (CM) of proximal tubule cells contain functionally active aquaporin 2 (AQP2), which can be transferred to other tubular epithelial cells [24,25]. EVs secreted by human kidney proximal tubule cells were also shown to modulate the regulation of purinergic signaling in collecting duct cells through increased extracellular ATP and downregulation of epithelial sodium channel (ENaC) [26]. Moreover, CM and isolated EVs from kidney epithelial cells were capable of inducing mesenchymal cells to acquire a more epithelial phenotype [28][29][30].…”

Section: Introductionmentioning

confidence: 99%

Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids

Lindoso

Yengej

Völlmy

et al. 2022

Preprint

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Advanced kidney in vitro models such as organoids or tubuloids still lack the intrinsic expression of various transport proteins needed for active secretory function. Extracellular vesicles (EVs), cell-derived structures that constitute the organ's microenvironment, are known to regulate various cellular processes, including kidney development and regeneration across the nephron. In this study, we propose a new application of renal tubular epithelial cell EVs as modulators for tubuloid functional maturation by increasing the levels of various differentiation markers such as organic anion transport 1 (OAT1), a protein involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters in tubuloids that resulted in improved cellular transport capacity. Next, a more in-depth proteomic data analysis demonstrated that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation process. Moreover, we demonstrated that EV-treated tubuloid-derived cells in a 3D tubular conformation as part of a bioartificial kidney can generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from renal tubular epithelial cells can phenotypically improve tubuloid maturation, thereby enhancing their potential as preclinical models and functional units in regenerative therapies.

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Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 10 publications

References 81 publications

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells

Extracellular Vesicles in Kidney Diseases: Moving Forward

Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids

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