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

ABSTRACT: The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on organic anion transporter 1 (OAT1), one of the most important proteins 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, resulting in improved OAT1 transport capacity. Next, a more in-depth proteomic data analysis showed that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation. Moreover, we demonstrated that the combination of EVs and tubuloid-derived cells can be used as part of a bioartificial kidney to generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from kidney tubular epithelial cells can phenotypically improve in vitro tubuloid maturation, thereby enhancing their potential as functional units in regenerative or renal replacement therapies.

SUBMITTER: Lindoso RS

PROVIDER: S-EPMC9284832 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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

Lindoso Rafael Soares RS Yousef Yengej Fjodor A FA Voellmy Franziska F Altelaar Maarten M Mancheño Juncosa Estela E Tsikari Theano T Ammerlaan Carola M E CME Van Balkom Bas W M BWM Rookmaaker Maarten B MB Verhaar Marianne C MC Masereeuw Rosalinde R

Journal of nanobiotechnology 20220715 1

The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on o ...[more]

PMID: 35841001

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Project description:Hypoxia is the key pathobiological trigger of tubular oxidative stress and cell death that drives the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). The mitochondrial-rich proximal tubular epithelial cells (PTEC) are uniquely sensitive to hypoxia and thus, are pivotal in propagating the sustained tubular loss of AKI-to-CKD transition. Here, we examined the role of PTEC-derived small extracellular vesicles (sEV) in propagating the 'wave of tubular death'. Ex vivo patient-derived PTEC were cultured under normoxia (21 % O2) and hypoxia (1 % O2) on Transwell inserts for isolation and analysis of sEV secreted from apical versus basolateral PTEC surfaces. Increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of hypoxic-apical sEV cargo identified distinct miRNAs linked with cellular injury pathways. In functional assays, hypoxic-apical sEV selectively induced ferroptotic cell death (↓glutathione peroxidase-4, ↑lipid peroxidation) in autologous PTEC compared with normoxic-apical sEV. The addition of ferroptosis inhibitors, ferrostatin-1 and baicalein, attenuated PTEC ferroptosis. RNAse A pretreatment of hypoxic-apical sEV also abrogated PTEC ferroptosis, demonstrating a role for sEV RNA in ferroptotic 'wave of death' signalling. In line with these in vitro findings, in situ immunolabelling of diagnostic kidney biopsies from AKI patients with clinical progression to CKD (AKI-to-CKD transition) showed evidence of ferroptosis propagation (increased numbers of ACSL4+ PTEC), while urine-derived sEV (usEV) from these 'AKI-to-CKD transition' patients triggered PTEC ferroptosis (↑lipid peroxidation) in functional studies. Our data establish PTEC-derived apical sEV and their intravesicular RNA as mediators of tubular lipid peroxidation and ferroptosis in hypoxic kidney injury. This concept of how tubular pathology is propagated from the initiating insult into a 'wave of death' provides novel therapeutic check-points for targeting AKI-to-CKD transition.

Project description:BackgroundAlbuminuria is a hallmark of diabetic kidney disease (DKD) that promotes its progression, leading to renal fibrosis. Renal macrophage function is complex and influenced by macrophage metabolic status. However, the metabolic state of diabetic renal macrophages and the impact of albuminuria on the macrophage metabolic state are poorly understood.MethodsExtracellular vesicles (EVs) from tubular epithelial cells (HK-2) were evaluated using transmission electron microscopy, nanoparticle tracking analysis and western blotting. Glycolytic enzyme expression in macrophages co-cultured with HSA-treated HK-2 cell-derived EVs was detected using RT-qPCR and western blotting. The potential role of EV-associated HIF-1α in the mediation of glycolysis was explored in HIF-1α siRNA pre-transfected macrophages co-cultured with HSA-treated HK-2 cell-derived EVs, and the extent of HIF-1α hydroxylation was measured using western blotting. Additionally, we injected db/db mice with EVs via the caudal vein twice a week for 4 weeks. Renal macrophages were isolated using CD11b microbeads, and immunohistofluorescence was applied to confirm the levels of glycolytic enzymes and HIF-1α in these macrophages.ResultsGlycolysis was activated in diabetic renal macrophages after co-culture with HSA-treated HK-2 cells. Moreover, HSA-treated HK-2 cell-derived EVs promoted macrophage glycolysis both in vivo and in vitro. Inhibition of glycolysis activation in macrophages using the glycolysis inhibitor 2-DG decreased the expression of both inflammatory and fibrotic genes. Mechanistically, EVs from HSA-stimulated HK-2 cells were found to accelerate macrophage glycolysis by stabilizing HIF-1α. We also found that several miRNAs and lncRNAs, which have been reported to stabilize HIF-1α expression, were increased in HSA-treated HK-2 cell-derived EVs.ConclusionOur study suggested that albuminuria induced renal macrophage glycolysis through tubular epithelial cell-derived EVs by stabilizing HIF-1α, indicating that regulation of macrophage glycolysis may offer a new treatment strategy for DKD patients, especially those with macroalbuminuria.

Project description:Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

Dataset Information

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

Publications

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

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