Vasopressin Regulates Extracellular Vesicle Uptake by Kidney Collecting Duct Cells

ECV uptake by mCCD_C11 cells is increased by desmopressin stimulation. (A) Flow cytometry data demonstrating no significant fluorescently loaded ECV uptake following desmopressin stimulation (3.16 ng/ml) for up to 8 hours (n=3). Fluorescent cells expressed as percentage of total cell number. (B) Flow cytometry data demonstrating ECV uptake following longer desmopressin stimulation. n=3; *P<0.05: 3.16 ng/ml desmopressin stimulation versus no stimulation. For (A) and (B) error bars represent SD. (C) Desmopressin (3.16 ng/ml for 96 hours)-stimulated mCCD_C11 cells had significantly increased fluorescence after incubation with labeled ECVs. Fluorescent cells expressed as percentage of total cell number (n=5). (D) NTA analyses of fluorescently loaded ECVs in the cell culture supernatant from control and desmopressin (3.16 ng/ml for 96 hours) stimulated cells presented as the area under the concentration curve for particle sizes between 20–100 nm (n=5). Desmopressin stimulation reduced the concentration of ECVs in the supernatant. (E) Fluorescence of control and desmopressin stimulated cells (3.16 ng/ml for 96 hours) exposed to fluorescently loaded ECVs in the absence and presence of tolvaptan (10 nM). Fluorescent cells expressed as percentage of total cell number (n=5). (F) Fluorescence of control and desmopressin-stimulated cells (3.16 ng/ml for 96 hours) exposed to fluorescently loaded ECVs in the absence and presence of endothelin-1 (10 pg/ml). Fluorescent cells expressed as percentage of total cell number (n=5). (C–F) Tukey plots. Bottom and top of the box are the first and third quartiles, and the band inside is the median. The whiskers are the lowest datum still within 1.5 interquartile range of the lower quartile and the highest datum still within 1.5 interquartile range of the upper quartile. *P<0.05. AUC, area under the curve.

ECVs deliver functional miRNA into desmopressin-stimulated mCCD_C11 cells. Change in gene expression when desmopressin stimulation (3.16 ng/ml for 96 hours) is compared with unstimulated cells in the absence of ECVs, with control HUVEC-derived ECVs or with miR-503 loaded ECVs. (A) Vascular endothelial growth factor A. (B) Fibroblast growth factor 2. (C) Cyclin E-1. (D) Cell division cycle 25A. Values are expressed as the difference between control values in unstimulated cells minus control values in desmopressin-stimulated cells, with 18S as endogenous control. Negative values indicate downregulation and positive values indicate upregulation of target genes by desmopressin without or with the stated ECVs. n=9; *P<0.05. All plots are Tukey plots.

Rat primary collecting duct cells take up ECVs after desmopressin stimulation. The cells express AQP2 protein (green), as detected by fluorescent antibody labeling. Fluorescently loaded ECVs (red) enter primary cells with desmopressin (3.16 ng/ml for 2 hours) treatment. DAPI-stained nuclei are blue.

ECV uptake by mCCD_C11 cells following desmopressin stimulation is mediated by cAMP and dynamin activity. (A) Desmopressin (3.16 ng/ml for 96 hours) stimulated ECV uptake, which was decreased by PKA inhibition (H-89–25 µM) of the cAMP pathway. (B) ECV uptake was increased by cAMP stimulation through forskolin (10 µM). (C) Dynasore (150 nM) inhibition of dynamin activity decreased ECV uptake by desmopressin-stimulated cells below that of the control cells. (A–C) Fluorescent cells expressed as percentage of total cell number. n=6; *P<0.05. All plots are Tukey plots.

ECV uptake is cell type specific. (A) NTA measurement of ECVs from different cell types before incubation with mCCD_C11 cells. (B) Comparing total cell fluorescence between control and desmopressin (3.16 ng/ml for 96 hours) stimulated mCCD_C11 cells after labeled ECV incubation from three cell types: mCCD_C11 cells (CCD), HK2 (human proximal tubular cell line), and RG1 (murine juxtaglomerular cell line). ECVs, 1×10⁸/ml, were added to all experiments. n=6; *P<0.05. (C) NTA analyses of cell culture supernatant incubated with different cell type derived ECVs (mCCD_C11, HK2, and RG1) from control and desmopressin (3.16 ng/ml for 96 hours) stimulated cells presented as the area under the concentration curve (AUC) for particles sized between 20 and 100 nm. Desmopressin stimulation reduced the concentration of mCCD_C11 and HK2 ECVs in the supernatant but not in cells incubated with RG1 ECVs (D) Polarized cells take up ECVs under desmopressin regulation. Total cell fluorescence of polarized mCCD_C11 cells stimulated with desmopressin (3.16 ng/ml for 96 hours) either apically or basolaterally compared with unstimulated cells. Labeled ECVs were applied to the apical compartment of Transwell. n=3; *P<0.05. (B–D) Tukey plots. CCD, collecting duct; HK2, proximal tubule; RG1, juxtaglomerular cell derived.

Vasopressin V2 receptor regulates urinary ECV excretion in mice and humans. (A) Urinary excretion of systemically adminstered ECVs in mice. Group 1: control group; urine ECV excretion following two IV injections of fluorescently loaded ECVs. Group 2: urine ECV excretion after two IV injections of labeled ECVs. Between injections, mice were treated with tolvaptan (0.3 mg/kg). n=5 per group; *P<0.05. Group 3: urine ECV excretion after two IV injections of labeled ECVs. Between injections, mice were treated with furosemide (1 mg/kg, n=4). ECV excretion expressed as percentage of the total number of injected ECVs excreted in the urine. Data expressed as a Tukey plot. (B) Twenty-four hour ECV excretion by a patient with central diabetes insipidus. Lines represent the time of desmopressin treatment (dashed line) and concentration of ECVs expressing nephron segment–specific proteins: glomerular (podocalyxin-like protein, PODX-L), proximal tubular (cubilin), and CD24 (pan-segment urinary ECV marker). ECV urine concentration measured by NTA and normalized by urinary creatinine concentration. All data are expressed as mean±SD. NTA measurements were taken in triplicate for each time point. AUC, area under the concentration curve.