Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells

Abstract

Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contains exosomes that are released from the entire length of the nephron and change in composition with kidney disease. Exosomes can shuttle information between non-renal cells via transfer of protein and RNA. In this study murine kidney collecting duct (mCCDC11) cells were used to demonstrate that exosomes can act as a signalling mechanism between cells. First, the release of exosomes by mCCDC11 cells was confirmed by multiple approaches. Following isopynic centrifugation, exosomal proteins flotillin-1 and TSG101 were identified in fractions consistent with exosomes. Electron microscopy demonstrated structures consistent in size and shape with exosomes. Exposure of mCCDC11 cells to the synthetic vasopressin analogue, desmopressin, did not affect exosomal flotillin-1 or TSG101 but increased aquaporin 2 (AQP2) in a dose- and time-dependent manner that was highly correlated with cellular AQP2 (exosomal AQP2 vs. cellular AQP2, Pearson correlation coefficient r = 0.93). To test whether the ratio of exosomal AQP2/flotillin-1 is under physiological control in vivo, rats were treated with desmopressin. The ratio of AQP2/flotillin-1 in the urinary exosome was significantly increased. Inter-cellular signalling by exosomes was demonstrated: exosomes from desmopressin-treated cells stimulated both AQP2 expression and water transport in untreated mCCDc11 cells (water flow across cells: control exosome treatment 52.8 ± 11 μl cm(-2); AQP2-containing exosomes 77.4 ± 4 μl cm(-2), P = 0.05, n = 4). In summary, the amount of AQP2 in exosomes released from collecting duct cells is physiologically regulated and exosomal AQP2 closely reflects cellular expression. Exosomes can transfer functional AQP2 between cells and this represents a novel physiological mechanism for cell-to-cell communication within the kidney.

Figure 1. mCCDC11 cells release exosomes

A, in the mCCDC11 cell culture medium, the exosomal marker proteins flotillin-1 and TSG101 localise to the ultracentrifugation pellet (pellet) rather than the supernatant. B, Western blot for aquaporin 2 (AQP2), flotillin-1 and TSG101 on fractions obtained following isopycnic centrifugation. The exosomal markers are present in fractions corresponding to a density of 1.10–1.15 g ml^-1. The positive control was unfractioned exosomes. C, structures of exosome size and shape are visible in the cell culture medium using transmission electron microscopy. Arrows indicate 2 exosomes. Bar, 100 nm.

Figure 2. Stimulation increases exosomal AQP2

A, Western blot for aquaporin 2 (AQP2), TSG101and flotillin-1 in desmopressin-stimulated cells and their exosomes. Cells were stimulated for 96 h with desmopressin (316 pg ml^-1, 1 ng ml^-1 or 3.16 ng ml^-1). Control was no desmopressin. AQP2 in exosomes was increased by desmopressin cell stimulation for 96 h but not 48 h. Cell and exosomal TSG101 and flotillin-1 was unchanged. AQP2 was also upregulated in the cell extracts at 96 h. 1–3 are replicates. B, treatment of mCCDC11cells with desmopressin increased the cell and exosome AQP2 content. AQP2 was upregulated in the exosomes and cells after 96 h of desmopressin stimulation. *p < 0.05, **P < 0.001. n = 4. AQP2 was quantified by Western blot and band densitometry. C, the AQP2 content of exosomes was closely correlated with cellular AQP2. AQP2 was quantified by Western blot and band densitometry. Each point represents a separate experiment. D, the ratio of AQP2:flotillin-1 in rat urinary exosomes was increased by subcutaneous injection of desmopressin (1 μg kg^-1) on day 3 and day 4. *P = 0.05, n = 3.

Figure 3. mCCDC11 cells exposed to AQP2-containing exosomes express functional AQP2

A, AQP2 protein expression was measured by Western blot and band densitometry. Cellular AQP2 expression (‘cells’) was measured after 48 h incubation with AQP2-containing exosomes (derived from mCCDC11cells treated with desmopressin (3.16 ng ml^-1) for 96 h); control exosomes derived from unstimulated cells or direct treatment with desmopressin (3.16 ng ml^-1). The concentration of AQP2 in the exosomes is also presented (‘exosomes’). **p < 0.02 vs. control. *p < 0.05 vs. control. n = 4. B, the water flow across mCCDC11monolayers after 48 h co-culture with exosomes from mCCDC11cells exposed to desmopressin (3.16 ng ml^-1) immediately before exosome isolation (desmopressin t = 0) or 96 h before exosome isolation (desmopressin t = –96 h). Water flow was determined by weighing the medium in the apical and basolateral compartments. Water flow is expressed as μl per area of monolayer. *P = 0.05 by paired analysis. n = 4.