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Biology (Basel)
2025 Mar 01;143:. doi: 10.3390/biology14030252.
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Small Extracellular Vesicles with a High Sphingomyelin Content Isolated from Hypertensive Diabetic db/db Mice Inhibits Calcium Mobilization and Augments Amiloride-Sensitive Epithelial Sodium Channel Activity.
Ramsay H
,
Yu L
,
Alousi FF
,
Alli AA
.
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Extracellular vesicles (EVs) contain bioactive lipids that play a key role in pathophysiology. We hypothesized that EVs released from salt-loaded hypertensive diabetic db/db mice have increased bioactive lipid content that inhibits intracellular calcium mobilization and increases the activity of renal epithelial sodium channels (ENaC). An enrichment of sphingomyelins (SMs) was found in small urinary EVs (uEVs) isolated from salt-loaded hypertensive diabetic db/db mice (n = 4) compared to non-salt loaded db/db mice with diabetes alone (n = 4). Both groups of mice were included in the same cohort to control for variability. Cultured mouse cortical collecting duct (mpkCCD) cells loaded with a calcium reporter dye and challenged with small uEVs from hypertensive diabetic db/db mice showed a decrease in calcium mobilization when compared to cells treated with small uEVs from diabetic db/db mice. The amiloride-sensitive transepithelial current was increased in mpkCCD cells treated with small uEVs with abundant sphingomyelin content from hypertensive diabetic db/db mice in a dose- and time-dependent manner. Similar results were observed in mpkCCD cells and Xenopus 2F3 cells treated with exogenous sphingomyelin in a time-dependent manner. Single-channel patch clamp studies showed a decrease in ENaC activity in cells transiently transfected with sphingomyelin synthase 1/2 specific siRNA compared to non-targeting siRNA. These data suggest EVs with high sphingomyelin content positively regulate renal ENaC activity in a mechanism involving an inhibition of calcium mobilization.
Figure 1. Quantification of sphingomyelin concentration in small urinary extracellular vesicles isolated from the urine of diabetic db/db mice and salt-loaded hypertensive diabetic db/db mice. EVs were isolated from separate collections of urine (30 mLs each) from diabetic db/db mice (N = 4) and salt-loaded hypertensive diabetic db/db mice (N = 4). The concentration of each uEV preparation was normalized after performing NanoSight analysis. The small uEVs were lysed in RIPA buffer and the number of sphingomyelins present in the samples was determined by performing an in vitro sphinomyelin fluorescent-based assay. N = 4 per group. A Student t test was performed to compare the two groups. * represents a p-value < 0.05.
Figure 2. Small urinary EVs from salt-loaded hypertensive diabetic db/db mice compared to diabetic db/db mice inhibit calcium influx in mpkCCD cells. (A) Summary analysis showing a transient influx in intracellular calcium mediated by ionomycin (Ion) after pretreating mpkCCD cells with small uEVs from each group; (B) Representative immunofluorescence images before (left panel) and after (right panel) calcium influx; (C) Summary graph of the relative fluorescent intensity in (B). N = 4 per group. A Student t test was performed to compare the two groups. uEVs refer to urinary extracellular vesicles. NS refers to normal salt (non-salt loaded diabetic db/db mice). HS refers to high salt (salt-loaded hypertensive diabetic db/db mice). * represents a p-value < 0.05.
Figure 3. Exogenous sphingomyelins (SM-6) (1 μM) augment amiloride-sensitive transepithelial current in mpkCCD cells and in Xenopus 2f3 cells. (A) transepithelial current in mpkCCD cells treated with SM-6 or VEH. (B) transepithelial current in 2f3 cells treated with SM-6 or VEH. At the end of the experiment for each cell type, 1 μM amiloride was administered on the apical side as a measure of amiloride-sensitive transepithelial current. Closed squares represent cells treated with vehicle and open circles represent cells treated with SM-6. N = 4 per group. A Student t test was performed to make comparisons between the two groups. VEH represents vehicle. SM-6 represents sphingomyelin-6. * represents a p-value < 0.05.
Figure 4. Patch clamp analysis of a highly selective cation channel in mouse mpkCCD cells transiently transfected with sphingomyelin synthase 1 and 2 specific siRNA or non-targeting control siRNA. mpkCCD cells transfected with sphingomyelin synthase 1 and 2 siRNA or non-targeting control siRNA were patched for ENaC single channel activity. (A) NPo as a measure of ENaC activity, (B) N, as a measure of the number of channels in a patch, (C) Po, as a measure of the open probability of the channel. The numbers at the top of the plots indicate the number of patches per group. (D) representative traces where C represents the closed state and O represents the open state. (E) Current voltage curve. (F) Representative Western blot for smgs1 (top blot) to assess siRNA mediated knockdown of Smgs1 and transfection efficiency in mpkCCD cells. The Western blot for actin (bottom blot) was used to assess lane loading. Densitometric analysis and plot of the Smgs1 protein band normalized to actin (right). siRNA sgms refers to sphingomyelin synthase 1/2 specific siRNA. siRNA ctrl refers to non-targeting control siRNA. NS refers to no significant difference between the groups. The number of experiments for each group is given at the top of panels A–C. For NPo and N, N = 24 in each group. A Student t test was performed to compare the two groups. * represents a p-value < 0.05.
Supplementary Figure 1. Characterization of small extracellular vesicles isolated from the urine of
diabetic db/db mice and salt-loaded hypertensive diabetic db/db mice (A) Nanoparticle tracking analysis
of small uEVs isolated from urine samples from diabetic db/db mice maintained on a normal salt diet and
from urine samples from hypertensive diabetic db/db mice maintained on a high salt diet. (B) Western
blot of the uEV marker CD9 from the two groups, (C) Western blot of the uEV marker caveolin-1 from the
two groups, (D) Western blot of the uEV marker flotillin from the two groups, (E) Western blot of the uEV
marker syntenin from the two groups, (F) transmission electron microscopy analysis of uEVs from a
pooled sample (n=4) of uEVs from dbdb mice maintained on a normal salt diet (left) or uEVs from
hypertensive diabetic db/db mice maintained on a high salt diet (right). 100,000x direct magnification.White arrows indicate uEVs from each group. +CTRL refers to positive control. NS refers to normal salt
diet and HS refers to high salt diet in which the mice were maintained during the study. N=4 for each
group. A student t test was performed to compare the two groups. A p-value <0.05 was considered
significant.
Supplementary Figure 2. Western blot and densitometric analysis of ENaC alpha protein
expression in mpkCCD cells treated with small uEVs isolated from db/db mice maintained on a
normal salt (NS) or high salt (HS) diet. (a) Western blot of ENaC alpha, (b) densitometric analysis
of the uncleaved and cleaved forms of ENaC alpha indicated by the arrows in panel (a).
Supplementary Figure 3. Full Western blot and densitometric analysis of CD9 (Figure S1-B),
Caveolin-1 (Figure S1-C), Flotillin (Figure S1-D), and syntenin (Figure S1-E)
Supplementary Figure 4. Full Western blot and densitometric analysis ofSMGS1 and actin (Figure
4F)
Supplementary Figure 5. Full Western blot and densitometric analysis of ENaC alpha 59 and Actin
