Question: 

The amiloride-sensitive epithelial sodium channel (ENaC) is essential for the maintenance of body sodium balance and the long term regulation of arterial blood pressure. In the kidney, ENaC is expressed in tubular epithelial cells of the aldosterone-sensitive distal nephron. ENaC regulation is highly complex and various hormones and local mediators are known to affect ENaC function via receptors localized in the basolateral membrane of epithelial cells. We hypothesized that the presence of peritubular endothelial cells may affect ENaC regulation. To investigate this, we co-cultured M-1 cells with renal microvascular endothelial cells and studied the effect of the co-culture conditions on transepithelial sodium transport.

Methodology: 

We used the M-1 mouse cortical collecting duct cell line known to endogenously express ENaC. M-1 cells were maintained in serum-free PC-1 culture medium and grown on permeable supports to reach confluence and form tight epithelial monolayers 9–10 days after seeding. At this stage microvascular endothelial glEND.2 cells were seeded on the opposite side of the permeable supports; these cells have to be maintained in culture medium containing 10% fetal bovine serum (FBS). In control experiments M-1 cells were exposed to 10% FBS in the absence of co-cultured glEND.2 cells. Transepithelial sodium transport via ENaC was studied by measuring the equivalent short circuit current (I_SC). Protein expression of abg ENaC was analyzed by Western Blotting and immunofluorescence staining using ENaC subunit specific antibodies.

Result: 

Surprisingly, control experiments in the absence of glEND.2 cells demonstrated that application of PC-1 medium containing 10% FBS to the basolateral side of M-1 monolayers reduced I_SC by about 90% within 24 hours. The reduction in I_SC was associated with a drop in transepithelial resistance. Moreover, basolateral application of FBS reduced the protein expression of b-, but not of a- and g-ENaC. Application of FBS to the apical side of M-1 cells did not cause a pronounced inhibition of I_SC suggesting that the inhibitory effect on ENaC is mediated by basolateral receptors. Inhibition of I_SC was preserved with heat-inactivated and charcoal-stripped FBS. Basolateral application of endotoxins and growth factors failed to mimic the effect of FBS. Interestingly, in the presence of co-cultured glEND.2 cells, the inhibitory effect of FBS on I_SC was attenuated.

Conclusion: 

FBS contains one or several heat-resistant mediators that can impair ENaC function in tubular epithelial cells possibly by activating basolateral membrane receptors which remain to be determined. Co-cultured endothelial cells appear to attenuate the inhibitory effect of FBS. Our data suggest that in vivo peritubular endothelial cells may protect tubular epithelial cells from circulating mediators with a potential inhibitory effect on transepithelial sodium transport.