Recent evidence suggests that the epithelial sodium channel (ENaC) is differentially regulated along the aldosterone sensitive distal nephron (ASDN) with a higher activity in the early parts of the ASDN than in its later parts (Loffing J & Korbmacher C. Pflügers Archiv 458:111–135, 2009). Previously we demonstrated that in outside-out patches of ENaC-expressing Xenopus laevis oocytes serum- and glucocorticoid-inducible kinase (SGK1) included in the pipette solution stimulates ENaC (Diakov A & Korbmacher C. J Biol Chem 279:38134–38142, 2004). The aim of the present study was to compare the effect of SGK1 on ENaC activity in outside-out patches from different parts of the ASDN, i.e. in the late distal convoluted tubule/early connecting tubule (DCT2/CNT) and in the late CNT/early collecting duct (CNT/CCD).

Methods: 

Using microdissected split open mouse tubules we determined the effect of constitutively active SGK1 included in the pipette solution on the amiloride-sensitive current (DI_ami) in the whole-cell configuration and subsequently in excised outside-out patches. From these values the ratio of outside-out DI_ami to whole-cell DI_ami was calculated for individual cells. Pipette solution contained either SGK1 (20 U/ml) or vehicle.

Results: 

We confirmed that the average baseline whole-cell DI_ami at a holding potential of -60 mV is larger in the DCT2/CNT (34 8± 90 pA, n=11) than in the CNT/CCD (38 ± 3.6 pA; n=10; p<0.01). Interestingly, we did not detect a significant stimulatory effect of SGK1 on whole-cell DI_ami which remained at 334 ± 54 pA (n=8) in the DCT2/CNT and at 32 ± 7.8 pA (n=10) in the CNT/CCD. This may be attributed to a limited diffusion of the SGK1 to the plasma membrane in the whole-cell configuration. In contrast, in the outside-out configuration SGK1 stimulated DI_ami in both parts of the ASDN. This is evidenced by a significant increase of the ratio of outside-out DI_ami to whole-cell DI_ami. In the DCT2/CNT segment SGK1 increased this ratio by about threefold from 0.91 ± 0.19% to 2.7 ± 0.54% (p<0.05). In the CNT/CCD, SGK1 also increased this ratio by a factor of about three, i.e. from 8.4 ± 3.6% to 26.7 ± 7.8% (p<0.05).

Conclusion: 

In outside-out patches from microdissected mouse tubules SGK1 acutely stimulates ENaC in a similar way as previously described for ENaC expressed in Xenopus laevis oocytes. The relative stimulatory effect of SGK1 on ENaC currents is similar in the DCT2/CNT to that in the CNT/CCD. Thus, a different responsiveness to an acute exposure to SGK1 cannot explain the regional differences of ENaC currents observed along the ASDN.