Ceramide inhibits epithelial sodium channels in A6 distal nephron cells

Several lines of evidence suggest that tumor necrosis factor α (TNF‐α) is closely related to both kidney allograft rejection and end‐stage renal disease and that ceramide acts as a messenger responsible for the signal transduction initiated by TNF‐α. The present study is to determine whether ceramide affects sodium transport by regulating the epithelial sodium channel (ENaC). Using patch‐clamp techniques and A6 distal nephron cells as a model, we found that application of C 2 ‐ceramide (50 μM), a membrane‐permeable analog of ceramide, to the apical bath reduced ENaC open probability from 0.48 ± 0.19 to 0.11 ± 0.07 in five cell‐attached patches (p < 0.01). In contrast, ENaC open probability was not affected by dihydroceramide (50 μM), an inactive analog of C 2 −ceramide. Application of sphingomyelinase (0.1 U/ml) to the apical bath increased cellular ceramide concentration identified by mouse monoclonal anti‐ceramide antibody using confocal microscopy, and reduced ENaC open probability from 0.36 ± 0.17 to 0.09 ± 0.06 in four cell‐attached patches (p < 0.05). Ceramide‐induced inhibition of ENaC appears to be independent of other cytosolic molecules because ceramide also inhibited ENaC activity in inside‐out patches. Recent studies have shown that ceramide can interfere the binding of pleckstrin homology domains to phosphatidylinositol 3,4,5‐trisphosphate (PIP 3 ) and PIP 3 stimulates ENaC in inside‐out patches and binds to γ‐ENaC. We found that ceramide abolished activation of ENaC by PIP 3 in inside‐out patches. These data together suggest that ceramide inhibits ENaC, probably by abolishing the interaction between ENaC and PIP 3 . Supported by NIH‐NIDDK grant R01DK067110 to HPM.