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Regulation of the Interaction of NCC and ENaCβ by SGK1
Author(s) -
Liu Melissa,
AlKhalili Otor,
Eaton Douglas,
Wynne Brandi,
Hoover Robert
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.969.22
Subject(s) - sgk1 , epithelial sodium channel , luciferase , aldosterone , chemistry , nedd4 , transfection , distal convoluted tubule , microbiology and biotechnology , biology , endocrinology , biochemistry , ubiquitin , gene , reabsorption , sodium , ubiquitin ligase , organic chemistry
Aldosterone and angiotensin II are the primary hormones mediating NCC and ENaC function, which regulate salt and water balance. Two downstream mediators include the aldosterone‐mediated activation of SGK1 and angiotensin II‐mediated activation of WNK4. Additionally, the negative regulator NEDD4‐2 is an important modulator of NCC and ENaC function. Previously, our laboratory has demonstrated that NCC and ENaC subunits (α, β or γ) are closely associated and interact in the distal convoluted tubule (DCT2); however, whether this interaction can be modulated has yet to be investigated. Herein, we investigate physiological parameters by which NCC and ENaCβ are augmented. We used a mammalian two‐hybrid (M2H) assay, using vectors (pBind/pAct with NCC and/or ENaCβ, empty pBind/pAct and SGK1, WNK4 and NEDD4‐2 expression vectors) which allow for activation of the luciferase reporter vector (firefly) when interaction occurs. COS‐7 cells were transfected and allowed to grow for 48 hours. Cells were then lysed and results of the M2H assay were determined by analysis of firefly and renilla luciferase; renilla was used as a transfection control. Data were normalized and shown as firefly/renilla luciferase values. Our data demonstrate that NCC and ENaCβ may not interact; however, with SGK1 overexpression, we observed an increase as compared to negative vectors only (70.4±20.28 vs. 6.36±1.37, p<0.0001, ANOVA, n=4). Interestingly, WNK4 and NEDD4‐2 did not seem to alter the baseline interaction of NCC and ENaCβ. These data suggest that increases in SGK1, possibly via aldosterone, may produce increased NCC and ENaCβ interaction.