Aldosterone Rapidly Activates Na⁺/H⁺ Exchange in M-1 Cortical Collecting Duct Cells via a PKC-MAPK Pathway

Abstract

Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na⁺/H⁺ exchanger (NHE)-mediated intracellular pH (pH_i) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH₄Cl was used to induce the acid load. Results: Aldosterone (10 nM) induced a rapid (<5 min) concentration-dependent increase in pH_i recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 nM). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µM) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µM) and cariporide (1 µM). The PKC inhibitor chelerythrine chloride (1 µM) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µM), a PKC_α inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µM), a PKC_δ antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µM) and dexamethasone (100 nM) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 nM) had no significant effect. MAPK inhibition using PD98059 (25 µM) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKC_α/MAPK pathway independent of the classical MR.

Protein kinase D1 modulates aldosterone-induced ENaC activity in a renal cortical collecting duct cell line

“…PKD1 phosphorylates IKK to promote NFB-dependent transcription in response to oxidative stress (26), PKD1 phosphorylates cJun to suppress its transcriptional activity in response to EGF (43) and PKD isoforms stabilize ERK1/2 activity to promote cell growth (27,44). ERK1/2 activation is the most frequently described signalling response to aldosterone (45,46), and the stabilization of ERK1/2 activation by aldosterone is also PKD1-dependent (47). Oestrogen promotes ERK1/2 activation, and activated ERK1/2 phosphorylates oestrogen receptor at Ser118, to promote its nuclear localization (28).…”

Mechanisms Underlying Rapid Aldosterone Effects in the Kidney

“…This rise is due to an ERK1/2-dependent rise in NHE1 activity (103,119). The resulting transient rise in cytosolic pH may facilitate the activation of pH-sensitive signaling intermediates that are involved in cell cycle progression, such as the ribosomal S6 kinase (RSK) family protein kinases (120).…”

Aldosterone as a renal growth factor

“…The abundance of EGFR in cells from the renal tubule is responsive to aldosteronedependent transcription [36] and expression of EGFR is necessary to permit the stimulation of rapid signalling responses by aldosterone in CHO cells [37]. The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 activation of ERK1/2 signalling by aldosterone in cells derived from the distal nephron has been described by several groups including our own [38,39] and this response has been linked to trans-activation of EGFR [40]. The nuclear localization of ERK1/2 occurs subsequent to its activation and is consistent with its role as a regulator of transcription factors.…”