Response to Role of Epithelial Sodium Channels in the Renal Myogenic Response?
We appreciate the comments from Loutzenhiser and Aaronson1 regarding our report2 that afferent arteriolar autoregulation was inhibited by amiloride or benzamil (bath; 10 μmol/L) and by perfusion with 5 μmol/L of amiloride. In both conditions, KCl-induced vasoconstriction was preserved, indicating intact voltage-dependent Ca2+ channel function. With immunofluorescent detection of the epithelial sodium channel (ENaC) subunits (α, β, and γ), the data support the hypothesis that ENaC-like proteins may function as mechanosensitive ion channel complexes transducing myogenic responses.
As noted, amiloride and benzamil can inhibit other channels and transporters, but they generally require concentrations greater than those used here. The IC50 for amiloride inhibiting the Na+/H+ exchanger is 84 μmol/L and 1100 μmol/L for the Na+/Ca2+ exchanger.3 Benzamil is 10 times more potent than amiloride for inhibiting sodium channels and the Na+/Ca2+ exchanger (IC50 ≈100.0 μmol/L) but is less potent toward the Na+/H+ exchanger (IC50 ≈1.0 mmol/L).3 The maximum amiloride and benzamil concentrations that we used (10 μmol/L) exceed the IC50 for sodium channels (submicromolar) but are far below the inhibitory concentrations for Na+/H+ antiporters or Na+/Ca2+ exchangers. We recognize that pharmacological selectivity is imperfect and that data interpretation must reflect such limitations. Our data support the hypothesis within the context of these drug actions.
The pharmacokinetics of amiloride-like agents for inhibiting ENaC activity were largely determined in vitro using epithelial cells or Xenopus oocytes expressing ENaC. The IC50 of amiloride on ENaC and other signaling mechanisms is influenced by many factors, such as membrane potential or pH.3 Amiloride binds to serum proteins effectively reducing drug concentrations in albumin-containing solutions. Notably, our preparation includes diffusion barriers that limit drug delivery to tissue and could reduce effective drug concentrations reaching the target. Compared with the original ENaC studies,4,5 many differences exist in experimental conditions (see Discussion in Reference 2), which could contribute to differences between studies. Nevertheless, we obtained comparable impairment of autoregulatory behavior using 2 inhibitors exhibiting markedly different effects on other transporters. This strengthens the likelihood that the effects observed reflect sodium channel involvement.
Indeed, some similarities exist between our work and that of Wang et al.6 Superfusion with 1.0 μmol/L of amiloride or benzamil did not inhibit autoregulation. We also observed that 10 μmol/L of benzamil decreased the arteriolar diameter by ≈21%1 compared with a ≈55% decline in hydronephrotic kidneys.6 However, benzamil enhanced myogenic responses in hydronephrotic kidneys6 and inhibited autoregulation in our study,2 suggesting that inhibition of autoregulation by amiloride and benzamil acts upstream of membrane depolarization, consistent with the original finding.4,5 The reasons for the discrepancies between our study2 and that of Wang et al6 are not clear.
Finally, positive immunostaining for all 3 of the ENaC subunits in preglomerular smooth muscle cells support the hypothesis that ENaC-like proteins are present in preglomerular smooth muscle, which could act as mechanosensors involved in autoregulatory responses. Tissue-specific differences could exist between immunoreactive ENaC-like proteins from epithelial and smooth muscle cells that would confer different pharmacokinetic properties. Nevertheless, detection of these proteins and the inhibitory action of amiloride and benzamil on autoregulation supports the hypothesis that ENaC may form a mechanosensitive complex triggering myogenic responses. We agree that direct electrophysiological approaches are needed to determine whether ENaC-like proteins form this complex.
Overall, the data support a role for ENaC-like proteins in autoregulatory signaling. Somewhat different findings from different laboratories and the recognition that pharmacological agents are imperfect underscore the need for additional, more focused studies to clarify the issues surrounding the ENaC hypothesis.
Sources of Funding
This study was supported by grants from the National Institutes of Health (DK 44628 and HL 074167). Z.G. is the recipient of a post-doctoral fellowship from the Greater Southeast Affiliate of the American Heart Association.
Loutzenhiser R, Aaronson PI. Role of epithelial sodium channels in the renal myogenic response. Hypertension. 2010; 55: e6.
Guan Z, Pollock JS, Cook AK, Hobbs JL, Inscho EW. Effect of epithelial sodium channel blockade on the myogenic response of rat juxtamedullary afferent arterioles. Hypertension. 2009; 54: 1062–1069.
Drummond HA, Gebremedhin D, Harder DR. Degenerin/epithelial Na+ channel proteins: components of a vascular mechanosensor. Hypertension. 2004; 44: 643–648.
Jernigan NL, Drummond HA. Vascular ENaC proteins are required for renal myogenic constriction. Am J Physiol Renal Physiol. 2005; 289: F891–F901.
Wang X, Takeya K, Aaronson PI, Loutzenhiser K, Loutzenhiser R. Effects of amiloride, benzamil, and alterations in extracellular Na+ on the rat afferent arteriole and its myogenic response. Am J Physiol Renal Physiol. 2008; 295: F272–F282.