Control of ENaC ubiquitination.

Ubiquitination influences the expression of the epithelial Na channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process.

High dietary K intake inhibits proximal tubule transport.

The impact of chronic dietary K loading on proximal tubule (PT) function was measured using free-flow micropuncture along with measurements of overall kidney function, including urine volume, glomerular filtration rate, and absolute and fractional Na and K excretion in the rat. Feeding animals a diet with 5% KCl [high K (HK)] for 7 days reduced glomerular filtration rate by 29%, increased urine volume by 77%, and increased absolute K excretion by 202% compared with rats on a 1% KCl [control K (CK)] diet. HK did not change absolute Na excretion but significantly increased fraction excretion of Na (1.

Expression of ENaC subunits in epithelia.

The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and activation of the channel, we quantified the expression of individual subunits of ENaC in rat kidneys and colon using calibrated Western blots. The estimated abundance for the three subunits differed by an order of magnitude with the order γENaC ∼ βENaC ≫ αENaC in both organs.

ENaC and ROMK channels in the connecting tubule regulate renal K+ secretion.

We measured the activities of epithelial Na channels (ENaC) and ROMK channels in the distal nephron of the mouse kidney and assessed their role in the process of K+ secretion under different physiological conditions. Under basal dietary conditions (0.5% K), ENaC activity, measured as amiloride-sensitive currents, was high in cells at the distal end of the distal convoluted tubule (DCT) and proximal end of the connecting tubule (CNT), a region we call the early CNT (CNTe).

Cleavage state of γENaC in mouse and rat kidneys.

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the γ subunit. Here, we investigated the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in Fisher rat thyroid cells by coexpressing the apical membrane-bound protease channel-activating protease 1 (CAP1; prostasin).