Acute cholesterol-induced anti-natriuretic effects: role of epithelial Na+ channel activity, protein levels, and processing

Abstract

The epithelial Na(+) channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na(+) handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1-3 weeks. Secondary compensation returned urine output and urinary Na(+) excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na(+) handling, which occur in part by effects on extracellular γ-ENaC cleavage.

FIGURE 1.

Enhanced dietary cholesterol causes sodium and water retention. Animals were fed a control (CONT) diet or ECD. Data summarized were averaged from weekly values. A, the ECD stimulated renal sodium reabsorption manifested as a marked decrease of Na⁺ excretion (U_NaV). These effects were observed by week 1 after the diet change and recovered toward control by the end of the study (week 10). B and C, urinary osmolarity and volume also decreased by the ECD. These changes persisted until the end of the study. n = 6–10 animals in each group (see “Experimental Procedures” for details). Statistically significant changes indicated by a p value. Error bars, S.E.

FIGURE 2.

Changes of animal body weight. Weight gain was computed in each animal as a function of time and normalized to the gain per day. Animal weight gain reached a plateau toward the end of the study. No significant differences were observed between the two groups throughout the study, although the ECD group exhibited a tendency toward a higher rate during weeks 2 and 3. n = 6–10 in each group. CONT, control; Error bars, S.E.

FIGURE 3.

ECD increases urinary and membrane cholesterol content but does not affect glomerular permeability. A, urinary total protein excretion (U_TPV) was unaffected by the ECD, suggesting that hypercholesterolemia did not impair filtration via glomerular injury. B, urinary creatinine excretion (U_CRV) was also unchanged by the ECD, further ruling out chronic changes of glomerular permeability, especially given the lack of changes to serum creatinine. C, urinary cholesterol excretion increased by the ECD, indicating that the ECD was sufficient in elevating circulating cholesterol levels despite the potential compensation in synthesis in the rat. D, ECD led to an increase of renal membrane cholesterol content which was significant by the end of the study. n = 4–10 in each group. CONT, control; Error bars, S.E.

FIGURE 4.

Absence of marked changes of blood pressure with ECD. A, SBP was unchanged in either group of animals during the first 3 weeks. A small increase was observed by the end of the study (see Table 2). A clear diurnal pattern was also observed, which was not modified by the ECD. B, mean arterial pressure was also unchanged during the first 3 weeks but exhibited a small (∼5 mm Hg) increase by week 10 (summarized in Table 2). These results rule out changes of pressure and GFR as the mechanism mediating the anti-natriuresis observed in weeks 1–3. n = 5 in each group. CONT, control.

FIGURE 5.

ECD does not affect organ weight except for liver. Heart, kidney, and lung weights were unchanged, consistent with the absence of marked changes of blood pressure. Liver weight was significantly elevated in the cholesterol diet at both weeks 3 (inset) and 10. n = 4–10 in each group. Error bars, S.E.

FIGURE 6.

ECD does not affect α-ENaC expression or processing. Western blot of renal homogenates probed with two α-ENaC antibodies. Effects at weeks 3 and 10 are summarized in A and B, respectively. Full-length α subunit migrated at ∼100 kDa and was detected with the N-terminal antigen antibody at both time points (A1 and B1) and the loop antigen antibody at week 10. The level of this subunit was low in contrast to that of other fragments and was unchanged by the ECD. Proteolytically cleaved α was the dominant observed protein and was only detected with the loop antibody (A2 and B2). This cleaved subunit migrated at 65 kDa, with a second protein detected at 37 kDa (see “Experimental Procedures”). ECD did not significantly affect levels of processed 65-kDa α at either 3 or 10 weeks. Data are normalized to GAPDH expression. n = 4–6 in each group. CONT, control; Error bars, S.E.

FIGURE 7.

ECD does not affect β-ENaC expression. β-ENaC migrated at ∼90 kDa as described previously. A second product, which was possibly intracellularly degraded β, migrated at 55 kDa. ECD was without effects on β at both 3 weeks (A) and 10 weeks (B). n = 4–6 in each group. CONT, control; Error bars, S.E.

FIGURE 8.

ECD enhanced γ-ENaC levels and cleavage. Effects at weeks 3 and 10 are summarized in A and B. Full-length γ migrated at 85–90 kDa and was poorly observed in all animals. A faint full-length band was detected in some animals (A1) and at higher exposures; however, these exposures were saturating to signal acquisition and were not used. A, cleaved γ migrated as a doublet with the main protein at 60 kDa. The levels of this γ increased with ECD after 3 weeks. B, at week 10, γ remained largely in the cleaved form, and two additional cleavage products were now clearly evident and migrated at 50–55 kDa. Cleaved γ levels were elevated by ∼2-fold when all three γ products were grouped (50–60 kDa multiband). When the levels of the two additional cleavage products were summarized separately (50–55 kDa), ECD caused an increase of ∼3-fold. Data were normalized to GAPDH. n = 4–6 in each group. CONT, control; Error bars, S.E.

FIGURE 9.

Dietary cholesterol enhances γ-ENaC-cleaving urinary proteases. Data are summarized as initial rates of fluorescence, a reflection of the initial rate of enzymatic cleavage. Data were averaged from each week and demonstrate the activity of urinary proteases capable of cleaving γ after RKRR (A) and RKRK (B). At week 1, ECD caused a peak increase in RKRR activity, which returned toward control by week 2. On the other hand, ECD caused a marked increase of RKRK activity by week 2, and this increase was sustained until the end of the study. These data suggest the presence of two distinct groups of proteases that can act on γ, which underlie the shift in molecular weight observed in Fig. 8. n = 4–6 for each group. CONT, control; Error bars, S.E.

FIGURE 10.

Dietary cholesterol increases ENaC open probability. Cell-attached patch clamp of principal cells in split open tubules examining ENaC activity. Recordings were carried out at an applied pipette potential of −60 mV (intracellular with respect to bath). The currents observed were dependent on the presence of Na⁺ in the pipette, were not observed with amiloride, and were of the size and kinetics expected for ENaC. A, representative traces demonstrating higher channel activity in the ECD. B, mean open probability was elevated in the cholesterol group. The increase in P_o was not accompanied by an increase in the number of active channels observed per patch, indicating that channel membrane density probably is not modified by ECD. n = 6 animals/group with 32–48 recordings in each group. CONT, control; Error bars, S.E.