Postprandial Effects on ENaC-Mediated Sodium Absorption

Abstract

Recent studies have suggested that postprandial increases in insulin directly contribute to reduced urinary sodium excretion. An abundance of research supports the ability of insulin to augment epithelial sodium channel (ENaC) transport. This study hypothesized that ENaC contributes to the increase in renal sodium reabsorption following a meal. To test this, we used fasted or 4 hour postprandial Sprague Dawley rats to analyze ENaC expression and activity. We also assessed total expression of additional sodium transporters (Na⁺-Cl^- cotransporter (NCC), Na⁺-K⁺-2Cl^- cotransporter (NKCC2), and Na⁺-K⁺-ATPase (NKA)) and circulating hormones involved in the renin-angiotensin-aldosterone system (RAAS). We found that after carbohydrate stimulus, ENaC open probability increased in split-open isolated collecting duct tubules, while ENaC protein levels remained unchanged. This was supported by a lack of change in phosphorylated Nedd4-2, an E3 ubiquitin ligase protein which regulates the number of ENaCs at the plasma membrane. Additionally, we found no differences in total expression of NCC, NKCC2, or NKA in the postprandial rats. Lastly, there were no significant changes in RAAS signaling between the stimulated and fasted rats, suggesting that acute hyperinsulinemia increases ENaC activity independent of the RAAS signaling cascade. These results demonstrate that insulin regulation of ENaC is a potential mechanism to preserve sodium and volume loss following a meal, and that this regulation is distinct from classical ENaC regulation by RAAS.

Figure 1

Blood glucose and plasma electrolytes following the meal stimulus. Summary graphs of (a) blood glucose, (b) plasma Na⁺, (c) K⁺, and (d) Cl⁻ concentrations in fasted and postprandial Sprague-Dawley rats. *p < 0.05, ***p < 0.001, respectively, N ≥ 5 rats.

Figure 2

Plasma insulin levels from fasted or postprandial rats. Relative serum insulin levels as determined by ELISA. The insulin levels of the postprandial rats are significantly increased compared to controls 4 hours after the meal stimulus. N = 5 rats, **p < 0.01.

Figure 3

ENaC activity in isolated cortical collecting duct (CCD) tubules. (a) Representative current traces from cell-attached patches containing ENaC recorded from the apical membrane of split-open CCD cells of fasted or postprandial Sprague-Dawley rats. Event probability histograms of the representative traces showing the distribution of open and closed states are depicted to the right of the trace. (b) Summary graphs of ENaC activity (NP_o), channel open probability (P_o) and the average number of channels per patch from isolated CCDs. *p < 0.05, **p < 0.01, respectively, N ≥ 5 rats.

Figure 4

Expression of ENaC subunits in fasted or postprandial rats. (a) Western blots of α-, β-, and γ-ENaC subunit expression. (b) Summary graphs of ENaC subunit expression after normalization to β-actin loading control. Each lane represents 1 rat, N = 5 rats in each group.

Figure 5

Total and phosphorylated Nedd4-2 expression in fasted or postprandial rats. (a) Western blots of Nedd4-2 and phosphorylated Nedd4-2 (pNedd4-2). (b) Summary graphs of band densitometry after normalization to β-actin loading control, and also the ratio of phosphorylated to total Nedd4-2. Each lane represents 1 rat, N = 5 rats in each group.

Figure 6

Expression of NCC, NKCC2 and αNKA in fasted or postprandial rats. (a) Western blots demonstrating NCC, NKCC2, and α-Na⁺/K⁺ ATPase (αNKA) expression. (b) Summary graphs of band densitometry after normalization to β-actin loading control. Each lane represents 1 rat, N = 5 rats in each group.

Figure 7

Angiotensin peptide plasma expression levels. Equilibrium levels of (a) Ang I (1–10) and Ang II (1–8), (b) And III (2–8), and Ang-(1–7), and (c) Ang IV (3–8) and Ang 1–5. There were no significant differences between fasted and postprandial rats; p-values are given for each graph. ACE, angiotensin converting enzyme; APA, aminopeptidase A; APN, aminopeptidase N.

Figure 8

Aldosterone levels and ACE and renin activity. (a) Summary graph of aldosterone level in the fasting and postprandial rats. (b) Ratio of aldosterone and Ang II (AA2 ratio), representative of changes in the adrenal response to Ang II. (c) The ratio of Ang II/Ang I demonstrating angiotensin-converting enzyme (ACE) activity and (d) the sum of Ang I and Ang II giving circulating plasma renin activity (PRA). There were no significant differences between fasted and postprandial rats; p values are given for each graph.