Low-salt diet induces claudin-3 expression and drives adaptive changes in collecting duct of claudin-3-deficient mice

Abstract

BackgroundRenal sodium reabsorption occurs via both transcellular and paracellular pathways. Tight junction proteins play a key role in mediating paracellular transport. The collecting duct (CD) is critical for the fine-tuning of Na+ balance and is sensitive to changes in dietary salt intake. A low-sodium diet, which increases endogenous aldosterone secretion, stimulates transcellular sodium transport via epithelial Na+ channels (ENaC) and Na,K-ATPase. We hypothesized that a low-sodium diet also modulates paracellular Na+ permeability by regulating the expression or function of claudin-3, a major tight junction protein in the CD, in order to limit the back-leak of reabsorbed sodium and preserve sodium balance. MethodsWe used in vivo mouse models and cultured mouse CD principal cells (mCCDcl1) to assess aldosterones effects on tight junction proteins. In mCCDcl1 cells, aldosterone-induced changes in claudin-3 expression and localization were evaluated via Western blotting and immunofluorescence, and Ussing chamber assays were used to assess paracellular Na+ and Cl- permeability after modulating claudin-3 expression. Wild-type and claudin-3 knockout mice were fed low (0.01%) or normal (0.18%) sodium diets for seven days. In subsets of low sodium diet mice, spironolactone (a mineralocorticoid receptor antagonist) was administered. ResultsIn mice, a low-sodium diet upregulates renal claudin-3 expression. Concordantly, in vitro studies using mCCDcl1 cells showed that aldosterone treatment increased claudin-3 protein levels and promoted its localization to the lateral membrane. Functional analyses demonstrated that claudin-3 overexpression reduced paracellular permeability to both Na+ and Cl-, while claudin-3 silencing increased it. Claudin-3 knockout mice subjected to a low-sodium diet exhibited compensatory upregulation of the - and {gamma}-subunits of ENaC, alongside increased expression of claudin-4, claudin-8, and claudin-10. This highlights an adaptive response that maintains sodium homeostasis in the absence of claudin-3. Importantly, this compensatory mechanism persists even under spironolactone treatment, suggesting that the adaptation of claudin-3-deficient mice occurs independently of mineralocorticoid receptor activation. ConclusionsOur findings demonstrate that aldosterone enhances claudin-3 expression, reinforcing the paracellular barrier to Na+ and complementing its classical role in transcellular Na+ transport. Under low-sodium conditions, claudin-3-deficient mice adapt through complementary mechanisms aimed at increasing sodium reabsorption via ENaC activation and upregulation of claudin-4 and claudin-8, both barrier-forming claudins that restrict paracellular sodium leakage in the CD. This is associated with increased claudin-10 abundance in the thick ascending limb of Henle, a pore-forming claudin that facilitates paracellular sodium permeability. This study advances our understanding of the complex control of renal sodium handling, revealing adaptive mechanisms in response to low-salt diet and claudin-3 deficiency.