The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant.