Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Molecular Mechanisms of Renal Magnesium Reabsorption

Magnesium is an essential cofactor in many cellular processes, and aberrations in magnesium homeostasis can have life-threatening consequences. The kidney plays a central role in maintaining serum magnesium within a narrow range (0.70 to 1.10 mmol/L). Along the proximal tubule and thick ascending limbs, magnesium reabsorption occurs via paracellular pathways. Members of the claudin family form the magnesium pores in these segments, and also regulate magnesium reabsorption by adjusting the transepithelial voltage that drives it. Along the distal convoluted tubule transcellular reabsorption via heteromeric TRPM6/7 channels predominates, though paracellular reabsorption may also occur. In this segment, the NaCl cotransporter plays a critical role in determining transcellular magnesium reabsorption. While the general machinery involved in renal magnesium reabsorption has been identified by studying genetic forms of magnesium imbalance, the mechanisms regulating it are poorly understood. This review discusses pathways of renal magnesium reabsorption by different segments of the nephron, emphasizing newer findings that provide insight into regulatory process, and outlining critical unanswered questions.

Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality

Absorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na+/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.

Na+-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na+ Recycling in Mouse Small Intestine

Many nutrients are absorbed via Na+ cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na+. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support Na+ cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient (cldn15−/−) mice, which have been shown to have decreased paracellular Na+ permeability. We measured glucose-induced currents (ΔIsc) under open- and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJNa) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in ΔIsc and unidirectional mucosal to serosal 22Na+ (∆JNaMS) flux in both wild-type and cldn15−/− mice. However, under open-circuit conditions, ΔIsc was observed but ∆JNaMS was strongly inhibited in wild-type but not in cldn15−/− mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na+ conductance was decreased and glucose-induced ∆JNaMS increment was observed under open-circuit conditions. We concluded that the Na+ which is absorbed by Na+-dependent glucose cotransport is recycled back into the lumen via paracellular Na+ conductance through claudin-15, which is driven by Na+ cotransport induced luminal negativity.

Passing the Vascular Barrier: Endothelial Signaling Processes Controlling Extravasation

A central function of the vascular endothelium is to serve as a barrier between the blood and the surrounding tissue of the body. At the same time, solutes and cells have to pass the endothelium to leave or to enter the bloodstream to maintain homeostasis. Under pathological conditions, for example, inflammation, permeability for fluid and cells is largely increased in the affected area, thereby facilitating host defense. To appropriately function as a regulated permeability filter, the endothelium uses various mechanisms to allow solutes and cells to pass the endothelial layer. These include transcellular and paracellular pathways of which the latter requires remodeling of intercellular junctions for its regulation. This review provides an overview on endothelial barrier regulation and focuses on the endothelial signaling mechanisms controlling the opening and closing of paracellular pathways for solutes and cells such as leukocytes and metastasizing tumor cells.

EB 2017 Article: Interpretation of the lactulose:mannitol test in rural Malawian children at risk for perturbations in intestinal permeability

The dual sugar absorption test, specifically the lactulose:mannitol test, is used to assess gut health. Lactulose absorption is said to represent gut damage and mannitol absorption is used as a measure of normal small bowel function and serves as normalizing factor for lactulose. A underappreciated limitation of this common understanding of the lactulose:mannitol test is that mannitol is not absorbed to any substantial extent by a transcellular process. Additionally, this interpretation of lactulose:mannitol is not consistent with current understanding of paracellular pathways, where three pathway types exist: pore, leak, and unrestricted. Pore and leak pathways are regulated biological constructions of the small bowel barrier, and unrestricted pathways represent micropathological damage. We analyzed 2334 lactulose:mannitol measurements rigorously collected from 622 young rural Malawian children at high risk for poor gut health in light of the pathway model. An alternative method of normalizing for gut length utilizing autopsy data is described. In our population, absorbed lactulose and mannitol are strongly correlated, r = 0.68 P <0.0001, suggesting lactulose and mannitol are traversing the gut barrier via the same pathways. Considering measurements where pore pathways predominate, mannitol flux is about 14 times that of lactulose. As more leak pathways are present, this differential flux mannitol:lactulose falls to 8:1 and when increased numbers of unrestricted pathways are present, the differential flux of mannitol:lactulose is 6:1. There was no substantial correlation between the lactulose:mannitol and linear growth. Given that mannitol will always pass through a given pathway at a rate at least equal to that of lactulose, and lactulose absorption is a composite measure of flux through both physiologic and pathologic pathways, we question the utility of the lactulose:mannitol test. We suggest using lactulose alone is as informative as lactulose:mannitol in a sugar absorption testing in subclinical gut inflammation. Impact statement Our work integrates the standard interpretation of the lactulose:mannitol test (L:M), with mechanistic insight of intestinal permeability. There are three paracellular pathways in the gut epithelium; pore, leak, and unrestricted. Using thousands of L:M measurements from rural Malawian children at risk for increased intestinal permeability, we predict the differential flux of L and M through the pathways. Our findings challenge the traditional notions that little L is absorbed through a normal epithelial barrier and that M is a normalizing factor for L. Our observations are consistent with pore pathways allowing only M to pass. And that substantial amounts of L and M pass through leak pathways which are normal, regulated, cell-junctional adaptations. So M is a composite measure of all pathways, and L is not a measure solely of pathologic gut damage. Using L alone as a probe will yield more information about gut health than L:M.