Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules.
ABSTRACT: Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.
Project description:The blood-testis barrier includes strands of tight junctions between somatic Sertoli cells that restricts solutes from crossing the paracellular space, creating a microenvironment within seminiferous tubules and providing immune privilege to meiotic and postmeiotic cells. Large cysts of germ cells transit the Sertoli cell tight junctions (SCTJs) without compromising their integrity. We used confocal microscopy to visualize SCTJ components during germ cell cyst migration across the SCTJs. Cysts become enclosed within a network of transient compartments fully bounded by old and new tight junctions. Dissolution of the old tight junctions releases the germ cells into the adluminal compartment, thus completing transit across the blood-testis barrier. Claudin 3, a tight junction protein, is transiently incorporated into new tight junctions and then replaced by claudin 11.
Project description:Claudins are tight junction membrane proteins that regulate paracellular permeability to ions and solutes in many physiological systems. The electric property of claudin is the most interesting and pertains to two important organ functions: the renal and sensorineural functions. The kidney comprises of three major segments of epithelial tubules with different paracellular permeabilities: the proximal tubule (PT), the thick acending limb of Henle's loop (TALH) and the collecting duct (CD). Claudins act as ion channels allowing selective permeation of Na(+) in the PT, Ca(2+) and Mg(2+) in the TALH and Cl(-) in the CD. The inner ear, on the other hand, expresses claudins as a barrier to block K(+) permeation between endolymph and perilymph. The permeability properties of claudins in different organs can be attributed to claudin interaction within the cell membrane and between neighboring cells. The first extracellular loop of claudins contains determinants of paracellular ionic permeability. While analogous to transmembrane ion channels in many ways, the biophysical and biochemical properties of claudin based paracellular channels remain to be fully characterized.
Project description:Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue- and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na(+), similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms.
Project description:Claudins constitute the major component of tight junctions and regulate paracellular permeability of epithelia. Claudin-10 occurs in two major isoforms that form paracellular channels with ion selectivity. We report on two families segregating an autosomal recessive disorder characterized by generalized anhidrosis, severe heat intolerance and mild kidney failure. All affected individuals carry a rare homozygous missense mutation c.144C>G, p.(N48K) specific for the claudin-10b isoform. Immunostaining of sweat glands from patients suggested that the disease is associated with reduced levels of claudin-10b in the plasma membranes and in canaliculi of the secretory portion. Expression of claudin-10b N48K in a 3D cell model of sweat secretion indicated perturbed paracellular Na+ transport. Analysis of paracellular permeability revealed that claudin-10b N48K maintained cation over anion selectivity but with a reduced general ion conductance. Furthermore, freeze fracture electron microscopy showed that claudin-10b N48K was associated with impaired tight junction strand formation and altered cis-oligomer formation. These data suggest that claudin-10b N48K causes anhidrosis and our findings are consistent with a combined effect from perturbed TJ function and increased degradation of claudin-10b N48K in the sweat glands. Furthermore, affected individuals present with Mg2+ retention, secondary hyperparathyroidism and mild kidney failure that suggest a disturbed reabsorption of cations in the kidneys. These renal-derived features recapitulate several phenotypic aspects detected in mice with kidney specific loss of both claudin-10 isoforms. Our study adds to the spectrum of phenotypes caused by tight junction proteins and demonstrates a pivotal role for claudin-10b in maintaining paracellular Na+ permeability for sweat production and kidney function.
Project description:The tight junction is an important subcellular organelle which plays a vital role in epithelial barrier function. Claudin, as the integral membrane component of tight junctions, creates a paracellular transport pathway for various ions to be reabsorbed by the kidneys. This review summarizes advances in claudin structure, function and pathophysiology in kidney diseases. Different claudin species confer selective paracellular permeability to each of three major renal tubular segments: the proximal tubule, the thick ascending limb of Henle's loop and the distal nephron. Defects in claudin function can cause a wide spectrum of kidney diseases, such as hypomagnesemia, hypercalciuria, kidney stones and hypertension. Studies using transgenic mouse models with claudin mutations have recapitulated several of these renal disease phenotypes and have elucidated the underlying biological mechanisms. Modern recording approaches based upon scanning ion conductance microscopy may resolve the biophysical nature of claudin transport function and provide novel insight into tight junction architecture.
Project description:Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. The paracellular pathway in the collecting duct of the kidney is a predominant route for transepithelial chloride reabsorption that determines the extracellular NaCl content and the blood pressure. However, the molecular mechanisms underlying the paracellular chloride reabsorption in the collecting duct are not understood. Here we showed that in mouse kidney collecting duct cells, claudin-4 functioned as a Cl(-) channel. A positively charged lysine residue at position 65 of claudin-4 was critical for its anion selectivity. Claudin-4 was observed to interact with claudin-8 using several criteria. In the collecting duct cells, the assembly of claudin-4 into TJ strands required its interaction with claudin-8. Depletion of claudin-8 resulted in the loss of paracellular chloride conductance, through a mechanism involving its recruitment of claudin-4 during TJ assembly. Together, our data show that claudin-4 interacts with claudin-8 and that their association is required for the anion-selective paracellular pathway in the collecting duct, suggesting a mechanism for coupling chloride reabsorption with sodium reabsorption in the collecting duct.
Project description:Claudins are discovered to be key players in renal epithelial physiology. They are involved in developmental, physiological, and pathophysiological differentiation. In the glomerular podocytes, claudin-1 is an important determinant of cell junction fate. In the proximal tubule, claudin-2 plays important roles in paracellular salt reabsorption. In the thick ascending limb, claudin-14, -16, and -19 regulate the paracellular reabsorption of calcium and magnesium. Recessive mutations in claudin-16 or -19 cause an inherited calcium and magnesium losing disease. Synonymous variants in claudin-14 have been associated with hypercalciuric nephrolithiasis by genome-wide association studies (GWASs). More importantly, claudin-14 gene expression can be regulated by extracellular calcium levels via the calcium sensing receptor. In the distal tubules, claudin-4 and -8 form paracellular chloride pathway to facilitate electrogenic sodium reabsorption. Aldosterone, WNK4, Cap1, and KLHL3 are powerful regulators of claudin and the paracellular chloride permeability. The lessons learned on claudins from the kidney will have a broader impact on tight junction biology in other epithelia and endothelia.
Project description:Efficient oxygen utilization in the kidney may be supported by paracellular epithelial transport, a form of passive diffusion that is driven by preexisting transepithelial electrochemical gradients. Claudins are tight-junction transmembrane proteins that act as paracellular ion channels in epithelial cells. In the proximal tubule (PT) of the kidney, claudin-2 mediates paracellular sodium reabsorption. Here, we used murine models to investigate the role of claudin-2 in maintaining energy efficiency in the kidney. We found that claudin-2-null mice conserve sodium to the same extent as WT mice, even during profound dietary sodium depletion, as a result of the upregulation of transcellular Na-K-2Cl transport activity in the thick ascending limb of Henle. We hypothesized that shifting sodium transport to transcellular pathways would lead to increased whole-kidney oxygen consumption. Indeed, compared with control animals, oxygen consumption in the kidneys of claudin-2-null mice was markedly increased, resulting in medullary hypoxia. Furthermore, tubular injury in kidneys subjected to bilateral renal ischemia-reperfusion injury was more severe in the absence of claudin-2. Our results indicate that paracellular transport in the PT is required for efficient utilization of oxygen in the service of sodium transport. We speculate that paracellular permeability may have evolved as a general strategy in epithelial tissues to maximize energy efficiency.
Project description:Claudins (CLDNs) play crucial roles in the formation of tight junctions. We have reported that abnormal expression of CLDN2 confers chemoresistance in the spheroids of human lung adenocarcinoma A549 cells. A food composition, which can reduce CLDN2 expression, may function to prevent the malignant progression. Here, we found that ethanol extract of Brazilian green propolis (EBGP) and kaempferide, a major component of EBGP, decrease CLDN2 expression. In the two-dimensional culture model, EBGP decreased the tight junctional localization of CLDN2 without affecting that of zonula occludens-1, an adaptor protein, and enhanced paracellular permeability to doxorubicin, a cytotoxic anticancer drug. EBGP reduced hypoxic stress, and enhanced the accumulation and sensitivity of doxorubicin in the spheroid of A549 cells. Kaempferide dose-dependently decreased CLDN2 expression, although dihydrokaempferide and pinocembrin did not. The phosphorylation of Akt, a regulatory factor of CLDN2 expression, was inhibited by kaempferide but not by dihydrokaempferide. The 2,3-double bond in the C ring may be important to inhibit Akt. Kaempferide decreased the mRNA level and promoter activity of CLDN2, indicating that it inhibits the transcription of CLDN2. In accordance with EBGP, kaempferide decreased the tight junctional localization of CLDN2 and increased a paracellular permeability to doxorubicin, suggesting that it diminished the paracellular barrier to small molecules. In addition, kaempferide reduced hypoxic stress, and enhanced the accumulation and sensitivity of doxorubicin in the spheroids. In contrast, dihydrokaempferide did not improve the sensitivity to doxorubicin. Further study is needed using an animal model, but we suggest that natural foods abundantly containing kaempferide are candidates for the prevention of the chemoresistance of lung adenocarcinoma.
Project description:In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henle's loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10-deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.