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The Role of Tight Junction Proteins in Water and Electrolyte Transport

Reviewer and Curator: Larry H. Bernstein, MD, FCAP 

This article is Part II of a series that explores the physiology, genomics, and the proteomics of water and electrolytes in human and mammalian function in health and disease.  In this portion of curation, we examine the role of special proteins at the tight junctions of cells, including the claudins.  Consistent with the exploration of cation homeostasis, the last featured article is one of the altered handling of calcium (Ca2+) in CHF, and the closely regulated calcium efflux by the sodium-calcium exchanger (NCX).

The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio).

Kwong RW, Kumai Y, Perry SF.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
PLoS One. 2013 Aug 14;8(8):e70764.
http://dx.doi.org/10.1371/journal.pone.0070764   eCollection 2013.

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio).

We observed that the half-time for saturation of water influx (K u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H(+)-ATPase-rich cells or Na(+)/K(+)-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation.

Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca(2+)-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

PMID:  23967101  PMCID: PMC3743848    http://www.ncbi.nlm.nih.gov/pubmed/23967101

The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio.

Kwong RW, Perry SF.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada. wkwong@uottawa.ca
Am J Physiol Regul Integr Comp Physiol. Apr 1, 2013; 304(7):R504-13. http://dx.doi.org/10.1152/ajpregu.00385.2012  Epub 2013 Jan 30.

The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac.

Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate-dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression.

Additionally, Na(+) efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na(+) levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na(+). Nevertheless, the elevated loss of Na(+) in the morphants was compensated by an increase in Na(+) uptake.

Notably, we observed that the increased Na(+) uptake in the morphants was attenuated in the presence of the selective Na(+)/Cl(-)-cotransporter (NCC) inhibitor metolazone, or during exposure to Cl(-)-free water. These results suggested that the increased Na(+) uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H(+)-ATPase inhibitor bafilomycin A1 was found to reduce Na(+) uptake in the morphants, suggesting that H(+)-ATPase activity was essential to provide a driving force for Na(+) uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na(+) handling in zebrafish.
PMID: 23364531   http://www.ncbi.nlm.nih.gov/pubmed/23364531

Evidence for a role of tight junctions in regulating sodium permeability in zebrafish (Danio rerio) acclimated to ion-poor water.

Kwong RW, Kumai Y, Perry SF.
Department of Biology, University of Ottawa, Ottawa, ON, Canada. wkwong@uottawa.ca
J Comp Physiol B. Feb 2013 ;183(2):203-13.
http://dx.doi.org/10.1007/s00360-012-0700-9  Epub 2012 Jul 29.

Freshwater teleosts are challenged by diffusive ion loss across permeable epithelia including gills and skin. Although the mechanisms regulating ion loss are poorly understood, a significant component is thought to involve paracellular efflux through pathways formed via tight junction proteins. The mammalian orthologue (claudin-4) of zebrafish (Danio rerio) tight junction protein, claudin-b, has been proposed to form a cation-selective barrier regulating the paracellular loss of Na(+).

The present study investigated the cellular localization and regulation of claudin-b, as well as its potential contribution to Na(+) homeostasis in adult zebrafish acclimated to ion-poor water. Using a green fluorescent protein-expressing line of transgenic zebrafish, we found that claudin-b was expressed along the lamellar epithelium as well as on the filament in the inter-lamellar regions. Co-localization of claudin-b and Na(+)/K(+)-ATPase was observed, suggesting its interaction with mitochondrion-rich cells. Claudin-b also appeared to be associated with other cell types, including the pavement cells. In the kidney, claudin-b was expressed predominantly in the collecting tubules. In addition,

exposure to ion-poor water caused a significant increase in claudin-b abundance as well as a decrease in Na(+) efflux, suggesting a possible role for claudin-b in regulating paracellular Na(+) loss. Interestingly, the whole-body uptake of a paracellular permeability marker, polyethylene glycol-400, increased significantly after prolonged exposure to ion-poor water, indicating that an increase in epithelial permeability is not necessarily coupled with an increase in passive Na(+) loss. Overall, our study suggests that in ion-poor conditions, claudin-b may contribute to a selective reduction in passive Na(+) loss in zebrafish.
PMID: 22843140   http://www.ncbi.nlm.nih.gov/pubmed/22843140

Claudin-16 and claudin-19 function in the thick ascending limb.

Hou J, Goodenough DA.
Washington University School of Medicine, Div Renal Diseases, St Louis, Missouri
Curr Opin Nephrol Hypertens. Sep 2010; 19(5):483-8. http://dx.doi.org/10.1097/MNH.0b013e32833b7125.

The thick ascending limb (TAL) of the loop of Henle is responsible for reabsorbing 25–40% of filtered Na⁺, 50–60% of filtered Mg²⁺ and 30–35% of filtered Ca²⁺. The dissociation of salt and water reabsorption in the TAL serves both to dilute the urine and to establish the corticomedullary osmolality gradient. Active transcellular salt reabsorption results in a lumen-positive transepithelial voltage that drives passive paracellular reabsorption of divalent cations. Claudins are the key components of the paracellular channel. The paracellular channels in the tight junction have properties of ion selectivity, pH dependence and anomalous mole fraction effects, similar to conventional transmembrane channels. Genetic mutations in claudin-16 and claudin-19 cause an inherited human renal disorder, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC).

In the TAL of Henle’s loop, the epithelial cells form a water-impermeable barrier, actively transport Na⁺ and Cl− via the transcellular route, and provide a paracellular pathway for the selective absorption of cations. Na+ K⁺ and Cl− enter the cell through the Na-K-2Cl cotransporter (NKCC2) in the luminal membrane. Na⁺ exits the cell through the Na⁺/K⁺-ATPase, in exchange for K⁺ entry. K⁺ is secreted into the lumen through the renal outer medullary potassium channel. Cl− leaves the cell through the basolateral Cl− channel, made up of two subunits, ClCKb and barttin. The polarized distribution of luminal K⁺ versus basolateral Cl− conductance generates a spontaneous voltage source _(Vsp) of +7−8mV , depending on active transcellular NaCl reabsorption. With continuous NaCl reabsorption along the axis of the TAL segment, the luminal fluid is diluted to 30–60mmol/l  and a large NaCl transepithelial chemical gradient develops at the end of the TAL. Because the paracellular permeability of the TAL is cation-selective (with a _PNa/PCl value between 2 and 4), the diffusion voltage _(Vdi) is superimposed onto the active transport voltage _(Vsp) and becomes the major source of the transepithelial voltage (V_te), which now increases up to +30mV.

Early in-vivo micropuncture studies have shown that approximately 50–60% of the filtered Mg²⁺ is reabsorbed in the TAL. The flux–voltage relationship indicates that Mg²⁺ is passively reabsorbed from the lumen to the peritubular space through the paracellular pathway in this segment, driven by a lumen positive _Vte.  Vte is made of the sum of Vsp ^and _Vdi. There are two prerequisites required for the paracellular Mg²⁺ reabsorption in the TAL: the lumen-positive _Vte as the driving force and the paracellular permeability for the divalent cation Mg²⁺.

Claudin-16 and claudin-19 underlie familial hypercalciuric hypomagnesemia with nephrocalcinosis

Claudin-16 and claudin-19 play a major role in the regulation of magnesium reabsorption in the thick ascending limb (TAL). This review describes recent findings of the physiological function of claudin-16 and claudin-19 underlying normal transport function for magnesium reabsorption in the TAL. Mutations in the genes encoding the tight junction proteins claudin-16 and claudin-19 cause the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. FHHNC, OMIM #248250, is a rare autosomal recessive tubular disorder. As a consequence of excessive renal Mg²⁺ and Ca²⁺ wasting, patients develop the characteristic triad of hypomagnesemia, hypercalciuria and nephrocalcinosis. Recurrent urinary tract infections and polyuria/polydipsia are frequent initial symptoms. Other clinical symptoms include nephrolithiasis, abdominal pain, convulsions, muscular tetany, and failure to thrive. Additional laboratory findings include elevated serum parathyroid hormone levels before the onset of chronic renal failure, incomplete distal tubular acidosis, hypocitraturia, and hyperuricemia. In contrast to hypomagnesemia and secondary hypocalcemia (HSH, OMIM #602014), FHHNC is generally complicated by end-stage renal failure in early childhood or adolescence.

Simon et al. used the positional cloning strategy and identified claudin-16 (formerly known as paracellin-1), which is mutated in patients with FHHNC. Most mutations reported to date in claudin-16 are missense mutations clustering in the first extracellular loop composing the putative ion selectivity filter. Konrad et al. have found mutations in another tight junction gene encoding claudin-19 from new cohorts of FHHNC patients (OMIM #248190). The renal tubular phenotypes are indistinguishable between patients with mutations in claudin-16 and those with mutations in claudin-19. Although claudin-16 and claudin-19 underlie FHHNC and paracellular Mg²⁺ reabsorption in the TAL, the transient receptor potential channel melastatin 6 (TRPM6) regulates the apical entry of Mg²⁺ into the distal convoluted tubule epithelia. Mutations in TRPM6 cause the HSH syndrome.

These above data suggested the hypothesis that claudin-16 and/or claudin-19 forms a selective paracellular Mg²⁺/Ca²⁺ channel, which was tested in a number of in-vitro studies. Ikari et al. transfected low-resistance Madin-Darby canine kidney (MDCK) cells with claudin-16 and reported that the Ca²⁺ flux in these cells was increased in the apical to basolateral direction, whereas the Ca²⁺ flux in the opposite direction remained unchanged. The Mg²⁺ flux was without any noticeable change. Kausalya et al.  transfected the high-resistance MDCK-C7 cell line and found that claudin-16 only moderately increased Mg²⁺ permeability without any directional preference. The effects of claudin-16 on Mg²⁺/Ca²⁺ permeation appeared so small (<50%) that the Mg²⁺/Ca²⁺ channel theory incompletely explains the dramatic effect of mutations in claudin-16 on Mg²⁺ and Ca²⁺ homoeostasis in FHHNC patients. However, , Hou et al.  transfected the anion-selective LLC-PK1 cell line with claudin-16 and found a large increase in Na⁺ permeability _(PNa) accompanied by a moderately enhanced Mg²⁺ permeability (PM_g). The permeability of claudin-16 to other alkali metal cations was found to be: K⁺ > Rb⁺ > Na+.  Yu et al. emphasized that these residue replacements can influence protein structures that may have impacts on ion permeability independent of amino acid charge.

The cation selectivity of the tight junction is vital for generating the lumen positive transepithelial potential in the TAL, which drives paracellular absorption of magnesium. Claudin-16 and claudin-19 require each other for assembly into tight junctions in the TAL. Heteromeric claudin-16 and claudin-19 interaction forms a cation selective tight junction paracellular channel. Loss of either claudin-16 or claudin-19 in the mouse kidney abolishes the cation selectivity for the TAL paracellular pathway, leading to excessive renal wasting of magnesium.

Claudins interact with each other both intracellularly and intercellularly: they copolymerize linearly within the plasma membrane of the cell, together with the integral protein occludin, to form the classical intramembrane fibrils or strands visible in freeze-fracture replicas. These intramembrane interactions (side-to-side) can involve one claudin protein (homomeric or homopolymeric) or different claudins (heteromeric or heteropolymeric). In the formation of the intercellular junction, claudins may interact head-to-head with claudins in an adjacent cell, generating both homotypic and heterotypic claudin–claudin interactions. Using the split-ubiquitin yeast 2-hybrid assay, Hou et al. found strong claudin-16 and claudin-19 heteromeric interaction. The point mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) or claudin-19 (L90P and G123R) that are known to cause human FHHNC disrupted the claudin-16 and claudin-19 heteromeric interaction. In mammalian cells such as the human embryonic kidney 293 cells, claudin-16 can be coimmunoprecipitated with claudin-19. Freeze-fracture replicas revealed the assembly of tight junction strands in L cells coexpressing claudin-16 and claudin-19, supporting their heteromeric interaction.

Coexpression of claudin-16 and claudin-19 in LLC-PK1 cells resulted in a dramatic upregulation of _PNa and down-regulation of _PCl, generating a highly cation-selective paracellular pathway. Certain FHHNC mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) or claudin-19 (L90P and G123R) that disrupted their heteromeric interaction abolished this physiological change. As claudin-16 colocalizes with claudin-19 in the TAL epithelia of the kidney, claudin-16 and claudin-19 association through heteromeric interactions confers cation selectivity to the tight junction in the TAL. Human FHHNC mutations in claudin-16 or claudin-19 that abolish the cation selectivity diminish the lumen-positive _Vdi as the driving force for Mg²⁺ and Ca²⁺ reabsorption, readily explaining the devastating phenotypes in FHHNC patients.  Hou et al. generated claudin-16 deficient mouse models using lentiviral transgenesis of siRNA to knock down claudin-16 expression by more than 99% in mouse kidneys. Claudin-16 knockdown mice show significantly reduced plasma Mg²⁺ levels and excessive urinary excretions (approximately four-fold) of Mg²⁺ and Ca²⁺. Calcium deposits are observed in the basement membranes of the medullary tubules and the interstitium in the kidney of claudin-16 knockdown mice. These phenotypes of claudin-16 knockdown mice recapitulate the symptoms in human FHHNC patients.

The paracellular reabsorption of Mg²⁺ and Ca²⁺ is driven by a lumen-positive _Vte made up of two components: _Vsp ^and _Vdi. When isolated TAL segments were perfused ex vivo with symmetrical NaCl solutions, there was no difference in _Vsp between claudin-16 knockdown and wild-type mice, indicating _Vsp was normal in claudin-16 knockdown. Blocking the NKCC2 channel with furosemide (thus dissipating _VSP), the cation selectivity _(PNa/PCl) was significantly reduced from3.1 ± 0.3 in wild type to 1.5 ± 0.1 in claudin-16 knockdown, resulting in the loss of _Vdi. When perfused with a NaCl gradient of 145mmol/l (bath) versus 30mmol/l (lumen), the resulting _Vdi was +18mV in wild type, but only +6.6mV in claudin-16 knockdown. Thus, the reduction in _Vdi accounted for a substantive loss of the driving force for Mg²⁺ and Ca²⁺ reabsorption.

Renal handling of Na⁺ in claudin-16 knockdown mice is more complex. In the early TAL segment, the transcellular and paracellular pathways form a current loop in which the currents traversing the two pathways are of equal size but opposite direction. Net luminal K⁺ secretion and basolateral Cl− absorption polarize the TAL epithelium and generate _Vsp. As the paracellular pathway is cation selective _{(PNa/PCl=2–4} , the majority of the current driven by _Vsp through the paracellular pathway is carried by Na⁺ moving from the lumen to the interstitium. Hebert et al. estimated that, for each Na⁺ absorbed through the trans-cellular pathway, one Na⁺ is absorbed through the paracellular pathway. With the loss of claudin-16 and the concomitant loss of paracellular cation selectivity, Na⁺ absorption through the paracellular pathway is reduced.  In the late TAL segment, dilution of NaCl in the luminal space creates an increasing chemical transepithelial gradient; back diffusion of Na⁺ through the cation-selective tight junction generates a lumen-positive _Vdi across the epithelium. The paracellular absorption of Na⁺ will be diminished when _Vdi ^equals _Vsp, and reversed when _Vdi exceeds Vsp. As an equilibrium potential, _Vdi blocks further Na⁺ backleak into the lumen. Without claudin-16, _Vdi will be markedly reduced well below normal, providing a driving force for substantial Na⁺ secretion. Indeed, claudin-16 knockdown mice had increased fractional excretion of Na⁺ (FEN_a) and developed hypotension and secondary hyperaldosteronism. The observed Na⁺ and volume loss are consistent with human FHHNC phenotypes. For example, polyuria and polydipsia are the most frequently reported symptoms from FHHNC patients.

Epithelial paracellular channels are increasingly understood to be formed from claudin oligomeric complexes. In the mouse TAL, claudin-16 and claudin-19 cooperate to form cation-selective paracellular channels required for normal levels of magnesium reabsorption. Different subsets of the claudin family of tight junction proteins are found distributed throughout the nephron, and understanding their roles in paracellular ion transport will be fundamental to understanding renal ion homeostasis.

Keywords: claudin; hypomagnesemia; thick ascending limb; tight junction; transepithelial voltage.     PMID: 20616717  PMCID: PMC3378375  http://www.ncbi.nlm.nih.gov/pubmed/20616717

Function and regulation of claudins in the thick ascending limb of Henle.

Günzel D, Yu AS.
Depart Clin Physiol, Charité, Campus Benjamin Franklin, Berlin, Germany.
Pflugers Arch. May 2009; 458(1):77-88.
http://dx.doi.org/10.1007/s00424-008-0589-z  Epub 2008 Sep 16.

The thick ascending limb (TAL) of Henle mediates transcellular reabsorption of NaCl while generating a lumen-positive voltage that drives passive paracellular reabsorption of divalent cations. Disturbance of paracellular reabsorption leads to Ca(2+) and Mg(2+) wasting in patients with the rare inherited disorder of familial hypercalciuric hypomagnesemia with nephrocalcinosis (FHHNC). Recent work has shown that the claudin family of tight junction proteins form paracellular pores and determine the ion selectivity of paracellular permeability. Importantly, FHHNC has been found to be caused by mutations in two of these genes, claudin-16 and claudin-19, and mice with knockdown of claudin-16 reproduce many of the features of FHHNC. Here, we review the physiology of TAL ion transport, present the current view of the role and mechanism of claudins in determining paracellular permeability, and discuss the possible pathogenic mechanisms responsible for FHHNC.

Tight junctions form the paracellular barrier in epithelia. Claudins are ~22 kDa proteins that were first identified by Mikio Furuse in the laboratory of the late Shoichiro Tsukita as proteins that copurified in a tight junction fraction from the chicken liver [23]. The observation that they were transmembrane proteins with 4 predicted membrane domains and 2 extracellular domains raised early on the possibility that they could play a key role in intercellular adhesion and formation of the paracellular barrier. In 1999, Richard Lifton’s group identified mutations in a novel gene, which they called paracellin, as the cause of familial hypercalciuric hypomagnesemia, an inherited disorder believed to be due to failure of paracellular reabsorption of divalent cations in the thick ascending limb of the renal tubule. Paracellin turned out to be a claudin family member (claudin-16). This suggested that claudins in general might be directly involved in regulating paracellular transport in all epithelia. This is now supported by numerous studies demonstrating that overexpressing or ablating expression of various claudin isoforms in cultured cell lines or in mice affects both the degree of paracellular permeability and its selectivity (vide infra). Furthermore, in mammals alone there are ~24 claudin genes and each exhibits a distinct tissue-specific, pattern of expression. Thus, the specific claudin isoform(s) expressed in each tissue might explain its paracellular permeability properties.

Each nephron segment expresses a unique set of multiple claudin isoforms, and each isoform is expressed in multiple segments, thus making a complicated picture which even varies between different species. The role of combinations of claudins in determining paracellular permeability properties has hardly been studied yet. In mouse, rabbit and cattle, the thick ascending limb of Henle’s loop is thought to express claudins 3, 10, 11, 16  in adulthood, and, at least in mouse, additionally claudin-6 during development. In addition, claudin-4 has been found in cattle and claudin-8 in rabbit. To date, the distribution of Claudin-19 has been investigated in mouse, rat, and man where its presence in the TAL was demonstrated.

The thick ascending limb (TAL) of Henle’s loop, working as “diluting segment” of the nephron, is characterized by two major properties: high transepithelial, resorptive transport of electrolytes and low permeability to water. Major players to achieve electrolyte transport are the apical Na⁺-K⁺-2Cl−symporter (NKCC2), the apical K⁺ channel ROMK, the basolateral Cl− channel (CLC-Kb) together with its subunit barttin and the basolateral Na⁺/K⁺-ATPase . The combined actions of these transport systems have been extensively reviewed and are therefore only briefly summarized here. Na⁺ and Cl− are resorbed by entering the cells apically through NKCC2 and leaving the cells basolaterally through the Na⁺/K⁺-ATPase and CLC-Kb, respectively. In contrast, K⁺ is either recycled across the apical membrane as it is entering through NKCC2 and leaving through ROMK, or even secreted, as it is also entering the cells basolaterally through the Na⁺/K⁺-ATPase. Taking these ion movements together, there is a net movement of positive charge from the basolateral to the apical side of the epithelium, giving rise to a lumen positive voltage (3 – 9 mV [11]; about 5 – 7 mV [30,31]; 7 – 8 mV [57]). Over the length of the TAL, luminal NaCl concentration decreases gradually to concentrations of 30 – 60 mM at the transition to the distal tubule, depending on the flow rate within the tubule (low flow rates resulting in low concentrations).

To keep up such a high gradient, the TAL epithelium has to be tight to water and various studies summarized by Burg and Good report water permeability values from 28 µm/s down to values indistinguishable from zero. Tight junctions of the TAL are, however, highly permeable to cations with PNa being about 2 – 2.7 fold, 2.5 fold or even up to 6 fold that of _PCl. Amongst the monovalent cations, a permeability sequence of PK > PNa > PRb = PLi > PCs > Porganic cation was observed which is similar to Eisenman sequence VIII or IX, indicating a strong interaction between the permeating ion and the paracellular pore that enables at least partial removal of the hydration shell (see below). As reviewed by Burg and Good, the transepithelial sodium and chloride permeabilities, estimated from radioisotope fluxes, are high (in the range of 10 – 63·10−⁶ cm/s) and the transepithelial electrical resistance is correspondingly low (21 – 25 W cm² ; 30 – 40 W cm² ; 11 – 34 W cm² . Blocking active transport by the application of furosemide or ouabain increases transepithelial resistance only slightly, indicating that the low values are primarily due to a very high paracellular permeability. Due to these properties of TAL epithelial cells, Na⁺ ions leak back into the lumen of the tubule, creating a diffusion (dilution) potential that adds another 10 – 15 mV to the lumen positive potential, so that considerable potential differences (25 mV ; 30 mV; cTAL 23 mV, mTAL 17 mV ) may be reached at very slow flow rates.

Considerable proportions of the initially filtrated Mg²⁺ (50 – 60%; 50 – 70%; 65 – 75%) and Ca²⁺ (20%; 30 – 35% are resorbed in the TAL. The transepithelial potential is considered to provide the driving force for the predominantly paracellular resorption of Mg²⁺ and Ca²⁺ as in many studies, transport of both divalent ions in the TAL has been found to be strictly voltage dependent (resorbtive at lumen positive potentials, zero at 0 mV and secretory at lumen negative potentials) and permeability considerable (PCa 7.7·10−⁶ cm/s, i.e. approximately 25% of PNa). There is however, some conflicting evidence, e.g. by Suki et al. and Friedman. Both studies used cTAL (cortical TAL) and found that decreasing the transepithelial potential by applying furosemide did either not alter the unidirectional lumen to bath Ca²⁺ flux (rabbit) or left a substantial net Ca²⁺ resorption (mouse). Similarly, Rocha et al. found that bath application of ouabain almost abolished the transepithelial potential, but hardly affected net Ca²⁺ resorption and conclude that (a) all segments of Henle’s loop are relatively impermeable to calcium and (b) net calcium resorption occurs in the thick ascending limb which cannot be explained by passive mechanisms.  Mandon et al. conclude that both Mg²⁺ and Ca²⁺ are transported in the cTAL but not in the mTAL (medullary TAL) of rat and mouse, although transepithelial potential differences were similar in both segments, and even if the transepithelial potential was experimentally elevated to values above 20 mV. Wittner et al. even found evidence that in mouse mTAL the passive permeability to divalent cations is very low and that Ca²⁺ and Mg²⁺ can be secreted into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. They conclude that this Ca²⁺ and Mg²⁺ transport is most probably of cellular origin. In contrast, in rabbit, both ions are transported along the whole length of the TAL.

Both, Mg²⁺ and Ca²⁺ resorption are modulated through the action of the basolateral Ca (and Mg) sensing receptor (CaSR) which is found along the entire nephron but especially in the loop of Henle, distal convoluted tubule (DCT) and the inner medullary collecting duct. Different modes of action on Ca²⁺ and Mg²⁺ homeostasis exist, such as an indirect action through the modulation of PTH secretion or direct effects on the cells expressing CaSR. In the TAL the latter model is based on the assumptions depicted above, i.e. that Mg²⁺ and Ca²⁺ are resorbed paracellularly, driven by the lumen positive potential, so that a reduction in NaCl resorption causes a reduction in driving force for Mg²⁺ and Ca²⁺ resorption. As reviewed by Hebert and Ward, CaSR is activated through an increase in basolateral Ca²⁺ and/or Mg²⁺ concentration which triggers an increase in the intracellular Ca²⁺ concentration. This reduces the activity of the adenylate cyclase which, in turn, inhibits transcellular transport of Na⁺ and Cl−. In addition, the increase in intracellular Ca²⁺ activates phospholipase A2 (PLA2) and thus increases the intracellular concentration of arachidonic acid and its derivative, 20-HETE. 20-HETE inhibits NKCC2, ROMK and the Na⁺/K⁺-ATPase and by this Mg²⁺ and Ca²⁺ resorption. In keeping with this hypothesis, mutations in CaSR affect Ca/Mg resorption. Inactivating mutations cause hypercalcemia, hypocalciuria, hypomagnesiuria and, in some patients hypermagnesemia. Conversely, activating mutations (gain of function mutations) lead to hypocalcemia, hypercalciuria, hypermagnesiuria and in up to 50% of the patients mild hypomagnesemia.

Bartter syndrome type I (mutations in NKCC2), and type II (mutations in ROMK) lead to hypercalciuria and thus cause nephrocalcinosis, but no hypomagnesemia is observed. Reports on hypermagnesiuria are conflicting: while Kleta and Bockenhauer link it to nephrocalcinosis seen in these patients, Rodriguez-Soriano states that patients with neonatal Bartter syndrome (i.e. type I or II) show a lack of hypermagnesiuria that may be explained by compensation in the DCT. Hypomagnesemia is occationally present in Bartter type III (CLC-Kb). However, here it is believed to be mainly due to effects on DCT, where CLC-Kb shows highest expression. Patients with Bartter syndrome IV (CLC-Kbsubunit barttin) may or may not present nephrocalcinosis, while Mg²⁺ homeostasis appears undisturbed. Interestingly, the largest effects on Mg²⁺-homeostasis are observed in Gitelman syndrome, a defect in the Na⁺/Cl− symport (NCC) predominantly found in the DCT, where Mg²⁺ is transported along the transcellular route. Affected patients suffer from hypomagnesemia, hypermagnesuria and hypocalciuria. The effect on Mg²⁺ in Gitelman syndrome is still poorly understood and possibly due to a concomitant down-regulation of TRPM6, the apical Mg²⁺ uptake channel in DCT.

More than 30 different claudin-16 mutations have now been reported in families with FHHNC. Because of the large number of unique mutations, it has not been possible to identify any clear qualitative correlation between the phenotype and individual mutations, although certain mutations are associated with greater severity of disease. In 2006, a second locus was identified, CLDN19, which encodes claudin-19. In the initial report, the phenotype appeared similar to that due to claudin-16 mutations, with the exception that there was a high prevalence of ocular abnormalities, including macular colobomata, nystagmus and myopia. Claudin-19 is normally expressed at high levels in the retina, but why it causes these ocular disorders is unknown.

In vitro studies of claudin function comprise inducible or non-inducible transfection of various cells lines with cDNA for claudins that are not endogenously expressed by the cell line used. Alternatively, cells can be transfected with siRNA directed against an endogenous claudin. In both cases, cells are then grown to confluence on permeable filter supports that allow measurement of transepithelial permeabilities. Before the results of permeability studies can be interpreted, however, several parameters have to be controlled.

First, special care has to be taken to make sure that the exogenous claudin is correctly inserted into the tight junction. This can be achieved e.g. by confocal laser scanning microscopy, colocalizing the claudin of interest  with a tight junction marker protein such as occludin.

Second, it has to be ensured that endogenous claudin expression remains unaffected, as permeability changes always result from the combined effects of alterations in endogenous and exogenous claudins.

Third, it has to be kept in mind that, typically, epithelia express several different claudins that act together to produce tissue specific permeability properties. Thus, ideally, a cell line should be chosen that provides a claudin background resembling that usually experienced by the claudin investigated. The latter two points may be the reason for contradicting results obtained in permeability studies expressing a specific claudin in different cell lines.

Studies of paracellular permeabilities can be divided into two groups, those employing electrophysiological measurements (e.g. determination of diffusion potentials), and those measuring ion or solute flux, using either radioactive isotopes or various analytical methods to determine the amount transported.

Although transepithelial conductances depend on paracellular permeabilities of the predominant ions in the bath solution, conductance changes alone cannot be used to predict ion permeabilities.

Firstly, conductances always depend on both ion and counter-ion, not on one ion species alone.

Secondly, transepithelial conductances are the sum of the conductances of the transcellular and paracellular pathways.

Thus, they only reflect paracellular permeability, if paracellular conductance dominates transepithelial conductance and if transcellular conductance remains constant throughout the experiment. This, however, is often not the case, as concentration changes of the ions investigated may affect transcellular conductance, e.g. by activating ion transporters or by inhibiting ion channels. Thirdly, the specific conductance of each solution employed may differ and has therefore to be assessed and taken into account. Thus, comparison of results from diffusion potential measurements or flux studies and conductance measurements may even yield contradicting results. For the same reasons, other methods based on pure conductance/resistance measurements, including the more sophisticated conductance scanning method or one-path impedance spectroscopy are not ion specific and do not allow the measurement of paracellular permeabilities to single ions.

In contrast to electrophysiological measurements, flux measurements are not limited to ions but can also be extended to uncharged molecules.  Flux measurements per se do not distinguish between transcellular or paracellular transport. Therefore, to estimate paracellular permeabilities, inhibition or at least estimation of the transcellular flux is necessary. Assuming that transcellular flux for energetic reasons is not easily reversible while paracellular flux is passive and thus generally assumed to be symmetric, the transcellular proportion is often estimated by calculating the difference between apical to basolateral and basolateral to apical fluxes. All flux measurements are very sensitive to the development of diffusion zones (“unstirred layers”) near the cells. These layers are depleted/enriched in the compound transported and thus alter the driving forces acting on these compounds, if bath solutions are not continually circulated. If ionic fluxes are investigated, transepithelial potentials may develop that diminish or completely inhibit the flux investigated.

All the techniques described above have been employed to investigate the function of claudin-16 and -19, especially with respect to their ability to increase paracellular permeability to divalent cations. The hypothesis, that claudin-16 (then called paracellin-1) may be a paracellular Mg²⁺ and Ca²⁺ pore was originally expressed by Simon et al. It was based on the findings that mutations in claudin-16 were the cause of the severe disturbance in Mg²⁺ and Ca²⁺ homeostasis in FHHNC patients together with the observations that claudin-16 is a tight junction protein located in the TAL, i.e. the nephron segment responsible for bulk Mg²⁺ resoption along the paracellular pathway. When, recently, it was found that claudin-19 mutations were underlying hitherto unexplained cases of FHHNC and that claudin-19 co-localized with claudin-16, the hypothesis was extended to claudin-19.

1. Cole DEC, Quamme GA. Inherited disorders of renal magnesium handling. J Am Soc Nephrol 2000;11:1937–1947. [PubMed: 11004227]
2. de Rouffignac C, Quamme G. Renal magnesium handling and its hormonal control. Physiol Rev 1994;74:305–322. [PubMed: 8171116]  
3. Meij IC, van den Heuvel LP, Knoers NV. Genetic disorders of magnesium homeostasis. BioMetals 2002;15:297–307. [PubMed: 12206395]
4. Satoh J, Romero MF. Mg²⁺ transport in the kidney. BioMetals 2002;15:285–295. [PubMed: 12206394]  
5. Schlingmann KP, Konrad M, Seyberth HW. Genetics of hereditary disorders of magnesium homeostasis. Pediatr Nephrol 2004;19:13–25. [PubMed: 14634861]  
6. Simon DB, Lu Y, Choate KA, et al. Paracellin-1, a renal tight junction protein required for paracellular Mg²⁺ resorption. Science 1999;285:103–106. [PubMed: 10390358]

PMID: 18795318  PMCID:  PMC2666100  http://www.ncbi.nlm.nih.gov/pubmed/18795318

Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis.

Breiderhoff T, Himmerkus N, Stuiver M, Mutig K, Will C, Meij IC et al.
Max Delbrück Center for Molec Med, Berlin, Germany. t.breiderhoff@mdc-berlin.de

Erratum in Proc Natl Acad Sci. 2012 Sep 11;109(37):15072.
Proc Natl Acad Sci. Aug 28, 2012; 109(35):14241-6.   http://dx.doi.org/10.1073/pnas.1203834109. Epub 2012 Aug 13.

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.

Whereas regulation of transporters and channels involved in trans-cellular ion transport has been characterized in much detail, the functional and molecular determinants of paracellular ion trans­port in the kidney remain incompletely understood. In the thick ascending limb (TAL) of Henle’s loop, both trans-cellular and paracellular ion transport pathways contribute to reabsorption of Na⁺, Cl⁻, Mg²⁺, and Ca²⁺. Na⁺ and Cl⁻ are reabsorbed mostly transcellularly by the concerted action of chan­nels and transporters. Mutations in five of the genes involved lead to Bartter syndrome, a disorder characterized by salt wasting and polyuria. Whereas Cl⁻ is transported exclusively transcellularly, ∼50% of the Na⁺ load, as well as Ca²⁺ and Mg²⁺, are reabsorbed via paracellular pathways. In the TAL, this paracellular route is highly cation-selective. The paracellular passage is largely controlled by the tight junction (TJ), a supramolecular structure of membrane-spanning proteins, their intracellular adapters, and scaffolding proteins. Claudins, a family comprising 27 members, are the main components of the TJ defining the permeability properties. They interact via their extracellular loops with corre­sponding claudins of the neighboring cell to allow or restrict pas­sage of specific solutes (5, 6). In the kidney, their expression pattern is closely related to the corresponding segment-specific solute reabsorption profile. Several claudins are expressed in the TAL, including claudin-16, -19, -10, -3, and -18 The importance of claudin-16 and -19 in this tissue is documented by mutations in CLDN16 and CLDN19, which cause familial hypomagnesemia, hypercalciuria, and nephrocalcinosis, an autosomal recessive dis­order that leads to end-stage renal disease. The relevance of CLDN16 for paracellular reabsorption of Mg²⁺ and Ca²⁺ was confirmed in mouse models with targeted gene disruption. In addition, claudin-14, expressed in the TAL of mice on a high-calcium diet, was identified as negative regulator of claudin-16 function (15), and sequence variants in CLDN14 have been asso­ciated with human kidney stone disease. The functional significance of claudin-10, which is also ex­pressed in the TAL, remains unclear. This TJ protein is expressed in two isoforms, claudin-10a and claudin-10b, which differ in their first extracellular loop. In cultured epithelial cells, heter-ologous expression of claudin-10a increases paracellular anion transport, whereas claudin-10b expression increases paracellular cation transport. Both isoforms are expressed differentially along the nephron, with claudin-10a found predominantly in cortical segments, whereas claudin-10b is enriched in the medullary region.  In the present study we generated a mouse model with a TAL-specific Cldn10 gene defect to query the role of this protein in renal paracellular in transport in vivo. We found that claudin-10 is crucial to paracellular Na⁺ handling in the TAL, and that its absence leads to a shift from paracellular sodium transport to paracellular hyperreabsorption of Ca²⁺ and Mg²⁺.

Analysis of claudin-10 expression in the kidney. (A) Western blot analysis of kidney membrane extracts from control (ctr) and cKO mice. A dramatic reduction in claudin-10 protein can be seen in kidneys of cKO mice. Levels of the TJ marker occludin are unchanged. (B) Gene expression analysis of Cldn10 variants on cDNA from isolated segments of the nephron. (C) Immunohistological detection of claudin-10 and markers for PCT (NHE3) and TAL (NKCC2) on sections from control mice (ctr) and cKO mice demonstrates no difference in the signal for claudin-10 in the PCT between WT and cKO. Claudin-10 is expressed in TAL tubules positive for NKCC2. No specific clau-din-10 staining is evident in the TAL of cKO mice. Claudin-10 is detected in TJs positive for ZO-1. This signal is absent in cKO mice, whereas ZO-1 staining is unchanged. (Scale bar: 25 μm.)

In control animals, claudin-10 is located mainly in the TAL, as documented by coimmunostaining with the Na⁺K⁺2Cl⁻ cotrans-porter (NKCC2). In this segment, a large portion of the claudin-10 immunofluorescence signal is located outside of the TJ; however, claudin-10 is present in the TJ, as demonstrated by colocalization with the TJ protein ZO-1. PCTs positive for the sodium-proton exchanger NHE-3 showed a considerably weaker signal restricted to the TJ area. Claudin-10 immunoreactivity was virtually absent in NKCC2-positive tubules of cKO mice, in line with the activity of Cre recombinase in this cell type. The immu-noreactivity of claudin-10 in PCTs of cKOs remained unchanged, however. ZO-1 staining in TAL sections of cKOs was unchanged compared with controls, indicating no unspecific effect on TJ structures. The TJ localization of claudin-16 and claudin-19 in medullary rays was similar in cKOs and controls.   To investigate the phenotypic consequences of renal claudin-10 deficiency, we per­formed a histological examination of the kidneys of 10-wk-old cKO mice and their respective controls. Kidneys from cKO mice contained extensive medullary calcium deposits, as revealed by von Kossa and alizarin red S staining. The deposits were found along the outer stripe of the outer medulla. The detection of extensive calcification suggests alterations in renal ion homeostasis in mice deficient for claudin-10.  Serum Na⁺ and Cl⁻ levels and their renal FE excretion rates were not different be­tween genotypes. In addition, serum creatinine and glomerular filtration rate were not altered compared with controls. Taken together, these findings indicate that calcium deposition does not nonpecifically affect overall glomerular or tubular function.

Fig 4. Gene expression analysis of renal claudins (A) and representative renal ion transporters and channels (B) by real-time PCR. Cldn10 deficiency results in differential gene expression of several genes. Values from cKO animals are shown relative to control mice (mean ± SEM). Wnk1, Wnk1-KS, Kcnj1, and Trpm6, n = 5/4; all other genes, n = 10/10. *P < 0.05; **P < 0.01; ***P < 0.001.    The thiazide-sensitive NaCl cotransporter NCC (Slc12a3), the protein involved in NaCl absorption in the DCT, and the respective inhibitory, kidney-specific kinase-defective KS-WNK1 were expressed at lower levels in the cKO mice. Taken together, these data suggest specific compensatory alterations in components of both paracellular and transcellular renal ion transport mechanisms in mice deficient in claudin-10 in the TAL.

Urinalysis demonstrated that the inhibition of TAL tubular transport by furosemide resulted in a completely differ­ent pattern of tubular Ca²⁺ and Mg²⁺ handling that identifies the TAL as the major nephron segment affected by claudin-10 deficiency.  Interestingly, the different effects on plasma Mg²⁺ and Ca²⁺ levels reflect the different major reabsorption sites of these ions. Some 60% of the filtered Mg²⁺ is reabsorbed in the TAL, compared with only 20% of the filtered Ca²⁺ load (20). Ca²⁺ hyperreabsorption in TAL seems to be balanced by reduced (proximal and) distal tubular Ca²⁺ transport. The hyperreabsorption of divalent cations in mice deficient in claudin-10 is in opposition to the loss of divalent cations seen in mouse models of claudin-16 deficiency and in human patients with mutation in CLDN16 or CLDN19. This finding indicates that claudins in the TAL have functions that differentially affect paracellular cation transport in this segment. Mice deficient for claudin-10b in the TAL exhibit decreased permeability for Na⁺ and increased permeability for Ca²⁺ and Mg²⁺, whereas in mice with claudin-16 or claudin-19 deficiency, decreased sodium per­meability in the TAL is paralleled by decreased reabsorption of Ca²⁺ and Mg²⁺.

1. Greger R (1981) Cation selectivity of the isolated perfused cortical thick ascending limb of Henle’s loop of rabbit kidney. Pflugers Arch 390:30–37.
2. Furuse M (2010) Molecular basis of the core structure of tight junctions. Cold Spring Harb Perspect Biol 2:a002907.
3. Konrad M, et al. (2006) Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular in­volvement. Am J Hum Genet 79:949–957.
4.  Simon DB, et al. (1999) Paracellin-1, a renal tight junction protein required for par-acellular Mg2⁺ resorption. Science 285:103–106.
5. Hou J, et al. (2007) Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium. J Biol Chem 282:17114–17122.
6. Himmerkus N, et al. (2008) Salt and acid-base metabolism in claudin-16 knockdown mice: Impact for the pathophysiology of FHHNC patients. Am J Physiol Renal Physiol 295:F1641–F1647.

 PMID: 22891322  PMCID: PMC3435183   http://www.ncbi.nlm.nih.gov/pubmed/22891322

Paracellin-1 is critical for magnesium and calcium reabsorption in the human thick ascending limb of Henle.

Blanchard A, Jeunemaitre X, Coudol P, Dechaux M, Froissart M, et al.
Université Pierre et Marie Curie, INSERM and Laboratoire de Génétique Moléculaire, Hôpital Universitaire Européen Georges Pompidou, Paris, France. blanch@ccr.jussieu.fr
Kidney Int. 2001 Jun; 59(6):2206-15.

A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and calcium reabsorption, since mutations of PCLN-1 are present in the hypomagnesemia hypercalciuria syndrome (HHS).
No functional experiments have demonstrated that TAL magnesium and calcium reabsorption were actually impaired in patients with HHS.
Genetic studies were performed in the kindred of two unrelated patients with HHS.

We found two yet undescribed mutations of PCLN-1 (Gly 162 Val, Ala 139 Val). In patients with HHS, renal magnesium and calcium reabsorptions were impaired as expected; NaCl renal conservation during NaCl deprivation and NaCl tubular reabsorption in diluting segment were intact. Furosemide infusion in CS markedly increased NaCl, Mg, and Ca urinary excretion rates. In HHS patients, furosemide similarly increased NaCl excretion, but failed to increase Mg and Ca excretion. Acute MgCl(2) infusion in CS and ERH patient provoked a dramatic increase in urinary calcium excretion without change in NaCl excretion. When combined with MgCl(2) infusion, furosemide infusion remained able to induce normal natriuretic response, but was unable to increase urinary magnesium and calcium excretion further. In HHS patients, calciuric response to MgCl(2) infusion was blunted.

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