Posts Tagged ‘urinary excretion’

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 (Vte), 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 (PMg). 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+ (FENa) 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 Clare 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 Clis 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+2Clcotrans-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 Cllevels 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.

In patients with HHS, levels of circulating renin and aldosterone were normal, suggesting normal blood and extracellular volume. In addition, HHS patient 2 was normally able to lower her sodium excretion below 10 mmol/day during sodium deprivation, and in HHS pa­tient 1, sodium reabsorption in the diluting segment was normal as assessed by hypotonic saline infusion.  After oral NH4Cl load: Minimal urinary pH was 5.8 (normal value <5.4), and maximal net acid excretion reached only 24 pmol/min (normal value >80). Both subjects had hypocitraturia. The latter data suggested in the two probands distal defect of urinary acidification, probably related to nephrocalcinosis.

Because the filtered load of calcium but not the filtered load of magnesium remains unchanged during acute magnesium infusion in humans, the increase in calcium excretion is a better index of the inhibitory effect of peritubular magnesium on renal tubular divalent cation transport.  Urinary sodium excretion remained almost constant in both subjects during MgCl2 infusion (data not shown). Accordingly, the FECa/FENa ratio, which should remain constant if sodium reabsorption was primarily affected, increased in the CS and EHR patient.  Before the furosemide infusion, serum ultrafilterable (UF) Ca concentrations were similar in patients with HHS and the controls. However, Ca excretion markedly differed and was approximately five times higher in HHS patients than in controls.

In the two patients with homozygous mutations in the PCLN-1 gene, an impairment in renal tubular magne­sium and calcium reabsorption with normal NaCl recla­recla­mation was demonstrated. Accordingly, comparative studies performed under baseline condition in one pa­tient with ERH and in HHS patients demonstrated that the magnesium and calcium excretion in HHS patients were inappropriately high when compared with serum magnesium and calcium concentrations. However, renal NaCl reabsorption in HHS patients was intact. There was no clinical evidence of extracellular fluid volume  contraction. Furthermore, basal circulating renin and aldosterone concentrations were normal and adapted to the normal Na intake. Finally, abnormal NaCl reclama­tion in the diluting segment of the nephron was excluded in one patient, while the other was able to adapt normally to a sodium deprived diet.

This study is the first to our knowledge to demonstrate that homozygous mutations of PCLN-1 result in a selective defect in paracellular Mg and Ca reabsorption in the TAL, with intact NaCl reabsorption ability at this site. In addition, the study supports a selective physiological effect of basolateral Mg(2+) and Ca(2+) concentration on TAL divalent cation paracellular permeability, that is, PCLN-1 activity.   PMID: 11380823   http://www.ncbi.nlm.nih.gov/pubmed/11380823

Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure

Elizabeth Juneman*, Reza Arsanjani, Hoang M Thai, Jordan Lancaster, Jeffrey B Madwed, Steven Goldman
Citation: Elizabeth Juneman, et al. (2013) Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure. J Cardio Vasc Med 1: 1-6

This study was designed to determine the potential therapeutic effects of a new sodium-hydrogen exchanger (NHE-1) inhibitor in the rat coronary artery ligation model of chronic heart failure. After the induction of acute myocardial infarction, rats were entered randomly dose dranging from 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg. Solid state micrometer hemodynamics, echocardiographic, and pressure-volume relationships were measured after 6 weeks of treatment. Treatment with this NHE- 1 inhibitor at 3 mg/kg increased (P< 0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13) while the 1 mg/kg dose decreased (P< 0.05) the infarct size in CHF rats from 21.7±1.4% (N=7) to 15.9±0.7% (N=3) and prevented (P< 0.05) dilatation of the left ventricle in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10). These study results suggest that this new NHE-1 inhibitor may be potentially useful in treating CHF with an improvement in maladaptive left ventricule remodeling. Because the mechanism of action of this agent is entirely different than the currently applied approach in treating CHF that focuses on aggressive neurohormonal blockade and because this agent does not adversely affect important hemodynamic variables, further investigations with this agent may be warranted.

Keywords: Congestive heart failure; Sodium/hydrogen exchange; Cardiovascular disease; Cardiovascular drugs; CHF: Chronic Heart Failure; NHE-1: Sodium-Hydrogen Exchanger; NCX: Sodium-Calcium Exchanger; Ca2+: Calcium; Na+: Sodium; Na+-K+ATPase: Sodium-Potassium ATPase; NKCC: Sodium-Potassium-Chloride co-transporter; MI: Myocardial Infarction; BI: Boehringer Ingelheim; LV: left Ventricle; EF: Ejection Fraction; LVD: left ventricular dysfunction; PV: Pressure-Volume; SE: Standard Error; ARB: Angiotensin Receptor Blocker; ACE: Angiotensin Converting Enzyme

Without reviewing the pathophysiology of CHF here, altered calcium (Ca2+) handling is a hallmark of CHF. Intracellular Ca2+ concentration is closely regulated by sodium-calcium exchanger (NCX) and Ca2+ efflux is dependent on the intracellular sodium (Na+) concentration and trans-sarcolemmal Na gradient. Multiple channels including sodium-potassium ATPase (Na+-K+ ATPase), sodium-hydrogen exporter (NHE), sodium-bicarbonate co-transporter, sodium-potassium-chloride co-transporter (NKCC), and sodium-magnesium exchanger are responsible for regulation of intracellular sodium in cardiac myocytes. The intracellular concentration of Na+ is significantly increased in heart failure, primarily due to influx of Na+. The NHE plays an integral part in rise of intracellular Na+ concentration and development of hypertrophy in heart failure. Because of its multifaceted role in myocardial function, there has been interest in examining the effects of NHE-1 inhibitors in heart failure.

In this study we report the physiologic responses of a new NHE-1 inhibitor, in a rodent model of heart failure. Previous evaluation of the pharmacokinetic properties of this agent in rat and dog revealed low clearance and robust oral bioavailability, suggesting a potential for once daily oral administration. This new compound was found to be potentially effective in preventing ischemic injury in isolated cells systems and in ischemic injury in isolated cells systems and in a Langendorff isolated heart preparation. Based on these encouraging a pharmacokinetic data, and the established preclinical roof of principle, the next step in new drug development was to test this inhibitor in an appropriate disease-relevant animal model. For this, we chose the rat coronary ligation model of CHF, which is the established model of chronic ischemic heart failure and well performed in our laboratory. The model with permanent occlusion of the left coronary artery is important because this model a similar to the clinical syndrome of CHF. This rat coronary artery model of CHF is the same model used in the classic study defining the beneficial use of angiotensin converting enzyme inhibition with captopril in the treatment of CHF. Thus results in this model have the potential to be predictive of the clinical response seen in patients.


In vivohemodynamic effect of NHE1: As noted previously by our laboratory, rats with severe CHF compared to Sham had changes (P< 0.05) in right ventricular weight, mean arterial pressure, tau, the time constant of LV relaxation, LV systolic pressure, LV end-diastolic pressure, +LVdP/dt, -LVdP/dt, dead volume and peak developed pressure. In this study, treatment resulted in no changes in body weight, chamber weight or hemodynamics. Because we stopped the lowest dose (0.3 mg/kg) there are only hemodynamic data with this dose in rats with CHF.

Echocardiographic changes in LV function and Dimensions with NHE1: Rats with CHF have decreases in EF accompanied by increases in LV systolic and diastolic dimensions. There was no change in anterior wallsystolic displacement. These data are consistent with other reports in this model showing that at 6 weeks after left coronary artery ligation, rats with large MIs have dilated left ventricles with LV remodeling and poor LV function (14,15). Treatment with the highest dose of 3 mg/kg increased (P< 0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13). Treatment with 1 mg/kg prevented maladaptive LV remodeling, it prevented (P< 0.05) dilatation of the LV in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10) with no change anterior wall thickening.

Pressure-Volume relationships:  Although there are no significant changes in the PV relationships for either the Sham or CHF rats, there is a trend for the PV loop in CHF to be shifted toward the pressure axis with treatment. These data are consistent with the trend toward decreases in LV dimensions seen with treatment in CHF rats.


This study can be viewed as a corollary of a pilot Phase II clinical trial to look for a signal of a beneficial physiologic effect of this new NHE-1 inhibitor in CHF. In terms of drug development, this is an appropriate approach, i.e., take an agent with a therapeutic focus, with an acceptable toxicology profile, alter its pharmacokinetics to improve its oral delivery and bioavailability and then study the drug in an appropriate animal model. The administration of this agent to rats with CHF after left coronary artery ligation resulted in a therapeutic benefit with an increase in EF and a decrease in infarct size in rats with the largest infarcts. There is a suggestion of the prevention of LV remodeling with decreases in LV end-diastolic and end-systolic dimensions accompanied by a similar trend in the PV loop with a shift toward the pressure axis. There were no changes in hemodynamics.

Importantly, the decrease in infarct size with no changes in hemodynamicswould positively affect LV remodeling by minimizing LV dilatation without changes in LV afterload. From a therapeutic perspective, an agent like this may be advantageous in the treatment of heart failure after MI. The lack of hemodynamic changes is not a clinical problem because we already have agents that decrease afterload and lower LV end-diastolic pressure such as angiotensin converting enzyme (ACE) inhibitors and angiotens in receptor blockers (ARBs). In treating CHF, we also have diuretics to control blood volume, which in turn reduces LV end-diastolic pressure. The other potential advantage of an NHE-1 inhibitor is that as opposed to our current use of aggressive neurohormonal blockade, this represents a different approach to treating CHF. This is attractive because we essentially have exhausted or maximized our effects of neurohumoral blockade and with no real new treatments for CHF introduced in the last 10-15 years, need to look for other approaches to treat CHF.

Drug development is obviously a complicated and expensive undertaking. In exploring this agent, we would proposea stepwise approach. In this case with an agent whose analogs have been studied extensively, our thought would be to perform a larger dose ranging study in CHF rats to define dose response curves for systolic function as well as obtain more information on pharmacokinetics, as well as diastolic function and structural changes.

An attractive aspect of this work is that we are examining an agent with a different mechanisms of action that current treatments for heart failure. The stimulus to study the agent in an animal model of heart failure was based on multifaceted roles of sodium-hydrogen exchangers on myocardial function. Nine isoforms of NHE have currently been identified, with NHE- 1 being the predominant isoform in the plasma membrane of the myocardium [3,24]. Because NHE is activated by intracellular acidosis, angiotensin II, and catecholamines, its activity is expectedly increased in heart failure. Inhibition of NHE-1 has previously been associated with decreased fibrosis, apoptosis, preserved contractility, and attenuation of hypertrophy and development of heart failure.

1. Baartscheer A, van Borren MMGJ (2008) Sodium Ion Transporters as New Therapeutic Targets in Heart Failure. Cardiovasc Hematol Agents Med Chem 6: 229-236.
2. Murphy E, Eisner DA (2009) Regulation of Intracellular and Mitochondrial Sodium in Health and Disease. Circ Res 104: 292-303
3. Despa S, Islam MA, Weber CR, Pogwizd SM, Bers DM (2002) Intracellular Na(+) Concentration is Elevated in Heart Failure but Na/K Pump Function is Unchanged. Circulation 105: 2543-2548.
4. Baartscheer A, Schumacher CA, van Borren MMGJ, Belterman CNW, Coronel R, et al. (2003) Increased Na+/H+-Exchange Activity is the Cause of Increased [Na+]i and Underlies Disturbed Calcium Handling in the Rabbit Pressure and Volume Overload Heart Failure Model. Cardiovasc Res 57: 1015-1024.
5.  Pieske B, Houser SR (2003) [Na+]i Handling in the Failing Human Heart. Cardiovasc Res 57: 874-886.
6.  Engelhardt S, Hein L, Keller U, Klämbt K, Lohse MJ (2002) Inhibition of Na(+)-H(+) Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in Beta(1)- Adrenergic Receptor Transgenic Mice. Circ Res 90: 814-819.
7.  Chen L, Chen CX, Gan XT, Beier N, Scholz W, et al. (2004) Inhibition and Reversal of Myocardial Infarction-Induced Hypertrophy and Heart Failure by NHE-1 Inhibition. Am J Physiol Heart Circ Physiol 286: 381-387.
8.  Marano G, Vergari A, Catalano L, Gaudi S, Palazzesi S, et al. (2004) Na+/ H+ Exchange Inhibition Attenuates Left Ventricular Remodeling and Preserves Systolic Function in Pressure-Overloaded Hearts. Br J Pharmacol 141: 526-532.
9. Goldman S, Raya TE (1995) Rat Infarct Model of Myocardial Infarction and Heart Failure. J Card Fail 1: 169-177.
10. Gaballa MA, Goldman S (2002) Ventricular Remodeling in Heart Failure. J Card Fail. 8: 476-485.
11. Pfeffer MA, Pfeffer JM, Steinberg C, Finn P (1985) Survival After Experimental Myocardial Infarction: Beneficial Effects of Long-Term Therapy with Captopril. Circulation 72: 406-412.
12. Raya TE, Gay RG, Aguirre M, Goldman S (1989) Importance of Venodilatation in Prevention of Left Ventricular Dilatation after Chronic Large Myocardial Infarction in Rats: A Comparison of Captopril and Hydralazine. Circ Res 64: 330-337.

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Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


pone.0070764.g006  Morpholino knockdown of aquaporin-1a1 reduces water influx.       NIHMS262281.html

nihms81087f1  Localization of claudin proteins in mammalian kidney.      F1.medium  intracellular Mg2+ in normal and Mg2+ depleted immortalized mouse distal convoluted tubule (MDCT) cells

F2.small  membrane voltage influences Mg2+ uptake in MDCT cells    pnas.1203834109fig04  Gene expression analysis of renal claudins

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