Phosphorylated claudin-16 interacts with Trpv5 and regulates transcellular calcium transport in the kidney.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca2+ pathway in the DCT and identify a molecular component in renal Ca2+ homeostasis under direct regulation of PTH.

Claudin-4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins.

Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here, we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 alone can form adhesive membrane interfaces without the accessory proteins that are present in vivo By controlling the molecular composition of the inner and outer leaflets of jetted vesicle membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins with ectodomains as small as 5 nm but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.

ILDR1 is important for paracellular water transport and urine concentration mechanism.

Whether the tight junction is permeable to water remains highly controversial. Here, we provide evidence that the tricellular tight junction is important for paracellular water permeation and that Ig-like domain containing receptor 1 (ILDR1) regulates its permeability. In the mouse kidney, ILDR1 is localized to tricellular tight junctions of the distal tubules. Genetic knockout of Ildr1 in the mouse kidney causes polyuria and polydipsia due to renal concentrating defects. Microperfusion of live renal distal tubules reveals that they are impermeable to water in normal animals but become highly permeable to water in Ildr1 knockout animals whereas paracellular ionic permeabilities in the Ildr1 knockout mouse renal tubules are not affected. Vasopressin cannot correct paracellular water loss in Ildr1 knockout animals despite normal effects on the transcellular aquaporin-2-dependent pathway. In cultured renal epithelial cells normally lacking the expression of Ildr1, overexpression of Ildr1 significantly reduces the paracellular water permeability. Together, our study provides a mechanism of how cells transport water and shows how such a mechanism may be exploited as a therapeutic approach to maintain water homeostasis.

Mosaic expression of claudins in thick ascending limbs of Henle results in spatial separation of paracellular Na+ and Mg2+ transport.

The thick ascending limb (TAL) of Henle's loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Förster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2.

Parathyroid hormone controls paracellular Ca2+ transport in the thick ascending limb by regulating the tight-junction protein Claudin14.

Renal Ca2+ reabsorption is essential for maintaining systemic Ca2+ homeostasis and is tightly regulated through the parathyroid hormone (PTH)/PTHrP receptor (PTH1R) signaling pathway. We investigated the role of PTH1R in the kidney by generating a mouse model with targeted deletion of PTH1R in the thick ascending limb of Henle (TAL) and in distal convoluted tubules (DCTs): Ksp-cre;Pth1rfl/fl Mutant mice exhibited hypercalciuria and had lower serum calcium and markedly increased serum PTH levels. Unexpectedly, proteins involved in transcellular Ca2+ reabsorption in DCTs were not decreased. However, claudin14 (Cldn14), an inhibitory factor of the paracellular Ca2+ transport in the TAL, was significantly increased. Analyses by flow cytometry as well as the use of Cldn14-lacZ knock-in reporter mice confirmed increased Cldn14 expression and promoter activity in the TAL of Ksp-cre;Pth1rfl/fl mice. Moreover, PTH treatment of HEK293 cells stably transfected with CLDN14-GFP, together with PTH1R, induced cytosolic translocation of CLDN14 from the tight junction. Furthermore, mice with high serum PTH levels, regardless of high or low serum calcium, demonstrated that PTH/PTH1R signaling exerts a suppressive effect on Cldn14. We therefore conclude that PTH1R signaling directly and indirectly regulates the paracellular Ca2+ transport pathway by modulating Cldn14 expression in the TAL. Finally, systemic deletion of Cldn14 completely rescued the hypercalciuric and lower serum calcium phenotype in Ksp-cre;Pth1rfl/fl mice, emphasizing the importance of PTH in inhibiting Cldn14. Consequently, suppressing CLDN14 could provide a potential treatment to correct urinary Ca2+ loss, particularly in patients with hypoparathyroidism.

Characterization of Membrane Patch-Ion Channel Probes for Scanning Ion Conductance Microscopy.

Integration of dual-barrel membrane patch-ion channel probes (MP-ICPs) to scanning ion conductance microscopy (SICM) holds promise of providing a revolutionized approach of spatially resolved chemical sensing. A series of experiments are performed to further the understanding of the system and to answer some fundamental questions, in preparation for future developments of this approach. First, MP-ICPs are constructed that contain different types of ion channels including transient receptor potential vanilloid 1 and large conductance Ca2+ -activated K+ channels to establish the generalizability of the methods. Next, the capability of the MP-ICP platforms in single ion channel activity measurements is proved. In addition, the interplay between the SICM barrel and the ICP barrel is studied. For ion channels gated by uncharged ligands, channel activity at the ICP barrel is unaffected by the SICM barrel potential; whereas for ion channels that are gated by charged ligands, enhanced channel activity can be obtained by biasing the SICM barrel at potentials with opposite polarity to the charge of the ligand molecules. Finally, a proof-of-principle experiment is performed and site-specific molecular/ionic flux sensing is demonstrated at single-ion-channel level, which show that the MP-ICP platform can be used to quantify local molecular/ionic concentrations.

KLHL3 regulates paracellular chloride transport in the kidney by ubiquitination of claudin-8.

A rare Mendelian syndrome--pseudohypoaldosteronism type II (PHA-II)--features hypertension, hyperkalemia, and metabolic acidosis. Genetic linkage studies and exome sequencing have identified four genes--with no lysine kinase 1 (wnk1), wnk4, Kelch-like 3 (KLHL3), and Cullin 3 (Cul3)--mutations of which all caused PHA-II phenotypes. The previous hypothesis was that the KLHL3-Cul3 ubiquitin complex acted on the wnk4-wnk1 kinase complex to regulate Na(+)/Cl(-) cotransporter (NCC) mediated salt reabsorption in the distal tubules of the kidney. Here, we report the identification of claudin-8 as a previously unidentified physiologic target for KLHL3 and provide an alternative explanation for the collecting duct's role in PHA-II. Using a tissue-specific KO approach, we have found that deletion of claudin-8 in the collecting duct of mouse kidney caused hypotension, hypokalemia, and metabolic alkalosis, an exact mirror image of PHA-II. Mechanistically, the phenotypes in claudin-8 KO animals were caused by disruption of the claudin-8 interaction with claudin-4, the paracellular chloride channel, and delocalization of claudin-4 from the tight junction. In mouse collecting duct cells, knockdown of KLHL3 profoundly increased the paracellular chloride permeability. Mechanistically, KLHL3 was directly bound to claudin-8, and this binding led to the ubiquitination and degradation of claudin-8. The dominant PHA-II mutation in KLHL3 impaired claudin-8 binding, ubiquitination, and degradation. These findings have attested to the concept that the paracellular pathway is physiologically regulated through the ubiquitination pathway, and its deregulation may lead to diseases of electrolyte and blood pressure imbalances.

Inducible Expression of Claudin-1 in Glomerular Podocytes Generates Aberrant Tight Junctions and Proteinuria through Slit Diaphragm Destabilization.

The tight junction (TJ) has a key role in regulating paracellular permeability to water and solutes in the kidney. However, the functional role of the TJ in the glomerular podocyte is unclear. In diabetic nephropathy, the gene expression of claudins, in particular claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit and the appearance of TJ-like structures between the foot processes. However, there is no definitive evidence to show slit diaphragm (SD) to TJ transition in vivo Here, we report the generation of a claudin-1 transgenic mouse model with doxycycline-inducible transgene expression specifically in the glomerular podocyte. We found that induction of claudin-1 gene expression in mature podocytes caused profound proteinuria, and with deep-etching freeze-fracture electron microscopy, we resolved the ultrastructural change in the claudin-1-induced SD-TJ transition. Notably, immunolabeling of kidney proteins revealed that claudin-1 induction destabilized the SD protein complex in podocytes, with significantly reduced expression and altered localization of nephrin and podocin proteins. Mechanistically, claudin-1 interacted with both nephrin and podocin through cis- and trans-associations in cultured cells. Furthermore, the rat puromycin aminonucleoside nephrosis model, previously suspected of undergoing SD-TJ transition, exhibited upregulated expression levels of claudin-1 mRNA and protein in podocytes. Together, our data attest to the novel concept that claudins and the TJ have essential roles in podocyte pathophysiology and that claudin interactions with SD components may facilitate SD-TJ transition that appears to be common to many nephrotic conditions.

Claudins in barrier and transport function-the kidney.

Claudins are discovered to be key players in renal epithelial physiology. They are involved in developmental, physiological, and pathophysiological differentiation. In the glomerular podocytes, claudin-1 is an important determinant of cell junction fate. In the proximal tubule, claudin-2 plays important roles in paracellular salt reabsorption. In the thick ascending limb, claudin-14, -16, and -19 regulate the paracellular reabsorption of calcium and magnesium. Recessive mutations in claudin-16 or -19 cause an inherited calcium and magnesium losing disease. Synonymous variants in claudin-14 have been associated with hypercalciuric nephrolithiasis by genome-wide association studies (GWASs). More importantly, claudin-14 gene expression can be regulated by extracellular calcium levels via the calcium sensing receptor. In the distal tubules, claudin-4 and -8 form paracellular chloride pathway to facilitate electrogenic sodium reabsorption. Aldosterone, WNK4, Cap1, and KLHL3 are powerful regulators of claudin and the paracellular chloride permeability. The lessons learned on claudins from the kidney will have a broader impact on tight junction biology in other epithelia and endothelia.

Quantitative Visualization of Nanoscale Ion Transport.

Understanding ion transport properties at various interfaces, especially at small length scales, is critical in advancing our knowledge of membrane materials and cell biology. Recently, we described potentiometric-scanning ion conductance microscopy (P-SICM) for ion-conductance measurement in polymer membranes and epithelial cell monolayers at discrete points in a sample. Here, we combine hopping mode techniques with P-SICM to allow simultaneous nanometer-scale conductance and topography mapping. First validated with standard synthetic membranes and then demonstrated in living epithelial cell monolayers under physiological conditions, this new method allows direct visualization of heterogeneous ion transport of biological samples for the first time. These advances provide a noncontact local probe, require no labeling, and present a new tool for quantifying intrinsic transport properties of a variety of biological samples.

The Cap1-claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation.

The paracellular pathway through the tight junction provides an important route for transepithelial chloride reabsorption in the kidney, which regulates extracellular salt content and blood pressure. Defects in paracellular chloride reabsorption may in theory cause deregulation of blood pressure. However, there is no evidence to prove this theory or to demonstrate the in vivo role of the paracellular pathway in renal chloride handling. Here, using a tissue-specific KO approach, we have revealed a chloride transport pathway in the kidney that requires the tight junction molecule claudin-4. The collecting duct-specific claudin-4 KO animals developed hypotension, hypochloremia, and metabolic alkalosis due to profound renal wasting of chloride. The claudin-4-mediated chloride conductance can be regulated endogenously by a protease-channel-activating protease 1 (cap1). Mechanistically, cap1 regulates claudin-4 intercellular interaction and membrane stability. A putative cap1 cleavage site has been identified in the second extracellular loop of claudin-4, mutation of which abolished its regulation by cap1. The cap1 effects on paracellular chloride permeation can be extended to other proteases such as trypsin, suggesting a general mechanism may also exist for proteases to regulate the tight junction permeabilities. Together, we have discovered a theory that paracellular chloride permeability is physiologically regulated and essential to renal salt homeostasis and blood pressure control.

Claudins and the kidney.

Claudins are tight junction membrane proteins that regulate paracellular permeability of renal epithelia to small ions, solutes, and water. Claudins interact within the cell membrane and between neighboring cells to form tight junction strands and constitute both the paracellular barrier and the pore. The first extracellular domain of claudins is thought to be the pore-lining domain and contains the determinants of charge selectivity. Multiple claudins are expressed in different nephron segments; such differential expression likely determines the permeability properties of each segment. Recent evidence has identified claudin-2 as constituting the cation-reabsorptive pathway in the proximal tubule; claudin-14, -16, and -19 as forming a complex that regulates calcium transport in the thick ascending limb of the loop of Henle; and claudin-4, -7, and -8 as determinants of collecting duct chloride permeability. Mutations in claudin-16 and -19 cause familial hypercalciuric hypomagnesemia with nephrocalcinosis. The roles of other claudins in kidney diseases remain to be fully elucidated.

Corticomedullary difference in the effects of dietary Ca²âº on tight junction properties in thick ascending limbs of Henle's loop.

The thick ascending limb of Henle's loop (TAL) drives an important part of the reabsorption of divalent cations. This reabsorption occurs via the paracellular pathway formed by the tight junction (TJ), which in the TAL shows cation selectivity. Claudins, a family of TJ proteins, determine the permeability and selectivity of this pathway. Mice were fed with normal or high-Ca(2+) diet, and effects on the reabsorptive properties of cortical and medullary TAL segments were analysed by tubule microdissection and microperfusion. Claudin expression was investigated by immunostaining and quantitative PCR. We show that the TAL adapted to high Ca(2+) load in a sub-segment-specific manner. In medullary TAL, transcellular NaCl transport was attenuated. The transepithelial voltage decreased from 10.9 ± 0.6 mV at control diet to 8.3 ± 0.5 mV at high Ca(2+) load, thereby reducing the driving force for Ca(2+) and Mg(2+) uptake. Cortical TAL showed a reduction in paracellular Ca(2+) and Mg(2+) permeabilities from 8.2 ± 0.7 to 6.2 ± 0.5 ∙ 10(-4) cm/s and from 4.8 ± 0.5 to 3.0 ± 0.2 · 10(-4) cm/s at control and high-Ca(2+) diet, respectively. Expression, localisation and regulation of claudins 10, 14, 16 and 19 differed along the corticomedullary axis: Towards the cortex, the main site of divalent cation reabsorption in TAL, high-Ca(2+) intake led to a strong upregulation of claudin-14 within TAL TJs while claudin-16 and -19 were unaltered. Towards the inner medulla, only claudin-10 was present in TAL TJ strands. In summary, high-Ca(2+) diet induced a reduction of divalent cation reabsorption via a diminution of NaCl transport and driving force in mTAL and via decreased paracellular permeabilities in cTAL. We reveal an important regulatory pattern along the corticomedullary axis and improve the understanding how the kidney disposes of detrimental excess Ca(2+).

Claudin-14 regulates renal Ca⁺⁺ transport in response to CaSR signalling via a novel microRNA pathway.

The paracellular claudin channel of the thick ascending limb (TAL) of Henle is critical for Ca(++) reabsorption in the kidney. Genome-wide association studies (GWASs) have identified claudin-14 associated with hypercalciuric nephrolithiasis. Here, we show that claudin-14 promoter activity and transcript are exclusively localized in the TAL. Under normal dietary condition, claudin-14 proteins are suppressed by two microRNA molecules (miR-9 and miR-374). Both microRNAs directly target the 3'-UTR of claudin-14 mRNA; induce its mRNA decay and translational repression in a synergistic manner. Through physical interaction, claudin-14 blocks the paracellular cation channel made of claudin-16 and -19, critical for Ca(++) reabsorption in the TAL. The transcript and protein levels of claudin-14 are upregulated by high Ca(++) diet, while downregulated by low Ca(++) diet. Claudin-14 knockout animals develop hypermagnesaemia, hypomagnesiuria, and hypocalciuria under high Ca(++) dietary condition. MiR-9 and miR-374 transcript levels are regulated by extracellular Ca(++) in a reciprocal manner as claudin-14. The Ca(++) sensing receptor (CaSR) acts upstream of the microRNA-claudin-14 axis. Together, these data have established a key regulatory role for claudin-14 in renal Ca(++) homeostasis.

Capturing Rare Conductance in Epithelia with Potentiometric-Scanning Ion Conductance Microscopy.

Tight junctions (TJs) are barrier forming structures of epithelia and can be described as tightly sealed intercellular spaces. Transport properties have been extensively studied for bicellular TJs (bTJs). Knowledge of the barrier functions of tricellular junctions (tTJs) are less well understood, due largely to a lack of proper techniques to locally measure discrete tTJ properties within a much larger area of epithelium. In this study, we use a nanoscale pipet to precisely locate tTJs within epithelia and measure the apparent local conductance of tTJs with a technique termed potentiometric scanning ion conductance microscopy (P-SICM). P-SICM shows the ability to differentiate transport through tTJs and bTJs, which was not possible with previous techniques and assays. We describe P-SICM investigations of both wild type and tricellulin overexpression Madin-Darby Canine Kidney (strain II, MDCKII) cells.

Claudins and mineral metabolism.

PURPOSE OF REVIEW: The tight junction conductance made of the claudin-based paracellular channel is important in the regulation of calcium and magnesium reabsorption in the kidney. This review describes recent findings of the structure, the function, and the physiologic regulation of claudin-14, claudin-16, and claudin-19 channels that through protein interactions confer calcium and magnesium permeability to the tight junction. RECENT FINDINGS: Mutations in two tight junction genes - claudin-16 and claudin-19 - cause the inherited renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. A recent genome-wide association study has identified claudin-14 as a major risk gene of hypercalciuric nephrolithiasis. The crystal structure of claudin-19 has recently been resolved allowing the reconstruction of a claudin assembly model from cis-dimers made of claudin-16 and claudin-19 interaction. MicroRNAs have been identified as novel regulators of the claudin-14 gene. The microRNA-claudin-14 operon is directly regulated by the Ca sensing receptor gene in response to hypercalcemia. SUMMARY: The paracellular pathway in the kidney is particularly important for mineral metabolism. Three claudin proteins - claudin-14, claudin-16, and claudin-19 - contribute to the structure and function of this paracellular pathway. Genetic mutations and gene expression changes in these claudins may lead to alteration of the paracellular permeability to calcium and magnesium, ultimately affecting renal mineral metabolism.

Paracellular transport in the collecting duct.

PURPOSE OF REVIEW: The paracellular pathway through the tight junction provides an important route for chloride reabsorption in the collecting duct of the kidney. This review describes recent findings of how defects in paracellular chloride permeation pathway may cause kidney diseases and how such a pathway may be regulated to maintain normal chloride homeostasis. RECENT FINDINGS: The tight junction in the collecting duct expresses two important claudin genes - claudin-4 and claudin-8. Transgenic knockout of either claudin gene causes hypotension, hypochloremia, and metabolic alkalosis in experimental animals. The claudin-4 mediated chloride permeability can be regulated by a protease endogenously expressed by the collecting duct cell - channel-activating protease 1. Channel-activating protease 1 regulates the intercellular interaction of claudin-4 and its membrane stability. Kelch-like 3, previously identified as a causal gene for Gordon's syndrome, also known as pseudohypoaldosteronism II, directly interacts with claudin-8 and regulates its ubiquitination and degradation. The dominant pseudohypoaldosteronism-II mutation (R528H) in Kelch-like 3 abolishes claudin-8 binding, ubiquitination, and degradation. SUMMARY: The paracellular chloride permeation pathway in the kidney is an important but understudied area in nephrology. It plays vital roles in renal salt handling and regulation of extracellular fluid volume and blood pressure. Two claudin proteins, claudin-4 and claudin-8, contribute to the function of this paracellular pathway. Deletion of either claudin protein from the collecting duct causes renal chloride reabsorption defects and low blood pressure. Claudins can be regulated on posttranslational levels by several mechanisms involving protease and ubiquitin ligase. Deregulation of claudins may cause human hypertension as exemplified in the Gordon's syndrome.

Epigenetic regulation of microRNAs controlling CLDN14 expression as a mechanism for renal calcium handling.

The kidney has a major role in extracellular calcium homeostasis. Multiple genetic linkage and association studies identified three tight junction genes from the kidney--claudin-14, -16, and -19--as critical for calcium imbalance diseases. Despite the compelling biologic evidence that the claudin-14/16/19 proteins form a regulated paracellular pathway for calcium reabsorption, approaches to regulate this transport pathway are largely unavailable, hindering the development of therapies to correct calcium transport abnormalities. Here, we report that treatment with histone deacetylase (HDAC) inhibitors downregulates renal CLDN14 mRNA and dramatically reduces urinary calcium excretion in mice. Furthermore, treatment of mice with HDAC inhibitors stimulated the transcription of renal microRNA-9 (miR-9) and miR-374 genes, which have been shown to repress the expression of claudin-14, the negative regulator of the paracellular pathway. With renal clearance and tubule perfusion techniques, we showed that HDAC inhibitors transiently increase the paracellular cation conductance in the thick ascending limb. Genetic ablation of claudin-14 or the use of a loop diuretic in mice abrogated HDAC inhibitor-induced hypocalciuria. The genetic mutations in the calcium-sensing receptor from patients with autosomal dominant hypocalcemia (ADH) repressed the transcription of miR-9 and miR-374 genes, and treatment with an HDAC inhibitor rescued the phenotypes of cell and animal models of ADH. Furthermore, systemic treatment of mice with antagomiRs against these miRs relieved claudin-14 gene silencing and caused an ADH-like phenotype. Together, our findings provide proof of concept for a novel therapeutic principle on the basis of epigenetic regulation of renal miRs to treat hypercalciuric diseases.

Claudin-14 underlies Ca⁺⁺-sensing receptor-mediated Ca⁺⁺ metabolism via NFAT-microRNA-based mechanisms.

Pathologic dysregulation of extracellular calcium metabolism is difficult to correct. The extracellular Ca(++)-sensing receptor (CaSR), a G protein-coupled receptor that regulates renal Ca(++) handling through changes in paracellular channel permeability in the thick ascending limb, has emerged as an effective pharmacological candidate for managing calcium metabolism. However, manipulation of CaSR at the systemic level causes promiscuous effects in the parathyroid glands, kidneys, and other tissues, and the mechanisms by which CaSR regulates paracellular transport in the kidney remain unknown. Here, we describe a CaSR-NFATc1-microRNA-claudin-14 signaling pathway in the kidney that underlies paracellular Ca(++) reabsorption through the tight junction. With CaSR-specific pharmacological reagents, we show that the in vivo gene expression of claudin-14 is regulated through a transcriptional mechanism mediated by NFATc1-microRNA and associated chromatin remodeling. Transgenic knockout and overexpression approaches showed that claudin-14 is required for CaSR-regulated renal Ca(++) metabolism. Together, our results define an important signaling cascade that, when dysregulated, may mediate Ca(++) imbalance through changes in tight junction permeability.

Scanning ion conductance microscopy measurement of paracellular channel conductance in tight junctions.

Elucidation of epithelial transport across transcellular or paracellular pathways promises to advance the present understanding of ion transport and enables regulation of cell junctions critical to the cell and molecular biology of the epithelium. Here, we demonstrate a new instrumental technique, potentiometric scanning ion conductance microscopy (P-SICM), that utilizes a nanoscale pipet to differentiate paracellular and transcellular transport processes at high spatial resolution. The technique is validated for well-defined polymer membranes and then employed to study wild type and claudin-deficient mutants of Madin-Darby Canine Kidney strain II (MDCKII) cells. Paracellular permeabilities conferred by claudin-2 are captured by P-SICM which demonstrates the utility to monitor apparent conductance at subcellular levels.