New insights into renal calcium-sensing receptor activation.

PURPOSE OF REVIEW: Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood. RECENT FINDINGS: Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation. SUMMARY: These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms.

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

AIM: Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. METHODS: To uncover these mechanisms, force development was measured ex vivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused ex vivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. RESULTS: Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. CONCLUSIONS: In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Single cell-resolved study of advanced murine MASH reveals a homeostatic pericyte signaling module.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.

Increased Slc34a2 expression and paracellular phosphate permeability contribute to high intestinal phosphate absorption in young mice.

AIM: Phosphorus is a critical constituent of bone as a component of hydroxyapatite. Bone mineral content accrues rapidly early in life necessitating a positive phosphorus balance, which could be established by a combination of increased renal reabsorption and intestinal absorption. Intestinal absorption can occur via a transcellular pathway mediated by the apical sodium-phosphate cotransporter, Slc34a2/NaPiIIb or via the paracellular pathway. We sought to determine how young mammals increase dietary phosphorus absorption from the small intestine to establish a positive phosphorus balance, a prerequisite for rapid bone growth. METHODS: The developmental expression profile of genes mediating phosphate absorption from the small intestine was determined in mice by qPCR and immunohistochemistry. Additionally, Ussing chamber studies were performed on small bowel of young (p7-p14) and older (8- to 17-week-old) mice to examine developmental changes in paracellular Pi permeability and transcellular Pi transport. RESULTS: Blood and urinary Pi levels were higher in young mice. Intestinal paracellular phosphate permeability of young mice was significantly increased relative to older mice across all intestinal segments. NaPiIIb expression was markedly increased in juvenile mice, in comparison to adult animals. Consistent with this, young mice had increased transcellular phosphate flux across the jejunum and ileum relative to older animals. Moreover, transcellular phosphate transport was attenuated by the NaPiIIb inhibitor NTX1942 in the jejunum and ileum of young mice. CONCLUSION: Our results are consistent with young mice increasing phosphate absorption via increasing paracellular permeability and the NaPiIIb-mediated transcellular pathway.

Maternal Epidermal Growth Factor Promotes Neonatal Claudin-2 Dependent Increases in Small Intestinal Calcium Permeability.

A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by µCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals.

Claudin-19 localizes to the thick ascending limb where its expression is required for junctional claudin-16 localization.

The kidney is critical for mineral homeostasis. Calcium and magnesium reabsorption in the renal thick ascending limb (TAL) involves claudin-16 (CLDN16) and claudin-19 (CLDN19) and pathogenic variants in either gene lead to familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) with severe calcium and magnesium wasting. While both CLDN16 and CLDN19 localize to the TAL, varying expression patterns in the renal tubule have been reported using different antibodies. We, therefore, studied the localization of CLDN19 in the kidneys of wild-type and Cldn19-deleted mice using three anti-CLDN19 antibodies and examined the role of Cldn19 deletion on CLDN16 and CLDN10 localization. We find that CLDN19 localizes to basolateral membrane domains of the medullary and cortical TAL but only to the tight junction of TALs in the outer stripe of outer medulla and cortex, where it colocalizes with CLDN16. Furthermore, in TALs from Cldn19-deleted mice, CLDN16 is expressed in basolateral membrane domains but not at the tight junction. In contrast, Cldn19 ablation does not change CLDN10 localization. These findings directly implicate CLDN19 in regulating permeability in the TAL by allowing junctional insertion of CLDN16 and may explain the shared renal phenotypic characteristics in FHHNC patients.

Effects of parathyroid hormone on renal tubular calcium and phosphate handling.

Central to the maintenance of calcium homeostasis is the regulated reabsorption of calcium along the nephron. To this end, parathyroid hormone (PTH) is released from the parathyroid gland in response to lowered plasma calcium levels. This hormone acts through the PTH 1 receptor along the nephron to increase urinary phosphate excretion and decrease urinary calcium excretion. In the proximal tubule, PTH inhibits phosphate reabsorption by reducing the abundance of sodium phosphate cotransporters in the apical membrane. PTH likely decreases calcium reabsorption from the proximal tubule, by reducing the reabsorption of sodium, an event necessary for the paracellular movement of calcium across this segment. In the thick ascending limb (TAL), PTH increases calcium permeability and may increase the electrical driving force thereby increasing calcium reabsorption in the TAL. Finally, in the distal convolution, PTH acts to increase transcellular calcium reabsorption by increasing the activity and abundance of the apically expressed calcium channel TRPV5.

Molecular mechanisms underlying paracellular calcium and magnesium reabsorption in the proximal tubule and thick ascending limb.

Calcium and magnesium are the most abundant divalent cations in the body. The plasma level is controlled by coordinated interaction between intestinal absorption, reabsorption in the kidney, and, for calcium at least, bone storage and exchange. The kidney adjusts urinary excretion of these ions in response to alterations in their systemic concentration. Free ionized and anion-complexed calcium and magnesium are filtered at the glomerulus. The majority (i.e., >85%) of filtered divalent cations are reabsorbed via paracellular pathways from the proximal tubule and thick ascending limb (TAL) of the loop of Henle. Interestingly, the largest fraction of filtered calcium is reabsorbed from the proximal tubule (65%), while the largest fraction of filtered magnesium is reclaimed from the TAL (60%). The paracellular pathways mediating these fluxes are composed of tight junctional pores formed by claudins. In the proximal tubule, claudin-2 and claudin-12 confer calcium permeability, while the exact identity of the magnesium pore remains to be determined. Claudin-16 and claudin-19 contribute to the calcium and magnesium permeable pathway in the TAL. In this review, we discuss the data supporting these conclusions and speculate as to why there is greater fractional calcium reabsorption from the proximal tubule and greater fractional magnesium reabsorption from the TAL.

The contribution of regulated colonic calcium absorption to the maintenance of calcium homeostasis.

Calcium absorption and secretion can occur along the length of the small and large intestine. To date, the focus of research into intestinal calcium absorption has been the small intestine, the site contributing the majority of intestinal calcium absorption. However, evidence that the colon contributes as much as 10% of enteral calcium transport has been available for decades. Transcellular calcium absorption and bidirectional paracellular calcium flux contributing to either net absorption or secretion have been observed in the colon, depending on the physiological state. Moreover, the calcium transport pathways contributing to colonic absorption or secretion are regulated by a variety of hormones, including calcitriol, plasma calcium and dietary factors, including prebiotics. Herein we review historical and recent research highlighting the role of colonic calcium transport in overall maintenance of calcium balance, and suggest these data are consistent with the colon being a site of significant regulated transepithelial calcium transport.

Differential parathyroid and kidney Ca2+-sensing receptor activation in autosomal dominant hypocalcemia 1.

BACKGROUND: Parathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of renal CaSRs attenuates Ca2+ transport and increases expression of the pore-blocking claudin-14. Patients with autosomal dominant hypocalcemia 1 (ADH1), due to activating CASR mutations, exhibit hypocalcemia but not always hypercalciuria (elevated Ca2+ in urine). The latter promotes nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body including the kidney harbor activating CASR mutations, it is not understood why only some ADH1 patients display hypercalciuria. METHODS: Activation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells. FINDINGS: Cldn14 showed blood Ca2+ concentration-dependent regulation, which was absent in mice with kidney-specific Casr deletion, indicating Cldn14 is a suitable marker for chronic CaSR activation in the kidney. Mice with a gain-of-function mutation in the Casr (Nuf) were hypocalcemic with low plasma PTH levels. However, renal CaSRs were not activated at baseline but only after normalizing blood Ca2+ levels. Similarly, significant hypercalciuria was not observed in a ADH1 patient until blood Ca2+ was normalized. In vitro experiments indicate that increased CaSR expression in the parathyroid relative to the kidney could contribute to tissue-specific CaSR activation thresholds. INTERPRETATION: Our findings suggest that parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even when an activating mutation is present. These findings identify a conceptually new mechanism of CaSR-dependent Ca2+ balance regulation that aid in explaining the spectrum of hypercalciuria in ADH1 patients. FUNDING: Erasmus+ 2018/E+/4458087, the Canadian Institutes for Health research, the Novo Nordisk Foundation, the Beckett Foundation, the Carlsberg Foundation and Independent Research Fund Denmark.

Detection of DZIP1L mutations by whole-exome sequencing in consanguineous families with polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported. METHODS: Four children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing. RESULTS: We identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L. CONCLUSIONS: In line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases. A higher resolution version of the Graphical abstract is available as Supplementary information.

Gentamicin Inhibits Ca2+ Channel TRPV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway.

BACKGROUND: Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal Ca2+ wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. METHODS: We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), as determined by patch clamp in HEK293 cells. RESULTS: Gentamicin increased urinary Ca2+ excretion in wild-type mice after acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/- mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and chronic gentamicin administration downregulated distal nephron Ca2+ transporters. CONCLUSIONS: Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into Ca2+ wasting in patients treated with gentamicin.

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles.

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Endothelial mineralocorticoid receptor ablation confers protection towards endothelial dysfunction in experimental diabetes in mice.

AIM: With diabetes comes a significant risk of macrovascular and microvascular complications. Circulating aldosterone levels increase in patients with diabetes. Aldosterone can directly affect vascular function via activation of the mineralocorticoid receptor (MR). We hypothesized that aldosterone via endothelial MR impairs endothelial function in a murine model of experimental diabetes. METHOD: Endothelial cell-specific mineralocorticoid receptor knockout MRflox/flox ; Tie2-Cre mice (ECMR-KO) and wild-type FVB littermates were subjected to an experimental type-1 diabetic model by low dose streptozotocin injections (55mg/kg/day) for five consecutive days. After 10 weeks of diabetes, second-order mesenteric resistance arteries were perfused ex vivo to evaluate vessel contractility and endothelial function. The effect of ex vivo incubation with aldosterone with and without the antagonist, spironolactone was determined. RESULTS: Diabetic ECMR-KO and wild-type mice had similar, elevated, plasma aldosterone concentration while only diabetic wild-type mice displayed elevated urine albumin excretion and cardiac and kidney hypertrophy at 10 weeks. There were no differences in contraction (Emax and EC50 ) to thromboxane receptor agonist (U46619) and elevated K+ between groups. Wild-type diabetic mice showed impaired acetylcholine (ACh)-dependent relaxation, while diabetic ECMR-KO mice had intact ACh-mediated relaxation. Aldosterone incubation ex vivo impaired ACh mediated relaxation and rendered responses similar to diabetic WT arteries. Direct, ex vivo aldosterone effects were absent in ECMR-KO animals. Ex vivo inhibitory effects of aldosterone on endothelial relaxation in arteries from WT were abolished by spironolactone. CONCLUSION: These findings show that endothelial cell mineralocorticoid receptor activation accounts for diabetes-induced systemic endothelial dysfunction in experimental diabetes and may explain the cardiovascular protection by MR antagonists in diabetes.

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

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

Activation of the calcium sensing receptor increases claudin-14 expression via a PLC -p38-Sp1 pathway.

Activation of the basolateral calcium sensing receptor (CaSR) in the renal tubular thick ascending limb (TAL) increases claudin-14 expression, which reduces paracellular calcium (Ca2+ ) permeability, thus increasing urinary Ca2+ excretion. However, the upstream signaling pathway contributing to altered CLDN14 gene expression is unknown. To delineate this pathway, we identified and then cloned the CaSR responsive region including the promoter of mouse Cldn14 into a luciferase reporter vector. This 1500 bp sequence upstream of the 5' UTR of Cldn14 variant 1, conferred increased reporter activity in the presence of high extracellular Ca2+ (5 mM) relative to a lower (0.5 mM) concentration. Assessment of Cldn14 reporter activity in response to increased extracellular Ca2+ in the presence or absence of specific inhibitors confirmed signaling through PLC and p38, but not JNK. Overexpression of SP1 attenuated Cldn14 reporter activity in response to CasR signaling. SP1 is expressed in the TAL and phosphorylation was attenuated by CaSR signaling. Finally, activating mutations in the CaSR increased Cldn14 reporter activity while a dominant negative mutation in the CaSR inhibited it. Together, these studies suggest that basolateral activation of the CASR leads to increased Cldn14 expression via a PLC- stimulated p38 pathway that prevents Sp1 mediated repression.