Genetic testing in children with nephrolithiasis and nephrocalcinosis.

BACKGROUND: Diagnosing genetic kidney disease has become more accessible with low-cost, rapid genetic testing. The study objectives were to determine genetic testing diagnostic yield and examine predictors of genetic diagnosis in children with nephrolithiasis/nephrocalcinosis (NL/NC). METHODS: This retrospective multicenter cross-sectional study was conducted on children ≤ 21 years old with NL/NC from pediatric nephrology/urology centers that underwent the Invitae Nephrolithiasis Panel 1/1/2019-9/30/2021. The diagnostic yield of the genetic panel was calculated. Bivariate and multiple logistic regression were performed to assess for predictors of positive genetic testing. RESULTS: One hundred and thirteen children (83 NL, 30 NC) from 7 centers were included. Genetic testing was positive in 32% overall (29% NL, 40% NC) with definite diagnoses (had pathogenic variants alone) made in 11.5%, probable diagnoses (carried a combination of pathogenic variants and variants of uncertain significance (VUS) in the same gene) made in 5.4%, and possible diagnoses (had VUS alone) made in 15.0%. Variants were found in 28 genes (most commonly HOGA1 in NL, SLC34A3 in NC) and 20 different conditions were identified. Compared to NL, those with NC were younger and had a higher proportion with developmental delay, hypercalcemia, low serum bicarbonate, hypophosphatemia, and chronic kidney disease. In multivariate analysis, low serum bicarbonate was associated with increased odds of genetic diagnosis (ß 2.2, OR 8.7, 95% CI 1.4-54.7, p = 0.02). CONCLUSIONS: Genetic testing was high-yield with definite, probable, or possible explanatory variants found in up to one-third of children with NL/NC and shows promise to improve clinical practice. A higher resolution version of the Graphical abstract is available as Supplementary information.

Lubiprostone is non-selective activator of cAMP-gated ion channels and Clc-2 has a minor role in its prosecretory effect in intestinal epithelial cells.

Loss of prosecretory Cl- channel CFTR activity is considered as the key cause of gastrointestinal disorders in cystic fibrosis including constipation and meconium ileus. Clc-2 is proposed as an alternative Cl- channel in intestinal epithelia that can compensate for CFTR loss-of-function. Lubiprostone is an FDA-approved drug with Clc-2 activation as its presumed mechanism of action. However, relative contribution of Clc-2 in intestinal Cl- secretion and the mechanism of action of lubiprostone remain controversial due to lack of selective Clc-2 inhibitors. Using recently identified selective Clc-2 inhibitor AK-42, we characterized the roles of Clc-2 in Cl- secretion in human intestinal epithelial T84 cells. Clc-2 inhibitor AK-42 had minimal (15%) inhibitory effect on secretory short-circuit current (Isc) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTRinh-172) caused 2-3 fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory Isc by 20%, whereas CFTRinh-172 caused 2-3 fold greater inhibition. In addition to increasing CFTR and Clc-2-mediated apical Cl- conductance, lubiprostone increased basolateral membrane K+ conductance, which was completely reversed by cAMP-activated K+ channel inhibitor BaCl2 All components of lubiprostone-induced secretion (Clc-2, CFTR and K+ channels) were inhibited by ~65% with the extracellular Ca2+-sensing receptor (CaSR) activator cinacalcet that stimulates cAMP hydrolysis. Lastly, EP4 prostaglandin receptor inhibitor GW627368 pretreatment inhibited lubiprostone-induced secretion by 40% without any effect on forskolin response. Our findings suggest that Clc-2 has minor role in cAMP-induced intestinal Cl- secretion; and lubiprostone is not a selective Clc-2 activator, but general activator of cAMP-gated ion channels in human intestinal epithelial cells. Significance Statement Cl- channel Clc-2 activation is the proposed mechanism of action of the FDA-approved constipation drug lubiprostone. Using first-in-class selective Clc-2 inhibitor AK-42, we showed that Clc-2 has minor contribution in intestinal Cl- secretion induced by lubiprostone and cAMP agonists. We also found that lubiprostone is a general activator of cAMP-gated ion channels in human intestinal epithelial cells (via EP4 receptors). Our findings clarify the roles of Clc-2 in intestinal Cl- secretion and elucidate the mechanism of action of approved-drug lubiprostone.

A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.

Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Bile acid diarrhea (BAD) is common with ileal resection, Crohn's disease, and diarrhea-predominant irritable bowel syndrome. Here, we demonstrate the efficacy of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (R)-benzopyrimido-pyrrolo-oxazine-dione-27 (BPO-27) in reducing bile acid-induced fluid and electrolyte secretion in colon. Short-circuit current measurements in human T84 colonic epithelial cells and planar colonic enteroid cultures showed a robust secretory response following mucosal but not serosal addition of chenodeoxycholic acid (CDCA) or its taurine conjugate, which was fully blocked by CFTR inhibitors, including (R)-BPO-27. (R)-BPO-27 also fully blocked CDCA-induced secretory current in murine colon. CFTR activation by CDCA primarily involved Ca2+ signaling. In closed colonic loops in vivo, luminal CDCA produced a robust secretory response, which was reduced by ∼70% by (R)-BPO-27 or in CFTR-deficient mice. In a rat model of BAD produced by intracolonic infusion of CDCA, (R)-BPO-27 reduced the elevation in stool water content by >55%. These results implicate CFTR activation in the colon as a major prosecretory mechanism of CDCA, a bile acid implicated in BAD, and support the potential therapeutic efficacy of CFTR inhibition in bile acid-associated diarrheas.-Duan, T., Cil, O., Tse, C. M., Sarker, R., Lin, R., Donowitz, M., Verkman, A. S. Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Interstitial cells of Cajal, which express the calcium-activated chloride channel transmembrane member 16A (TMEM16A), are an important determinant of gastrointestinal (GI) motility. We previously identified the acylaminocycloalkylthiophene class of TMEM16A inhibitors, which, following medicinal chemistry, gave analog 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide (TMinh-23) with 30 nM half-maximal inhibitory concentration. Here, we tested the efficacy of TMinh-23 for inhibition of GI motility in mice. In isolated murine gastric antrum, TMinh-23 strongly inhibited spontaneous and carbachol-stimulated rhythmic contractions. Pharmacokinetic analysis showed predicted therapeutic concentrations of TMinh-23 for at least 4 h following a single oral or intraperitoneal dose at 10 mg/kg. Gastric emptying, as assessed following an oral bolus of phenol red or independently by [99mTc]-diethylenetriamine pentaacetic acid scintigraphy, was reduced by TMinh-23 by ∼60% at 20 min. Interestingly, there was little effect of TMinh-23 on baseline whole-gut transit time or time to diarrhea induced by castor oil. Consequent to the delay in gastric emptying, TMinh-23 administration significantly reduced the elevation in blood sugar in mice following an oral but not intraperitoneal glucose load. These results provide pharmacological evidence for involvement of TMEM16A in gastric emptying and suggest the utility of TMEM16A inhibition in disorders of accelerated gastric emptying, such as dumping syndrome, and potentially for improving glucose tolerance in diabetes mellitus/metabolic syndrome and enhancing satiety in obesity.-Cil, O., Anderson, M. O., Yen, R., Kelleher, B., Huynh, T. L., Seo, Y., Nilsen, S. P., Turner, J. R., Verkman, A. S. Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.

Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler's diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC50 down to ∼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 mg/kg. (R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas, (R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.-Cil, O., Phuan, P.-W., Gillespie, A. M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N. C., Lin, R., Donowitz, M., Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.

Small-Molecule Inhibitors of Pendrin Potentiate the Diuretic Action of Furosemide.

Pendrin is a Cl-/HCO3- exchanger expressed in type B and non-A, non-B intercalated cells in the distal nephron, where it facilitates Cl- absorption and is involved in Na+ absorption and acid-base balance. Pendrin-knockout mice show no fluid-electrolyte abnormalities under baseline conditions, although mice with double knockout of pendrin and the Na+/Cl- cotransporter (NCC) manifest profound salt wasting. Thus, pendrin may attenuate diuretic-induced salt loss, but this function remains unconfirmed. To clarify the physiologic role of pendrin under conditions not confounded by gene knockout, and to test the potential utility of pendrin inhibitors for diuretic therapy, we tested in mice a small-molecule pendrin inhibitor identified from a high-throughput screen. In vitro, a pyrazole-thiophenesulfonamide, PDSinh-C01, inhibited Cl-/anion exchange mediated by mouse pendrin with a 50% inhibitory concentration of 1-3 µM, without affecting other major kidney tubule transporters. Administration of PDSinh-C01 to mice at predicted therapeutic doses, determined from serum and urine pharmacokinetics, did not affect urine output, osmolality, salt excretion, or acid-base balance. However, in mice treated acutely with furosemide, administration of PDSinh-C01 produced a 30% increase in urine output, with increased Na+ and Cl- excretion. In mice treated long term with furosemide, in which renal pendrin is upregulated, PDSinh-C01 produced a 60% increase in urine output. Our findings clarify the role of pendrin in kidney function and suggest pendrin inhibition as a novel approach to potentiate the action of loop diuretics. Such combination therapy might enhance diuresis and salt excretion for treatment of hypertension and edema, perhaps including diuretic-resistant edema.

Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats.

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20 µM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 h, and a urine concentration of 20-40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors.

Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 µM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.

Genetic abnormalities and prognosis in patients with congenital and infantile nephrotic syndrome.

BACKGROUND: Congenital nephrotic syndrome (CNS) and infantile nephrotic syndrome (INS) are caused primarily by mutations in genes that encode structural and regulatory proteins of the glomerular filtration barrier. The aim of this study was to determine genotype-phenotype correlations and prognosis in patients with CNS and INS. METHODS: NPHS1, NPHS2, LAMB2 and the eighth and ninth exons of WT1 were sequenced in 80 and 22 patients with CNS and INS, respectively. Genotype-phenotype correlations and survival were evaluated. RESULTS: Causative mutations were identified in 64.7 % of patients, of which NPHS1 mutations were the most common (37.4 %). The mutation detection rate was twofold higher in CNS patients than in INS patients (72.5 vs. 36.2 %). The most commonly mutated gene in CNS patients was NPHS1 (46.3 %) versus NPHS2 (13.6 %) and WT1 (13.6 %) in INS patients. NPHS2 mutations, female patients with NPHS1 mutations, and NPHS1 mutations affecting the transmembrane or intracellular domains of nephrin were associated with longer survival. CONCLUSIONS: Based on our present findings, the likelihood of identification of a genetic cause decreases with increasing age at diagnosis. The underlying genetic abnormality should be identified as early as possible, as this knowledge will facilitate clinicians in their prognostic prediction and enable patients to receive appropriate genetic counseling.

Small-molecule inhibitors of urea transporters.

Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or 'urearetics,' in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done.

MCP1 2518 A/G polymorphism affects progression of childhood focal segmental glomerulosclerosis.

Monocyte chemoattractant protein-1 (MCP-1) is a highly specific chemokine for monocytes and plays roles in pathogenesis of various renal diseases. The aim of this study is to investigate the effect of MCP1 2518 A/G polymorphism on the incidence and clinical course of focal segmental glomerulosclerosis (FSGS) in children. MCP1 2518 A/G genotype was identified by PCR-RFLP in 60 biopsy-proven FSGS patients, 76 steroid sensitive nephrotic syndrome (SSNS) patients, and 96 healthy children. MCP-1 levels in urine and serum were measured by ELISA in all patients and the correlations of genotype with MCP-1 levels and clinical outcome were evaluated. The genotype frequencies for MCP1 were similar in all groups. The percentage of patients who develop chronic renal failure was higher in patients with AA allele compared to GA or GG alleles (46% vs. 35% respectively, p < 0.01, Odds ratio: 1.59). Serum MCP-1 levels were similar in all groups, whereas urinary MCP-1 levels of the patients with FSGS (1680 pg/mg creatinine) were significantly higher than that of patients with SSNS (365 pg/mg creatinine, p < 0.05) and healthy controls (348 pg/mg creatinine; p < 0.05). Urinary MCP-1 levels were correlated with the degree of proteinuria in FSGS group (r = 0.529, p = 0.016). Our results suggest that the AA genotype might be a risk factor for the progression of renal disease in FSGS and MCP1 genotyping may help the physicians to predict prognosis in these patients.

A novel mutation in protein C gene (PROC) causing severe phenotype in neonatal period.

Homozygous protein C deficiency is among rare causes of thrombophilia. Herein, we present a neonate with purpura fulminans, disseminated intravascular coagulation and severe intracranial hemorrhage who was found to have plasma protein C level of 4%. The molecular work-up revealed a novel homozygous mutation of T903C (amino acid position Leu 270 Pro) located in a catalytic domain region of PROC gene. Asymptomatic course in patients with low but measurable levels of protein C levels has been reported, which is different than observed in our patient who had a very severe course despite plasma protein C level of 4%.

Repurposing calcium-sensing receptor activator drug cinacalcet for ADPKD treatment.

ADPKD is characterized by progressive cyst formation and enlargement leading to kidney failure. Tolvaptan is currently the only FDA-approved treatment for ADPKD; however, it can cause serious adverse effects including hepatotoxicity. There remains an unmet clinical need for effective and safe treatments for ADPKD. The extracellular Ca2+-sensing receptor (CaSR) is a regulator of epithelial ion transport. FDA-approved CaSR activator cinacalcet can reduce cAMP-induced Cl- and fluid secretion in various epithelial cells by activating phosphodiesterases (PDE) that hydrolyze cAMP. Since elevated cAMP is a key mechanism of ADPKD progression by promoting cell proliferation, cyst formation and enlargement (via Cl- and fluid secretion), here we tested efficacy of cinacalcet in cell and animal models of ADPKD. Cinacalcet treatment reduced cAMP-induced Cl- secretion and CFTR activity in MDCK cells as suggested by ∼70 % lower short-circuit current (Isc) changes in response to forskolin and CFTRinh-172, respectively. Cinacalcet treatment inhibited forskolin-induced cAMP elevation by 60 % in MDCK cells, and its effect was completely reversed by IBMX (PDE inhibitor). In MDCK cells treated with forskolin, cinacalcet treatment concentration-dependently reduced cell proliferation, cyst formation and cyst enlargement by up to 50 % without affecting cell viability. Cinacalcet treatment (20 mg/kg/day for 7 days, subcutaneous) reduced renal cyst index in a mouse model of ADPKD (Pkd1flox/flox;Ksp-Cre) by 20 %. Lastly, cinacalcet treatment reduced cyst enlargement and cell proliferation in human ADPKD cells by 60 %. Considering its efficacy as shown here, and favorable safety profile including extensive post-approval data, cinacalcet can be repurposed as a novel ADPKD treatment.

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Substituted 4-methylcoumarin inhibitors of SLC26A3 (DRA) for treatment of constipation and hyperoxaluria.

SLC26A3, also known as downregulated in adenoma (DRA), is an anion (Cl-, HCO3- and oxalate) exchanger in the luminal membrane of intestinal epithelial cells. Loss of DRA function in mice and humans causes congenital chloride-losing diarrhea and reduces urinary excretion of oxalate, a major constituent of kidney stones. Thus, inhibition of DRA is a potential treatment approach for constipation and calcium oxalate kidney stones. High-throughput screening previously identified 4,8-dimethylcoumarins (4a-4c) as DRA inhibitors, with lead candidate 4b having an IC50 of 40-50 nM for DRA inhibition. Here, we explored the effects of varying substituents at the 8-position, and replacing 8-methyl by 5-methyl (4e-4h). A focused library of 17 substituted compounds (4d-4t) was synthesized with good yield and purity. Compounds were tested for DRA inhibition potency using Fischer rat thyroid cells stably expressing DRA and a halide-sensitive YFP. Structure-activity analysis revealed that 8-bromo- (4m-4p) and 8-fluoro-coumarins (4q-4t) were slightly less potent than the corresponding 8-chloro analogs, demonstrating that the size of methyl or chloro substituents at the coumarin 8 position affects the potency. An analog containing 8-chlorocoumarin (4k) had ∼2-fold improved potency (IC50 25 nM) compared with the original lead candidate 4b. 5,8-Dimethylcoumarins were active against DRA, but with much lower potency than 4,8-disubstituted coumarins. In mice, orally administered 4k at 10 mg kg-1 reduced constipation and normalized stool water content in a loperamide-induced constipation model with comparable efficacy to 4b. Pharmacokinetic analysis of orally administered 4k at 10 mg kg-1 in mice indicated serum levels of >10 µM for at least six hours after single dose. This study expands SAR knowledge of 4,8-disubstituted coumarin inhibitors of DRA as novel drug candidates for constipation and kidney stones.

Calcium-sensing receptor activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas.

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl- channels, and basolateral membrane K+ channels. 30 µM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Calcium-sensing receptor (CaSR) modulates ocular surface chloride transport and its inhibition promotes ocular surface hydration.

PURPOSE: Ocular surface hydration is critical for eye health and its impairment can lead to dry eye disease. Extracellular calcium-sensing receptor (CaSR) is regulator of ion transport in epithelial cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. CFTR is also a major ion channel in ocular surface epithelia, however the roles of CaSR in ocular surface are not well studied. This study aims to investigate expression and functional roles of CaSR in ocular surface. METHODS: CaSR immunostaining was performed in mouse and human cornea and conjunctiva. Ocular surface potential difference (OSPD) and tear fluid volume measurements were performed in mice with topically applied cinacalcet (CaSR activator) and NPS-2143 (CaSR inhibitor). RESULTS: CaSR is expressed in corneal and conjunctival epithelia of mice and humans. Topically administered CaSR activator cinacalcet inhibits cAMP agonist forskolin-induced Cl- secretion and CFTR activity up to 90 %. CaSR inhibitor NPS-2143 stimulates CFTR-mediated Cl- secretion in mouse ocular surface, after which cAMP agonist forskolin had minimal additional secretory effects. Single dose NPS-2143 treatment (as an eye drop) increases tear fluid volume in mice by ∼60 % compared to vehicle treatment. NPS-2143 effect on tear volume lasts at least 8 h after single dose. CONCLUSIONS: CaSR is a key regulator of ocular surface ion transport and CaSR inhibition promotes Cl- and tear secretion in the ocular surface. If they are found to be effective in in dry eye models, CaSR inhibitors (currently in clinical development) can potentially be repurposed as novel prosecretory treatments for dry eye disease.

DRA involvement in linaclotide stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Crypt and Villus Enterochromaffin Cells are Distinct Stress Sensors in the Gut.

The crypt-villus structure of the small intestine serves as an essential protective barrier, with its integrity monitored by the gut's sensory system. Enterochromaffin (EC) cells, which are rare sensory epithelial cells that release serotonin (5-HT), surveil the mucosal environment and signal both within and outside the gut. However, it remains unclear whether EC cells in intestinal crypts and villi respond to different stimuli and elicit distinct responses. In this study, we introduce a new reporter mouse model to observe the release and propagation of serotonin in live intestines. Using this system, we show that crypt EC cells exhibit two modes of serotonin release: transient receptor potential A1 (TRPA1)-dependent tonic serotonin release that controls basal ionic secretion, and irritant-evoked serotonin release that activates gut sensory neurons. Furthermore, we find that a thick protective mucus layer prevents TRPA1 receptors on crypt EC cells from responding to luminal irritants such as reactive electrophiles; if this mucus layer is compromised, then crypt EC cells become susceptible to activation by luminal irritants. On the other hand, villus EC cells detect oxidative stress through TRPM2 channels and co-release serotonin and ATP to activate nearby gut sensory fibers. Our work highlights the physiological importance of intestinal architecture and differential TRP channel expression in sensing noxious stimuli that elicit nausea and/or pain sensations in the gut.