Pendrin is a Cl
/HCO
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, PDS
-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 PDS
-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 PDS
-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, PDS
-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.
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.
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Atypical hemolytic uremic syndrome (aHUS) is a rare disease caused by genetic abnormalities, infections, autoimmune diseases, drugs, and malignancies. Anti-C5 monoclonal antibody eculizumab is the ...mainstay of treatment of aHUS caused by the genetic defects of the alternative complement pathway. However, the utility of eculizumab in non-genetic forms of aHUS and the timing of treatment discontinuation remain controversial. Here, we report successful short-term eculizumab use in two young adult patients with aHUS due to rare infectious and autoimmune etiologies: Lemierre's syndrome and post-infectious glomerulonephritis, respectively. Eculizumab was rapidly discontinued in both patients with no aHUS recurrence during long-term follow-up. Considering its favorable safety profile with appropriate meningococcal prophylaxis, eculizumab can be considered as a treatment option for non-genetic aHUS.
Glomerular disease is a common cause for proteinuria and chronic kidney disease leading to end-stage renal disease requiring dialysis or kidney transplantation in children. Nephrotic syndrome in ...children is diagnosed by the presence of a triad of proteinuria, hypoalbuminemia, and edema. Minimal change disease is the most common histopathological finding in children and adolescents with nephrotic syndrome. Focal segmental sclerosis is also found in children and is the most common pathological finding in patients with monogenic causes of nephrotic syndrome. Current classification system for nephrotic syndrome is based on response to steroid therapy as a majority of patients develop steroid sensitive nephrotic syndrome regardless of histopathological diagnosis or the presence of genetic mutations. Recent studies investigating the genetics of nephrotic syndrome have shed light on the pathophysiology and mechanisms of proteinuria in nephrotic syndrome. Gene mutations have been identified in several subcellular compartments of the glomerular podocyte and play a critical role in mitochondrial function, actin cytoskeleton dynamics, cell-matrix interactions, slit diaphragm, and podocyte integrity. A subset of genetic mutations are known to cause nephrotic syndrome that is responsive to immunosuppressive therapy but clinical data are limited with respect to renal prognosis and disease progression in a majority of patients. To date, more than 50 genes have been identified as causative factors in nephrotic syndrome in children and adults. As genetic testing becomes more prevalent and affordable, we expect rapid advances in our understanding of mechanisms of proteinuria and genetic diagnosis will help direct future therapy for individual patients.
ABSTRACT
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‐pyrrolooxazinedione (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 μg/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 μg/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. FASEB J. 31, 751–760 (2017). http://www.fasebj.org
ABSTRACT
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‐cycloheptabthiophene‐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. FASEB J. 33, 11247–11257 (2019). www.fasebj.org
Mutations of PKD1 and PKD2 account for 85 and 15% of cases of autosomal dominant polycystic kidney disease (ADPKD), respectively. Clinically, PKD1 is more severe than PKD2, with a median age at ESRD ...of 53.4 versus 72.7 yr. In this study, we explored whether a family history of renal disease severity predicts the mutated gene in ADPKD. We examined the renal function (estimated GFR and age at ESRD) of 484 affected members from 90 families who had ADPKD and whose underlying genotype was known. We found that the presence of at least one affected family member who developed ESRD at age < or =55 was highly predictive of a PKD1 mutation (positive predictive value 100%; sensitivity 72%). In contrast, the presence of at least one affected family member who continued to have sufficient renal function or developed ESRD at age >70 was highly predictive of a PKD2 mutation (positive predictive value 100%; sensitivity 74%). These data suggest that close attention to the family history of renal disease severity in ADPKD may provide a simple means of predicting the mutated gene, which has prognostic implications.
Diarrhea is a major side effect of ErbB receptor tyrosine kinase inhibitors (TKIs) in cancer chemotherapy. Here, we show that the primary mechanism of ErbB TKI diarrhea is activation of basolateral ...membrane potassium (K+) channels and apical membrane chloride (Cl-) channels in intestinal epithelia and demonstrate the efficacy of channel blockers in a rat model of TKI diarrhea. Short-circuit current in colonic epithelial cells showed that the TKIs gefitinib, lapatinib, and afatinib do not affect basal secretion but amplify carbachol-stimulated secretion by 2- to 3-fold. Mechanistic studies with the second-generation TKI afatinib showed that the amplifying effect on Cl- secretion was Ca2+ and cAMP independent, was blocked by CF transmembrane conductance regulator (CFTR) and K+ channel inhibitors, and involved EGFR binding and ERK signaling. Afatinib-amplified activation of basolateral K+ and apical Cl- channels was demonstrated by selective membrane permeabilization, ion substitution, and channel inhibitors. Rats that were administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from approximately 60% to greater than 80%, which was reduced by up to 75% by the K+ channel inhibitors clotrimazole or senicapoc or the CFTR inhibitor (R)-BPO-27. These results indicate a mechanism for TKI diarrhea involving K+ and Cl- channel activation and support the therapeutic efficacy of channel inhibitors.
Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calcium-activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth muscle tissue, ...electrically excitable cells, and some tumors. TMEM16A inhibitors have been proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and possibly cancer. Herein we report, by screening, the discovery of 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A and analysis of 48 synthesized analogs (10ab-10bw) of the original AACT compound (10aa). Structure-activity studies indicated the importance of benzene substituted as 2- or 4-methyl, or 4-fluoro, and defined the significance of thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (10bm), which contains an unusual bromodifluoroacetamide at the thiophene 2-position, had IC
of ∼30 nM, ∼3.6-fold more potent than the most potent previously reported TMEM16A inhibitor 4 (Ani9), and >10-fold improved metabolic stability. Direct and reversible inhibition of TMEM16A by 10bm was demonstrated by patch-clamp analysis. AACTs may be useful as pharmacological tools to study TMEM16A function and as potential drug development candidates.
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