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

Pendrin is a Cl/HCO3 2 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, PDSinhC01, inhibited Cl/anion exchange mediated by mouse pendrin with a 50% inhibitory concentration of 1–3mM,without affecting othermajor kidney tubule transporters. Administration of PDSinh-C01 tomice at predicted therapeutic doses, determined from serum and urine pharmacokinetics, did not affect urine output, osmolality, salt excretion, or acid-base balance. However, inmice 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 loopdiuretics. Such combination therapy might enhance diuresis and salt excretion for treatment of hypertension and edema, perhaps including diuretic-resistant edema. J Am Soc Nephrol 27: ccc–ccc, 2016. doi: 10.1681/ASN.2015121312 Pendrin (Slc26a4) is aCl/anion (HCO3 , I, SCN) exchanger whose loss of function in humans with Pendred syndrome causes early-onset sensorineural hearing loss, which is sometimes associated with thyroid and vestibular abnormalities.1 Pendrin is expressed primarily in the kidney, thyroid gland, inner ear, and inflamed airways.2,3 In the kidney, pendrin is expressed in the apical membrane of type B and non-A, non-B intercalated cells in the cortical collecting duct (CCD) and the connecting tubule (CNT).4 Pendrin functions primarily in renal Cl absorption andHCO3 2 secretion though its Cl/HCO3 2 exchange function; secondary effects of pendrin activity on electrochemical driving forces and autocrine factors, such as luminal ATP and HCO3 , may account for pendrin involvement in renal Na absorption through effects on epithelial sodium channel (ENaC) expression/function and activity of the Slc4a8 Na-dependent Cl/HCO3 2 exchanger (NDCBE).4 Pendrin expression is upregulated by a wide variety of stimuli, including aldosterone and salt depletion.5,6 Humans with Pendred syndrome have normal urinary concentrating function, but a child with Pendred syndromewas reported to have enhanced diuretic response to thiazides7; Received December 17, 2015. Accepted March 18, 2016. O.C. and P.M.H. contributed equally to this work. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Alan S. Verkman, Departments of Medicine and Physiology, University of California, San Francisco, 1246 Health Sciences East Tower, Box 0521, San Francisco, CA 941430521. Email: Alan.Verkman@ucsf.edu Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2016 ISSN : 1046-6673/2712-ccc 1 in addition, limited data suggest a protective role of pendrin loss of function mutations in hypertension.8 Phenotypestudies inknockoutmicehavesuggestedpendrinasa target for development of a new class of diuretics for treatment of salt-sensitive hypertension and edema. Compared with wild-type mice, pendrin knockoutmice show reduced BPon a salt-restricted diet,9,10 relative inability to excrete anHCO3 2 load,6 and a reduced pressor response to aldosterone.5 Pendrin-overexpressing mice manifest salt-sensitive hypertension.11 The most remarkable phenotype emerging from knockout studies is profound salt wasting in mice lacking pendrin and the Na/Cl cotransporter (NCC) under conditions where the single-knockoutmice do notmanifest salt wasting or volume depletion.12 However, because results in knockout mice are confounded by compensatory changes in the expression of other renal salt and water transporters, the precise role of pendrin in renal function remains unclear, as does the therapeutic utility of pharmacologic pendrin inhibition. Here, to clarify the role of pendrin under conditions not confounded by gene knockout, as well as the therapeutic potential ofpendrin inhibitors,we tested thediuretic efficacyof pendrin inhibitors in mice. The inhibitors were identified in a high-throughput screen against human pendrin, which was recently reported.13 Here, compounds were characterized and optimized for inhibition of murine pendrin and were tested in mice alone and in combination with shortor long-term diuretic therapy. Although no effect of pendrin inhibition alone was seen, as predicted from data in pendrin knockout mice and humans with Pendred syndrome, we found significant potentiation of the diuretic response to furosemide.

Pendrin (Slc26a4) is a Cl 2 /anion (HCO 3 2 , I 2 , SCN 2 ) exchanger whose loss of function in humans with Pendred syndrome causes early-onset sensorineural hearing loss, which is sometimes associated with thyroid and vestibular abnormalities. 1 Pendrin is expressed primarily in the kidney, thyroid gland, inner ear, and inflamed airways. 2,3In the kidney, pendrin is expressed in the apical membrane of type B and non-A, non-B intercalated cells in the cortical collecting duct (CCD) and the connecting tubule (CNT). 4Pendrin functions primarily in renal Cl 2 absorption and HCO 3  2 secretion though its Cl 2 /HCO 3 2 exchange function; secondary effects of pendrin activity on electrochemical driving forces and autocrine factors, such as luminal ATP and HCO 3 2 , may account for pendrin involvement in renal Na + absorption through effects on epithelial sodium channel (ENaC) expression/function and activity of the Slc4a8 Na + -dependent Cl 2 /HCO 3 2 exchanger (NDCBE). 4Pendrin expression is upregulated by a wide variety of stimuli, including aldosterone and salt depletion. 5,6Humans with Pendred syndrome have normal urinary concentrating function, but a child with Pendred syndrome was reported to have enhanced diuretic response to thiazides 7 ; in addition, limited data suggest a protective role of pendrin loss of function mutations in hypertension. 8henotype studies in knockout mice have suggested pendrin as a target for development of a new class of diuretics for treatment of salt-sensitive hypertension and edema.Compared with wild-type mice, pendrin knockout mice show reduced BP on a salt-restricted diet, 9,10 relative inability to excrete an HCO 3  2 load, 6 and a reduced pressor response to aldosterone. 5Pendrin-overexpressing mice manifest salt-sensitive hypertension. 11The most remarkable phenotype emerging from knockout studies is profound salt wasting in mice lacking pendrin and the Na + /Cl 2 cotransporter (NCC) under conditions where the single-knockout mice do not manifest salt wasting or volume depletion. 12However, because results in knockout mice are confounded by compensatory changes in the expression of other renal salt and water transporters, the precise role of pendrin in renal function remains unclear, as does the therapeutic utility of pharmacologic pendrin inhibition.
Here, to clarify the role of pendrin under conditions not confounded by gene knockout, as well as the therapeutic potential of pendrin inhibitors, we tested the diuretic efficacy of pendrin inhibitors in mice.The inhibitors were identified in a high-throughput screen against human pendrin, which was recently reported. 13Here, compounds were characterized and optimized for inhibition of murine pendrin and were tested in mice alone and in combination with short-or long-term diuretic therapy.Although no effect of pendrin inhibition alone was seen, as predicted from data in pendrin knockout mice and humans with Pendred syndrome, we found significant potentiation of the diuretic response to furosemide.

Characterization of Pendrin Inhibitors
As reported separately, a cell-based functional high-throughput screen of 36,000 synthetic small molecules against human pendrin revealed several chemical classes of small-molecule inhibitors.After structure-activity studies and testing on murine pendrin, a pyrazole-thiophenesulfonamide, PDS inh -C01, was selected for comprehensive analysis (Figure 1A), with corroborative analysis done for a chemically unrelated tetrahydropyrazolopyridine pendrin inhibitor, PDS inh -A01 (Supplemental Figure 1).PDS inh -C01 is composed of a thiophene with a sulfonamide group and a pyrazole heterocycle linked at the 3 and 5 positions, respectively.Structureactivity studies showed that changing the pyrazole from the 5 to the 4 position abolished activity and that a sulfonamide group at the 2 or 3 position was needed for inhibition activity.Limited substituents on the pyrazole were studied, with 39, 49-dimethyl giving the most potent compounds, followed by 39-methyl and trifluoromethyl.Substitution on the sulfonamide affected activity, with electron-neutral rings (such as tetrahydro-naphthalene and 2-ethylphenyl) giving best activity, whereas halide-substituted phenyl ring reduced activity.
Functional studies of pendrin-mediated Cl 2 exchange for I 2 , SCN 2 , and NO 3 2 were done in Fischer rat thyroid (FRT) cells stably expressing murine pendrin and a yellow fluorescent protein (YFP) halide-sensing fluorescent indicator (Figure 1B).Addition of I 2 , SCN 2 , or NO 3 2 to the extracellular solution cause YFP fluorescence quenching in pendrin-expressing cells, with near-zero quenching in cells expressing YFP alone.Pendrin inhibition by PDS inh -C01 reduced the rate of fluorescence quenching in a concentration-dependent manner (Figure 1C).Pendrin-mediated Cl 2 /HCO 3 2 exchange was measured from the kinetics of intracellular pH, using 29,79-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein fluorescence as a cytoplasmic pH sensor, following extracellular addition of gluconate in HCO 3

Pharmacokinetics of PDS inh -C01 in Mice
Pharmacokinetics measurements were done to guide studies of diuretic efficacy.A liquid chromatography/mass spectrometry (LC/MS) method was developed to measure PDS inh -C01 concentrations in mouse blood and urine.Figure 2, A and C, shows original LC/MS data and linear standard curves in plasma and urine in which known amounts of PDS inh -C01 were added to plasma and urine from untreated mice.Figure 2, B and D, summarizes PDS inh -C01 concentrations in plasma and urine after bolus intraperitoneal (IP) administration of 10 mg/kg PDS inh -C01, showing predicted therapeutic concentrations for several hours.
Pendrin Inhibition Alone Does Not Affect Fluid-Electrolyte and Acid-Base Balance PDS inh -C01 was administered to mice by IP injection as done in the pharmacokinetics measurements.Figure 3A shows similar 3-hour urine volume and osmolality in two different strains of mice treated with vehicle or PDS inh -C01, even at a very high dose of 50 mg/kg.PDS inh -C01 administration did not significantly change urine pH (Figure 3B) or blood gas values (Figure 3C), nor did it affect 3-hour urinary electrolyte excretion (Figure 3D).In addition, the chemically unrelated pendrin inhibitor PDS inh -A01 did not affect 3-hour urine volume and osmolality when given alone to mice (Supplemental Figure 3).

Pendrin Inhibition Potentiates Diuretic Action of Furosemide
Because pendrin inhibitors alone did not produce a diuretic response in mice, we tested whether pendrin inhibition might augment the diuretic response to furosemide, a loop diuretic that increases salt delivery to the pendrin-expressing CNT and CCD.Mice were administered furosemide and PDS inh -C01 (or vehicle) IP at time zero, and urine was collected for the next 3 hours.Figure 4A shows that PDS inh -C01 (10 mg/kg) significantly increased urine volume by approximately 30% at each dose of furosemide tested, without effect on urine osmolality.The diuretic effect was significantly greater than that produced by maximal furosemide (50 mg/kg).Increasing PDS inh -C01 dose to 50 mg/kg did not further potentiate the furosemide effect.PDS inh -C01, when given with 20 mg/kg furosemide, did not affect urine pH (Figure 4B) but produced a compensated metabolic alkalosis (Figure 4C).PDS inh -C01 increased 3-hour urinary Na + and Cl 2 excretion, with no significant effect on K + excretion (Figure 4D).To rule out an inhibitory effect of furosemide on pendrin activity that could confound the physiologic data, in vitro measurements showed no effect of furosemide on pendrin activity (Figure 4E).PDS inh -A01 had a similar effect on 3-hour urine volume and osmolality in furosemide-treated mice (Supplemental Figure 3).
Because long-term loop diuretic treatment upregulates renal pendrin expression, 14 which might potentiate the diuretic efficacy of pendrin inhibition, we studied the action of PDS inh -C01 in a long-term furosemide treatment model (Figure 5A).After 8 days of furosemide treatment, PDS inh -C01 further potentiated the furosemide effect.Figure 5B shows an approximately 60% increase in urine volume after PDS inh -C01 in the mice receiving long-term furosemide, without effect on urine osmolality.PDS inh -C01 significantly increased urinary Na + , K + , and Cl 2 excretion (Figure 5C).

Pendrin Inhibitors Reduce the Diuretic Action of Hydrochlorothiazide
Motivated by published data on pendrin/ NCC doubleknockout mice, 12 we investigated whether pendrin inhibitors might augment the diuretic effect of hydrochlorothiazide (HCTZ).As done in the acute furosemide study, mice were treated with HCTZ (20 mg/kg) alone or together with PDS inh -C01.Figure 6A shows that, unexpectedly, acute pendrin inhibition reduced the diuretic effect of HCTZ, increasing urine osmolality (Figure 6A) and reducing electrolyte excretion compared with HCTZ alone (Figure 6B).Similarly, PDS inh -A01 treatment reduced urine volume and increased urine osmolality in HCTZ-treated mice (Supplemental Figure 3).Possible reasons for this unanticipated finding are discussed below.Figure 6C shows that HCTZ does not inhibit pendrin directly, nor does PDS inh -C01 inhibit NCC, the major target of HCTZ.Additional experiments confirmed that HCTZ and PDS inh -C01 do not inactivate one another (Supplemental Figure 4).

DISCUSSION
We found that short-term administration of two distinct chemical classes of pendrin inhibitors did not produce a diuretic response in mice when given alone but potentiated the diuresis produced by furosemide, with a greater effect found in mice given long-term furosemide.The lack of effect of pendrin inhibition alone agrees with the lack of renal effect of pendrin knockout in mice and of pendrin loss of function in humans with Pendred syndrome.The potentiation of the diuretic action of furosemide by short-term pendrin inhibition supports the idea that pendrin limits diuretic efficacy by absorption of excess salt delivered to the pendrin-expressing distal nephron.The proportionately greater potentiation of pendrin inhibition with long-term furosemide treatment can be explained on the basis of pendrin upregulation with long-term furosemide treatment, 14 likely in part from increased aldosterone release. 10Our findings clarify the role of pendrin in normal renal physiology and support the potential efficacy of pendrin inhibitors to augment the diuretic efficacy of furosemide.The potentiation of the diuretic action of furosemide by pendrin inhibition is likely due to reduced absorption of excess Cl 2 delivered to the distal nephron after furosemide inhibition of Na + /K + /2Cl 2 cotransport by NKCC2 in the thick ascending limb of Henle, as diagrammed in Figure 7. Pendrin inhibition, together with short-term furosemide administration, increased Na + and Cl 2 excretion, without significant effect on K + excretion.In mice given longterm furosemide treatment, in which greater potentiation by pendrin inhibition was seen, K + excretion increased significantly, as did Na + and Cl 2 excretion.Distal fluid delivery is an important determinant of active K + secretion in CNT and CCD with increased flow, which reduces luminal K + concentration and increases the electrochemical driving force for K + secretion across the apical membrane.Various diuretics that increase distal fluid delivery produce different degrees of hypokalemia.For example, we previously reported that sustained inhibition of urea transporters by dimethylthiourea, without inhibition of salt transporters, increased urinary K + excretion in the short-term setting 15 but caused hypokalemia with long-term administration, albeit to a lesser degree than that produced by furosemide. 16Our findings here that pendrin inhibition does not increase K + excretion with short-term furosemide treatment, but does with long-term furosemide treatment, can be explained by the greater diuresis and distal fluid delivery in the long-term setting.HCTZ was reported to inhibit pendrin and NDCBE-dependent electroneutral Na + absorption without promoting K + secretion in isolated CCD of mice on an Na + -depleted diet. 17Because pendrin inhibitors augmented the kaliuretic response to furosemide in our study, further studies are needed to assess the role of pendrin in distal K + secretion.
As a Cl 2 /HCO 3 2 exchanger, pendrin inhibition could, in principal, reduce HCO 3 2 secretion by pendrin-expressing tubule segments and hence can cause relative urinary acidification and metabolic alkalosis.However, we found no effect of pendrin inhibition on urine pH when given alone or with furosemide.Blood gas analysis showed that pendrin inhibition produced compensated metabolic alkalosis when given with furosemide but had no effect when given alone.Earlier studies showed that pendrin knockout mice have unchanged urinary salt excretion but decreased urine pH and increased serum HCO 3  2 , 18 which may be related to compensatory changes in the expression of other HCO 3 2 transporters in pendrin knockout mice.Furosemide produces urinary acidification by increasing titratable acid, H + , and ammonium excretion 19,20 and can cause Cl 2 depletion alkalosis.During the maintenance phase of Cl 2 depletion alkalosis, pendrin activity is increased and limits the alkalosis by secreting HCO 3  2 into the lumen in exchange for Cl 2 . 21,22In the short-term setting studied here, furosemide slightly increased serum HCO 3 2 (20.5 mmol/L with versus 17.7 mmol/L without furosemide), which was further increased with pendrin inhibition (23.0 mmol/L), with compensatory increased pCO 2 .The mildly increased serum HCO 3 2 with pendrin inhibition and furosemide may be due to inhibition of attenuating effect of pendrin on development of the Cl 2 depletion alkalosis.
An unanticipated finding of our study was the reduced diuretic action of HCTZ with pendrin inhibition by two chemically unrelated pendrin inhibitors.The original idea that pendrin inhibition can augment thiazide-induced diuresis was based on the finding that double-knockout mice lacking pendrin and NCC manifested profound salt wasting, whereas mice lacking pendrin and NCC individually showed no fluid-electrolyte abnormalities. 12The difference in results between the prior knockout study and the inhibition study here may be related to confounding effects of altered expression of renal salt and water transporters produced by gene knockout.For example, pendrin knockout mice show a tendency for NCC upregulation, 23 and NCC knockout mice show distal nephron hypertrophy, significant pendrin overexpression, and apical displacement of pendrin. 24Pendrin or NCC overexpression may thus compensate for the loss of NCC or pendrin, respectively, in the knockout mice. 4The knockout mice may also manifest altered expression of other transporters and hormonal factors.Two pendrin and one NDCBE are thought to function in parallel to produce electroneutral Na + and Cl 2 absorption in intercalated cells. 25This mechanism is sensitive to HCTZ, although NCC expression is absent in CNT and CCD, which suggests the presence of additional target(s) of HCTZ in the distal nephron. 17CTZ-sensitive Na + and Cl 2 transport in CCD is abolished in NDCBE knockout mice, 17 and here we found that HCTZ does not have a direct inhibitory effect on pendrin activity.HCTZ action on NDCBE and other transporters in CCD warrants further investigation.An additional complicating factor is the regulation of ENaC by luminal HCO 3   2 9   in which pendrin and other HCO 3 2 transporters are involved.Inhibition of NCC and other target(s) by HCTZ, together with pendrin inhibition by pendrin inhibitors, might activate ENaC and consequently reduce the diuretic response to HCTZ.
The pharmacokinetics experiments showed that PDS inh -C01 administration produced urine concentrations over 3 hours close to its 50% inhibitory concentration for pendrin inhibition.However, it is difficult to estimate the compound concentration in the luminal fluid of the CNT and CCD based on urinary concentration.In addition, because PDS inh -C01 likely acts at a cytoplasmic site, its intracellular concentration is difficult to predict and would depend on inhibitor concentrations in blood and tubule fluid and apical and basolateral membrane transport mechanisms.
Pharmacologic inhibition of pendrin may have many clinical indications in renal and extrarenal disorders.In addition to its effects on Cl 2 homeostasis, a recent study showed that pendrin is expressed in rodent adrenal medulla and modulates catecholamine release, 26 raising the possibility of pendrin inhibition as a potential therapy in some forms of hypertension.The potentiation of loop diuretic action may be useful in diureticrefractory edema, as occurs in congestive heart failure associated with distal-nephron hypertrophy. 27,28Although pendrin expression in the kidney has not been investigated in humans or experimental animals with congestive heart failure, we predict it to be increased because of increased aldosterone.Outside of the kidney, pendrin inhibition may be beneficial for asthma and other inflammatory lung diseases 2,3,29 and has potential application to thyroid disorders.
There are no prior reports on the biologic effects of the pyrazole-thiophenesulfonamide chemical scaffold studied here.Computational modeling suggested that this scaffold is a potential DNA gyrase inhibitor. 30The physico-chemical properties of the pyrazolethiophenesulfonamide scaffold include the presence of multiple hydrogen bond acceptors, molecular mass of 408 Da, logP value of 4.0, and topologic polar surface area of 78.9 Å 2 .These values are within the guidelines for good oral bioavailability, 31,32 but marginal for absorption, distribution, metabolism, elimination, and toxicity. 33,34he pyrazole-thiophenesulfonamide scaffold does not belong to promiscuous binders known as pan-assay interference compound molecules. 35Although the presence of a thiophene ring has been associated with metabolic toxicity, 2,5-disubstituted thiophenes as found in PDS inh -C01 typically do not have this liability. 36Further optimization of this scaffold through structural modifications will be required to achieve a pharmacologic profile suitable for lead selection.
On the basis of the known tissue distribution and physiologic roles of pendrin, several potential adverse effects of pendrin inhibition are possible and will requireinvestigation in the preclinical development of a small-molecule pendrin inhibitor.Humans with Pendred syndrome develop profound deafness early in life as a consequence of altered inner-ear fluid dynamics producing irreversible anatomic abnormalities.Whether pharmacologic inhibition of pendrin could affect inner ear function is unknown, as is whether pendrin inhibition might interfere with thyroid hormone production in some patients.

Materials
PDS i n h -C01 and PDS i n h -A01 were purchased from ChemDiv (San Diego, CA).Other

Cell Culture
FRT cells were cultured in Kaign's modified Ham's F12 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 18 mg/ml myo-inositol, and 45 mg/ml ascorbic acid.FRT cells stably expressing murine pendrin (from plasmid) and EYFP-H148Q/I152L/F46L (referred to as YFP) (by lentivirus) were generated by limiting dilution and selection using 0.5 mg/ml G418.COS-7 fibroblasts were cultured in DMEM-H21 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin and transiently transfected with cDNAs encoding YFP and various membrane transporters using Lipofectamine 2000.For NCC activity, cells expressing YFP and NCC were equilibrated for 1 hour in Na + gluconate-substituted PBS and then subjected to a 70-mM I 2 gradient.A EYFP-H148Q/V163S/F46L fluorescence quenching assay in FRT cells was used to measure NKCC1 activity in which cells that had been equilibrated in gluconatesubstituted PBS buffer were exposed to a Cl 2 -containing solution containing 75 mM Na + and 75 mM K + .Amiloride-sensitive ENaC activity was measured by short-circuit current analysis of human bronchial epithelial cell cultures. 13Specificity assays were done with 25 mM PDS inh -C01.

Pharmacokinetics
Female CD-1 mice (8-10 weeks) were injected with 10 mg/kg PDS inh -C01 (in saline containing 5% DMSO and 10% Kolliphor HS) IP, and blood was collected by orbital puncture at 15, 30, 60, 150, and 240 minutes.Blood was centrifuged at 5000 rpm for 15 minutes to separate plasma.Urine was collected in metabolic cages.Plasma and urine samples (60 ml) were mixed with 300-ml acetonitrile and centrifuged at 13,000 rpm for 20 minutes, and 90 ml of the supernatant was taken for LC/MS (Waters 2695 and Micromass ZQ).The solvent system consisted of a linear gradient from 5% to 95% acetonitrile over 16 minutes.

Mouse Diuresis Studies
In short-term studies, mice (both CD-1 and C57Bl/6 strains) were injected IP with 10 or 50 mg/kg PDS inh -C01.In some experiments, mice were treated with 10 mg/kg PDS inh -C01 or PDS inh -A01 together with furosemide (5, 10, 20, or 50 mg/kg IP) or HCTZ (20 mg/kg IP).To minimize the variability in urine sampling, mice were placed in individual metabolic cages after bladders were emptied by gentle abdominal massage.At the end of the 3-hour urine collection period, bladders were emptied again by abdominal massage and the urine was mixed with that collected in the metabolic cages.This final urine was used for measurement of volume and osmolality (freezing point depression osmometry; Micro-osmometer; Precision Systems, Natick, MA).In long-term studies, mice were injected with 20 mg/kg furosemide (IP) twice a day for 8 days and then administered PDS inh -C01 (10 mg/kg) or vehicle at the time of the final furosemide dose.Urine was collected for 3 hours as described above.

Serum and Urine Chemistries and Blood Gas Analysis
Sodium and potassium concentrations in 3-hour collected urine samples were measured by flame photometry (PFP7 Clinical Flame Photometer; Bibby Scientific Ltd., Stone, Staffordshire, UK), and chloride concentration was measured by Idexx Laboratories Inc. (Sacramento, CA).For blood gas analysis, arterial blood was collected from the abdominal aorta under isoflurane anesthesia 3 hours after treatment.Blood gas was analyzed by iSTAT1 with CG4+ cartridges (Abbott Laboratories, Abbott Park, IL).Urine pH was measured on freshly collected urine samples using an AB15 pH Meter (Thermo Fisher Scientific, Pittsburgh, PA).

Statistical Analyses
Experiments with two groups were analyzed with a t test; when there were three or more groups, analysis was done using one-way ANOVA and post hoc Newman-Keuls multiple-comparisons test (GraphPad Prism 5; GraphPad Sotfware Inc., La Jolla, CA).P,0.05 was considered to represent statistically significant differences.

Figure 2 .
Figure 2. PDS inh -C01 pharmacokinetics in mice.(A) Inhibitor concentration assayed in mouse plasma by LC/MS after organic-phase extraction.Representative original LC/ MS data from plasma containing known (added) amounts of PDS inh -C01 and deduced calibration curve of integrated peak signal versus PDS inh -C01 concentration.(B) Plasma concentrations of PDS inh -C01 after single IP injection of 10 mg/kg PDS inh -C01.Original LC/MS data (right) and deduced time course of PDS inh -C01 concentration (left, mean6SEM, three mice).(C) Representative LC/MS data from urine containing known (added) amounts of PDS inh -C01 and deduced calibration curve of integrated peak signal versus PDS inh -C01 concentration.(D) Urine concentrations of PDS inh -C01 and original LC/MS data (right) from study in B (mean6SEM, three mice).

Figure 3 .
Figure 3. Acute effects of pendrin inhibition on fluid, electrolyte, and acid-base balance.(A) Three-hour urine volume and osmolality in CD1 (left two panels) and C57Bl/6 (right two panels) mice after IP administration of 10 or 50 mg/kg PDS inh -C01 at time zero (mean6SEM, three to six mice per group).(B) Time course of urine pH in PDS inh -C01-treated or vehicle-treated mice (mean6SEM, three mice per group).(C) Blood gas analysis of aortic blood collected 3 hours after PDS inh -C01 or vehicle treatment (mean6SEM, three to four mice per group).(D) Three-hour urinary Na + , K + , and Cl 2 excretion in PDS inh -C01-treated or vehicle-treated mice (mean6SEM, three to six mice per group).t test used for analysis.ns, not significant.

Figure 4 .
Figure 4. Pendrin inhibitor potentiates the acute diuretic efficacy of furosemide.(A) Three-hour urine volume and osmolality after IP administration of 10 or 50 mg/kg PDS inh -C01 at time zero, together with different amounts of furosemide (mean6SEM, three to six mice per group).*P,0.05;***P,0.001.ns, not significant, one-way ANOVA with post hoc Newman-Keuls test.(B) Time course of urinary pH in mice administered 20 mg/kg furosemide without or with PDS inh -C01 (mean6SEM, six mice per group).(C) Blood gas analysis in aortic blood collected at 3 hours in mice treated as in B (mean6SEM, three to four mice per group).(D) Three-hour urinary Na + , K + , and Cl 2 excretion in mice treated as in B (mean6SEM, four to six mice per group).t test used for analysis.*P,0.05.ns, not significant.(E) Assay of murine pendrin activity in FRT cells, showing no inhibition by 25 mM furosemide.

Figure 6 .
Figure 6.Pendrin inhibitor reduces the diuretic efficacy of HCTZ.(A) Three-hour urine volume and osmolality after IP administration of 10 mg/kg PDS inh -C01 without or with 20 mg/kg HCTZ (or vehicle) at time zero (mean6SEM, five to six mice per group).(B) Three-hour urinary Na + , K + , and Cl 2 excretion in the same animals (mean6SEM, five to six mice per group).*P,0.05;**P,0.01***P,0.001.ns, not significant, one-way ANOVA with post hoc Newman-Keuls test.(C) Assays of murine pendrin (left) and NCC (Slc12a3, right) in transfected FRT cells showing no inhibition of pendrin by 25 mM HCTZ or of NCC by 25 mM PDS inh -C01 (with 25 mM HCTZ producing full inhibition as positive control).

Figure 7 .
Figure 7. Proposed diuretic mechanism of pendrin inhibitor.See text for explanations.Thicknesses/lengths of the blue arrows represent water and salt delivery.
AnimalsAnimal experiments were approved by the University of California, San Francisco, Institutional Animal Care and Use Committee.Mice were provided with standard rodent chow (PicoLab Rodent Diet 20; Lab Diet, St. Louis, MO) and water ad libitum during all experiments.