small-molecule inhibitors of pendrin potentiate the diuretic action … · 2017. 8. 28. · j am...

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Small-Molecule Inhibitors of Pendrin Potentiate theDiuretic Action of Furosemide

Onur Cil, Peter M. Haggie, Puay-wah Phuan, Joseph-Anthony Tan, and Alan S. Verkman

Departments of Medicine and Physiology, University of California San Francisco, San Francisco, California

ABSTRACTPendrin is a Cl2/HCO3

2 exchanger expressed in type B and non-A, non-B intercalated cells in the distalnephron, where it facilitates Cl2 absorption and is involved in Na+ absorption and acid-base balance.Pendrin-knockout mice show no fluid-electrolyte abnormalities under baseline conditions, although micewith double knockout of pendrin and the Na+/Cl2 cotransporter (NCC) manifest profound salt wasting.Thus, pendrin may attenuate diuretic-induced salt loss, but this function remains unconfirmed. To clarifythe physiologic role of pendrin under conditions not confounded by gene knockout, and to test thepotential utility of pendrin inhibitors for diuretic therapy, we tested in mice a small-molecule pendrininhibitor identified from a high-throughput screen. In vitro, a pyrazole-thiophenesulfonamide, PDSinh-C01, inhibited Cl2/anion exchange mediated by mouse pendrin with a 50% inhibitory concentration of1–3mM,without affecting othermajor kidney tubule transporters. Administration of PDSinh-C01 tomice atpredicted therapeutic doses, determined from serum and urine pharmacokinetics, did not affect urineoutput, 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 Cl2 ex-cretion. In mice treated long term with furosemide, in which renal pendrin is upregulated, PDSinh-C01produced a 60% increase in urine output. Our findings clarify the role of pendrin in kidney function andsuggest pendrin inhibition as a novel approach to potentiate the action of loopdiuretics. Such combinationtherapy might enhance diuresis and salt excretion for treatment of hypertension and edema, perhapsincluding diuretic-resistant edema.

J Am Soc Nephrol 27: ccc–ccc, 2016. doi: 10.1681/ASN.2015121312

Pendrin (Slc26a4) is aCl2/anion (HCO32, I2, SCN2)

exchanger whose loss of function in humanswith Pendred syndrome causes early-onset senso-rineural hearing loss, which is sometimes associ-ated with thyroid and vestibular abnormalities.1

Pendrin is expressed primarily in the kidney, thy-roid gland, inner ear, and inflamed airways.2,3 Inthe kidney, pendrin is expressed in the apical mem-brane of type B and non-A, non-B intercalated cellsin the cortical collecting duct (CCD) and the con-necting tubule (CNT).4 Pendrin functions primarilyin renal Cl2 absorption andHCO3

2 secretion thoughits Cl2/HCO3

2 exchange function; secondary effectsof pendrin activity on electrochemical driving forcesand autocrine factors, such as luminal ATP andHCO3

2, may account for pendrin involvement inrenal Na+ absorption through effects on epithelialsodium channel (ENaC) expression/function and

activity of the Slc4a8 Na+-dependent Cl2/HCO32

exchanger (NDCBE).4 Pendrin expression is upre-gulated by a wide variety of stimuli, including aldo-sterone and salt depletion.5,6 Humans with Pendredsyndrome have normal urinary concentrating func-tion, but a child with Pendred syndromewas report-ed 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 atwww.jasn.org.

Correspondence: Dr. Alan S. Verkman, Departments of Medi-cine and Physiology, University of California, San Francisco, 1246Health Sciences East Tower, Box 0521, San Francisco, CA 94143-0521. Email: [email protected]

Copyright © 2016 by the American Society of Nephrology

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in addition, limited data suggest a protective role of pendrinloss of function mutations in hypertension.8

Phenotypestudies inknockoutmicehavesuggestedpendrinasatarget for development of a new class of diuretics for treatment ofsalt-sensitive hypertension and edema. Compared with wild-typemice, pendrin knockoutmice show reduced BPon a salt-restricteddiet,9,10 relative inability to excrete anHCO3

2 load,6 and a reducedpressor response to aldosterone.5 Pendrin-overexpressing micemanifest salt-sensitive hypertension.11 The most remarkable phe-notype emerging from knockout studies is profound salt wastingin mice lacking pendrin and the Na+/Cl2 cotransporter (NCC)under conditions where the single-knockoutmice do notmanifestsalt wasting or volume depletion.12 However, because results inknockout mice are confounded by compensatory changes in theexpression of other renal salt and water transporters, the preciserole of pendrin in renal function remains unclear, as does thetherapeutic utility of pharmacologic pendrin inhibition.

Here, to clarify the role of pendrin under conditions notconfounded by gene knockout, as well as the therapeuticpotential ofpendrin inhibitors,we tested thediuretic efficacyofpendrin inhibitors in mice. The inhibitors were identified ina high-throughput screen against human pendrin, which wasrecently reported.13 Here, compounds were characterized andoptimized for inhibition of murine pendrin and were tested inmice alone and in combination with short- or long-term di-uretic therapy. Although no effect of pendrin inhibition alonewas seen, as predicted from data in pendrin knockout miceand humans with Pendred syndrome, we found significantpotentiation of the diuretic response to furosemide.

RESULTS

Characterization of Pendrin InhibitorsAs reported separately, a cell-based functional high-throughputscreen of 36,000 synthetic small molecules against humanpendrin revealed several chemical classes of small-molecule inhib-itors.After structure-activity studiesandtestingonmurinependrin,a pyrazole-thiophenesulfonamide, PDSinh-C01, was selected forcomprehensive analysis (Figure 1A), with corroborative analysisdone for a chemically unrelated tetrahydropyrazolopyridine pen-drin inhibitor, PDSinh-A01 (Supplemental Figure 1). PDSinh-C01 iscomposed of a thiophenewith a sulfonamide group and a pyrazoleheterocycle linked at the 3 and 5 positions, respectively. Structure-activity studies showed that changing the pyrazole from the 5 to the4 position abolished activity and that a sulfonamide group at the2 or 3 position was needed for inhibition activity. Limited substit-uents on the pyrazole were studied, with 39, 49-dimethyl giving themost potent compounds, followed by 39-methyl and trifluoro-methyl. Substitution on the sulfonamide affected activity,with electron-neutral rings (such as tetrahydro-naphthalene and2-ethylphenyl) giving best activity, whereas halide-substitutedphenyl ring reduced activity.

Functional studies of pendrin-mediated Cl2 exchange forI2, SCN2, and NO3

2 were done in Fischer rat thyroid (FRT)

cells stably expressing murine pendrin and a yellow fluores-cent protein (YFP) halide-sensing fluorescent indicator (Figure1B). Addition of I2, SCN2, orNO3

2 to the extracellular solutioncause YFP fluorescence quenching in pendrin-expressing cells,with near-zero quenching in cells expressing YFP alone. Pendrininhibition by PDSinh-C01 reduced the rate of fluorescencequenching in a concentration-dependent manner (Figure 1C).Pendrin-mediated Cl2/HCO3

2 exchange was measured fromthe kinetics of intracellular pH, using 29,79-Bis-(2-Carboxy-ethyl)-5-(and-6)-Carboxyfluorescein fluorescence as a cytoplas-mic pH sensor, following extracellular addition of gluconate inHCO3

2/CO2-containingbuffer todriveCl2efflux,HCO3

2 influx,and consequent cytoplasmic alkalinization (Figure 1D). PDSinh-C01 reduced the kinetics of Cl2/HCO3

2 exchange, the activity ofpendrin of relevance to kidney function, in a concentration-dependent manner with a 50% inhibitory concentration of ap-proximately 1.2mM(Figure 1E). In selectivity studies, PDSinh-C01at 25 mM did not significantly inhibit the transport activities ofSlc4a1 (anion exchanger 1 [AE1]), Slc26a3 (chloride anion ex-changer [CLD]/downregulated-in-adenoma [DRA]), Na-K-Clcotransporter (NKCC1;Slc12a2), or ENaC (Figure 1F). PDSinh-C01 inhibition of pendrin was reversible, slow (suggesting anintracellular cite of action), noncompetitive, and not affectedby pH or high extracellular Cl2 (Supplemental Figure 2).

Pharmacokinetics of PDSinh-C01 in MicePharmacokinetics measurements were done to guide studiesof diuretic efficacy. A liquid chromatography/mass spectrometry(LC/MS) method was developed to measure PDSinh-C01 concen-trations in mouse blood and urine. Figure 2, A and C, showsoriginal LC/MSdata and linear standard curves inplasmaandurineinwhichknownamounts of PDSinh-C01were added toplasma andurine fromuntreatedmice. Figure 2, B andD, summarizes PDSinh-C01 concentrations in plasma and urine after bolus intraperitoneal(IP) administration of 10 mg/kg PDSinh-C01, showing predictedtherapeutic concentrations for several hours.

Pendrin Inhibition Alone Does Not Affect Fluid-Electrolyte and Acid-Base BalancePDSinh-C01 was administered to mice by IP injection as donein the pharmacokinetics measurements. Figure 3A shows sim-ilar 3-hour urine volume and osmolality in two differentstrains of mice treated with vehicle or PDSinh-C01, even at avery high dose of 50 mg/kg. PDSinh-C01 administration did notsignificantly 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 in-hibitor PDSinh-A01 did not affect 3-hour urine volume and os-molality when given alone to mice (Supplemental Figure 3).

Pendrin Inhibition Potentiates Diuretic Action ofFurosemideBecause pendrin inhibitors alone did not produce a diureticresponse in mice, we tested whether pendrin inhibition mightaugment the diuretic response to furosemide, a loop diuretic

2 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2016


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that increases salt delivery to the pendrin-expressing CNTandCCD.Micewere administered furosemide and PDSinh-C01 (orvehicle) IP at time zero, and urine was collected for the next 3hours. Figure 4A shows that PDSinh-C01 (10 mg/kg) signifi-cantly increased urine volume by approximately 30% at eachdose of furosemide tested, without effect on urine osmolality.The diuretic effect was significantly greater than that producedby maximal furosemide (50 mg/kg). Increasing PDSinh-C01dose to 50 mg/kg did not further potentiate the furosemideeffect. PDSinh-C01, when givenwith 20mg/kg furosemide, didnot affect urine pH (Figure 4B) but produced a compensatedmetabolic alkalosis (Figure 4C). PDSinh-C01 increased 3-hoururinary Na+ and Cl2 excretion, with no significant effect on K+

excretion (Figure 4D). To rule out an inhibitory effect of furose-mide on pendrin activity that could confound the physiologicdata, in vitro measurements showed no effect of furosemide onpendrin activity (Figure 4E). PDSinh-A01 had a similar effect on3-hour urine volume and osmolality in furosemide-treated mice(Supplemental Figure 3).

Because long-term loop diuretic treatment upregulatesrenal pendrin expression,14 which might potentiate the

diuretic efficacy of pendrin inhibition, we studied the actionof PDSinh-C01 in a long-term furosemide treatment model(Figure 5A). After 8 days of furosemide treatment, PDSinh-C01 further potentiated the furosemide effect. Figure 5Bshows an approximately 60% increase in urine volume afterPDSinh-C01 in the mice receiving long-term furosemide,without effect on urine osmolality. PDSinh-C01 significantlyincreased urinary Na+, K+, and Cl2 excretion (Figure 5C).

Pendrin Inhibitors Reduce the Diuretic Action ofHydrochlorothiazideMotivated by published data on pendrin/ NCC double-knockout mice,12 we investigated whether pendrin inhibitorsmight augment the diuretic effect of hydrochlorothiazide(HCTZ). As done in the acute furosemide study, mice weretreated with HCTZ (20mg/kg) alone or together with PDSinh-C01. Figure 6A shows that, unexpectedly, acute pendrin in-hibition reduced the diuretic effect of HCTZ, increasing urineosmolality (Figure 6A) and reducing electrolyte excretioncompared with HCTZ alone (Figure 6B). Similarly, PDSinh-A01 treatment reduced urine volume and increased urine

Figure 1. Small-molecule inhibitor of murine pendrin. (A) Chemical structure and structure-activity analysis of PDSinh-C01. (B) Pendrin-mediated Cl2/anion exchange. Diagram (top) and representative data (bottom) of FRT cells coexpressing murine pendrin and a YFPhalide sensing protein showing quenching of intracellular YFP fluorescence by iodide. (C) Concentration dependence of pendrin-facilitated Cl2/anion exchange by PDSinh-C01 (mean6SEM). (D) Pendrin-mediated Cl2/HCO3

2 exchange in BCECF-loaded FRT cellsexpressing murine pendrin. Diagram of assay (top) and representative data (bottom). Cl2/HCO3

2 is initiated by addition of gluconate-containing solution to drive Cl2 efflux, HCO3

2 influx, and cytoplasmic alkalinization. (E) Concentration dependence of pendrin-facilitatedCl2/HCO3

2 exchange by PDSinh-C01 (mean6SEM). (F) Specificity studies showing no significant inhibition of AE1 (Slc4a1), NKCC1(Slc12a2), CLD (Slc26a3), and ENaC by 25 mM PDSinh-C01 (mean6SEM). 4,49-Diisothiocyano-2,29-stilbenedisulfonic acid (DIDS) was usedat 500 mM to inhibit AE1 and bumetanide at 25 mM to inhibit NKCC1. BCECF, 29,79-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein.

J Am Soc Nephrol 27: ccc–ccc, 2016 Pendrin Inhibitors as Diuretics 3

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osmolality in HCTZ-treated mice (Supplemental Figure 3).Possible reasons for this unanticipated finding are discussedbelow. Figure 6C shows that HCTZ does not inhibit pendrindirectly, nor does PDSinh-C01 inhibit NCC, the major target ofHCTZ. Additional experiments confirmed that HCTZ andPDSinh-C01donot inactivate one another (Supplemental Figure 4).

DISCUSSION

We found that short-term administration of two distinctchemical classes of pendrin inhibitors did not produce adiuretic response inmice when given alone but potentiated thediuresis produced by furosemide, with a greater effect found inmice given long-term furosemide. The lack of effect of pendrininhibition alone agrees with the lack of renal effect of pendrinknockout in mice and of pendrin loss of function in humanswith Pendred syndrome. The potentiation of the diureticaction of furosemide by short-term pendrin inhibition sup-ports the idea that pendrin limits diuretic efficacy by absorp-tion of excess salt delivered to the pendrin-expressing distalnephron. The proportionately greater potentiation of pendrininhibition with long-term furosemide treatment can be

explained on the basis of pendrin upregula-tionwith long-termfurosemide treatment,14

likely in part from increased aldosteronerelease.10 Our findings clarify the role ofpendrin in normal renal physiology andsupport the potential efficacy of pendrininhibitors to augment the diuretic efficacyof furosemide.

The potentiation of the diuretic actionoffurosemide by pendrin inhibition is likelydue to reduced absorption of excess Cl2

delivered to the distal nephron after furo-semide inhibition of Na+/K+/2Cl2 cotrans-port by NKCC2 in the thick ascending limbof Henle, as diagrammed in Figure 7. Pen-drin inhibition, together with short-termfurosemide administration, increased Na+

and Cl2 excretion, without significant ef-fect on K+ excretion. In mice given long-term furosemide treatment, in whichgreater potentiation by pendrin inhibitionwas seen, K+ excretion increased signifi-cantly, as did Na+ and Cl2 excretion. Distalfluid delivery is an important determinantof active K+ secretion in CNT and CCDwith increased flow, which reduces luminalK+ concentration and increases the electro-chemical driving force for K+ secretionacross the apical membrane. Various di-uretics that increase distal fluid deliveryproduce 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 setting15 but caused hypokalemiawith long-term administration, albeit to a lesser degree thanthat produced by furosemide.16 Our findings here that pen-drin inhibition does not increase K+ excretionwith short-termfurosemide treatment, but does with long-term furosemidetreatment, can be explained by the greater diuresis and distalfluid delivery in the long-term setting. HCTZ was reported toinhibit pendrin and NDCBE-dependent electroneutral Na+

absorption without promoting K+ secretion in isolated CCDof mice on an Na+-depleted diet.17 Because pendrin inhibitorsaugmented the kaliuretic response to furosemide in our study,further studies are needed to assess the role of pendrin in distalK+ secretion.

As a Cl2/HCO32 exchanger, pendrin inhibition could, in

principal, reduce HCO32 secretion by pendrin-expressing tu-

bule segments and hence can cause relative urinary acidifica-tion and metabolic alkalosis. However, we found no effect ofpendrin inhibition on urine pH when given alone or withfurosemide. Blood gas analysis showed that pendrin inhibitionproduced compensated metabolic alkalosis when given withfurosemide but had no effect when given alone. Earlier studies

Figure 2. PDSinh-C01 pharmacokinetics in mice. (A) Inhibitor concentration assayed inmouse plasma by LC/MS after organic-phase extraction. Representative original LC/MS data from plasma containing known (added) amounts of PDSinh-C01 and de-duced calibration curve of integrated peak signal versus PDSinh-C01 concentration. (B)Plasma concentrations of PDSinh-C01 after single IP injection of 10 mg/kg PDSinh-C01.Original LC/MS data (right) and deduced time course of PDSinh-C01 concentration(left, mean6SEM, three mice). (C) Representative LC/MS data from urine containingknown (added) amounts of PDSinh-C01 and deduced calibration curve of integratedpeak signal versus PDSinh-C01 concentration. (D) Urine concentrations of PDSinh-C01and original LC/MS data (right) from study in B (mean6SEM, three mice).





showed that pendrin knockout mice have unchanged urinarysalt excretion but decreased urine pH and increased serumHCO3

2,18 which may be related to compensatory changes inthe expression of other HCO3

2 transporters in pendrinknockout mice. Furosemide produces urinary acidificationby increasing titratable acid, H+, and ammonium excre-tion19,20 and can cause Cl2 depletion alkalosis. During themaintenance phase of Cl2 depletion alkalosis, pendrin activityis increased and limits the alkalosis by secreting HCO3

2 intothe lumen in exchange for Cl2.21,22 In the short-term settingstudied here, furosemide slightly increased serum HCO3

2

(20.5 mmol/L with versus 17.7 mmol/L without furosemide),which was further increased with pendrin inhibition (23.0mmol/L), with compensatory increased pCO2. The mildlyincreased serum HCO3

2 with pendrin inhibition and furose-mide may be due to inhibition of attenuating effect of pendrinon development of the Cl2 depletion alkalosis.

An unanticipated finding of our study was the reduceddiuretic action of HCTZ with pendrin inhibition by twochemically unrelated pendrin inhibitors. The original idea thatpendrin inhibition can augment thiazide-induced diuresiswas based on the finding that double-knockout mice lackingpendrin and NCCmanifested profound salt wasting, whereasmice lacking pendrin and NCC individually showed no

fluid-electrolyte abnormalities.12 The dif-ference in results between the prior knock-out study and the inhibition study heremaybe related to confounding effects of alteredexpression of renal salt and water trans-porters produced by gene knockout. Forexample, pendrin knockout mice show atendency for NCC upregulation,23 andNCC knockout mice show distal nephronhypertrophy, significant pendrin overex-pression, and apical displacement of pen-drin.24 Pendrin or NCC overexpressionmay thus compensate for the loss of NCCor pendrin, respectively, in the knockoutmice.4 The knockoutmicemay alsomanifestaltered expression of other transporters andhormonal factors. Two pendrin and oneNDCBE are thought to function in parallelto produce electroneutral Na+ and Cl2 ab-sorption in intercalated cells.25 This mecha-nism is sensitive to HCTZ, although NCCexpression is absent in CNT and CCD,which suggests the presence of additionaltarget(s) of HCTZ in the distal nephron.17

HCTZ-sensitive Na+ and Cl2 transport inCCD is abolished in NDCBE knockoutmice,17 and here we found that HCTZdoes not have a direct inhibitory effect onpendrin activity. HCTZ action on NDCBEand other transporters in CCD warrantsfurther investigation. An additional com-

plicating factor is the regulation of ENaC by luminal HCO32 9

in which pendrin and other HCO32 transporters are involved.

Inhibition of NCC and other target(s) by HCTZ, together withpendrin inhibition by pendrin inhibitors, might activateENaC and consequently reduce the diuretic response to HCTZ.

The pharmacokinetics experiments showed that PDSinh-C01 administration produced urine concentrations over 3hours close to its 50% inhibitory concentration for pendrininhibition. However, it is difficult to estimate the compoundconcentration in the luminal fluid of the CNTand CCD basedon urinary concentration. In addition, because PDSinh-C01likely acts at a cytoplasmic site, its intracellular concentrationis difficult to predict and would depend on inhibitor concen-trations in blood and tubule fluid and apical and basolateralmembrane transport mechanisms.

Pharmacologic inhibition of pendrin may have many clinicalindications in renal and extrarenal disorders. In addition to itseffects onCl2homeostasis, a recent study showed that pendrin isexpressed in rodent adrenal medulla and modulates catechol-amine release,26 raising the possibility of pendrin inhibitionas a potential therapy in some forms of hypertension. The po-tentiation of loop diuretic action may be useful in diuretic-refractory edema, as occurs in congestive heart failure associatedwith distal-nephron hypertrophy.27,28 Although pendrin

Figure 3. Acute effects of pendrin inhibition on fluid, electrolyte, and acid-basebalance. (A) Three-hour urine volume and osmolality in CD1 (left two panels) andC57Bl/6 (right two panels) mice after IP administration of 10 or 50 mg/kg PDSinh-C01 attime zero (mean6SEM, three to six mice per group). (B) Time course of urine pH inPDSinh-C01–treated or vehicle-treated mice (mean6SEM, three mice per group).(C) Blood gas analysis of aortic blood collected 3 hours after PDSinh-C01 or vehicletreatment (mean6SEM, three to four mice per group). (D) Three-hour urinary Na+,K+, and Cl2 excretion in PDSinh-C01–treated or vehicle-treated mice (mean6SEM,three to six mice per group). t test used for analysis. ns, not significant.



expression in the kidney has not been inves-tigated in humans or experimental animalswith congestive heart failure, we predict it tobe increased because of increased aldoste-rone. Outside of the kidney, pendrin inhibi-tion may be beneficial for asthma and otherinflammatory lung diseases2,3,29 and has po-tential application to thyroid disorders.

There are no prior reports on the biologiceffects of the pyrazole-thiophenesulfonamidechemical scaffold studied here. Computa-tional modeling suggested that this scaffoldis a potential DNA gyrase inhibitor.30 Thephysico-chemical properties of the pyrazole-thiophenesulfonamide scaffold includethe presence of multiple hydrogen bond ac-ceptors, molecular mass of 408 Da, logPvalue of 4.0, and topologic polar surfacearea of 78.9 Å2. These values are within theguidelines for good oral bioavailability,31,32

but marginal for absorption, distribution,metabolism, elimination, and toxicity.33,34

The pyrazole-thiophenesulfonamide scaffolddoes not belong to promiscuous bindersknown as pan-assay interference com-pound molecules.35 Although the presenceof a thiophene ring has been associatedwith metabolic toxicity, 2,5-disubstitutedthiophenes as found in PDSinh-C01 typi-cally do not have this liability.36 Furtheroptimization of this scaffold through struc-tural modifications will be required toachieve a pharmacologic profile suitablefor lead selection.

On the basis of the known tissue distribu-tion and physiologic roles of pendrin, severalpotential adverse effects of pendrin inhibitionarepossibleandwill requireinvestigationinthepreclinical development of a small-moleculependrin inhibitor. Humans with Pendredsyndrome develop profound deafness earlyin life as a consequence of altered inner-earfluid dynamics producing irreversible ana-tomicabnormalities.Whetherpharmacologicinhibition of pendrin could affect inner earfunction is unknown, as is whether pendrininhibition might interfere with thyroid hor-mone production in some patients.

CONCISE METHODS

MaterialsPDSinh-C01 and PDSinh-A01 were pur-

chased from ChemDiv (San Diego, CA). Other

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/kgPDSinh-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-wayANOVA with post hoc Newman–Keuls test. (B) Time course of urinary pH in miceadministered 20 mg/kg furosemide without or with PDSinh-C01 (mean6SEM, six miceper group). (C) Blood gas analysis in aortic blood collected at 3 hours in mice treatedas in B (mean6SEM, three to four mice per group). (D) Three-hour urinary Na+, K+, andCl2 excretion in mice treated as in B (mean6SEM, four to six mice per group). t testused for analysis. *P,0.05. ns, not significant. (E) Assay of murine pendrin activity inFRT cells, showing no inhibition by 25 mM furosemide.

Figure 5. Pendrin inhibitor potentiates the chronic diuretic efficacy of furosemide. (A)Experimental protocol. (B) Three-hour urine volume and osmolality after IP adminis-tration of 10 mg/kg PDSinh-C01 without or with 20 mg/kg furosemide (or vehicle) attime zero (mean6SEM, three mice per group). (C) Three-hour urinary Na+, K+, and Cl2

excretion in the same animals (mean6SEM, three mice per group). *P,0.05;**P,0.01; ***P,0.001. ns, not significant, one-way ANOVA with post hoc Newman–Keuls test.



chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless

otherwise stated. A plasmid expressing murine pendrin was generated

in pcDNA3.1 (Invitrogen, Carlsbad, CA). A plasmid encoding murine

Slc12a3 (NCC) was purchased from Dharmacon (Lafayette, CO), and

plasmids encoding murine Slc4a1 (AE1) and myc-tagged murine

Slc26a3 (CLD/DRA) were from Origene (Rockville, MD). A plasmid

encoding human NKCC1 (94% sequence similarity to murine

NKCC1) was provided by B. Forbush (Yale University, New Haven,

CT).

Cell CultureFRT cells were cultured in Kaign’s modified

Ham’s F12 medium supplemented with 10%

FBS, 2 mM L-glutamine, 100 U/ml penicillin,

100mg/ml streptomycin, 18mg/mlmyo-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 sup-

plemented with 10% FBS, 2 mM L-glutamine,

100 U/ml penicillin, and 100mg/ml streptomycin

and transiently transfected with cDNAs encoding

YFP and various membrane transporters using

Lipofectamine 2000.

Assays of Pendrin FunctionPendrin-mediated exchange ofCl2with SCN2, I2,

or NO32 was measured from the kinetics of YFP

fluorescence quenching in a BMG FLUOstar

Omega plate reader. Pendrin/YFP-expressing FRT

cells were washed in PBS and transport measured

following a 70mMgradient of SCN2, I2, orNO32.

Pendrin-mediated Cl2/HCO32 exchange was

measured from the kinetics of cytoplasmic pH

using 29,79-Bis-(2-Carboxyethyl)-5-(and-6)-

Carboxyfluorescein-AM (Invitrogen). Pendrin-

expressing FRT cells in HCO32-containing buffer

(in mM: 120 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4,

10 glucose, 5 HEPES, 25 NaHCO32; pH 7.4;

95% O2/5% CO2 equilibrated) were exposed

to a Cl2-free buffer (gluconate replacing Cl2) to

drive Cl2 efflux and HCO32 influx.

Functional Assays of Other KidneyTubule Ion TransportersYFP-based fluorescence quenching assays in

COS-7 cells were established to measure AE1

(Slc4a1), CLD (Slc26a3), and NCC (Slc12a3)

activities. ForAE1assay, cells expressingYFPand

AE1were equilibrated in PBS and then subjected

to a 70-mM SCN2 gradient (SCN2 replacing

Cl2). For CLD assay, cells expressing YFP and

CLDwere subjected to a 102-mMSCN2 gradient.

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 I2 gradient. A EYFP-H148Q/V163S/F46L fluo-

rescence quenching assay in FRT cells was used to measure NKCC1

activity in which cells that had been equilibrated in gluconate-

substituted PBS buffer were exposed to a Cl2-containing solution con-

taining 75 mM Na+ and 75 mM K+. Amiloride-sensitive ENaC activity

was measured by short-circuit current analysis of human bronchial

epithelial cell cultures.13 Specificity assays were done with 25 mM

PDSinh-C01.

Figure 6. Pendrin inhibitor reduces the diuretic efficacy of HCTZ. (A) Three-hour urinevolume and osmolality after IP administration of 10 mg/kg PDSinh-C01 without or with20 mg/kg HCTZ (or vehicle) at time zero (mean6SEM, five to six mice per group). (B)Three-hour urinary Na+, K+, and Cl2 excretion in the same animals (mean6SEM, fiveto six mice per group). *P,0.05; **P,0.01 ***P,0.001. ns, not significant, one-wayANOVA with post hoc Newman–Keuls test. (C) Assays of murine pendrin (left) andNCC (Slc12a3, right) in transfected FRT cells showing no inhibition of pendrin by25 mM HCTZ or of NCC by 25 mM PDSinh-C01 (with 25 mM HCTZ producing full in-hibition as positive control).

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 theUniversity of California,

San Francisco, Institutional AnimalCare andUseCommittee.Mice

were provided with standard rodent chow (PicoLab Rodent Diet

20; Lab Diet, St. Louis, MO) and water ad libitum during all

experiments.

PharmacokineticsFemale CD-1mice (8–10weeks) were injected with 10mg/kg PDSinh-

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 sep-

arate plasma. Urine was collected in metabolic cages. Plasma and

urine samples (60ml) weremixed with 300-ml acetonitrile and centri-

fuged 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 StudiesIn short-term studies, mice (both CD-1 and C57Bl/6 strains) were

injected IP with 10 or 50 mg/kg PDSinh-C01. In some experiments,

mice were treated with 10 mg/kg PDSinh-C01 or PDSinh-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 stud-

ies, mice were injected with 20 mg/kg furosemide (IP) twice a day for

8 days and then administered PDSinh-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 AnalysisSodium 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 AnalysesExperiments 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 consid-

ered to represent statistically significant differences.

ACKNOWLEDGMENTS

This work was supported by grants DK72517, DK101373, DK35124,

EB00415, EY13574, and DK99803 from the National Institutes of

Health and grant R613 from the Cystic Fibrosis Foundation.

Some parts of this study were presented at the American Society of

Nephrology Kidney Week on November 3–8, 2015 (San Diego, CA).

DISCLOSURESNone.

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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2015121312/-/DCSupplemental.





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