BASIC RESEARCH www.jasn.org Small-Molecule Inhibitors of Pendrin Potentiate the Diuretic 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 ABSTRACT 2 2 Pendrin is a Cl /HCO3 exchanger expressed in type B and non-A, non-B intercalated cells in the distal 2 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 2 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- 2 C01, inhibited Cl /anion exchange mediated by mouse pendrin with a 50% inhibitory concentration of 1–3 mM, 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, + 2 administration of PDSinh-C01 produced a 30% increase in urine output, with increased Na and Cl ex- cretion. 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. J Am Soc Nephrol 27: 3706–3714, 2016. doi: 10.1681/ASN.2015121312 2 2 2 2 + 2 2 Pendrin (Slc26a4) is a Cl /anion (HCO3 ,I ,SCN ) activity of the Slc4a8 Na -dependent Cl /HCO3 exchanger whose loss of function in humans exchanger (NDCBE).4 Pendrin expression is upre- with Pendred syndrome causes early-onset senso- gulated by a wide variety of stimuli, including aldo- rineural hearing loss, which is sometimes associ- sterone and salt depletion.5,6 Humans with Pendred ated with thyroid and vestibular abnormalities.1 syndrome have normal urinary concentrating func- Pendrin is expressed primarily in the kidney, thy- tion, but a child with Pendred syndrome was report- roid gland, inner ear, and inflamed airways.2,3 In ed to have enhanced diuretic response to thiazides7; the kidney, pendrin is expressed in the apical mem- brane of type B and non-A, non-B intercalated cells in the cortical collecting duct (CCD) and the con- Received December 17, 2015. Accepted March 18, 2016. necting tubule (CNT).4 Pendrin functions primarily O.C. and P.M.H. contributed equally to this work. 2 2 in renal Cl absorption and HCO3 secretion though 2 2 Published online ahead of print. Publication date available at its Cl /HCO3 exchange function; secondary effects www.jasn.org. of pendrin activity on electrochemical driving forces Correspondence: Dr. Alan S. Verkman, Departments of Medi- and autocrine factors, such as luminal ATP and cine and Physiology, University of California, San Francisco, 1246 2 HCO3 , may account for pendrin involvement in Health Sciences East Tower, Box 0521, San Francisco, CA 94143- renal Na+ absorption through effects on epithelial 0521. Email: [email protected] sodium channel (ENaC) expression/function and Copyright © 2016 by the American Society of Nephrology 3706 ISSN : 1046-6673/2712-3706 JAmSocNephrol27: 3706–3714, 2016 www.jasn.org BASIC RESEARCH in addition, limited data suggest a protective role of pendrin cells stably expressing murine pendrin and a yellow fluores- loss of function mutations in hypertension.8 cent protein (YFP) halide-sensing fluorescent indicator (Figure 2 2 2 Phenotype studies in knockout mice have suggested pendrin as a 1B). Addition of I ,SCN ,orNO3 to the extracellular solution target for development of a new class of diuretics for treatment of cause YFP fluorescence quenching in pendrin-expressing cells, salt-sensitive hypertension and edema. Compared with wild-type with near-zero quenching in cells expressing YFP alone. Pendrin mice, pendrin knockout mice show reduced BP on a salt-restricted inhibition by PDSinh-C01 reduced the rate of fluorescence 2 9,10 6 diet, relative inability to excrete an HCO3 load, and a reduced quenching in a concentration-dependent manner (Figure 1C). 2 2 5 pressor response to aldosterone. Pendrin-overexpressing mice Pendrin-mediated Cl /HCO3 exchange was measured from manifest salt-sensitive hypertension.11 Themostremarkablephe- the kinetics of intracellular pH, using 29,79-Bis-(2-Carboxy- notype emerging from knockout studies is profound salt wasting ethyl)-5-(and-6)-Carboxyfluorescein fluorescence as a cytoplas- 2 in mice lacking pendrin and the Na+/Cl cotransporter (NCC) mic pH sensor, following extracellular addition of gluconate in 2 2 2 under conditions where the single-knockout mice do not manifest HCO3 /CO2-containing buffer to drive Cl efflux, HCO3 influx, 12 salt wasting or volume depletion. However, because results in and consequent cytoplasmic alkalinization (Figure 1D). PDSinh- 2 2 knockout mice are confounded by compensatory changes in the C01 reduced the kinetics of Cl /HCO3 exchange, the activity of expression of other renal salt and water transporters, the precise pendrin of relevance to kidney function, in a concentration- role of pendrin in renal function remains unclear, as does the dependent manner with a 50% inhibitory concentration of ap- therapeutic utility of pharmacologic pendrin inhibition. proximately 1.2 mM (Figure 1E). In selectivity studies, PDSinh-C01 Here, to clarify the role of pendrin under conditions not at 25 mM did not significantly inhibit the transport activities of confounded by gene knockout, as well as the therapeutic Slc4a1 (anion exchanger 1 [AE1]), Slc26a3 (chloride anion ex- potential of pendrin inhibitors, we tested the diureticefficacyof changer [CLD]/downregulated-in-adenoma [DRA]), Na-K-Cl pendrin inhibitors in mice. The inhibitors were identified in cotransporter (NKCC1;Slc12a2), or ENaC (Figure 1F). PDSinh- a high-throughput screen against human pendrin, which was C01 inhibition of pendrin was reversible, slow (suggesting an recently reported.13 Here, compounds were characterized and intracellular cite of action), noncompetitive, and not affected 2 optimized for inhibition of murine pendrin and were tested in by pH or high extracellular Cl (Supplemental Figure 2). mice alone and in combination with short- or long-term di- uretic therapy. Although no effect of pendrin inhibition alone Pharmacokinetics of PDSinh-C01 in Mice was seen, as predicted from data in pendrin knockout mice Pharmacokinetics measurements were done to guide studies and humans with Pendred syndrome, we found significant of diuretic efficacy. A liquid chromatography/mass spectrometry potentiation of the diuretic response to furosemide. (LC/MS) method was developed to measure PDSinh-C01 concen- trations in mouse blood and urine. Figure 2, A and C, shows original LC/MS data and linear standard curves in plasma and urine RESULTS in which known amounts of PDSinh-C01 were added to plasma and urine from untreated mice. Figure 2, B and D, summarizes PDSinh- Characterization of Pendrin Inhibitors C01 concentrations in plasma and urine after bolus intraperitoneal As reported separately, a cell-based functional high-throughput (IP) administration of 10 mg/kg PDSinh-C01, showing predicted screen of 36,000 synthetic small molecules against human therapeutic concentrations for several hours. pendrin revealed several chemical classes of small-molecule inhib- itors. After structure-activity studies and testing on murine pendrin, Pendrin Inhibition Alone Does Not Affect Fluid- a pyrazole-thiophenesulfonamide, PDSinh-C01, was selected for Electrolyte and Acid-Base Balance comprehensive analysis (Figure 1A), with corroborative analysis PDSinh-C01 was administered to mice by IP injection as done done for a chemically unrelated tetrahydropyrazolopyridine pen- in the pharmacokinetics measurements. Figure 3A shows sim- drin inhibitor, PDSinh-A01 (Supplemental Figure 1). PDSinh-C01 is ilar 3-hour urine volume and osmolality in two different composed of a thiophene with a sulfonamide group and a pyrazole strains of mice treated with vehicle or PDSinh-C01, even at a heterocycle linked at the 3 and 5 positions, respectively. Structure- very high dose of 50 mg/kg. PDSinh-C01 administration did not activity studies showed that changing the pyrazole from the 5 to the significantly change urine pH (Figure 3B) or blood gas values 4 position abolished activity and that a sulfonamide group at the (Figure 3C), nor did it affect 3-hour urinary electrolyte excretion 2 or 3 position was needed for inhibition activity. Limited substit- (Figure 3D). In addition, the chemically unrelated pendrin in- uentsonthepyrazolewerestudied,with39,49-dimethyl giving the hibitor PDSinh-A01 did not affect 3-hour urine volume and os- most potent compounds, followed by 39-methyl and trifluoro- molality when given alone to mice (Supplemental Figure 3). methyl.