Chlorthalidone Is Superior to Potassium Citrate in Reducing Calcium Phosphate Stones and Increasing Bone Quality in Hypercalciuric Stone-Forming Rats

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Chlorthalidone Is Superior to Potassium Citrate in Reducing Calcium Phosphate Stones and Increasing Bone Quality in Hypercalciuric Stone-Forming Rats BASIC RESEARCH www.jasn.org Chlorthalidone Is Superior to Potassium Citrate in Reducing Calcium Phosphate Stones and Increasing Bone Quality in Hypercalciuric Stone-Forming Rats Nancy S. Krieger,1 John R. Asplin,2 Ignacio Granja,2 Felix M. Ramos,1 Courtney Flotteron,1 Luojing Chen,1 Tong Tong Wu,3 Marc D. Grynpas,4 and David A. Bushinsky1 1Division of Nephrology, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York; 2Litholink Corporation, Laboratory Corporation of America Holdings, Chicago, Illinois; 3Department of Biostatistics and Computational Biology, University of Rochester School of Medicine, Rochester, New York; and 4Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada ABSTRACT Background The pathophysiology of genetic hypercalciuric stone-forming rats parallels that of human idiopathic hypercalciuria. In this model, all animals form calcium phosphate stones. We previously found that chlorthalidone, but not potassium citrate, decreased stone formation in these rats. Methods To test whether chlorthalidone and potassium citrate combined would reduce calcium phos- phate stone formation more than either medication alone, four groups of rats were fed a fixed amount of a normal calcium and phosphorus diet, supplemented with potassium chloride (as control), potassium citrate, chlorthalidone (with potassium chloride to equalize potassium intake), or potassium citrate plus chlorthalidone. We measured urine every 6 weeks and assessed stone formation and bone quality at 18 weeks. Results Potassium citrate reduced urine calcium compared with controls, chlorthalidone reduced it fur- ther, and potassium citrate plus chlorthalidone reduced it even more. Chlorthalidone increased urine citrate and potassium citrate increased it even more; the combination did not increase it further. Potassium citrate, alone or with chlorthalidone, increased urine calcium phosphate supersaturation, but chlorthali- done did not. All control rats formed stones. Potassium citrate did not alter stone formation. No stones formed with chlorthalidone, and rats given potassium citrate plus chlorthalidone had some stones but fewer than controls. Rats given chlorthalidone with or without potassium citrate had higher bone mineral density and better mechanical properties than controls, whereas those given potassium citrate did not. Conclusions In genetic hypercalciuric stone-forming rats, chlorthalidone is superior to potassium citrate alone or combined with chlorthalidone in reducing calcium phosphate stone formation and improving bone quality. JASN 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018101066 Idiopathic hypercalciuria (IH), an excess of urinary calcium (Ca) without a demonstrable metabolic cause, is the most common metabolic abnormality Received October 31, 2018. Accepted March 14, 2019. 1–3 in patients who form Ca-based kidney stones. Published online ahead of print. Publication date available at Elevated levels of urinary Ca increase the probabil- www.jasn.org. ity for nucleation and growth of calcium oxalate Correspondence: Dr. Nancy S. Krieger, Division of Nephrology, (CaOx) or calcium hydrogen phosphate (brushite) Department of Medicine, University of Rochester School of crystals into clinically significant kidney stones.4 Medicine and Dentistry, 601 Elmwood Avenue, Box 675, Ro- Patients with IH generally have normal serum Ca, chester, NY 14642. Email: [email protected] normal or elevated serum 1,25–dihydroxyvitamin Copyright © 2019 by the American Society of Nephrology JASN 30: ccc–ccc,2019 ISSN : 1046-6673/3007-ccc 1 BASIC RESEARCH www.jasn.org D , normal or elevated serum parathyroid hormone, and low 3 Significance Statement bone mineral density (BMD).4,5 IH exhibits a polygenic mode of inheritance.3,6,7 Genetic hypercalciuric stone-forming rats, which universally and To study this disorder we generated a strain of rats, the spontaneously form calcium phosphate stones, have a pathophys- genetic hypercalciuric stone-forming (GHS) rats to model hu- iology resembling that of human idiopathic hypercalciuria. The authors previously demonstrated that chlorthalidone, but not po- man IH. Selectively inbred for over 111 generations, GHS rats tassium citrate, decreased stone formation in this rat model. In this excrete approximately ten times the normal urine Ca of their study, they investigated whether chlorthalidone and potassium parent Sprague–Dawley rats.8,9 When fed a normal Ca diet, citrate combined would reduce calcium phosphate stone formation all form calcium phosphate (CaP) kidney stones.10 We have more than either medication alone. They found that chlorthalidone shownthathypercalciuriainGHSratsispolygenic.11,12 was more effective than potassium citrate alone or combined with chlorthalidone in reducing stone formation and increasing me- Like patients with IH, these rats have increased intestinal chanical strength and bone quality. However, replication of these Ca absorption,10,13 decreased renal Ca reabsorption,14 findings in patients with nephrolithiasis is needed before concluding and increased bone resorption,15 leading to increased urine that chlorthalidone alone is more efficacious in this regard than Ca excretion and CaP stone formation10,16,17 as well as a de- potassium citrate alone or in combination with chlorthalidone. 18,19 crease in BMD. Serum 1,25-dihydroxyvitamin D3 levels 13,20,21 are normal. imaging in a Faxitron. Femurs, humeri, tibiae, and vertebral Two important strategies that are used to decrease recurrent columns were harvested and prepared for BMD, histologic stone formation in humans are the use of potassium citrate studies, and mechanical testing. The University of Rochester 22,23 (KCit) or thiazide diuretics, alone or in combination. In Committee for Animal Resources approved all procedures. humans both KCit and thiazides alone have been shown to decrease stone formation24; however, there is a paucity of Urine and Serum Chemistries data directly comparing the efficacy of these two medications Urine Ca, magnesium, phosphorus, ammonium, and creati- in combination to prevent recurrent stone formation. We nine were measured spectrophotometrically using a Beckman have previously shown that giving GHS rats thiazides (specif- AU autoanalyzer (Beckman Coulter, Brea, CA). Urine potas- ically chlorthalidone [CTD]) decreases urine Ca, reduces sium, chloride, and sodium were measured by ion-specific urine CaP supersaturation, and decreases stone formation.25 electrodes on the Beckman AU and urine pH using a glass We also found that CTD improves BMD and bone quality in electrode. Urine citrate, oxalate, and sulfate were measured GHS rats.26 In a separate study we observed that giving by ion chromatography using a Dionex ICS 2000 system GHS rats KCit also decreases urine Ca, but increases CaP su- (Dionex Corp., Sunnyvale, CA). Oxalate was measured enzy- persaturation and does not decrease stone formation.27 In this matically using oxalate oxidase. All urine solutes were mea- study, we tested the hypothesis that CTD and KCit combined sured at 6, 12, and 18 weeks and a mean value for each time would more effectively reduce CaP stone formation and im- period as well as an overall mean was calculated. Serum Ca and prove BMD and bone quality in GHS rats than either treat- phosphorus were determined colorimetrically (BioVision, ment alone. Milpitas,CA).SerumparathyroidhormoneandReceptor activator of NF-kB ligand were determined by enzyme im- munoassay (Immutopics, San Clemente, CA and R&D Sys- METHODS tems, Minneapolis, MN). All of these methods have been used previously.16,26–30 Study Protocol Three-month-old GHS rats from the 111th generation were Urine Supersaturation randomly divided into four groups (each n=10) and housed Urine supersaturation with respect to CaOx and CaP solid individually in metabolic cages. All rats were fed a fixed phases were calculated from solute measurements using the amount of a normal Ca (1.2% Ca) and phosphorus computer program EQUIL2,31 as we have done previ- (0.65%) diet, supplemented with either potassium chloride ously.10,27,30,32–34 (4 mmol/d), KCit (4 mmol/d), CTD (1.25 mg/d) plus potas- sium chloride (to keep potassium intake constant), or KCit plus Kidney Stone Formation CTD, and had free access to deionized, distilled water. At weeks Kidneys and ureters were removed from each rat en bloc, fro- 6, 12, and 18, each rat was weighed and 24-hour urine was zen, and imaged in a Faxitron radiography device (Tucson, collected over 4 days, with two collections in thymol for pH, AZ) to determine extent of kidney stone formation. Three uric acid, and chloride, and two collections in hydrogen chlo- observers blinded to treatment scored all radiographs on a ride for all other measurements. Each rat received an intraper- scale ranging from 0 (no stones) to 4 (extensive stones). itoneal injection of 1% calcein green at 10 and 2 days before being euthanized for dynamic histomorphometry. At 18 weeks, Dual-Energy X-Ray Absorptiometry rats were euthanized and blood was collected via cardiac punc- Dual-energy x-ray absorptiometry with a Lunar PIXImus Bone ture. Kidneys, ureters, and bladders were removed for x-ray Densitometer (Lunar GE, Mississauga, Canada) was used to 2 JASN JASN 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH determine tissue density and mineral content. The areal BMD, Biomechanical Properties bone mineral content, and bone area were measured. Biomechanics of femurs were assessed using an Instron
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