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

Home , Calcium oxalate, Potassium citrate

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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- BASIC RESEARCH 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 further, 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 chlorthalidone 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: 1163–1173, 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

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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 potassium 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

1164 JASN JASN 30: 1163–1173, 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 4465 mechanical testing instrument (Instron, Burlington, Canada) Microcomputed Tomography and Labview 5.0 data acquisition software (National Instru- Microcomputed tomography with an 1174 compact Micro-CT ments, Austin, TX) to deﬁne a load-displacement curve. (Skyscan, Kontich, Belgium) was used to measure volumetric Ultimate load, stiffness, ultimate displacement, and energy bone mineral density (vBMD) and microarchitecture of the to break were calculated from the load-displacement curve. mid-diaphysis of right femurs and L6 vertebrae. For vertebrae, Data were normalized for specimen geometry to create a stress- density was measured between the growth plates, and for fe- strain curve to derive ultimate stress, failure strain, toughness, murs, 1 mm above and below the midshaft of the bone. Mea- and the Young modulus. sured parameters include femoral vBMD, anterior-posterior diameter, trabecular vBMD, bone volume (BV/TV), trabecular Three-Point Bending number (Tb.N), trabecular thickness (Tb.Th), and trabecular Three-point bending was performed on the right femurs to test separation (Tb.Sp). the strength of the cortical bone.

Tissue-Level Remodeling Vertebral Compression Tissue-level remodeling was assessed via histomorphometry Vertebral compressionwas measured on excised L6 vertebrae. on both mineralized (undecalcified) bone and unmineralized Vertebral compression does not result in complete fracture; (decalcified) bone. Stained sections were viewed microscopi- the failure point was determined by an 8%–10% reduction cally and results quantified using computer software. in load.

Undecalcified Histomorphometry Femoral Neck Fracture Undecalcified histomorphometry differentiates between min- The proximal end of the femurs was subjected to femoral neck eralized and demineralized tissue in bones fixed in 70% fracture using an Instron 4465 to test the mechanical properties ethanol. Sections were used for static and dynamic histomor- of cortical and trabecular bone combined. phometric analysis. Cross-sections of the left distal tibiae were used for back-scattered electron microscopy. Degree of Mineralization Back-scattered electron microscopy on both right tibiae and Static Histomorphometry left distal tibiae cross-section samples was done with a scanning Sectionsof undecalcified right tibiae were stained with Goldner electron microscope (XL300ESEM; Philips). Images were trichrome and quantified using the Bioquant Osteo 11.2.6 MIR taken using a solid-state back-scattered electron microscopy software (Bioquant Image Analysis, Nashville, TN). Trabecular detector (FEI Company, Hillsboro, OR). bone was analyzed in the proximal tibia metaphysis region. Parameters measured include percent BV/TV, percent bone Statistical Analyses surface, Tb.N, Tb.Sp, Tb.Th, osteoid volume, percent osteoid Urine analytes, serum values, and stone formation, expressed volume per bone volume, osteoid surface, percent osteoid sur- as mean6SEM, were compared among the four treatment face per bone surface, and osteoid width. groups (KCl, KCit, KCl+CTD, and KCit+CTD) by ANOVA with Bonferroni correction for pairwise comparison among Dynamic Histomorphometry the treatment groups (Statistica; StatSoft, Tulsa, OK). The Sections of calcein-labeled undecalcified right tibiae were main effects of the two drugs (KCit and CTD) and their in- used for dynamic histomorphometry and quantified using teraction effects at 18 weeks were tested by comparing to po- Bioquant Osteo 11.2.6 MIR software. Parameters measured tassium chloride (as control group) using linear regression include percent mineralized surface per bone surface, min- models on urine analytes, serum values, and stone formation eral apposition rate, bone formation rate normalized over (SAS v9.4; StataCorp., Cary, NC), assuming normally distrib- bone surface, and bone formation rate normalized over bone uted. Bone parameters were compared by t test using the SPSS volume. Statistics 20 program (SPSS, Chicago, IL) and expressed as mean6SD. P,0.05 was considered significant. Decalcified Histology Sections of decalcified left tibiae were stained for tartrate-re- sistant acid phosphatase to measure osteoclasts for assessment RESULTS of bone resorption and quantified using Bioquant Osteo 11.2.6 software. Trabecular bone of proximal tibial metaphysis was Urine and Serum analyzed for osteoclast surface, percent osteoclast surface, At the end of the study mean overall urine Ca for the entire 18- number of osteoclasts, and number of osteoclasts per mm week study was significantly decreased in male rats fed KCit, osteoclast surface. CTD decreased urine Ca further, and KCit+CTD decreased it

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Urine Ca Urine Citrate Urine P 30 300 40 * o# # * o * 30 o * o * 20 * *o# 200 20 mg/d 10 100 10

0 0 0

KCI KCit KCI KCit KCI KCit

KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD

Figure 1. Urine Ca, citrate, and phosphorus (P) were significantly altered by KCit, CTD or KCit+CTD. Rat diets were supplemented with either potassium chloride (KCl; 4 mmol/d) as a control, KCit (4 mmol/d), CTD (1.25 mg/d) plus potassium chloride or KCit+CTD. Twenty-four hour urine collections were done at 6, 12, and 18 weeks for analysis of solute levels as described in Methods and an overall mean of all three collections was calculated. Results are mean6SEM for ten rats per group. *P,0.05 versus potassium chloride; oP,0.05 versus KCit alone; #P,0.05 versus CTD alone. even more (Figure 1). Mean overall urine citrate was increased Mean overall urine oxalate was increased by KCit; however, by both KCit and CTD alone; KCit+CTD was not different oxalate was decreased with CTD and with KCit+CTD (Figure 2). from KCit alone. Urine phosphorus was elevated by KCit but Mean overall urine ammonium (NH4) was decreased by KCit not by CTD or KCit+CTD, although urine phosphorus with and increased by CTD; the combination of KCit+CTD increased KCit+CTD was greater than CTD alone. NH4 compared with KCit but decreased it compared with CTD Although KCit alone and CTD alone each significantly alone. Mean overall urine pH was increased by KCit, decreased decreased urine Ca, there was no significant drug interac- by CTD, and the combination was not different than KCit alone. tion betweenKCitandCTD whenurine Ca measurementsat There was a no significant drug interaction at 18 weeks 18 weeks were analyzed (P=0.55). KCit alone and CTD (P=0.18) between the effects of KCit and CTD on urine ox- aloneincreasedurinecitrateandtherewasasignificant alate. Although KCit decreased and CTD increased urine negative interaction effect when rats received both ammonium, there was a significant negative interaction ef- KCit+CTD (P=0.007). Only KCit alone significantly in- fect (P=0.001) when both KCit and CTD were given. Al- creased urine phosphorus and there was a significant neg- though KCit increased and CTD decreased urine pH, there ative interaction effect (P=0.02) when both KCit and CTD was a significant positive interaction effect (P=0.02) on pH were given. when both KCit and CTD were given.

Urine Ox Urine NH4 Urine pH 4 1.5 9 *o * *# 3 8 * 1.0 *o *o * 2 o# 7 * o mmol/d mmol/d * 0.5 1 6

0 0.0 5

KCI KCit KCI KCit KCI KCit

KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD

Figure 2. Urine oxalate (Ox), NH4, and pH were signiﬁcantly altered by KCit, CTD or KCit+CTD. Rat diets were supplemented with either potassium chloride (KCl; 4 mmol/d) as a control, KCit (4 mmol/d), CTD (1.25 mg/d) plus potassium chloride, or KCit+CTD. Twenty-four hour urine collections were done at 6, 12, and 18 weeks for analysis of solute levels as described in Methods and an overall mean of all three collections was calculated. Results are mean6SEM for ten rats per group. *P,0.05 versus potassium chloride; oP,0.05 versus KCit alone; #P,0.05 versus CTD alone.

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CaP SS CaOx SS UA SS 25 30 0.8 # * # *o 20 * o 0.6 20 * 15 0.4 o 10 10 0.2 * *# 5 0.0 0 0

KCI KCit KCI KCit KCI KCit

KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD

Figure 3. Urine supersaturation (SS) of CaP and CaOx were differentially regulated by KCit, CTD or KCit+CTD. Rat diets were supplemented with either potassium chloride (KCl; 4 mmol/d) as a control, KCit (4 mmol/d), CTD (1.25 mg/d) plus potassium chloride, or KCit+CTD. Twenty-four hour urine collections were done at 6, 12, and 18 weeks for analysis of solute levels as described in Methods. These values were used to calculate relative supersaturation and an overall mean of all three collections was calculated. Values for relative supersaturation are unitless. Results are mean6SEM for ten rats per group. *P,0.05 versus potassium chloride; oP,0.05 versus KCit alone; #P,0.05 versus CTD alone.

Mean overall urine supersaturation with respect to CaP was radiographs from all the rats, done by three independent and increased by KCit but not CTD, whereas KCit+CTD was not blinded reviewers, is shown in Figure 4, B and C. Significant different from KCit alone (Figure 3). Supersaturation with re- calcification was found in all rats fed the potassium chloride spect to CaOx was decreased only by CTD, whereas KCit and control diet. Potassium citrate had no effect on stone formation KCit+CTD were not different from potassium chloride alone. whereas CTD completely prevented stone formation. Stones There was no significant interaction between KCit and CTD were present with CTD+KCit and were significantly fewer than for either CaP supersaturation (P=0.19) or CaOx supersatu- those given KCit alone but significantly greater than those given ration (P=0.50) at 18 weeks. CTD. There was no significant interaction between KCit and At 18 weeks on each of the diets, serum values for sodium, CTD with respect to calcification score (P=0.18). potassium, bicarbonate, Ca, phosphorus, creatinine, and parathyroid hormone were not different between groups (Table 1). Bone Serum chloride was significantly decreased by CTD compared Microcomputed tomography analysis demonstrated that tra- with potassium chloride alone or KCit alone. Receptor activa- becular BV/TV in the L6 vertebrae was increased in both CTD tor of NF-kBligandwassignificantly increased only by and KCit+CTD compared with potassium chloride and KCit CTD+KCit compared with potassium chloride. alone groups (Figure 5A). Tb.Th in L6 was increased by CTD compared with potassium chloride. Tb.N in L6 was increased Stone Formation by CTD and KCit+CTD compared with potassium chloride Representative radiographs of kidneys at 18 weeks on the in- and KCit. Tb.Sp in L6 was decreased by CTD and CTD+KCit dicated diets are shown in Figure 4A. Quantitation of the compared with potassium chloride and KCit.

Table 1. Serum measurements after 18 weeks in GHS rats fed KCl, KCit, CTD, or KCit+CTD Solute KCl KCit KCl+CTD KCit+CTD Sodium, mmol/d 149.362.2 150.061.9 127.0614.4 147.461.6 Potassium, mmol/d 4.3760.09 4.7660.29 4.0460.04 4.6260.45 Chloride, mmol/d 105.662.0 104.761.5 96.562.5a,b 99.061.4 Bicarbonate, mmol/d 25.460.9 28.860.7 27.561.0 28.960.7 Ca, mg/dl 10.260.2 10.460.2 10.060.4 10.860.3 Phosphate, mg/dl 5.4760.17 6.3360.27 5.9060.38 6.5460.41 Creatinine, mg/d 0.3060.03 0.2960.01 0.2560.01 0.2760.01 PTH, pg/ml 612.26179.4 669.46180.9 168.7622.8 350.7664.5 RANKL, pg/ml 31.167.0 45.169.6 61.1616.0 102.1627.9a Results are mean6SEM for nine to ten rats per group. There were no signiﬁcant differences comparing KCl+CTD to KCit+CTD. KCl, potassium chloride; PTH, parathyroid hormone; RANKL, Receptor activator of NF-kB ligand. aP,0.05 versus KCl alone bP,0.05 versus KCit alone.

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A B calcification score KCI KCit 6

o# 4 *

KCI+CTD KCit+CTD 2

*o 0

Representative X-rays KCI KCit

KCI+CTD KCit+CTD C Presence of Calcification 25 kidneys with stones 12 rats with stones 20 10

8 15 6 10 4 5 2

0 0

KCI KCit

KCI+CTD KCit+CTD

Figure 4. Only CTD decreased kidney stones and calcification. At the conclusion of the 18-week study the extent of kidney stones and calcification were determined by three observers as described in Methods. (A) Representative x-rays of kidneys from rats receiving potassium chloride (KCl), KCit, CTD, or KCit+CTD. (B) Quantitation of stone formation and calcification in all rats (mean6SEM, n=10/group). (C) The number of kidneys (left+right) that contain any calcification (open bars) and the number of rats that exhibit any calcification (closed bars). *P,0.05 versus potassium chloride; oP,0.05 versus KCit alone; #P,0.05 versus CTD alone.

Cortical bone from the femur of rats fed CTD or KCit+CTD ultimate stress and larger modulus compared with potas- demonstrated increased volumetric BMD (grams per centi- sium chloride. meter squared) compared with KCit alone, whereas only For dynamic undecalcified histomorphometry, the percent CTD increased cross-sectional bone area compared with mineralized surface per bone surface measurement was lower KCit (Figure 5B). Both CTD and KCit+CTD increased cortical in CTD compared with KCit (Table 3). Analysis of osteoclasts thickness compared with potassium chloride or KCit, whereas in decalcified bones demonstrated no differences between the mean cortical thickness was increased by CTD compared with groups. potassium chloride and KCit. Vertebral compression tests indicate that CTD and KCit+CTD supported a larger ultimate load compared DISCUSSION with the potassium chloride control (Table 2). KCit+CTD had a larger fail load compared with potassium chloride. IH is the most common metabolic abnormality in patients who There were no differences in material properties of verte- form Ca-containing kidney stones. The increase in urinary bral bone with any treatments. The results from three- Ca excretion increases supersaturation with respect to Ca-con- point bending tests indicate that cortical bone from CTD taining solid phases and enhances the probability for nucle- fedratssupportedalargerultimateloadcomparedwith ation and growth of crystals into clinically significant kidney potassium chloride and KCit (Table 2). The cortical mate- stones. Clinically the two main pharmacologic thera- rial properties demonstrated that KCit+CTD had a larger pies to treat Ca-containing kidney stones are KCit and thiazide

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A B BV/TV (%) Tb. Th. (mm) Cortical vBMD (g/cm3) Bone Area (mm2) 50 0.10 1.6 o o o * 8.0 * o o 40 * 0.09 1.5 7.0 30 0.08 1.4

20 6.0 0.07 1.3

KCI KCit KCI KCit KCI KCit KCI KCit

KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD

Tb. N. (1/mm) Tb. Sp. (mm) Cs. Th. (mm) Mean Th. (mm) 6 0.9 0.4 0.7 o 5 o o *o *o * * * o 0.8 0.3 *o * 4 0.6 0.2 0.7 3 0.1 0.5 2 0.6 0.0

KCI KCit KCI KCit KCI KCit KCI KCit

KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD KCI+CTDKCit+CTD

Figure 5. Increased trabecular and cortical bone density in response to CTD after 18 weeks. Rat diets were supplemented with either potassium chloride (KCl; 4 mmol/d) as a control, KCit (4 mmol/d), CTD (1.25 mg/d) plus potassium chloride, or KCit+CTD. At the conclusion of the 18-week study, bones were collected from all rats and analyzed as described in Methods. (A) Percent BV/TV, Tb.Th, Tb.N, and Tb.Sp of L6 vertebrae are presented. (B) Cortical vBMD, bone area, cortical thickness (Cs.Th), and mean thickness (Th) of femoral cortical bone are presented. Results are mean6SD for n=10 bones/group. *P,0.05 versus potassium chloride; oP,0.05 versus KCit alone; there were no significant differences comparing KCl+CTD to KCit+CTD. diuretics.22,23,35 A number of studies have demonstrated that bone resorption45 and increases the glomerular filtrate pH both KCit36–39 and thiazide diuretics40–43 are each effective in leading to a direct increase in tubular Ca reabsorption and a reducing kidney stone formation. This study tests the hypoth- decrease in urinary Ca excretion. The increase in urine phos- esis that combined therapy with KCit and CTD would more phorus may be secondary to citrate binding intestinal Ca, thus effectively decrease CaP kidney stone formation and increase decreasing the availability of Ca to bind intestinal phosphorus, bone quality in GHS rats compared with either treatment allowing more phosphorus to be absorbed and excreted. Sim- alone. The results clearly demonstrate that in GHS rats, CTD ilarly, the increase in urinary oxalate may be secondary to in- reduces kidney stone formation and improves bone density testinal citrate binding Ca, freeing oxalate for absorption and and quality better than the KCit or the combination of subsequent excretion. In addition, there appears to be a close KCit+CTD in this rodent model of IH. relationship and interaction between the murine anion trans- Similar to what we have demonstrated previously in GHS porter Slc26a6, which has specificity for chloride/oxalate rats,27 administration of KCit led to an increase in urinary exchange, and the citrate transporter NaDC-1.46 NaDC-1 en- citrate, a reduction in urinary Ca and an increase in urinary hances Slc26a6 transport activity, and there is a reciprocal in- phosphorus and oxalate. Citrate is absorbed in the intestine, hibition of NaDC-1 by Slc26a6 to regulate oxalate/citrate which leads to systemic alkalization and a subsequent increase homeostasis. in citrate excretion.44 The mechanism by which citrate reduces As expected, the administration of CTD to GHS rats led to a urine Ca is multifactorial and the contribution of each factor reduction in urinary Ca excretion and an increase in urine to the reduction in urine Ca is not known.27 In the intestine, citrate excretion.25 This reduction in urinary Ca is due to in- citrate binds Ca leading to a reduction in intestinal Ca absorp- creased renal tubular Ca reabsorption.14 We have previously tion and subsequent urinary Ca excretion. The systemic alka- shown that in both rats25 and humans,47 this decrease in uri- lization induced by the intestinal absorption of citrate reduces nary Ca persists as the result of a thiazide-induced reduction in

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Table 2. Mechanical properties of trabecular (vertebral compression) and cortical (three- point bending) bone from GHS rats fed KCl, KCit, CTD, or KCit+CTD Bone Properties KCl KCit KCl+CTD KCIT+CTD Vertebral compression Structural properties Ultimate load, N 163.569.1 176.3651.4 213.1634.8a 217642.5a Fail load, N 146.768.4 157.9647.1 190.6631.7 194.8638.1a Energy to fail, mJ 95.8625.8 136.3695.3 151.2646.1 156.6645.4 Stiffness, N/mm 254.4669.4 289.56116.4 286.2666.9 297.96121.4 Material properties Ultimate stress, MPa 74.4613.2 72.4622.1 69.769.1 78.868.6 Fail stress, MPa 66.8611.9 64.8620.2 62.368.3 73.263.1 Fail strain 12.862.7 14.766.0 15.162.3 16.165.5 Energy to fail, mJ/mm3 5.561.3 7.265.1 6.862.4 7.162.7 Modulus, MPa 908.16313.3 928.86390.6 782.26227.9 8056316.1 Three-point bending Structural properties Ultimate load 169.9611.2 176.9618.5 19468.5a,b 185.8612.6 Fail load 125.4636 125.3638.4 117.4630.3 103.4621 Energy to fail 155.4641.2 139.3634.7 152.4626.9 153.2638.6 Stiffness 332.2617.2 333.7639.4 349.9614.3 348.8635.4 Material properties Ultimate stress 126.3615.2 133.9616.3 142.9611.3 152.3616.7a Fail stress 93.5628.6 94.5629 86.3621.9 79.369.2 Fail strain 11.962.2 10.961.79 11.261.71 1362.3 Energy to fail 10.862.4 1062.1 10.761.7 11.461.9 Modulus 2602.56330.2 2646.76344 26966256.3 3089.76485.1a Results are mean6SD for nine to ten rats per group. There were no signiﬁcant differences comparing KCl+CTD to KCit+CTD. KCl, potassium chloride. aP,0.05 versus KCl alone. bP,0.05 versus KCit alone. intestinal Ca absorption. The fall in urine oxalate excretion in The initial CaPsolidphaseissimilar to the type of stones that the CTD-treated rats may be secondary to this reduction the GHS rats spontaneously form.10 CTD alone reduced su- in intestinal Ca absorption leading to greater luminal Ca persaturation with respect to CaP and to CaOx. Because GHS and more CaOx binding in the intestine. rats fed a standard diet form only CaP stones, the reduction in

Table 3. Histomorphometry analyses of bones from GHS rats fed KCl, KCit, CTD, or KCit+CTD Histomorphometry KCl KCit KCl+CTD KCit+CTD Undecalcified static OV/BV, % 0.8060.6 0.6760.4 0.2760.40 0.2160.21 OS/BS, % 0.1160.09 0.0760.04 0.0360.04 0.0260.02 Osteoid width, mm 2.0061.04 2.2660.54 2.3360.54 2.7761.38 Dynamic MS/BS, % 0.1160.07 0.260.08 0.160.05a 0.1660.08 MAR, mm/d 1.5360.58 1.5860.32 1.2660.62 1.4760.57 BFR/BS, mm3/mm2 per d 0.1860.16 0.3260.15 0.1560.10 0.2460.14 Decalcified Oc.S, mm 0.3360.26 0.260.22 0.2360.15 0.2960.2 Oc.S/BS, % 0.0560.04 0.0360.03 0.0360.02 0.0560.04 N.Oc 4.7063.9 464.5 4.263.7 4.262.9 N.Oc/BS (1/mm) 0.6860.54 0.3860.29 0.4960.35 0.660.5 N.Oc/Oc.s (1/mm) 14.7162.5 15.863.8 13.7664.9 13.4862.2 There were no significant differences comparing all groups to KCl alone or comparing KCl+CTD to KCit+CTD. Results are mean6SD for eight to ten rats per group. There were no significant differences comparing KCl+CTD to KCit+CTD. KCl, potassium chloride; OV/BV, % osteoid volume/bone volume; OS/BS, % osteoid surface/bone surface; MS/BS, mineralized surface/bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate/bone surface; Oc.S, osteoclast surface; Oc.S/BS, osteoclast surface/bone surface; N.Oc, number of osteoclasts; N.Oc/BS, number of osteoclasts/bone surface; N.Oc/Oc.s, number of osteoclasts/ osteoclast surface. aP,0.05 versus KCit alone.

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CaP supersaturation may guide stone formation. Supporting CTD and KCit+CTD increased both cortical and trabecular this hypothesis we have previously shown that CaP stone for- structural properties of the bone more than KCit alone. CTD mation closely follows urine supersaturation with respect to increased the ultimate load of both cortical and trabecular the CaP solid phase in GHS rats. When we reduced dietary bone. Additionally, the KCit+CTD group was effective in im- phosphate in GHS rats, we could eliminate stone formation proving the ultimate stress and modulus in the cortical bone when CaP supersaturation was less than approximately 3.8.10 compared with the control. This agrees with the microcom- Similarly, in this study, there was universal stone formation puted tomography results as CTD, without or with KCit, also at a CaP supersaturation of 4.1 and none at a supersaturation led to a larger vBMD and BV/TV compared with KCit or po- of 3.2 (Figure 3). Here KCit, with or without CTD, led to a tassium chloride alone. We previously found that CTD im- significant increase in supersaturation with respect to the CaP proved Tb.N and BV/TV in GHS rats.25,26 These results were solid phase. This suggests that the increase in monohydrogen confirmed in this study, further supporting the observation phosphate secondary to the increase in urine pH more than that CTD improves bone quality. The results presented here offsets the fall in urine Ca and the increase in urine citrate also indicate that a combination of KCit+CTD led to improved induced by the alkali loading. Stone formation was not altered mechanical strength and bone quality compared with the po- with KCit alone but was reduced somewhat when CTD was tassium chloride control or KCit, but was not different com- added to KCit. Thus, the lowest numerical value of CaP su- pared with CTD alone. persaturation, induced by CTD alone, led to the fewest stones. There have been a number of human studies on the effect This suggests that, at least in GHS rats, CTD is superior to of thiazides and/or KCit on bone that have concluded that KCit, either alone or in combination with CTD, in lowering each treatment has a favorable effect on BMD.35 Adams supersaturation with respect to CaP and in suppressing CaP et al.50 gave thiazides to five hypercalciuric, osteoporotic males stone formation. and found an increase in BMD after 9 months. In a study using In considering a direct interaction between the effects of bone biopsies before and after thiazide administration to hy- KCit and the effects of CTD, there were differences in some, but percalciuric stone formers, Steiniche et al. found a reduction not all, urine parameters that demonstrated a significant in- in bone turnover after 6 months of treatment.51 Sakhaee et al. teraction effect when rats received both KCit+CTD. There studied eight patients with hypercalciuria and a slight eleva- was a significant negative interaction effect on urine citrate, tion in parathyroid hormone and found that after 1 year, the urine phosphorus, and urine ammonium when both drugs use of thiazide diuretics reduced the secondary hyperparathy- were given. There was a significant positive interaction effect roidism and reduced fractional resorption surfaces.49 Thia- on urine pH when both drugs were given. There was no sig- zides are commonly prescribed as treatment for hypertension. nificant interaction on urine Ca or oxalate when both drugs In a meta-analysis encompassing 21 observational studies of were given. However, with respect to prevention of stone for- almost 400,000 patients, the use of thiazide diuretics was mation, the importance of a drug interaction between KCit and associated with a significant 24% reduction in the risk of hip CTD on these individual urine parameters is not clear, as there fracture.52 Pak et al.53 studied the effect of KCit on vertebral was no significant interaction for the differences in urine CaOx BMD in patients with Ca urolithiasis and found increased supersaturation, CaP supersaturation, or kidney calcification vertebral BMD after 11 months of treatment. Vescini et al.54 when both drugs were given. examined the long-term effects of KCit on distal radius BMD In this study, KCit led to an increase in urinary pH whereas in patients with hypercalciuria and also concluded that long CTD led to a fall in urinary pH. In the vast majority of humans term treatment with KCit improved the BMD. These studies with Ca stones treated with a standard dose of the alkali KCit, did not examine the mechanical properties of the bone. A urine pH increases.22,23 Whether urinary supersaturation in- recent study utilizing computed tomography scans to deter- creases with respect to the CaP and CaOx solid phases depends mine BMD found that both thiazides and KCit improved on whether citrate excretion increases and Ca excretion de- BMD.55 In our study, we again demonstrate that CTD im- creases sufficiently to offset the effect of an increase in pH.48 proves BMD and the mechanical properties of bone,26 consis- The relative benefit versus risk of citrate in humans with CaP tent with observations in humans.47 However, our results with stones is frequently debated in the kidney stone community. KCit in GHS rats, in which we find no improvement in bone However, there are no prospective trials of stone prevention in density or quality, differ from the observations in humans. CaP stone disease, so this is one of the reasons we pursued this Perhapswithlongertreatment,asinthehumanstudies, study in GHS rats. KCit would be efficacious in improving bone density and qual- A direct consequence of hypercalciuria and subsequent ity in the GHS rats. stone formation is a loss of bone mineral, potentially leading In conclusion, in GHS rats who universally spontaneously to an increase in fracture risk in patients with IH.49 We found form CaP stones, a diet containing CTD alone is superior to that bones from rats given CTD demonstrated increased bone KCit alone or KCit+CTD in reducing stone formation, increas- strength and improved mechanical properties compared with ing BMD, and improving bone quality. If these findings in KCit, suggesting that bone quality was significantly improved GHS rats are replicated in patients with nephrolithiasis, treat- with CTD but not KCit. Overall mechanical testing found that ment of recurrent hypercalciuric Ca stone formation with

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CTD alone is preferable to treatment with KCit or the combi- 9. Bushinsky DA, Frick KK, Nehrke K: Genetic hypercalciuric stone-form- nation of KCit+CTD both to decrease stone formation and to ing rats. Curr Opin Nephrol Hypertens 15: 403–418, 2006 maintain BMDand quality. Whether adding KCit to CTD when 10. Bushinsky DA, Parker WR, Asplin JR: Calcium phosphate supersaturation regulates stone formation in genetic hypercalciuric stone-forming treating CaOx stones is better than either drug alone remains to rats. Kidney Int 57: 550–560, 2000 be studied. 11. Hoopes RR Jr., Reid R, Sen S, Szpirer C, Dixon P, Pannett AA, et al.: Quantitative trait loci for hypercalciuria in a rat model of kidney stone disease. JAmSocNephrol14: 1844–1850, 2003 12. Hoopes RR Jr., Middleton FA, Sen S, Hueber PA, Reid R, Bushinsky DA, ACKNOWLEDGMENTS et al.: Isolation and confirmation of a calcium excretion quantitative trait locus on chromosome 1 in genetic hypercalciuric stone-forming con- genic rats. JAmSocNephrol17: 1292–1304, 2006 Dr. Bushinsky and Dr. Asplin designed the study. Mr. Ramos, 13. Li XQ, Tembe V, Horwitz GM, Bushinsky DA, Favus MJ: Increased in- Ms. Flotteron, Mr. Granja and Dr. Chen carried out the experiments. testinal vitamin D receptor in genetic hypercalciuric rats. A cause of Dr. Krieger, Dr. Asplin, Dr. Wu, Dr. Grynpas, and Dr. Bushinsky intestinal calcium hyperabsorption. JClinInvest91: 661–667, 1993 analyzed the data. Dr. Krieger made the figures. Dr. Krieger, Dr. Asplin, 14. Tsuruoka S, Bushinsky DA, Schwartz GJ: Defective renal calcium reabsorption in genetic hypercalciuric rats. Kidney Int 51: 1540–1547, Dr. Grynpas, and Dr. Bushinsky drafted and revised the paper, all 1997 fi authors approved the nal version of the manuscript. 15. Krieger NS, Stathopoulos VM, Bushinsky DA: Increased sensitivity to

This work was supported by grant RO1 DK075462 from the 1,25(OH)2D3 in bone from genetic hypercalciuric rats. Am J Physiol National Institutes of Health to Dr. Bushinsky. 271: C130–C135, 1996 16. Asplin JR, Bushinsky DA, Singharetnam W, Riordon D, Parks JH, Coe FL: Relationship between supersaturation and crystal inhibition in hypercalciuric rats. Kidney Int 51: 640–645, 1997 DISCLOSURES 17. Bushinsky DA, Grynpas MD, Nilsson EL, Nakagawa Y, Coe FL: Stone formation in genetic hypercalciuric rats. Kidney Int 48: 1705–1713, Dr. Krieger reports stock, stock options, spouse is a consultant for Tricida, 1995 stock from Amgen, spouse-speaking fees from Sanoﬁ/Genzyme, spouse is a 18. Grynpas M, Waldman S, Holmyard D, Bushinsky DA: Genetic hyper- consultant from Relypsa/Vifor/Fresenius, spouse is an adjudicator for adverse calciuric stone-forming rats have a primary decrease in BMD and events from Novo Nordisk/Covance, grants from National Institutes of strength. J Bone Miner Res 24: 1420–1426, 2009 Health, grants from Renal Research Institute. Dr. Asplin is an employee of 19. Ng AH, Frick KK, Krieger NS, Asplin JR, Cohen-McFarlane M,

Litholink Corporation. Dr. Ramos has nothing to disclose. Dr. Flotteron has Culbertson CD, et al.: 1,25(OH)2D3-induces a mineralization defect and nothing to disclose. Dr. Granja is an employee of Litholink Corporation. loss of bone mineral density in genetic hypercalciuric stone-forming Dr. Chen has nothing to disclose. Dr. Wu has nothing to disclose. rats. Calcif Tissue Int 94: 531–543, 2014 Dr. Grynpas has nothing to disclose. Dr. Bushinsky reports personal fees 20. Yao J, Kathpalia P, Bushinsky DA, Favus MJ: Hyperresponsiveness of and consultant, stock and stock options from Tricida, personal fees and stock vitamin D receptor gene expression to 1,25-dihydroxyvitamin D3. A from Amgen, speaking fees from Sanoﬁ/Genzyme, personal fees and consul- new characteristic of genetic hypercalciuric stone-forming rats. JClin tant from Relypsa/Vifor/Fresenius, personal fees from Novo Nordisk/ Invest 101: 2223–2232, 1998 Covance, grants from National Institutes of Health, grants from Renal 21. Karnauskas AJ, van Leeuwen JP, van den Bemd GJ, Kathpalia PP, Research Institute. DeLuca HF, Bushinsky DA, et al.: Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats. J Bone Miner Res 20: 447–454, 2005 22. Coe FL, Worcester EM, Evan AP: Idiopathic hypercalciuria and forma- REFERENCES tion of calcium renal stones. Nat Rev Nephrol 12: 519–533, 2016 23. Zisman AL: Effectiveness of treatment modalities on kidney stone re- 1. Bose A, Monk RD, Bushinsky DA: Kidney stones. In: Williams Text- currence. Clin J Am Soc Nephrol 12: 1699–1708, 2017 book of Endocrinology, 13th Ed., edited by Melmed S, Polonsky KS, 24. Fink HA, Wilt TJ, Eidman KE, Garimella PS, MacDonald R, Rutks IR, Larsen PR, Kronenberg HM, Philadelphia, Elsevier, 2016, pp 1365– et al.: Medical management to prevent recurrent nephrolithiasis in 1384 adults: A systematic review for an American College of physicians 2. Monk RD, Bushinsky DA: Nephrolithiasis and nephrocalcinosis. In: clinical guideline. Ann Intern Med 158: 535–543, 2013 Comprehensive Clinical Nephrology, 5th Ed., edited by Johnson RJ, 25. Bushinsky DA, Asplin JR: Thiazides reduce brushite, but not calcium Frehally J, Floege J, Philadelphia, Elsevier, 2015, pp 688–702 oxalate, supersaturation, and stone formation in genetic hypercalciuric 3. Moe OW, Bushinsky DA: Genetic hypercalciuria: A major risk factor in stone-forming rats. J Am Soc Nephrol 16: 417–424, 2005 kidney stones. In: Genetics of Bone Biology and Skeletal Disease,ed- 26. Bushinsky DA, Willett T, Asplin JR, Culbertson C, Che SPY, Grynpas M: ited by Thakker RV, Whyte MP, Eisman JA, Igarashi T, London, UK, Chlorthalidone improves vertebral bone quality in genetic hyper- Elsevier, 2013, pp 585–604 calciuric stone-forming rats. J Bone Miner Res 26: 1904–1912, 2011 4. Bushinsky DA, Coe FL, Moe OW: Nephrolithiasis. In: The Kidney,9thEd., 27. Krieger NS, Asplin JR, Frick KK, Granja I, Culbertson CD, Ng A, et al.: edited by Brenner BM, Philadelphia, W.B. Saunders, 2012, pp 1455–1507 Effect of potassium citrate on calcium phosphate stones in a model of 5. Moe OW, Bonny O: Genetic hypercalciuria. J Am Soc Nephrol 16: 729– hypercalciuria. JAmSocNephrol26: 3001–3008, 2015 745, 2005 28. Asplin JR, Donahue SE, Lindeman C, Michalenka A, Strutz KL, 6. Stechman MJ, Loh NY, Thakker RV: Genetics of hypercalciuric neph- Bushinsky DA: Thiosulfate reduces calcium phosphate nephrolithiasis. rolithiasis: Renal stone disease. Ann N Y Acad Sci 1116: 461–484, 2007 JAmSocNephrol20: 1246–1253, 2009 7. Monico CG, Milliner DS: Genetic determinants of urolithiasis. Nat Rev 29. Frick KK, Asplin JR, Favus MJ, Culbertson C, Krieger NS, Bushinsky DA:

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