Effect of Potassium Citrate on Calcium Phosphate Stones in a Model of Hypercalciuria

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† † Nancy S. Krieger,* John R. Asplin, Kevin K. Frick,* Ignacio Granja, ‡ ‡ Christopher D. Culbertson,* Adeline Ng, Marc D. Grynpas, and David A. Bushinsky*

*Division of Nephrology, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York; †Litholink Corporation, Laboratory Corporation of America Holdings, Chicago, Illinois; and ‡Laboratory Medicine and Pathobiology Department, University of Toronto, Toronto, Ontario, Canada

ABSTRACT Potassium citrate is prescribed to decrease stone recurrence in patients with calcium nephrolithiasis. Citrate binds intestinal and urine calcium and increases urine pH. Citrate, metabolized to bicarbonate, should decrease calcium excretion by reducing bone resorption and increasing renal calcium reabsorption. However, citrate binding to intestinal calcium may increase absorption and renal excretion of both phosphate and oxalate. Thus, the effect of potassium citrate on urine calcium oxalate and calcium phosphate supersaturation and stone formation is complex and difficult to predict. To study the effects of potassium citrate on urine supersaturation and stone formation, we utilized 95th-generation inbred genetic hypercalciuric stone-forming rats. Rats were fed a fixed amount of a normal calcium (1.2%) diet supplemented with potassium citrate or potassium chloride (each 4 mmol/d) for 18 weeks. Urine was collected at 6, 12, and 18 weeks. At 18 weeks, stone formation was visualized by radiography. Urine citrate, phosphate, oxalate, and pH levels were higher and urine calcium level was lower in rats fed potassium citrate. Furthermore, calcium oxalate and calcium phosphate supersaturation were higher with potassium citrate; however, uric acid supersaturation was lower. Both groups had similar numbers of exclusively calcium phosphate stones. Thus, potassium citrate effectively raises urine citrate levels and lowers urine calcium levels; however, the increases in urine pH, oxalate, and phosphate levels lead to increased calcium oxalate and calcium phosphate supersaturation. Potassium citrate induces complex changes in urine chemistries and resultant supersaturation, which may not be beneficial in preventing calcium phosphate stone formation.

J Am Soc Nephrol 26: ccc–ccc, 2015. doi: 10.1681/ASN.2014121223

Hypercalciuria is the most common metabolic ab- The annual incidence of kidney stones in indus- normality observed in patients who form calcium- trialized nations exceeds 1 per 1000 persons, with a based kidney stones.1–3 The elevated levels of urinary lifetime risk of about 7% in women and about 11% calcium increase the probability for nucleation and in men.7 After an initial episode of nephrolithiasis, growth of calcium oxalate (CaOx) and/or calcium hy- approximately 60%–80% of patients form at least drogen phosphate (CaHPO4, brushite) crystals into one recurrent stone. Among the strategies to decrease clinically significant kidney stones.1 Calcium phosphate (CaP)–containing stones may be brushite, apatite, or less well defined crystal forms. Patients with Received December 15, 2014. Accepted February 2, 2015. fi idiopathic hypercalciuria (IH), de ned as excessive Published online ahead of print. Publication date available at urinary calcium without a demonstrable metabolic www.jasn.org. cause, generally have normal serum calcium, normal Correspondence: Dr. Nancy S. Krieger, Division of Nephrology, or elevated serum 1,25-dihydroxyvitamin D3, normal Department of Medicine, University of Rochester School of Medi- or elevated serum parathyroid hormone (PTH), nor- cine and Dentistry, 601 Elmwood Avenue, Box 675, Rochester, NY mal or low serum phosphate, and low bone mass.1,4,5 14642. Email: [email protected] – IH exhibits a polygenic mode of inheritance.4 6 Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: ccc–ccc, 2015 ISSN : 1046-6673/2612-ccc 1 BASIC RESEARCH www.jasn.org stone recurrence is the use of potassium citrate (K-cit).8–13 Oral The GHS rats were generated by selectively inbreeding citrate would be expected to decrease calcium available for Sprague-Dawley rats for increased urinary calcium excre- absorption,14,15 decrease bone resorption, and increase renal tion.24–48 When fed a standard, ample calcium diet, each GHS tubular calcium reabsorption, all of which would lower uri- rat now consistently excretes approximately 10-fold more urinary nary calcium. Increased urinary citrate would sequester uri- calcium than Sprague-Dawley controls.24–49 Like patients with nary calcium preventing the binding to calcium phosphate IH, GHS rats have normal serum calcium,24 increased intestinal (CaP) and/or oxalate, potentially leading to a reduction in calcium absorption26 and enhanced bone resorption,49 decreased stone formation. However, oral citrate should also increase renal tubule calcium reabsorption,36 and normal serum 1,25- urinary pH, which potentially could increase CaP stone for- dihydroxyvitamin D3 levels25,26,30,41,42 in addition to decreased mation. In vitro, citrate has direct inhibitory effects on CaOx bone mineral density.39,47 Hypercalciuria is a polygenic trait in crystal nucleation and growth (reviewed by Ryall16). Thus, it GHS rats,44 as it is in humans.4–6 When fed a standard, ample is difficult to predict the precise effects of K-cit on urine calcium diet, all GHS rats develop kidney stones,27,28,31,32 which supersaturation with respect to the common solid phases of are composed of CaP.27,31,33,34 Addition of hydroxyproline to the CaP and ultimately stone formation. diet of GHS rats results in CaOx stone formation.37,38,40 In patients, the addition of dietary K-cit appeared to To determine the effects of K-cit on urine solute excretion, decrease recurrent stone formation.8–13 In a randomized, supersaturation with respect to the common stone solid phases, double-blind, placebo-controlled study in humans, K-cit de- and CaP stone formation, GHS rats were fed a normal calcium creased recurrent stone formation.17 Pearle et al.18 published a diet without hydroxyproline with K-cit or potassium chloride meta-analysis of several treatments for prevention of recurrent (KCl), as control. stone formation, including thiazide diuretics and citrate. They identified three studies (including Pak et al.13 and Barcelo et al.17) using citrate supplements; stone recurrence decreased RESULTS in two studies but not in the third. A formal meta-analysis was not possible because one study reported only rate of stone Urine Solute Excretion formation and not patient numbers. K-cit treatment has All rats consistently ate their full allotment of food so that solute been reported to be effective in patients refractory to thiazide intake,exceptforcitrate,chloride,andK,wassimilarforallratsin treatment.13,19 both groups. K-cit induced a reduction in overall mean urinary Citrate has been used after lithotripsy to prevent stone calcium (K-cit, 16.160.5 mg/d versus KCl, 18.660.3 mg/d; recurrence. In a randomized controlled study, potassium and P,0.001), and urinary calcium was reduced with K-cit at 6 sodium citrate appeared to reduce the risk of stone recurrence and 12 weeks but not at 18 weeks (Figure 1). K-cit led to an after extracorporeal shockwave lithotripsy or percutaneous increase in both overall mean urinary phosphate (K-cit, 2961 nephrolithotomy.20 In another randomized controlled study, mg/d versus KCl, 2061mg/d;P,0.001) and overall mean uri- K-cit appeared effective in reducing lower caliceal calculi after nary oxalate (K-cit, 1.1160.04 mg/d versus KCl, 0.6260.02, shockwave lithotripsy.21 Neither of these studies was placebo P,0.001) and the increase in both urinary phosphate and uri- controlled. Current recommendations of the American nary oxalate was observed at each time point (Figure 1). Urological Association include citrate therapy to limit stone recurrence.22 However, in all human studies using stone formation as the primary endpoint, patients were chosen for having CaOx stones or calcium lithiasis. Therefore, CaP stone–forming patients were excluded or would be only a minority of the study participants. No prospective controlled trial has studied the effectiveness of citrate in reducing stone formation specifically in patients with CaP stones, and this remains an important clinical question.23 Because CaP stone formers are characterized by alkaline urine compared with CaOx stone formers, the effect of citrate on CaP supersaturation and stone formation are difficult to predict. The alkali load would increase urine pH, increasing CaP supersaturation, whereas an increase in citrate and reduction in urinary calcium would lower CaP supersaturation; however, the net effect is uncertain. Given the uncertainty in the effect of citrate on Figure 1. Urinary calcium (Ca) was decreased and urinary, phos- prevention of recurrent CaP stone disease, and the absence of phate (P), and oxalate (Ox) were increased with K-cit. Urine was human studies, we used the 95th generation of the genetic hyper- collected for 24 hours at 6, 12, and 18 weeks to determine solute calciuric stone–forming (GHS) rats to study the effect of K-cit on levels as described in the Concise Methods. Values are mean6SEM. CaP stone formation. *K-cit different from KCl, same time period, P,0.05.

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As expected, overall mean urinarycitrate waselevated in rats fed K-cit (K-cit, 18263.4 mg/d versus KCl, 11062.7 mg/d; P,0.001) (Table 1). K-cit increased the mean overall urinary pH (K-cit, 7.5160.04 versus KCl, 6.7160.05; P,0.001). K-cit reduced mean overall urinary NH4 (K-cit, 0.2560.01 mmol/d versus KCl, 0.4160.01 mmol/d; P,0.001). K-cit increased the mean overall urinary sulfate (K-cit, 1.0860.02 mEq/d versus KCl, 0.8660.02 mEq/d; P,0.001). Each of these changes was present at each time point. Overall mean urinary uric acid excretion did not differ (K-cit, 1.660.1 mg/d versus KCl, 2.06 0.2 mg/d; P=NS). K-cit resulted in a slightly lower overall mean urinary sodium (K-cit, 2.1660.03 mmol/d versus KCl, 2.3660.04; P,0.001), which was significant at 6 and 18 weeks but not at 12 weeks (Table 1). K-cit resulted in a slightly but significantly lower mean 6 6 Figure 2. Urinary supersaturation (SS) of CaP and CaOx were overall urinary K (K-cit, 4.8 0.04 mmol/d versus KCl, 5.3 0.08 increased and uric acid SS was decreased with K-cit. Urine was P, fi mmol/d; 0.001) that was signi cant at each time period. As collected for 24 hours at 6, 12, and 18 weeks to determine solute expected, the rats receiving K-cit excreted less chloride than the levels that were used to calculate relative supersaturation as rats receiving KCl (K-cit, 2.260.07 mEq/d versus KCl, 6.460.15 described in the Concise Methods. Values for relative supersat- mEq/d; P,0.001), which was significant at each 6-week time uration are mean6SEM and are unitless. *K-cit different from KCl, period. K-cit did not alter the overall mean urine volume (K-cit, same time period, P,0.05. 47.361.6 ml/d versus KCl, 53.062.7 ml/d; P=NS) or the overall mean urinary creatinine (K-cit, 12.8560.17 mg/d versus KCl, 12.7460.22 mg/d; P=NS). Stone Formation Urine Supersaturation K-cit did not alter the number of stones formed or extent of fi K-cit led to a significant increase in the urinary supersaturation calci cation in the kidneys (Figure 4). All rats formed stones of fi with respect to both CaP (K-cit, 8.560.6 versus KCl, 4.960.2; similar size and density and had similar degrees of calci cation P,0.001) and CaOx (K-cit, 9.860.5 versus KCl, 5.560.2; within the kidney and collecting system. P,0.001) and a reduction of supersaturation with respect to Visible stones were dissected from representative kidneys of uric acid (K-cit, 0.00360.0005 versus KCl, 0.02260.003; both groups and analyzed for crystal morphology. The P,0.001) (Figure 2). Each of these differences was significant diffraction patterns for all analyzed samples were congruent at each time period. with the diffraction pattern of biologic apatite, a CaP stone (Figure 5, top). With use of transmission electron microscopy Serum Levels (TEM), all kidney stone crystals had a rod- or needle-like Serum levels of calcium and PTH did not differ between K-cit– shape, similar to the morphology of biologic apatite crystals fed and KCl-fed rats, while serum phosphate levels were de- (Figure 5, bottom) and unlike the octahedral crystal morphol- creased in K-cit–fed rats (Figure 3). ogy of CaOx.

Table 1. Urinary citrate, pH and sulfate were increased while NH4, chloride, sodium and K were the same or decreased with K-cit compared to KCl 6wk 12wk 18wk Variable KCl K-cit KCl K-cit KCl K-cit Urinary citrate (mg/d) 104.263.5 175.264.6a 99.063.4 172.964.4a 126.562.4 199.565.7a Urinary pH 6.8560.06 7.6560.03a 6.7360.08 7.5260.03a 6.5360.08 7.3460.1a a a a Urinary NH4 (mmol/d) 0.4360.02 0.2360.03 0.4460.01 0.3160.02 0.3860.02 0.2060.01 a a a Urinary SO4 (mEq/d) 0.9560.04 1.1760.04 0.9060.03 1.1060.03 0.7360.02 0.9560.03 Urinary uric acid (mg/d) 2.460.1 2.160.0 1.760.1 1.460.2 2.060.5 1.560.2 Urinary sodium (mmol/d) 2.360.1 2.160.1a 2.460.1 2.360.0 2.360.0 2.160.1a Urinary potassium (mmol/d) 5.5460.14 4.8060.09a 5.3760.15 4.8360.06a 5.0760.07 4.6660.08a Urinary chloride (mmol/d) 5.860.07 1.860.04a 5.960.1 2.160.04a 7.560.2 2.760.07a Urine volume (ml) 50.163.8 47.962.7 53.963.1 50.262.3 55.566.6 43.663.4 Urinary creatinine (mg/d) 13.660.5 13.660.2 12.360.2 12.660.3 12.360.3 12.360.2 Values are expressed as the mean6SEM. Urine was collected for 24 hr at 6, 12, and 18 wk to determine solute levels as described in the Concise Methods. aK-cit different from KCl, same time period, P,0.05.

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Figure 3. K-cit decreased serum phosphate levels but did not change serum Ca or PTH. At the conclusion of the 18-week study, these levels were determined as described in the Concise Methods. Values are mean6SEM. *K-cit different from KCl, same time period, P,0.05.

DISCUSSION

Hypercalciuria is the most common metabolic abnormality observed in patients who form Ca-based kidney stones.1–3 Most patients with hypercalciuria have IH. The increased excretion of urinary calcium enhances the probability for nucleation and growth of CaOx and/or CaP crystals into clinically significant kidney stones.1 In patients with IH, K-cit is often used to prevent recurrent stone formation.8–13 However, no prospective controlled studies in humans have shown the efficacy of K-cit in preventing recurrent CaP stone formation.23 Citrate has a complex effect on urine solute excretion, and its effect on urine super- fi fi Figure 4. Kidney stone formation and calci cation was not dif- saturation with respect to CaP stone formation is dif cult to ferent in rats fed KCl or K-cit. At the conclusion of the 18-week predict. Some studies have shown that CaOx stones may trans- study, the extent of kidney stones and calcification was de- form into CaP stones over time, and use of citrate therapy may termined by three observers as described in the Concise Methods. explain this phenomenon.50,51 However, no definitive studies Extent of kidney calcification was also determined as described in have yet proved or refuted the role of citrate in transformation the Concise Methods. (A) Representative radiographs of kidneys of CaOx to CaP stone disease. from rats receiving KCl or K-cit. Calcification scores for the images In this study using GHS rats, the provision of K-cit led to an shown are provided as a reference. (B) Quantitation of stone increase in urine citrate excretion and K-cit also effectively formation and calcification in all rats receiving KCl or K-cit. Values 6 decreased urinary calcium. The mechanism by which K-cit are mean SEM. reduces urinary calcium is almost certainly multifactorial. While citrate itself is readily absorbed in the intestine,52 it also found an increase in urinary oxalate, sulfate, and decreases calcium absorption and urinary calcium excre- phosphorus and a decrease in serum phosphorus in GHS tion.14,15 Increased urinary citrate would also bind urinary rats treated with citrate, alterations not previously reported in calcium, removing the calcium from the pool available for humans treated with citrate. The binding of intestinal calcium binding with phosphate or oxalate. In addition, citrate metab- by citrate could decrease intestinal binding of calcium to olism to bicarbonate results in systemic alkalinization, which oxalate, potentially allowing greater absorption of oxalate. In directly decreases bone resorption53 and increases renal tubule the kidney, oxalate is transported in the proximal tubule.54,55 calcium reabsorption, thereby reducing the level of calcium in The murine anion transporter Slc26a6, found in renal proxi- urine. The decrease in urinary calcium by itself would favor- mal tubule and intestine, has specificity for chloride/oxalate ably reduce urinary supersaturation with respect to calcium- exchange.56–58 Knockout of Slc26a6 in mice leads to hyper- containing kidney stones. oxaluria,59,60 hypocitraturia,61 and CaOx stone formation.59 As seen in humans, citrate therapy lowered urine calcium In perfusion experiments the presence of sulfate or bicarbon- and raised urinary citrate and pH in the GHS rat. However, we ate inhibited oxalate transport, suggesting competitive

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exchange of extracellular chloride for intracellular sulfate but not chloride or oxalate.64 Further studies will be necessary to determine why urinary sulfate increased in the GHS rats fed K-cit. Inthisstudy,theprovisionofK-citledtoanincreaseinurinary pH, which will reduce the solubility of CaP. Conversely, the uric acid solid phase, which may be a nidus on which calcium stones form, is more soluble in alkaline urine. K-cit did not alter urinary volume, indicating that any change in urinary supersaturation was not the result of differences in urine volume. K-cit did not alter urinary creatinine excretion, indicating that administration of this drug did not affect muscle mass over this period. Zerwekh et al.65 fed Sprague-Dawley rats a low- or high-casein diet, with KCl or K-cit (4 mEq potassium per day) for 8 weeks. Urinary pH did not increase with K-cit. Compared with the low-casein diet+KCl, urinary calcium excretion increased with the high-casein diet and K-cit reduced this increased urinary calcium. K-cit did not alter urinary phosphate or oxalate. In the current study, we used a 3-fold higher dose of K-cit and observed a significant increase in urinary pH, urinary oxalate, and urinary phosphate and a decrease in urinary calcium. In this study we found that while K-cit effectively decreased urinary calcium it also increased urinary phosphate and oxalate and urinary pH, resulting in an actual increase in urinary Figure 5. Analysis of kidney stone crystals demonstrated CaP supersaturation with respect to the CaP and CaOx solid phases. stones with both KCl and K-cit. Upper panels: Representative Despite the increase in CaOx supersaturation, analysis of stones electron diffraction pattern of a stone from a rat fed KCl (left) and from each group found TEM and diffraction patterns indicating a stone from a rat fed K-cit (right) showing the typical configuration that all of the stones were composed of CaP. Supersaturation of of biologic apatite. Lower panels: TEM images from the same uric acid was decreased with K-cit, but this supersaturation is so stones used for the diffraction patterns: a stone from a rat fed KCl low in either case that there is little chance of uric acid stone (left) and a stone from a rat fed K-cit (right). formation. Stone formation in the GHS rats, which form only CaP stones on this diet, was not altered by K-cit. Of note, the inhibition among these anions.55 Co-expression of Slc26a6 and higher supersaturation of CaP in the citrate-treated rats did not the citrate transporter NaDC-1 indicates that these transporters lead to an increase in stone formation. This may reflect an interact.61 NaDC-1 enhanced Slc26a6 transport activity, while increase in urine crystal inhibitory activity due to the higher Slc26a6 inhibited NaDC-1.61 These data suggest a close relation- urinary citrate concentration.66 Further studies are necessary to ship between oxalate and citrate transport. In this study we determine whether the provision of K-cit to GHS rats fed hy- found that administration of K-cit led to a significant increase droxyproline, which causes them to form only CaOx stones, in urine oxalate excretion, and this increase would increase uri- would affect urinary supersaturation or stone formation. nary supersaturation with respect to CaOx. Thus, the provision of K-cit induces complex changes in The binding of intestinal calcium by citrate will also allow urine chemistries, some favorable and some deleterious to the greaterabsorptionofphosphate.Renalphosphatereabsorptionis goal of decreasing supersaturation with respect to CaP and regulated by the type II Na+-coupled phosphate cotransporter in CaOx. In this model of CaP stone formation, citrate was not the proximal tubule, which is suppressed by extracellular acid- effective in reducing stone burden. This study suggests that ification.62 The systemic and urinary alkalinization induced by further studies should be done in human CaP stone formers to K-cit would increase renal tubular phosphate reabsorption; determine the effects of K-cit on urine supersaturation with however, the increase in PTH, although not statistically signifi- respect to CaP and stone formation. cant, might account for the increase in urine phosphate and decrease in serum phosphate, which was found in this study. Urine sulfate increased in GHS rats fed K-cit. The anion CONCISE METHODS transporter Slc26a2, which demonstrates substrate specificity for sulfate, oxalate, and chloride, is also present in the proximal Animals tubule.63 Mutation of SLC26A2 in humans can cause recessive The GHS rats were derived from Sprague–Dawley rats (Charles River chondrodysplasia, as a result of abnormal sulfate transport.63 Laboratories, Kingston, NY) by successively inbreeding the most hyper- When expressed in Xenopus oocytes, acidic extracellular pH in- calciuric progeny of each generation.24–27,36–39,49 Eight-week-old male hibited anion exchange, while acidic intracellular pH activated GHS rats from the 95th generation were used in this study.

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Experimental Conditions on Formvar coated grids, and viewed in an FEI Tecnai 20 TEM (FEI At the startof the study (day 0), 22 GHS rats were placed in metaboliccages Co., Hillsboro, OR). Images and diffraction patterns were collected by and fed a normal calcium diet, 13 g/d (1.2% calcium; Harlan-Teklad, an AMT 16000 camera. Diffraction pattern analysis was done by Indianapolis, IN). K-cit, 4 mmol/d (11 rats), or KCl, 4 mmol/d, as a calculating the d-spacings by comparison to a gold pattern collected control (11 rats) was added to the diet to provide equivalent amounts under the same conditions. of the anions citrate and chloride. All rats had free access to deionized, distilled water. At 6-week intervals (6, 12, and 18 weeks), urine from Statistical Analyses each rat was collected for four 24-hour periods. For two collections, Values were compared by t test with a conventional computer pro- urine was acidified with HCl; for another two collections urine was gram (Statistica; StatSoft, Tulsa, OK). Values are expressed as mean6 collected in thymol. Collections in thymol were used for pH, uric acid, SEM. P#0.05 was considered to represent a statistically significant and chloride, and collections in HCl were used for all other measure- difference. ments. After 18 weeks, all rats were euthanized and their blood collected, and the kidneys quickly removed. Any animal that ate ,10 g of food per day or drank ,15 ml of water per day would have ACKNOWLEDGMENTS been excluded from further analysis; however, all rats met the pre- specified criteria during the entire study. The University of Rochester Committee for Animal Resources approved all procedures. This work was supported by grant R01-DK075462 from the National Institutes of Health. Urine and Serum Chemistries Urinary calcium, magnesium, phosphate, ammonium, and creatinine DISCLOSURES were measured spectrophotometrically using a Beckman CX5 Pro autoanalyzer (Beckman Coulter, Brea, CA). Urinary potassium, chloride, None. and sodium were measured by ion-specific electrodes on the Beckman CX5. Urinary pH was measured using a glass electrode, and citrate, REFERENCES oxalate,andsulfatewere measured by ionchromatography usingaDionex ICS 2000 system (Dionex Corp., Sunnyvale, CA). All urine solutes were measured at 6, 12, and 18 weeks, and a mean value for each time period 1. Bushinsky DA, Coe FL, Moe OW: Nephrolithiasis. In: The Kidney, edited by Brenner BM, Philadelphia, W.B. Saunders, 2012, pp 1455– and an overall mean value were calculated. Serum calcium and 1507 phosphate were determined colorimetrically (BioVision, Milpitas, 2. Monk RD, Bushinsky DA: Kidney stones. In: Williams Textbook of CA). 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