CJASN ePress. Published on July 22, 2011 as doi: 10.2215/CJN.00480111

Role of the Calcium-Sensing Receptor in Reducing the Risk for Calcium Stones

Kirsten Y. Renkema,*† Rene´J. M. Bindels,* and Joost G. J. Hoenderop*

Summary -The tight control of blood Ca2؉ levels within a narrow range is essential for the performance of vital physio

logic functions. Muscle contraction, neuronal excitation, and intracellular signaling processes acquisitively * 2؉ 2؉ Department of require Ca . It is the concerted action of intestine, bone, and kidney that controls the Ca balance through Physiology, Nijmegen 2؉ the regulation of intestinal absorption, bone (de)mineralization, and renal excretion of Ca , respectively. Centre for Molecular Along the nephron, fine-tuning of blood Ca2؉ levels takes place by Ca2؉ reabsorption. The calciotropic hor- Life Sciences, Radboud mones regulate Ca2؉ transport processes, leading to whole-body Ca2؉ homeostasis and, importantly, preserv- University Nijmegen ,2؉ 2؉ Medical Centre ing a constant Ca concentration in the blood. Defects in renal Ca handling can lead to hypercalciuria, Nijmegen, The consecutive kidney stone formation, and obstructive nephropathy. Here we give an overview of the key play- Netherlands; ؉ ers involved in normal Ca2 management and describe the in-depth investigations on a renal hypercalciuric †Department of Medical model of disease, the Trpv5 knockout mouse, which naturally displays molecular adaptations that prevent Genetics, University ,Ca2؉ precipitation in the kidney. Medical Center Utrecht Utrecht, The Clin J Am Soc Nephrol 6: 2076–2082, 2011. doi: 10.2215/CJN.00480111 Netherlands

Correspondence: Prof. Introduction transport is taking place in the TALH as well is still Dr. J. G. J. Hoenderop, ϩ Maintenance of a normal calcium (Ca2 ) balance is questionable and needs further investigation. A three- Department of Physiology (286), accomplished by the concerted action of the intestine, step process, similar to the duodenal route, mediates Radboud University ϩ ϩ kidney, and bone. Disturbances in the Ca2 balance active Ca2 reabsorption in the DCT and CNT, in- Nijmegen Medical ϩ can result in symptoms including kidney stones, os- volving cell entry via the epithelial Ca2 channel Centre (RUNMC), P.O. ϩ teoporosis, and rickets. Ca2 is absorbed from the diet TRPV5, cytosolic transport via calbindin-D , and Box 9101, 6500 HB 28K Nijmegen, The in multiple segments of the small intestine (1). In the extrusion into the peritubular capillaries by the Netherlands. Phone: 2ϩ ϩ 2ϩ jejunum and ileum, passive absorption of Ca takes Na -Ca -exchanger (NCX1) and PMCA1B (1). 0031 24 36 13426; ϩ place in a paracellular manner along with sodium and TRPV5- and TRPV6-mediated active entry of Ca2 Fax: 0031 24 36 ϩ water uptake. Besides that, active Ca2 absorption is from the lumen into the cytosol of intestinal and 16413; E-mail: J.Hoenderop@fysiol. hormonally regulated and mainly occurs in the duo- renal epithelial cells, respectively, is tightly con- ϩ umcn.nl denum. The transcellular Ca2 uptake process in- trolled by a complex network of interacting pro- volves luminal entry into the cell through the epi- teins and calciotropics hormones, representing the ϩ ϩ thelial Ca2 channel, transient receptor potential rate-limiting step in transcellular Ca2 (re)absorp- ϩ ϩ vanilloid member 6 (TRPV6) (2). Ca2 is subse- tion and, thereby, fine-tuning overall body Ca2 ϩ quently bound by Ca2 binding proteins (calbin- balance.

din-D9K) and shuttled to the basolateral side of the cell. The transcellular route is fulfilled by active Classic Calciotropic Hormones in Control ϩ ϩ Ca2 extrusion to the blood via the plasma mem- Ca2 homeostasis is preserved by the classically ϩ brane Ca2 -ATPase (PMCA1B). In the circulation, recognized calciotropic hormones vitamin D 2ϩ 45% of Ca is present as the free ionized form, while (1,25(OH)2D3), parathyroid hormone (PTH), and cal- 45% is bound to proteins. The remaining 10% forms citonin (4). Central to this process is the G-protein ϩ complexes with anions, including citrate, sulfate, and coupled Ca2 -sensing receptor (CaSR) in the parathy- ϩ phosphate. The majority of Ca2 is stored in the skel- roid glands that detects changes in blood ionized ϩ eton. Taking a closer look at the kidney, it is appreci- Ca2 levels (5). In response to an alteration in the ϩ ϩ ated that blood Ca2 is filtered at the glomerulus and blood Ca2 levels, the CaSR modulates PTH release thereby enters the lumen of the renal tubule. Along into the circulation. Specifically, the activation of the ϩ ϩ the course of the nephron passive, paracellular Ca2 CaSR by increased circulating Ca2 levels suppresses reabsorption occurs in the proximal tubule (PT) and PTH release and stimulates the secretion of calcitonin the thick ascending limb of Henle (TALH) (1). A small from the thyroid gland. Calcitonin reduces osteoclast- ϩ (10 to 15%), highly regulated quantity of Ca2 is mediated bone resorption and thereby promotes a ϩ reabsorbed in the distal convoluted tubule (DCT) and decrease in blood Ca2 levels (6). Gain-of-function the connecting tubule (CNT) in an active transcellular mutations in the CASR gene lower blood PTH levels, ϩ ϩ transport process (3). Whether transcellular Ca2 hampering Ca2 (re)absorption and thereby evoking

2076 Copyright © 2011 by the American Society of Nephrology www.cjasn.org Vol 6 August, 2011 Clin J Am Soc Nephrol 6: 2076–2082, August, 2011 CaSR and renal calcium stones, Renkema, Bindels, and Hoenderop 2077

ϩ hypocalcemia in humans (7). A hypocalcemic state does exerts an effect on renal Ca2 handling via TRPV5 upregu- not activate the CaSR, leading to PTH secretion. PTH stim- lation at the transcriptional level in rats, independent of 2ϩ 2ϩ ulates active Ca reabsorption, thereby enabling Ca 1,25(OH)2D3 (17). Furthermore, estrogen deficiency is ϩ ϩ retention in the body. Furthermore, PTH enhances Ca2 known to result in a negative Ca2 balance and bone loss mobilization from bone and the conversion of inact- in postmenopausal women (18). These results partly ex- ϩ ive vitamin D to its biologically active form— plain gender differences in renal Ca2 handling (19). The

1,25(OH)2D3—by the renal cytochrome P450 enzyme tissue kallikrein (TK) hormone is secreted into the pro- ␣ ␣ 25-hydroxyvitamin D3-1 -hydroxylase (1 -OHase) in urine by renal tubular epithelial cells. The TK knockout ϩ the kidney. Previous studies demonstrated that mouse demonstrates renal Ca2 wasting (20). In 2006,

1,25(OH)2D3 facilitates vitamin D receptor (VDR)– Gkika et al. described the mechanism through which TK ϩ mediated gene transcription that evokes transcription of stimulates TRPV5-mediated active Ca2 reabsorption. TK ϩ ϩ Ca2 transporter encoding genes in Ca2 transporting activates the bradykinin receptor, thereby stimulating the cells, established by the interaction of the 1,25(OH)2D3- phospholipase C/1,2-diacylglycerol/protein kinase C VDR complex with vitamin D responsive elements (PLC/DAG/PKC) pathway (21). As a result, TRPV5 chan- (VDREs) in the promoter regions of target genes (8). Mul- nels show an increased expression on the plasma mem- ϩ tiple intestinal and renal Ca2 transporter encoding genes brane, probably due to an inhibition of TRPV5 endocytosis. 2ϩ were shown to contain VDREs, indicating 1,25(OH)2D3 as Hence, TK regulates the Ca balance in a positive direc- ϩ ϩ an important stimulator of Ca2 (re)absorption (9). As a tion and thus preserves Ca2 in the body. The antiaging consequence, a lack of 1,25(OH)2D3 could cause distur- hormone Klotho is a transmembrane protein with an ex- ϩ bances in the Ca2 balance. For example, bone diseases like tracellular domain that is secreted into blood, urine, and osteoporosis, osteomalacia, and rickets occur as a conse- cerebrospinal fluid. Chang et al. demonstrated a stimula- ϩ quence of vitamin D deficiency, occurring during malnu- tory effect of Klotho on TRPV5-mediated active Ca2 ϩ trition, aging, and menopause. Humans with a defect in transport (22). Klotho was shown to alter the Ca2 perme- VDR function show bone abnormalities known as vitamin ability of the renal luminal membrane by the removal of D–dependent rickets type II (10). In the past, genetically alpha2,6-linked sialic acids (23). Thereby, Klotho can reg- modified mouse models were generated in which the ulate the cell surface retention of functional TRPV5, sub- 2ϩ 1,25(OH)2D3 endocrine system was inactivated. Two re- stantiating the important role of Klotho in Ca homeosta- search groups independently created the 1␣-OHase knock- sis. Moreover, the phenotypic characterization of the Klotho out mouse (11,12). Phenotypic analysis showed that these knockout mouse revealed hypercalciuria, hypervitamino- mice were unable to generate 1,25(OH)2D3, leading to se- sis D, infertility, short life span, and bone aberrations vere hypocalcemia, secondary , de- clearly overlapping with symptoms presented by Trpv5 ϩ creased bone mineral density, rickets, and growth retarda- knockout mice (24). Although large variations in Ca2 ϩ ϩ tion. Intestinal and renal Ca2 transporters were intake produce only small alterations in blood Ca2 levels, downregulated, whereas repletion with 1,25(OH)2D3 led to due to the tight control by the above-mentioned key play- ϩ ϩ the restoration of renal and intestinal Ca2 transporter ers, previous investigations demonstrated that high Ca2 ϩ ϩ expression levels and normalization of blood Ca2 levels intake is an important regulator of renal Ca2 transport

(13). These experiments confirmed the essential role of protein expression as well, specifically in a 1,25(OH)2D3- 2ϩ Ca transport proteins in 1,25(OH)2D3-mediated active deficient state, thereby contributing to the maintenance of ϩ ϩ Ca2 (re)absorption. Similarly, VDR knockout mice dis- a normal body Ca2 balance (13). played hypocalcemia, bone degradation, increased blood PTH levels, and hypervitaminosis D, due to a lack of VDR-mediated gene transcription (14). Altogether, these Model of Renal Hypercalciuria: Trpv5 Knockout Mice investigations underlined that absence of 1,25(OH)2D3 ϩ leads to severe disturbances of the Ca2 balance and that Genetic ablation of Trpv5 in mice allowed us to investi- ϩ 1,25(OH) D is of crucial importance to Ca2 homeostasis. gate the requirement of normal TRPV5 channel function- 2 3 2ϩ Furthermore, it was previously demonstrated that inacti- ing in Ca homeostasis. Mice lacking TRPV5 epithelial 2ϩ vating CASR gene mutations result in elevated blood PTH Ca channels in the DCT/CNT segment of the nephron ϩ and Ca2 levels, parathyroid hyperplasia, bone abnormal- displayed a significant hypercalciuria, excreting 6 to 10 2ϩ ities, and retarded growth in humans (15). Mice genetically times more Ca compared with control littermate mice ablated for the CASR gene demonstrated similar symp- (25). Further phenotypic characterization showed that toms (16). Together, the CaSR and the calciotropic hor- blood 1,25(OH)2D3 levels were significantly elevated and ϩ mones are crucial for maintenance of blood Ca2 levels normocalcemia was maintained. Bone mineral density was decreased and bone thickness was diminished. Intestinal within a narrow physiologic range. ϩ Ca2 transporters were significantly upregulated, and ab- sorption experiments, in which the mice were challenged ؉ ϩ ϩ Novel Regulators of the Ca2 Balance with radioactive Ca2 , showed significant Ca2 hyperab- In recent years, new key factors shown to be essential for sorption in the Trpv5 knockout mice. This indicated an ϩ body Ca2 homeostasis have been identified. Testosterone important molecular mechanism maintaining a normal ϩ ϩ ϩ was demonstrated to inhibit Ca2 reabsorption in vitro as Ca2 balance despite severe renal Ca2 wasting in the well as in vivo, whereas estrogen, produced in the female knockout mice, possibly involving the observed hypervi- ϩ ovaries, promotes Ca2 (re)absorption and bone mineral- taminosis D. By generating the Trpv5/1␣-OHase double ization processes. Van Abel et al. revealed that estrogen knockout mice, we assessed that additional gene inactiva- 2078 Clinical Journal of the American Society of Nephrology

tion of 1␣-OHase in Trpv5 knockout mice resulted in un- We hypothesized that both polyuria and increased urinary detectable blood 1,25(OH)2D3 levels (26). Thereby, the hy- acidification might promote the excretion of large amounts ϩ pervitaminosis D presented by Trpv5 knockout mice was of Ca2 without being precipitated in the renal collecting ϩ abolished, leading to a downregulation of intestinal Ca2 duct system. Further investigations provided insight into ϩ transporters, a drop in blood Ca2 levels, and a more the underlying molecular mechanisms applicable in kid- ϩ severe bone degradation compared to the Trpv5 knockout ney stone prevention (32). The crystallization of Ca2 - mice. In summary, these data demonstrated that the up- phosphate occurs via the conversion of phosphate to its 2ϩ 2Ϫ regulation of intestinal Ca transporters and the resulting divalent form (HPO4 ) in an alkaline, rather than in an ϩ Ca2 hyperabsorption in Trpv5 knockout mice were due to acidic, environment. From this can be hypothesized that a ϩ a secondary hypervitaminosis D. Thus, Trpv5 knockout decrease in urinary pH can prevent Ca2 -phosphate crys- mice maintained normocalcemia in the presence of renal tal formation (33). Acid/base transport processes take ϩ Ca2 wasting. Here we showed that a tight control of place along different nephron segments to control tubular 2ϩ active Ca transport by 1,25(OH)2D3 is of utmost impor- fluid pH levels (34). Final urinary pH is assessed in the ϩ tance in maintaining a normal extracellular Ca2 balance, intercalated cells of the renal CD, where fine-tuning of ϩ especially during a disturbed Ca2 balance, as present in urinary acidification takes place. The vacuolar proton ϩ Trpv5 knockout mice. pump H -ATPase, located in the type A intercalated cells ϩ of the CD, is mainly responsible for urinary H excretion. In 2009, we described how the exposure to high (5.0 mM) The Renal CaSR 2ϩ ϩ It is clear that the kidney plays a key role in the main- Ca concentrations significantly enhanced H -ATPase ϩ ϩ tenance of a normal Ca2 balance, reabsorbing more Ca2 activity in CD cells (32). This stimulatory effect was absent 2ϩ in CD cells from Atp6v1b1 knockout mice that were genet- in a hypocalcemic state, while excreting Ca during hy- ϩ ically ablated for the CD-specific B1 subunit of H -ATPase percalcemia. The CaSR in the parathyroid glands regulates ϩ ϩ PTH release, which is crucial for Ca2 homeostasis (5). The (35). Moreover, the CaSR agonist neomycin increased H - identification of the CaSR in the kidney indicated an im- ATPase activity as well, indicating the modulation of uri- 2ϩ nary acid excretion by CaSR activation. These findings portant regulatory function of this receptor in renal Ca 2ϩ handling as well (27). The CaSR is expressed at multiple suggested that an increased luminal Ca concentration, as consistently present in the renal DCT, CNT, and CD of sites along the nephron, and its cellular localization de- ϩ pends upon the region of the nephron where it is ex- Trpv5 knockout mice, stimulates H -ATPase activity via pressed. Together with the parathyroid CaSR, the renal apical CaSR activation. Interestingly, additional gene abla- CaSR shows to be of crucial importance for whole-body tion of Atp6v1b1 in Trpv5 knockout mice resulted in nor- 2ϩ malization of urinary pH levels and led to the tubular Ca handling. In the proximal tubule (PT) the CaSR is ϩ ϩ 2 activated by increased urinary Ca2 levels, which inhibits precipitation of Ca -phosphate in the medullary CD (Figure 1) (32). From previous studies it was known that PTH-induced phosphate transporter-retrieval from the 2ϩ apical membrane and results in increased phosphate reab- the formation of alkaline urine increases the risk for Ca - sorption. Hereby, excess loss of phosphate into the filtrate phosphate precipitation (33). Therefore, renal stones com- ϩ that contains elevated Ca2 concentrations can be avoided, monly occur in distal patients, who ϩ eventually preventing renal Ca2 -phosphate precipitation display a urinary acidification defect and concomitant hy- (28). In the TAL, approximately 20% to 25% of the filtered percalciuria (36). Furthermore, treatment with acetazol- ϩ Ca2 is reabsorbed (29). Since the TAL is impermeable to amide, a carbonic anhydrase inhibitor resulting in the al- 2ϩ kalization of the urine, increases the risk to drug-induced water, the Ca concentration at the basolateral side of the 2ϩ TAL cells consequently rises. This evokes an increase in the renal Ca -phosphate stone formation (37,38). Our obser- reabsorption of NaCl and other solutes along the loop of vations unequivocally revealed that the naturally occur- Henle. The CaSR is highly expressed at the basolateral ring increased urinary acidification through CaSR activa- tion in the distal nephron in Trpv5 knockout mice is an membrane of the TAL, where it senses the extracellular ϩ ϩ 2 Ca2 concentration (30). This permits a negative feedback essential adaptive mechanism preventing renal Ca - ϩ mechanism of the reabsorbed Ca2 onto the cell, attenuat- phosphate precipitation in a hypercalciuric state (Figure 2). ϩ ing Ca2 reabsorption and preventing hypercalcemia (31). In the (CD), the CaSR is expressed at the apical membrane Diluting the Urine Prevents Renal Stone Formation in principal and intercalated cells (30). We introduce a Urine concentration takes place in the CD where the novel regulatory function of the CaSR in the collecting 2ϩ vasopressin-regulated aquaporin-2 (AQP2) water channels duct, important for the prevention of renal Ca phosphate are localized in the apical membrane of principal cells (39). precipitation. Increased diuresis diminishes the risk for renal crystal ϩ precipitation by reducing urinary Ca2 levels. A urinary CaSR Activation Decreases the Risk to Renal Stone concentration defect was demonstrated in Trpv5 knockout Formation by Urinary pH Regulation mice and was further characterized by a significant de- Besides a robust hypercalciuria, a significant polyuria crease in urinary osmolarity and the occurrence of poly- and a decrease in urinary pH were consistently demon- uria, caused by downregulation of renal AQP2 expression strated in Trpv5 knockout mice (25). Furthermore, hyper- (Figure 2). Interestingly, medullary membrane protein phosphaturia was present in these mice, predisposing fractions, isolated from control and Trpv5 knockout mice, ϩ them to an increased risk of Ca2 -phosphate precipitation. revealed a significant downregulation of AQP2 proteins in ϩ ϩ Strikingly, no renal Ca2 -containing stones were observed. the knockouts. Sands and coworkers linked Ca2 and wa- Clin J Am Soc Nephrol 6: 2076–2082, August, 2011 CaSR and renal calcium stones, Renkema, Bindels, and Hoenderop 2079

Figure 1. | Nephrolithiasis in Trpv5/Atp6v1b1 knockout mice. (A) Transmission electron microscopy image of a tubular precipitate in the medullary collecting duct of a 1-week-old Trpv5/Atp6v1b1 knockout mouse (ϫ2500). (B) Scanning transmission electron microscopy image of (1) a precipitate, x-ray maps separately depicting (2) Ca2ϩ and (3) phosphorus content. (C) Energy-dispersive x-ray (EDX) microanalysis of the renal precipitate (arrowhead in B) demonstrating the presence of Ca2ϩ and phosphorus.

increase their urinary volume by an average of only 0.3 L/24 h, and given the observation that Trpv5/Atp6v1b1 double knockout mice increased their urinary volume fur- ther, adequate hydration is insufficient to prevent stone formation (41). Therefore, the initiation of additional ad- aptations, like increased urinary acidification, is highly ϩ important to decrease the risk to Ca2 -phosphate stone formation. Furthermore, other putative target mecha- nisms should be studied. Urinary molecules that retard ϩ the formation of Ca2 -containing stones should be of great research interest, in which citrate is already a known urinary stone inhibitor in clinical practice (42,43). Therapeutic strategies for nephrolithiasis are dependent on the type of stones occurring and the primary cause of the ϩ urinary Ca2 loss. Thiazide diuretics are often prescribed, ϩ as these drugs have a Ca2 -sparing effect and thereby ϩ lower the urinary Ca2 levels (44). A possible concomitant Figure 2. | Model of renal CaSR signaling in the collecting duct. The positive effect of thiazide treatment on bone mineral den- CaSR is localized at the apical side of principal cells and intercalated sity was proposed. However, due to contradictory research cells of the collecting duct. AQP2 proteins are responsible for water outcomes, this needs further investigation (45,46). Novel ϩ reabsorption, whereas Hϩ-ATPase proteins pump Hϩ ions into the therapies hampering Ca2 -phosphate stone formation urine. During hypercalciuria the CaSR is activated, leading to AQP2 could imply the stimulation of urinary acidification. Fur- downregulation and evoking polyuria. Furthermore, the CaSR trig- ther research would be necessary to acquire insight into the ϩ gers urinary acidification by increasing H -ATPase activity. Both possible working mechanism of such an intervention in 2ϩ polyuria and increased urinary acidification prevent renal Ca - humans. phosphate precipitation. Ca2ϩ, calcium; CaSR, calcium-sensing re- ceptor; AQP2, aquaporin-2. Hypercalciuria and Nephrolithiasis: A ter homeostasis, suggesting an important functional role Socioeconomic Problem for the renal CaSR (40). The expression of the CaSR along Hypercalciuria forms the main risk factor for renal stone the apical membrane of the CD cells was shown to facili- formation (47). Bound to urinary solutes like phosphate ϩ tate the activation of the receptor during a hypercalciuric and oxalate, Ca2 can precipitate in the urine and the renal state. This underlines the mechanism by which CaSR-me- tissue once sufficiently supersaturated. Most renal stones ϩ ϩ diated downregulation of AQP2 expression evokes poly- contain Ca2 , with the majority classified as Ca2 -oxalate ϩ uria, assisting the excretion of large amounts of Ca2 . The stones (48). Nephrolithiasis forms a worldwide health and ϩ physiologic connection between Ca2 and water balance socioeconomic problem, occurring in every geographical, established an important mechanism by which renal stone cultural, or racial group. In the United States (US), over 5% formation is hampered during a hypercalciuric state, em- of the population develops a clinically significant episode phasizing the importance of sufficient hydration in people of during life, with a large economic at risk for kidney stones. Given that humans are able to impact (49). Underlying causal factors for nephrolithiasis 2080 Clinical Journal of the American Society of Nephrology

include genetic predisposition, dietary habits, urinary tract Future Directions infection, and disorders characterized by a disturbance in The investigations described here demonstrate that nor- ϩ body ion handling (50). Patients suffering from renal stone mal renal Ca2 handling is fundamental in the prevention ϩ disease experience renal colic (severe pain), obstruction of of disturbances in the Ca2 balance, greatly underlined by urine flow, hematuria, and slowly progressing tissue dam- characterization of the unique hypercalciuric Trpv5 knock- age, whereas kidney stones form a risk factor for chronic out mouse model. Interestingly, the underlying mecha- kidney disease (51). New advances in treatment technolo- nisms that explained polyuria and increased urinary acid- gies are under current investigation, although shock-wave ification were established in these mice, revealing a crucial ϩ lithotripsy and surgery are the common therapies at the role for the renal CaSR in prevention of Ca2 -phosphate moment, depending on the size of the kidney stone. The stone formation. In humans, the effects of hypercalciuria chance for recurrence is 60% to 75% and, therefore, phar- on urinary volume and pH remain to be elucidated, pro- macologic treatment, lifestyle, and dietary modifications viding important knowledge for the development of novel are implemented in individuals at risk. Further investiga- therapeutic strategies and screening possibilities for the tions are necessary to elucidate the genetic and environ- benefit of kidney stone patients. Investigations should fo- ϩ mental risk factors for hypercalciuria-related nephrolithia- cus on local therapeutic targets like Ca2 , acid/base and sis and will permit the fine-tuning of therapeutic strategies water transporting proteins, and the renal CaSR. Future that are currently employed. therapeutic strategies that modulate urinary volume and pH might involve calcimimetic compounds that locally Genetic Susceptibility to Renal Stone Formation activate the CaSR in the collecting duct. Additionally, pH- Individuals with a positive family history have an in- sensing receptors were recently identified in the kidney creased risk for the development of kidney stones (52). (58). Via these proton sensors, alterations in urinary and This strongly suggests that genetic factors are involved in blood pH are monitored, after which downstream signal- the pathogenesis of hypercalciuria (50). As there are a ing evokes a tight regulation of body pH. As urinary pH is 2ϩ myriad of potential disturbances in the Ca balance that an important parameter for renal stone formation, these can cause hypercalciuria, with many genes encoding pro- pH sensors might be important therapeutic targets in the 2ϩ teins involved in Ca homeostasis, hypercalciuria is very prevention of kidney stones as well. Whether urinary pH ϩ heterogeneous. Thus, many candidate genes were hypoth- modulation can influence Ca2 -oxalate stone formation esized to be involved in the pathogenesis of hypercalciuria. has been debatable so far. Previous investigations sug- ϩ Trpv5, as well as Trpv6, gene ablation in mice leads to gested that an acidic environment stimulates Ca2 -oxalate 2ϩ hypercalciuria and, thus, these epithelial Ca channels are precipitation, whereas others showed no effect of pH (48). important candidate genes possibly involved in the patho- Altogether, these investigations are very important for the genesis of hypercalciuria. To date, mutation analysis of the understanding of the processes that take place in the body human TRPV5 gene has not revealed a primary role for during a hypercalciuric state and also raise new issues 2ϩ defects in this epithelial Ca channel in idiopathic hyper- concerning applicability in clinical practice that require calciuria (53). However, the involvement of TRPV5 or follow-up. TRPV6 gene defects in hypercalciuria has not been defin- itively excluded. Specific single nucleotide polymorphisms (SNPs) or haplotypes of the encoding genes may modulate Disclosures None. channel activity and might therefore be responsible for ϩ altered renal Ca2 excretion. Unusual haplotype differ- ences were identified in the TRPV6 gene among world- References wide populations (54). It was suggested that a specific 1. Hoenderop JG, Nilius B, Bindels RJ: Calcium absorption TRPV6 haplotype comprising three nonsynonymous SNPs across epithelia. Physiol Rev 85: 373–422, 2005 (C157R, M378V, and M681T) resulted in a selective advan- 2. Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, tage during human history. This might indicate a different Brown EM, Hediger MA: Molecular cloning and characteriza- TRPV6 channel function related to specific haplotypes. tion of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274: 22739–22746, 1999 However, functional characterization of these SNPs in the 3. Friedman PA: Mechanisms of renal calcium transport. Exp TRPV6 channel by patch-clamp analysis revealed no sig- Nephrol 8: 343–350, 2000 nificant differences in biophysical channel function (55). 4. Nemeth EF: Regulation of cytosolic calcium by extracellular Recently, Shakhssalim and coworkers associated CASR divalent cations in C-cells and parathyroid cells. Cell Cal- cium 11: 323–327, 1990 gene polymorphisms with recurrent calcium kidney stone 5. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, formation (56). The complex and heterogeneous nature of Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC: Cloning hypercalciuria and nephrolithiasis requires investigations and characterization of an extracellular Ca2ϩ-sensing recep- in large, well defined patient cohorts to identify associated tor from bovine parathyroid. Nature 366: 575–580, 1993 gene defects. Linkage analysis and revolutionary next-gener- 6. Fudge NJ, Kovacs CS: Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm ation sequencing techniques form powerful tools to identify that the CaSR regulates calcitonin release in vivo. BMC disease-causing genes. Reed et al. followed a genome-wide Physiol 4: 5, 2004 linkage approach in three families with hypercalciuria. Sig- 7. Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, nificant linkage was found for a locus on chromosome Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall- Taylor P, Brown EM, Thakker RV: A familial syndrome of hy- 1q23.4–1q24, in which a hypothetical human ortholog of the pocalcemia with hypercalciuria due to mutations in the calci- rat soluble adenylate cyclase was identified (57). However, um-sensing receptor. N Engl J Med 335: 1115–1122, 1996 no causative relationship was identified. 8. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thomp- Clin J Am Soc Nephrol 6: 2076–2082, August, 2011 CaSR and renal calcium stones, Renkema, Bindels, and Hoenderop 2081

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