Calcium-sensing receptor gene polymorphisms in patients with calcium urolithiasis: a systematic review

Abstract

Background: Calcium-sensing receptor gene (CaSR) might be a strong component of the complex genetic background regulating calcium excretion and stone formation in kidney. The polymorphisms in different parts of the gene could be associated with calcium stone formation. Objective: The individual studies indicate a correlation between CaSR gene polymorphisms and urolithiasis but not sufficient to draw a precise result. We aimed to search literature to come to a reliable conclusion through a systematic review. Materials and methods: We have serached individiual studies regarding the issue and high quality reviews. We assessed all the studies to draw a conclusion. Conclusion: The genetic background of the neprolithiasis is an interesting issue deserving to be disscussed on. Regading genes of which, CaSR gene polymorphisms is the leading one, are studied in many individual studies but they are not sufficient to have a precise conclusion. Well-oriented and documented, phenotypically homogenous large cohort studies are needed for further evaluation.

• Calcium
• calcium-sensing receptor
• gene expression
• gene polymorphism
• kidney stones

Introduction

Urolithiasis is one of the most prevalent disorders in Urology practice which is estimated to be at 1–15% with the probability of having a stone varying according to age, gender, race, and geographic location. In the United States, the prevalence of stone disease has been estimated at 10–15%.1–3 The most common component of urinary calculi is calcium, which is a major constituent of nearly 75% of stones. Calcium oxalate makes up about 60% of all stones; mixed calcium oxalate and hydroxyapatite, 20%; and brushite stones, 2%.

The calcium-sensing receptor (CaSR) is the key controller of extracellular calcium homeostasis via its effects on regulation of parathyroid hormone (PTH) secretion and renal calcium reabsorption. The CaSR is a G-protein-coupled membrane receptor that allows parathyroid and kidney tubular cells to regulate parathyroid hormone (PTH) secretion and tubular calcium reabsorption according to the plasma calcium concentration. The CaSR molecule comprises a large extracellular domain, a transmembrane domain, and an intracellular tail. Calcium binding to the extracellular domain stimulates the interaction of transmembrane and intracellular domains with G-proteins and activates a complex network of intracellular messengers, leading to phospholipase C-dependent inositol 1,4,5-triphosphate production and thus calcium release from intracellular stores as self-maintained low frequency repetitive calcium spikes. Through this cascade of events, CaSR activation inhibits PTH secretion and calcium reabsorption in the thick ascending limb. Thus, CaSR activation increases urinary calcium excretion by both a direct and a PTH-mediated effect on kidney tubular cells. While the activation of CaSR especially in thick ascending limb results in hypercalciuria which forms the main risk factor for renal stone formation, CaSR activation in collecting ducts of nephron decreases the risk to renal stone formation by urinary pH regulation.

Calcium-hypercalciuria

The regulation of calcium (Ca⁺²) metabolism is accomplished by the concerted action of the intestine, kidney, and bone. Ca⁺² is absorbed from the diet in multiple segment of intestine.4 Thirty percent to 40% of dietary calcium is absorbed from intestine, with most being absorbed in the small intestine, and only approximately 10% absorbed in the colon.4 Calcium is absorbed in the ionic state, and incomplete calcium absorption due in part to formation of soluble calcium complexes in the intestinal lumen. Substances that complex calcium such as phosphate, citrate, oxalate, and sulfate reduce the availability of ionic calcium for absorption.5 The active vitamin D is the most potent stimulator of intestinal calcium absorption. The conversion of 25-hydroxyvitamin D₃ to 1,25 (OH)₂D₃ is succeeded in kidney with the stimulation of parathyroid hormone (PTH). A decrease in serum calcium increases secretion of PTH and it stimulates the enzyme 1α-hydroxylase, which is located in the mitochondria of the proximal renal tubule. PTH is a crucial hormone in maintaining serum calcium concentration within normal range. In response to serum calcium levels, extracellular CaSR regulates PTH secretion and renal tubular calcium reabsorption.6 PTH stimulates osteoclasts, further raising serum calcium. At the kidney, PTH enhances renal calcium reabsorption and reduces renal tubular reabsorption of phosphate. It also stimulates synthesis of 1,25(OH)₂D₃, which leads to enhanced intestinal calcium and phosphate absorption. PTH does not affect intestinal absorption directly.

Hypercalciuria is the most common abnormality identified in calcium stone formers seen in up to 40% of the stone formers.7–10 The majority of Ca⁺⁺ reabsorption takes place paracellularly along the proximal tubule (PT: 60–65% of filtered Ca⁺⁺) and the thick ascending limb of Henle’s loop (TALH: 25–30% of filtered Ca⁺⁺). The distal convoluted tubule (DCT) reabsorbs the remaining 8–10% of filtered Ca⁺⁺ transcellularly.11 High urinary calcium concentrations lead to increased urinary saturation of salts.12 Additionally, citrate, which is the common urinary inhibitor is reduced by the way of complication in hypercalciuric statement of urine.13 Intestine, bone and kidney are the main sites regulating calcium transport and any dysregulation of these sites result in hypercalciuria. Although the classic classification, which is formed on the basis of unique pathophysiologic abnormalities, is used widely with its utility in simplifying the understanding and treatment of metabolic derangements, many have argued that hypercalciuria is associated with multiple, interrelated disturbances that cannot be simply separated into a specific organ system.8

Calcium sensing receptor in kidney

Since the CaSR is expressed at multiple sites along the nephron, it is reasonable to discuss its function according to its cellular localization depends upon the region of the nephron where it is expressed. It was illustrated in Figure 1.14

Figure 1. Intrarenal localization and roles of the CaSR. Notes: Cellular polarity of the CaSR is apical [in the proximal tubule and outer/inner medullary collecting duct (OMCD/IMCD)] and basolateral [in the thick ascending limb (TAL) and, occasionally, in the cortical collecting duct (CCD)]. Species differences exist in the distal convoluted tubule (DCT)/connecting segment (CNT), where receptor expression can be detected apically and/or basolaterally/intracellularly. PCT/PST, proximal convoluted/straight tubule; tDL/tAL, thin discending/ascending limb; MTAL/CTAL, medullary/cortical thick ascending limb; JGA, juxtaglomerular apparatus; TRPV5, transient receptor potential vanilloid 5; PMCA, plasma membrane Ca2_-ATPase; VitD, vitamin D; VDR, vitamin D receptor; NKCC2, Na_-K_-2Cl_ cotransporter 2; ROMK, renal outer medullary potassium K_ channel; PTH, parathyroid hormone; AQP2, aquaporin 2.

CaSR in proximal convoluted tubule

CaSR is expressed on the apical membrane of the proximal tubule cells and responds to the luminal calcium.15 PTH results in phosphate excretion in proximal tubules by degradation of phosphate reabsorption carriers (NPT2c and NPT2a) in subapical vesicles derived from brush border. Microperfusion studies in single proximal tubules isolated from mice demonstrated that CaSR stimulates phosphate reabsorption by inhibition of cyclic AMP production and PTH-mediated internalization of phosphate reabsorption carriers, NPT2c and NPT2a.16 Studies conducted in proximal tubule-derived cell lines also suggest that 1α-hydroxylase activity is inhibited in the presence of high Ca²⁺.17 In a study carried out with a murine model in which the full-length CaSR has been ablated have shown that CaSR diminishes the response to 1,25(OH)₂D₃ independently of PTH actions.18 Thus CaSR exerts a tight control on circulating 1,25(OH)₂D₃ both at the level of its synthesis (in the proximal tubule) and in modulating its effects. Conversely, 1,25(OH)₂D₃,19 PTH, and dietary phosphate modulate both CaSR gene and protein expression in the proximal tubule,20 suggesting the existence of a local feedback loop for the regulation of Ca²⁺ and P_i excretion independently of systemic changes in calciotropic hormones.

CaSR in TAL of Henle’s loop

The loop of Henle consists of four segments; the thin descending limb (DLH), the thin ascending limb (ALH), the medullary thick ascending limb (mTALH), and the cortical thick ascending limb (cTALH). CaSR is expressed on the basolateral membrane of the thick ascending limb of Henle loop.15 About 20–25% of the filtered calcium is reabsorbed in the loop of Henle, largely by the cortical (CTAL) and, to a lesser extent, by the medullary (MTAL) thick ascending limb, through both transcellular and paracellular routes.21 The apical Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) and the renal outer medullary potassium K⁺(ROMK) channel generate the “driving force” which is crucial for calcium and sodium reabsorption.22 In in-vitro studies on microdissected or isolated microperfused tubules from laboratory animals, CaSR activation by serum-interstitial calcium enhanced the intracellular production of arachidonic acid and 20-hydroxyeicosatetraenoic acid (HETE) that inactivates the potassium ROMK channel and the Na-K-2Cl cotransporter.23 The inactivation of these carriers inhibits the passive reabsorption of sodium, potassium and chloride and blocks the potassium recycling to the lumen through the specific ROMK channel.23 In thick ascending limbs isolated from dogs, CaSR also inhibited the phosphorylation of claudin-16 through a mechanism mediated by the decrease in the protein kinase A activity. The unphosphorilated form of claudin-16 was not expressed in tight junctions and its absence reduced the tight junction permeability to calcium and magnesium, thus amplifying the calciuric effect of CaSR.24 Figure 2 demonstrates the activities of CaSR in a cell model from the thick ascending limb.25

Figure 2. CaSR activities in a cell model from the thick ascending limb (TAL). Notes: CaSR decreases paracellular calcium reabsorption by inhibition of renal outer medullary potassium channel (ROMK)-mediated potassium recycling to the lumen that supports the luminal positive electric gradient. This gradient is the driving force for the passive paracellular calcium reabsorption in the TAL. AA, arachidonic acid; 20-HETE, 20-hydroxyeicosatetraenoic acid; PKA, protein kinase A; PLA2, phospholipase A2.

While NaCl reabsorption through NKCC2 is electroneutral (NKCC2 translocates 1 Na⁺, 1 K⁺, and 2 Cl⁻ ions from the lumen into the cell), apical K⁺ represents the rate-limiting step of this process and K⁺ ions back-diffuse into the lumen through the ROMK channels.21 Na⁺ and Cl⁻ accumulated inside the cell are then transported into the bloodstream through basolateral Na⁺-K⁺-ATPase and Cl⁻ channels, respectively. Overall, these processes yield a net cellular reabsorption of NaCl and the generation of a lumen-positive transepithelial potential difference, which drives nonselective cation reabsorption (largely Ca²⁺ and Mg²⁺ but also Na⁺) through the paracellular route.21

During hypercalcemia, activation of the basolateral CaSR inhibits ROMK channels26 which contribute to the recycling of K⁺ into the lumen of the TAL.27 This action of hypercalcemia limits the rate of Na⁺-K⁺-2Cl⁻ cotransport by reducing the availability of luminal K⁺. Thus the greater the hypercalcemia, the greater is the inhibition of ROMK and NKCC2 and the faster the dissipation of the lumen-positive transepithelial voltage. Additionally, microperfusion experiments in mice single tubules evidenced that CaSR activation suppressed TRPV5 calcium channel expression in the apical membrane of TAL cells28 and transcellular PTH-induced calcium reabsorption in the cortical tract of the TAL.29

In the MTAL water permeability is minimal because of the absence of luminal aquaporins, and water reabsorption does not follow paracellular divalent cation movements. The reduced permeability of this nephron segment would produce a rapid increase in basolateral NaCl and/or Ca²⁺/Mg²⁺ concentration, which could inhibit NKCC2, ROMK, and, possibly, CLN-16.

CaSR in distal convoluted tubule

Distal tubule accounts for 15% of total Ca²⁺ reabsorbed by the kidney. CaSR is located on the basolateral membrane of distal convoluted tubular cells. In cultured dog cells from this tubular segment CaSR was found to reduce the active calcium reabsorption by interfering with the calcium pump function through a phospholipase C-dependent mechanism. The signaling pathway for this effect requires activation of Gq proteins.30 In DCT the transepithelial PD is against Ca²⁺ reabsorption and the paracellular permeability of Ca²⁺ ions is very low.31 Ca²⁺ reabsorption is an active, transcellular process, which is regulated by PTH and 1,25(OH)₂D₃.31,32 CaSR reduces calcium pump activity (PMCA) and calcium active reabsorption in distal convoluted tubule (Figure 3).33

Figure 3. CaSR in distal convoluted tubule. CaSR reduces calcium pump activity (PMCA) and calcium active reabsorption.

CaSR in collecting ducts

CaSR is represented on the apical membrane of principal and intercalated cells in collecting ducts of nephron segment. Experiments in hypercalciuric knockout mice for the TRPV5 calcium channel showed that CaSR stimulated proton-secreting H-pump in the type A intercalated cells and free water excretion by inhibition of aquaporin (AQP2) membrane expression in principal cells.34,35

The genetic background of stone formation

The development of calcium stone crystallization is a multifactorial disease in which genetic and environmental factors are included. Since 1960s, it is a well known fact that having stone-forming family relatives is a predisposition to have urolithiasis.36–38 In a study conducted by Goldfarb DS et al.,39 stone production was observed higher in monozygous than dizygous twins born in Vietnam. The proportion of kidney stone susceptibility attributable to genetic causes39 was 56% indicating nephrolithiasis is a complex disease on which genetic and environmental factors have impact. That is why we should consider it as a complex disease like diabetes, hypertension and ischemic heart disease. It was also demonstrated in these family studies that nephrolithiasis transmission pattern was not Mendelian, complex polygenic pattern was available.

Recently, great importance is attributed to epigenetic modifications which is defined as regulation of gene expression without altering the DNA sequence. Epigenetic changes involve DNA methylation at cytosine-guanine sequences-CpG sites, histone posttranslational medications (histone methylation acetylation, phosphorylation, ubiquitination, simulation, and propionylation), and microRNAs.40

The major aforementioned genes predisposing to susceptibility to renal stone forming are Claudin 14 (CLDN14), CaSR, Osteopontine (OPN), and Vit-D receptor (VDR). Because the genetic background of stone formation is a complicated issue including single-candidate genes as well as epigenetic process, it is not simple to identify which gene is more responsible from stone formation. Furthermore, environmental and dietary factors are also important that they might modify gene expressions at different biological levels, eventually influencing the phenotypic effects of gene products.

Calcium sensing receptor single nucleotide gene polymorphisms

Several studies have examined the possible impact of single nucleotide polymorphisms (SNPs) in the CaSR on Ca²⁺ homeostasis and kidney stone forming. In the CaSR gene, exon 1 is in the 50 untranslated region also including promoters, exon 2 presents the transcription starting site codon, and exon 7 encodes for the transmembrane domain and the intracellular tail.

One study found that CaSR SNPs and related haplotypes (a haplotype in this setting is a sequence of SNPs on a single strand of DNA) were a determinant of the normal range of serum Ca⁺² concentration in the population.41 This normal range is greater that measured in any given individual. Indeed, certain haplotypes of SNPs at positions 986, 990, and 1011 in the CaSR’s C tail were significantly associated with higher or lower serum ionized Ca⁺² concentration within the normal range, and accounted for 17% of the variation in the normal range.41 In some cases, other groups have had discordant results with regard to the A986S polymorphism,42 perhaps because of small sample size or populations with differing frequencies of the SNPs. Clearly additional studies with larger sample sizes in well-characterized populations are needed.

The impact of CaSR SNPs on parameters related to renal function/dysfunction has also been studied. Having two glycine residues at position 990, instead of the more common alanine, has been associated with (1) lower PTH levels in hemodialysis patients,43 (2) hypercalciuria,44 and (3) greater suppressibility of PTH during induced hypercalcemia in hemodialysis patients.45 In another study, there was a greater risk of stone disease in patients with PHPT and the ACG haplotype at positions 986, 990, and 1011.46 Some associations of SNPs with traits relevant to the kidney have involved noncoding SNPs (those present in introns or the promoter of the CaSR gene), perhaps by modifying receptor expression. In African–Americans in the Indianapolis area, three SNPs were associated with systolic blood pressure and with urinary calcium excretion.47 These same investigators observed an interaction between CaSR, the CLCNKB (the basolateral chloride channel in the thick limb), and NKCC genes that contributed to variation in diastolic blood pressure, perhaps through changes in sodium and/or calcium transport in the TAL.48 These results indicate that the SNPs within the CaSR and other genes with which it interacts in the kidney could be a fruitful avenue of investigation. The availability of very large databases, with 1,000,000 SNPs for genomewide association studies (GWAS) should be very helpful in this regard. Table 1 demonstrates the associated studies regarding calcium sensing receptor gene polymorphisms in patients with nephrolithiasis.

Table 1. The associated studies regarding calcium sensing receptor gene polymorphisms in patients with nephrolithiasis.

SNP	Location	Ethnicity	Patients	Noun	Analysis
Arg990Gly	Exon 7	Italian Italian Canadian Iranian European	PHPT PHPT SF and CNTs SF and CNTs General population	94 237 899 99 20,611	AS AS HWDA AS GWAS
Ala986Ser	Exon 7	Iranian	SF and CNTs	99	AS
rs17251221	Intron 4	Taiwanese	SF and CNTs	480	AS
rs7652589	5′UTR	Italian Italian	SF and CNTs PHPT	676 332	AS AS
rs1501899	Intron 1	Italian Italian	SF and CNTs PHPT	676 332	AS AS

Notes: 5′UTR, 5′untranslated region; AS, association study; CNTs, controls; GWAS, genome-wide association study; HWDA, Hardy Weinberg disequilibrium analysis; PHPT, primary hyperparathyroidism; SF, stone formers; SNP, single-nucleotide polymorphism.

Conclusions

The findings of individual studies indicate that CaSR gene polymorphisms are implicated in the development of nephrolithiasis through different mechanisms. However, representing a cause and effect relationship for a suspected gene is not such simple. Many other factors, of which nutrition and environment are the most prominent, may include in the issue making it a quite complicated problem. The reasonable approach to the problem should be based on well-designed studies in which nutritional and lifestyle factors are described detailed with a large cohort examined.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.