A Single Nucleotide Polymorphism (rs4236480) in TRPV5 Calcium Channel Gene Is Associated with Stone Multiplicity in Calcium Nephrolithiasis Patients

Nephrolithiasis is characterized by calcification of stones in the kidneys from an unknown cause. Animal models demonstrated the functional roles of the transient receptor potential vanilloid member 5 (TRPV5) gene in calcium renal reabsorption and hypercalciuria. Therefore, TRPV5 was suggested to be involved in calcium homeostasis. However, whether genetic polymorphisms of TRPV5 are associated with kidney stone multiplicity or recurrence is unclear. In this study, 365 Taiwanese kidney-stone patients were recruited. Both biochemical data and DNA samples were collected. Genotyping was performed by a TaqMan allelic discrimination assay. We found that a TRPV5 polymorphism (rs4236480) was observed to be associated with stone multiplicity of calcium nephrolithiasis, as the risk of stone multiplicity was higher in patients with the TT+CT genotype than in patients with the CC genotype (p = 0.0271). In summary, despite the complexity of nephrolithiasis and the potential association of numerous calcium homeostatic absorption/reabsorption factors, TRPV5 plays an important role in the pathogenesis of calcium nephrolithiasis.


Introduction
Nephrolithiasis is a disease caused by calcifications that form stones in the kidney. The major component of these stones is calcium. Nephrolithiasis is a globally common disorder, and the prevalence and incidence of nephrolithiasis have been dramatically increasing worldwide. This phenomenon has placed heavy burdens on national economics and healthcare systems [1,2]. Many factors were suggested to be risk factors for nephrolithiasis, such as a family's health history, environmental exposure, for example, to steel manufacturing [3], dietary intake [4][5][6][7], a low fluid intake [8], and hypercalciuria [9][10][11][12]. Genetic polymorphisms were also reported to be risk factors causing kidney-stone formation and elevating nephrolithiasis recurrence. For example, several genetic variations of the calcium-sensing receptor (CASR) gene were reported to be associated with an increase in recurrent calcium kidney-stone formation [13,14]. Genetic polymorphisms of Orai1, a major subunit of the store-operated calcium channel, were also reported to be associated with nephrolithiasis recurrence [15]. In addition, levels of the vitamin D receptor (VDR) and its genetic polymorphisms were also indicated to be associated with idiopathic hypercalciuria (IH) [16,17].
The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) is considered the gatekeeper channel for activating calcium transport and reabsorption [18,19]. TRPV5 channel was found to be associated with urinary calcium (re)absorption in protein expression and channel voltage experiments [20,21]. This channel is expressed in the epithelia of renal connecting tubules and distal convoluted tubules, which are major parts of the kidneys involved in calcium homeostasis [22][23][24]. A previous study indicated that a TRPV5 genetic polymorphism has a conservative calcium effect in African subjects, and an animal model showed that genetic knockdown in mice produced serious hypercalciuria, which is a major risk factor for kidney-stone formation [18,19]. Despite the study results of Renkema et al. not supporting a functional role for TRPV5 in the pathogenesis of renal hypercalciuria, they still suggested that the TRPV5 gene should not be excluded as a potential risk factor for hypercalciuria [25].
The goal of this study was to investigate associations of the single-nucleotide polymorphism (SNP), rs4236480, of TRPV5 with the stone number (multiplicity) and recurrence rate in a Taiwanese population.

Patient Recruitment and Study
Methods. There were 365 renal-stone patients who fulfilled the diagnostic criteria for nephrolithiasis and were enrolled in this study. Sample collection was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH) and patients were recruited after signing informed consent. Subjects were excluded if they had a history of renal tubular acidosis, primary and secondary hyperparathyroidism, renal failure, gout, chronic diarrhea, or cancer. We excluded patients with other stone compositions and only recruited patients with idiopathic calcium nephrolithiasis.
Each patient's radiographic and echographic examinations of renal stones were retrieved along with clinical laboratory data, such as age, sex, and family history of nephrolithiasis. Renal-stone samples were obtained by either spontaneous stone passage or surgical intervention. Moreover, in stone number examination, kidney-stone patients with a single stone were allocated to single stone group, while kidney-stone patients having stone numbers exceeding one were allocated to multiple stone group.
The past medical history for renal-stone was retrospectively traced back as far as the patient's medical record was available. Patients who had two or more symptomatic episodes within at least a 6-month interval were classified to the recurrent group; those with only one episode were allocated to the nonrecurrent group. Due to the retrospective nature of the study, some subjects did not have clear past medical history documented in their medical records. Those patients who did not have clear documentations to classify them into either single or recurrent groups were excluded from the analysis for recurrence.

DNA Extraction.
DNA was derived from whole-blood samples which were collected from patients. Blood cells were first treated with 0.5% sodium dodecylsulfate lysis buffer and then with protease K (1 mg/mL) for 4 h at 60 ∘ C to digest nuclear proteins. A Gentra (QIAGEN, Valencia, CA)  [27]. Functionally this SNP represents a nonsynonymous polymorphism in intracellular N-and Ctails [25]. Genotyping was performed using the TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA). In short, a polymerase chain reaction (PCR) using a 96-well microplate with an ABI 9700 thermal cycler (Applied Biosystems) was performed for the purpose of target sequence enlargement. After the PCR, the fluorescence was measured and analyzed using system SDS software version 1.2.3 (Applied Biosystems). We followed the protocol which was previously reported [26].

Statistical Analysis.
Statistical software R (version 3.0.3) and R package Bioconductor [28,29] were used for data cleaning and statistical analyses. Genotyping and allelic frequencies between phenotypes were statistically analyzed by the likelihood ratio test. Each p value was adjusted for age and sex. A p value of <0.05 was considered statistically significant.

Demographic and Clinical Features of Stone-Bearing
Patients. We recruited 365 kidney-stone patients for this study. The mean age of patients was 54.3 ± 12.0 (standard deviation) years, and 68.5% of patients were male ( Table 1). As shown in the HapMap project database, rs4236480 of TRPV5 has a 10% minor allelic frequency among a Han Chinese population from Beijing, China [27]. A graphical outline of the rs4236480 location is shown in Figure 1.

The TRPV5 Genetic Polymorphism (rs4236480) Is Significantly Associated with Stone Multiplicity in Calcium
Mediators of Inflammation 3 rs4236480 5 UTR 3 UTR Figure 1: Graphical overview of the genotyped human TRPV5 gene.  The value was adjusted for sex and age.
Nephrolithiasis Patients. In order to investigate the association between stone multiplicity and genotypes, we further examined whether the TRPV5 SNP rs4236480 genotypes were associated with the stone number of calcium nephrolithiasis patients. The T allele of the rs4236480 polymorphism is a risk allele that appeared to have a dominant genetic effect upon the C allele ( Table 2). The risk for stone multiplicity was higher in patients who carried at least one minor allele than in the wild-type group, and a significant association between rs4236480 and kidney-stone multiplicity was observed (genotype model, = 0.0372; dominant model, = 0.0271; log additive model, = 0.0167) ( Table 2).

Lack of an Association of the TRPV5 Genetic Polymorphism (rs4236480) with the Risk of Stone Recurrence in Patients with Calcium Nephrolithiasis.
We suspected that SNP genotypes (of rs4236480) were associated with stone recurrence in calcium nephrolithiasis patients. However, the association of genotypes with stone recurrence of calcium nephrolithiasis did not reach statistical significance (Table 3).

No Association between the rs4236480 Polymorphism of TRPV5 and Biochemical Data of Nephrolithiasis Patients.
To better understand the interrelation between rs4236480 and clinical manifestation risk factors, biochemical data of serum/urine levels of calcium, uric acid, and creatinine were analyzed using urine samples obtained from patients on the spot and with creatinine correction. No significance was found among these clinical parameters (Table 4), and a subgroup analysis also produced no significant results (Table 5).

Discussion
We recruited 365 nephrolithiasis patients to conduct an association study between the TRPV5 genetic polymorphism, rs4236480, and stone multiplicity/recurrence in calcium nephrolithiasis patients. We found a significant association between rs4236480 and stone multiplicity. To our knowledge, this is the first study to report a significant association of the TRPV5 genetic polymorphism, rs4236480, with the types of kidney-stone multiplicity in Taiwanese calcium nephrolithiasis patients. Genetic and environmental risk factors influence body levels of calcium ions considerably and thus contribute to calcium nephrolithiasis [30,31]. Calcium is the predominant constituent of kidney-stone precipitates, as nearly 80% of stones are made of calcium, either mostly calcium oxalate or less frequently calcium phosphate [32][33][34]. High levels of urinary calcium are predominantly urine chemical aberrations in recurrent kidney-stone formers. Subsequently, hypercalciuria is considered to be the main risk factor for renal-stone formation. A family history of nephrolithiasis increases an individual's predisposition for a higher risk of kidney-stone formation [35,36], which substantially implies a susceptibility to genetic causes. Polymorphisms in multiple genes have been recently reported to be associated with the renal stone formation, that include calcium-sensing receptor (CASR), vitamin D receptor (VDR), and osteopontin (OPN),  The value corresponding to the likelihood ratio test was obtained from a comparison with the null model and was adjusted for sex and age. 1 UCa/UCr, urinary calcium-creatinine ratio. OR: odds ratio; CI: confidence interval.
inositol 1,4,5-trisphosphate (IP3) 3-kinase C (ITPKC), and ORAI1 and claudin 14 (CLDN14) genes [15,26,37]. TRPV5 is expressed in renal tubules and is regarded as a gatekeeper for calcium ion reabsorption [38,39]. TRPV5 knockdown mice exhibited severe hypercalciuria [18]. This gene downregulates urinary reabsorption of calcium and exaggerates urinary excretion of calcium in genetic hypercalciuric stone-forming rats [21]. In a previous study, DNA sequence analysis of the coding region of TRPV5 showed a novel T-to-C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P) [40]. In the same report, after comparing wild-type mice to heterozygous and homozygous mutant mice, there was evidence of hypercalciuria, polyuria, and a more-acidic urinary pH [40].
Limited clinical studies about the association between TRPV5 genotypes and hypercalciuria are available. The first report investigated four TRPV5 polymorphisms in Americans of African descent, where four nonsynonymous SNPs, A8V, R154H (rs4236480), A563T, and L712F, were investigated [19]. Only one variation was found to be significant: A563T of TRPV5 inferred a higher calcium ion reabsorption ability among African Americans, giving people of African descent a conservative superiority for calcium influx over other ethnicities [19]. Another report with 20 French hypercalciuria patients found nonsignificant functional impacts of three TRPV5 SNPs (A8V, R154H, and A561T) [25] compared to wild-type normal TRPV5 [25]. However, the limited set of human subjects' assigned may cause false-negative results. Our results suggest the role of gene polymorphism in increasing the presence of multiple stone numbers in patients carrying this nonsynonymous variation. The results may support a role of TRPV5 in the development of calcium nephrolithiasis in Taiwanese population.
There were a few limitations to this study. First, we observed no association between the rs4236480 polymorphism and increased serum and/or urine levels of calcium in this cohort of Taiwanese patients. Recent genome wide studies have shown how tremendous cumulative factors contribute to serum/urine calcium concentration. O'Seaghdha et al. provided evidence that indicated several polymorphisms in vital genes for calcium homeostasis [41,42]. Due to polygenic nature of calcium nephrolithiasis, it is difficult to detect a genetic association between one gene and urine/serum calcium concentration with a modest sample size. Additionally, the analysis for stone recurrence generated a null result. It might be, however, a false-negative result from our recurrence analysis due to the limitation of retrospective data collection. A conceivable justification for our current results was the difference in the genetic context of the cohort population and the possible polygenic complexity of nephrolithiasis disease.

Conclusions
In conclusion, our results indicated a significant association between rs4236480 and kidney-stone multiplicity and 6 Mediators of Inflammation showed that rs4236480 is a relevant susceptibility indicator for stone multiplicity in nephrolithiasis patients.