Research ArticleStudy of the Association between ITPKC GeneticPolymorphisms and Calcium Nephrolithiasis

Wei-Chih Kan12 Yii-Her Chou3 Siou-Jin Chiu3 Yu-Wen Hsu4 Hsing-Fang Lu4

Wenli Hsu5 and Wei-Chiao Chang45678

1 Department of Nephrology Chi-Mei Medical Center Tainan 710 Taiwan2Medical Laboratory Science and Biotechnology Chung Hwa University of Medical Technology Tainan 717 Taiwan3Department of Urology College of Medicine Kaohsiung Medical University Kaohsiung 807 Taiwan4Department of Clinical Pharmacy Taipei Medical University Taipei 110 Taiwan5 Cancer Center Kaohsiung Medical University Hospital Kaohsiung 807 Taiwan6Department of Clinical Medicine Kaohsiung Medical University Kaohsiung 807 Taiwan7Master Program for Clinical Pharmacogenomics and Pharmacoproteomics School of Pharmacy Taipei Medical UniversityTaipei 110 Taiwan

8Department of Pharmacy Taipei Medical University Wanfang Hospital Taipei 116 Taiwan

Correspondence should be addressed to Wei-Chiao Chang wcctmuedutw

Received 26 November 2013 Accepted 15 January 2014 Published 3 March 2014

Academic Editor Shuen-Iu Hung

Copyright copy 2014 Wei-Chih Kan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nephrolithiasis is a multifactorial disease caused by environmental hormonal and genetic factors Genetic polymorphisms ofORAI1 which codes for the main subunit of the store-operated calcium (SOC) channel were reported to be associated with therisk and recurrence of calcium nephrolithiasis Inositol 145-trisphosphate (IP3) 3-kinase C (ITPKC) is a negative regulator ofthe SOC channel-mediated signaling pathway We investigated the association between calcium containing nephrolithiasis andgenetic variants of ITPKC gene in Taiwanese patients 365 patients were recruited in this study Eight tagging single nucleotidepolymorphisms of ITPKC were selected for genotyping ITPKC genotypes were determined by TaqMan assay ITPKC plasmidswere transfected into cells to evaluate the intracellular calcium mobilization Our results indicated that rs2607420 CC genotype inthe intron region of the ITPKC gene is associated with a lower eGFR by both Modification of Diet in Renal Diseases (119875 = 00405)and Cockcroft-Gault (119875 = 00215) equations in patients with calcium nephrolithiasis Our results identify a novel polymorphismfor renal function and highlight the importance of ITPKC as a key molecule to regulate calcium signaling

1 Introduction

Urolithiasis is a global problem affecting almost all popula-tions in theworld In developed countries the prevalence rateof urolithiasis was reported to be 4sim20 In Taiwan theprevalence was reported to be 96 [1] The lifetime risk ofurolithiasis is about 10sim15 in the developed world but therisk was as high as 20sim25 in the Middle East [2] Further-more in 20sim75 of patients the disease recurs within 10years of the first episode [3] Consequently urolithiasis causesa burden on society and significantly influences patientsrsquoquality of life Previous epidemiological studies described anassociation between obesity and nephrolithiasis [4]

Urolithiasis often involves the formation of stones con-taining calcium compounds mainly calcium oxalate andcalcium phosphate which account for 70sim80 of reportedcases of urolithiasis Calcium urolithiasis is thought tohave a physicochemical origin involving processes such asnucleation growth aggregation and retention of crystals inthe urine The crystals include inorganic (eg calcium uricacid phosphate and citrate) and organic substances (theTamm-Horsfall glycoprotein and osteopontin) [5] Calciumnephrolithiasis is a type of calcium metabolism disorderSeveral studies indicated that the crystals may injure renalepithelial cells through inflammatory reactions and apopto-sis resulting in stone formation [6ndash8] It is thought to be

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 397826 6 pageshttpdxdoiorg1011552014397826

2 BioMed Research International

5998400UTR 3

998400UTR

rs11673492rs28493229

rs7257602rs7251246

rs890934 rs10420685rs2607420

rs2290692

Figure 1 Graphical overview of the genotyped human ITPKC gene

a multifactorial disease influenced by environmental hor-monal and genetic factors

Our previous study indicated that genetic polymorphismsof ORAI1 which codes for the main subunit of the store-operated calcium (SOC) channel were associated with therisk and recurrence of calcium nephrolithiasis in a Taiwanesepopulation [9] Inositol 145-trisphosphate (IP

3) 3-kinase C

(ITPKC) is a negative regulator of the SOC channel-mediatednuclear factor of activated T cell (NFAT) signaling pathwayand is involved in immune modulation via phosphorylationof IP3[10 11] A functional single-nucleotide polymorphism

(SNP) of ITPKC (rs28493229)was found to be associatedwithsusceptibility to Kawasaki disease and coronary artery lesionformation [11] The purpose of this study was to determinewhether SNPs (rs11673492 rs28493229 rs7257602 rs7251246rs890934 rs10420685 rs2607420 and rs2290692) of ITPKCare associated with the recurrence stone number or kidneyfunction of patients with nephrolithiasis

2 Material and Methods

21 Patients and Methods We enrolled 365 patients whofulfilled the diagnostic criteria for nephrolithiasis at Kaoh-siung Medical University Hospital (KMUH) Radiographicand echographic documentation of urinary stones in thesepatients were collected Stone samples were obtained eitherfrom spontaneous passage or by surgical manipulation Wealso collected clinical information such as age gender familyhistory of nephrolithiasis and episodes of stone recurrenceThe past history of the stone episode was traced back to thewhole life as far as patients could remember Patients with atleast 2 symptomatic episodes (at least 6 months apart) or newstones after treatmentwere classified into the recurrent groupand those with only 1 episode were classified into the singlegroup All subjects provided informed consent The studyprotocol conformed to the Declaration of Helsinki and thestudy was approved by the Institute Review Board of KMUH

22 DNA Extraction DNA was extracted from blood sam-ples collected from subjects Blood cells were first treatedwith 05 sodium dodecyl sulfate lysis buffer and thenwith protease K (1mgmL) for 4 h at 60∘C to digest thenuclear proteins Total DNA was harvested using a Gentra(QIAGEN Inc Valencia CA) extraction kit and 70 alcoholprecipitation as in our previous study [12]

23 Genotyping We selected seven tagging SNPs(rs11673492 rs7257602 rs7251246 rs890934 rs10420685

rs2607420 and rs2290692) with a minor allele frequencygreater than 10 in the Han Chinese Beijing population fromthe HapMap database (httphapmapncbinlmnihgov)In addition we included rs28493229 in this study whichresulted from its association with the inflammation whichhas been demonstrated The ITPKC gene structure wasshown in Figure 1 Genotyping was carried out using theTaqMan allelic discrimination assay (Applied BiosystemsFoster City CA) Briefly we performed a polymerase chainreaction (PCR) using a 96-well microplate with an ABI 9700thermal cycler (Applied Biosystems) The thermal cycleconditions were as follows denaturing at 95∘C for 10minfollowed by 40 cycles of denaturing at 92∘C for 15 s andannealing and extending at 60∘C for 1min After the PCRthe fluorescence was measured and analyzed using SystemSDS software version 123 (Applied Biosystems Foster CityCA)

24 Calcium Imaging Intracellular Ca2+ ([Ca2+]i) responseswere induced by application of thapsigargin (TG) (Sigma-Aldrich St Louis MO) in ITPKC-overexpressing HEK293cells according to the previously described methods [13]Before the experiments cells were stained with 1 120583M Fluo-4-AM (Molecular Probes Eugene OR) at 37∘C for 20minand then washed with BSS buffer (54mMKCl 55mM D-glucose 1mMMgSO

4 130mMNaCl 20mM Hepes at pH

74 and 2mMCaCl2) [Ca2+]i concentrations were estimated

based on the ratio of fluorescence intensities emitted uponexcitation with consecutive 3-s pulses of 488 nm light ata resolution of 1376 times 1038 pixels using an OlympusCellandR IX81 fluorescence microscope (Olympus Essex UK)equipped with anMT 20 illumination system (Olympus) andUPLanApo 10x objective lens The [Ca2+]i concentration wasestimated based on calibration curves as follows A Ca2+calibration curve was created using a Ca2+ calibration bufferkit (Molecular Probes) This experiment was repeated fivetimes [Ca2+]i was calculated from Fluo-4 excited at 488 nmand imaged using an Olympus CellandR IX81 fluorescencemicroscope and UPLanApo 10x objective lens at 20∘C Fluo-4 signals were calibrated by measuring the fluorescenceintensity from microcuvettes containing 10mMK

2-EGTA

(pH 720) buffered to various [Ca2+]i levelsTheCa2+ concen-tration was calculated using the following formula [Ca2+]i =KD times ((119865 minus 119865min)(119865max minus 119865)) Plotting the fluorescenceintensity versus [Ca2+]

119894yielded the calibration curve with the

formula of [Ca2+]i = KD times ((119865minus119865min)(119865max minus119865)) where KDis 345 nM 119865 is the Fluo-4 intensity 119865max is 640 and 119865min is217 for Fluo-4

BioMed Research International 3

Table 1 Basal characteristics of patients with nephrolithiasis and ofnormal controls

Characteristic Patients with nephrolithiasisNumber of subjects 365Gender male number () 250 (685)Age (years)a 543 plusmn 120

Range 24sim86aMean plusmn SD

25 Statistical Analysis SAS 91 for Windows (Cary NC)was used for all statistical analyses Statistical differences ingenotypes and allelic frequencies between cases and controlswere assessed by 1205942 test A 119875 value of lt005 was consideredto be statistically significant

3 Results

31 Demographic and Clinical Characteristics of Subjects Weincluded 365 nephrolithiasis patients in this study Table 1shows the demographic characteristics of subjects The meanage of patients plusmn standard deviation was 543 plusmn 120 yearsand 685 of patients were male (Table 1) In total 8 taggingSNPs of ITPKC were selected in this study from the HapMapdatabase (httphapmapncbinlmnihgov)These SNPs hadat least 10 minimum allele frequencies (MAFs) in a HanChinese Beijing population A graphical overview of thegenotyped polymorphisms is shown in Figure 1

32 Association Study of ITPKC Genetic Polymorphisms withthe Risk of Stone Numbers and Recurrence in Patients withCalcium Nephrolithiasis Among cases 113 patients had hadrecurrent episodes and 116 patients had had a single episodeWe tested whether the rs11673492 rs28493229 rs7257602rs7251246 rs890934 rs10420685 rs2607420 and rs2290692genotypes were associated with the stone number or recur-rence of calcium nephrolithiasis As shown in Table 2 wefound no significant association of genotypes or allelicfrequencies with stone numbers No significant associationwas noted between genotypes and the recurrence of calciumnephrolithiasis (Table 2)

33 Associations of ITPKC Genetic Polymorphisms with theEstimated Glomerular Filtration Rate (eGFR) Previous stud-ies indicated that renal stones may cause renal impairmentand decrease renal function [14]Therefore we further calcu-latedModification ofDiet in RenalDiseases- (MDRD-) basedand Cockcroft-Gault- (C-G-) based eGFRs which are widelyused tools to predict the renal function of nephrolithiasispatients [15 16] We tested the relationship between ITPKCgenetic polymorphisms and renal function Among the SNPsof ITPKC we tested rs7251246 had a significant association(119875 = 00456) with a lower eGFR as calculated by C-G whilethe rs2607420 CC genotype had a significant correlation witha lower eGFR as calculated by both MDRD (119875 = 00405) andC-G (119875 = 00215) (Table 3)

1000

800

600

400

200

0

0 5 10 15 20

Time (min)

TG

pcDNAITPKC

[Ca2+

] i(n

M)

Ca2+-free 2mM Ca2+

Figure 2 Effects of ITPKC on calcium influx in HEK293 cellsThapsigargin (TG 2 120583M) was applied in a Ca2+-free BSS solutionThe extracellular Ca2+ concentration abruptly increased from 0 to2mM which triggered the store-operated (SOC) channel influx

34 Effects of ITPKC Overexpression in Calcium Signaling inHEK293 Cells The mechanism by which ITPKC influencescellular signaling is not clear Thus we transfected ITPKCplasmids into cells and evaluated the intracellular calciummobilization As shown in Figure 2 overexpression of ITPKCslightly increased the calcium release (first calcium peak)Importantly the sustained calcium influx was reduced (sec-ond calcium peak)

4 Discussion

Nephrolithiasis is caused by a variety of conditions includingmetabolic disorders and anatomical defects It is consid-ered a metabolic disorder commonly associated with type2 diabetes obesity dyslipidemia and hypertension Mostcases of nephrolithiasis are idiopathic in such cases there isundoubtedly a genetic predisposition but both environmen-tal and lifestyle factors may play important roles [17] Almost40 of patients who develop a stone for the first time willdevelop a second stone within 3 years of the first episode ifprophylactic measures are not taken The physicochemicaltheory of stone formation considers urine a supersaturatedsolution in which homogeneous or heterogeneous nucleationcan lead to initiation of crystal formation which can thenaggregate and grow into a stone [17] A well-known theoryis Randallrsquos plaques this theory proposes that subepithelialinterstitial calcium-based deposits act as nuclei for stoneformation These plaques which are composed of apatite(calcium phosphate not calcium oxalate) originate adjacentto the thin limbs of the loops of Henle As they grow insize they may injure the renal papillary duct epitheliumand serve as sites for intratubular adhesion and growth ofcalcium oxalate crystals [17] A positive family history ofkidney stones is strongly associated with an increased risk

4 BioMed Research International

Table 2 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical medical records data of patients with kidneystones

SNP Genotype Stone numbers ()119875 value Stone frequency ()

119875 valueMultiple Single Recurrence Nonrecurrence

rs11673492TT 12 (97) 10 (81)

0905011 (97) 10 (87)

04025CT 58 (468) 59 (476) 46 (407) 57 (496)CC 54 (435) 55 (443) 56 (496) 48 (417)

rs28493229 CG 18 (148) 10 (81) 01032 16 (144) 9 (79) 01198GG 104 (852) 113 (919) 95 (856) 105 (921)

rs7257602GG 32 (258) 24 (192)

0370527 (239) 23 (198)

07665AG 55 (444) 65 (520) 54 (478) 58 (500)AA 37 (298) 36 (288) 32 (283) 35 (302)

rs7251246GG 38 (307) 30 (240)

0263429 (257) 33 (285)

07521GA 50 (403) 63 (504) 52 (460) 55 (474)AA 36 (290) 32 (256) 32 (283) 28 (241)

rs890934TT 23 (185) 29 (232)

0391923 (203) 24 (207)

09975GT 58 (468) 62 (496) 55 (487) 56 (483)GG 43 (347) 34 (272) 35 (310) 36 (310)

rs10420685GG 7 (56) 5 (40)

078426 (53) 6 (52)

08333AG 43 (347) 41 (331) 37 (327) 42 (365)AA 74 (597) 78 (629) 70 (620) 67 (583)

rs2607420CC 10 (81) 9 (72)

0961810 (89) 9 (78)

09191CT 51 (415) 52 (416) 43 (384) 47 (405)TT 62 (504) 64 (512) 59 (527) 60 (517)

rs2290692GG 38 (307) 28 (224)

0216128 (248) 32 (276)

08602CG 50 (403) 63 (504) 53 (469) 54 (465)CC 36 (290) 34 (272) 32 (283) 30 (259)

of stone formation the relative risk is 2sim3 times higher insuch individuals than in individuals without a family historyof kidney stones [6]

Calcium nephrolithiasis is a type of calcium metabolicdisorder and most renal stones contain calcium Formationof stones may also result from injury and inflammation of therenal tubular epithelium Recent study indicated that crystalsdeposition or formation inside the kidney not only directlycauses renal epithelial cell damage but also may induceimmune response [18] Crystals act as a stimulant that triggerinnate immunity and lead to generate several cytokines suchas IL-1120573 and IL-18 that drive inflammatory response [18]Genetic factors that regulate calciummetabolism and inflam-mation are possibly linked with nephrolithiasis Geneticpolymorphisms of CLDN14 CASR OPN ORAI1 and VDRwere reported to be involved in calcium nephrolithiasis inhumans [19ndash23] Our previous study indicated that geneticpolymorphisms of ORAI1 are associated with susceptibilityto calcium urolithiasis [6] ORAI1 is a pore subunit of theSOC channel involved in different physiological functionsincluding immune responses and inflammatory reactions[24] Indeed calcium influx through the SOC channel canregulate the secretion of proinflammatory molecules suchas arachidonic acid and leukotriene C

4[25] ITPKC is an

upstream gene of the SOC channel that may influence its

activation via IP3phosphorylation which in turn regu-

lates immune responses [26] Therefore we supposed thatgenetic variant of ITPKC may contribute to influence thecalcium influx and furthermore may induce the activationof T cells which provoke inflammatory reaction By usingfluorescence-based calcium detection we provide the firstevidence to support a functional role of ITPKC in calciumsignaling (Figure 2)

In this study we found no significant associationsbetween genotypes of ITPKC (rs11673492 rs28493229rs7257602 rs7251246 rs890934 rs10420685 rs2607420and rs2290692) and calcium nephrolithiasis However wecannot rule out the possibility that other low-frequencygenetic polymorphisms of ITPKC may contribute to calciumnephrolithiasis Importantly we found that rs2607420 wassignificantly associated with kidney function (MDRD andC-G) Although we had no evidence to prove that rs2607420the intronic SNP directly affects the splicing efficiency orinfluences the affinity of transcription factor binding sitehowever another intronic SNP of ITPKC rs28493229 haddemonstrated that the variant of rs28493229 is associatedwith the mRNA expression level of ITPKC [11] Thus thevariant of rs2607420 may result in enhancing the risk ofrenal injury through changing ITPKC expression levelEven so the effects of ITPKC on calcium nephrolithiasis

BioMed Research International 5

Table 3 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical biochemical data of patients with kidney stones

SNP Genotype Sample number () MDRD119875 value C-G

119875 value(mLmin173m2) (mLmin)

rs11673492TT 32 (88) 776 plusmn 160

01471714 plusmn 192

02342CT 164 (451) 849 plusmn 299 851 plusmn 330

CC 168 (461) 840 plusmn 259 830 plusmn 282

rs28493229CC 0 (00) mdash

05622mdash

08885CG 42 (125) 829 plusmn 264 832 plusmn 293

GG 295 (875) 869 plusmn 343 821 plusmn 367

rs7257602GG 92 (253) 805 plusmn 285

02855779 plusmn 292

04558AG 164 (450) 868 plusmn 283 856 plusmn 293

AA 108 (297) 801 plusmn 247 823 plusmn 312

rs7251246GG 96 (264) 825 plusmn 224

00990843 plusmn 289

00456lowastGA 164 (450) 876 plusmn 299 874 plusmn 307

AA 104 (286) 767 plusmn 273 732 plusmn 283

rs890934TT 77 (211) 779 plusmn 290

04380744 plusmn 233

01932GT 180 (493) 845 plusmn 289 840 plusmn 327

GG 108 (296) 844 plusmn 236 860 plusmn 291

rs10420685GG 16 (44) 909 plusmn 125

02153895 plusmn 211

01545AG 126 (346) 865 plusmn 264 877 plusmn 317

AA 222 (610) 802 plusmn 285 789 plusmn 293

rs2607420CC 27 (74) 690 plusmn 164

00405lowast686 plusmn 182

00215lowastCT 143 (393) 872 plusmn 266 892 plusmn 316

TT 194 (533) 816 plusmn 279 792 plusmn 283

rs2290692GG 93 (194) 831 plusmn 226

04016848 plusmn 293

00784CG 164 (342) 858 plusmn 279 867 plusmn 308

CC 107 (464) 789 plusmn 309 741 plusmn 285

lowastSignificant (119875 lt 005) values are in bold

and kidney functions are still unclear More research isneeded to determine the molecular basis of ITPKC and thepathogenesis of calcium nephrolithiasis

In conclusion we found that rs2607420 in the intronregion of the ITPKC gene is associate with estimated crea-tinine clearance in patients with calcium nephrolithiasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wei-Chih Kan and Yii-Her Chou contributed equally to thepaper

Acknowledgments

This work was supported by grants from Chi-Mei MedicalCenter Kaohsiung Medical University Research Foundation(101CM-KMU-08) and Taipei Medical University (TMU101-AE1-B14)

References

[1] Y-H Lee W-C Huang J-Y Tsai et al ldquoEpidemiologicalstudies on the prevalence of upper urinary calculi in TaiwanrdquoUrologia Internationalis vol 68 no 3 pp 172ndash177 2002

[2] C Y C Pak ldquoKidney stonesrdquoThe Lancet vol 351 no 9118 pp1797ndash1801 1998

[3] A Basiri N Shakhssalim A R Khoshdel et al ldquoFamilialrelations and recurrence pattern in nephrolithiasis new wordsabout old subjectsrdquoUrology Journal vol 7 no 2 pp 81ndash86 2010

[4] M J Semins A D ShoreM AMakary TMagnuson R Johnsand B R Matlaga ldquoThe association of increasing body massindex and kidney stone diseaserdquo Journal of Urology vol 183 no2 pp 571ndash575 2010

[5] D R Basavaraj C S Biyani A J Browning and J J CartledgeldquoThe role of urinary kidney stone inhibitors and promoters inthe pathogenesis of Calcium containing renal stonesrdquoEAU-EBUUpdate Series vol 5 no 3 pp 126ndash136 2007

[6] W G Robertson M Peacock and B E C Nordin ldquoCalciumcrystalluria in recurrent renal-stone formersrdquo The Lancet vol2 no 7610 pp 21ndash24 1969

[7] MW Bigelow J HWiessner J G Kleinman andN SMandelldquoCalcium oxalate-crystal membrane interactions dependenceon membrane lipid compositionrdquo Journal of Urology vol 155no 3 pp 1094ndash1098 1996

[8] S R Khan and D J Kok ldquoModulators of urinary stoneformationrdquo Frontiers in Bioscience vol 9 pp 1450ndash1482 2004

6 BioMed Research International

[9] Y-H Chou S-H H Juo Y-C Chiu et al ldquoA polymorphismof the ORAI1 gene is associated with the risk and recurrence ofcalcium nephrolithiasisrdquo Journal of Urology vol 185 no 5 pp1742ndash1746 2011

[10] K Sauer and M P Cooke ldquoRegulation of immune cell devel-opment through soluble inositol-1345- tetrakisphosphaterdquoNature Reviews Immunology vol 10 no 4 pp 257ndash271 2010

[11] Y Onouchi T Gunji J C Burns et al ldquoITPKC functionalpolymorphism associated with Kawasaki disease susceptibilityand formation of coronary artery aneurysmsrdquo Nature Geneticsvol 40 no 1 pp 35ndash42 2008

[12] K D Yang J-C Chang H Chuang et al ldquoGene-gene and gene-environment interactions on IgE production in prenatal stagerdquoAllergy vol 65 no 6 pp 731ndash739 2010

[13] W L Hsu M H Tsai M W Lin et al ldquoDifferential effectsof arsenic on calcium signaling in primary keratinocytes andmalignant (HSC-1) cellsrdquo Cell Calcium vol 52 no 2 pp 161ndash169 2012

[14] G Gambaro S Favaro and A DrsquoAngelo ldquoRisk for renal failurein nephrolithiasisrdquoAmerican Journal of Kidney Diseases vol 37no 2 pp 233ndash243 2001

[15] D W Cockcroft and M H Gault ldquoPrediction of creatinineclearance from serum creatininerdquoNephron vol 16 no 1 pp 31ndash41 1976

[16] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[17] N Johri B Cooper W Robertson S Choong D Rickardsand R Unwin ldquoAn update and practical guide to renal stonemanagementrdquo Nephron vol 116 no 3 pp c159ndashc171 2010

[18] S R Mulay A Evan and H J Anders ldquoMolecular mechanismsof crystal-related kidney inflammation and injury Implicationsfor cholesterol embolism crystalline nephropathies and kidneystone diseaserdquo in Nephrology Dialysis Transplantation Euro-pean Renal Association 2013

[19] J A Sayer ldquoRenal stone diseaserdquo Nephron vol 118 no 1 ppp35ndashp44 2011

[20] G Thorleifsson H Holm V Edvardsson et al ldquoSequencevariants in the CLDN14 gene associate with kidney stones andbone mineral densityrdquo Nature Genetics vol 41 no 8 pp 926ndash930 2009

[21] L G Ferreira A C Pereira and I P Heilberg ldquoVitamin Dreceptor and calcium-sensing receptor gene polymorphisms inhypercalciuric stone-forming patientsrdquo Nephron vol 114 no 2pp c135ndashc144 2010

[22] J E Zerwekh M R Hughes B Y Reed et al ldquoEvidencefor normal vitamin D receptor messenger ribonucleic acidand genotype in absorptive hypercalciuriardquo Journal of ClinicalEndocrinology and Metabolism vol 80 no 10 pp 2960ndash29651995

[23] S Nishio M Hatanaka H Takeda T Iseda H Iwata andM Yokoyama ldquoAnalysis of urinary concentrations of calciumphosphate crystal- associated proteins 1205722-HS-glycoproteinprothrombin F1 and osteopontinrdquo Journal of the AmericanSociety of Nephrology vol 10 supplement 14 pp S394ndashS3961999

[24] A Braun D Varga-Szabo C Kleinschnitz et al ldquoOrai1(CRACM1) is the platelet SOC channel and essential forpathological thrombus formationrdquo Blood vol 113 no 9 pp2056ndash2063 2009

[25] W-C Chang and A B Parekh ldquoClose functional couplingbetween Ca2+ release-activated Ca2+ channels arachidonic acidrelease and leukotriene C

4secretionrdquoThe Journal of Biological

Chemistry vol 279 no 29 pp 29994ndash29999 2004[26] H-C Kuo and W-C Chang ldquoGenetic polymorphisms in

Kawasaki diseaserdquo Acta Pharmacologica Sinica vol 32 no 10pp 1193ndash1198 2011

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2 BioMed Research International

5998400UTR 3

998400UTR

rs11673492rs28493229

rs7257602rs7251246

rs890934 rs10420685rs2607420

rs2290692

Figure 1 Graphical overview of the genotyped human ITPKC gene

a multifactorial disease influenced by environmental hor-monal and genetic factors

Our previous study indicated that genetic polymorphismsof ORAI1 which codes for the main subunit of the store-operated calcium (SOC) channel were associated with therisk and recurrence of calcium nephrolithiasis in a Taiwanesepopulation [9] Inositol 145-trisphosphate (IP

3) 3-kinase C

(ITPKC) is a negative regulator of the SOC channel-mediatednuclear factor of activated T cell (NFAT) signaling pathwayand is involved in immune modulation via phosphorylationof IP3[10 11] A functional single-nucleotide polymorphism

(SNP) of ITPKC (rs28493229)was found to be associatedwithsusceptibility to Kawasaki disease and coronary artery lesionformation [11] The purpose of this study was to determinewhether SNPs (rs11673492 rs28493229 rs7257602 rs7251246rs890934 rs10420685 rs2607420 and rs2290692) of ITPKCare associated with the recurrence stone number or kidneyfunction of patients with nephrolithiasis

2 Material and Methods

21 Patients and Methods We enrolled 365 patients whofulfilled the diagnostic criteria for nephrolithiasis at Kaoh-siung Medical University Hospital (KMUH) Radiographicand echographic documentation of urinary stones in thesepatients were collected Stone samples were obtained eitherfrom spontaneous passage or by surgical manipulation Wealso collected clinical information such as age gender familyhistory of nephrolithiasis and episodes of stone recurrenceThe past history of the stone episode was traced back to thewhole life as far as patients could remember Patients with atleast 2 symptomatic episodes (at least 6 months apart) or newstones after treatmentwere classified into the recurrent groupand those with only 1 episode were classified into the singlegroup All subjects provided informed consent The studyprotocol conformed to the Declaration of Helsinki and thestudy was approved by the Institute Review Board of KMUH

22 DNA Extraction DNA was extracted from blood sam-ples collected from subjects Blood cells were first treatedwith 05 sodium dodecyl sulfate lysis buffer and thenwith protease K (1mgmL) for 4 h at 60∘C to digest thenuclear proteins Total DNA was harvested using a Gentra(QIAGEN Inc Valencia CA) extraction kit and 70 alcoholprecipitation as in our previous study [12]

23 Genotyping We selected seven tagging SNPs(rs11673492 rs7257602 rs7251246 rs890934 rs10420685

rs2607420 and rs2290692) with a minor allele frequencygreater than 10 in the Han Chinese Beijing population fromthe HapMap database (httphapmapncbinlmnihgov)In addition we included rs28493229 in this study whichresulted from its association with the inflammation whichhas been demonstrated The ITPKC gene structure wasshown in Figure 1 Genotyping was carried out using theTaqMan allelic discrimination assay (Applied BiosystemsFoster City CA) Briefly we performed a polymerase chainreaction (PCR) using a 96-well microplate with an ABI 9700thermal cycler (Applied Biosystems) The thermal cycleconditions were as follows denaturing at 95∘C for 10minfollowed by 40 cycles of denaturing at 92∘C for 15 s andannealing and extending at 60∘C for 1min After the PCRthe fluorescence was measured and analyzed using SystemSDS software version 123 (Applied Biosystems Foster CityCA)

24 Calcium Imaging Intracellular Ca2+ ([Ca2+]i) responseswere induced by application of thapsigargin (TG) (Sigma-Aldrich St Louis MO) in ITPKC-overexpressing HEK293cells according to the previously described methods [13]Before the experiments cells were stained with 1 120583M Fluo-4-AM (Molecular Probes Eugene OR) at 37∘C for 20minand then washed with BSS buffer (54mMKCl 55mM D-glucose 1mMMgSO

4 130mMNaCl 20mM Hepes at pH

74 and 2mMCaCl2) [Ca2+]i concentrations were estimated

based on the ratio of fluorescence intensities emitted uponexcitation with consecutive 3-s pulses of 488 nm light ata resolution of 1376 times 1038 pixels using an OlympusCellandR IX81 fluorescence microscope (Olympus Essex UK)equipped with anMT 20 illumination system (Olympus) andUPLanApo 10x objective lens The [Ca2+]i concentration wasestimated based on calibration curves as follows A Ca2+calibration curve was created using a Ca2+ calibration bufferkit (Molecular Probes) This experiment was repeated fivetimes [Ca2+]i was calculated from Fluo-4 excited at 488 nmand imaged using an Olympus CellandR IX81 fluorescencemicroscope and UPLanApo 10x objective lens at 20∘C Fluo-4 signals were calibrated by measuring the fluorescenceintensity from microcuvettes containing 10mMK

2-EGTA

(pH 720) buffered to various [Ca2+]i levelsTheCa2+ concen-tration was calculated using the following formula [Ca2+]i =KD times ((119865 minus 119865min)(119865max minus 119865)) Plotting the fluorescenceintensity versus [Ca2+]

119894yielded the calibration curve with the

formula of [Ca2+]i = KD times ((119865minus119865min)(119865max minus119865)) where KDis 345 nM 119865 is the Fluo-4 intensity 119865max is 640 and 119865min is217 for Fluo-4

BioMed Research International 3

Table 1 Basal characteristics of patients with nephrolithiasis and ofnormal controls

Characteristic Patients with nephrolithiasisNumber of subjects 365Gender male number () 250 (685)Age (years)a 543 plusmn 120

Range 24sim86aMean plusmn SD

25 Statistical Analysis SAS 91 for Windows (Cary NC)was used for all statistical analyses Statistical differences ingenotypes and allelic frequencies between cases and controlswere assessed by 1205942 test A 119875 value of lt005 was consideredto be statistically significant

3 Results

31 Demographic and Clinical Characteristics of Subjects Weincluded 365 nephrolithiasis patients in this study Table 1shows the demographic characteristics of subjects The meanage of patients plusmn standard deviation was 543 plusmn 120 yearsand 685 of patients were male (Table 1) In total 8 taggingSNPs of ITPKC were selected in this study from the HapMapdatabase (httphapmapncbinlmnihgov)These SNPs hadat least 10 minimum allele frequencies (MAFs) in a HanChinese Beijing population A graphical overview of thegenotyped polymorphisms is shown in Figure 1

32 Association Study of ITPKC Genetic Polymorphisms withthe Risk of Stone Numbers and Recurrence in Patients withCalcium Nephrolithiasis Among cases 113 patients had hadrecurrent episodes and 116 patients had had a single episodeWe tested whether the rs11673492 rs28493229 rs7257602rs7251246 rs890934 rs10420685 rs2607420 and rs2290692genotypes were associated with the stone number or recur-rence of calcium nephrolithiasis As shown in Table 2 wefound no significant association of genotypes or allelicfrequencies with stone numbers No significant associationwas noted between genotypes and the recurrence of calciumnephrolithiasis (Table 2)

33 Associations of ITPKC Genetic Polymorphisms with theEstimated Glomerular Filtration Rate (eGFR) Previous stud-ies indicated that renal stones may cause renal impairmentand decrease renal function [14]Therefore we further calcu-latedModification ofDiet in RenalDiseases- (MDRD-) basedand Cockcroft-Gault- (C-G-) based eGFRs which are widelyused tools to predict the renal function of nephrolithiasispatients [15 16] We tested the relationship between ITPKCgenetic polymorphisms and renal function Among the SNPsof ITPKC we tested rs7251246 had a significant association(119875 = 00456) with a lower eGFR as calculated by C-G whilethe rs2607420 CC genotype had a significant correlation witha lower eGFR as calculated by both MDRD (119875 = 00405) andC-G (119875 = 00215) (Table 3)

1000

800

600

400

200

0

0 5 10 15 20

Time (min)

TG

pcDNAITPKC

[Ca2+

] i(n

M)

Ca2+-free 2mM Ca2+

Figure 2 Effects of ITPKC on calcium influx in HEK293 cellsThapsigargin (TG 2 120583M) was applied in a Ca2+-free BSS solutionThe extracellular Ca2+ concentration abruptly increased from 0 to2mM which triggered the store-operated (SOC) channel influx

34 Effects of ITPKC Overexpression in Calcium Signaling inHEK293 Cells The mechanism by which ITPKC influencescellular signaling is not clear Thus we transfected ITPKCplasmids into cells and evaluated the intracellular calciummobilization As shown in Figure 2 overexpression of ITPKCslightly increased the calcium release (first calcium peak)Importantly the sustained calcium influx was reduced (sec-ond calcium peak)

4 Discussion

Nephrolithiasis is caused by a variety of conditions includingmetabolic disorders and anatomical defects It is consid-ered a metabolic disorder commonly associated with type2 diabetes obesity dyslipidemia and hypertension Mostcases of nephrolithiasis are idiopathic in such cases there isundoubtedly a genetic predisposition but both environmen-tal and lifestyle factors may play important roles [17] Almost40 of patients who develop a stone for the first time willdevelop a second stone within 3 years of the first episode ifprophylactic measures are not taken The physicochemicaltheory of stone formation considers urine a supersaturatedsolution in which homogeneous or heterogeneous nucleationcan lead to initiation of crystal formation which can thenaggregate and grow into a stone [17] A well-known theoryis Randallrsquos plaques this theory proposes that subepithelialinterstitial calcium-based deposits act as nuclei for stoneformation These plaques which are composed of apatite(calcium phosphate not calcium oxalate) originate adjacentto the thin limbs of the loops of Henle As they grow insize they may injure the renal papillary duct epitheliumand serve as sites for intratubular adhesion and growth ofcalcium oxalate crystals [17] A positive family history ofkidney stones is strongly associated with an increased risk

4 BioMed Research International

Table 2 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical medical records data of patients with kidneystones

SNP Genotype Stone numbers ()119875 value Stone frequency ()

119875 valueMultiple Single Recurrence Nonrecurrence

rs11673492TT 12 (97) 10 (81)

0905011 (97) 10 (87)

04025CT 58 (468) 59 (476) 46 (407) 57 (496)CC 54 (435) 55 (443) 56 (496) 48 (417)

rs28493229 CG 18 (148) 10 (81) 01032 16 (144) 9 (79) 01198GG 104 (852) 113 (919) 95 (856) 105 (921)

rs7257602GG 32 (258) 24 (192)

0370527 (239) 23 (198)

07665AG 55 (444) 65 (520) 54 (478) 58 (500)AA 37 (298) 36 (288) 32 (283) 35 (302)

rs7251246GG 38 (307) 30 (240)

0263429 (257) 33 (285)

07521GA 50 (403) 63 (504) 52 (460) 55 (474)AA 36 (290) 32 (256) 32 (283) 28 (241)

rs890934TT 23 (185) 29 (232)

0391923 (203) 24 (207)

09975GT 58 (468) 62 (496) 55 (487) 56 (483)GG 43 (347) 34 (272) 35 (310) 36 (310)

rs10420685GG 7 (56) 5 (40)

078426 (53) 6 (52)

08333AG 43 (347) 41 (331) 37 (327) 42 (365)AA 74 (597) 78 (629) 70 (620) 67 (583)

rs2607420CC 10 (81) 9 (72)

0961810 (89) 9 (78)

09191CT 51 (415) 52 (416) 43 (384) 47 (405)TT 62 (504) 64 (512) 59 (527) 60 (517)

rs2290692GG 38 (307) 28 (224)

0216128 (248) 32 (276)

08602CG 50 (403) 63 (504) 53 (469) 54 (465)CC 36 (290) 34 (272) 32 (283) 30 (259)

of stone formation the relative risk is 2sim3 times higher insuch individuals than in individuals without a family historyof kidney stones [6]

Calcium nephrolithiasis is a type of calcium metabolicdisorder and most renal stones contain calcium Formationof stones may also result from injury and inflammation of therenal tubular epithelium Recent study indicated that crystalsdeposition or formation inside the kidney not only directlycauses renal epithelial cell damage but also may induceimmune response [18] Crystals act as a stimulant that triggerinnate immunity and lead to generate several cytokines suchas IL-1120573 and IL-18 that drive inflammatory response [18]Genetic factors that regulate calciummetabolism and inflam-mation are possibly linked with nephrolithiasis Geneticpolymorphisms of CLDN14 CASR OPN ORAI1 and VDRwere reported to be involved in calcium nephrolithiasis inhumans [19ndash23] Our previous study indicated that geneticpolymorphisms of ORAI1 are associated with susceptibilityto calcium urolithiasis [6] ORAI1 is a pore subunit of theSOC channel involved in different physiological functionsincluding immune responses and inflammatory reactions[24] Indeed calcium influx through the SOC channel canregulate the secretion of proinflammatory molecules suchas arachidonic acid and leukotriene C

4[25] ITPKC is an

upstream gene of the SOC channel that may influence its

activation via IP3phosphorylation which in turn regu-

lates immune responses [26] Therefore we supposed thatgenetic variant of ITPKC may contribute to influence thecalcium influx and furthermore may induce the activationof T cells which provoke inflammatory reaction By usingfluorescence-based calcium detection we provide the firstevidence to support a functional role of ITPKC in calciumsignaling (Figure 2)

In this study we found no significant associationsbetween genotypes of ITPKC (rs11673492 rs28493229rs7257602 rs7251246 rs890934 rs10420685 rs2607420and rs2290692) and calcium nephrolithiasis However wecannot rule out the possibility that other low-frequencygenetic polymorphisms of ITPKC may contribute to calciumnephrolithiasis Importantly we found that rs2607420 wassignificantly associated with kidney function (MDRD andC-G) Although we had no evidence to prove that rs2607420the intronic SNP directly affects the splicing efficiency orinfluences the affinity of transcription factor binding sitehowever another intronic SNP of ITPKC rs28493229 haddemonstrated that the variant of rs28493229 is associatedwith the mRNA expression level of ITPKC [11] Thus thevariant of rs2607420 may result in enhancing the risk ofrenal injury through changing ITPKC expression levelEven so the effects of ITPKC on calcium nephrolithiasis

BioMed Research International 5

Table 3 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical biochemical data of patients with kidney stones

SNP Genotype Sample number () MDRD119875 value C-G

119875 value(mLmin173m2) (mLmin)

rs11673492TT 32 (88) 776 plusmn 160

01471714 plusmn 192

02342CT 164 (451) 849 plusmn 299 851 plusmn 330

CC 168 (461) 840 plusmn 259 830 plusmn 282

rs28493229CC 0 (00) mdash

05622mdash

08885CG 42 (125) 829 plusmn 264 832 plusmn 293

GG 295 (875) 869 plusmn 343 821 plusmn 367

rs7257602GG 92 (253) 805 plusmn 285

02855779 plusmn 292

04558AG 164 (450) 868 plusmn 283 856 plusmn 293

AA 108 (297) 801 plusmn 247 823 plusmn 312

rs7251246GG 96 (264) 825 plusmn 224

00990843 plusmn 289

00456lowastGA 164 (450) 876 plusmn 299 874 plusmn 307

AA 104 (286) 767 plusmn 273 732 plusmn 283

rs890934TT 77 (211) 779 plusmn 290

04380744 plusmn 233

01932GT 180 (493) 845 plusmn 289 840 plusmn 327

GG 108 (296) 844 plusmn 236 860 plusmn 291

rs10420685GG 16 (44) 909 plusmn 125

02153895 plusmn 211

01545AG 126 (346) 865 plusmn 264 877 plusmn 317

AA 222 (610) 802 plusmn 285 789 plusmn 293

rs2607420CC 27 (74) 690 plusmn 164

00405lowast686 plusmn 182

00215lowastCT 143 (393) 872 plusmn 266 892 plusmn 316

TT 194 (533) 816 plusmn 279 792 plusmn 283

rs2290692GG 93 (194) 831 plusmn 226

04016848 plusmn 293

00784CG 164 (342) 858 plusmn 279 867 plusmn 308

CC 107 (464) 789 plusmn 309 741 plusmn 285

lowastSignificant (119875 lt 005) values are in bold

and kidney functions are still unclear More research isneeded to determine the molecular basis of ITPKC and thepathogenesis of calcium nephrolithiasis

In conclusion we found that rs2607420 in the intronregion of the ITPKC gene is associate with estimated crea-tinine clearance in patients with calcium nephrolithiasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wei-Chih Kan and Yii-Her Chou contributed equally to thepaper

Acknowledgments

This work was supported by grants from Chi-Mei MedicalCenter Kaohsiung Medical University Research Foundation(101CM-KMU-08) and Taipei Medical University (TMU101-AE1-B14)

References

[1] Y-H Lee W-C Huang J-Y Tsai et al ldquoEpidemiologicalstudies on the prevalence of upper urinary calculi in TaiwanrdquoUrologia Internationalis vol 68 no 3 pp 172ndash177 2002

[2] C Y C Pak ldquoKidney stonesrdquoThe Lancet vol 351 no 9118 pp1797ndash1801 1998

[3] A Basiri N Shakhssalim A R Khoshdel et al ldquoFamilialrelations and recurrence pattern in nephrolithiasis new wordsabout old subjectsrdquoUrology Journal vol 7 no 2 pp 81ndash86 2010

[4] M J Semins A D ShoreM AMakary TMagnuson R Johnsand B R Matlaga ldquoThe association of increasing body massindex and kidney stone diseaserdquo Journal of Urology vol 183 no2 pp 571ndash575 2010

[5] D R Basavaraj C S Biyani A J Browning and J J CartledgeldquoThe role of urinary kidney stone inhibitors and promoters inthe pathogenesis of Calcium containing renal stonesrdquoEAU-EBUUpdate Series vol 5 no 3 pp 126ndash136 2007

[6] W G Robertson M Peacock and B E C Nordin ldquoCalciumcrystalluria in recurrent renal-stone formersrdquo The Lancet vol2 no 7610 pp 21ndash24 1969

[7] MW Bigelow J HWiessner J G Kleinman andN SMandelldquoCalcium oxalate-crystal membrane interactions dependenceon membrane lipid compositionrdquo Journal of Urology vol 155no 3 pp 1094ndash1098 1996

[8] S R Khan and D J Kok ldquoModulators of urinary stoneformationrdquo Frontiers in Bioscience vol 9 pp 1450ndash1482 2004

6 BioMed Research International

[9] Y-H Chou S-H H Juo Y-C Chiu et al ldquoA polymorphismof the ORAI1 gene is associated with the risk and recurrence ofcalcium nephrolithiasisrdquo Journal of Urology vol 185 no 5 pp1742ndash1746 2011

[10] K Sauer and M P Cooke ldquoRegulation of immune cell devel-opment through soluble inositol-1345- tetrakisphosphaterdquoNature Reviews Immunology vol 10 no 4 pp 257ndash271 2010

[11] Y Onouchi T Gunji J C Burns et al ldquoITPKC functionalpolymorphism associated with Kawasaki disease susceptibilityand formation of coronary artery aneurysmsrdquo Nature Geneticsvol 40 no 1 pp 35ndash42 2008

[12] K D Yang J-C Chang H Chuang et al ldquoGene-gene and gene-environment interactions on IgE production in prenatal stagerdquoAllergy vol 65 no 6 pp 731ndash739 2010

[13] W L Hsu M H Tsai M W Lin et al ldquoDifferential effectsof arsenic on calcium signaling in primary keratinocytes andmalignant (HSC-1) cellsrdquo Cell Calcium vol 52 no 2 pp 161ndash169 2012

[14] G Gambaro S Favaro and A DrsquoAngelo ldquoRisk for renal failurein nephrolithiasisrdquoAmerican Journal of Kidney Diseases vol 37no 2 pp 233ndash243 2001

[15] D W Cockcroft and M H Gault ldquoPrediction of creatinineclearance from serum creatininerdquoNephron vol 16 no 1 pp 31ndash41 1976

[16] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[17] N Johri B Cooper W Robertson S Choong D Rickardsand R Unwin ldquoAn update and practical guide to renal stonemanagementrdquo Nephron vol 116 no 3 pp c159ndashc171 2010

[18] S R Mulay A Evan and H J Anders ldquoMolecular mechanismsof crystal-related kidney inflammation and injury Implicationsfor cholesterol embolism crystalline nephropathies and kidneystone diseaserdquo in Nephrology Dialysis Transplantation Euro-pean Renal Association 2013

[19] J A Sayer ldquoRenal stone diseaserdquo Nephron vol 118 no 1 ppp35ndashp44 2011

[20] G Thorleifsson H Holm V Edvardsson et al ldquoSequencevariants in the CLDN14 gene associate with kidney stones andbone mineral densityrdquo Nature Genetics vol 41 no 8 pp 926ndash930 2009

[21] L G Ferreira A C Pereira and I P Heilberg ldquoVitamin Dreceptor and calcium-sensing receptor gene polymorphisms inhypercalciuric stone-forming patientsrdquo Nephron vol 114 no 2pp c135ndashc144 2010

[22] J E Zerwekh M R Hughes B Y Reed et al ldquoEvidencefor normal vitamin D receptor messenger ribonucleic acidand genotype in absorptive hypercalciuriardquo Journal of ClinicalEndocrinology and Metabolism vol 80 no 10 pp 2960ndash29651995

[23] S Nishio M Hatanaka H Takeda T Iseda H Iwata andM Yokoyama ldquoAnalysis of urinary concentrations of calciumphosphate crystal- associated proteins 1205722-HS-glycoproteinprothrombin F1 and osteopontinrdquo Journal of the AmericanSociety of Nephrology vol 10 supplement 14 pp S394ndashS3961999

[24] A Braun D Varga-Szabo C Kleinschnitz et al ldquoOrai1(CRACM1) is the platelet SOC channel and essential forpathological thrombus formationrdquo Blood vol 113 no 9 pp2056ndash2063 2009

[25] W-C Chang and A B Parekh ldquoClose functional couplingbetween Ca2+ release-activated Ca2+ channels arachidonic acidrelease and leukotriene C

4secretionrdquoThe Journal of Biological

Chemistry vol 279 no 29 pp 29994ndash29999 2004[26] H-C Kuo and W-C Chang ldquoGenetic polymorphisms in

Kawasaki diseaserdquo Acta Pharmacologica Sinica vol 32 no 10pp 1193ndash1198 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 3

Table 1 Basal characteristics of patients with nephrolithiasis and ofnormal controls

Characteristic Patients with nephrolithiasisNumber of subjects 365Gender male number () 250 (685)Age (years)a 543 plusmn 120

Range 24sim86aMean plusmn SD

25 Statistical Analysis SAS 91 for Windows (Cary NC)was used for all statistical analyses Statistical differences ingenotypes and allelic frequencies between cases and controlswere assessed by 1205942 test A 119875 value of lt005 was consideredto be statistically significant

3 Results

31 Demographic and Clinical Characteristics of Subjects Weincluded 365 nephrolithiasis patients in this study Table 1shows the demographic characteristics of subjects The meanage of patients plusmn standard deviation was 543 plusmn 120 yearsand 685 of patients were male (Table 1) In total 8 taggingSNPs of ITPKC were selected in this study from the HapMapdatabase (httphapmapncbinlmnihgov)These SNPs hadat least 10 minimum allele frequencies (MAFs) in a HanChinese Beijing population A graphical overview of thegenotyped polymorphisms is shown in Figure 1

32 Association Study of ITPKC Genetic Polymorphisms withthe Risk of Stone Numbers and Recurrence in Patients withCalcium Nephrolithiasis Among cases 113 patients had hadrecurrent episodes and 116 patients had had a single episodeWe tested whether the rs11673492 rs28493229 rs7257602rs7251246 rs890934 rs10420685 rs2607420 and rs2290692genotypes were associated with the stone number or recur-rence of calcium nephrolithiasis As shown in Table 2 wefound no significant association of genotypes or allelicfrequencies with stone numbers No significant associationwas noted between genotypes and the recurrence of calciumnephrolithiasis (Table 2)

33 Associations of ITPKC Genetic Polymorphisms with theEstimated Glomerular Filtration Rate (eGFR) Previous stud-ies indicated that renal stones may cause renal impairmentand decrease renal function [14]Therefore we further calcu-latedModification ofDiet in RenalDiseases- (MDRD-) basedand Cockcroft-Gault- (C-G-) based eGFRs which are widelyused tools to predict the renal function of nephrolithiasispatients [15 16] We tested the relationship between ITPKCgenetic polymorphisms and renal function Among the SNPsof ITPKC we tested rs7251246 had a significant association(119875 = 00456) with a lower eGFR as calculated by C-G whilethe rs2607420 CC genotype had a significant correlation witha lower eGFR as calculated by both MDRD (119875 = 00405) andC-G (119875 = 00215) (Table 3)

1000

800

600

400

200

0

0 5 10 15 20

Time (min)

TG

pcDNAITPKC

[Ca2+

] i(n

M)

Ca2+-free 2mM Ca2+

Figure 2 Effects of ITPKC on calcium influx in HEK293 cellsThapsigargin (TG 2 120583M) was applied in a Ca2+-free BSS solutionThe extracellular Ca2+ concentration abruptly increased from 0 to2mM which triggered the store-operated (SOC) channel influx

34 Effects of ITPKC Overexpression in Calcium Signaling inHEK293 Cells The mechanism by which ITPKC influencescellular signaling is not clear Thus we transfected ITPKCplasmids into cells and evaluated the intracellular calciummobilization As shown in Figure 2 overexpression of ITPKCslightly increased the calcium release (first calcium peak)Importantly the sustained calcium influx was reduced (sec-ond calcium peak)

4 Discussion

Nephrolithiasis is caused by a variety of conditions includingmetabolic disorders and anatomical defects It is consid-ered a metabolic disorder commonly associated with type2 diabetes obesity dyslipidemia and hypertension Mostcases of nephrolithiasis are idiopathic in such cases there isundoubtedly a genetic predisposition but both environmen-tal and lifestyle factors may play important roles [17] Almost40 of patients who develop a stone for the first time willdevelop a second stone within 3 years of the first episode ifprophylactic measures are not taken The physicochemicaltheory of stone formation considers urine a supersaturatedsolution in which homogeneous or heterogeneous nucleationcan lead to initiation of crystal formation which can thenaggregate and grow into a stone [17] A well-known theoryis Randallrsquos plaques this theory proposes that subepithelialinterstitial calcium-based deposits act as nuclei for stoneformation These plaques which are composed of apatite(calcium phosphate not calcium oxalate) originate adjacentto the thin limbs of the loops of Henle As they grow insize they may injure the renal papillary duct epitheliumand serve as sites for intratubular adhesion and growth ofcalcium oxalate crystals [17] A positive family history ofkidney stones is strongly associated with an increased risk

4 BioMed Research International

Table 2 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical medical records data of patients with kidneystones

SNP Genotype Stone numbers ()119875 value Stone frequency ()

119875 valueMultiple Single Recurrence Nonrecurrence

rs11673492TT 12 (97) 10 (81)

0905011 (97) 10 (87)

04025CT 58 (468) 59 (476) 46 (407) 57 (496)CC 54 (435) 55 (443) 56 (496) 48 (417)

rs28493229 CG 18 (148) 10 (81) 01032 16 (144) 9 (79) 01198GG 104 (852) 113 (919) 95 (856) 105 (921)

rs7257602GG 32 (258) 24 (192)

0370527 (239) 23 (198)

07665AG 55 (444) 65 (520) 54 (478) 58 (500)AA 37 (298) 36 (288) 32 (283) 35 (302)

rs7251246GG 38 (307) 30 (240)

0263429 (257) 33 (285)

07521GA 50 (403) 63 (504) 52 (460) 55 (474)AA 36 (290) 32 (256) 32 (283) 28 (241)

rs890934TT 23 (185) 29 (232)

0391923 (203) 24 (207)

09975GT 58 (468) 62 (496) 55 (487) 56 (483)GG 43 (347) 34 (272) 35 (310) 36 (310)

rs10420685GG 7 (56) 5 (40)

078426 (53) 6 (52)

08333AG 43 (347) 41 (331) 37 (327) 42 (365)AA 74 (597) 78 (629) 70 (620) 67 (583)

rs2607420CC 10 (81) 9 (72)

0961810 (89) 9 (78)

09191CT 51 (415) 52 (416) 43 (384) 47 (405)TT 62 (504) 64 (512) 59 (527) 60 (517)

rs2290692GG 38 (307) 28 (224)

0216128 (248) 32 (276)

08602CG 50 (403) 63 (504) 53 (469) 54 (465)CC 36 (290) 34 (272) 32 (283) 30 (259)

of stone formation the relative risk is 2sim3 times higher insuch individuals than in individuals without a family historyof kidney stones [6]

Calcium nephrolithiasis is a type of calcium metabolicdisorder and most renal stones contain calcium Formationof stones may also result from injury and inflammation of therenal tubular epithelium Recent study indicated that crystalsdeposition or formation inside the kidney not only directlycauses renal epithelial cell damage but also may induceimmune response [18] Crystals act as a stimulant that triggerinnate immunity and lead to generate several cytokines suchas IL-1120573 and IL-18 that drive inflammatory response [18]Genetic factors that regulate calciummetabolism and inflam-mation are possibly linked with nephrolithiasis Geneticpolymorphisms of CLDN14 CASR OPN ORAI1 and VDRwere reported to be involved in calcium nephrolithiasis inhumans [19ndash23] Our previous study indicated that geneticpolymorphisms of ORAI1 are associated with susceptibilityto calcium urolithiasis [6] ORAI1 is a pore subunit of theSOC channel involved in different physiological functionsincluding immune responses and inflammatory reactions[24] Indeed calcium influx through the SOC channel canregulate the secretion of proinflammatory molecules suchas arachidonic acid and leukotriene C

4[25] ITPKC is an

upstream gene of the SOC channel that may influence its

activation via IP3phosphorylation which in turn regu-

lates immune responses [26] Therefore we supposed thatgenetic variant of ITPKC may contribute to influence thecalcium influx and furthermore may induce the activationof T cells which provoke inflammatory reaction By usingfluorescence-based calcium detection we provide the firstevidence to support a functional role of ITPKC in calciumsignaling (Figure 2)

In this study we found no significant associationsbetween genotypes of ITPKC (rs11673492 rs28493229rs7257602 rs7251246 rs890934 rs10420685 rs2607420and rs2290692) and calcium nephrolithiasis However wecannot rule out the possibility that other low-frequencygenetic polymorphisms of ITPKC may contribute to calciumnephrolithiasis Importantly we found that rs2607420 wassignificantly associated with kidney function (MDRD andC-G) Although we had no evidence to prove that rs2607420the intronic SNP directly affects the splicing efficiency orinfluences the affinity of transcription factor binding sitehowever another intronic SNP of ITPKC rs28493229 haddemonstrated that the variant of rs28493229 is associatedwith the mRNA expression level of ITPKC [11] Thus thevariant of rs2607420 may result in enhancing the risk ofrenal injury through changing ITPKC expression levelEven so the effects of ITPKC on calcium nephrolithiasis

BioMed Research International 5

Table 3 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical biochemical data of patients with kidney stones

SNP Genotype Sample number () MDRD119875 value C-G

119875 value(mLmin173m2) (mLmin)

rs11673492TT 32 (88) 776 plusmn 160

01471714 plusmn 192

02342CT 164 (451) 849 plusmn 299 851 plusmn 330

CC 168 (461) 840 plusmn 259 830 plusmn 282

rs28493229CC 0 (00) mdash

05622mdash

08885CG 42 (125) 829 plusmn 264 832 plusmn 293

GG 295 (875) 869 plusmn 343 821 plusmn 367

rs7257602GG 92 (253) 805 plusmn 285

02855779 plusmn 292

04558AG 164 (450) 868 plusmn 283 856 plusmn 293

AA 108 (297) 801 plusmn 247 823 plusmn 312

rs7251246GG 96 (264) 825 plusmn 224

00990843 plusmn 289

00456lowastGA 164 (450) 876 plusmn 299 874 plusmn 307

AA 104 (286) 767 plusmn 273 732 plusmn 283

rs890934TT 77 (211) 779 plusmn 290

04380744 plusmn 233

01932GT 180 (493) 845 plusmn 289 840 plusmn 327

GG 108 (296) 844 plusmn 236 860 plusmn 291

rs10420685GG 16 (44) 909 plusmn 125

02153895 plusmn 211

01545AG 126 (346) 865 plusmn 264 877 plusmn 317

AA 222 (610) 802 plusmn 285 789 plusmn 293

rs2607420CC 27 (74) 690 plusmn 164

00405lowast686 plusmn 182

00215lowastCT 143 (393) 872 plusmn 266 892 plusmn 316

TT 194 (533) 816 plusmn 279 792 plusmn 283

rs2290692GG 93 (194) 831 plusmn 226

04016848 plusmn 293

00784CG 164 (342) 858 plusmn 279 867 plusmn 308

CC 107 (464) 789 plusmn 309 741 plusmn 285

lowastSignificant (119875 lt 005) values are in bold

and kidney functions are still unclear More research isneeded to determine the molecular basis of ITPKC and thepathogenesis of calcium nephrolithiasis

In conclusion we found that rs2607420 in the intronregion of the ITPKC gene is associate with estimated crea-tinine clearance in patients with calcium nephrolithiasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wei-Chih Kan and Yii-Her Chou contributed equally to thepaper

Acknowledgments

This work was supported by grants from Chi-Mei MedicalCenter Kaohsiung Medical University Research Foundation(101CM-KMU-08) and Taipei Medical University (TMU101-AE1-B14)

References

[1] Y-H Lee W-C Huang J-Y Tsai et al ldquoEpidemiologicalstudies on the prevalence of upper urinary calculi in TaiwanrdquoUrologia Internationalis vol 68 no 3 pp 172ndash177 2002

[2] C Y C Pak ldquoKidney stonesrdquoThe Lancet vol 351 no 9118 pp1797ndash1801 1998

[3] A Basiri N Shakhssalim A R Khoshdel et al ldquoFamilialrelations and recurrence pattern in nephrolithiasis new wordsabout old subjectsrdquoUrology Journal vol 7 no 2 pp 81ndash86 2010

[4] M J Semins A D ShoreM AMakary TMagnuson R Johnsand B R Matlaga ldquoThe association of increasing body massindex and kidney stone diseaserdquo Journal of Urology vol 183 no2 pp 571ndash575 2010

[5] D R Basavaraj C S Biyani A J Browning and J J CartledgeldquoThe role of urinary kidney stone inhibitors and promoters inthe pathogenesis of Calcium containing renal stonesrdquoEAU-EBUUpdate Series vol 5 no 3 pp 126ndash136 2007

[6] W G Robertson M Peacock and B E C Nordin ldquoCalciumcrystalluria in recurrent renal-stone formersrdquo The Lancet vol2 no 7610 pp 21ndash24 1969

[7] MW Bigelow J HWiessner J G Kleinman andN SMandelldquoCalcium oxalate-crystal membrane interactions dependenceon membrane lipid compositionrdquo Journal of Urology vol 155no 3 pp 1094ndash1098 1996

[8] S R Khan and D J Kok ldquoModulators of urinary stoneformationrdquo Frontiers in Bioscience vol 9 pp 1450ndash1482 2004

6 BioMed Research International

[9] Y-H Chou S-H H Juo Y-C Chiu et al ldquoA polymorphismof the ORAI1 gene is associated with the risk and recurrence ofcalcium nephrolithiasisrdquo Journal of Urology vol 185 no 5 pp1742ndash1746 2011

[10] K Sauer and M P Cooke ldquoRegulation of immune cell devel-opment through soluble inositol-1345- tetrakisphosphaterdquoNature Reviews Immunology vol 10 no 4 pp 257ndash271 2010

[11] Y Onouchi T Gunji J C Burns et al ldquoITPKC functionalpolymorphism associated with Kawasaki disease susceptibilityand formation of coronary artery aneurysmsrdquo Nature Geneticsvol 40 no 1 pp 35ndash42 2008

[12] K D Yang J-C Chang H Chuang et al ldquoGene-gene and gene-environment interactions on IgE production in prenatal stagerdquoAllergy vol 65 no 6 pp 731ndash739 2010

[13] W L Hsu M H Tsai M W Lin et al ldquoDifferential effectsof arsenic on calcium signaling in primary keratinocytes andmalignant (HSC-1) cellsrdquo Cell Calcium vol 52 no 2 pp 161ndash169 2012

[14] G Gambaro S Favaro and A DrsquoAngelo ldquoRisk for renal failurein nephrolithiasisrdquoAmerican Journal of Kidney Diseases vol 37no 2 pp 233ndash243 2001

[15] D W Cockcroft and M H Gault ldquoPrediction of creatinineclearance from serum creatininerdquoNephron vol 16 no 1 pp 31ndash41 1976

[16] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[17] N Johri B Cooper W Robertson S Choong D Rickardsand R Unwin ldquoAn update and practical guide to renal stonemanagementrdquo Nephron vol 116 no 3 pp c159ndashc171 2010

[18] S R Mulay A Evan and H J Anders ldquoMolecular mechanismsof crystal-related kidney inflammation and injury Implicationsfor cholesterol embolism crystalline nephropathies and kidneystone diseaserdquo in Nephrology Dialysis Transplantation Euro-pean Renal Association 2013

[19] J A Sayer ldquoRenal stone diseaserdquo Nephron vol 118 no 1 ppp35ndashp44 2011

[20] G Thorleifsson H Holm V Edvardsson et al ldquoSequencevariants in the CLDN14 gene associate with kidney stones andbone mineral densityrdquo Nature Genetics vol 41 no 8 pp 926ndash930 2009

[21] L G Ferreira A C Pereira and I P Heilberg ldquoVitamin Dreceptor and calcium-sensing receptor gene polymorphisms inhypercalciuric stone-forming patientsrdquo Nephron vol 114 no 2pp c135ndashc144 2010

[22] J E Zerwekh M R Hughes B Y Reed et al ldquoEvidencefor normal vitamin D receptor messenger ribonucleic acidand genotype in absorptive hypercalciuriardquo Journal of ClinicalEndocrinology and Metabolism vol 80 no 10 pp 2960ndash29651995

[23] S Nishio M Hatanaka H Takeda T Iseda H Iwata andM Yokoyama ldquoAnalysis of urinary concentrations of calciumphosphate crystal- associated proteins 1205722-HS-glycoproteinprothrombin F1 and osteopontinrdquo Journal of the AmericanSociety of Nephrology vol 10 supplement 14 pp S394ndashS3961999

[24] A Braun D Varga-Szabo C Kleinschnitz et al ldquoOrai1(CRACM1) is the platelet SOC channel and essential forpathological thrombus formationrdquo Blood vol 113 no 9 pp2056ndash2063 2009

[25] W-C Chang and A B Parekh ldquoClose functional couplingbetween Ca2+ release-activated Ca2+ channels arachidonic acidrelease and leukotriene C

4secretionrdquoThe Journal of Biological

Chemistry vol 279 no 29 pp 29994ndash29999 2004[26] H-C Kuo and W-C Chang ldquoGenetic polymorphisms in

Kawasaki diseaserdquo Acta Pharmacologica Sinica vol 32 no 10pp 1193ndash1198 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

Table 2 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical medical records data of patients with kidneystones

SNP Genotype Stone numbers ()119875 value Stone frequency ()

119875 valueMultiple Single Recurrence Nonrecurrence

rs11673492TT 12 (97) 10 (81)

0905011 (97) 10 (87)

04025CT 58 (468) 59 (476) 46 (407) 57 (496)CC 54 (435) 55 (443) 56 (496) 48 (417)

rs28493229 CG 18 (148) 10 (81) 01032 16 (144) 9 (79) 01198GG 104 (852) 113 (919) 95 (856) 105 (921)

rs7257602GG 32 (258) 24 (192)

0370527 (239) 23 (198)

07665AG 55 (444) 65 (520) 54 (478) 58 (500)AA 37 (298) 36 (288) 32 (283) 35 (302)

rs7251246GG 38 (307) 30 (240)

0263429 (257) 33 (285)

07521GA 50 (403) 63 (504) 52 (460) 55 (474)AA 36 (290) 32 (256) 32 (283) 28 (241)

rs890934TT 23 (185) 29 (232)

0391923 (203) 24 (207)

09975GT 58 (468) 62 (496) 55 (487) 56 (483)GG 43 (347) 34 (272) 35 (310) 36 (310)

rs10420685GG 7 (56) 5 (40)

078426 (53) 6 (52)

08333AG 43 (347) 41 (331) 37 (327) 42 (365)AA 74 (597) 78 (629) 70 (620) 67 (583)

rs2607420CC 10 (81) 9 (72)

0961810 (89) 9 (78)

09191CT 51 (415) 52 (416) 43 (384) 47 (405)TT 62 (504) 64 (512) 59 (527) 60 (517)

rs2290692GG 38 (307) 28 (224)

0216128 (248) 32 (276)

08602CG 50 (403) 63 (504) 53 (469) 54 (465)CC 36 (290) 34 (272) 32 (283) 30 (259)

of stone formation the relative risk is 2sim3 times higher insuch individuals than in individuals without a family historyof kidney stones [6]

Calcium nephrolithiasis is a type of calcium metabolicdisorder and most renal stones contain calcium Formationof stones may also result from injury and inflammation of therenal tubular epithelium Recent study indicated that crystalsdeposition or formation inside the kidney not only directlycauses renal epithelial cell damage but also may induceimmune response [18] Crystals act as a stimulant that triggerinnate immunity and lead to generate several cytokines suchas IL-1120573 and IL-18 that drive inflammatory response [18]Genetic factors that regulate calciummetabolism and inflam-mation are possibly linked with nephrolithiasis Geneticpolymorphisms of CLDN14 CASR OPN ORAI1 and VDRwere reported to be involved in calcium nephrolithiasis inhumans [19ndash23] Our previous study indicated that geneticpolymorphisms of ORAI1 are associated with susceptibilityto calcium urolithiasis [6] ORAI1 is a pore subunit of theSOC channel involved in different physiological functionsincluding immune responses and inflammatory reactions[24] Indeed calcium influx through the SOC channel canregulate the secretion of proinflammatory molecules suchas arachidonic acid and leukotriene C

4[25] ITPKC is an

upstream gene of the SOC channel that may influence its

activation via IP3phosphorylation which in turn regu-

lates immune responses [26] Therefore we supposed thatgenetic variant of ITPKC may contribute to influence thecalcium influx and furthermore may induce the activationof T cells which provoke inflammatory reaction By usingfluorescence-based calcium detection we provide the firstevidence to support a functional role of ITPKC in calciumsignaling (Figure 2)

In this study we found no significant associationsbetween genotypes of ITPKC (rs11673492 rs28493229rs7257602 rs7251246 rs890934 rs10420685 rs2607420and rs2290692) and calcium nephrolithiasis However wecannot rule out the possibility that other low-frequencygenetic polymorphisms of ITPKC may contribute to calciumnephrolithiasis Importantly we found that rs2607420 wassignificantly associated with kidney function (MDRD andC-G) Although we had no evidence to prove that rs2607420the intronic SNP directly affects the splicing efficiency orinfluences the affinity of transcription factor binding sitehowever another intronic SNP of ITPKC rs28493229 haddemonstrated that the variant of rs28493229 is associatedwith the mRNA expression level of ITPKC [11] Thus thevariant of rs2607420 may result in enhancing the risk ofrenal injury through changing ITPKC expression levelEven so the effects of ITPKC on calcium nephrolithiasis

BioMed Research International 5

Table 3 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical biochemical data of patients with kidney stones

SNP Genotype Sample number () MDRD119875 value C-G

119875 value(mLmin173m2) (mLmin)

rs11673492TT 32 (88) 776 plusmn 160

01471714 plusmn 192

02342CT 164 (451) 849 plusmn 299 851 plusmn 330

CC 168 (461) 840 plusmn 259 830 plusmn 282

rs28493229CC 0 (00) mdash

05622mdash

08885CG 42 (125) 829 plusmn 264 832 plusmn 293

GG 295 (875) 869 plusmn 343 821 plusmn 367

rs7257602GG 92 (253) 805 plusmn 285

02855779 plusmn 292

04558AG 164 (450) 868 plusmn 283 856 plusmn 293

AA 108 (297) 801 plusmn 247 823 plusmn 312

rs7251246GG 96 (264) 825 plusmn 224

00990843 plusmn 289

00456lowastGA 164 (450) 876 plusmn 299 874 plusmn 307

AA 104 (286) 767 plusmn 273 732 plusmn 283

rs890934TT 77 (211) 779 plusmn 290

04380744 plusmn 233

01932GT 180 (493) 845 plusmn 289 840 plusmn 327

GG 108 (296) 844 plusmn 236 860 plusmn 291

rs10420685GG 16 (44) 909 plusmn 125

02153895 plusmn 211

01545AG 126 (346) 865 plusmn 264 877 plusmn 317

AA 222 (610) 802 plusmn 285 789 plusmn 293

rs2607420CC 27 (74) 690 plusmn 164

00405lowast686 plusmn 182

00215lowastCT 143 (393) 872 plusmn 266 892 plusmn 316

TT 194 (533) 816 plusmn 279 792 plusmn 283

rs2290692GG 93 (194) 831 plusmn 226

04016848 plusmn 293

00784CG 164 (342) 858 plusmn 279 867 plusmn 308

CC 107 (464) 789 plusmn 309 741 plusmn 285

lowastSignificant (119875 lt 005) values are in bold

and kidney functions are still unclear More research isneeded to determine the molecular basis of ITPKC and thepathogenesis of calcium nephrolithiasis

In conclusion we found that rs2607420 in the intronregion of the ITPKC gene is associate with estimated crea-tinine clearance in patients with calcium nephrolithiasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wei-Chih Kan and Yii-Her Chou contributed equally to thepaper

Acknowledgments

This work was supported by grants from Chi-Mei MedicalCenter Kaohsiung Medical University Research Foundation(101CM-KMU-08) and Taipei Medical University (TMU101-AE1-B14)

References

[1] Y-H Lee W-C Huang J-Y Tsai et al ldquoEpidemiologicalstudies on the prevalence of upper urinary calculi in TaiwanrdquoUrologia Internationalis vol 68 no 3 pp 172ndash177 2002

[2] C Y C Pak ldquoKidney stonesrdquoThe Lancet vol 351 no 9118 pp1797ndash1801 1998

[3] A Basiri N Shakhssalim A R Khoshdel et al ldquoFamilialrelations and recurrence pattern in nephrolithiasis new wordsabout old subjectsrdquoUrology Journal vol 7 no 2 pp 81ndash86 2010

[4] M J Semins A D ShoreM AMakary TMagnuson R Johnsand B R Matlaga ldquoThe association of increasing body massindex and kidney stone diseaserdquo Journal of Urology vol 183 no2 pp 571ndash575 2010

[5] D R Basavaraj C S Biyani A J Browning and J J CartledgeldquoThe role of urinary kidney stone inhibitors and promoters inthe pathogenesis of Calcium containing renal stonesrdquoEAU-EBUUpdate Series vol 5 no 3 pp 126ndash136 2007

[6] W G Robertson M Peacock and B E C Nordin ldquoCalciumcrystalluria in recurrent renal-stone formersrdquo The Lancet vol2 no 7610 pp 21ndash24 1969

[7] MW Bigelow J HWiessner J G Kleinman andN SMandelldquoCalcium oxalate-crystal membrane interactions dependenceon membrane lipid compositionrdquo Journal of Urology vol 155no 3 pp 1094ndash1098 1996

[8] S R Khan and D J Kok ldquoModulators of urinary stoneformationrdquo Frontiers in Bioscience vol 9 pp 1450ndash1482 2004

6 BioMed Research International

[9] Y-H Chou S-H H Juo Y-C Chiu et al ldquoA polymorphismof the ORAI1 gene is associated with the risk and recurrence ofcalcium nephrolithiasisrdquo Journal of Urology vol 185 no 5 pp1742ndash1746 2011

[10] K Sauer and M P Cooke ldquoRegulation of immune cell devel-opment through soluble inositol-1345- tetrakisphosphaterdquoNature Reviews Immunology vol 10 no 4 pp 257ndash271 2010

[11] Y Onouchi T Gunji J C Burns et al ldquoITPKC functionalpolymorphism associated with Kawasaki disease susceptibilityand formation of coronary artery aneurysmsrdquo Nature Geneticsvol 40 no 1 pp 35ndash42 2008

[12] K D Yang J-C Chang H Chuang et al ldquoGene-gene and gene-environment interactions on IgE production in prenatal stagerdquoAllergy vol 65 no 6 pp 731ndash739 2010

[13] W L Hsu M H Tsai M W Lin et al ldquoDifferential effectsof arsenic on calcium signaling in primary keratinocytes andmalignant (HSC-1) cellsrdquo Cell Calcium vol 52 no 2 pp 161ndash169 2012

[14] G Gambaro S Favaro and A DrsquoAngelo ldquoRisk for renal failurein nephrolithiasisrdquoAmerican Journal of Kidney Diseases vol 37no 2 pp 233ndash243 2001

[15] D W Cockcroft and M H Gault ldquoPrediction of creatinineclearance from serum creatininerdquoNephron vol 16 no 1 pp 31ndash41 1976

[16] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[17] N Johri B Cooper W Robertson S Choong D Rickardsand R Unwin ldquoAn update and practical guide to renal stonemanagementrdquo Nephron vol 116 no 3 pp c159ndashc171 2010

[18] S R Mulay A Evan and H J Anders ldquoMolecular mechanismsof crystal-related kidney inflammation and injury Implicationsfor cholesterol embolism crystalline nephropathies and kidneystone diseaserdquo in Nephrology Dialysis Transplantation Euro-pean Renal Association 2013

[19] J A Sayer ldquoRenal stone diseaserdquo Nephron vol 118 no 1 ppp35ndashp44 2011

[20] G Thorleifsson H Holm V Edvardsson et al ldquoSequencevariants in the CLDN14 gene associate with kidney stones andbone mineral densityrdquo Nature Genetics vol 41 no 8 pp 926ndash930 2009

[21] L G Ferreira A C Pereira and I P Heilberg ldquoVitamin Dreceptor and calcium-sensing receptor gene polymorphisms inhypercalciuric stone-forming patientsrdquo Nephron vol 114 no 2pp c135ndashc144 2010

[22] J E Zerwekh M R Hughes B Y Reed et al ldquoEvidencefor normal vitamin D receptor messenger ribonucleic acidand genotype in absorptive hypercalciuriardquo Journal of ClinicalEndocrinology and Metabolism vol 80 no 10 pp 2960ndash29651995

[23] S Nishio M Hatanaka H Takeda T Iseda H Iwata andM Yokoyama ldquoAnalysis of urinary concentrations of calciumphosphate crystal- associated proteins 1205722-HS-glycoproteinprothrombin F1 and osteopontinrdquo Journal of the AmericanSociety of Nephrology vol 10 supplement 14 pp S394ndashS3961999

[24] A Braun D Varga-Szabo C Kleinschnitz et al ldquoOrai1(CRACM1) is the platelet SOC channel and essential forpathological thrombus formationrdquo Blood vol 113 no 9 pp2056ndash2063 2009

[25] W-C Chang and A B Parekh ldquoClose functional couplingbetween Ca2+ release-activated Ca2+ channels arachidonic acidrelease and leukotriene C

4secretionrdquoThe Journal of Biological

Chemistry vol 279 no 29 pp 29994ndash29999 2004[26] H-C Kuo and W-C Chang ldquoGenetic polymorphisms in

Kawasaki diseaserdquo Acta Pharmacologica Sinica vol 32 no 10pp 1193ndash1198 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

Table 3 Association analysis of ITPKC single-nucleotide polymorphisms (SNPs) and clinical biochemical data of patients with kidney stones

SNP Genotype Sample number () MDRD119875 value C-G

119875 value(mLmin173m2) (mLmin)

rs11673492TT 32 (88) 776 plusmn 160

01471714 plusmn 192

02342CT 164 (451) 849 plusmn 299 851 plusmn 330

CC 168 (461) 840 plusmn 259 830 plusmn 282

rs28493229CC 0 (00) mdash

05622mdash

08885CG 42 (125) 829 plusmn 264 832 plusmn 293

GG 295 (875) 869 plusmn 343 821 plusmn 367

rs7257602GG 92 (253) 805 plusmn 285

02855779 plusmn 292

04558AG 164 (450) 868 plusmn 283 856 plusmn 293

AA 108 (297) 801 plusmn 247 823 plusmn 312

rs7251246GG 96 (264) 825 plusmn 224

00990843 plusmn 289

00456lowastGA 164 (450) 876 plusmn 299 874 plusmn 307

AA 104 (286) 767 plusmn 273 732 plusmn 283

rs890934TT 77 (211) 779 plusmn 290

04380744 plusmn 233

01932GT 180 (493) 845 plusmn 289 840 plusmn 327

GG 108 (296) 844 plusmn 236 860 plusmn 291

rs10420685GG 16 (44) 909 plusmn 125

02153895 plusmn 211

01545AG 126 (346) 865 plusmn 264 877 plusmn 317

AA 222 (610) 802 plusmn 285 789 plusmn 293

rs2607420CC 27 (74) 690 plusmn 164

00405lowast686 plusmn 182

00215lowastCT 143 (393) 872 plusmn 266 892 plusmn 316

TT 194 (533) 816 plusmn 279 792 plusmn 283

rs2290692GG 93 (194) 831 plusmn 226

04016848 plusmn 293

00784CG 164 (342) 858 plusmn 279 867 plusmn 308

CC 107 (464) 789 plusmn 309 741 plusmn 285

lowastSignificant (119875 lt 005) values are in bold

and kidney functions are still unclear More research isneeded to determine the molecular basis of ITPKC and thepathogenesis of calcium nephrolithiasis

In conclusion we found that rs2607420 in the intronregion of the ITPKC gene is associate with estimated crea-tinine clearance in patients with calcium nephrolithiasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wei-Chih Kan and Yii-Her Chou contributed equally to thepaper

Acknowledgments

This work was supported by grants from Chi-Mei MedicalCenter Kaohsiung Medical University Research Foundation(101CM-KMU-08) and Taipei Medical University (TMU101-AE1-B14)

References

[1] Y-H Lee W-C Huang J-Y Tsai et al ldquoEpidemiologicalstudies on the prevalence of upper urinary calculi in TaiwanrdquoUrologia Internationalis vol 68 no 3 pp 172ndash177 2002

[2] C Y C Pak ldquoKidney stonesrdquoThe Lancet vol 351 no 9118 pp1797ndash1801 1998

[3] A Basiri N Shakhssalim A R Khoshdel et al ldquoFamilialrelations and recurrence pattern in nephrolithiasis new wordsabout old subjectsrdquoUrology Journal vol 7 no 2 pp 81ndash86 2010

[4] M J Semins A D ShoreM AMakary TMagnuson R Johnsand B R Matlaga ldquoThe association of increasing body massindex and kidney stone diseaserdquo Journal of Urology vol 183 no2 pp 571ndash575 2010

[5] D R Basavaraj C S Biyani A J Browning and J J CartledgeldquoThe role of urinary kidney stone inhibitors and promoters inthe pathogenesis of Calcium containing renal stonesrdquoEAU-EBUUpdate Series vol 5 no 3 pp 126ndash136 2007

[6] W G Robertson M Peacock and B E C Nordin ldquoCalciumcrystalluria in recurrent renal-stone formersrdquo The Lancet vol2 no 7610 pp 21ndash24 1969

[7] MW Bigelow J HWiessner J G Kleinman andN SMandelldquoCalcium oxalate-crystal membrane interactions dependenceon membrane lipid compositionrdquo Journal of Urology vol 155no 3 pp 1094ndash1098 1996

[8] S R Khan and D J Kok ldquoModulators of urinary stoneformationrdquo Frontiers in Bioscience vol 9 pp 1450ndash1482 2004

6 BioMed Research International

[9] Y-H Chou S-H H Juo Y-C Chiu et al ldquoA polymorphismof the ORAI1 gene is associated with the risk and recurrence ofcalcium nephrolithiasisrdquo Journal of Urology vol 185 no 5 pp1742ndash1746 2011

[10] K Sauer and M P Cooke ldquoRegulation of immune cell devel-opment through soluble inositol-1345- tetrakisphosphaterdquoNature Reviews Immunology vol 10 no 4 pp 257ndash271 2010

[11] Y Onouchi T Gunji J C Burns et al ldquoITPKC functionalpolymorphism associated with Kawasaki disease susceptibilityand formation of coronary artery aneurysmsrdquo Nature Geneticsvol 40 no 1 pp 35ndash42 2008

[12] K D Yang J-C Chang H Chuang et al ldquoGene-gene and gene-environment interactions on IgE production in prenatal stagerdquoAllergy vol 65 no 6 pp 731ndash739 2010

[13] W L Hsu M H Tsai M W Lin et al ldquoDifferential effectsof arsenic on calcium signaling in primary keratinocytes andmalignant (HSC-1) cellsrdquo Cell Calcium vol 52 no 2 pp 161ndash169 2012

[14] G Gambaro S Favaro and A DrsquoAngelo ldquoRisk for renal failurein nephrolithiasisrdquoAmerican Journal of Kidney Diseases vol 37no 2 pp 233ndash243 2001

[15] D W Cockcroft and M H Gault ldquoPrediction of creatinineclearance from serum creatininerdquoNephron vol 16 no 1 pp 31ndash41 1976

[16] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[17] N Johri B Cooper W Robertson S Choong D Rickardsand R Unwin ldquoAn update and practical guide to renal stonemanagementrdquo Nephron vol 116 no 3 pp c159ndashc171 2010

[18] S R Mulay A Evan and H J Anders ldquoMolecular mechanismsof crystal-related kidney inflammation and injury Implicationsfor cholesterol embolism crystalline nephropathies and kidneystone diseaserdquo in Nephrology Dialysis Transplantation Euro-pean Renal Association 2013

[19] J A Sayer ldquoRenal stone diseaserdquo Nephron vol 118 no 1 ppp35ndashp44 2011

[20] G Thorleifsson H Holm V Edvardsson et al ldquoSequencevariants in the CLDN14 gene associate with kidney stones andbone mineral densityrdquo Nature Genetics vol 41 no 8 pp 926ndash930 2009

[21] L G Ferreira A C Pereira and I P Heilberg ldquoVitamin Dreceptor and calcium-sensing receptor gene polymorphisms inhypercalciuric stone-forming patientsrdquo Nephron vol 114 no 2pp c135ndashc144 2010

[22] J E Zerwekh M R Hughes B Y Reed et al ldquoEvidencefor normal vitamin D receptor messenger ribonucleic acidand genotype in absorptive hypercalciuriardquo Journal of ClinicalEndocrinology and Metabolism vol 80 no 10 pp 2960ndash29651995

[23] S Nishio M Hatanaka H Takeda T Iseda H Iwata andM Yokoyama ldquoAnalysis of urinary concentrations of calciumphosphate crystal- associated proteins 1205722-HS-glycoproteinprothrombin F1 and osteopontinrdquo Journal of the AmericanSociety of Nephrology vol 10 supplement 14 pp S394ndashS3961999

[24] A Braun D Varga-Szabo C Kleinschnitz et al ldquoOrai1(CRACM1) is the platelet SOC channel and essential forpathological thrombus formationrdquo Blood vol 113 no 9 pp2056ndash2063 2009

[25] W-C Chang and A B Parekh ldquoClose functional couplingbetween Ca2+ release-activated Ca2+ channels arachidonic acidrelease and leukotriene C

4secretionrdquoThe Journal of Biological

Chemistry vol 279 no 29 pp 29994ndash29999 2004[26] H-C Kuo and W-C Chang ldquoGenetic polymorphisms in

Kawasaki diseaserdquo Acta Pharmacologica Sinica vol 32 no 10pp 1193ndash1198 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 BioMed Research International

[9] Y-H Chou S-H H Juo Y-C Chiu et al ldquoA polymorphismof the ORAI1 gene is associated with the risk and recurrence ofcalcium nephrolithiasisrdquo Journal of Urology vol 185 no 5 pp1742ndash1746 2011

[10] K Sauer and M P Cooke ldquoRegulation of immune cell devel-opment through soluble inositol-1345- tetrakisphosphaterdquoNature Reviews Immunology vol 10 no 4 pp 257ndash271 2010

[11] Y Onouchi T Gunji J C Burns et al ldquoITPKC functionalpolymorphism associated with Kawasaki disease susceptibilityand formation of coronary artery aneurysmsrdquo Nature Geneticsvol 40 no 1 pp 35ndash42 2008

[12] K D Yang J-C Chang H Chuang et al ldquoGene-gene and gene-environment interactions on IgE production in prenatal stagerdquoAllergy vol 65 no 6 pp 731ndash739 2010

[13] W L Hsu M H Tsai M W Lin et al ldquoDifferential effectsof arsenic on calcium signaling in primary keratinocytes andmalignant (HSC-1) cellsrdquo Cell Calcium vol 52 no 2 pp 161ndash169 2012

[14] G Gambaro S Favaro and A DrsquoAngelo ldquoRisk for renal failurein nephrolithiasisrdquoAmerican Journal of Kidney Diseases vol 37no 2 pp 233ndash243 2001

[15] D W Cockcroft and M H Gault ldquoPrediction of creatinineclearance from serum creatininerdquoNephron vol 16 no 1 pp 31ndash41 1976

[16] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[17] N Johri B Cooper W Robertson S Choong D Rickardsand R Unwin ldquoAn update and practical guide to renal stonemanagementrdquo Nephron vol 116 no 3 pp c159ndashc171 2010

[18] S R Mulay A Evan and H J Anders ldquoMolecular mechanismsof crystal-related kidney inflammation and injury Implicationsfor cholesterol embolism crystalline nephropathies and kidneystone diseaserdquo in Nephrology Dialysis Transplantation Euro-pean Renal Association 2013

[19] J A Sayer ldquoRenal stone diseaserdquo Nephron vol 118 no 1 ppp35ndashp44 2011

[20] G Thorleifsson H Holm V Edvardsson et al ldquoSequencevariants in the CLDN14 gene associate with kidney stones andbone mineral densityrdquo Nature Genetics vol 41 no 8 pp 926ndash930 2009

[21] L G Ferreira A C Pereira and I P Heilberg ldquoVitamin Dreceptor and calcium-sensing receptor gene polymorphisms inhypercalciuric stone-forming patientsrdquo Nephron vol 114 no 2pp c135ndashc144 2010

[22] J E Zerwekh M R Hughes B Y Reed et al ldquoEvidencefor normal vitamin D receptor messenger ribonucleic acidand genotype in absorptive hypercalciuriardquo Journal of ClinicalEndocrinology and Metabolism vol 80 no 10 pp 2960ndash29651995

[23] S Nishio M Hatanaka H Takeda T Iseda H Iwata andM Yokoyama ldquoAnalysis of urinary concentrations of calciumphosphate crystal- associated proteins 1205722-HS-glycoproteinprothrombin F1 and osteopontinrdquo Journal of the AmericanSociety of Nephrology vol 10 supplement 14 pp S394ndashS3961999

[24] A Braun D Varga-Szabo C Kleinschnitz et al ldquoOrai1(CRACM1) is the platelet SOC channel and essential forpathological thrombus formationrdquo Blood vol 113 no 9 pp2056ndash2063 2009

[25] W-C Chang and A B Parekh ldquoClose functional couplingbetween Ca2+ release-activated Ca2+ channels arachidonic acidrelease and leukotriene C

4secretionrdquoThe Journal of Biological

Chemistry vol 279 no 29 pp 29994ndash29999 2004[26] H-C Kuo and W-C Chang ldquoGenetic polymorphisms in

Kawasaki diseaserdquo Acta Pharmacologica Sinica vol 32 no 10pp 1193ndash1198 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom