STUDIO DEL CALCIUM SENSING RECEPTOR E POSSIBILE RUOLO NELLA CALCOLOSI RENALE DI CALCIO

Background: The calcium-sensing receptor (CaSR) is a candidate gene of calcium nephrolithiasis. Previously, studies found an association between the CaSR gene region including P1 and P2 promoters and calcium nephrolithiasis. Particularly, the rs7652589 and rs1501899 SNPs, localized up and downstream of promoters, were associated to calcium nephrolithiasis in normocitraturic patients with a p-value of 0.0009 and 0.0004 respectively. The aims of this study were to identified some other CaSR gene promoter SNPs in CaSR promoter region associated with calcium nephrolithiasis and to test their effects on CaSR expression in tubular kidney tissue. Methods: By bioinformatic approch, SNPs were selected as located in P1 or P2 or modifing transcription factors binding sites. SNPs genotyping was performed by Taqman genotyping assays of rs7648041, rs7648044, rs6776158 and rs1048213 in 167 idiopathic calcium stone formers and 213 healthy controls, matched for age, bodi mass index and gender. In 109 normal kidney medulla tissues CaSR mRNA level was evaluated by real-time PCR and SNPs associated to calcium nephrolithiasis (rs7652589, rs1501899 and rs6776158) were genotyped. The influence of rs6776158 SNP on P1 transcription regulation was tested by luciferase assay. Results: A fine mapping of the CaSR gene promoter region has been performed to identify SNPs with a possible role in CaSR gene regulation. In stone formers and, controls, SNPs localized in CaSR regolatory were genotyped. The data showed that SNP rs6776158, localized in P1 (159 bp upstream the transcription start site), resulted associated with the disease. Particularly, rs6776158 minor allele showed a higher frequency in stone formers than in controls (37.8% vs 26.4%, p=0.00084). The homozygous people for polimorfic allele have a risk to develop the disease 3 times higher than other genotypes (O.R.=2.968). CaSR mRNA was measured in kidney medulla tissues and was related to the kidney stones associated SNPs. A decreased CaSR mRNA was found in homozygous subjects for the minor allele of rs6776158 and rs1501899 vs heterozygous and homozygous for the common allele (rs6776158: n=95, 2.753±0.22 vs n=14, 1.69±0.33, p=0.0157; rs1501899: n=95, 2.753±0.22 vs n=14, 1.69±0.33, p=0.0157). No variation was found with rs7652589. In HEK-293 and HKC-8 cells, luciferase assays confirmed that rs6776158 polimorfic allele decrease the P1 activity. Conclusions: These findings confirmed the association of calcium nephrolithiasis with CaSR promoter region. Particularly, the polimorfic allele rs6776158 was more frequent in stone formers than in controls. Moreover, omozigous subjet for the polimorfic allele have a risk 3 times higher to show the disease and a reduction of kidney CaSR mRNA expression. For this reason we hypothesize that a reduction of CaSR expression can predispose to calcium nephrolithiasis.