Giuseppe Vezzoli

Influence of Calcium-Sensing Receptor Gene on Urinary Calcium Excretion in Stone-Forming Patients

Calcium-sensing receptor (CaSR) is a plasma membrane protein that regulates tubular reabsorption of Ca. To establish its role in idiopathic hypercalciuria, the association of urinary Ca excretion with the polymorphisms of CASR gene has been studied in healthy subjects and in hypercalciuric and normocalciuric Ca stone formers. CASR exon 7 single nucleotide polymorphisms (SNP), G/T at codon 986, G/A at codon 990, and C/G at codon 1011, were evaluated by PCR amplification and direct sequencing in 97 normocalciuric stone formers, 134 hypercalciuric stone formers, and 101 normocalciuric healthy controls. Four haplotypes were defined on the basis of CASR gene SNP: haplotype 1 was characterized by the most frequent sequence; haplotypes 2, 3, or 4 by the presence of a single polymorphic variant at codon 986, 990, or 1011, respectively. The relative risk of hypercalciuria was calculated with multinomial logistic regression and was significantly increased only in individuals carrying haplotype 3 (Odds ratio, 13.0 [95% confidence interval, 1.7 to 99.4]). Accordingly, Ca excretion was higher in subjects bearing haplotype 3, whereas those bearing haplotype 2 showed a slight increase of plasma Ca concentration. Multiple regression analysis showed that haplotype 3 explained 4.1% of the total variance of Ca excretion and 12.6% of the variance explained by the variables considered in the study. In conclusion, CASR gene could be a component of the complex genetic background regulating Ca excretion. Arg990Gly polymorphism could facilitate activation of CaSR and increase Ca excretion and susceptibility to idiopathic hypercalciuria.

Effects of a low-salt diet on idiopathic hypercalciuria in calcium-oxalate stone formers: a 3-mo randomized controlled trial

BACKGROUND A direct relation exists between sodium and calcium excretion, but randomized studies evaluating the sustained effect of a low-salt diet on idiopathic hypercalciuria, one of the main risk factors for calcium-oxalate stone formation, are still lacking. OBJECTIVE Our goal was to evaluate the effect of a low-salt diet on urinary calcium excretion in patients affected by idiopathic calcium nephrolithiasis. DESIGN Patients affected by idiopathic calcium stone disease and hypercalciuria (>300 mg Ca/d in men and >250 mg Ca/d in women) were randomly assigned to receive either water therapy alone (control diet) or water therapy and a low-salt diet (low-sodium diet) for 3 mo. Twenty-four-hour urine samples were obtained twice from all patients: one sample at baseline on a free diet and one sample after 3 mo of treatment. RESULTS A total of 210 patients were randomly assigned to receive a control diet (n = 102) or a low-sodium diet (n = 108); 13 patients (2 on the control diet, 11 on the low-sodium diet) withdrew from the trial. At the follow-up visit, patients on the low-sodium diet had lower urinary sodium (mean +/- SD: 68 +/- 43 mmol/d at 3 mo compared with 228 +/- 57 mmol/d at baseline; P < 0.001). Concomitant with this change, they showed lower urinary calcium (271 +/- 86 mg/d at 3 mo compared with 361 +/- 129 mg/d on the control diet, P < 0.001) and lower oxalate excretion (28 +/- 8 mg/d at 3 mo compared with 32 +/- 10 mg/d on the control diet, P = 0.001). Urinary calcium was within the normal range in 61.9% of the patients on the low-salt diet and in 34.0% of those on the control diet (difference: +27.9%; 95% CI: +14.4%, +41.3%; P < 0.001). CONCLUSION A low-salt diet can reduce calcium excretion in hypercalciuric stone formers. This trial was registered at clinicaltrials.gov as NCT01005082.

Calcium kidney stones are associated with a haplotype of the Calcium-sensing receptor gene regulatory region

BACKGROUND Calcium-sensing receptor gene (CaSR) is a candidate to explain susceptibility to calcium kidney stones. Thus, we studied CaSR gene single-nucleotide polymorphisms (SNPs) and haplotypes associated with stones. METHODS Four hundred and sixty-three calcium stone formers and 213 healthy controls were genotyped for 21 SNPs mapping the whole CaSR gene. CaSR gene structure was studied. SNPs and haplotypes were analysed for association with stones. RESULTS Three haplotype blocks were identified in the CaSR gene. The first block was characterized by six SNPs and included gene promoters. The rs7652589 and rs1501899 SNPs and the CATTCA haplotype of the first block were significantly more frequent in normocitraturic calcium kidney stone formers than controls. The risk of stones was increased in normocitraturic homozygous patients and heterozygotes for the CATTCA haplotype. The rate of stones was higher in stone formers with the CATTCA haplotype. In a three-generation family, calcium stones were associated with the CATTCA haplotype. The bioinformatic analysis identified a new site for the octamer-binding transcription factor 1 in the presence of the variant alleles at the rs7652589 and rs1501899 SNPs. This transcription factor may downregulate the transcription of vitamin D-dependent genes and the CaSR expression. Conclusion. SNPs and CATTCA haplotype of the CaSR gene first block is associated with kidney stones in normocitraturic patients.

Polymorphisms at the regulatory regions of the CASR gene influence stone risk in primary hyperparathyroidism

BACKGROUND AND OBJECTIVE Single nucleotide polymorphisms (SNPs) of the calcium-sensing receptor (CASR) gene at the regulatory region were associated with idiopathic calcium nephrolithiasis. To confirm their association with nephrolithiasis, we tested patients with primary hyperparathyroidism (PHPT). DESIGN A genotype-phenotype association study. METHODS In all, 332 PHPT patients and 453 healthy controls were genotyped for the rs7652589 (G>A) and rs1501899 (G>A) SNPs sited in the noncoding regulatory region of the CASR gene. Allele, haplotype, and diplotype distribution were compared between PHPT patients and controls, and in stone forming and stone-free PHPT patients. RESULTS The allele frequency at rs7652589 and rs1501899 SNPs was similar in PHPT patients and controls. The A minor alleles at these two SNPs were more frequent in stone forming (n=157) than in stone-free (n=175) PHPT patients (rs7652589: 36.9 vs 27.1%, P=0.007; rs1501899: 37.1 vs 26.4%, P=0.003). Accordingly, homozygous or heterozygous PHPT patients for the AA haplotype (n=174, AA/AA or AA/GG diplotype) had an increased stone risk (odds ratio 1.83, 95% confidence interval 1.2-2.9, P=0.008). Furthermore, these PHPT patients had higher serum concentrations of ionized calcium and parathyroid hormone (1.50 ± 0.015 mmol/l and 183 ± 12.2 pg/ml) than patients with the GG/GG diplotype (n=145, 1.47 ± 0.011 mmol/l (P=0.04) and 150 ± 11.4 pg/ml (P=0.049)). Using a logistic regression model, the increase in stone risk in PHPT patients was predicted by AA/AA or AA/GG diplotype, the highest tertile of serum ionized calcium values and the lowest tertile of age. CONCLUSIONS Polymorphisms located in the regulatory region of the CASR gene may increase susceptibility of the PHPT patients to kidney stone production.

Abnormal red-cell calcium pump in patients with idiopathic hypercalciuria.

Idiopathic hypercalciuria is a common disorder whose inheritance suggests an enzyme abnormality in calcium transport. We measured calcium-magnesium-ATPase activity in erythrocytes from 38 patients (mean age [+/- SEM], 40 +/- 2.1 years) with idiopathic hypercalciuria (24-hour urinary calcium excretion greater than or equal to 0.1 mmol per kilogram of body weight) and a history of multiple calcium oxalate kidney stones. As compared with 41 healthy controls, the patients with hypercalciuria had increased erythrocyte-membrane calcium-magnesium-ATPase activity (64.2 +/- 2.19 vs. 51.6 +/- 1.91 nmol of ATP split per milligram per minute; P less than 0.01) and increased sodium-potassium pump activity (6866 +/- 233 vs. 6096 +/- 228 mumol of sodium per liter of red cells per hour; P less than 0.05). No significant difference between the two groups was found in erythrocyte sodium-potassium cotransport, sodium-lithium countertransport, or potassium content. In 66 patients with kidney stones (38 with hypercalciuria and 28 with normal calcium excretion), 24-hour urinary calcium excretion correlated with calcium-magnesium-ATPase activity (r = 0.46, P less than 0.001). Erythrocyte calcium-magnesium-ATPase activity remained unchanged in eight subjects studied after four months on a low-calcium diet. A study of 30 healthy families found significant correlations between mean values in parents and those in offspring for calcium-magnesium-ATPase (r = 0.68, P less than 0.001) and urinary calcium excretion (r = 0.45, P less than 0.02), with no significant correlations between parents with respect to these measures (r = 0.27 and r = 0.08, respectively). We conclude that abnormalities in erythrocyte calcium-magnesium-ATPase activity may represent an inherited defect in calcium transport related to the cause of idiopathic hypercalciuria.

Genetics and calcium nephrolithiasis

Calcium nephrolithiasis is one of the most prevalent uronephrologic disorders in the western countries. Studies in families and twins evidenced a genetic predisposition to calcium nephrolithiasis. Family-based or case-control studies of single-candidate genes evidenced the possible involvement of calcium-sensing receptor (CASR), vitamin D receptor (VDR), and osteopontin (OPN) gene polymorphisms in stone formation. The only high-throughput genome-wide association study identified claudin 14 (CLDN14) gene as a possible major gene of nephrolithiasis. Specific phenotypes were related with these genes: CASR gene in normocitraturic patients, VDR gene in hypocitraturic patients with severe clinical course, and CLDN14 gene in hypercalciuric patients. The pathogenetic weight of these genes remains unclear, but an alteration of their expression may occur in stone formers. Technological skills, accurate clinical examination, and a detailed phenotype description are the basis to get new insight about the genetic basis of nephrolithiasis.

Update on Primary Hypercalciuria From a Genetic Perspective

PURPOSE This review provides a brief update on genetic studies of primary hypercalciuria. We consider their possible implications for the pathogenesis and complications of primary hypercalciuria. MATERIALS AND METHODS Using the PubMed, MEDLINE and Scopus databases we reviewed the literature on pathogenesis and the complications of hypercalciuria, giving particular attention to genetic studies in humans. RESULTS Primary hypercalciuria is a defect occurring in 5% to 10% of the general population and it is most commonly detected in patients with calcium kidney stones or osteoporosis. In children it is associated with hematuria, renal stones or nocturnal enuresis. Although high penetrance, autosomal dominant inheritance cannot be ruled out, hypercalciuria is probably a polygenic disorder. A number of genes have been suggested as candidates in the pathogenesis of common idiopathic calcium nephrolithiasis and hypercalciuria, ie soluble adenylate cyclase, calcium sensing receptor, vitamin D receptor, chloride channel-5, sodium-phosphate cotransporter-2 and claudin-16. These genes may also have a role in complications of hypercalciuria. CONCLUSIONS The classic distinction among absorptive, renal and resorptive hypercalciuria seems insufficient to explain the many cellular and tissue modifications observed in patients with primary hypercalciuria. The condition seems to be a separate disorder, characterized by altered calcium transport in the intestine, kidney and bone, and caused by various combinations of multiple genetic and dietary changes.

Calcium ATPase in erythrocytes of spontaneously hypertensive rats of the milan strain

Calcium (Ca)-dependent ATPase activity was determined in erythrocyte membrane ghosts from normal and spontaneously hypertensive rats of the Milan strain in order to detect changes in enzyme activity which had previously been shown in the spontaneously hypertensive rat strain. Activity was similar in control and hypertensive rats in the absence of calmodulin. In contrast, activity in the presence of saturating amounts of calmodulin was significantly lower in the hypertensive rats. At the Vmax (free Ca2+ concentration 10 mumol/l) the decrease was about 30% (70.1 +/- 8.94 versus 49.1 +/- 4.75 nmol of ATP split/mg of ghost proteins per min; P less than 0.05). The affinity of the ATP-dependent Ca2+ pump for Ca2+ (Km about 1 mumol/l) was not altered in the hypertensive rats. It is possible that deficient Ca ATPase activity sustains an increase of intracellular free Ca in cells of hypertensive rats concomitant with the intracellular sodium (Na) decrease typical of this strain.

Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor

Previous studies have demonstrated a gain-of-function of the calcium-sensing receptor (CASR) gene R990G polymorphism. In this study, activation of the R990G CASR stably transfected in HEK-293 (HEK-990G) cells compared with that of the common variant (HEK-wild-type (WT)) by increasing concentrations of CaCl(2) or calcimimetic R-568 caused significantly higher intracellular free calcium concentration ([Ca(2+)](i)) and lower Ca-EC(50). Moreover, the [Ca(2+)](i) oscillation percentage was higher with a larger sinusoidal pattern in HEK-990G. R-568 induced a shift of the oscillatory events from 4 to 2  mmol/l extracellular calcium concentration in HEK-990G cells and increased the sinusoidal oscillation percentage in comparison with HEK-WT. Preincubation with thapsigargin or phospholipase C inhibitors completely prevented oscillations in both cell lines, consistent with the involvement of the inositol trisphosphate pathway, while protein kinase C inhibitor prevented oscillations in HEK-WT cells only. Finally, CaCl(2) and R-568 caused a significant increase in p44/42 extracellular signaling-regulated kinase phosphorylation, with the mean Ca-EC(50) values being significantly lower in HEK-990G. Our findings demonstrated that the 990G allele is associated with high sensitivity to R-568, which provided new evidence for differences in CASR signaling.

Decreased transcriptional activity of calcium-sensing receptor gene promoter 1 is associated with calcium nephrolithiasis

BACKGROUND CaSR gene is a candidate for calcium nephrolithiasis. Single-nucleotide polymorphisms (SNPs) encompassing its regulatory region were associated with calcium nephrolithiasis. AIMS We tested SNPs in the CaSR gene regulatory region associated with calcium nephrolithiasis and their effects in kidney. SUBJECTS AND METHODS One hundred sixty-seven idiopathic calcium stone formers and 214 healthy controls were genotyped for four CaSR gene SNPs identified by bioinformatics analysis as modifying transcription factor binding sites. Strontium excretion after an oral load was tested in 55 stone formers. Transcriptional activity induced by variant alleles at CaSR gene promoters was compared by luciferase reporter gene assay in HEK-293 and HKC-8 cells. CaSR and claudin-14 mRNA levels were measured by real-time PCR in 107 normal kidney medulla samples and compared in patients with different CaSR genotype. RESULTS Only rs6776158 (A>G), located in the promoter 1, was associated with nephrolithiasis. Its minor G allele was more frequent in stone formers than controls (37.8% vs 26.4%, P = .001). A reduced strontium excretion was observed in GG homozygous stone formers. Luciferase fluorescent activity was lower in cells transfected with the promoter 1 including G allele at rs6776158 than cells transfected with the A allele. CaSR mRNA levels were lower in kidney medulla samples from homozygous carriers for the G allele at rs6776158 than carriers for the A allele. Claudin-14 mRNA levels were also lower in GG homozygous subjects. CONCLUSIONS Minor allele at rs6776158 may predispose to calcium stones by decreasing transcriptional activity of the CaSR gene promoter 1 and CaSR expression in kidney tubules.