Associations between renal sodium‐citrate cotransporter (hNaDC‐1) gene polymorphism and urinary citrate excretion in recurrent renal calcium stone formers and normal controls

Abstract

Purpose: Urinary citrate is a potent inhibitor of renal stone formation. Its excretion is regulated by Na + /dicarboxylate cotransporter-1 (NaDC-1), which is expressed on the apical membrane of renal proximal tubules. Many patients with calcium urolithiasis exhibit hypocitraturia, however, the mechanisms are not perfectly understood. We examined whether or not the I550V polymorphism in human NaDC-1 gene (hNaDC-1) influenced urinary citrate excretion. Materials and methods: I550V polymorphism was investigated in 105 patients with recurrent renal calcium stone formation (RSF) and 107 age-matched healthy volunteers with non-renal stone formation (NSF), using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis and two 24-h urine samples. Results: Overall and in the RSF groups, subjects with a BB (homozygous for the digested Bcl-I allele) genotype exhibited a significantly lower urinary citrate excretion level than subjects with a bb (homozygous for the undigested allele) genotype. Genotype distributions between subjects with hypocitraturia and normocitraturia were significantly different, with the BB genotype being more frequently observed in subjects with hypocitraturia -both overall and in each of the RSF and NSF groups. Although the BB genotype was observed more frequently in the RSF group than in the NSF group, no statistical differences among the distributions of the three genotypes (BB, Bb [heterozygous] and bb) were observed between the RSF and NSF groups. Conclusion: These results suggest that the B allele of I550V polymorphism of hNaDC-1 may be associated with a reduction in urinary citrate excretion and contribute to hypocitraturia in recurrent renal stone formers.

The<i> I</i>550<i>V</i> polymorphism in the renal human sodium/dicarboxylate cotransporter 1 (<i>hNaDC</i>-1) gene is associated with the risk for urolithiasis in adults from Southeastern, Mexico

“…This SNP has been associated with the risk of hypocitraturia in recurrent renal stone formers [13]. However, those results have not yet been reproduced in any other population.…”

“…Implication of the SNP I550V-hNaDC-1 in the mechanism of stone formation might be due to the isoleucine-to-valine alteration at position 550 in hNaDC-1 is located in the last trans membrane domain and the C-terminal half, which contains amino acid residues that determine substrate recognition and cation affinity; therefore, altering the function for transferring citrate. It is also possible that the rate of transcription or stability, of hNaDC-1 is modified by the SNP [13]. It is worth to note that the intestinal absorption of citrate, is the main source of plasma citrate [18].…”

“…However, those results have not yet been reproduced in any other population. Also, such study found no direct association between the I550V SNP and UL [13], nor the influence of the SNP on either the rate of gene expression or the NaDC-1 transport activity have been proven through in vitro studies [12]. Based on the above and in order to provide evidence to help determine the role performed by this SNP in the genetic risk of UL, this study focuses on evaluating the association of I550V SNP in hNaDC-1 gene with the genetic risk for develop hypocitraturia and/or urolithiasis in a population of adult subjects from Southeastern Mexico, where UL is highly prevalent and considered endemic.…”

“…The prepared samples were placed in an Applied Biosystems Veriti thermocycler with the following PCR conditions: initial denaturation for 1 min at 94˚C, 30 cycles of denaturation for 1 min at 94˚C, annealing for 1 min at 59˚C and extension for 1 min at 72˚C and finally 10 min final extension at 72˚C, followed by cooling for 5 min at 4˚C. The amplified DNA was incubated at 50˚C overnight in the presence of the restriction endonuclease Bcl I (Bacillus caldolyticus I) [13]. Each restriction reaction contained 10 μl of amplified DNA, 7.62 μl of sterile H 2 O, 2 μl of buffer C, 0.2 BSA and 1.8u of Bcl I restriction enzyme Promega Cat # R4654).…”

“…When cut, the 520 bp fragment produces two fragments, one with 354 bp and one with 166 bp. After incubation, the samples were again subject to polyacrylamide gel electrophoresis, from which genotypes were identified, according to Okamoto et al [13].…”