Kidney stones, hypercalciuria, and recent insights into proximal tubule calcium reabsorption.

PURPOSE OF REVIEW: Most kidney stones are composed of calcium, and the greatest risk factor for kidney stone formation is hypercalciuria. Patients who form kidney stones often have reduced calcium reabsorption from the proximal tubule, and increasing this reabsorption is a goal of some dietary and pharmacological treatment strategies to prevent kidney stone recurrence. However, until recently, little was known about the molecular mechanism that mediates calcium reabsorption from the proximal tubule. This review summarizes newly uncovered key insights and discusses how they may inform the treatment of kidney stone formers. RECENT FINDINGS: Studies examining claudin-2 and claudin-12 single and double knockout mice, combined with cell culture models, support complementary independent roles for these tight junction proteins in contributing paracellular calcium permeability to the proximal tubule. Moreover, a family with a coding variation in claudin-2 causing hypercalciuria and kidney stones have been reported, and reanalysis of Genome Wide Association Study (GWAS) data demonstrates an association between noncoding variations in CLDN2 and kidney stone formation. SUMMARY: The current work begins to delineate the molecular mechanisms whereby calcium is reabsorbed from the proximal tubule and suggests a role for altered claudin-2 mediated calcium reabsorption in the pathogenesis of hypercalciuria and kidney stone formation.

Tea intake and risk of kidney stones: A mendelian randomization study.

OBJECTIVES: Observational studies indicate that tea intake is associated with a decreased risk of kidney stones. Here we performed a mendelian randomization (MR) analysis to evaluate whether this association is causal. METHODS: Forty-four independent genetic variants strongly associated with tea intake were identified from a large genome-wide association study, including 448 060 individuals of the UK Biobank. We additionally obtained genome-wide association study summary statistics for kidney stones from the FinnGen consortium (5985 cases and 253 943 controls) and UK Biobank (6536 cases and 388 508 controls). Random-effect inverse variance weighted regression was used to evaluate causal estimates. The random-effect inverse variance weighted estimates based on the FinnGen consortium and UK Biobank were meta-analyzed using fixed-effects meta-analysis. Other MR methods, including MR-Egger, weighted median, weighted mode, and MR-Pleiotropy RESidual Sum and Outlier, were also performed to test the robustness of our results. RESULTS: In a combined sample of 12 521 cases and 642 451 controls, the inverse variance weighted analysis indicated that genetically predicted tea intake was causally associated with a decreased risk of kidney stones (odds ratio = 0.47; 95% CI, 0.34-0.66; P < 0.001). This association was consistent in other MR methods. CONCLUSIONS: This study suggests that tea intake may be causally associated with a decreased risk of kidney stones.

Coffee and Caffeine Consumption and Risk of Kidney Stones: A Mendelian Randomization Study.

RATIONALE & OBJECTIVE: Coffee and caffeine consumption have been associated with a lower risk of kidney stones in observational studies. We conducted a Mendelian randomization study to assess the causal nature of these associations. STUDY DESIGN: Mendelian randomization analysis. SETTING & PARTICIPANTS: Independent genetic variants associated with coffee and caffeine consumption at the genome-wide significance level were selected from previously published meta-analyses as instrumental variables. Summary-level data for kidney stones were obtained from the UK Biobank study (6,536 cases and 388,508 noncases) and the FinnGen consortium (3,856 cases and 172,757 noncases). EXPOSURE: Genetically predicted coffee and caffeine consumption. OUTCOME: Clinically diagnosed kidney stones. ANALYTICAL APPROACH: Mendelian randomization methods were used to calculate causal estimates. Estimates from the 2 sources were combined using the fixed-effects meta-analysis methods. RESULTS: Genetically predicted coffee and caffeine consumption was associated with a lower risk of kidney stones in the UK Biobank study, and the associations were directionally similar in the FinnGen consortium. The combined odds ratio of kidney stones was 0.60 (95% CI, 0.46-0.79; P < 0.001) per a genetically predicted 50% increase in coffee consumption and 0.81 (95% CI, 0.69-0.94; P = 0.005) per a genetically predicted 80-mg increase in caffeine consumption. LIMITATIONS: Genetic influence on kidney stone risk via pathways not involving coffee or caffeine. CONCLUSIONS: Using genetic data, this study provides evidence that higher coffee and caffeine consumption may cause a reduction in kidney stones.

Klotho gene polymorphism in renal stone formers from Northwestern India.

Klotho gene is an important gene involved in calcium homeostasis, and polymorphisms of this gene may render the individual prone to renal stone formation. We evaluated G395A single nucleotide polymorphisms (SNPs) of Klotho gene at rs1207568 in renal stone patients of North India. This was a prospective study involving 150 patients of renal stone disease (aged 15-60 years) and 100 age- and sex-matched controls. The DNA was isolated and subjected to polymerase chain reaction (PCR) for identifying the G395A Klotho SNPs at rs1207568. Confronting two pair primers were used, and gel electrophoresis showing two bands at 175,252 bp was considered as GG genotype, three bands at 121,175 and 252 bp as GA and two bands at 121 and 252 bp as AA genotype. The association between genotype and cases was evaluated by using Chi-square test and logistic regression analysis. Cases and controls were well matched for age (40.65 vs 42.06, p = 0.063) and sex (p = 0.420). Significantly high proportion of patients with renal stones had GG genotype as compared to controls (odds ratio (OR) 2.37(1.39,4.03), p = 0.001). None of the participants (cases and controls) had homozygous recessive AA genotype. The risk of stone formation was significantly higher in the population carrying G allele {OR 1.94 (1.225-3.073), p 0.004}. Mean serum calcium was higher in stone formers with GG genotype as compared to those with GA genotype (9.16 mg/dl vs 8.91 mg/dl; p = 0.06). GG genotype of G396A Klotho gene SNPs is associated with renal stone formation. The G allele carrier is twice at risk of renal stone formation. The absence of AA genotype in north-western Indian population remains a curiosity.

Genetic variants of calcium and vitamin D metabolism in kidney stone disease.

Kidney stone disease (nephrolithiasis) is a major clinical and economic health burden with a heritability of ~45-60%. We present genome-wide association studies in British and Japanese populations and a trans-ethnic meta-analysis that include 12,123 cases and 417,378 controls, and identify 20 nephrolithiasis-associated loci, seven of which are previously unreported. A CYP24A1 locus is predicted to affect vitamin D metabolism and five loci, DGKD, DGKH, WDR72, GPIC1, and BCR, are predicted to influence calcium-sensing receptor (CaSR) signaling. In a validation cohort of only nephrolithiasis patients, the CYP24A1-associated locus correlates with serum calcium concentration and a number of nephrolithiasis episodes while the DGKD-associated locus correlates with urinary calcium excretion. In vitro, DGKD knockdown impairs CaSR-signal transduction, an effect rectified with the calcimimetic cinacalcet. Our findings indicate that studies of genotype-guided precision-medicine approaches, including withholding vitamin D supplementation and targeting vitamin D activation or CaSR-signaling pathways in patients with recurrent kidney stones, are warranted.

[A young girl with recurrent calculosis and hypercalcemia].

Mutations of the CYP24A1 gene are associated with alterations in the activity of the enzyme 25-OH-D-24-hydroxylase, resulting in dysfunction of the metabolism of vitamin D. This enzymatic deficiency may cause hypercalcemia, low parathyroid hormone levels, hypercalciuria, nephrolithiasis and nephrocalcinosis. The clinical case of a young woman with recurrent renal lithiasis, hypercalcemia and hypercalciuria is described. These features are linked to deficiency of the enzyme 25-OH-D-24-hydroxylase, therefore to a biallelic mutation of the CYP24A1 gene.

Vitamin D and kidney stone disease.

PURPOSE OF REVIEW: Vitamin D is important in maintaining calcium homeostasis, but its role in kidney stone disease and its effect on stone formation are still not clear. RECENT FINDINGS: Kidney stone formers tend to experience enhanced intestinal calcium absorption, increased urinary calcium excretion, and excessive bone mineral loss. Although direct actions of active vitamin D have been implicated in all these processes, the effect of nutritional vitamin D (vitamin D2 or vitamin D3) use on calcium balance among stone formers is still not clear. In addition, the safety of nutritional vitamin D use in the stone forming population is also not established, considering the potential effect of its use on raising urinary calcium. However, most of the observational studies do not support a significant association between higher nutritional vitamin D store and increased risk of stone formation. Short-term nutritional vitamin D repletion in stone formers with vitamin D deficiency also does not appear to increase urinary calcium excretion. SUMMARY: The effect of nutritional vitamin D use in stone formers is still not clear. As vitamin D deficiency is highly prevalent among stone formers, future prospective studies are needed to establish the biological effect, as well as the safety and efficacy of nutritional vitamin D therapy in this unique patient population.

Effect of bolus and divided feeding on urine ions and supersaturation in genetic hypercalciuric stone-forming rats.

Because urine ion excretion varies throughout the day, clinicians monitor 24 h urine samples to measure ion excretion and supersaturation in kidney stone patients. However, these results are averages and may not reflect maximal supersaturation which drives stone formation. We measured ion excretion and saturation in genetic hypercalciuric stone-forming rats on both a normal or low calcium diet over 0-3, 3-6 and 6-24 h using two feeding protocols, where the daily food allotment was fed either as a bolus or divided into three portions. With a normal calcium diet, urine calcium, oxalate, volume, and calcium oxalate supersaturation were significantly greater on the bolus compared to the divided feeds in the prandial and postprandial periods. Bolus eaters also excreted more calcium and oxalate and had increased volume over 24 h. Maximal calcium oxalate supersaturation was greater during the initial time periods than during the entire 24 h, regardless of the feeding schedule. With the low calcium diet, the effect of bolus feeding was reduced. Thus, urine ion excretion and supersaturation vary with the type of feeding. If these results are confirmed in man, it suggests that eating as a bolus may result in greater prandial and postprandial calcium oxalate supersaturation. This may increase growth on Randall's plaques and promote stone disease.

Effect of cinacalcet on urine calcium excretion and supersaturation in genetic hypercalciuric stone-forming rats.

Idiopathic hypercalciuria is the most common metabolic abnormality in patients with nephrolithiasis. Through successive inbreeding, we have developed a strain of rats whose urine calcium (UCa) excretion is approximately 8-10-fold greater than that of control rats and who spontaneously form kidney stones. We have termed these rats genetic hypercalciuric stone-forming (GHS) rats. The physiology of the hypercalciuria in the GHS rats closely parallels that of man. We have recently shown that the GHS rat kidneys have an increased number of receptors for calcium (CaR) compared to Sprague-Dawley rats, the strain of rats originally bred to develop the GHS rats. Calcimimetics, such as cinacalcet (Cin), increase the sensitivity of the CaR to Ca. The effects of Cin on UCa are complex and difficult to predict. We tested the hypothesis that Cin would alter urinary (U) Ca and supersaturation with respect to calcium hydrogen phosphate (CaHPO(4)) and calcium oxalate (CaOx). GHS or control rats were fed a normal Ca diet (0.6% Ca) for 28 days with Cin (30 mg/kg/24 h) added to the diet of half of each group for the last 14 days. The protocol was then repeated while the rats were fed a low Ca (0.02% Ca) diet. We found that Cin led to a marked reduction in circulating parathyroid hormone and a modest reduction in serum Ca. Cin did not alter UCa when the GHS rats were fed the normal Ca diet but lowered UCa when they were fed the low Ca diet. However, Cin did not alter U supersaturation with respect to either CaOx or CaHPO(4) on either diet. If these findings in GHS rats can be confirmed in man, it suggests that Cin would not be an effective agent in the treatment of human idiopathic hypercalciuria and resultant stone formation.

A twin study of genetic and dietary influences on nephrolithiasis: a report from the Vietnam Era Twin (VET) Registry.

BACKGROUND: Nephrolithiasis is a complex phenotype that is influenced by both genetic and environmental factors. We conducted a large twin study to examine genetic and nongenetic factors associated with stones. METHODS: The VET Registry includes approximately 7500 male-male twin pairs born between 1939 to 1955 with both twins having served in the military from 1965 to 1975. In 1990, a mail and telephone health survey was sent to 11,959 VET Registry members; 8870 (74.2%) provided responses. The survey included a question asking if the individual had ever been told of having a kidney stone by a physician. Detailed dietary habits were elicited. In a classic twin study analysis, we compared concordance rates in monozygotic (MZ) and dizygotic (DZ) twins. We also conducted a cotwin control study of dietary risk factors in twins discordant for stones. RESULTS: Among dizygotic twins, there were 17 concordant pairs and 162 discordant pairs for kidney stones. Among monozygotic twins, there were 39 concordant pairs and 163 discordant pairs. The proband concordance rate in MZ twins (32.4%) was significantly greater than the rate in DZ twins (17.3%) (chi(2)= 12.8; P < 0.001), consistent with a genetic influence. The heritability of the risk for stones was 56%. In the multivariate analysis of twin pairs discordant for kidney stones, we found a protective dose-response pattern of coffee drinking (P= 0.03); those who drank 5 or more cups of coffee were half as likely to develop kidney stones as those who did not drink coffee (OR = 0.4, 95% CI 0.2, 0.9). Those who drank at least 1 cup of milk per day were half as likely to report kidney stones (OR = 0.5, 95% CI 0.3, 0.8). There were also marginally significant protective effects of increasing numbers of cups of tea per day and frequent consumption of fruits and vegetables. Other factors such as the use of calcium supplements, alcohol drinking, consumption of solid dairy products, and the amount of animal protein consumed were not significantly related to kidney stones in the multivariate model. CONCLUSION: These results confirm that nephrolithiasis is at least in part a heritable disease. Coffee, and perhaps tea, fruits, and vegetables were found to be protective for stone disease. This is the first twin study of kidney stones, and represents a new approach to elucidating the relative roles of genetic and environmental factors associated with stone formation.

Identification of a novel splice site mutation of CLCN5 gene and characterization of a new alternative 5' UTR end of ClC-5 mRNA in human renal tissue and leukocytes.

Mutations in the CLCN5 gene have been detected in Dent's disease and its phenotypic variants (X-linked recessive nephrolithiasis, X-linked recessive hypophosphatemic rickets, and idiopathic low-molecular-weight proteinuria of Japanese children). Dent's disease is a tubular disorder characterized by low-molecular-weight proteinuria, and nephrolithiasis associated with nephrocalcinosis and hypercalciuria. ClC-5 is the first chloride channel for which a definitive role in the trafficking and acidification-dependent recycling of apical membrane proteins has been established. In the course of CLCN5 SSCP analysis in patients with hypercalciuric nephrolithiasis, we detected a novel mutation at intron 2 of the CLCN5 gene, a T-to-G substitution, located 17 bp upstream of the AG acceptor site. To determine the effect of IVS2-17 T>G mutation on the correct splicing of intron 2, we studied ClC-5 transcripts in a patient's peripheral blood leukocytes by means of quantitative comparative RT/PCR, and found a new ClC-5 5' UTR isoform characterized by the untranslated exon 1b and by retention of intron 1b. This new isoform--isoform B1--was not correlated with mutation since it was detected also in control leukocytes and in renal tissues of kidney donors, thus confirming its physiological role. By RACE analysis we determined the putative transcriptional start site which is located at intron 1a, 251 nt upstream of the first nucleotide of the untranslated exon 1b. ORF analysis revealed that intron 1b retention in isoform B1 stabilizes the initiation of translation to the AGT at position 297 of the ClC-5 cDNA coding region.

Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation.

The human hereditary disorder Dent's disease is linked to loss-of-function mutations of the chloride channel ClC-5. Many of these mutations involve insertion of premature stop codons, resulting in truncation of the protein. We determined whether the functional activity of ClC-5 could be restored by coexpression of the truncated protein (containing the NH2-terminal region) with its complementary "missing" COOH-terminal region. Split channel constructs for ClC-5, consisting of complementary N and C protein regions, were created at an arbitrary site in the COOH-terminal region (V655) and at four Dent's disease mutation sites (R347, Y617, R648, and R704). Coexpression of complementary fragments for the split channel at V655 produced currents with anion and pH sensitivity similar to those of wild-type ClC-5. Channel activity was similarly restored when complementary split channel constructs made for Dent's mutation R648 were coexpressed, but no ClC-5 currents were found when split channels for mutations R347, Y617, or R704 were coexpressed. Immunoblot and immunofluorescence studies of COS-7 cells revealed that N or C protein fragments could be transiently expressed and detected in the plasma membrane, even in split channels that failed to show functional activity. The results suggest that ClC-5 channel activity can be restored for specific Dent's mutations by expression of the missing portion of the ClC-5 molecule.

Effects of allopurinol on renal stone formation and osteopontin expression in a rat urolithiasis model.

BACKGROUND: The inhibitory effect of allopurinol on calcium oxalate urolithiasis has been reported, but its effect on stone matrix proteins has not been studied in vivo. To clarify the effect of allopurinol on the matrix, we investigated its effect on the expression of osteopontin (OPN), which we previously identified as an important stone matrix protein. METHODS: Control rats were not treated. Rats of the stone group were given ethylene glycol (EG) and vitamin D(3), while the allopurinol groups (low-dose group and high-dose group) were treated with allopurinol in addition to receiving EG and vitamin D(3). RESULTS: The rate of renal stone formation was lower in the allopurinol groups than in the stone group. This was associated with a low expression of OPN mRNA in allopurinol-treated rats relative to that in the stone group. CONCLUSION: Allopurinol was effective in preventing calcium oxalate stone formation and reduced OPN expression in rats. Our results suggest that allopurinol prevents renal stone formation by acting against not only the control of oxalate but also OPN expression.

Alendronate decreases urine calcium and supersaturation in genetic hypercalciuric rats.

BACKGROUND: The mechanism of excess urine calcium excretion in human idiopathic hypercalciuria (IH) has not been determined but may be secondary to enhanced intestinal calcium absorption, decreased renal calcium reabsorption, and/or enhanced bone demineralization. We have developed a strain of genetic hypercalciuric stone-forming (GHS) rats as an animal model of human IH. When these GHS rats are placed on a low-calcium diet (LCD), urinary calcium (UCa) excretion exceeds dietary calcium intake, suggesting that bone may contribute to the excess UCa excretion. We used the GHS rats to test the hypothesis that bone contributes to the persistent IH when they are fed an LCD by determining if alendronate (Aln), which inhibits bone resorption, would decrease UCa excretion. METHODS: GHS rats (N = 16) and the parent strain (Ctl, N = 16) were fed 13 g/day of a normal (1.2%) calcium diet (NCD) for seven days and were then switched to a LCD (0. 02%) for seven days. Ctl and GHS rats in each group were then continued on LCD for an additional seven days, with or without injection of Aln (50 micrograms/kg/24 hrs). UCa excretion was measured daily during the last five days of each seven-day period. To determine the effects of Aln on urine supersaturation, the experiment was repeated. All relevant ions were measured, and supersaturation with respect to calcium oxalate and calcium hydrogen phosphate was determined at the end of each period. RESULTS: UCa was greater in GHS than in Ctl on NCD (7.4 +/- 0.5 mg/24 hrs vs. 1.2 +/- 0.1, GHS vs. Ctl, P < 0.01) and on LCD (3.9 +/- 0.2 mg/24 hrs vs. 0. 7 +/- 0.1, GHS vs. Ctl, P < 0.01). LCD provides 2.6 mg of calcium/24 hrs, indicating that GHS rats are excreting more calcium than they are consuming. On LCD, Aln caused a significant decrease in UCa in GHS rats and brought GHS UCa well below calcium intake. Aln caused a marked decrease in calcium oxalate and calcium hydrogen phosphate supersaturation. CONCLUSION: Thus, on a LCD, there is a significant contribution of bone calcium to the increased UCa in this model of IH. Aln is effective in decreasing both UCa and supersaturation. The Aln-induced decrease in urine supersaturation should be beneficial in preventing stone formation in humans, if these results, observed in a short-term study using the hypercalciuric stone-forming rat can be confirmed in longer term human studies.

Heritable syndrome of pseudoxanthoma elasticum with abnormal phosphorus and vitamin D metabolism.

A patient with pseudoxanthoma elasticum was documented to be hyperphosphatemic and mildly hypercalcemic for six years. Complications included metastatic calcification, absorptive hypercalciuria, and renal insufficiency. The 1,25-dihydroxyvitamin D value was elevated, despite normal serum parathyroid hormone values, high serum phosphate levels, and renal insufficiency. Either increased dietary calcium or prednisone seemed to suppress the 1,25-dihydroxyvitamin D value. Nephrolithiasis or abnormalities suggestive of pseudoxanthoma elasticum occurred in the patient's father, daughter, and several siblings, suggesting a distinct familial syndrome in which connective tissue changes are accompanied by abnormalities of phosphorus and vitamin D metabolism that may resemble those in the syndrome of familial tumoral calcinosis. Nine similar cases were described before 1970.