Understanding the gut-kidney axis in nephrolithiasis: an analysis of the gut microbiota composition and functionality of stone formers.

OBJECTIVES: The involvement of the gut microbiota in the pathogenesis of calcium nephrolithiasis has been hypothesised since the discovery of the oxalate-degrading activity of Oxalobacter formigenes, but never comprehensively studied with metagenomics. The aim of this case-control study was to compare the faecal microbiota composition and functionality between recurrent idiopathic calcium stone formers (SFs) and controls. DESIGN: Faecal samples were collected from 52 SFs and 48 controls (mean age 48±11). The microbiota composition was analysed through 16S rRNA microbial profiling approach. Ten samples (five SFs, five controls) were also analysed with deep shotgun metagenomics sequencing, with focus on oxalate-degrading microbial metabolic pathways. Dietary habits, assessed through a food-frequency questionnaire, and 24-hour urinary excretion of prolithogenic and antilithogenic factors, including calcium and oxalate, were compared between SFs and controls, and considered as covariates in the comparison of microbiota profiles. RESULTS: SFs exhibited lower faecal microbial diversity than controls (Chao1 index 1460±363vs 1658±297, fully adjusted p=0.02 with stepwise backward regression analysis). At multivariate analyses, three taxa (Faecalibacterium, Enterobacter, Dorea) were significantly less represented in faecal samples of SFs. The Oxalobacter abundance was not different between groups. Faecal samples from SFs exhibited a significantly lower bacterial representation of genes involved in oxalate degradation, with inverse correlation with 24-hour oxalate excretion (r=-0.87, p=0.002). The oxalate-degrading genes were represented in several bacterial species, whose cumulative abundance was inversely correlated with oxaluria (r=-0.85, p=0.02). CONCLUSIONS: Idiopathic calcium SFs exhibited altered gut microbiota composition and functionality that could contribute to nephrolithiasis physiopathology.

Copy-number disorders are a common cause of congenital kidney malformations.

We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

Twenty-five years of idiopathic calcium nephrolithiasis: has anything changed?

Idiopathic calcium nephrolithiasis (ICN) is a disease whose prevalence is rising. Our aim was to assess whether lifestyle indicators and habits of calcium stone formers in Italy have changed over the last 25 years, trying to establish a connection with the diffusion of Internet access. Therefore we examined the database of the Stone Clinic of Parma University Hospital and extracted 1952 (1192 M, 760 F) patients with ICN who underwent a full clinical and laboratory evaluation from 1986 to 2010. Laboratory evaluation included data on urinary 24-h volume, pH, sodium, potassium, chloride, calcium, phosphate, uric acid, magnesium, oxalate, and citrate. Patients were split in three groups on a chronological basis, according to official EUROSTAT-ISTAT data of Internet connection among families in Italy: Group 1, pre-Internet era (1986-1998, 853 patients); Group 2, narrow-band era (1999-2004, 467 patients); Group 3, broad-band era (2005-2010, 632 patients). Over the time we found a significant increase in water intake (1.37 vs. 1.78 L in men and 1.21 vs. 1.55 L in women, Group 1 vs. Group 3, p-trend<0.001) and a decrease in urinary sodium and chloride for both genders and calcium and magnesium only for males, while females experienced a slight increase in oxalate excretion. Supersaturation indexes for calcium and uric acid stones dramatically fell for both genders. The percentage of stone formers performing physical activity significantly rise (41% Group 3 vs. 8% Group 1, p<0.001) and we also found a trend of reduction in mean blood pressure. Therefore, the lifestyle of Italian idiopathic calcium stone formers has changed over the last 25 years, and the rising Internet access may have played a great role in driving this change.

Hippuric acid in 24 h urine collections as a biomarker of fruits and vegetables intake in kidney stone formers.

This work aimed to underline the prospects of hippuric acid, a product of the metabolism of polyphenols, as a new biomarker of fruits and vegetables intake associated with lithogenic risk. Biochemical parameters of lithogenic risk and hippuric acid were measured in the 24 h urine collections of a cohort of 696 Italian kidney stone formers divided into two subgroups according to their different dietary habits. The link between lithogenic risk parameters and hippuric acid was assessed and this compound was revealed as a valuable biomarker of fruits and vegetables intake in kidney stone formers. A cut-off value of urinary excretion of hippuric acid, 300 mg/24 h, was set as the threshold of discrimination between low and high intake of fruits and vegetables for these patients. These results highlight the importance of monitoring of the excretion hippuric acid in urine to address proper dietary guidelines for the management of stone former patients.

Influence of lean and fat mass on bone mineral density and on urinary stone risk factors in healthy women.

BACKGROUND: The role of body composition (lean mass and fat mass) on urine chemistries and bone quality is still debated. Our aim was therefore to determine the effect of lean mass and fat mass on urine composition and bone mineral density (BMD) in a cohort of healthy females. MATERIALS AND METHODS: 78 female volunteers (mean age 46 ± 6 years) were enrolled at the Stone Clinic of Parma University Hospital and subdued to 24-hour urine collection for lithogenic risk profile, DEXA, and 3-day dietary diary. We defined two mathematical indexes derived from body composition measurement (index of lean mass-ILM, and index of fat mass-IFM) and the cohort was split using the median value of each index, obtaining groups differing only for lean or fat mass. We then analyzed differences in urine composition, dietary intakes and BMD. RESULTS: The women with high values of ILM had significantly higher excretion of creatinine (991 ± 194 vs 1138 ± 191 mg/day, p = 0.001), potassium (47 ± 13 vs 60 ± 18 mEq/day, p < 0.001), phosphorus (520 ± 174 vs 665 ± 186 mg/day, p < 0.001), magnesium (66 ± 20 vs 85 ± 26 mg/day, p < 0.001), citrate (620 ± 178 vs 807 ± 323 mg/day, p = 0.002) and oxalate (21 ± 7 vs 27 ± 11 mg/day, p = 0.015) and a significantly better BMD values in limbs than other women with low values of ILM. The women with high values of IFM had similar urine composition to other women with low values of IFM, but significantly better BMD in axial sites. No differences in dietary habits were found in both analyses. CONCLUSIONS: Lean mass seems to significantly influence urine composition both in terms of lithogenesis promoters and inhibitors, while fat mass does not. Lean mass influences bone quality only in limb skeleton, while fat mass influences bone quality only in axial sites.

Dietary habits in women with recurrent idiopathic calcium nephrolithiasis.

BACKGROUND: Nutrition has been widely recognized to influence the risk of kidney stone formation. Therefore the aim of our study was to assess: a) whether usual diet of women with idiopathic calcium nephrolithiasis (ICN) living in Parma (Northern-Italy) is different compared to healthy controls, b) how their diet differs from Italian National guidelines and c) whether it is related to nephrolithiasis clinical course. METHODS: 143 women with recurrent ICN (mean age 43 ± 13 ys) and 170 healthy women (mean age 42 ± 11 ys) were enrolled. All women completed a food frequency questionnaire for the last 60-days and a 3-day dietary diary analysed with a dedicated software. RESULTS: Stone formers showed a higher consumption of sausages, ham, meat and sweets than healthy controls (43.1% vs 11.1%, 29.4% vs 13.9%, 21.6% vs 4.2%, 66.7% vs 18.1%, p < 0.001). The 3-day diary analysis showed an intake of calories, carbohydrates, lipids and non-discretionary sodium about 10% higher than healthy controls (p < 0.001). Finally, after dividing the population into 3 age groups (≤30, 31-40, > 40 years), the differences described above were amplified in the class ≤30 years, where nephrolithiasis presented a more serious course (shorter recurrence interval, greater stone-rate). In this age group the intake of fruit and vegetables was notably lower than guideline recommendations. CONCLUSIONS: We conclude that the usual diet of women with recurrent ICN is different from controls and characterized by low intake of fruits and vegetables and higher consumption of simple sugars and foods with high protein and salt content. This dietary imbalance could play a role in the ICN pathogenesis, especially in younger women.

Idiopathic calcium nephrolithiasis with pure calcium oxalate composition: clinical correlates of the calcium oxalate dihydrate/monohydrate (COD/COM) stone ratio.

Pure calcium oxalate is the most frequent type of idiopathic kidney stone composition. Fourier transform infrared spectroscopy (FT-IR) allows to detect the ratio of calcium oxalate dihydrate (COD) and monohydrate (COM) crystals in stones, but the clinical significance of this parameter remains uncertain. The objective of this observational study was to verify the association of clinical and laboratory parameters of kidney stone disease with COD/COM ratio in a group of 465 (322 M, age 46 ± 14) patients suffering from idiopathic calcium nephrolithiasis with pure calcium oxalate stones (≥ 97%). Each participant underwent a complete clinical examination, serum chemistry, 24-h urine collection for the determination of the profile of lithogenic risk, and had stones analyzed by FT-IR. Most (62%) of the stones had a COD/COM ratio ≤ 0.25, and the urine chemistry of the corresponding patients showed a low prevalence of urinary metabolic abnormalities. With increasing COD/COM ratio intervals (0-0.25, 0.26-0.50, 0.51-0.75, 0.76-1), a significant association was observed for the number of urological procedures, serum calcium, 24-h urinary calcium excretion, prevalence of hypercalciuria and relative calcium oxalate supersaturation, and a negative trend was detected for the age of the first stone episode (all p values < 0.05). A linear regression model showed that the only parameters significantly associated with COD/COM ratio were 24-h urinary calcium excretion (standardized ß = 0.464, p < 0.001) and urine pH (standardized ß = 0.103, p = 0.013). In pure calcium oxalate idiopathic stones, COD/COM ratio may reflect the presence of urinary metabolic risk factors, and represent a guide for the prescription of urinary analyses.

Insights about urinary hippuric and citric acid as biomarkers of fruit and vegetable intake in patients with kidney stones: The role of age and sex.

OBJECTIVES: Urinary hippuric acid (HA) and citrate can represent useful biomarkers of fruit and vegetable (FAV) intake in nephrolithiasis. However, their clinical significance across the life span has been poorly investigated. The aim of this study was to investigate the association between the two biomarkers with FAV intake across different age groups and sexes in a large group of stone formers (SFs). METHODS: SFs undergoing baseline 24-h urinary collection for metabolic profile of lithogenic risk at our institution were consecutively enrolled for a 6-y time span (N = 1185; 625 men). HA and citrate excretions were determined by ion chromatography and ultraviolet method, respectively. SFs completed a food frequency questionnaire on the intake of FAV. Stepwise logistic regression was applied to investigate factors associated with very low FAV (≤1 servings/d) and analysis of covariance to compare citrate and HA excretion across age groups and sexes. RESULTS: Very low FAV intake prevalence declined with age (Ptrend < 0.001), and was inversely associated with HA and citrate excretion (P < 0.001) in a stepwise logistic regression model. A significant increasing trend was verified for both biomarkers across age groups until the age of 65 for HA (P < 0.001) and 55 for citrate (P < 0.001). Citrate excretion significantly declined after the age of 65, and was higher in women than men in adult age groups, regardless of FAV intake. CONCLUSIONS: Both urinary citrate and HA were positively associated with FAV intake in SFs. However, unlike HA, citrate excretion was significantly influenced by the female sex and by older age.

Dietary treatment of nephrolithiasis.

The prevalence of idiopathic nephrolithiasis is increasing in rich countries. Dietary manipulation could contribute to the prevention of both its first appearance and the recurrence of the disease. The target of dietary treatment is to decrease the "urinary lithogenic risk factors" such as low urine volume, hypercalciuria, hyperoxaluria, hyperuricosuria, hyperphosphaturia, hypocitraturia, hypomagnesuria and excessively alkaline or acid urinary pH. Due to the lack of randomized controlled trials focused on this problem, there is not ample evidence to confidently recommend dietary changes. Despite this, numerous recent and past experiences support modification of diet as having a primary role in the prevention of nephrolithiasis. In particular, it is recommended to limit animal protein and salt intake, to consume milk and derivatives in amounts corresponding to calcium intake of about 1200 mg/day and to assume fiber (40 g/day), vegetables and fruit daily avoiding foods with high oxalate content. Furthermore, vitamin C intake not exceeding 1500 mg/day plays a protective role as well as avoiding vitamin B6 deficiency and abstaining, if possible, from vitamin D supplements. Lastly, it is recommended to drink enough water to bring the urinary volume up to at least 2 L/day and, as much as possible, to use fresh or frozen products rather than prepacked or precooked foods which are often too rich in sodium chloride.

Determinants of calcium and oxalate excretion in subjects with calcium nephrolithiasis: the role of metabolic syndrome traits.

BACKGROUND: The association of metabolic syndrome (MetS) traits with urinary calcium (UCE) or oxalate excretion (UOE) is uncertain in calcium stone formers (CSFs). Our aim was to investigate this association in a large group of Caucasian CSFs. METHODS: We retrospectively reviewed data of CSFs evaluated at our Kidney Stone Clinic from 1984 to 2015. Data on body mass index (BMI), MetS traits defined according to international consensus, family history of urolithiasis, anti-hypertensive treatments, calcemia, renal function, and 24-h urinary profile of lithogenic risk were collected. The association between MetS traits and UCE or UOE was tested with multivariate linear regression models accounting for a long list of potential confounders. RESULTS: We included 3003 CSFs, aged 44 ± 14 years. The prevalence of hypertension, diabetes, overweight (BMI ≥ 25 kg/m2) and dyslipidemia was 17, 2, 42 and 38%, respectively. Median values of UCE and UOE were 211 mg/24 h (IQR 143-296) and 28 mg/24 h (IQR 22-34), respectively. At a multivariate model, including age, sex, date of examination, drug treatments, family history, renal function, blood calcium and urinary factors as covariates, hypertension was a significant positive determinant of UCE (ß ± SE 0.23 ± 0.07, p = 0.003), but overweight, dyslipidemia and diabetes were not. No MetS trait was significantly associated with UOE in multivariate models. CONCLUSIONS: In a large group of Caucasian CSFs, hypertension was the only MetS trait significantly associated with UCE, while no MetS trait was associated with oxalate excretion.

Comparison of two diets for the prevention of recurrent stones in idiopathic hypercalciuria.

BACKGROUND: A low-calcium diet is recommended to prevent recurrent stones in patients with idiopathic hypercalciuria, yet long-term data on the efficacy of a low-calcium diet are lacking. Recently, the efficacy of a low-calcium diet has been questioned, and greater emphasis has been placed on reducing the intake of animal protein and salt, but again, long-term data are unavailable. METHODS: We conducted a five-year randomized trial comparing the effect of two diets in 120 men with recurrent calcium oxalate stones and hypercalciuria. Sixty men were assigned to a diet containing a normal amount of calcium (30 mmol per day) but reduced amounts of animal protein (52 g per day) and salt (50 mmol of sodium chloride per day); the other 60 men were assigned to the traditional low-calcium diet, which contained 10 mmol of calcium per day. RESULTS: At five years, 12 of the 60 men on the normal-calcium, low-animal-protein, low-salt diet and 23 of the 60 men on the low-calcium diet had had relapses. The unadjusted relative risk of a recurrence for the group on the first diet, as compared with the group on the second diet, was 0.49 (95 percent confidence interval, 0.24 to 0.98; P=0.04). During follow-up, urinary calcium levels dropped significantly in both groups by approximately 170 mg per day (4.2 mmol per day). However, urinary oxalate excretion increased in the men on the low-calcium diet (by an average of 5.4 mg per day [60 micromol per day]) but decreased in those on the normal-calcium, low-animal-protein, low-salt diet (by an average of 7.2 mg per day [80 micromol per day]). CONCLUSIONS: In men with recurrent calcium oxalate stones and hypercalciuria, restricted intake of animal protein and salt, combined with a normal calcium intake, provides greater protection than the traditional low-calcium diet.

Calcium urolithiasis course in young stone formers is influenced by the strength of family history: results from a retrospective study.

The role of the strength of family history of stones (FHS), i.e., degree of relatives with the disease, on the course of calcium urolithiasis (CU) is not fully understood, particularly in young patients where genetic background has the greatest influence on disease expression. Thus, with a retrospective cross-sectional design, we examined baseline clinical parameters and urinary chemistries of 369 subjects (196 M) with CU and 96 controls (41 M) aged between 15 and 25 at the time of the first visit at our stone clinic. Subjects with metabolic syndrome traits, known causes of CU or CU onset before the age of 15 were excluded. Clinical and metabolic parameters were compared among stone formers (SF) and controls, stratified by gender, the presence and type of FHS determined through the kinship coefficient of relatives with stones. No significant differences in clinical course were found between SF with and without FHS, except for the presence of bilateral stones (OR 2.01, 95% CI 1.20-3.39, p < 0.01). A significant age-, sex- and disease duration-adjusted trend for a higher number of colics (p for trend = 0.001), number of stones (p for trend = 0.002), stone rate (p for trend = 0.003) and the presence of retained stones (OR 1.60, 95% CI 1.14-2.21, p = 0.006) was detected with increasing FHS strength. Urinary chemistries were unaffected by FHS in both SF and controls, except for a higher calcium excretion in females with FHS (p < 0.05). The type of FHS, thus, significantly influences the clinical course of CU in young SF, mainly irrespective of urinary factors.

The influence of maternal and paternal history on stone composition and clinical course of calcium nephrolithiasis in subjects aged between 15 and 25.

Our aim was to compare the influence of maternal history of stones (MHS) and paternal history of stones (PHS) on composition of calculi and disease course in a group of patients with calcium nephrolithiasis (CN) aged between 15 and 25, the age range with the maximal influence of family history on disease expression. One-hundred thirty-five patients (68 F) with CN and one stone-forming parent were retrospectively selected from the database of our outpatient stone clinic, and categorized according to MHS or PHS. Data about stone disease course and composition of passed calculi, determined by chemical analysis or Fourier-transformed infrared spectrophotometry, were collected together with information on blood chemistry and 24-h urinary profile of lithogenic risk. The characteristics of disease course and stone composition were compared using logistic regression tests adjusted for age, sex, and BMI or analysis of covariance where appropriate. Patients with MHS (n = 46) had significantly higher urinary calcium/creatinine ratio and ammonium, a higher prevalence of urological treatments (57 vs 27 %, p < 0.001) and mixed calcium oxalate/calcium phosphate stone composition (69 vs 35 %, p = 0.002) than those with PHS. At multivariate logistic regression models, MHS was independently associated with urological treatments (OR 4.5, 95 %CI 1.9-10.7, p < 0.001) and the formation of calculi with mixed calcium oxalate/calcium phosphate composition (OR 5.8, 95 %CI 1.9-17.9, p = 0.002). The method of stone analysis did not affect this result. In conclusion, in subjects aged 15-25, MHS is associated with mixed calcium stones and with a higher risk for urological procedures, and should be, therefore, considered in the management of urolithiasis.

Family history influences clinical course of idiopathic calcium nephrolithiasis: case-control study of a large cohort of Italian patients.

AIM: To evaluate the contribution of family history of stones (FHS), up to second-degree relatives, on clinical course (age of first renal colic, bilateral disease, retained calculi, recurrences, urological procedures) of idiopathic calcium nephrolithiasis (ICN) and urinary parameters of lithogenic risk, a case-control study was carried out. METHOD: Clinical records of 2080 patients with ICN were evaluated and categorized according to FHS. Data about clinical course of disease and urinary parameters of lithogenic risk were collected. Student's t, Chi square tests and binary logistic regression were applied to discriminate groups with vs. without FHS. RESULTS: FHS was associated with an earlier onset of stone disease (age at first episode 32 ± 13 vs. 37 ± 15 years, p <0.001), particularly in females. After adjustment for multiple covariates, FHS was significantly associated to a higher risk of recurrence [odds ratio (OR) 1.2, 95 % confidence interval (1.1-1.4), p = 0.04], retained stones [OR 1.3, CI (1.1-1.5), p = 0.004], bilateral stones [OR 1.2, 95 % CI (1.1-1.5), p = 0.022] and urological procedures [OR 1.2, 95 % CI (1.1-1.5), p = 0.034]. FHS did not influence urinary parameters of lithogenic risk, except for calcium excretion in females. CONCLUSION: A positive family history influences the clinical course of ICN with some significant gender-related differences, substantially irrespective of urinary parameters of lithogenic risk.

Idiopathic Calcium Nephrolithiasis and Hypovitaminosis D: A Case-control Study.

OBJECTIVE: To investigate the association between vitamin D deficiency (25-hydroxyvitamin D <20 ng/mL) and idiopathic calcium nephrolithiasis (ICN). METHODS: A total of 884 patients with ICN (363 males, mean age of 51 ± 14) and 967 controls (162 males, mean age of 59 ± 15) from an area with no food fortification policy were considered following a case-control study design. Patients were enrolled at a third-level outpatient stone clinic. Controls were selected from a laboratory database after exclusion of those with nephrolithiasis, bone, endocrine, liver, and kidney diseases. Serum 25-hydroxyvitamin-D (25-OH-D), date of test, presence/history of diabetes, and cardiovascular disease including hypertension were recorded for all subjects. Serum parathormone, calcium, phosphorus, and urinary factors of lithogenic risk were available in stone formers (SF). After univariate statistical analysis, propensity score matching with conditional logistic regression was used to control for the possible effects of covariates. RESULTS: The prevalence of 25-OH-D <20 ng/mL was 56% in SF and 44% in controls (P <.001), with median levels of 18 ng/mL [interquartile range (IQR) of 12-24)] versus 23 ng/mL (IQR of 14-30) (age and sex adjusted P <.001). After a fully adjusted conditional logistic regression analysis, performed on propensity-matched cohorts (442 SF, 442 controls), there was a statistically significant association between vitamin D deficiency and odds of nephrolithiasis (estimated odds ratio of 2.29, confidence interval 95% 1.74-3.02, P <.001). 25-OH-D levels were not different in hypercalciuric and normocalciuric SF (median and IQR of 18 ng/mL and 13-23 vs 19 ng/mL and 13-26, respectively, P = .2). CONCLUSIONS: SF have lower serum 25-OH-D levels than controls. The role of hypovitaminosis D in the onset of ICN should be better reconsidered.

Medical treatment of nephrolithiasis.

The medical treatment of nephrolithiasis is aimed in particular at the prevention of relapses, even though in some cases, such as cystine or uric acid lithiasis, the calculi also can be dissolved on site. When the diagnosis and metabolic profile have been performed correctly, medical treatment is effective in a large number of patients. The greatest difficulty is the patient's compliance with the most suitable prevention measures and the frequency of follow-up controls. This compliance can be influenced significantly by the amount of time that the doctor spends to explain the origin of the disease. This article reviews the main methods available for the medical treatment of various nephrolithiasis types, namely water intake, diet, and drugs, supplying the relevant information about the mechanism of action, metabolic consequences, indications, evidence provided from studies, dosage, efficacy, and side-effects. Finally, brief simplified guidelines are given for the medical treatment of stone disease caused by calcium oxalate or calcium phosphate, uric acid, cystine, and struvite.

Crystalline aggregation in vitro: interaction between urinary macromolecules and the micromolecular environment.

The formation of calcium oxalate (CaOx) crystals, their growth and above all their agglomeration tendency are considered to be decisive factors in the renal stone formation. The aim of this study was to verify whether the formation in vitro of crystalline macroaggregates, induced by increasing loads of oxalate, is different in normal subjects as opposed to CaOx stone formers, free from urinary metabolic abnormalities. The possible interference of the urinary micromolecular environment on the activities of macromolecules with molecular weight greater than 10,000 Daltons was also evaluated. Formation of crystalline macroaggregates took place in controls at a relative CaOx supersaturation equivalent to 32.6 +/- 12.9 and in stone formers at 34 +/- 12.3 (NS); the CaOx RS aggregation--CaOx RS nucleation delta was 11.4 +/- 12.2 in controls and 15.8 +/- 13.9 in stone formers (NS). Leaving aside the stone disease variable, the urine characterized by high baseline CaOx saturation, high ionic strength and high levels of citrate, magnesium and calcium, seemed particularly predisposed to macroaggregates formation; the opposite was true for urine characterized by a totally different micromolecular environment. A very close relationship was found between the CaOx RS aggregation--CaOx RS nucleation delta in urine filtered at 0.22 mu and in metastable solutions containing macromolecules in similar concentrations to those found in the native urine (r = 0.74, P < 0.0001). In conclusion the formation of crystalline macroaggregates in vitro does not present differences in normal subjects compared to CaOx stone formers free from urinary metabolic abnormalities. The urinary macromolecules seem to participate in the formation of the crystalline macroaggregates and their action seems to be affected by the urinary micromolecular environment and, even when they are re-suspended in a completely different medium, their action seems to be preserved intact.

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.

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.

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.