Molecular Basis, Diagnostic Challenges and Therapeutic Approaches of Bartter and Gitelman Syndromes: A Primer for Clinicians.

Gitelman and Bartter syndromes are rare inherited diseases that belong to the category of renal tubulopathies. The genes associated with these pathologies encode electrolyte transport proteins located in the nephron, particularly in the Distal Convoluted Tubule and Ascending Loop of Henle. Therefore, both syndromes are characterized by alterations in the secretion and reabsorption processes that occur in these regions. Patients suffer from deficiencies in the concentration of electrolytes in the blood and urine, which leads to different systemic consequences related to these salt-wasting processes. The main clinical features of both syndromes are hypokalemia, hypochloremia, metabolic alkalosis, hyperreninemia and hyperaldosteronism. Despite having a different molecular etiology, Gitelman and Bartter syndromes share a relevant number of clinical symptoms, and they have similar therapeutic approaches. The main basis of their treatment consists of electrolytes supplements accompanied by dietary changes. Specifically for Bartter syndrome, the use of non-steroidal anti-inflammatory drugs is also strongly supported. This review aims to address the latest diagnostic challenges and therapeutic approaches, as well as relevant recent research on the biology of the proteins involved in disease. Finally, we highlight several objectives to continue advancing in the characterization of both etiologies.

Effects of bodybuilding supplements on the kidney: A population-based incidence study of biopsy pathology and clinical characteristics among middle eastern men.

BACKGROUND: The incidence of kidney diseases among bodybuilders is unknown. METHODS: Between January 2011 and December 2019, the Iraqi Kurdistan 15 to 39 year old male population averaged 1,100,000 with approximately 56,000 total participants and 25,000 regular participants (those training more than 1 year). Annual age specific incidence rates (ASIR) with (95% confidence intervals) per 100,000 bodybuilders were compared with the general age-matched male population. RESULTS: Fifteen male participants had kidney biopsies. Among regular participants, diagnoses were: focal segmental glomerulosclerosis (FSGS), 2; membranous glomerulonephritis (MGN), 2; post-infectious glomeruonephritis (PIGN), 1; tubulointerstitial nephritis (TIN), 1; and nephrocalcinosis, 2. Acute tubular necrosis (ATN) was diagnosed in 5 regular participants and 2 participants training less than 1 year. Among regular participants, anabolic steroid use was self-reported in 26% and veterinary grade vitamin D injections in 2.6%. ASIR for FSGS, MGN, PIGN, and TIN among regular participants was not statistically different than the general population. ASIR of FSGS adjusted for anabolic steroid use was 3.4 (- 1.3 to 8.1), a rate overlapping with FSGS in the general population at 2.0 (1.2 to 2.8). ATN presented as exertional muscle injury with myoglobinuria among new participants. Nevertheless, ASIR for ATN among total participants at 1.4 (0.4 to 2.4) was not significantly different than for the general population at 0.3 (0.1 to 0.5). Nephrocalcinosis was only diagnosed among bodybuilders at a 9-year cumulative rate of one per 314 vitamin D injectors. CONCLUSIONS: Kidney disease rates among bodybuilders were not significantly different than for the general population, except for nephrocalcinosis that was caused by injections of veterinary grade vitamin D compounds.

Oral administration of oxalate-enriched spinach extract as an improved methodology for the induction of dietary hyperoxaluric nephrocalcinosis in experimental rats.

Experimental induction of hyperoxaluria by ethylene glycol (EG) administration is disapproved as it causes metabolic acidosis while the oral administration of chemically synthesized potassium oxalate (KOx) diet does not mimic our natural system. Since existing models comprise limitations, this study is aimed to develop an improved model for the induction of dietary hyperoxaluria, and nephrocalcinosis in experimental rats by administration of naturally available oxalate rich diet. Male albino Wistar rats were divided into five groups. Group I, control; group II rats received 0.75% EG, group III rats fed with 5% KOx diet and group IV and V rats were administered with spinach extract of 250 and 500 mg soluble oxalate/day respectively, for 28 d. Urine and serum biochemistry were analyzed. After the experimental period, rats were sacrificed, liver and kidney tissue homogenates were used for antioxidant and lipid peroxidation assay. Relative change in expression of kidney injury molecule-1 (KIM-1) and crystal modulators genes in kidney tissues were evaluated. Tissue damage was assessed by histology studies of liver and kidney. Experimental group rats developed hyperoxaluria and crystalluria. Urine parameters, serum biochemistry, antioxidant profile, lipid peroxidation levels and gene expression analysis of experimental group II and III rats reflected acute kidney damage compared to group V rats. Histopathology results showed moderate hyperplasia in liver and severe interstitial inflammation in kidneys of group II and III than group V rats. Ingestion of naturally available oxalate enriched spinach extract successfully induced dietary hyperoxaluria and nephrocalcinosis in rats with minimal kidney damage.

The effect of cinacalcet on bone remodeling and renal function in transplant patients with persistent hyperparathyroidism.

BACKGROUND: Parathyroidectomy is associated with renal functional losses in transplant patients; cinacalcet offers an attractive alternative. METHODS: We performed a prospective observational study in 58 patients with persisting hyperparathyroidism after renal transplantation (Ca≥2.6 mmol/L) and impaired renal transplant function (estimated glomerular filtration rate [eGFR] <50 mL/min). The patients received 30 to 90 mg cinacalcet for 12 months with the target to normalize serum Ca. We measured parathyroid hormone (PTH), serum Ca, serum phosphorus, alkaline phosphatase, bone-specific alkaline phosphatase, osteocalcin, and telopeptide at 0, 1, 2, 3, 6, 9, and 12 months of cinacalcet treatment. Fractional excretion of calcium and phosphorus (n=24) were monitored at 0 and 1 month. RESULTS: At inclusion, creatinine was 181±70 µmol/L, eGFR 43±19 mL/min, PTH 371±279 pg/mL, and Ca 2.73±0.22 mmol/L. We observed nephrocalcinosis in 58% of biopsied patients at enrollment. After cinacalcet, Ca decreased significantly and normalized at nearly any measurement. Phosphorus increased significantly at months 1, 9, and 12. PTH decreased significantly, but only at months 9 and 12 and did not normalize. Bone-specific alkaline phosphatase increased significantly (>normal) by month 12. eGFR decreased and serum creatinine increased at all time points. The Δ(creatinine) % increase correlated significantly with the Δ(PTH) % decrease at month 1 and 12. Telopeptide and alkaline phosphatase correlated with PTH and telopeptide also correlated with serum creatinine. CONCLUSION: Calcium-phosphorus homeostasis in hypercalcemic renal transplant patients normalizes under cinacalcet and PTH decreases, albeit not to normal. The renal functional decline could be PTH mediated, analogous to the effects observed after parathyroidectomy.

Consumption of krill protein concentrate prevents early renal injury and nephrocalcinosis in female Sprague-Dawley rats.

Female Sprague-Dawley rats provide an animal model for studying the role of nutrition in renal health due to their sensitivity to diet-induced alterations in kidney function. Nephrocalcinosis, a common renal abnormality found in rats, has been implicated in subsequent renal failure. Simple dietary manipulations, such as changing the source of dietary protein, may influence nephrocalcinosis. This study evaluates the consumption of krill protein concentrate (KPC), a novel and high-quality protein, on renal and bone health. Young female Sprague-Dawley rats (n = 10/group) were individually housed in metabolic cages and fed ad libitum diets consisting of 10% crude protein supplied as KPC or casein for 4 weeks. Diets were isocaloric, isonitrogenous, and matched for calcium (Ca) and phosphorus (P). Urinary n-acetyl glucosaminidase (NAG) was measured and kidney histology performed to assess kidney damage. Biomarkers of kidney function were determined by calorimetric assays. Ca and P balance and bone concentrations were measured using inductively coupled plasma mass spectrometry. Femoral strength was determined by three-point bend testing. Rats fed KPC had lower (P = 0.005) urinary NAG levels and minimal microtubular Ca deposition compared to rats fed casein. There was a tendency (P < 0.06) for higher glomerular filtration rates and lower proteinuria, and higher (P = 0.03) urinary output in rats fed KPC compared to casein. There were no differences in Ca and P balance or bone measurements of total bone mineral content, Ca, P or strength between rats fed KPC and casein. Based on the study results, KPC prevented early renal injury leading to nephrocalcinosis and potential bone loss.

AA amyloidosis due to chronic oxalate arthritis and vasculitis in a patient with secondary oxalosis after jejunoileal bypass surgery.

We report a case of a woman with secondary oxalosis after jejunoileal bypass surgery for obesity, who presented with oxalate stone disease and renal insufficiency requiring dialysis. Thirty years after surgery, longstanding osteoarticular symptoms were recognized as oxalate arthritis. Eventually, she also developed oxalate vasculitis, which improved with corticoid treatment and intensification of dialysis. Work-up for kidney transplantation revealed AA amyloidosis on gastric and colonic biopsies. Since no other cause of chronic inflammation could be identified, it was concluded that the amyloidosis was secondary to oxalate arthritis and vasculitis. To our knowledge, this is the first report on this association.

The effect of L-arginine methyl ester on indices of free radical involvement in a rat model of experimental nephrocalcinosis.

The aim of this study was to test the effect of L: -arginine methyl ester (L-Arg) on indices of free radical involvement in a rat model of experimental nephrocalcinosis. Twenty-eight Sprague-Dawley rats were randomized into four groups of seven. The first group (G1), the sham-control group received pure distilled drinking water. The second group (G2) received drinking water containing 0.7% ethylene glycol (EG) in distilled water for 3 weeks. The third group (G3) received drinking water containing 0.7% EG in distilled water for 3 weeks and L-Arg was administered for 3 weeks. The fourth group (G4) received drinking water containing 0.7% EG in distilled water for 3 weeks and L-NAME was administered for 3 weeks. Urine and aortic blood was collected to determine some parameters. The kidneys were also removed for histological examination. The increase in blood urea nitrogen, serum creatinine, K(+), Mg(2+ )and uric acid were mild in group 3 compared with the groups 2 and 4. The urinary concentrations of Na(+), K(+), Mg(2+) and uric acid were noticed to be similar among the groups. However, Ca(2+ )and oxalate excretion were significantly higher in groups 2, 3 and 4 than in group 1. The mean values of SOD, CAT and GSH-Px values were significantly increased in group 3 when compared to groups 2 and 4. Presence of aggregated urinary crystals was clearer in experimental groups compared to group 1. The tubular dilatation, epithelial degeneration and lymphocytic infiltration were significantly found in groups 2 and 4. Mild tissue damage was observed in L-Arg-pretreated rats. Under polarized light microscope intense crystals in the cortex and medulla were observed in the kidney of group 2 and 4 and moderate crystals were noticed in group 3. In conclusion, L-Arg supplementation may decrease free radicals and tubulary membrane injury in nephrocalcinosis due to infiltrating leukocytes and decreased antioxidant enzyme activities in rats fed with EG diet.

Renal failure and nephrocalcinosis associated with oral sodium phosphate bowel cleansing: clinical patterns and renal biopsy findings.

Acute renal failure (ARF) is rarely reported after bowel preparation with sodium phosphate. We report a patient with mild Crohn disease (in remission), without history of renal disease, and with normal baseline renal function, who developed ARF 14 days after bowel preparation for colonoscopy with oral sodium phosphate. A renal biopsy showed multifocal calcium phosphate deposition in the renal tubules against a background of diffuse chronic tubulointerstitial injury. Review of the literature suggested 2 distinct patterns of ARF in the context of sodium phosphate bowel cleansing. One pattern is characterized by ARF, which develops a few hours or days after sodium phosphate administration, as a component of a systemic syndrome associated with severe hyperphosphatemia and hypocalcemia. Correction of these electrolyte abnormalities was frequently associated with rapid recovery of renal function. The cause of ARF in this context was not clear because the favorable outcome negated the need for renal biopsy. In the second pattern, exemplified by the current patient, ARF was identified incidentally. These patients did not have any features of an acute syndrome immediately after sodium phosphate administration and presented much later (usually weeks) with mild, nonspecific symptoms. At the time of presentation, the serum calcium and phosphate levels were normal. The renal biopsies in each of these patients showed nephrocalcinosis as the possible cause of ARF. The renal failure improved at least partially in most of these patients, but persisted in rare cases.

Nephrocalcinosis caused by dietary calcium:phosphorus imbalance in female rats develops rapidly and is irreversible.

Female rats fed the standardized AIN-76A diet develop kidney calcium deposits (nephrocalcinosis, NC). A low dietary Ca:P molar ratio is a primary factor in this disorder. The AIN-93G diet has a lower P content and higher Ca:P molar ratio and lowers the incidence of NC. To examine the early stages of NC induced by dietary Ca:P imbalance and the potential reversibility of this disorder, weanling female Sprague-Dawley rats were fed a modified AIN-93G test diet containing Ca:P at AIN-76A levels (NC-inducing diet) for 0.5-16 wk before necropsy, or were switched to AIN-93G control diet after 0.5-4 wk until necropsy at 16 wk. A dramatic increase in incidence and severity of NC was noted after 2 wk of feeding the test diet. NC was not reversible by switching to the control diet. As little as 0.5 wk of exposure to the test diet followed by 15.5 wk of consuming the control diet resulted in increased incidence and severity of NC compared with 16 wk of consuming the control diet. Short-term (as little as several days) feeding of an NC-inducing diet to female rats can lead to NC even if they are switched to an optimal diet.

1alpha-Hydroxylase gene ablation and Pi supplementation inhibit renal calcification in mice homozygous for the disrupted Npt2a gene.

Disruption of the major renal Na-phosphate (Pi) cotransporter gene Npt2a in mice leads to a substantial decrease in renal brush-border membrane Na-Pi cotransport, hypophosphatemia, and appropriate adaptive increases in renal 25-hydroxyvitamin D3-1alpha-hydroxylase (1alphaOHase) activity and the serum concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D]. The latter is associated with increased intestinal Ca absorption, hypercalcemia, hypercalciuria, and renal calcification in Npt2-/- mice. To determine the contribution of elevated serum 1,25(OH)2D levels to the development of hypercalciuria and nephrocalcinosis in Npt2-/- mice, we examined the effects of 1alphaOHase gene ablation and long-term Pi supplementation on urinary Ca excretion and renal calcification by microcomputed tomography. We show that the urinary Ca/creatinine ratio is significantly decreased in Npt2-/-/1alphaOHase-/- mice compared with Npt2-/- mice. In addition, renal calcification, determined by estimating the calcified volume to total renal volume (CV/TV), is reduced by 80% in Npt2-/-/1alphaOHase-/- mice compared with that in Npt2-/- mice. In Npt2-/- mice derived from dams fed a 1% Pi diet and maintained on the same diet, we observed a significant decrease in urinary Ca/creatinine that was also associated with 80% reduction in CV/TV when compared with counterparts fed a 0.6% diet. Taken together, the present data demonstrate that both 1alphaOHase gene ablation and Pi supplementation inhibit renal calcification in Npt2-/- mice and that 1,25(OH)2D is essential for the development of hypercalciuria and nephrocalcinosis in the mutant strain.

Diet and kidney diseases in rats.

Diet-associated kidney diseases of rats includes nephropathy in both sexes and nephrocalcinosis in females. High protein content of diets appears to be the major cause for severe nephropathy and changing the source of protein to one such as soy protein, restricting caloric intake, or modifying the diet to decrease protein consumption could decrease the severity of nephropathy. The NTP-2000 diet with lower protein content than most diets decreases the severity of nephropathy and increases the survival of Fischer 344 rats without substantial changes in growth patterns and body weights. Nephrocalcinosis, characterized by mineralization of renal tubules at the corticomedullary junction, has been reported in young and adult female rats of most strains and stocks suggesting a major contribution of female sex hormones to the development of this lesion. Calcium (Ca), phosphorous (P), magnesium (Mg), and chloride (Cl) imbalances, especially a Ca:P ratio of less than 1.0 in diet, are considered to be associated with this lesion. Most commercial diets commonly used for toxicology studies have a Ca:P molar ratio of less than 1.0. Increasing the Ca:P molar ratio to more than 1.0 and closer to 1.3 in the AIN-93 purified diet and NTP-2000 nonpurified diet prevents the development of this lesion. Genetics will predispose rats to some diseases and environmental factors will influence the severity of these diseases. Diet is one of the most important environmental factors. Diets balanced for nutrients without excesses could markedly improve the health of rats used in chronic studies leading to substantial increases in survival and thereby accomplish the objective of chronic toxicity and carcinogenicity studies.

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

Effect of thiazide on established furosemide-induced nephrocalcinosis in the young rat.

A previous study on furosemide-induced nephrocalcinosis (NC) showed only partial resolution of the calcifications after discontinuation of the diuretic. We investigated whether treatment with chlorothiazide (CTZ) will expedite the resolution of established furosemide-induced NC. Seventy-eight weanling male Sprague-Dawley rats were divided into eight groups. Three groups were studied for 1 week: A, control; B, furosemide 40 mg/kg per 24 h; C, CTZ 100 mg/kg per 24 h. Five groups were studied for 5 weeks: D, control; E, F, G, furosemide 40 mg/kg per 24 h for 1 week followed by 4 weeks of observation (E), CTZ 50 mg/kg per 24 h (F), and CTZ 100 mg/kg per 24 h (G) and; and CTZ 100 mg/kg per 24 h (H) for 5 weeks. At the end of each study period urine and blood were collected, one kidney was studied histologically and the contralateral ashed for quantitative calcium (Ca) analysis. Animals in group B developed NC with a kidney Ca content of 1,844 +/- 203 micrograms/g dry tissue compared with group A 248 +/- 86 (P < 0.05) and group C 256 +/- 56 (P < 0.05). There were no differences among the three groups with regard to creatinine clearance, urine phosphate (P) or Ca excretion, although the latter tended to be lower in group C. Animals in group E showed a reduction in the magnitude of NC, with kidney Ca of 550 +/- 398 micrograms/g dry tissue, which was lower than in group B (P < 0.05) but still higher than in groups D (140 +/- 27) (P < 0.05) or H (162 +/- 63) (P < 0.05). Kidney Ca content in groups F (497 +/- 142) and G (489 +/- 271 micrograms/g dry tissue) was similar to that in group E. There were no differences among the five groups with regard to creatinine clearance or urine P excretion. Urine Ca excretion was significantly lower in groups F and G than groups D and E. We conclude that once established, NC caused by furosemide is not affected by CTZ therapy in spite of the anticalciuric property of the latter.

Diminished kidney function and nephrocalcinosis in rats fed a magnesium-deficient diet.

The effect of a magnesium-deficient diet on kidney function was studied in young male rats. The rats were fed a purified diet with a magnesium content of either 20.5 (control diet) or 2.6 mmol/kg (magnesium-deficient diet) for 21 d. In rats fed the magnesium-deficient diet, kidney wet and dry weights were significantly increased, and calcium and phosphorus concentrations in the kidney were significantly higher than in rats fed the control diet. Upon histological examination, an increase in the mesangial matrix of the glomeruli and injury to the brush border of the proximal tubules were observed in rats fed the magnesium-deficient diet. Also, a deposition of calcium was observed in the tubules of the corticomedullary junction and medulla of these rats. Total protein and albumin concentrations in serum were significantly decreased in rats fed the magnesium-deficient diet. Urinary albumin excretion was significantly higher, and N-acetyl-beta-D-glucosaminidase activity in the urine was significantly increased in rats fed the magnesium-deficient diet. These findings indicate diminished glomerular and proximal tubular functions. We suggest that a magnesium-deficient diet not only induces nephrocalcinosis, but it also diminishes kidney function.

X-linked hypophosphatemia: normal renal function despite medullary nephrocalcinosis 25 years after transient vitamin D2-induced renal azotemia.

Nephrocalcinosis (NC) detected by ultrasound is a recognized abnormality for some patients with X-linked hypophosphatemia (XLH) who received vitamin D2 and inorganic phosphate therapy, but is commonly observed in XLH patients treated with 1,25-dihydroxyvitamin D3 and inorganic phosphate supplementation. Nevertheless, long-term follow-up of kidney function in XLH patients with NC detected ultrasonographically has not been reported. We investigated two women with XLH, ages 31 (patient 1) and 39 (patient 2) years, each of whom had suffered at least one documented episode of vitamin D2-induced hypercalcemia and renal azotemia during childhood. Patient 2 had also been treated with inorganic phosphate. No medications for XLH had been taken during adulthood. Renal ultrasonography at our institution demonstrated marked bilateral medullary NC in both women. No other explanation was found for their NC that apparently occurred several decades earlier from medical therapy for XLH. Detailed studies (including creatinine clearance, beta2-microglobulin excretion, and fasting urinary osmolality and acidification) revealed no impairment of kidney function in either patient. Our findings indicate that subradiographic medullary NC acquired during medical therapy for XLH may persist for decades, but with no adverse renal sequelae. Definitive (long-term) assessment of kidney function in the XLH population with NC, however, will be necessary to fully understand the risk of current medical treatment for this most common heritable form of rickets.

Inbred strains of rats have differential sensitivity to dietary phosphorus-induced nephrocalcinosis.

The degree of nephrocalcinosis after increasing the dietary phosphorus concentration from 0.2 to 0.5 g/100 g was measured in weanling female rats of 10 inbred strains. Based on kidney calcium concentrations and histological kidney calcification scores, there were considerable strain differences in nephrocalcinogenesis; 86% of the strain variability in nephrocalcinosis was attributable to genetic factors. Two strains with the most extreme nephrocalcinogenic responses were retested and the strain difference was found to be reproducible. Mean plasma phosphorus concentrations after phosphorus feeding were lower in the sensitive strain than in the insensitive strain. The high phosphorus diet produced greater urinary phosphorus concentrations, with the increase being greater in the sensitive strain. The strain difference in the response of urinary phosphorus concentrations after raising dietary phosphorus level may determine the strain difference in phosphorus-induced nephrocalcinosis. After consuming the high phosphorus diet, RP rats housed in groups in solid-floored cages had significantly higher degrees of nephrocalcinosis than their counterparts housed individually in metabolism cages with wire-mesh bases.

Dietary fluoride prevents phosphorus-induced nephrocalcinosis in female rats.

The effect of dietary fluoride (F) on nephrocalcinosis was studied in young, female rats. Nephrocalcinosis was induced by a diet rich in phosphorus (P). F in the diet effectively counteracted P-induced nephrocalcinosis in a dose-dependent fashion. The feeding of increasing amounts of F caused decreasing calcium (Ca) and F concentrations in kidney. This suggests that the amount of Ca in kidney determines F accumulation in this organ, rather than F intake. Increasing amounts of F in the diet caused increasing rates of urinary and fecal excretion and whole-body retention of F. Dietary F did not influence urinary and fecal excretion and plasma concentrations of Ca, magnesium (Mg), and P. The metabolic basis for the protective effect of F against the development of nephrocalcinosis remains to be established.