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When extrapolating data from animal toxicological studies a default factor (dUF) of 100 is applied to derive a heath based guidance value. The UF takes into account the interspecies differences (ID) and the intraspecies variability (IV). When re-evaluating the safety of phosphates used as food additives nephrocalcinosis was identified as the critical endpoint. The underlying mechanism for nephrocalcinosis was attributed to the precipitation of calcium phosphate in the kidney, depending on its solubility, irrespective of the species and the population. Based on the mechanism, the volume of primary urine, for which the glomerular filtration rate (GFR) was used as a proxy, was considered to be the only parameter relevant for ID and IV. Median value of GFR in rats was 4.0 ml/min/kg bw. In humans it was 1.6 ml/min/kg bw in healthy adults and 0.9 in elderly. These values were calculated from the distribution of the GFR data from 8 studies in rats (n = 191), 16 studies in adults (n = 1540) and 5 studies in elderly (n = 2608). Multiplying the distribution of the ratio rat/healthy humans (ID) with the distribution of the ratio healthy humans/elderly human (IV) resulted in a phosphate specific factor of 4.5 (3.3-6.7) (median; 25th - 75th percentile)., (Copyright © 2020. Published by Elsevier Inc.)

Background: Reduced kidney volume (KV) following prematurity is a proxy for reduced nephron number and is associated with the development of hypertension and end-stage renal disease in adults. We investigated whether extreme prematurity affects KV, function, and blood pressure in school-aged children and if nephrocalcinosis (NC) developed during the neonatal period had additional effects., Methods: We investigated 60 children at a mean age of 7.7 years: 20 born extremely preterm (EPT < 28 weeks gestational age with NC (NC+)), 20 born EPT without NC (NC-), and 19 born as full-term infants (control). We measured KV by ultrasound, collected blood and urine samples to evaluate renal function, and measured office and 24-h ambulatory blood pressure (ABPM)., Results: Children born EPT had significantly smaller kidneys (EPT (NC+ NC-) vs control (estimated difference, 11.8 (CI - 21.51 to - 2.09 ml), p = 0.018) and lower but normal cystatin C-based glomerular filtration rate compared with control (estimated difference, - 10.11 (CI - 0.69 to - 19.5), p = 0.035). KV and function were not different between NC+ and NC- groups. Change in KV in relation to BSA (KV/BSA) from the neonatal period to school age showed significantly more EPT children with neonatal NC having a negative evolution of KV (p = 0.01). Blood pressure was normal and not different between the 3 groups. Fifty percent of EPT had a less than 10% day-to-night decline in ABPM., Conclusions: Kidney growth and volume is affected by EPT birth with NC being a potential aggravating factor. Circadian blood pressure regulation seems abnormal in EPT-born children.

Williams-Beuren syndrome is a rare neurodevelopmental disorder, characterized by congenital heart defects, abnormal facial features, mental retardation with specific cognitive and behavioral profile, growth hormone deficiency, renal and skeletal anomalies, inguinal hernia, infantile hypercalcaemia. We report a case with Williams-Beuren syndrome associated with a single kidney and nephrocalcinosis complicated by hypercalcaemia. A male infant, aged 20 months presented growth retardation associated with a psychomotor impairment, dysmorphic features and nephrocalcinosis. He had also hypercalciuria and hypercalcemia. Echocardiography was normal. DMSA renal scintigraphy showed a single functioning kidney. The FISH generated one ELN signal in 20 metaphases read and found the presence of ELN deletion, with compatible Williams-Beuren syndrome.

Background: Nephrocalcinosis (NC) is an important clinical problem seen in critically ill preterm neonates treated with loop diuretics. No reliable animal models are available to study the pathogenesis of NC in preterm infants. The purpose of this study was to develop a reproducible and clinically relevant animal model of NC for these patients and to explore the impact of extracellular fluid (ECF) volume contraction induced by sodium and chloride depletion in this process., Methods: Three-week-old weanling Sprague-Dawley rats were fed diets deficient in either chloride or sodium or both. A subgroup of rats from each dietary group was injected daily with furosemide (40 mg/kg i.p.)., Results: Rats fed a control diet, with or without furosemide, or a chloride-depleted diet alone, did not develop NC. By contrast, 50% of the rats injected with furosemide and fed the chloride-depleted diet developed NC. Moreover, 94% of the rats fed the combined sodium- and chloride-depleted diet developed NC, independently of furosemide use. NC was associated with the development of severe ECF volume contraction; hypochloremic, hypokalemic, metabolic alkalosis; increased phosphaturia; and growth retardation., Conclusion: Severe ECF volume contraction induced by chronic sodium and chloride depletion appears to play an important role in the pathogenesis of NC.

Renal injury in early life may lead to hypertension and renal disease in adulthood. In this prospective study, we estimated renal glomerular and tubular function and kidney length (KL) during the first 2 years of life of preterm infants with nephrocalcinosis (NC) associated with prematurity. The study cohort comprised 107 preterm children, 63 with NC and 44 control subjects without NC who were matched for gender, gestational age and birth weight. Kidney function was estimated based on measurements of serum creatinine (Scr), estimated glomerular filtration rate (eGFR), fractional excretion (FE) of sodium (Na), potassium (K), phosphate (P), magnesium (Mg) and uric acid (UA) and on the ratios of urinary Ca, oxalate (UOx) and citrate (UCit) to urinary creatinine (UCa/Ucr, UOx/Ucr and UCit/Ucr, respectively) calculated from morning urine collections. KL was measured by ultrasonography. Measurements were made at 40 weeks postmenstrual age and at 3, 6, 12 and 24 months of age. At 3 and 6 months, the NC group had higher UCa/Ucr, FEK and FEUA than the control group; at 12 months, only the UCa/Ucr and FEUA was still higher. The UCa/UCit ratio was higher in the NC group. Scr and eGFR did not differ between the groups at any time point. The NC group had a shorter KL up to 12 months of life (left kidney) or 24 months (right kidney). Based on these results, we conclude that NC in the preterm infants enrolled in our study was associated with impaired renal tubular function and a shorter KL in the first year of life.

The aim of the study was to evaluate the natural course of nephrocalcinosis (NC) in preterm neonates and the effect of NC on blood pressure and renal glomerular and tubular function. In a prospective observational study of 201 preterm neonates (gestational age <32 weeks) NC was present at term in 83 patients (41%), who were subsequently examined at 6, 12, and 24 months, and until August 2000 annually (with a maximum of 4 years) if NC persisted. Examination consisted of blood pressure measurement, renal ultrasonography, and glomerular and tubular function tests. The probability that NC, when present at term, would persist for 15 and 30 months was 34% [21-45, 95% confidence interval (CI)] and 15% (5-25, 95% CI) (Kaplan-Meier), respectively. Urinary tract infection did not occur more frequently in patients with NC (2.5%) than patients without NC at term (4.4%). Systolic and diastolic blood pressures above the 95th percentile were found in 39% and 48% of patients at 1 year and 30% and 34% at 2 years ( P<0.001). Mean glomerular filtration rate (GFR) (inulin clearance) at 1 and 2 years was 92 and 102 ml/min per 1.73 m(2), respectively. TP/GFR and excretion of alpha(1)-microglobulin were normal. The desmopressin test was impaired in 4 of 30 patients at 1 year and 2 of 25 at 2 years. It was concluded that while proximal tubular function is unaffected in children with neonatal NC, high blood pressure and impaired glomerular and distal tubular function might occur more frequently than in healthy children. Although no relationship can be proven between NC and hypertension or diminished renal function in this study, these results justify a large follow-up study with matched controlled study groups.

We carried out a retrospective survey on 152 children and adolescents with nephrocalcinosis (NC) in 22 German centers of pediatric nephrology. Etiology, clinical manifestations, growth and development, sonographic appearance of NC and renal function were analyzed. The median age at the time of diagnosis was 3.3 (range 0.1-21) years and the median duration of follow-up was 4.1 years. In 34% of children NC was associated with idiopathic hypercalciuria (IHC) and in 32% with various hereditary tubular disorders. In 9% NC was observed subsequent to prophylactic bolus administration of vitamin D in infancy. A positive family history was found in 36%. Clinical manifestations were mainly failure to thrive during the 1st year of life (46%), psychomotor/mental retardation (28%) and urinary tract infection (34%). In 14% nephrolithiasis was associated. During the follow-up the proportion of patients with the most severe degree of NC (stages 2b or 3) increased from 40% to 55% and that of hypercalciuria decreased from 79% to 52%. Body height was <2 standard deviation scores (SDS) of normal in 41% at the time of diagnosis and in 32% at the last observation; the increase in relative height was significant only for IHC. Glomerular filtration rate (GFR) and urinary concentration capacity changed only slightly with time. At the last investigation GFR was <50 ml/min/1.73 m2 in 6% and concentration capacity <800 mosmol/kg in 48% of patients. The degree of NC was negatively correlated with GFR and concentration capacity.

To assess the impact of prematurity-associated nephrocalcinosis on kidney function later in life, 20 premature children with neonatal nephrocalcinosis and 20 controls, matched for birth weight and postnatal age but without nephrocalcinosis, were examined (birth weight 905+/-209 vs. 957+/-226 g; study age 4.7+/-1.1 vs. 4.6+/-0.9 years). Distal tubular acidification capacity was measured with the oral acetazolamide test, in which the response was abnormal in 1 out of the 20 children with a history of nephrocalcinosis, but in none of the controls. Urinary calcium and beta(2)-microglobulin excretion were higher in the children with nephrocalcinosis, but no differences were found in fractional excretion of sodium and potassium or tubular reabsorption of phosphate. Estimated creatinine clearance was not different between the groups. Of the 6 children with nephrocalcinosis lasting beyond 2 years of age, 5 had had chronic lung disease neonatally and exhibited a tendency for compensated respiratory acidosis at the time of the examination. Neonatal nephrocalcinosis seems to lead to some signs of renal tubular dysfunction in early childhood of preterm infants. Glomerular function, however, appears not to be specifically disturbed by nephrocalcinosis.

Dent's disease, which is a renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent's disease.

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.

Loss-of-function mutations of the ClC-5 chloride channel lead to Dent's disease, a syndrome characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. We show that ClC-5 is expressed in renal proximal tubule cells, which normally endocytose proteins passing the glomerular filter. Expression is highest below the brush border in a region densely packed with endocytotic vesicles, where ClC-5 colocalizes with the H+-ATPase and with internalized proteins early after uptake. In intercalated cells of the collecting duct it again localizes to apical intracellular vesicles and colocalizes with the proton pump in alpha-intercalated cells. In transfected cells, ClC-5 colocalizes with endocytosed alpha2-macroglobulin. Cotransfection with a GTPase-deficient rab5 mutant leads to enlarged early endosomes that stain for ClC-5. We suggest that ClC-5 may be essential for proximal tubular endocytosis by providing an electrical shunt necessary for the efficient acidification of vesicles in the endocytotic pathway, explaining the proteinuria observed in Dent's disease.

Objective. The purpose of this study was to induce nephrocalcinosis (NC) in rabbits with phosphate, vitamin D, oxalate and furosemide, to determine the effect on renal function and to correlate detection on ultrasound (US) and computed tomography (CT) with pathology. Materials and methods. Seventy-five immature New Zealand white rabbits were divided into five groups of 15. In each group, 5 animals were controls and 10 were given oral phosphate, furosemide, vitamin D or oxalate. Unilateral nephrectomy was performed at 3-6 weeks, and 5 rabbits of each test group were withdrawn from the substance. Weekly US was performed as well as US, CT and measurement of serum creatinine at the time of nephrectomy and prior to planned demise. Results. A total of 140 kidneys in 75 rabbits had both pathological and US correlation, with CT correlation in 126. Forty rabbits developed nephrocalcinosis with early (post nephrectomy at 3-6 weeks) or late (post demise at 12-20 weeks) pathological correlation obtained in 53 kidneys. Forty-one of these kidneys were from test animals: 23 developed NC early, 18 late. Twelve controls developed NC: 4 early, 8 late. Comparing US and CT to pathology, the sensitivity was 96% for US, 64% for CT. Specificity was 85% for US and 96% for CT. In 109 kidneys, information on serum creatinine level was available to correlate with pathology. The mean creatinine level was 138 mmol/l for those with NC and 118 mmol/l for those without NC (P<0.001). Conclusion. In this study, the presence of NC was significantly associated with increasing serum creatinine. Overall, US was more sensitive and CT was more specific in the detection of NC.

We studied 25 patients treated with oral phosphate and 1,25(OH)2 vitamin D for 0.5-15 y (mean, 7 y) to determine the incidence of nephrocalcinosis and its relationship to treatment, renal function, and growth. During the follow-up period, 3 hypercalcaemic and 13 random hypercalciuric episodes were documented. Creatinine clearances and urine concentration tests were normal in all patients. One patient's 24-h urine specimen indicated hypercalciuria. Kidney ultrasonography revealed nephrocalcinosis in 80% of the patients, but its severity was not significantly related to the dose of calcitriol or of phosphate, the duration of treatment, the age at which treatment was started, or growth. None of the 9 untreated affected family members had nephrocalcinosis. Longitudinal studies revealed that the greatest loss in height velocity occurred during the first 2 y of life. Our study shows that nephrocalcinosis is a common complication of phosphate and 1,25(OH)2D3 treatment, but it is not necessarily associated with impaired renal function. Although the treatment failed to prevent a decrease in height velocity during infancy, it effectively maintained height velocity after 2 y of age.

Primary hyperparathyroidism in childhood is rare. Long-standing hypercalcaemia is reported to result in severe and irreversible renal damage. However, renal complications of hyperparathyroidism, particularly nephrocalcinosis are uncommon. We report a 12-year-old girl presenting after three years with extensive nephrocalcinosis and with rapid recovery of renal function.

Nephrocalcinosis has been observed in premature infants treated with furosemide. To see whether furosemide-induced renal calcium accumulation is reflected in easily accessible extrarenal cells, we measured cytosolic free calcium ([Ca2+]i) in blood mononuclear cells and kidney tissue calcium of guinea pigs chronically treated with furosemide. At week 0, the mean [Ca2+]i in blood mononuclear cells using the fluorescent indicator quin2 was 105 +/- 4 nM. After 2 weeks of treatment with furosemide, the high dosage (10 or 20 mg/kg/day) caused a significant increase in [Ca2+]i. There was no change in cell volume after 1 or 2 weeks of treatment with furosemide at this dosage. The kidney total calcium concentration and histological calcium accumulation increased with increasing furosemide dosages. These observations suggest that calcium accumulation in blood mononuclear cells may reflect the calcium accumulation in the kidney after furosemide treatment.

The question was addressed whether dietary phosphorus-induced nephrocalcinosis in rats is associated with impaired kidney function. Weanling female rats were fed purified diets containing either 0.4 or 0.6% (wt/wt) phosphorus for 28 d. The diet containing 0.6% phosphorus produced marked kidney calcification, as determined both by chemical analysis of kidney calcium and histological examination in kidney sections. Histological examination did not show calcification in stomach, lung, heart or thoracic aorta, which are predisposition sites of metastatic calcification in secondary renal hyperparathyroidism. In rats fed the 0.6% phosphorus diet, phosphorus retention and urinary excretion were greater compared with rats fed the 0.4% phosphorus diet. The following indicators of kidney function were examined: water intake, urinary volume, urine and plasma osmolality, urine and plasma creatinine, urine and plasma urea, urea and creatinine clearance and urinary albumin excretion. Of these indicators, only urinary albumin excretion was significantly increased in rats fed the nephrocalcinogenic diet. In a further experiment, the increase of urinary albumin was reproduced. After pooling the results of the two experiments, in individual rats fed the 0.6% phosphorus diet, the concentration of kidney calcium was found to be positively related with kidney weight expressed relative to body weight (r = 0.82, n = 22) and with albumin excretion in urine (r = 0.79, n = 28). The increased weight of calcinotic kidneys was mainly due to both calcium deposition and tubular hyperplasia. It is concluded that dietary phosphorus-induced nephrocalcinosis is associated with impaired kidney function in rats.

Single-nephron and whole-kidney function were studied in female rats with corticomedullary nephrocalcinosis, and in animals where the lesion had been prevented either by a dietary magnesium supplement or by using a diet with a calcium:phosphorus ratio in excess of 1. At the single-nephron level, rats with nephrocalcinosis had prolonged tubular fluid transit times. Proximal transit time was 19.42 +/- 1.98 (mean +/- S.E. of mean) vs. 11.58 +/- 0.19 s for controls; distal transit time was 62.64 +/- 9.16 vs. 31.50 +/- 1.03 s for controls. Although single-nephron function is altered in nephrocalcinosis, data obtained from rats in metabolism cages indicate that whole-kidney function is largely unaffected by the lesion.