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Furosemide (F)-induced nephrocalcinosis (NC) has been traditionally described in low birth weight premature infants. To investigate the role of age on F-induced nephrocalcinosis we studied 24 Sprague-Dawley male rats grouped by age and F therapy vs. control as follows: A (4-week-old control), B (4-week-old + F), C (6-week-old control), D (6-week-old + F), E (10-week-old control), F (10-week-old + F). The rats were placed in metabolic cages for measurement of urine output, food and water intake. At day 14 they were anesthetized, exsanguinated and their kidneys harvested. Renal calcium deposition was assessed using NC score (scale 0-4) and quantitative calcium analysis in the contralateral kidney. Treated animals gained less weight and had higher urine output and fluid intake than the age-matched controls demonstrating the diuretic effect of furosemide. Control groups A, C, and E scored 0 histologically compared with B 2.75 +/- 0.50, D 2.00 +/- 0.58, and F 3.00 +/- 0.82 (p < 0.05 in all three paired groups). Kidney calcium content (micrograms/g dry weight) in B was 2,815.68 +/- 1,553.77 vs. A 202.58 +/- 32.02 (p = 0.04); D 1,574.05 +/- 540.21 vs. C 212.22 +/- 30.91 (p = 0.02); F 2,591.40 +/- 1,269.80 vs. E 210.38 +/- 26.79 (p = 0.02). There was no difference in the magnitude of NC among the three treated groups themselves. To determine the possible effect of age on timing of onset of NC additional 30 4-week-old and 30 10-week-old rats were studied. All 60 rats received furosemide. Six rats from each group were sacrificed on days 1, 3, 5, 7 and 11. In both groups, significant calcifications were seen already on day 3 and maximum calcification noted between days 3 and 5. We conclude that in this model the development of NC occurs within a few days of furosemide administration and that this phenomenon is not age dependent but rather reflects a property of the loop diuretic itself.

Objective: Furosemide treatment in the human neonate is associated with sodium depletion, growth retardation, hypercalciuria and nephrocalcinosis. Dietary sodium intake is known to directly influence urinary calcium excretion. The objectives of this study were to create a rat model of furosemide-induced nephrocalcinosis and to test the effects of dietary sodium supplementation on growth, electrolyte balance, calciuria, and renal calcifications., Methods: Initially, 18 weanling Sprague-Dawley rats were randomly divided into three groups. Groups A (control) and B were fed a basal diet. Group C was fed a sodium-enriched diet. Groups B and C received furosemide (40 mg/kg) intraperitoneally daily for 28 days. At the end of the study, serum, urine, and kidney samples were obtained for biochemical and histologic analyses. The three groups were then compared for differences in growth, electrolyte homeostasis, calcium excretion and nephrocalcinosis. Subsequently an additional 15 rats were studied to confirm our findings regarding urinary calcium excretion and kidney calcifications., Results: Treatment with furosemide without sodium supplementation (group B) resulted in decreased weight gain compared with group A (137.5 +/- 12.9 vs 154.0 +/- 10.6 g; p < 0.05), hypokalemia (3.7 +/- 0.1 vs. 4.4 +/- 0.4 mEq/l; p < 0.05), and nephrocalcinosis (187.1 +/- 155 vs. 18.8 +/- 6.9 micrograms Ca/g dry kidney; p < 0.05). Sodium supplementation (group C) normalized weight gain and corrected electrolyte abnormalities without increasing calciuria or nephrocalcinosis., Conclusions: We conclude that in this animal model, chronic furosemide treatment results in growth failure and development of nephrocalcinosis. Sodium supplementation protects against the deleterious effects of furosemide on weight gain and electrolyte homeostasis with no adverse effect on nephrocalcinosis.

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.