Nephrol Dial Transplant (2002) 17: 1396–1401
Original Article
Reduced bone mass in children with idiopathic hypercalciuria and in their asymptomatic mothers Michael Freundlich1, Evelyn Alonzo2, Ezequiel Bellorin-Font2 and Jose R. Weisinger3 1
University of Miami School of Medicine, Miami, Florida, USA, 2Clinical Research Center, Centro Nacional de Dialisis y Trasplante Renal, MSAS, Caracas, Venezuela and 3Universidad Central de Venezuela and Head, Division of Nephrology, Hospital Universitario de Caracas, Venezuela
Abstract Background. Patients with nephrolithiasis and idiopathic hypercalciuria (IH) may exhibit reduced bone mineral density (BMD). Most studies measuring BMD in IH patients employing dual-energy X-ray absorptiometry (DEXA) have been performed in adults, and no study has been conducted in NorthAmerican children. Optimal bone mineral accretion during childhood and adolescence is critical to the attainment of a healthy adult skeleton. Bone mineral accretion and eventual adult peak bone mass are largely dependent on genetic factors. Hypercalciuria is also frequently linked to genetic determinants. Therefore, we carried out a cross-sectional evaluation of bone mineral metabolism in children with IH, and in their asymptomatic premenopausal mothers. Methods. Quantitative BMD using DEXA was performed in 21 children with IH and in their asymptomatic mothers. Bone resorption was assessed by measuring the urinary concentrations of pyridinoline and deoxypiridinoline. Simultaneous calcium-modulating hormonal determinations, including serum intact immunoreactive parathyroid hormone and 1,25(OH)2D3, were performed. The expression of interleukin-1a (IL-1a) by peripheral blood mononuclear cells (PBMCs) was determined by polymerase chain reaction. Results. Reduced BMD values were observed in eight children (38%) and in seven mothers (33%). The children of osteopenic mothers exhibited significantly reduced BMD Z-score values of lumbar spine (P-0.05) when compared with children of mothers with normal BMD. Bone resorption markers were normal in most children with IH. Hypercalciuria was detected in five out of 20 (25%) asymptomatic mothers and it correlated (rs0.81) to femoral BMD in mothers
Correspondence and offprint requests to: Michael Freundlich, MD, 3435 Hayes Street, Hollywood, FL 33021, USA. Email:
[email protected] #
with osteopenia. The expression of IL-1a mRNA by PBMCs from IH children did not differ from controls. Conclusions. Reduced BMD was detected in a large proportion of children with IH. Hypercalciuria and reduced BMD were uncovered in a substantial number of their otherwise healthy asymptomatic mothers. The diminished BMD in adults with IH may start early in life, could be influenced by genetic factors, and may represent a risk factor for osteoporosis later in life. Keywords: bone densitometry; children; hypercalciuria; nephrolithiasis; osteopenia; osteoporosis
Introduction Idiopathic hypercalciuria (IH) is a frequent metabolic disorder occurring in children and adults, characterized by excessive urinary calcium excretion and normal serum calcium levels in the absence of other hypercalciuric conditions such as vitamin D intoxication, endogenous or exogenous glucocorticoid excess, sarcoidosis, immobilization, or hyperthyroidism [1]. Besides constituting the most common cause of urolithiasis in all age groups, IH is a frequent cause of microscopic haematuria in children [2]. Other symptoms such as dysuria, frequency of urination, pyuria, enuresis, recurrent urinary tract infections, and abdominal colicky pains, are frequently recognized in children with documented IH [3]. Excessive intestinal absorption of calcium or a renal calcium leak due to a tubular disturbance as well as increased mobilization of bone calcium have been suggested as important factors in the pathophysiology of hypercalciuria [4]. Recent studies have suggested that different interleukins may promote excessive bone resorption in patients with IH and may contribute to their reduced bone mineral mass [5]. Diminished bone mineral density (BMD) has been reported in children with IH by some investigators [6,7], but not by others
2002 European Renal Association–European Dialysis and Transplant Association
Bone densitometry in hypercalciuric children
[8]. However, all reported patients, in whom BMD was assessed by dual-energy X-ray absorptiometry (DEXA), are from geographic areas other than the USA [7], where different constitutional and environmental factors may impact bone mineral mass measurements. The few reported studies in US children [6,8] have employed single photon absorptiometry of the forearm, which analyses a skeletal site with less metabolic activity and physiologic relevance. Several studies have suggested that IH is inherited by an autosomal dominant pattern of transmission and linkage analysis has mapped a gene defect in some patients with absorptive hypercalciuria [9], but multiple factors and genes probably determine ultimate urinary calcium excretion [10]. At the same time, bone mineralization throughout childhood and adolescence and final adult peak bone mass may also be determined by genetic factors [11]. Therefore, following the identification of IH in a group of children, we decided to evaluate BMD in both the children and in their asymptomatic mothers. In addition, biochemical markers of bone metabolism and the expression of interleukin-1a (IL-1a) were measured in the serum of these children with IH.
Subjects and methods
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(iPTH) was determined by an immunoradiometric assay [12], 1,25(OH)2D3 (calcitriol) by a radioreceptor assay [13], and urinary pyridinoline (PYD) and deoxypiridinoline (DPD) by high-pressure liquid chromatography [14,15]. Paediatric control values for urinary PYD and DPD were obtained from morning urine samples collected at our paediatric clinic from healthy children and from children evaluated for reasons not affecting growth, bone metabolism, or renal function (ns45). All IH patients and normal children were well nourished, consumed an unrestricted habitual diet, and had normal age-adjusted weight (range 10–95 percentile, median 50 percentile) and height (range 10–90 percentile, median 50 percentile). All children and mothers were residents of the South Florida area (USA). All clinical studies were completed and specimens obtained at the Pediatric Nephrology Outpatient Clinic located in South Florida. The expression of IL-1a by peripheral blood mononuclear cells (PBMCs) was determined by the polymerase chain reaction (PCR) as described previously [5]. In brief, adherent cell supernatants from PBMCs were obtained by Ficoll Hypaque density gradients, which contained )95% monocytes. Following RNA extraction, stranded cDNA synthesis was performed and subjected to PCR mix and then amplified. The efficiency of reversed transcription was evaluated using human b-actin as an internal control. Cytokine-specific primers for IL-1a and human b-actin were obtained from Clontech (Palo Alto, CA). PCR products were separated on agarose gel and the intensity of the bands read in an imaging densitometer model GS-670 (Bio-Rad, Hercules, CA).
Patients BMD analysis Twenty-two, all white, hypercalciuric children (15 males, seven females), including two siblings, and their mothers were identified at our Pediatric Nephrology Clinic located in South Florida, USA. Although all patients with IH and their parents followed at our center during the study period were offered to participate, in most instances only the mothers agreed to participate anduor they were the sole caretakers of their children. Therefore, only mothers were included in the present study. Except for five children who presented with nephrolithiasis, all other patients were referred for evaluation of one or more of the following symptoms: haematuria (gross or microscopic), dysuria, urinary urgency-frequency, enuresis, or recurrent urinary tract infections. All children had normal glomerular filtration rates (118"17 mlu minu1.73 m2) ranging from 96 to 163 mluminu1.73 m2. None had evidence of nephrocalcinosis on routine renal sonography. Hypercalciuria was defined in children as a urinary calcium excretion )4 mgukg body weightu24 h and in adults )250 mgu24 h [1,8]. All mothers were premenopausal and none reported previous kidney stones or urinary symptoms, bone fractures, malignancy, or endocrine diseases that could affect bone metabolism. Family histories, including paternal background when available, were negative for other renal disorders or chronic renal failure. Neither the children nor the mothers were receiving any drug that could affect bone homeostasis, including vitamin D supplements. A written informed consent was obtained from participating mothers.
Laboratory methods Urinary calcium and creatinine were determined by photometric methods. Serum intact parathyroid hormone
BMD of the lumbar spine (L2–L4) and the femoral neck were determined by DEXA with a DPX-L bone densitometer (Lunar Corporation, Madison, WI). Periodic phantom scans consistently revealed coefficient of variation values (precision) of -1%. BMD values are presented as standard deviation scores (SDS) and are reported as Z scores in the children (SD values compared with age and gender-matched control means) and as T scores in the mothers (matched for young adult reference mean values), both calculated from the manufacturer’s database. Reference values in US children and adolescents were obtained by the manufacturer in normal females (ns879) and males (ns736); ages 5–19 years [16]. In adults, a score value less than 1 was considered osteopenia and greater than 2.5 was defined as osteoporosis [17 ].
Statistical analysis Results are presented as mean"SD except when otherwise indicated. In order to preserve the statistical independence of the data, one of the siblings, chosen at random, was excluded from the analysis. Thus, the results presented below are based on data from 21 mother–child pairs. Comparisons between two groups of data were carried out by the Student’s t-test for unpaired observations and by the Mann–Whitney test, as appropriate. Correlations between two variables were obtained by the Pearson linear regression analysis or by the Spearman rank correlation coefficient. Reported P values (one- or two-tailed) of -0.05 were considered significant.
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Table 1. Clinical data in children with IH and in their asymptomatic mothers
Children All (ns21) BMD less than 1 SDS (ns8) BMD normal (ns13) Mothers All (ns21) BMD less than 1 SDS (ns7) BMD normal (ns14)
Age (years)
Gender FuM
Urinary calcium
BMD spine (SDS)
BMD hip (SDS)
PTH (pguml)
1,25(OH)2D3 (pguml)
9.6"4.0 8.0"3.5 10.6"4.1
7u14 3u5 4u9
6.2"3.0y 7.1"4.6y 5.6"1.2y
0.1"1.3 1.80"0.6 0.50"0.9
0.32"1.37 1.71"0.4 0.53"1.0
25.4"16.4 23.3"4.9 25.4"13.3
57"23 61"36 54.6"19
175"107z 196"78z 164"121z
0.16"1.0 0.92"0.23 0.5"0.8
0.3"0.9 1.23"0.2 0.1"0.7
39.7"5.0 38.3"7.0 40"3.7
ymgukg body weightu24 h. zmgu24 h.
Results The mean age of the children was 9.6"4.0 years (range 4–16) and that of their corresponding mothers was 39.7"5.0 years (range 26– 48). All children, as an including criterion in this study, displayed elevated urinary calcium excretion (6.2"3.0 mgukg body weightu24 h). Urinary pH values on random specimens ranged from 5 to 7. The distribution of urinary calcium excretion in the mothers ranged from 45 to 394 mgu24 h (175"107 mgu24 h) and in five out of 21 (24%) was consistent with hypercalciuria (327"54 mgu24 h). Measured BMD values with a SDS less than 1 at the lumbar spine or femoral neck were detected in eight children (38%) and in seven mothers (33%) (Table 1). Children of mothers with BMD values considered osteopenic (ns7) displayed significantly more negative Z-score values in the lumbar spine (mean"SEM, 0.94"0.47 vs 0.32"0.33, P-0.05) compared with those of mothers with normal BMD. Similarly, femoral neck Z-score values were also lower and in the osteopenic range in these children of osteopenic mothers, (mean"SEM, 1.06"0.32 vs 0.07"0.42) and in three of those seven children BMD Z scores were less than 1 (Figure 1). Children with low or normal BMD were of comparable ages (8.0"3.5 vs 10.6"4.1 years, respectively, Ps0.14) but their urinary calcium excretion was higher (7.1"4.6 vs 5.6"1.2 mgukg body weightu24 h, Ps0.19, low vs normal BMD, respectively). A negative correlation was observed between urinary calcium and BMD in those with normal femoral BMD (rs0.48, one-tailed P-0.05); these correlations were not significant in children with low femoral, low spinal, and normal spinal BMD. In children with nephrolithiasis, two out of five displayed osteopenia, and in their corresponding mothers two out of five had hypercalciuria and one out of five disclosed osteopenia. In the children with IH, urinary PYD and DPD concentrations (nmolummol creatinine) stratified by age (GIs2–10 years, GIIs)10 years) were not significantly higher than age-matched controls (PYD, GI 306"120 vs 281"85; GII 207"113 vs 259"103)
Fig. 1. BMD, presented as mean"SEM, of spine and femoral neck in children (ns7) of osteopenic mothers (dark bars) compared with children (ns13) of mothers with normal BMD (light bars). Z scores (values compared with age-matched normal controls) in children of osteopenic mothers were lower at both lumbar spine (P-0.05) and femoral hip locations.
(DPD, GI 88"41 vs 76"25; GII, 52"31 vs 69"29) and except for two high values, all fell within 2 SD of those controls. Serum iPTH concentrations (normal range 11–54 pguml) were normal (25.4"11.4, range 5–49 pguml) in all children and did not correlate to their BMD. Serum 1,25(OH)2D3 values (normal range in infants and children 15–90 pguml) were normal in all except in one child (57"23, range 27–99 pguml) and did not correlate to BMD either. Urinary calcium excretion was similar in mothers with low as compared with normal BMD (196"78 vs 164"121 mgu24 h, respectively) and did not correlate to either spinal (rs0.14) nor femoral BMD (rs0.15). However, in the mothers with low femoral BMD (ns6), the correlation between urinary calcium excretion and BMD was strong and nearly reached statistical significance (rs0.81, Ps0.058). In mothers with normal femoral, and low or normal spinal BMD, urinary calcium and BMD correlated weaker. Urinary calcium excretion of mothers (mgu24 h) and that of children (mgukg body weightu24 h) did not correlate as a group (rs0.23), but became more apparent when estimated in the children–mother pair (ns7)
Bone densitometry in hypercalciuric children
corresponding to the mothers with a BMD less than 1 SD (rs0.63, Ps0.1). The expression of IL-1a mRNA in unstimulated PBMCs from the IH children revealed band density measurements comparable with controls (2.02"1.24 vs 2.09"1.16).
Discussion This study demonstrates diminished BMD in 38% of children with IH, as well as in some of their asymptomatic mothers. In addition, hypercalciuria was uncovered in a number of otherwise healthy mothers. Biochemical markers of bone metabolism and bone resorption were normal in most children. Earlier studies utilizing single photon densitometry of the radial bone, a site of less physiologic relevance, reported either diminished [6] or normal [8] BMD. Studies performed in other countries employing DEXA methodology, similar to that employed by us, have reported diminished BMD at the spinal site in 30% of patients [7]. Measuring both spinal and femoral sites, we observed reduced BMD in a slightly higher proportion (38%) of studied children. Within this group of IH children, three out of eight (38%) patients with reduced BMD were between 10 and 16 years old, a critical period for bone mineral accretion during which 40–50% of peak bone mass is accumulated [18]. Failure to attain an optimal peak bone mass could be associated with lower bone density later in life, a major risk factor for future osteoporosis [18,22]. The negative relationship between urinary calcium excretion and femoral BMD, not reported previously in children with IH, raises interesting considerations. Earlier studies suggested a role of secondary hyperparathyroidism [19] with its consequent excessive bone resorption as a potential cause for the reduced BMD in some patients. Employing a highly sensitive iPTH assay we found these values to be normal. Furthermore, no correlation was found between the serum PTH and the BMD values at neither studied site. These results are in agreement with most studies in which PTH levels were normal or relatively low in patients with IH [4,7]. Serum 1,25(OH)2D3 values were also normal in virtually all children. Elevated concentrations have been reported but most patients display normal or slightly elevated serum 1,25(OH)2D3 concentrations [4 ]. A higher sensitivity to 1,25(OH)2D3 leading to higher rates of calcium efflux from the bone was demonstrated in genetic hypercalciuric rats [20], but whether similar abnormalities leading to eventual osteopenia take place in the human bone of patients with IH, remains unknown. Recent studies in patients with IH [21] have demonstrated a reduction in the hypercalciuria and in the concentration of urinary markers of bone resorption following the administration of alendronate, a bisphosphonate capable of blocking excessive bone resorption. These observations provide further support to the central role of bone
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calcium in contributing to the increased urinary excretion of calcium in IH [4]. Other factors like weight, height, race, and nutritional status may also influence BMD [22]. Malnutrition occurs frequently in children of developing countries, where some of the previous studies were performed. In this study, all the children were well nourished and had normal height and weight for their age, and consumed an unrestricted habitual diet. Other mechanisms involved in bone resorption need to be considered. Monocyte-derived cytokines play a key role in bone remodelling, and recent studies have suggested a role for IL-1a, IL-6, and TNF-a in promoting bone resorption in adult patients with IH [5]. However, we were unable to demonstrate similar results in the IH children tested in the present study. Whether alterations in stimulated cytokines become evident only later in life remains to be determined. The relatively small number of children with osteopenia tested in this study may be another explanation for the absence of a statistically significant difference, especially when considering the large SD values of the IL-1a mRNA determinations. When bone is resorbed, DPD and PYD, two mature cross-linking amino acids present in bone type I collagen, are released into the circulation and excreted in the urine, and their measurement has been considered a useful marker of bone resorption [23]. The urinary concentrations of DPD and PYD in our IH children were not different than those obtained in a group of healthy age-matched controls. Measuring other markers of bone resorption, Garcia-Nieto et al. [7] did not find any abnormalities in their children with IH and osteopenia either. As in growing children, the urinary excretion of DPD and PYD can be ;3–6-fold higher than in adults [23], proper interpretation of results requires age-adjusted controls. The finding of normal urinary markers of bone resorption in the patients with reduced BMD may have several possible explanations. First, more intense and more prolonged rates of bone resorption, resulting in lower BMD measurements, may be required to result in abnormally higher urinary concentrations of these markers. Our patients with reduced BMD Z scores would be categorized as having osteopenia with measurements between 1 and 2.5 SDS rather than overt osteoporosis where values are less than 2.5 SDS [17]. Secondly, intraindividual variations may range from 15 to 50%, and single measurements of these urinary markers seem to have limited utility and exhibit poor correlations to BMD measurements [24]. Thirdly, the reduced BMD may be only in part the result of excessive bone resorption, as diminished bone formation and defective mineralization have also been observed in patients with IH [25]. Thus, future studies employing more reliable serum biochemical markers of bone resorption and formation in children with IH are needed. Of particular interest were the findings of low BMD in some of the mothers of IH children. While reduced BMD has been described previously in adults with IH, all the reported patients were kidney stone
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formers [4]. None of the mothers of our children with IH had a previous history of nephrolithiasis or any known metabolic disorder potentially affecting bone mineralization. Although urinary calcium excretion did not correlate to BMD, and as a group was similar in mothers with normal or low BMD, six out of seven mothers with osteopenia displayed urinary calcium excretion rates )140 mgug creatinine, a lower cut-off point utilized to define hypercalciuria in adults [26]. In a recent large epidemiologic study, normal women of similar age to our studied mothers had an average urinary calcium excretion rate of 186 mgu24 h, and the authors called for a reassessment of the traditional definition of hypercalciuria [27]. According to this parameter, eight out of 21 (38%) asymptomatic mothers in our study would have been classified as having hypercalciuria. In mothers with low femoral BMD, the correlation of BMD measurements and calciuria (rs0.81) was strong and nearly reached statistical significance. In a larger group of women with IH, we indeed were able to display these relationships more significantly [5]. The elevated urinary calcium excretion detected in asymptomatic mothers suggests a possible role of heredity. A family history of kidney stones can be obtained in as many as 45% of patients with IH and several studies have suggested an autosomal dominant pattern of transmission [10]. The lack of known progressive renal disease in other family members, as well as the absence of nephrocalcinosis in all patients, militates against X-linked recessive hypercalciuric nephrolithiasis [10]. The findings of lower BMD values observed in the children of mothers with osteopenia (ns7 ) are of further interest (Figure 1). In this group of children, three out of seven displayed BMD values less than 1 Z score. These observations may indicate a potential role of genetic factors influencing BMD [11]. Family studies have indicated heritability estimates for bone mass in the order of 60–80% [28]. The group of children with IH consisted of seven females and 15 males. We did not include fathers in this study because of the high proportion of mothers as the sole family caretakers, and the father’s wide unavailability. In our study, five out of eight children with reduced BMD were males. As the heritability of BMD from mother to son is not as strong as to daughters [28], future studies ideally should also include fathers. It has been suggested that allelic differences in the vitamin D receptor (VDR) gene encoding the VDR may be responsible for the number of VDRs present in the intestine and bone, which ultimately affect intestinal calcium absorption and peak bone mass [29]. Recently, linkage of markers for the VDR locus to nephrolithiasis and hypercalciuria was reported in a large cohort of families [30]. Whether the genes involved in determining bone mass may also play a role in affecting urinary calcium excretion in patients with IH remains unknown. Further studies including gene mapping and cloning
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should help us understand further the genetics governing both, excessive urinary calcium excretion and bone mineral turnover in patients with IH [10], and should help answer whether they may just represent the chance concurrence of two unrelated illnesses. In summary, we have demonstrated diminished BMD in children with untreated IH as well as osteopenia in a substantial proportion of their asymptomatic mothers. The osteopenia in these asymptomatic and premenopausal mothers, may be relevant for future further diminished BMD with its consequent risk of eventual osteoporosis. These observations suggest that the reduced bone mass observed in adults with IH may start early in life, could be influenced by genetic determinants, and might represent an important risk factor for osteoporosis. Whether specific pharmacological interventions in children with IH will eventually result in higher peak bone mass and normal adult BMD, remains to be studied. Following the identification of children with IH, measurement of their BMD and screening for hypercalciuria and abnormal BMD in their mothers, seems appropriate. Acknowledgements. This work was supported, in part, by grant # G-9700080 of the Consejo Nacional de Investigaciones Cientificas y Tecnologicas de Venezuela (CONICIT). Portions of this study were presented at the 30th Annual Meeting of the American Society of Nephrology, November 2–5, 1997, San Antonio, Texas, and published in abstract form (J Am Soc Nephrol 1997; 8: 551A). Marilyn E. Freundlich provided expert administrative support.
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1401 23. Rauch F, Schonau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994; 102: 94–97 24. Seibel MJ, Baylink DJ, Farley JR et al. Basic science and clinical utility of biochemical markers of bone turnover—a congress report. Exp Clin Endocrinol Diabetes 1997; 105: 125–133 25. Malluche HH, Tshoepe W, Ritz E, Meyer-Sabellek W, Massry SG. Abnormal bone histology in idiopathic hypercalciuria. J Clin Endocrinol Metab 1980; 50: 654–658 26. Coe FL, Parks JH. Nephrolithiasis: Pathogenesis and Treatment, 2nd edn. Year Book Medical Publishers Inc., Chicago, 1988; 108–115 27. Curhan GC, Willett WC, Speizer FE, Stampfer MJ. Twentyfour-hour urine chemistries and the risk of kidney stones among women and men. Kidney Int 2001; 59: 2290–2298 28. McKay HA, Bailey DA, Wilkinson AA, Houston CS. Familial comparison of bone density at the proximal femur and spine. Bone Miner 1994; 24: 95–107 29. Morrison NA, Qi JC, Tokita A. Prediction of bone density from vitamin D receptor alleles. Nature 1995; 367: 284–287 30. Scott P, Ouimet D, Valiquette L et al. Suggestive evidence for a susceptibility gene near the vitamin D receptor locus in idiopathic calcium stone formation. J Am Soc Nephrol 1999; 10: 1007–1013 Received for publication: 12.10.01 Accepted in revised form: 29.3.02
