Idiopathic Hypercalciuria and Nephrolithiasis

C H A P T E R

71 Idiopathic Hypercalciuria and Nephrolithiasis Murray J. Favus, Fredric L. Coe University of Chicago Pritzker School of Medicine, Chicago, IL, USA

INTRODUCTION The inclusion of a review of idiopathic hypercalciuria (IH) in a book devoted to vitamin D is entirely appropriate given that all of the metabolic changes of IH can be reproduced in healthy volunteers by the addition of doses of calcitriol that are so small as to not cause hypercalcemia. Hypercalciuria occurs in 5 to 7% of children and adults and is the single most common cause of calcium oxalate kidney stones. The chapter focuses on IH as a major cause of hypercalciuria and nephrolithiasis and the potential role of vitamin D. Less frequent causes of hypercalcemia and hypercalciuria may also promote renal stone formation and are discussed in Chapters 45 and 72. Idiopathic hypercalciuria is characterized by normocalcemia in the absence of known systemic causes of hypercalciuria. Intestinal calcium (Ca) absorption is almost always increased, and serum 1,25(OH)2D levels are elevated in some but not all patients. Serum parathyroid hormone (PTH) levels are elevated in less than 5%. The pathogenesis of IH is unknown but several models have been offered from observations in patients including: a primary increase in intestinal Ca absorption; a primary overproduction of 1,25(OH)2D; and a primary renal tubular Ca transport defect or “renal leak” of Ca. Evidence for each model can be found in some patients with IH, suggesting the disorder may be heterogeneous. One-half to two-thirds of IH patients have elevated serum 1,25(OH)2D levels. The remainder with normal serum 1,25(OH)2D levels cannot be distinguished from those with elevated levels as intestinal Ca absorption is just as high, and negative Ca balance may develop during low Ca intake. Of particular importance is the observation that all of the changes in Ca metabolism characteristic of IH can be induced by the administration of small doses of 1,25(OH)2D3 to healthy volunteers.

Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10071-X

An animal model of genetic hypercalciuria has been developed in Sprague-Dawley rats by breeding hypercalciuric males and females. The hypercalciuria is now present in all offspring and all form Ca-containing kidney stones. The hypercalciuria in genetic hypercalciuric stone-forming (GHS) rats is due to increased intestinal Ca absorption and bone resorption and decreased renal Ca reabsorption. Elevated vitamin D receptor (VDR) content in intestinal mucosa, renal tubules, and bone cells strongly supports the concept that the genetic hypercalciuria is a state of excess vitamin D receptor. A post-transcriptional dysregulation of VDR is suggested by increased VDR mRNA and VDR protein that has normal binding affinity for 1,25(OH)2D3. The nature of the genetic defect in GHS rats and in human IH remains unknown.

IDIOPATHIC HYPERCALCIURIA Overview Hypercalciuria is common among patients with Ca oxalate nephrolithiasis and is thought to contribute to stone formation by increasing the state of urine supersaturation with respect to Ca and oxalate. Flocks [1] first commented on the frequency of hypercalciuria among patients with Ca stones; however, it was not until the mid-1950s that Albright and Henneman [2,3] defined the condition of IH as hypercalciuria with normal serum Ca, no systemic illness, and no clinical skeletal disease. The definition of hypercalciuria is arbitrary and based on the distribution of urine Ca excretion values among unselected populations of healthy men and women in Western countries [4,5]. The distribution of urine Ca forms a continuum that is clustered about a mean with a long tail of higher values. IH patients are those whose urine Ca exceeds the arbitrary upper limit of normal,

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which is most commonly defined as greater than 300 mg/24 h for men, greater than 250 mg/24 h for women, or greater than 4 mg/kg body weight or 140 mg Ca per gram urine creatinine for either sex [6]. Using these definitions, hypercalciuria is found in about 50% of calcium oxalate stone-formers [6,7] and is the most common cause of normocalcemic hypercalciuric stone formation [7]. The diagnosis of IH requires the exclusion of known causes of normocalcemic hypercalciuria (see Table 71.1). Surveys of stone-formers attending kidney stone clinics report a high proportion with kidney stone formation among first-degree relatives [8,9]. A genetic basis of IH was further suggested by subsequent surveys [10e12] that revealed a strong familial occurrence of IH with high rates of vertical and horizontal penetrance (see Fig. 71.1) consistent with an autosomal dominant mode of inheritance. IH also occurs in children with the same frequency of occurrence as in adults [13]. That hypercalciuria can have a genetic origin has been clearly demonstrated by breeding experiments in which the offspring of spontaneously hypercalciuric male and female SpragueDawley rats are intensely hypercalciuric [14e16]. Other human hypercalciuric genetic disorders have been described (Chapter 63), but they differ from IH in having a renal phosphate leak [17] that may lead to rickets including renal tubular acidosis [18] and X-linked recessive stone formation with early renal failure [19]. Idiopathic hypercalciuria is a common disorder that affects 5 to 7% of otherwise healthy men and women [4]. If 50% of all stone-formers have IH [6,7], and the frequency of stone disease among adults is 0.5%, then 80 to 90% of IH is asymptomatic and never associated with stone formation. The increased frequency of osteopenia in IH patients (see “Low bone mass,” below) suggests that hypercalciuria may be an important pathogenetic factor for development of low bone mass even among those who do not form stones.

TABLE 71.1

Causes of Normocalcemic Hypercalciuria

Paget’s disease Sarcoidosis Hyperthyroidism Renal tubular acidosis Cushing’s syndrome

Pathogenesis of Human Idiopathic Hypercalciuria Renal Histopathology in Calcium Oxalate Nephrolithiasis Interstitial crystal deposition at or near the tips of papillae is found in 100% of kidneys of Ca oxalate stone-formers who have IH and no systemic cause of hypercalciuria or other cause of stone formation (Table 71.1). Less frequently (43%), non-stone-formers may have such papillary depositions [20]. On biopsy, these lesions first described by Randall [21] have recently been found to be composed of Ca phosphate (apatite) and contain no Ca oxalate [22]. The plaques originate in the basement membrane of the thin loops of Henle and spread through the interstitium to just beneath the urothelium. There is no inflammatory cell infiltrate to suggest tissue reaction or cell injury. On transmission electron microscopy, lesion particles are laminated microspheres composed of apatite crystals alternating with organic matrix that comes together to form a syncytium. The syncytium extends to the sub-urothelial space and can be seen from the urolithelial side as the white plaque Randall described. Osteopontin has been found in the plaque and may play a role in possible tissue injury response or mineral deposition [23]. The absence of Ca phosphate or Ca oxalate crystal deposition within the renal tubule lumen in IH stoneformers strongly suggests that the Ca phosphate plaque appears to serve as a site onto which Ca oxalate crystals attach, grow, and form clinical Ca oxalate stones [22,24]. The role of hypercalciuria in the development of the Ca phosphate interstitial lesions of Randall’s plaques remains unknown; however, the Ca phosphate plaques are rather specific for Ca oxalate stone-formers, as the interstitial lesions are absent in intestinal bypass patients who form Ca oxalate stones [22]. Sub-urothelial plaque is also found in stone-formers with primary hyperparathyroidism, ileostomy, and small bowel resection, and in brushite stone-formers. Brushite (CaHPO4 2H2O) is a unique form of Ca phosphate stone that has a tendency to recur if patients are not aggressively treated. In five patients with primary hyperparathyroidism and Ca phosphate stones, renal cortical and papillary histopathology showed both dilation and plugging of ducts and papillary deformity characteristic of Ca phosphate stone-formers. Interstitial plaque and stone anchoring characteristic of IH Ca oxalate stoneformers were also present. Thus, the two different pathogenetic processes of stone formation may occur in the same individual [25,26].

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Increased Intestinal Calcium Absorption Normally, the quantity of Ca absorbed is determined by dietary Ca intake and the efficiency of intestinal Ca

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FIGURE 71.1

Family pedigrees of nine probands with idiopathic hypercalciuria. Solid circles and solid squares are females and males with hypercalciuria; S is stone formation; * indicates children (younger than age 20). Arrows indicate probands from each family. Dashed symbols are relatives who were not studied. Hypercalciuria occurred in 11 of 24 siblings, 7 of 16 offspring, and 1 of 3 parents of probands. Reprinted by permission of The New England Journal of Medicine [10]. Copyright 1979, Massachusetts Medical Society.

absorption [27]. Absorption of Ca across the intestine is the sum of two transepithelial transport processes: a non-saturable paracellular pathway and a saturable, cellular active transport system [28,29] (see also Chapters 19 and 34). Absorption via the paracellular path is diffusional and driven by the lumen-toblood Ca gradient [27]. The cellular pathway is vitamin D dependent and is regulated by the ambient concentration of 1,25(OH)2D. Thus, intraluminal Ca concentration and tissue 1,25(OH)2D levels are the driving forces for Ca translocation via the paracellular and cellular pathways, respectively. Increased intestinal Ca absorption has been found in most patients with IH [30e38]. Using either a single oral dose of Ca isotope to measure fecal isotope excretion or double Ca isotope administration in which the intravenous dose adjusts for isotope distribution, IH patients have an increase in the Ca absorptive flux (Fig. 71.2). External Ca balance studies conducted while IH patients and normal non-stone-formers ingested diets containing comparable amounts of Ca show net intestinal Ca absorption rates to be greater in IH

patients [39]. Biopsies of proximal intestinal mucosae following oral Ca isotopic administration reveal increased mucosal accumulation of isotope compared

FIGURE 71.2 Intestinal Ca absorption in healthy volunteers and patients with IH. Absorption rates are expressed as percentages of dietary Ca absorbed as calculated from the appearance of Ca isotopes in blood or fecal collections. Values are means (horizontal bar)
2 standard deviations. Names indicate references: Birge [31]; Wills [32]; Pak [35]; Kaplan [36]; and Shen [37].

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to normocalciuric non-stone-formers [40]. Thus, by all techniques used, IH is characterized by increased intestinal Ca absorption. Elevated 1,25(OH)2D Kaplan and colleagues [36] first reported elevated serum levels of 1,25(OH)2D in a group of patients with IH. Subsequently, others have confirmed that, on average, serum 1,25(OH)2D levels are higher in IH (Fig. 71.3). Increased in vivo conversion of tritiated 25hydroxyvitamin D3 (3H-25(OH)D3) to 3H-1,25(OH)2D3 with normal metabolic clearance [41] in a group of IH patients with elevated serum 1,25(OH)2D levels indicate that the increase in serum 1,25(OH)2D levels in some IH patients results from increased production by increased conversion of 25(OH)D to 1,25(OH)2D. Of note is the considerable overlap of serum 1,25(OH)2D levels between IH patients and non-stone-formers (Fig. 71.3). Thus, for about 50% of IH patients, increased intestinal Ca transport may be driven by increased circulating 1,25(OH)2D. For the remainder, other mechanisms of increased Ca absorption must be considered in the presence of normal circulating 1,25(OH)2D. The mechanism whereby 1,25(OH)2D production is increased in IH is unknown. The major regulators of renal proximal tubule mitochondrial 25-hydroxyvitamin D 1a-hydroxylase (1a-hydroxylase) activity include PTH, phosphate depletion, and insulin-like growth factor-I (IGF-I) (see Chapters 3 and 45). However, only 5% of IH patients have elevated circulating PTH levels [35,42], and urinary cAMP levels,

a surrogate measure of PTH, are normal in most patients [35,43,44]. Mild hypophosphatemia with reduced renal tubular phosphate reabsorption has been described in as many as one-third of IH patients [36,37,42,45]. A strong inverse association between serum 1,25(OH)2D levels and renal tubular phosphate reabsorption has been reported [37,44]. As elevated PTH or hypophosphatemia accompany elevated serum 1,25(OH)2D in only a minority of patients; the cause of increased serum 1,25(OH)2D in most patients with IH remains unknown. Detailed studies of IGF-I have not been performed. The description of mutations in the q23.3eq24 region of the first chromosome in three kindreds with absorptive IH [46] involves a region containing a gene that is analogous to the rat soluble adenylate cyclase gene. This first description of specific base pair substitutions suggests the possibility of a gene defect associated with IH that may involve altered receptor signaling. Whether this mutation alters functions related to the regulation of the 1a-hydroxylase remains to be determined. However, caution has been expressed in accepting this report as conclusively demonstrating that the substitutions or a mutation of this gene causes IH [47]. Serum 1,25(OH)2D values in normal subjects and IH patients overlap extensively in each series reported (Fig. 71.3). Kaplan et al. [36] found that in patients with absorptive IH (defined as normal fasting urine Ca and normal or elevated serum 1,25(OH)2D) intestinal Ca absorption measured by fecal excretion of orally administered 47Ca was increased out of proportion to the simultaneously measured serum 1,25(OH)2D concentration (Fig. 71.4B). In contrast, a strong positive correlation between intestinal Ca absorption and serum 1,25(OH)2D is found in normal volunteers, normocalciuric stone-formers, patients with primary hyperparathyroidism, and IH patients with fasting hypercalciuria (Fig. 71.4A). The high intestinal Ca absorption rates with either normal or elevated serum 1,25(OH)2D levels suggest that the pathogenesis of IH is heterogeneous, with at least one phenotype resulting from 1,25(OH)2D overproduction. Decreased Renal Calcium Reabsorption

FIGURE 71.3 Plot of means
2 SD of serum 1,25(OH)2D in IH patients and non-stone-formers. Horizontal bar is mean of group. Names indicate references: Kaplan [36]; Shen [37]; Insogna [41]; Coe [43]; Gray [45]; Van Den Berg [72]; and Breslau [73].

A defect in the tubular reabsorption of Ca, a so-called renal leak of Ca, has been postulated as a primary event in the development and maintenance of hypercalciuria in IH. Two reports [48,49] found a greater fraction of filtered Ca excreted in the urine of IH patients compared to non-stone-formers. The values were calculated from inulin clearance or creatinine clearance and used blood ionized Ca as an estimate of ultrafilterable Ca. Although urinary sodium (Na) excretion is a major determinant of Ca excretion in normal and IH patients, there is no evidence that patients over-ingest or over-excrete Na.

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FIGURE 71.4 Relationship of calcium absorption to 1,25(OH)2D levels. (A) Fractional intestinal absorption of oral

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Ca versus serum 1,25(OH)2D level in normal controls (open circles), normocalciuric stone-formers (NN, filled circles), IH stone-formers who have fasting hypercalciuria and elevated PTH (RH, filled squares), and patients with primary hyperparathyroidism (PHPT, open squares). (B) Fractional calcium absorption of IH patients with absorptive hypercalciuria (normal fasting urine Ca) superimposed on the 95% confidence limits for the relationship in controls. Reproduced from [36] by copyright permission of the American Society for Clinical Investigation.

Hydrochlorothiazide and acetazolamide increase urine Ca, Na, and magnesium (Mg) excretion in IH compared to normals [50], suggesting a generalized defect in proximal tubule electrolyte and water transport in IH patients. Studies of the patterns and timing of urine Ca excretion in IH stone-formers and controls while fed three meals daily demonstrate that hyperabsorption of dietary Ca is rapidly followed by enhanced excretion of Ca into the urine with the bulk of 24-hour urine Ca excretion occurring postprandially during the waking hours. The marked increase in urine Ca excretion after meals is due to decreased fractional tubular Ca reabsorption in IH and control subjects with greater changes in IH subjects. The increased Ca excretion is independent of sodium excretion, and serum PTH levels do not differ between control and IH subjects and cannot explain the greater prandial fall in tubule Ca reabsorption in IH. Serum magnesium and phosphorus levels in IH are below controls throughout the day, and tubule phosphate reabsorption is lower in IH after meals. The studies indicate that the primary mechanism whereby postprandial urine Ca increases is reduced tubule Ca reabsorption, and IH differ from controls in the magnitude of the response [51]. In IH stone-formers, the reduction in postprandial proximal tubule reabsorption of sodium and Ca is matched by increased distal reabsorption so that urine sodium excretion is not different between normals and IH. Distal Ca reabsorption is not sufficiently increased to match Ca delivery, so hypercalciuria results. Urine Ca excretion and overall renal fractional Ca reabsorption are high in IH when adjusted for distal Ca delivery, which strongly suggests a reduction in both distal

as well as proximal Ca reabsorption. These new findings indicate that IH results from a multi-site, presumably genetic-mediated alteration in tubule transport. The increased Ca delivery into the distal nephron may be involved in apatite plaque and stone formation through deposition of apatite within the papillary interstitium [52]. Ca oxalate stone formation depends on urine ion concentrations and state of supersaturation, which are heavily influenced by an imbalance between water excretion and the excretion of the insoluble stoneforming salts and subsequent high concentrations that supersaturate the urine and inner medullary collecting duct (IMCD) fluid. Activation of the IMCD apical membrane calcium-sensing receptor (CaSR) has been postulated to modulate urine volume, and the VDR is a regulator of CaSR gene expression. However, CaSR activation does not appear to be a key regulator in protecting against stone formation [53]. The CaSR is described in detail in Chapter 24. Low Bone Mass Abnormal skeletal metabolism in IH has been demonstrated by low bone mineral density of the distal radius [54,55] and lumbar spine [56e58] and by lower skeletal Ca content by neutron activation analysis [59]. Reports differ as to possible pathogenesis, with low bone density found only in those with renal leak hypercalciuria in one study [55], and in those with absorptive hypercalciuria in another study [57]. Information on bone dynamics is limited to one early study in which 47Ca labeling showed increased bone turnover, with bone resorption and formation both enhanced [60]. Two studies of bone histology showed reduced bone apposition rate,

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delayed mineralization of osteoid seams, and prolonged mineralization lag time and formation period [61,62]. These observations suggest defective mineralization, which may be caused by hypophosphatemia in some patients. The observations are also consistent with a defect in osteoblastic function. Measurements of biochemical markers of bone turnover reveal increased urine hydroxyproline excretion in unselected IH patients [63] and increased serum osteocalcin in IH patients with renal but not absorptive hypercalciuria [64]. Whether the low bone density is a result of the lifelong hypercalciuria, habitual low Ca intake, or a genetic defect in osteoblast or osteoclast function independent of urine Ca excretion remains to be determined. In a study of 59 subjects from 11 families with at least one member a hypercalciuric Ca oxalate stone-former [65], lumbar spine and femoral neck bone density z-scores varied inversely with urine Ca and urine ammonium in the stone-formers but not in the non-stone-formers (Fig. 71.5). There were no correlations of z-score for bone turnover markers or serum 1,25(OH)2D levels. Ca consumption was lower in stone-formers, suggesting that the admonition to ingest a low Ca diet to avoid more stones in fact predisposes to bone loss. A 3-year follow-up of BMDs of the same subjects showed that the changes over time in BMD femoral neck z-score and to a lesser extent in spine z-score can be predicted by the baseline 24-hour urine calcium excretion in hypercalciuric stone-formers and their first-degree relatives (Fig. 71.6). The strong predictive value of urine Ca excretion strengthens the concept that BMD changes in IH are indeed linked closely with hypercalciuria itself through mechanisms that are yet to be determined. Markers of bone turnover, serum 1,25(OH)2D levels and urine ammonium and sulfate did not predict bone loss, nor did Ca intake. Early identification of those stone-formers at greatest risk of bone loss may provide greater incentive for the treating physician to monitor BMD over time and provide dietary and pharmacologic intervention as needed. In the current study, bone turnover markers including serum bone-specific alkaline phosphatase (BSAP), urine hydroxyproline (OHP), and urine collagen breakdown products, were normal and not useful in predicting bone loss. Other studies of IH bone disease have reported either normal or slightly elevated turnover markers; however, the inconsistency of the bone marker data across studies of IH patients differs from the highenormal or modestly elevated bone marker levels found in untreated postmenopausal women. In general, metabolic bone diseases with high bone turnover are associated with high rates of bone resorption and formation rates that lag behind, thereby permitting net bone loss. In IH bone disease, levels of bone turnover markers

are not elevated systematically and suggest that bone formation may be suppressed and bone resorption may not be elevated. Therefore, bone turnover rates may not play as critical a role in creating low bone mass in IH as it does in postmenopausal osteoporosis and other metabolic bone disorders. Cvijetic et al. [67] followed BMD over time in 34 male recurrent stone-formers, nine of whom were hypercalciuric, and compared them to 30 normal male subjects. One year later BMDs of the spine and femoral neck were not different between the groups. Significant correlations were noted between loss of BMD at the femoral neck and diet calcium intake, urinary uric acid excretion, and age of the subjects. The 1-year data are consistent with IH bone disease being a state of low bone turnover that may require some time to manifest low bone mass and increased fracture risk. The well-documented low bone mass in IH patients is associated with increased fracture risk [68]. Reduction of urine Ca during thiazide therapy has been studied in a small number of IH patients and found to be effective in improving bone mass (see “Thiazides,” below). Genetic Contributions to Stone Disease Genetic studies have been conducted on small numbers of subjects often with familial nephrolithiasis, and allelic variants of VDR and CaSR have covaried with stones in some studies but not others. However, the associations are weak, and reproducibility has been difficult. A recent genome-wide association study has yielded potential new important insights [69]. The study of 3773 cases and 42 510 controls from Iceland and the Netherlands reveals common, synonymous variants in the CLDN14 gene that are associated with kidney stones. The claudin genes encode a family of proteins important in tight junction formation and function in a number of tissues as diverse as the kidney and cochlear structures of the inner ear. Sixty-two percent of the general population are homozygous for rs219780[C] and this variant increases the stone risk 1.64-fold compared to noncarriers. The same variants were also found to associate with reduced BMD at the hip. These findings suggest that reduced claudin protein function in kidney and perhaps intestine may alter Ca transport and predispose to Ca kidney stone formation. The data also suggest that both Ca stone formation and low BMD are specifically mediated through the variants in CLDN14 rather than a more general relationship between the two phenotypes. However, the presence and location of CLDN14 expression in bone is unknown, and an alternative explanation is that altered renal Ca transport and urine Ca losses are proximate events in development of low bone mass independent of a direct involvement of the CLDN14 gene in bone. In the kidney CLDN14 is expressed in the proximal convoluted tubule and loop

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FIGURE 71.5 Femoral neck and lumbar spine bone density z-scores and urine calcium excretion in IH stone-formers and non-stone-formers. (A). Non-stone-formers femoral neck z-scores. (B). Non-stone-formers lumbar spine z-scores. (C). IH femoral neck z-scores. (D). IH lumbar spine z-scores. Men are solid circles and women are sold triangles. For IH, BMD at both sites varied inversely with urine calcium. Reproduced from [65] with permission from Nature Publications Group.

FIGURE 71.6 Relationship between change in bone density z-score and urine calcium excretion in idiopathic hypercalciuria. Left panel: Change in femoral neck z-score over 3 years inversely correlated (r ¼ 0.37, p ¼ 0.02) with initial urine calcium excretion (mg/day). Right panel: Change in lumbar spine z-score inversely correlated (r ¼ 0.28; p ¼ 0.08) with urine calcium excretion. Ellipses of containment are 1.0 standard deviation. Reproduced from [66] with permission from Nature Publications Group.

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of Henle and has been shown to selectively decrease cation paracellular permeability [70]. If defective CLDN14 increases proximal convoluted tubule cation paracellular permeability, then greater distal Ca delivery may be anticipated, where transcellular transport of monovalent potassium ion creates an electrical gradient that favors retention of Ca within the tubule lumen and increased urine Ca excretion. It would be predicted that altered CLDN14 protein would have little or no effect on distal Ca reabsorption. Further studies of CLDN14 in well-characterized phenotypes of kidney stone-formers will be informative. Proposed Pathogenetic Models of Idiopathic Hypercalciuria On the basis of the consistent increase in intestinal Ca absorption, normal or elevated serum 1,25(OH)2D levels, and normal or elevated fasting urinary Ca, Pak and colleagues [71] separated IH into three groups: absorptive, renal, and resorptive. In the first, primary intestinal Ca hyperabsorption (Fig. 71.7A) would transiently raise postprandial serum Ca above normal and increase ultrafilterable Ca. Postprandial hypercalcemia would transiently suppress PTH secretion, resulting in reduced tubular Ca reabsorption and hypercalciuria.

In the second model, a primary renal tubular leak of Ca (Fig. 71.7B) would cause hypercalciuria and a transient reduction in serum Ca. Secondary hyperparathyroidism would normalize serum Ca and increase proximal tubule 1,25(OH)2D synthesis, which would stimulate intestinal Ca absorption. PTH secretion would then decline to the extent that serum Ca is normalized. This scenario predicts that serum 1,25(OH)2D would be elevated in renal IH and normal or elevated in absorptive IH [72,73]. A third possibility is based on a primary overproduction of 1,25(OH)2D3 that increases intestinal Ca absorption and bone resorption (Fig. 71.7C) while PTH remains normal and fasting urine Ca excretion may be normal or elevated. Tests of the Models Knowledge of the pathophysiology of IH is fundamental to developing rational therapy for the prevention of recurrent kidney stones. If the model of primary intestinal overabsorption were correct, then dietary Ca restriction would reduce the amount of Ca absorbed and Ca excreted in the urine without altering bone mass. If a renal leak of Ca were the primary event, or if urinary Ca originates from bone rather than diet, then restricting dietary Ca will have little effect on

FIGURE 71.7 Three proposed models of IH. (A) Absorptive IH with primary intestinal overabsorption, postprandial hypercalcemia, suppressed PTH, and normal fasting urine Ca. Serum 1,25(OH)2D is normal. (B) Primary renal tubular leak of Ca leads to a transient decrease in serum Ca and elevated PTH with secondary increases in serum 1,25(OH)2D and intestinal Ca absorption. Fasting urine Ca is elevated. (C) Primary overproduction of 1,25(OH)2D increases serum 1,25(OH)2D and stimulates intestinal Ca absorption and bone resorption. Serum PTH is normal or decreased, and fasting urine Ca is normal or elevated.

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urinary Ca excretion, while worsening Ca balance and promoting bone loss. Testing certain predictions has assessed the accuracy of the absorptive and renal models. FASTING SERUM PTH AND URINE CA

If repeated episodes of postprandial hypercalcemia suppress PTH secretion sufficiently to cause chronic hypoparathyroidism, fasting serum PTH and urine cAMP would be low and fasting urine Ca elevated. Transient suppression of PTH would permit normal serum PTH, urine cAMP, and fasting urine Ca. In contrast, renal IH requires increased PTH and urine cAMP and increased fasting urine Ca [74]. As existing PTH radioimmunoassays do not differentiate normal from low values, most IH patients have been found to have PTH levels in the normal range. The recent study of prandial urine Ca excretion in IH subjects found serum PTH to be within the normal range and slightly lower than controls, which argues against a regulatory role of PTH in the decreased renal tubule Ca reabsorption [51,52]. Although normal fasting urine Ca is not unusual among IH patients, only 5% have elevated PTH and, therefore, fail to meet the criteria for renal IH [35,71]. Thus, a primary renal leak of Ca with a secondary increase in PTH cannot account for fasting hypercalciuria in a majority of patients. About 24% of patients meet the criteria of absorptive hypercalciuria by reducing urine Ca during fasting to maintain neutral Ca balance [72]. EXTERNAL CA BALANCE

The relationship between net intestinal Ca absorption and 24-hour urine Ca excretion calculated from 6-day balance studies is different in IH patients compared to normal subjects (Fig. 71.8) [75e81]. In non-stoneformers, urinary Ca excretion is positively correlated with net absorption, and overall Ca balance is positive when net absorption is greater than 200 mg per 24 h (see 95% confidence limits calculated from balance studies on normal subjects in Fig. 71.8). Net Ca absorption tends to be greater in IH patients, and for every level of net absorption, 24-hour urine Ca excretion is higher in the patients compared to healthy subjects. In IH patients, a greater portion of absorbed Ca is excreted in the urine. In normal subjects, net Ca absorption exceeds urine Ca excretion, and balance is positive when net absorption exceeds 200 mg/24 h. In contrast, almost 50% of the IH patients have urine Ca excretion in excess of net absorption and are in negative Ca balance, even when allowance is made for some variability in the balance data (
50 mg). Thus, at all levels of net Ca absorption, negative Ca balance (above the zero balance or above the line of identity) is common in IH patients but not in healthy subjects. Negative Ca

FIGURE 71.8

Urinary Ca excretion as a function of net intestinal Ca absorption. Data are derived from 6-day external mineral balance studies. Solid lines indicate the 95% confidence limits about the mean regression line derived from the data on 195 adult non-stone-formers. Individual balance studies performed on 51 patients with IH are shown as open circles. The dashed line represents equivalent rates of urinary Ca excretion and net intestinal Ca absorption (the line of identity). Normal values are from [50,63,77,78]. Values from patients are from [50,62,80e84] and J. Lemann (personal communication, 1992). Adapted from [110].

balance in the presence of adequate Ca intake is incompatible with a primary hyperabsorption of dietary Ca or absorptive hypercalciuria and cannot, by itself, account for the hypercalciuria. URINE CA AND CA BALANCE DURING LOW-CA DIET

The hypothesis of primary intestinal Ca overabsorption predicts that dietary Ca restriction would reduce the amount of Ca absorbed and would therefore reduce urinary Ca excretion. Like normal subjects, IH patients would be in positive or neutral Ca balance when net absorption is above 200 mg/24 h (Fig. 71.8). A low-Ca diet would reduce urine Ca excretion through an increase in PTH secretion, which would promote distal tubular Ca reabsorption. In contrast, patients with a primary renal Ca leak would be unable to conserve urine Ca at any level of Ca intake and would maintain an excessive or inappropriately high urine Ca excretion even during low-Ca diet. As a result, Ca balance during low-Ca diet would shift from positive or neutral to negative or become more negative. Serum PTH would be expected to increase to high levels during a low-Ca diet. To test whether the responses of IH patients fit these predictions, Coe et al. [43] fed a low-Ca diet (2 mg/kg/day) to nine normal volunteers and 26 unselected IH stone-formers. After 10 days on the

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detectable bone loss. The data also suggest that some patients with IH may have diet-dependent hypercalciuria, whereas others have diet-independent hypercalciuria. The two proposed mechanisms cannot be readily distinguished by any clear discontinuity in the distribution of urine Ca values, and serum PTH and 1,25(OH)2D levels do not predict the urine Ca responses during a low-Ca diet. Patients with the highest urine Ca and most extreme negative Ca balance had serum PTH and 1,25(OH)2D levels that were not different from patients who conserved Ca to the levels found in normal subjects. ROLE OF 1,25(OH)2D EXCESS

FIGURE 71.9 Calcium intake and urinary excretion in patients with IH and normal subjects. Urine Ca excretion (UCa) and Ca intake (Cal) are during a low-Ca diet. Mean Ca intakes for patients and controls (2.29
0.15 versus 2.31
0.05 mg/kg/day) were not different. Mean urine excretion rates during low Ca intake and values of Cal-CaE (an index of Ca balance) differed significantly between normals and IH patients. Subjects and patients are arranged in ascending order of urinary Ca excretion. Reprinted by permission of the publisher from [43]. Copyright 1982 by Excerpta Medica Inc.

diet, urine Ca excretion decreased to 2.0 mg/kg body weight or less in both patients and controls, but 17 of the 26 IH patients (Fig. 71.9) showed values greater than the highest value in normal controls. In patients, urine Ca excretion (CaE) ranged from normal to persistently high levels. It exceeded Ca intake (CaI) (Fig. 71.9, CaI e CaE) in 11 of the 26 patients and none of the non-stone-forming controls. Thus, almost 50% of the patients had more Ca in the urine than was provided by the diet and were clearly in negative Ca balance. The results indicate that a chronic low-Ca diet may be detrimental for some patients, as the inability to conserve urine Ca would eventually lead to clinically

The majority of patients are classified as having absorptive hypercalciuria [71,74], yet negative Ca balance during low-Ca diet [43] without elevated PTH, or 1,25(OH)2D, is not predicted by the absorptive model (Fig. 71.7A). Patients who meet the criteria of renal hypercalciuria tend to have higher serum 1,25(OH)2D levels, but only a small portion have elevated PTH levels. Further, serum 1,25(OH)2D levels do not predict whether patients will be classified as absorptive or renal, and at least one-third of patients have normal serum 1,25(OH)2D levels despite intestinal Ca hyperabsorption. For them, the mechanism of intestinal Ca hyperabsorption remains unexplained. One study of ten IH stone-formers and ten age-matched normal subjects found a two-fold elevation of peripheral blood monocyte (PBM) VDR [82]. As PBM may be stimulated to differentiate into mature osteoclasts, in part mediated by VDR, the results suggest that elevated tissue VDR concentrations in the presence of normal circulating 1,25(OH)2D levels may mediate increased vitamin D biologic actions. The model of primary vitamin D excess (Fig. 71.7C) is supported by elevated 1,25(OH)2D production rates and enhanced biological actions of 1,25(OH)2D, including increased intestinal Ca absorption and bone resorption. Creation of a mild form of 1,25(OH)2D excess was achieved by the administration of pharmacological doses of 1,25(OH)2D3 (3.0 mg/day) to healthy men for 10 days while Ca intake varied from low (160 mg) to normal (372 mg) or high (880 mg) [83e85]. Increased urine Ca excretion and net intestinal Ca absorption led to negative Ca balance as calculated from 6-day metabolic balance studies (Fig. 71.10). Dietary Ca strongly influenced the response to 1,25(OH)2D3, as Ca balance was more negative during low Ca intake, and the increase in urine Ca resulted primarily from accelerated bone resorption. At lowenormal or normal Ca intake, 1,25(OH)2D3 administration increased urine Ca and net intestinal Ca absorption, and Ca balance remained neutral. During normal-Ca diet, 1,25(OH)2D3 administration maintained neutral or positive Ca balance.

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FIGURE 71.10 Intestinal Ca absorption, urine Ca excretion, and Ca balance in normal men receiving 1,25(OH)2D3 (hatched bars) or controls (open bars) at varying levels of dietary Ca. Values (mg/24 h) are means
SEM for six men per group. For Ca balance, values above the horizontal line indicate positive balance and those below the line, negative balance. Data from [84] and [85]. Reprinted with permission from [83].

Thus, 3 mg/day of 1,25(OH)2D3, which was insufficient to cause hypercalcemia, during the 10-day study has profound effects on intestinal Ca absorption, urine Ca excretion, and Ca balance. Further, 1,25(OH)2D3 administration caused negative Ca balance only during low Ca intake. l,25(OH)2D3-induced changes in Ca balance in normal subjects are similar to those observed in IH patients at comparable levels of Ca intake. In other experiments, ketoconazole administration to IH patients inhibited renal 1,25(OH)2D biosynthesis [73] and decreased serum 1,25(OH)2D levels, intestinal Ca absorption, and urine Ca excretion. Although ketoconazole has many actions in multiple tissues, it appears that by inhibiting 1a-hydroxylase activity, it can reverse the Ca metabolic defects in IH patients. The results of the effects of 1,25(OH)2D3 treatment and the response to ketoconazole provide further support for a primary 1,25(OH)2D excess in at least some patients with IH. The nature of the disordered regulation of renal 1,25(OH)2D production or action remains to be determined, as neither elevated PTH nor hypophosphatemia were present in responders or were absent in nonresponders to ketoconazole.

GENETIC HYPERCALCIURIC RATS Clinical studies that test the proposed models of human IH including primary intestinal Ca overabsorption, decreased renal Ca reabsorption, and excess vitamin D have been complicated by difficulty in

controlling for potential variables such as genetic and environmental factors that may influence dietary patterns. The availability of an animal model of IH would permit the testing of the three hypotheses under conditions that exclude or control for genetic and dietary influences. The strong familial occurrence of IH in humans and the high frequency of elevated urine Ca in adult men and women suggested that spontaneous hypercalciuria might also be found in animals.

Establishment of a Colony of Genetically Hypercalciuric Rats The distribution of urine Ca excretion in a population of male Sprague-Dawley (SD) rats fed a normal-Ca diet (0.8% Ca) followed a non-Gaussian distribution, which was similar to that found in a population of healthy humans in that values were clustered about the mean with a long tail of higher values [14]. Hypercalciuria (in mg Ca/24 hour) defined as urine Ca greater than two standard deviations above the mean value identified 5 to 10% of male and female rats as spontaneously hypercalciuric. Mating males and females with the most severe hypercalciuria resulted in offspring with hypercalciuria. The most hypercalciuric offspring were used for repeated matings, creating a colony with hypercalciuria that has increased in intensity and frequency with each successive generation [15]. By the twentieth generation, over 95% of males and females were hypercalciuric. By the fortieth generation, mean urine Ca

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excretion was 7.0
0.3 mg/24 h compared to the stable mean excretion of less than 0.75 mg/24 h by wild-type SD rats [87]. Hypercalciuria is detectable soon after weaning at about 6 weeks (50 g body weight) and persists lifelong. Weight and growth of the hypercalciuric rats are comparable to wild-type SD rats obtained from the same supplier that provided the original spontaneously hypercalciuric animals. No anatomical or structural abnormalities have been identified in GHS rats; however, by 18 weeks of age 100% of the animals have multiple bilateral Ca-containing kidney stones in the upper and lower urinary tracts [87]. No stones are found in the kidney or urinary tract of wild-type SD rats. In a first approach to mapping the genes contributing to hypercalciuria in GHS rats, quantitative trait locus (QTL) mapping of F2 rats from a cross between GHS and normocalciuric WKY rats was undertaken [88]. A Ca excretion QTL (hypercalciuria 1 (HC1)) was identified with a logarithm of odds (LOD) score of 2.91 on chromosome 1. The microarray data for the HC1 congenic rats clearly demonstrate the perturbation of Ca metabolism and transport pathways defined in the GHS rats. The HC1 QTL region contains at least one gene that contributes either directly or indirectly to hypercalciuria in the GHS rat model, which was previously estimated to contribute 7% of the variation in Ca excretion. To date a putative HC1 gene or other genes that may contribute to the hypercalciuria has yet to be identified.

Serum and Urine Chemistries Serum Ca and Mg are within the normal range in the colony now referred to as genetic hypercalciuric stoneforming (GHS) rats [15]. Serum phosphate is lower in female rats, and there is no difference between GHS and wild-type males and females. Serum PTH levels in GHS rats are not different from controls. Urine volumes are greater in the GHS rats.

Renal Ca Handling During an adequate Ca diet, urine Ca excretion was greater in the prandial (0e3 h) and postprandial (3e6 h) periods when the GHS rats were offered their daily food as a bolus compared to when given three equal portions. Total 24-hour urine Ca excretion was greater in the GHS rats fed as a bolus compared to Ca excretion by rats given a similar amount of food in divided doses [90]. Of particular note is that the bolusfed rats excreted more Ca in the urine over the entire 24 hours than excreted by rats fed the same amount of Ca in three divided portions. Urine Ca excretion increased after meals in GHS and NC rats due to a fall in the fractional reabsorption of Ca and fell further in the GHS rats just as the idiopathic stone-formers had

greater reductions in renal tubule Ca reabsorption than normal controls. If peak supersaturation drives Ca oxalate or Ca phosphate stone formation, then it would be predicted that less stone formation would appear in GHS rats fed in a divided manner. Calcimimetics, such as cinacalcet (Cin), are small organic molecules that act as allosteric activators of the CaSR, which increases sensitivity of the CaSR to serum Ca and substantially lowers PTH levels. While Cin reduced serum PTH and Ca in the GHS and control rats, it did not alter urine supersaturation with respect to CaOxalate or phosphate (CaHPO4) in either strain [91]. Similar responses have been observed in humans treated with Cin. In rats fed a diet low in Ca (0.02% Ca), serum PTH was lower in GHS rats than in NC rats, suggesting that even during reduced dietary Ca intake, hypercalciuria in GHS rats persisted and was driven more by enhanced intestinal Ca absorption and/or bone resorption than by reduced renal tubular Ca reabsorption.

Mineral Balance Six-day external balance studies performed while the animals were fed a normal-Ca diet showed the animals to be in positive balance for Ca, Mg, and phosphorus [15] with greater net Ca absorption in GHS rats. The GHS rats maintained positive Ca balance because the increased urine Ca excretion was matched by a greater net intestinal Ca absorption.

Intestinal Calcium Transport To investigate the mechanism of the increased net Ca absorption, segments of proximal duodenum were mounted in vitro in modified Ussing chambers, and transepithelial bidirectional fluxes of Ca were measured in the absence of electrochemical gradients [22]. Under these conditions [15], duodenal segments from GHS rats had a five-fold increase in the mucosal-to-serosal (absorptive) transepithelial flux of Ca (Jms), whereas the secretory flux of Ca from serosa to mucosa (Jsm) was only mildly increased compared to wild type (Table 71.2). As Ca Jms was 10 to 12 times higher than Ca Jsm, changes in Jsm had a non-significant effect on net Ca absorption (Ca Jnet).

Serum 1,25(OH)2D Circulating 1,25(OH)2D levels were lower in the fourth-generation GHS rats; however, the differences disappeared by the tenth generation (at 190 g, mean
SD serum 1,25(OH)2D was 135
12 versus 174
19 pg/ml (P,NS), and no subsequent differences in serum 1,25(OH)2D levels have been observed [16]). In vitro

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1371

The increased intestinal Ca transport and normal serum 1,25(OH)2D levels in GHS rats suggested either that Ca transport was being stimulated by an unidentified, vitamin D-independent process or that 1,25(OH)2D action was being amplified at the level of vitamin D target tissues. As 1,25(OH)2D stimulates Ca

transport by binding to the vitamin D receptor (VDR) that in turn upregulates vitamin-D-dependent genes that encode for proteins involved in transepithelial Ca transport, and because the biological actions of 1,25(OH)2D are directly related to the tissue VDR content [92e94], VDR binding in intestinal epithelial cells was measured. Duodenal cytosolic fractions prepared in high-potassium buffer from male GHS rats bound more 3 H-1,25(OH)2D3 than comparable fractions from wildtype control rats [16] (Fig. 71.12). Cytosolic fractions from kidney cortex and from splenic monocytes also exhibited greater specific binding of 1,25(OH)2D3. Scatchard analysis of the specific binding curves revealed a single class of VDR-binding sites in tissues from both wild-type and GHS rats. The number of VDR-binding sites in GHS rat duodenal cells was double that found in cells from wild-type rats (536
73 versus 243
42 fmol/mg protein; n ¼ 8 and n ¼ 14; p < 0.001), with comparable affinity of the receptor for its ligand (0.33
0.01 versus 0.49
0.01 nM; non-significant). A two-fold increase in VDR-binding sites was also found in GHS rat renal cortical homogenates [16]. Using Western blotting, homogenates of duodenal mucosa from GHS rats contained a band at 50 kDa that comigrated with duodenal extracts from wild-type rats and with recombinant human VDR. The bands from the GHS rat tissues were more intense compared to controls, confirming that the increase in specific 3 H-1,25(OH)2D3 binding was due to an increase in VDR protein. Northern analysis of RNA extracts from GHS and wild-type rat tissues revealed a single species of VDR mRNA at 4.4 kb with no difference in migration between the two groups [16]. Duodenal extracts from

FIGURE 71.11 Duodenal Ca net flux (Jnet) as a function of serum 1,25(OH)2D for hypercalciuric and normocalciuric male (open and filled squares, respectively) and female (open and filled circles, respectively) rats. Jnet and serum 1,25(OH)2D were correlated for male and female normocalciuric rats (r ¼ 0.789, n ¼ 12, p < 0.001, solid line) and for male and female GHS rats (r ¼ 0.500, n ¼ 17, p < 0.03, dotted line). The regressions were different (F ratio ¼ 5.469, p < 0.015). Reproduced from [15] by copyright permission of the American Society for Clinical Investigation.

FIGURE 71.12 Specific binding of 3H-1,25(OH)2D3 to duodenal cytosolic fractions (VDR) prepared from GHS rats (filled circles) and wild-type controls (open circles) while fed a normal Ca diet. Values are means
SEM for four observations per concentration point. *, p < 0.05; **, p < 0.01; ***, p < 0.005 versus controls. Reproduced from [16] by copyright permission of the American Society for Clinical Investigation.

TABLE 71.2

In vitro Bidirectional Duodenal Calcium Active Transport*

Flux

NM

GHM

NF

GHF

Jms

51 þ 12

264 þ 27

29 þ 9

258 þ 40

Jsm

11 þ 2

19 þ 2

14 þ 2

23 þ 2

Jnet

40 þ 11

245 þ 28

14 þ 8

235 þ 40

* Values are means
SE for 5 to 11 rats per group. NM and NF are normocalciuric (wild-type) male and female rats, respectively. GHM and GHF are genetic hypercalciuric male and female rats, respectively. Jms and Jsm are mucosal-to-serosal and serosal-tomucosal fluxes of Ca, respectively. Jnet is net Ca absorption, where Jnet ¼ Jms e Jsm. Adapted from [16] and reproduced with permission from The Journal of Clinical Investigation, by copyright permission of the American Society for Clinical Investigation.

duodenal net flux (Jnet, equal to Jms  Jsm) for Ca was positively correlated with serum 1,25(OH)2D in normocalciuric and GHS male and female rats (Fig. 71.11). However, the regression coefficients were different for the wildtype and GHS rats, with the latter having a steeper slope. Ca Jnet was greater in GHS rats with serum 1,25(OH)2D levels comparable to the wild-type rats, strongly suggesting that duodenal Ca-transporting cells in GH rats are more sensitive to 1,25(OH)2D.

Vitamin D Receptor

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GHS rats contained less VDR mRNA than controls. Estimates of duodenal cell transcription rates using standard nuclear run-on assays found no clear difference between GHS rats and controls [16]. The in vivo halflife of the VDR mRNA in GHS rat duodenum was comparable to that of controls (6 h). Administration of a small dose of 1,25(OH)2D3 (30 ng as a single dose) resulted in a significant elevation of VDR message and prolongation of message half-life in GHS rats but not controls [95,96]. Thus, in GHS rat intestine, the increased VDR level is not due to an increase in VDR gene transcription. The data are consistent with either an increase in VDR mRNA translation efficiency or changes that result in a prolongation of the VDR half-life. The increased accumulation of the vitamin-D-dependent calbindin-D9K found in GHS rat duodenum [16] is evidence that the increased level of VDR is functional and that the increased Ca transport is likely a vitaminD-mediated process. VDR protein levels in GHS rat duodenum and kidney (Fig. 71.13) in GHS rats from generation 80e90 are greater [97] than reported from earlier generations [16,95,96]. In the recent studies [97], VDR protein levels were elevated 9.9-fold in jejunum and 6.2-fold in ileum from GHS rats compared with NC rats (Fig. 71.13A). GHS rats had elevated VDR protein levels in duodenum, jejunum, ileum, and kidney cortex. In contrast to the initial measurements of VDR mRNA in the early generations of offspring [16,95,96], the current study of generations 80e90 [97] reveals significant increases in VDR gene expression. While the early generations of GHS rats had low levels of VDR mRNA by Northern blotting, the most recent generations of GHS rats have duodenal VDR mRNA levels that are increased 3.1-fold above controls [97]. The increases in VDR gene expressions from the early to the recent generations accompanies the increases in urine Ca excretion that ranged from 2.0 mg per 24 h in the early

generations to 8 to 12 mg/24 h in the current generations (control rats are 0.5 to 1.0 mg/24 h urine Ca). Whether the increases in urine Ca excretion are mediated by the greater expressions of VDR is plausible but remains to be determined. Compared to VDR gene exression rates, duodenal VDR protein levels are now increased 6.3-fold. In GHS rat kidney, VDR mRNA and protein levels were raised 2.5- and 5.5fold, respectively. The greater magnitude increase in VDR protein compared with mRNA in GHS rat duodenum and kidney supports previous observations [16,96] that increased VDR levels may be due to a combination of enhanced transcriptional regulation, greater efficiency of VDR translational events, and prolonged half-life and stability of the VDR protein. Indeed, prolonged half-life of the GHS rat duodenal and renal cortical VDR protein was described in a previous in vivo study [96]. To assess the genetic molecular basis for the elevated VDR, kidney genomic DNA from four GHS and two NC rats were subjected to DNA sequencing. The results revealed no mutation, polymorphism, or splicing variant in the 50 UTR exons, exons, intron/exon boundary regions, or 30 UTR exons of the VDR. There was no difference in the DNA sequence of the proximal 2 kb of the VDR proximal promoter region between the two groups. Therefore, the higher mRNA levels of VDR in GHS rats may not result from mutation or allelic variation in the cDNA or proximal promoter regions. Nevertheless, it is possible that differences exist further upstream than 2 kb. Snail nuclear protein has been recognized as a negative regulator of VDR in neoplastic cells and tissues [98]. In GHS rats, Snail mRNA levels were suppressed 7.5-fold in duodenum, 2.2-fold in jejunum, 2.0-fold in ileum, and 6.7-fold in kidney, with only the duodenal and kidney suppressions being statistically significant [97]. In colon cancer cells, upregulated Snail is inversely

FIGURE 71.13 VDR protein and mRNA levels in GHS rat intestine and kidney. (A) Representative immunoblot of VDR in total nuclear protein extracts from GHS and NC (control) rat intestine and kidney. Lanes 1e4 are NC and lanes 5e8 are from GHS rats. Lanes are: duodenum (1 and 5); jejunum (2 and 6); ileum (3 and 7); and kidney cortex (4 and 8). (B) VDR and GAPDH mRNA by real-time PCR for GHS and NC intestinal segments and kidney. Results are individual values with mean as horizontal bars (n ¼ 4 per group). *, p ¼ 0.027; **, p ¼ 0.034; and ***, p ¼ 0.036. Reproduced from [97] by copyright permission of the American Society for Bone and Mineral Research.

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correlated with cellular dedifferentiation and low VDR expression. The mechanism whereby suppression of Snail in duodenum and kidney of GHS rats may enhance VDR expression is centered on the interaction of Snail and E-box binding sites within the VDR proximal promoter [97]. In the GHS rats, we postulate that suppression of Snail removes tonic repression of VDR and permits its overexpression. The cause of reduced Snail expression in GHS rat tissues is not known; however, Snail binding to the VDR promoter in NC was weaker in GHS rats (Fig. 71.13). These results indicate that under the pathologic condition in GHS rats, lower levels of Snail reduced repression of VDR expression. Major questions remain as to the genetic basis of the reduced Snail levels and increased VDR activity. The cumulative evidence suggests that the primary genetic defect does not directly involve the VDR gene.

Low Bone Density In vitro studies of bone resorption using neonatal calvariae from normal and GHS rats show that Ca efflux (a measure of bone resorption) increases in a dose-dependent manner in the presence of 1,25(OH)2D3 or PTH [99]. The doseeresponse curve is much steeper for 1,25(OH)2D3 in calvariae from GHS rats, whereas the doseeresponse curves for PTH-stimulated Ca efflux are not different between control and GHS calvariae. Western blotting showed a four-fold increase in VDR protein from GHS neonatal rat calvariae [99]. Thus, the increase in target tissue VDR exerts biological actions that increase l,25(OH)2D3-dependent bone resorption, which likely contributes to the hypercalciuria. In studies of bone composition, GHS rats fed a diet high in Ca (2.0% Ca) had reduced cortical (humerus) and trabecular (L1eL5 vertebrae) BMDs, whereas lowcalcium diet (LCD) reduced BMDs to a similar extent in both GHS and NC rats [100]. In GHS rats fed HCD, trabecular volume and thickness decreased, whereas LCD increased both osteoid surface and volume 20fold. GHS rats fed HCD had no change in vertebral strength (failure stress), ductibility (failure strain), stiffness (modulus), or toughness, whereas in the humerus, there was reduced ductibility and toughness and an increase in modulus, indicating that the defect in mechanical properties is mainly manifested in cortical, rather than trabecular, bone. GHS rat cortical bone is more mineralized than trabecular bone and LCD decreased the mineralization profile. While fed an adequate Ca diet (0.6%), GHS rats have reduced BMD with reduced trabecular volume, mineralized volume, and thickness, and their bones are more brittle and fracture prone, indicating that GHS rats have an intrinsic disorder of bone that is not secondary to diet.

1373

Response to Low-calcium Diet To test whether the hypercalciuria in GHS rats is the result of a primary overabsorption of dietary Ca, GHS and wild-type control rats were fed diets either normal (0.6% Ca) or low (0.02% Ca) with respect to Ca. During the LCD, urine Ca excretion decreased in both groups (Fig. 71.14); however, urine Ca remained higher in GHS rats and resulted in negative Ca balance [101]. The inability of GHS rats to conserve Ca during low Ca intake excludes overabsorption of dietary Ca as the sole cause of hypercalciuria in GHS rats.

Summary of Pathogenesis in the Genetic Hypercalciuric Rat Figure 71.15 summarizes current knowledge of the pathogenesis of hypercalciuria in the GHS rats. Breeding by selection for hypercalciuria has emphasized a trait in the offspring that likely involves the expression of several genes for full phenotypic expression. To date, none of the genes has been identified. Studies demonstrate increased VDR concentration in intestine, kidney, and bone which may be part of the primary event(s) and cause the hypercalciuria; however, a secondary adaptive increase in VDR to compensate for urinary Ca losses has not been excluded. The elevated VDR gene expression is accompanied by suppression of SNAIL gene, which is known to suppress VDR gene expression. The role of the suppressed SNAIL is not known, but appears to be pivotal in permitting VDR to increase and hence the GHS rats phenotype. Further information is required regarding the renal handling of Ca in GHS rats and whether the GHS genotype results in a primary defect in renal Ca transport.

FIGURE 71.14 Daily urine Ca excretion in nineteenth-generation GHS rats (open symbols) or wild-type control rats (filled symbols) fed a normal Ca diet (NCD, 0.6% Ca, triangles) during days 1e10 followed by either continuation of the NCD (triangles) or feeding of a low Ca diet (LCD, 0.02% Ca, circles). Rats were pair-fed to 13 g of diet per day. Reprinted with permission from [101].

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Breeding for hypercalciuria

Genomic events

Vitamin D receptor

Intestinal Ca absorption

Bone resorption

?

Renal tubular Ca transport

Hypercalciuria

FIGURE 71.15 Proposed series of events that result from breeding selection for hypercalciuric rats. The renal handling of Ca by GHS rats and the role of increased VDR content, if any, in the transport process remain unknown.

CURRENT VIEW OF HUMAN GENETIC HYPERCALCIURIA Striking similarities in Ca metabolism between GHS rats, IH patients, and human volunteers treated with 1,25(OH)2D3 (Table 71.3) strongly support a primary role of excess 1,25(OH)2D biological action in the pathogenesis of human IH. When deprived of dietary Ca, few IH patients conserve Ca to the extent that normals do (Fig. 71.9). The renal IH model predicts ongoing urinary losses of Ca independent of Ca intake, and negative Ca balance during a low-calcium diet. However, most patients have normal, not elevated PTH, as renal IH would require. Therefore, the absorptive and renal models of hypercalciuria cannot explain the response of most patients to a low-Ca diet. In nonstone-formers, 1,25(OH)2D3 administration changes

TABLE 71.3

Pathophysiology of Genetic Hypercalciuria

Parameter

Human

Human

GHS rats

Serum Ca

N

N

N

Serum phosphate

N

N-D

N

Serum 1,25(OH)2D

I

N-I

N

Urinary Ca on NCD

I

I

I

Urinary Ca on LCD

I

N-I

I

Intestinal Ca absorption

I

I

I

Ca balance on NCD

Pos-N

N-Neg

Pos

Ca balance on LCD

N-Neg

N-Neg

Neg

Values for human controls are responses to treatment with 3 mg 1,25(OH)2D3 daily for 7 days compared to pretreatment. GHS, genetic hypercalciuric stoneforming; NCD, normal Ca diet; LCD, low Ca diet; N, normal; I, increased; D, decreased; Pos, positive; Neg, negative.

urine Ca and Ca balance to those observed in a majority of IH patients who have either normal or elevated serum 1,25(OH)2D levels. For some patients, elevated serum 1,25(OH)2D3 increases in intestinal Ca hyperabsorption and urine Ca excretion, and causes negative Ca balance during low Ca intake. The source of 1,25(OH)2D excess is more elusive in patients with normal serum 1,25(OH)2D levels. They may be more similar to the GHS rats in that both have normal serum 1,25(OH)2D, increased intestinal absorption and bone resorption during a low-Ca diet, and low bone density. Whether these changes in human IH are due to increased intestinal, renal, and bone cell VDR content that can amplify the biological actions of normal circulating 1,25(OH)2D levels remains to be determined.

THERAPEUTICS OF IDIOPATHIC HYPERCALCIURIA AND EFFECTS ON CALCIUM METABOLISM Dietary Calcium Restriction Hypercalciuria promotes urine calcium oxalate supersaturation and increases spontaneous crystal formation [102]. The goals of preventive therapy are to reduce Ca oxalate supersaturation by increasing urine volume and decreasing urine Ca excretion. If the pathophysiologic role of 1,25(OH)2D excess or VDR excess is borne out, then ideal therapy may eventually include either a specific 1,25(OH)2D antagonist or an inhibitor of VDR function. In the absence of such agents, therapies will continue to concentrate on lowering urine Ca excretion using thiazide agents to enhance renal tubular reabsorption. Restriction of dietary Ca does not reduce urine Ca oxalate supersaturation but can promote bone loss. Increasing dietary Ca intake may reduce urine oxalate excretion but the rise in urine Ca excretion prevents a decrease in urine Ca oxalate supersaturation. Since the description of IH, dietary Ca restriction has been recommended to lower urine Ca. Dietary Ca restriction or the use of Ca-binding resin to prevent absorption [103] could be efficacious for patients with primary intestinal Ca hyperabsorption (absorptive hypercalciuria). However, it appears that many patients develop negative Ca balance during low Ca intake. For them, chronic dietary Ca restriction and negative Ca balance would eventually cause bone loss, osteoporosis, and increased fracture risk. Reports of lower bone density in IH patients suggest that Ca restriction may only worsen the existing reduction in bone mass. Therefore, treatment with Ca restriction requires knowledge that the patient will likely not conserve urine Ca and will develop negative Ca balance.

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SUMMARY

Thiazides Thiazide and the related chlorthalidone diuretics reduce urine Ca excretion by inducing an NaCl diuresis, which causes volume contraction and decreased Ca delivery to the distal tubule segments [104]. These agents also stimulate distal tubule Ca reabsorption through a direct interaction with the tubule cells [103e105]. Thiazides may decrease or have no effect [34,38,106] on intestinal Ca transport in IH patients, and serum 1,25(OH)2D and PTH levels are not changed by thiazides. In one study, IH patients treated with chlorthalidone for 6 months improved Ca balance to or toward positive by decreasing both urine Ca and intestinal Ca absorption [107], with urine Ca declining to a greater extent than intestinal absorption. The epidemiological studies suggesting that chronic thiazide therapy reduces fracture risk [108,109] may result from druginduced improvement in Ca balance [106] and reduced bone turnover and improved mineralization [64]. The effects of thiazide on urine Ca and bone metabolism are accompanied by a decrease in new Ca stone formation compared to placebo controls [110]. The beneficial effect of thiazide is evident during the second and third year of therapy, when stone recurrence is reduced by about 50 to 80%. The reduction in new stone formation is due to a decrease in urine Ca oxalate supersaturation, as urine Ca declines while oxalate is unchanged. As thiazides can reduce urine Ca excretion and stone formation rates in all forms of IH [111], knowledge of the pathogenesis of IH in each patient may not be required prior to selecting thiazide therapy. Additional management includes adequate hydration to reach 2.0 liters of urine per 24 h. Decreased urine oxalate can decrease urine Ca oxalate supersaturation; however, a low-oxalate diet is difficult for patients to follow, and reduction in intestinal oxalate absorption enhances Ca absorption and urine Ca excretion, resulting in no net change in urine Ca oxalate supersaturation. Urine Ca excretion may be reduced by sustained alkalinization of the urine through increasing citrate delivery. Potassium citrate in multiple divided doses can raise urine pH and reduce urine Ca by creating a soluble Ca citrate complex that lessens Ca available for complexation with oxalate.

RISK OF STONE FORMATION USING VITAMIN D ANALOGS A growing research interest in the cell differentiation and immune modulator effects of vitamin D and analogs may result in their use in a variety of disorders [112e114] (also see chapters in Section IX “Analogs”). However, the development of hypercalciuria and hypercalcemia

may limit the use of the naturally occurring vitamin D metabolites, as well as synthetic analogs [115,116]. While some vitamin D analogs are reported to have little or no hypercalcemic action, hypercalcemia and hypercalciuria may appear at higher doses through the classic vitamin D actions on intestine, kidney, and bone [114,115]. LowCa diets have had only modest beneficial effects to limit hypercalciuria and hypercalcemia and could promote bone loss. It remains to be determined whether the newer vitamin D analogs with less calcemic activity will, in practice, cause less calciuria and a lower risk of kidney stone formation. Until such actions of the vitamin D analogs are known, standard approaches to minimize stone formation should be followed. These include (1) assuring sufficient fluid intake to maintain at least 1.5 to 2.0 liters urine output per day; and (2) if necessary, increasing urine citrate excretion to normal in those with low citrate [102]. While discontinuation or reduction in vitamin D analog treatment would reverse the hypercalciuria, the beneficial effect of vitamin D analog therapy to control tumor growth may well be greater than the relative importance of reversing hypercalciuria and preventing kidney stones. The addition of a thiazide may avoid or minimize hypercalciuria, but hypercalcemia may occur because of thiazide-induced Ca retention.

SUMMARY Idiopathic hypercalciuria (IH) affects 5 to 7% of adults and children and is the single most common cause of Ca oxalate kidney stone formation and also causes low bone mass. All of the metabolic features of IH can be reproduced by the administration of calcitriol to normal adults. There is also evidence of 1,25(OH)2D3 overproduction in some IH subjects. Excess 1,25(OH)2D3 action appears responsible for the intestinal Ca hyperabsorption, increased bone resorption, and decreased renal tubule Ca reabsorption. Serum 1,25(OH)2D3 is elevated in about 60% of patients. Patients with normal serum 1,25(OH)2D3 levels have comparable elevations in intestinal Ca absorption, and the possibility of elevated tissue vitamin D receptor found in GHS rats is suggested by one study in IH subjects which found elevated peripheral blood monocyte levels [82]. Further, GHS rats and IH patients share changes in Ca transport in intestine, bone, and kidney. The GHS model of human IH permits studies of the cellular and molecular mechanisms of hypercalciuria found in humans. In this model, increased levels of VDR protein in duodenum, kidney, and bone may explain the hyperabsorption of Ca, increased bone resorption, and decreased renal tubule Ca reabsorption even in the presence of normal circulating concentrations of 1,25D.

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[116]

VIII. DISORDERS

1379 T. Naveh-Many, J. Silver, Effects of calcitriol, 22-oxa-calcitriol, and calcipotriol on serum calcium and parathyroid hormone gene expression, Endocrinology 133 (1993) 2724e2728. A.J. Brown, J. Finch, M. Grieff, C. Ritter, N. Kubodera, Y. Nishii, et al., The mechanism for the disparate actions of calcitriol and 22-oxacaltriol in the intestine, Endocrinology 133 (1993) 1158e1164.