J Am Soc Nephrol 14: 1082–1095, 2003
REVIEW
Molecular Mechanisms of Primary Hypercalciuria
KEVIN K. FRICK and DAVID A. BUSHINSKY
Department of Medicine, Nephrology Unit, University of Rochester School of Medicine and Dentistry,
Rochester, New York.
Nephrolithiasis, with a lifetime incidence of up to 13% (1–9),
results in significant morbidity as well as substantial economic
costs, not only directly from medical treatment but also indi-
rectly through time lost from work. Approximately 70% of
kidney stones are composed of calcium, generally combined
with oxalate and/or phosphate (1,7). Hypercalciuria is the most
consistent metabolic abnormality found in patients with cal-
cium nephrolithiasis (1–10). Indeed, idiopathic hypercalciuria
(IH), excess calcium excretion with no identifiable metabolic
cause, is found in up to 40% of stone-formers (11) but has an
incidence of less than 10% in the overall population (12). The
elevation in urinary calcium leads to increased supersaturation
with respect to a solid phase, generally calcium oxalate or
calcium phosphate, which increases the propensity to kidney
stone formation (13).
Idiopathic hypercalciuria is an inherited metabolic abnor-
mality (14 –17). In pediatric patients with nephrolithiasis, 73%
had a family history of kidney stones in at least one first-order
or second-order relative, as opposed to a prevalence of 22% in
a control population of pediatric renal and urologic patients
(18). Of the patients with hypercalciuria, the prevalence of
nephrolithiasis in the family history was 69% (18). Coe et al.
(19) found a strong inheritance pattern in patients with neph-
rolithiasis that they conjectured was autosomal dominant. In
support of a genetic basis for hypercalciuria, we have selec-
tively bred a strain of rats for this disorder. After almost 60
generations of inbreeding, all of the rats are hypercalciuric:
they excrete approximately 8 to 10 times as much calcium as
control animals and almost uniformly form kidney stones
(13,16,17,20 –31) (Figure 1). The ability to select for this trait,
hypercalciuria, solidifies the genetic nature of this disorder.
In a normal, nonpregnant, adult, intestinal calcium absorp-
tion is precisely balanced by urine calcium excretion so that the
total amount of body calcium remains constant. Consuming a
typical diet of 20 mmol of calcium per day, approximately 16
mmol are lost to fecal excretion, indicating that 4 mmol are
absorbed through the intestine (Figure 2) (9,32,33). The pri-
mary reservoir of body calcium, the skeleton, contains about 20
Correspondence to Dr. Kevin K. Frick, Research Assistant Professor of Med-
icine, University of Rochester School of Medicine and Dentistry, 601 Elm-
wood Avenue, Box 675, Rochester, NY 14642. Phone: 585-275-3180; Fax:
585-442-9201; E-Mail: Kevin_Frick@URMC.Rochester.edu
1046-6673/1404-1082
Journal of the American Society of Nephrology
Copyright © 2003 by the American Society of Nephrology
DOI: 10.1097/01.ASN.0000062960.26868.17
mol of calcium, of which about 14 mmol per day are ex-
changed through balanced bone formation and resorption. Ex-
tracellular fluid includes another 25 mmol of calcium. The
kidney filters approximately 270 mmol of calcium per day, of
which all but 4 mmol are reabsorbed (3,4). It is logical to
assume that idiopathic hypercalciuria is caused by dysregula-
tion of calcium transport at sites where large fluxes of calcium
must be precisely controlled: these sites are the intestine,
kidney, and bone.
If one could understand the molecular mechanism(s) by
which primary hypercalciuria occurs, clinical investigators
could then screen families of stone formers before the onset of
overt disease and possibly direct treatment at the specific
underlying molecular defect(s) in calcium transport. Compli-
cating the search for genes responsible for hypercalciuria are
the following caveats: mutations in several pathways can be
responsible for hypercalciuria; expression of more than one
gene may need to be altered to cause a discernible phenotype;
and penetrance of the mutation(s) may be incomplete. A single
genetic abnormality responsible for idiopathic hypercalciuria is
unlikely, as there appears to be a continuum in the rates of
calcium excretion between normal and hypercalciuric humans
(7,34) and control and inbred hypercalciuric rats (16,17).
This focus of this review will be the delineation of potential
mechanisms responsible for idiopathic hypercalciuria by study-
ing selected known genetic disorders resulting in excess urine
calcium excretion. We will exclude hypercalciuria resulting
from metabolic abnormalities for which there is no known
genetic defect for calcium transport, such as primary hyper-
parathyroidism, malignancy with production of PTHrP, renal
tubular acidosis, vitamin D toxicity, immobilization, hyperthy-
roidism, and Paget disease (9). We will partition our discussion
into disorders affecting the intestine, the kidney, and the bone,
as these sites are responsible for regulation of calcium ho-
meostasis and molecular defects in these sites of calcium
transport can contribute to hypercalciuria and subsequent stone
formation. Although this is an organ-based separation, we must
recognize that calcium transport pathways are often general-
ized and not limited to one anatomic site. In addition, as studies
of the genetic hypercalciuric rats have shown (see below),
hypercalciuria may involve a dysregulation of multiple calcium
transport systems.
Intestinal Calcium Transport
Approximately 90% of calcium absorption occurs in the
small intestine, whereas the remaining 10% occurs in the
cecum and ascending colon (35). Intestinal calcium absorption
J Am Soc Nephrol 14: 1082–1095, 2003
Figure 1. Urine calcium as a function of generation number in female
genetic hypercalciuric stone (GHS)–forming rats. The most hypercal-
ciuric rats of each successive generation were selectively inbred. Data
represents urine calcium excretion (mean ⫾ SEM) at the indicated
generation number. Calcium excretion increased as a linear function
of generation number over the first 30 generations and then stabilized
at a level eight to ten times that of controls, whose calcium excretion
did not vary over time (13,16,17,20 –31).
Figure 2. Anatomic sites of calcium flux. The figure indicates the
major sites of calcium flux in the body with average amounts of daily
calcium flux indicated. Dietary (D) calcium (Ca) undergoes net ab-
sorption (␣) through the intestine into the extracellular fluid (ECF).
Daily bone resorption (Br) and formation (Bf) are equal. The filtered
load (FL) of calcium is substantially reabsorbed (fr, fractional reab-
sorption) in the kidney, resulting in urine (U) calcium excretion that
is virtually identical to the amount absorbed in the intestine (151).
proceeds through two pathways: a nonsaturable paracellular
pathway thought to predominate when the diet is replete in
calcium, and a saturable vitamin D-dependent transcellular
pathway, which becomes the major pathway when dietary
calcium is limited (Figure 3) (36). The active, transcellular
pathway is downregulated by a diet replete in calcium (37).
Luminal calcium enters the enterocyte at the microvillus border
of the apical membrane via facilitated translocation through an
epithelial calcium channel, either CaT1 (38) or ECaC (39);
diffusion of calcium through the cell is facilitated by binding to
calbindin D9k (40), and extrusion of calcium through the ba-
solateral membrane against an electrochemical gradient is
achieved by plasma membrane Ca2⫹-ATPase (PMCA) (41,42).
Molecular Mechanisms of Primary Hypercalciuria 1083
Figure 3. Absorption of calcium in the small intestine. Calcium from
the diet is absorbed by transcellular mechanisms, especially when
dietary calcium is limited. Calcium enters the enterocyte through an
apical calcium channel (both CaT1 and ECaC are present) and dif-
fuses through the cytoplasm bound to calbindin D9k. At the basolateral
surface, calcium is extruded from the cell by plasma membrane
Ca2⫹-ATPase (PMCA) (32,33).
CaT1 was isolated from a rat duodenal cDNA library using
expression cloning in Xenopus oocytes and assaying 45Ca2⫹
uptake (38). The 727–amino acid protein encoded has se-
quence and structural homology to the calcium store-operated
channels, which include TRP and TRP-like, important in Dro-
sophila vision; mammalian homologs include the capsaicin
receptor, VR1 (43). As expressed in oocytes, the Km for Ca2⫹
was 0.44 mM, consistent with an estimated intestinal [Ca2⫹] of
1 to 5 mM after a calcium-containing meal (38). Rat tissue
screening with Northern blots indicates that a 3.0-kb band of
hybridization to CaT1 was abundant in small intestine and a
6.5-kb band was present in small amounts in thymus, brain,
and adrenal gland; no CaT1 hybridization was detected in
kidney. In situ hybridization to rat intestinal segments indi-
cated a gradient of expression with traversal of the intestine: in
the small intestine, CaT1 RNA was present at highest levels in
duodenum, to a lesser degree in the proximal jejunum, and
absent from the ileum. In the large intestine, CaT1 RNA was
abundant in the cecum while present only at low levels in the
colon. In all segments, CaT1 RNA was consistently expressed
at higher levels in villus tips than in crypts. Barley et al. have
isolated a cDNA probe, ECAC2, from human duodenum that is
90% identical to rat CaT1 and distinct from human ECAC1
(44) and which they conclude is the human homologue to CaT1
(45). In biopsy samples from 20 healthy volunteers, duodenal
RNA levels for ECAC2 correlated with levels of hybridization
for calbindin D9k (r ⫽ 0.48; P ⬍ 0.05) and PMCA1 (r ⫽ 0.83;
P ⬍ 0.001) but not with 1,25(OH)2D3 or 25(OH)D3 levels (45),
consistent with the finding that rat CaT1 RNA levels are not
responsive to treatment with 1,25(OH)2D3 for 15 h (38). How-
ever, two strains of vitamin D-receptor deficient (VDR-KO)
mice both showed RNA levels of CaT1 in duodenum that were
less than 10% of levels in WT controls (46).
CaT1 shares sequence homology to the epithelial calcium
1084 Journal of the American Society of Nephrology
transporter ECaC (39,47), which was isolated by expression
cloning from a rabbit connecting tubule/cortical collecting duct
cDNA library (39). ECaC RNA is present in duodenum at very
high levels and is found as well in the jejunum, kidney, and
placenta (39). In the rabbit duodenum, ECaC protein is present
in high levels in villus tips, whereas it is virtually absent from
crypts, a pattern similar to that seen for CaT1 (48). Double-
immunofluorescent labeling of serial sections of duodenum
indicated that ECaC, calbindin D9k, and PMCA colocalized to
the same cells, with ECaC found apically, PMCA basolater-
ally, and calbindin D9k distributed throughout the cells (48).
Whereas RNA abundance suggests that CaT1 is the principal
epithelial calcium channel in duodenum, ECaC RNA levels are
also decreased in VDR-KO mice in duodenum (46).
Human diseases in which upregulation of intestinal calcium
absorption is clearly the primary lesion are rare. Hypersensi-
tivity to 1,25(OH)2D3 or its metabolites can lead to increased
intestinal absorption (49). Scott et al. (50) have found linkage
of IH with an apparent absorptive component to microsatellite
markers near the VDR locus in a cohort of 47 French-Canadian
pedigrees. Jackman et al. (51) have also found a polymorphism
in the VDR gene in 19 patients with hypercalciuria and a
family history of nephrolithiasis. However, other studies have
found no linkage to VDR in different kindreds with hypercal-
ciuria (52,53). Imamura et al. (54) described two unrelated
children with increased intestinal absorption of calcium, each
with deletion of 4q33-qter and substitution of unknown chro-
mosomal segments. However, human ECaC1 maps to 7q31.1-
q31.2 (55), and ECAC2 is nearby at 7q34-q35 (45). The gene
for PCMA1 is located at 12q21–23 (56), and calbindin-D9k is
on the X chromosome (57). Reed et al. (58) have mapped a
defect in three kindreds with increased intestinal absorption of
calcium to 1q23.3-q24, and they have identified a group of
base substitutions in a putative gene within that locus, four of
which increased the relative risk of disease 2.2-fold to 3.5-fold
(59). The sequence has pronounced homology to the rat soluble
adenylate cyclase gene and transcripts were detected, although
the functionality of this gene has yet to be established (59). It
may be of interest that rat CaT1 has a potential protein kinase
A phosphorylation site near the amino terminus that is absent
from the corresponding region of ECaC (38).
Genetic or acquired renal phosphate wasting will lead to
hypophosphatemia and an increase in 1,25(OH)2D3, resulting
in excess intestinal calcium absorption and hypercalciuria.
Hereditary hypophosphatemic rickets with hypercalciuria
(HHRH) is an example of this pathophysiology (60). Patients
with this disease have decreased reabsorption of phosphate,
high serum levels of 1,25(OH)2D3, and enhanced intestinal
absorption of calcium. These symptoms are similar to the
phenotype of mice with constructed deletion of the gene for the
kidney-specific Na-Pi cotransporter Npt2, although the bone
phenotype is different; while HHRH patients display rickets
and osteomalacia, the Npt2 knockout (KO) mice show a delay
in bone mineralization at 21 d after birth and an increase in
number of trabeculae and decrease in marrow space at 115 d
(61). Npt2 KO mice display higher levels of duodenal mRNA
for ECaC, CaT1, and calbindin D9k, consistent with their
J Am Soc Nephrol 14: 1082–1095, 2003
known increase in intestinal calcium absorption (62). However,
mapping of the Npt2 locus in HHRH kindreds revealed no
mutations that cosegregated with the disease (63,64). To screen
for mutations in Npt2, Prie et al. (65) sequenced the gene from
20 patients with urolithiasis or bone demineralization associ-
ated with idiopathic hypophosphatemia and reduced phosphate
reabsorption. Two patients with Npt2 mutations were identi-
fied, one with a substitution of phenylalanine for arginine 48
(exon 3), and the second with a methionine for valine 147
substitution (exon 5); both patients were heterozygous for these
mutations. Each of these mutations lies in regions conserved
between human, rat, rabbit, mouse, opossum, and flounder.
Sequencing of exons 3 and 5 in 120 controls excluded these
mutations as common polymorphisms. Microinjection of either
mutant RNA into Xenopus oocytes induced a smaller phos-
phate current than did injection of wild-type Npt2 RNA. Co-
injection of mutant RNA plus wild-type RNA led to a smaller
current than wild-type RNA alone, consistent with the domi-
nant negative effect of Npt2 mutation inferred from the geno-
types of the patients.
Another class of molecular defects resulting in hypophos-
phatemia includes X-linked hypophosphatemic rickets/osteo-
malacia (XLR) and its mouse homolog, hyp; autosomal dom-
inant hypophosphatemic rickets/osteomalacia (ADHR); and
tumor-induced rickets/osteomalacia (TIO or OHO) (66). These
hypophosphatemias are characterized by normocalcemia and
normocalciuria and low to normal circulating levels of
1,25(OH)2D3 (66). In 1995, a gene defective in XLH was
discovered and named PHEX, for phosphate-regulating gene
with homologies to endopeptidases, on the X-chromosome
(67). This gene bears the hallmarks of a membrane-bound
metalloproteinase, and more than 150 PHEX mutations have
been documented (68). It was hypothesized that PHEX was
necessary for inactivation of an unidentified peptide, which
decreased renal phosphate reabsorption, and was termed
“phosphatonin”. ADHR and TIO were hypothesized to result
from dysregulated expression of phosphatonin (69). The gene
for ADHR was linked to chromosome 12p13 (70) and identi-
fied as a member of the fibroblast growth factor family,
FGF-23 (71); FGF-23 was also identified as the substance
overproduced by tumors in TIO (72). FGF-23 inhibits renal
epithelial cell phosphate uptake in vitro (73). Mutations found
in patients with ADHR have been shown to alter proteolytic
cleavage sites of FGF-23, which do not allow it to be inacti-
vated (72) by PHEX (73). Serum levels of FGF-23 are elevated
in TIO and XLR (74). A second potential phosphatonin pro-
duced by tumors causing osteomalacia has been identified as
frizzled-related protein-4 (69,75), a member of the family of
Wnt receptors. The mechanism by which frizzled-related pro-
tein-4 overproduction results in hyperphosphaturia is not yet
clear. Other phosphatonins are known to exist: targeted disrup-
tion of the Na⫹/H⫹ exchanger regulatory factor NHERF-1, a
protein that binds both NHE3 and Npt2, results in hyperphos-
phaturia and hypercalciuria (76). Although the total amount of
Npt2 was not affected in NHERF-1 null mice, immunostaining
revealed that, rather than being located on the apical surface,
Npt2 was internalized in vesicles.
J Am Soc Nephrol 14: 1082–1095, 2003
To date, the role of the phosphatonins, FGF-23 and frizzled-
related protein-4, have not been studied in HHRH. Although
HHRH is inherited as an autosomal recessive (60) the known
mutations in phosphatonin pathways have X-linked or autoso-
mal dominant inheritance (75).
Renal Calcium Transport
Calcium Transport in the Proximal Tubule. Urinary
calcium excretion must equal net intestinal calcium absorption
to maintain calcium homeostasis. The kidney filters approxi-
mately 270 mmol of calcium, of which more than 98% must be
reabsorbed to maintain neutral calcium balance (9,32,33). Any
disorder of renal calcium reabsorption leads to hypercalciuria
and the potential for stone formation. Approximately 70% of
reabsorption occurs in the proximal tubule, predominantly
through paracellular pathways with salt and water carrying
calcium from the lumen to the interstitium through the trans-
port mechanism of solvent drag (Figure 4).
Dent Disease (X-Linked Recessive Nephrolithiasis). Dent
and Friedman (77) reported a form of Fanconi syndrome (gen-
eralized proximal tubule transport defects) with hypercalciuria,
low–molecular weight proteinuria, nephrolithiasis, and nephro-
calcinosis. Positional cloning revealed that this disease, as well
as X-linked recessive nephrolithiasis, X-linked hypophos-
phatemic rickets, and idiopathic low–molecular weight pro-
teinuria of Japanese children, are all the results of defects in the
CLC-5 chloride channel (78 – 81). The CLC family of chloride
channels encompasses nine members, including CLC-Kb, in-
volved in some cases of Bartter disease (see below) (82,83) and
CLC-1, the muscle chloride channel, mutated in Thomsen
myotonia (84). These channels are voltage-gated and out-
wardly rectifying, with a high selectivity for chloride. Lutckx
et al. (85) have found by immunohistochemistry that CLC-5 is
localized to the S3 segment of the proximal tubule and to
Figure 4. Reabsorption of calcium in the proximal tubule. Approxi-
mately 70% of filtered calcium is reabsorbed in the proximal tubule
through a paracellular pathway predominantly by solvent drag
(32,33).
Molecular Mechanisms of Primary Hypercalciuria 1085
mTAL. Subsequent subcellular fractionation studies indicate
that CLC-5 is localized to endosomes (85).
To examine the biologic effects of decreased CLC-5,
Luyckx et al. (86) designed a hammerhead ribozyme specific
for CLC-5 and produced transgenic knockdown mice (RZ). By
Western blotting, kidney membranes from RZ mice had an
80% reduction in levels of CLC-5. There was no difference in
serum chemistries between RZ and controls. Urine calcium/
creatinine ratios were significantly higher in male RZ mice as
compared with gender-matched controls, whereas calcium ex-
cretion in female RZ mice was not significantly different from
female controls. Both groups of female mice had greater cal-
cium excretion than the males. When fed a low-calcium diet,
calcium excretion in the male RZ mice was not different from
control male mice. As the mice aged, urine calcium excretion
fell and normalized by 18 wk, hampering efforts to determine
whether the hypercalciuria in the RZ mice caused nephrolithi-
asis or osteopenia.
To further examine the phenotype resulting from CLC-5
deficiency, two other groups made total knockouts of CLC-5 in
mice, producing animals with somewhat divergent phenotypes.
Piwon et al. (87) disrupted exon 5 and eliminated exon 6 of
CLC-5 and substantiated that CLC-5 protein was undetectable
by Western blots in kidney, liver, intestine, or testis. Both
CLC-5 ⫺/y (male knockout) and ⫺/⫺ (female knockout) an-
imals were normocalcemic, normocalciuric, and hyperphos-
phaturic. Serum 25(OH)D3 and 1,25(OH)2D3 levels were re-
duced in both ⫺/y and ⫺/⫺ animals, whereas PTH levels were
not significantly increased. However, urine from ⫺/y and ⫺/⫺
animals contained higher levels of PTH relative to creatinine
(PTH/Cr) and 25(OH)D3/Cr, suggesting urinary loss of calci-
um-mobilizing proteins. SDS-PAGE analysis of the urine from
both the male and female knockouts demonstrated increased
levels of many urinary proteins, including vitamin D– binding
protein (DBP), albumin, and retinol-binding protein, suggest-
ing that CLC-5 is needed for endocytic retention of small
proteins, an established property of proximal tubule. Gunther et
al. (88) localized CLC-5 in the proximal tubule by immuno-
cytochemistry; although diffuse CLC-5 staining can be seen
throughout the cell, higher concentrations are seen in vesicles
adjacent to the brush border membrane, a region known to be
important for endocytic activity. CLC-5 colocalizes with H⫹-
ATPase, and this pairing is important for acidification of
endocytotic vesicles (88); CLC-5 is thought to act as a shunt
allowing diffusion of Cl⫺ into the endosome, decreasing the
accumulation of positive charges and allowing for greater
acidification. Endocytosis in proximal tubule also requires
megalin. Atlhough most megalin knockout animals die, the
uncommon survivors show low–molecular weight proteinuria,
including loss of vitamin D– binding protein (89,90). The uri-
nary loss of vitamin D results in growth retardation and exces-
sive osteoblastic and osteoclastic activity (89). A kidney-tar-
geted megalin knockout is viable; these animals have
hypocalcemia and osteomalacia (91). With loss of CLC-5, the
amount of megalin in the proximal tubule was reduced in ⫺/y
animals relative to ⫹/y (87). The phosphate transporter Npt2,
localized to brush border, was also downregulated in the prox-
1086 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 1082–1095, 2003

imal tubule of ⫺/y animals, consistent with the observed in a delicate balance between too little and too much
hyperphosphaturia (87). However, a low-Pi diet increased the 1,25(OH)2D3 (84,93). The latter would increase intestinal ab-
expression of Npt2. When PTH was administered to Pi-de- sorption of calcium, resulting in hypercalciuria. CLC-5 has
pleted animals, Npt2 was internalized within 15 min in WT also been detected in mouse intestine (93) and here could play
animals, but internalization took 1 h in ⫺/y animals. In the a role in calcium absorption by controlling endocytosis of
proximal tubule, PTH binds to megalin and is endocytosed for epithelial calcium channels. Furthermore, it is not clear if all
degradation; in ⫺/y proximal tubule, the delayed degradation cellular CLC-5 is associated with endosomes or whether it may
causes a luminal gradient of PTH to form, such that S3 seg- also play a role in other chloride circuits, analogous to CLC-Kb
ments are exposed to higher PTH levels and show higher Npt2 in Bartter type 3 (see below).
internalization than do S1 segments (87). Calcium Transport in the Thick Ascending Limb of
Wang et al. (92) also inactivated CLC-5 by insertion of a Henle’s Loop. Twenty percent of filtered calcium is reab-
neomycin resistance cassette between exon 5 and exon 6; ⫺/y sorbed in the thick ascending limb of the loop of Henle (TAL),
mice failed to produce CLC-5 RNA or protein. All ⫺/y and via both paracellular and transcellular processes (Figure 5). In
⫺/⫺ animals were normocalcemic, hypercalciuric, proteinuric, this segment, a lumen-positive voltage generated by the Na/K/
and ⫺/y (males) were hyperphosphaturic. These animals had 2Cl transporter (NKCC2/BSC-1) provides the driving force for
an elevation in both urinary amino acids and low–molecular paracellular transport of calcium. Inhibition of NKCC2 by loop
weight proteins, including DBP and Clara cell protein, due to diuretics (bumetanide, furosemide) (94,95) causes a decrease
impaired endocytosis. About 7% of ⫺/y mice had spinal de- in the lumen-positive voltage resulting in decreased paracellu-
formities and backward growth of teeth consistent with abnor- lar calcium reabsorption leading to hypercalciuria. The study
mal calcium metabolism and skeletal growth. Thus these ani- of inherited tubulopathies, particularly Bartter syndrome (96 –
mals closely mimicked the clinical presentation of patients 98), has proven valuable for understanding the mechanisms of
with Dent disease. ion transport in the kidney.
Silva et al. (83) have examined the effects of vitamin D
deficiency and thyroparathyroidectomy (TPTX) in rats on
CLC-5 RNA and protein levels. The combination of vitamin D
deficiency and TPTX caused a fourfold to fivefold increase in
urinary Ca/Cr, which was normalized by restoration of PTH.
Surprisingly, serum Ca was not affected by vitamin D defi-
ciency or TPTX. CLC-5 RNA levels from the renal cortex
were decreased by about 45% in the vitamin D– deficient
TPTX rats, and CLC-5 protein was not detected; PTH repletion
restored the levels of CLC-5 RNA and protein. No changes in
CLC-5 RNA or protein were seen in renal medulla. These
studies suggest that PTH regulates CLC-5.
It is not clear how a loss of CLC-5 function leads to
hypercalciuria; however, abnormal regulation of PTH and
1,25(OH)2D3 synthesis appear important. The diminished re-
cycling of luminal PTH receptors leads to increased local
concentrations of PTH, as shown by the higher levels of Npt2
internalization in S3 segment as compared with S1 segment of
the proximal tubule (92). As has been observed, the decreased
numbers of cell surface phosphate transporters should inhibit
reabsorption of phosphate and lead to hyperphosphaturia.
However, as PTH induces calcium reabsorption, higher local
PTH concentrations alone would not explain the observed
hypercalciuria. With loss of functional CLC-5, the decrease of Figure 5. Reabsorption of calcium in the thick ascending limb of the
small protein reuptake profoundly affects vitamin D metabo- loop of Henle. Approximately 20% of filtered calcium is reabsorbed
lism. The majority of circulating 1,25(OH)2D3 is protein- in the thick ascending limb of the loop of Henle. Paracellular reab-
bound and lost when the vitamin D– binding protein (DBP) is sorption is driven by a lumen-positive voltage created through sodi-
not conserved as has previously been shown in the megalin um/potassium/2 chloride reabsorption. Sodium, potassium, and chlo-
ride enter the cell through the bumetamide-sensitive transporter
knockout mice (89,91). In addition, the proximal tubule is the
NKCC2, driven by low intracellular concentrations of Na⫹ and Cl⫺
site of conversion of 25(OH)D3 to 1,25(OH)2D3 and uptake of
maintained by the activity of Na⫹/K⫹-ATPase and the chloride chan-
25(OH)D3 bound to DBP by proximal tubule cells is essential nel CLC-Kb. Potassium reenters the tubular lumen through the
for this conversion. The enzyme responsible for the hydroxy- ROMK channel, creating the lumen-positive voltage that drives cal-
lation, 1-␣-hydroxylase, is upregulated by PTH. CLC-5 knock- cium through the tight junctions, composed largely of paracellin-1
out animals have higher levels of 1-␣-hydroxylase mRNA (PCLN-1), into the blood. The calcium-sensing receptor (CaSR) reg-
(84). It has thus been postulated that the loss of CLC-5 results ulates ROMK and thus calcium reabsorption (32,33).
J Am Soc Nephrol 14: 1082–1095, 2003
Bartter Syndrome. Bartter syndrome was first described
as hypokalemic, hypochloremic metabolic alkalosis (96). The
primary mechanism for this disorder is a failure to adequately
reabsorb sodium in the thick ascending limb of Henle’s loop.
The syndrome varies widely in the degree of severity; the most
severely affected individuals present before birth with polyhy-
dramnios as a consequence of fetal polyuria and are often
delivered prematurely (99). Severe hypercalciuria often results
in rapid progression of nephrocalcinosis. Other patients with
Bartter syndrome may be asymptomatic into adulthood. Al-
though the condition usually appears sporadically, it can also
be inherited as an autosomal recessive trait (100). The loop
diuretics mimic the urinary effects of Bartter syndrome (sodi-
um wasting, kaliuresis. hypercalciuria) by inhibiting NKCC2
(94), and some Bartter syndrome patients have an impaired
response to furosemide (101); it was therefore a logical exten-
sion to look for NKCC2 defects in Bartter syndrome patients.
Simon et al. (102) demonstrated that nine children with Bartter
syndrome from four families had mutations in NKCC2, many
of which would introduce premature truncation of the protein.
The driving force for sodium and chloride uptake in the thick
ascending limb is derived from the low intracellular concen-
trations of these ions maintained by basolateral Na/K-ATPase
and chloride channels (CLC-Kb), and the activity of NKCC2 is
dependent on the presence of luminal potassium (Figure 5).
Recycling of potassium ions by the ATP-regulated potassium
channel ROMK provides the requisite K⫹ and generates the
lumen-positive potential that drives paracellular calcium trans-
port (98). In type 1 Bartter syndrome, there is a mutation of
NKCC2; in type 2 Bartter syndrome, there is a mutation in the
potassium channel ROMK (103,104); in type 3, there is a
mutation in the chloride channel CLC-Kb (105,106). Each of
these mutations, in NKCC2, ROMK, or CLC-Kb, leads to a
decrease in the lumen-positive voltage and a reduction in
calcium reabsorption; however, while all of these patients are
hypercalciuric, the type 3 patients rarely present with nephro-
calcinosis (105,106).
The calcium-sensing receptor (CaSR), present in TAL as
well as the proximal tubule, DCT, and CCD (107,108), mon-
itors serum calcium levels and regulates renal tubular calcium
reabsorption. Inactivating mutations of the calcium-sensing
receptor cause familial hypocalciuric hypercalcemia (FHH)
(109). Hebert et al. (110) offer a useful model on the regulation
of renal calcium reabsorption by the CaSR. In the presence of
elevated basolateral levels of calcium, where reabsorption
should be minimized, Gi coupled to the CaSR induces a re-
duction in intracellular cAMP levels, which in turn limit the
activation of NKCC2. The calcium-sensing receptor can also
activate phospholipase A2 to produce arachidonic acid, the
metabolites of which, including 20-HETE, inhibit NKCC2 and
ROMK. In support of this mechanism, Pearce et al. have found
six kindreds with an autosomal dominant inheritance of hy-
pocalcemia and hypercalciuria who proved to have activating
mutations in the calcium-sensing receptor (111,112). Vezzoli
et al. (113) examined four single-nucleotide polymorphisms
(SNP) in exon 7 of the calcium-sensing receptor and found one
SNP associated with a 13-fold higher risk of hypercalciuria.
Molecular Mechanisms of Primary Hypercalciuria 1087
Mutations in the paracellular channel that resulted in re-
duced calcium reabsorption would also be expected to lead to
hypercalciuria. Paracellin-1 (PCLN-1) was identified by Simon
et al. (114) as the principal protein in the tight junctions of the
TAL and is a member of the claudin family of tight junction
proteins (claudin-16) (115). Mutations in PCLN-1 were found
in cases of familial hypomagnesemia with hypercalciuria and
nephrocalcinosis (FHHNC, also known as hypomagnesemia
hypercalciuria syndrome, HHS) (116). Two unrelated patients
with HHS and missense mutations in PCLN-1 were confirmed
to have defective reabsorption of magnesium and calcium
without excessive loss of sodium (117). Furosemide infusion
increased sodium excretion sixfold both in controls and in the
2 HHS patients; however, Mg2⫹ and Ca2⫹ excretion increased
twofold and ninefold, respectively, in controls but did not
change in patients with HHS. MgCl2 infusion elicited a sixfold
increase in fractional excretion of calcium in the controls but
did not induce hypercalciuria in the patients with HHS (117).
These data support the hypothesis that primary sequence alter-
ations in PCLN-1 affect calcium and magnesium reabsorption.
Screening of 25 European families with FHHNC, including
33 affected individuals and their nonaffected relatives, re-
vealed that 94% of the affected patients had mutations in
PCLN-1, with 48% of those being a missense Leu151Phe
mutation (116). The common genetic abnormality and the
finding that many of these patients lived in a single geographic
region (Germany or Eastern Europe) suggested that the muta-
tion occurred in a common ancestor many generations ago
(founder effect) (116). An alternative, but less likely explana-
tion is that DNA sequences around Leu151 are unstable. In 13
of 23 families, there was an increased incidence of hypercal-
ciuria and nephrolithiasis in heterozygotic individuals not af-
fected by FHHNC, suggesting that heterozygotes have a partial
defect in paracellular calcium transport (gene dosage effect)
(116).
Calcium Transport in the Distal Tubule. The remaining
8% of filtered calcium is reabsorbed in the distal convoluted
tubule and connecting tubule; reabsorption in these segments is
predominantly active, transcellular transport under hormonal
regulation (Figure 6) (9,32,33,47,98,118). Similar to transport
in the intestine, in the distal convoluted and connecting tubule,
calcium enters the cell at the apical surface through a calcium
channel and binds to the calcium binding protein calbindin,
which serves as a shuttle to transport calcium across the cell.
At the basolateral surface, calcium is extruded against an
electrochemical gradient. However, the molecular species of
the proteins involved in renal calcium transport differs from
those in the intestine.
The identity of the apical calcium channel in distal tubule
remains somewhat controversial; Hoenderop et al. (119) did
not detect CaT1 RNA in human kidney RNA using RT-PCR
but did find abundant ECaC RNA, while Peng et al. (120,121)
found evidence of CaT1 RNA in human kidney RNA as well
as ECaC, localized to distal nephron. Abundance studies using
real-time PCR suggest that CaT1 RNA is more abundant than
ECaC RNA in kidney (122). Fractionation studies with isolated
rat tubules indicate that CaT1 RNA is found primarily in
1088 Journal of the American Society of Nephrology
Figure 6. Reabsorption of calcium in the distal convoluted tubule.
Approximately 8% of filtered calcium is reabsorbed by the distal
tubule. Calcium enters the cell through the ECaC calcium channel and
diffuses through the cytosol bound to calbindin D28k. Calcium extru-
sion into the blood occurs through the Na⫹/Ca2⫹ exchanger (NCX)
and plasma membrane Ca2⫹-ATPase (PMCA) (32,33).
mTAL (123), while immunohistochemistry indicates that
ECaC expression begins in the second segment of the distal
convoluted tubule and extends throughout the DCT but not into
the cortical collecting duct (124).
In the distal tubule, calbindin D28k acts as the principal
calcium shuttle (47,118). Calbindin D9k and parvalbumin, an-
other calcium-binding protein, are found only at the basolateral
membrane and may play a role in the extrusion of calcium from
the basolateral surface of the cell (125). Calbindin D28K knock-
out mice (126) exhibit excessive hypercalciuria when fed a
high-calcium diet (127). Basolateral calcium transport occurs
via both the plasma membrane Na⫹/Ca2⫹ exchanger (NCX)
and Ca2⫹-ATPase (PMCA), which have been estimated to
transport 70% and 30% of calcium respectively (128,129).
Calcium transport in the distal tubule is of particular interest
because it is regulated by parathyroid hormone (PTH) and
perhaps by 1,25(OH)2D3. Although apical calcium entry is
generally considered to be the rate-limiting step in reabsorp-
tion, these calcitropic hormones may affect one or several
components of the pathway.
Hoenderop et al. (130) have examined the effects of
1,25(OH)2D3 depletion and repletion on ECaC expression.
Rats were made vitamin D– deficient and hypocalcemic by
feeding a nonrachitogenic D-deficient diet, followed by a vi-
tamin D-deficient, Ca-free diet for 2 wk. This regimen led to
drop in plasma Ca2⫹ and a decline in 1,25(OH)2D3; injection
of 1,25(OH)2D3 led to rapid repletion. Vitamin D deficiency
caused a decline in mRNA for ECaC and reduced levels of
immunofluorescent labeling; restoration of vitamin D levels
led to upregulation of ECaC RNA and protein. Vitamin D
repletion also resulted in upregulation of RNA and protein for
calbindin-D28k; ECaC and calbindin-D28k colocalize to cells in
the DCT and CNT.
J Am Soc Nephrol 14: 1082–1095, 2003
Bone Resorption and Hypercalciuria. Many patients
with hypercalciuria, especially if they are consuming a low-
calcium diet, excrete more calcium than they absorb (1–10).
The source of the additional urinary calcium must be the
skeleton, by far the largest repository of calcium in the body.
Several studies have confirmed that patients with nephrolithi-
asis generally have a reduction in the density of their skeletons
compared with age-matched and gender-matched controls
(131–136). Pietschmann et al. (131) examined bone density in
120 patients with nephrolithiasis and found lower spinal bone
mineral density (BMD) in those who were hypercalciuric com-
pared with those who were normocalciuric. Jaeger et al. (132)
assayed BMD in 110 male Swiss idiopathic stone formers and
compared the results with 234 controls. Stone formers were
slightly shorter, had a higher BMI (body mass index), but a
significantly lower BMD at the tibial diaphysis and the tibial
epiphysis compared with the controls. There was no difference
between normocalciuric and hypercalciuric stone formers at
these sites. Ten of seventeen patients on a low-calcium diet for
at least 1 yr had a decreased BMD and 27 stone formers
reported fractures as compared with none of the controls.
Giannini et al. (133) found that 49 patients with recurrent
stones and IH had a lower lumbar spine Z-score than normal
controls, and they also had significantly higher bone alkaline
phosphatase levels and lower blood pH than controls. Misael
da Silva et al. (134) examined bone formation and resorption
parameters in 40 nephrolithiasis patients and classified ten as
osteopenic. Forty-five percent of hypercalciuric patients were
classified as osteopenic; they had about 20% less bone mass
than normocalciuric controls. While bone volume was not
different between controls and hypercalciuric patients, the hy-
percalciuric patients had increased osteoid thickness, a greater
percentage of eroded surface, and increased osteoclast and
osteoblast surface as a fraction of bone surface. Mineralization
lag time was also greater in hypercalciuric patients as com-
pared with controls. Tasca et al. (135) have found a more
negative Z-score in L1-L2 in hypercalciuric patients than in
controls.
In an attempt to preserve bone in patients with hypercalci-
uric nephrolithiasis and osteopenia, the bisphosphonate, etidr-
onate, was administered to seven male patients (136). Patients
were also advised to eat more calcium and less animal protein
and salt. At 1 yr, patients, each of whom received treatment,
had a significantly higher spinal (L2-L4) T-score, although no
difference was seen at 2 yr. Unfortunately, compliance was
poor in the study; patients showed no net increase in calcium
uptake, no decrease in protein consumption, and a significant
increase in NaCl consumption. Taken as a whole, these data
indicate that patients with hypercalciuria are at risk for bone
loss; as a consequence their bone density, as a proxy for
calcium balance, should be monitored. A low-calcium diet is
not effective in reducing the risk of stone recurrence and poses
a substantial risk to maintenance of bone health (137,138).
Genetic Hypercalciuric Stone-Forming Rats
To model idiopathic hypercalciuria and spontaneous stone
formation in humans, we have developed an animal model of
J Am Soc Nephrol 14: 1082–1095, 2003
hypercalciuria and nephrolithiasis (13,16,17,20 –31). Given ev-
idence for a genetic predisposition to hypercalciuria in both
humans and rats (14,19,139,140), Bushinsky and coworkers
screened adult male and female Sprague-Dawley rats for hy-
percalciuria and used the animals with the highest urinary
calcium excretion to breed the next generation, followed by
subsequent selection and inbreeding of their most hypercalci-
uric offspring, repeating the selection for almost 60 generations
(16,20 –30). By the thirtieth generation, the GHS rats (for
genetic hypercalciuric stone-forming) were excreting nearly
ten times as much calcium as simultaneously studied control
female rats (Figure 1) (13,16,17,20 –31). The rats were found
to have defects in calcium transport in the intestine, kidneys,
and bone, similar to abnormalities found in patients with idio-
pathic hypercalciuria (1–10).
Intestinal Calcium Absorption. Bushinsky and Favus (20)
studied the rate of intestinal calcium transport in GHS rats.
Fourth generation GHS rats not only exhibited increased uri-
nary calcium excretion but also had significantly elevated net
intestinal calcium absorption. Net duodenal calcium absorp-
tion, measured in vitro as well as in vivo, was greater in the
GHS rats despite lower 1,25(OH)2D3 levels in GHS males
compared with normocalciuric males, and no difference in
1,25(OH)2D3 levels among the females. When the female
control and GHS rats were placed on a low-calcium diet, there
was an increase in serum levels of 1,25(OH)2D3 in both
groups. However, there was a greater increase in net intestinal
calcium absorption in the GHS rats, even though 1,25(OH)2D3
did not increase as much as in controls. These results suggested
that the GHS rats were similar to the majority of humans with
idiopathic hypercalciuria; that is, there was an increase in both
urine calcium excretion and intestinal calcium absorption with
normal to only slightly elevated 1,25(OH)2D3 levels (141,142).
The Vitamin D Receptor. The finding of increased intes-
tinal calcium absorption without an elevation in 1,25(OH)2D3
levels led Li et al. to hypothesize that alteration of the receptor
for vitamin D might be responsible for the abnormal regulation
of calcium by enterocytes (22,143). The intensity of
1,25(OH)2D3 action correlates with receptor number and sat-
uration (144,145) both in rats in vivo (146 –148) and in cell
culture studies in vitro (149,150). The vitamin D receptor-rich
cytosolic fractions from GHS rat proximal duodenum bound
more [3H]1,25(OH)2D3 than similar fractions prepared from
normocalciuric controls (22). Using Scatchard analysis, we
demonstrated that this increase in binding of 1,25(OH)2D3 by
the vitamin D receptor was due to an increase in the number of
intestinal binding sites rather than enhanced affinity of the
vitamin D receptor for its ligand. Northern analysis of GHS
and control rat mRNA revealed no increased expression of the
vitamin D receptor gene to account for the increase in receptor
number. Gene transcription of the vitamin D receptor was
comparable for both groups of rats, as was synthesis of the
vitamin D-dependent calcium-binding protein, calbindin D9K.
Using western blot analysis, however, more calbindin D9K was
detected in intestinal protein from the GHS rats than from
controls. There is thus an increase in 1,25(OH)2D3 action in
GHS rats despite normal serum levels of 1,25(OH)2D3.
Molecular Mechanisms of Primary Hypercalciuria 1089
Yao et al. (30) found that the vitamin D receptor in the GHS
rats hyperresponded to minimal doses of 1,25(OH)2D3. The
hyperresponsiveness occurred through an increase in vitamin D
receptor stability without involving alterations in VDR gene
transcription, de novo protein synthesis, or mRNA sequence.
1,25(OH)2D3 administration also led to an increase in duodenal
and renal calbindin mRNA levels in GHS rats, whereas levels
were either suppressed or unchanged in wild-type animals.
Thus this hyperresponsiveness appears to be of functional
significance in that it affects vitamin D receptor-responsive
genes in 1,25(OH)2D3 target tissues.
Response to a Low-Calcium Diet. To determine if, in
addition to enhanced intestinal calcium absorption, other
mechanisms were contributing to hypercalciuria in the GHS
rats, nineteenth-generation GHS and normocalciuric control
rats were placed on a diet nearly devoid of calcium (0.02%)
and compared with similar rats eating a normal calcium (0.6%)
diet (21). On the normal-calcium diet, both groups of rats were
in positive calcium balance, despite marked hypercalciuria in
the GHS rats. On both the normal-calcium and low-calcium
diets, intestinal calcium absorption was greater in the GHS rats
compared with controls. When placed on the low-calcium diet,
both normal and GHS rats had a decrease in urinary calcium
excretion; however, urinary calcium excretion in the GHS rats
was eight times greater than that in controls. This persistent
calcium excretion in the GHS rats when fed a low-calcium diet
placed many of them, but not the controls, in negative calcium
balance.
Defective Renal Tubular Calcium Reabsorption. To de-
termine if GHS rats have a defect in renal calcium reabsorption
Tsuruoka et al. (28) performed 14C-inulin clearance studies on
female GHS and control rats. Some GHS and control rats were
fed standard rat chow, and others were fed a similar amount of
a low-calcium diet. Each rat was parathyroidectomized and
infused with calcium chloride to maintain normal concentra-
tion of serum calcium. After equilibration, urine was collected
for three periods with blood ultrafiltrable calcium and 14C-
inulin levels obtained at the midpoint. We found that both GHS
and control rats had similar GFR and the same ultrafiltrable
calcium concentrations resulting in similar filtered loads of
calcium (28). Despite the consistency of calcium presented to
the proximal tubule, the GHS rats had approximately three
times the fractional calcium and urinary calcium excretion
compared with control rats. The results were similar whether
the rats were fed a normal-calcium or a low-calcium diet.
Primary Bone Resorption. The increased sensitivity to
1,25(OH)2D3 observed in enterocytes, which is presumably
due to the increase in number of vitamin D receptors (22), may
be important in bone, as well. To determine if GHS rat bones
are more sensitive to exogenous 1,25(OH)2D3 compared with
bone from control rats, we cultured calvariae from neonatal
GHS and control rats with or without 1,25(OH)2D3 or PTH for
48 h (25). There was significant stimulation of calcium efflux
from GHS calvariae at 1 and 10 nM 1,25(OH)2D3, while
control calvariae showed no significant response to
1,25(OH)2D3 at any concentration tested. In contrast, PTH
induced a similar degree of bone resorption in control and GHS
1090 Journal of the American Society of Nephrology
rat calvariae. Immunoblot analysis demonstrated a fourfold
increase in the level of vitamin D receptors in GHS rat cal-
variae compared with control calvariae, similar to the increased
intestinal receptors described previously (22,30). There was no
comparable change in vitamin D receptor RNA levels as mea-
sured by slot blot analysis, suggesting the altered regulation of
the vitamin D receptor occurs posttranscriptionally. We then
determined if alendronate, an inhibitor of bone resorption,
would decrease urine calcium excretion by retarding bone
resorption (29). On the low-calcium diet, the urine calcium of
the GHS rats exceeded their calcium intake, indicating that
some of the urine calcium was from bone mineral stores.
Alendronate caused a significant decrease in urine calcium in
the GHS rats and brought urine calcium well below calcium
intake. Thus there is a significant contribution of bone to the
hypercalciuria in the GHS rat, which is consistent with the
observation that patients with nephrolithiasis often have de-
creased bone mineral density.
There is little doubt that one of the primary mechanisms of
hypercalciuria in the GHS rats is intestinal hyperabsorption of
calcium due to an increased sensitivity to 1,25(OH)2D3. The
persistent hypercalciuria on a diet essentially free of calcium
provides evidence for an additional mechanisms contributing
to the excess urine calcium excretion. We have shown evi-
dence for both primary bone dissolution apparently due to
augmented vitamin D receptor number in GHS rat osteoblasts
leading to enhanced bone resorption and a primary defect in
renal tubular reabsorption of calcium. Though the renal tubular
defect would necessitate bone resorption to maintain normal
serum calcium levels, both the renal and bone defects have
been shown to be independent of each other (25,28,29). The
independent defects in calcium handling by the intestine, kid-
ney, and bone suggest a systemic defect in calcium handling
resulting in hypercalciuria. The gradual increase in calcium
excretion with subsequent generations suggests that at least
several genes are responsible for the hypercalciuria (Figure 1).
Human studies demonstrate that there appears to be an asso-
ciation between the hypercalciuria and genes localized on
chromosome 1 (58). The identity of the specific genes involved
is not currently known.
Acknowledgments
This work was supported in part by grants AR 46289, DK 57716,
and DK 56788 from the National Institutes of Health.
Glossary of Terms
Calbindins: a family of cytoplasmic Ca2⫹ binding proteins
CaSR: calcium-sensing receptor
CaT1: Calcium transporter 1, homologous but not identical
to ECaC; the human counterpart is ECAC2
CLC: chloride channel
ECaC: Epithelial calcium channel, so named in analogy to
the epithelial sodium channel ENaC; also known as TRPV
GHS: genetic hypercalciuric stone-forming rat strain
NCX: Na⫹/Ca2⫹ exchanger
NKCC2: Na⫹/K⫹/2 Cl⫺ transporter
Npt2: Na⫹/Pi cotransporter
J Am Soc Nephrol 14: 1082–1095, 2003
Null Mutation Terminology: Introduction of an altered
transgene into the germline of mice can, by the process of
homologous recombination, led to disruption of the native
gene, producing a null mutation, which is also known as a
knockout animal. Animals with a null mutation for both
copies of the allele (homozygotes) are designated by the ge-
notype ⫺/⫺. Animals with a null mutation for one copy of the
allele (heterozygotes) are designated by the genotype ⫺/⫹. If
the disrupted gene is on the X chromosome, male knockouts
have genotype ⫺/y, while female knockouts are ⫺/⫺
Paracellular absorption: transport through cell-to-cell
junctions
PCLN1: paracellin-1, a major component of TAL tight
junctions
PMCA: plasma membrane calcium ATPase
ROMK: outwardly rectifying membrane K⫹ channel
Transcellular absorption: transport through the cytoplasm
of the cell
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