18
Article
Calcium Regulation and Management of Hypo- and
Hypercalcemia
Silvia M. Titan, MD, PhD
Nephrology & Hypertension Division, Mayo Clinic, Rochester, Minnesota
Nephrology Division, Faculdade de Medicina da Universidade de S~ao Paulo, S~ao Paulo, SP, Brazil
Rosa M. A. Moyse s, MD, PhD
Laboratorio de Investigaç~ao Medica 16 do Hospital das Clınicas da Faculdade de Medicina da Universidade de S~ao
Paulo, S~ao Paulo, SP, Brazil
Learning Objectives
1. To identify the main processes related to calcium transport in
the kidney, as well as the main transporters and hormones
mediating these processes
2. To recognize new knowledge related to the role of claudins in
calcium tubular transport
3. To recognize new knowledge related to the role of CaSRs in the
kidneys
4. To recognize causes of hypo and hypercalcemia related to
kidney disease
Physiology
Approximately 60% of calcium in the blood is not bound to albu-
min and is filtered at the glomerulus, either as free ionic calcium
or complexed with bicarbonate, citrate, or phosphate. Under physi-
ologic conditions, of the total amount filtered (filtered load of cal-
cium), only a minor percentage is excreted. Therefore, although
the glomerular filtration rate (GFR) is a major determinant of the
urinary calcium excretion, it is the tubular handling of calcium the
one responsible for fine tuning of the final amount excreted. About
65% of the filtered calcium is reabsorbed at the proximal tubule,
with the remaining 20%, 10%, and 5% handled in the thick
ascending loop of Henle (TALH), distal tubule, and connecting
tubule, respectively (1).
In the proximal tubule, calcium transport occurs mainly
through a paracellular pathway due to a favorable electrochemical
gradient secondary to sodium and chloride reabsorption (solvent
drag) and to a lesser extent, transcellular transport. The paracellular
transport is facilitated by claudin-2, and mutations in this protein
are associated with hypercalciuria in mice (2,3) and implicated in
the risk of kidney stones in human (4). Curry et al. (4) recently
showed that claudin-2-null mice have decreased renal tubular reab-
sorption and papillary nephrocalcinosis. The increased distal delivery
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Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
of calcium to the loop of Henle predisposes to nephrocalcinosis, and
nephrolithiasis. In addition, these animals have increased net intesti-
nal absorption, due to reduced intestinal calcium secretion. The authors
also evaluated common genetic variants using a genome-wide associa-
tion study in 11,130 kidney stone cases and 187,639 controls and
found that nine single nucleotide polymorphisms in the noncoding
region of the claudin-2 gene (CLDN2) were associated with nephro-
lithiasis risk (4). Further analysis suggested that these single nucleotide
polymorphisms (SNPs) are able to decrease claudin-2 expression.
Therefore, this paper corroborates the role of claudin-2 in nephro-
lithiasis and identifies a potential target for therapy in the future.
In the TALH, calcium transport is also paracellular and depen-
dent on the gradient created by active Na1 and Cl2 transport
through Na1-K1-2Cl2 cotransporter (NKCC2) in the luminal
membrane, Na1/K1-ATPase in the basolateral membrane and K1
recirculation through renal outer medullary potassium channel in
the apical membrane. The molecular basis for the paracellular trans-
port in the TALH was not clear until recently, when mutations in
claudins 16 and 19 causing familial hypomagnesemia with hypercal-
ciuria and nephrocalcinosis were identified (5,6). Subsequently,
claudin-14 was reported to modulate calcium transport by reducing
cation permeability of claudin-16 (7), and mutations in claudin-14
have been associated with increased risk of kidney stones and
reduced bone mineral density in a genome-wide association study
(8). Acquired defects related to claudins may also be responsible for
disturbances in calcium and magnesium transport. In a recent publi-
cation, antibodies against claudin-16 have been described in a
patient presenting severe magnesium wasting, hypocalcemia, and
tubulo-interstitial chronic nephropathy (9). Intravenous administra-
tion of his serum caused severe urinary waste in rats. Of note, the
patient received diagnosis 3 years later of a renal carcinoma expres-
sing high levels of claudin-16 mRNA. Claudins are a family of at
least 26 proteins that interact with each other either in the same cell
or across cells, forming tight junctions (10). Claudins have four
transmembrane domains and the first domain is related to paracellu-
lar permeability, acting as barrier or pore depending on its proper-
ties. The kidney expresses many different forms of claudins,
predominantly in the tubule but also in the epithelial cell of Bow-
man capsule and in podocyte.
Copyright # 2023 by the American Society of Nephrology
Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
4 Claudins are directly involved in the molecular
basis of paracellular transport of calcium (and
magnesium) in the proximal tubule, through
claudin-2, and in the TALH, through claudins 14,
16, and 19. Claudin mutations have been associ-
ated with increased risk of kidney stones (claudins
2 and 14), familial hypomagnesemia, hypercal-
ciuria and nephrocalcinosis (claudins 16 and 19),
and decreased bone mineral density (claudin 14).
In the distal tubule, Ca21 reabsorption is an active transcellular
process, involving the permeable ion channel (TRPV5) at the apical
membrane, intracellular binding to calbindins and extrusion to the
circulation through Na1/Ca21 exchanger, plasma membrane Ca21-
ATPase (PMCA), and Na1-Ca21-K1 exchanger (NaCKX) localized
at the basolateral membrane. KCTD1 gene codes for the potassium
channel tetramerization domain containing 1 protein and promotes
differentiation of TAL and DCT cells. KCTD1 has been shown to
modulate magnesium and calcium transport in these segments by
reducing NCC cotransporter expression in the DCT and by reduc-
ing NKCC2/claudin-16/claudin-19 and increasing claudin-14
expression in the TAL (11). At the collecting duct, calcium is not
reabsorbed. Figure 1 summarizes the molecular basis for calcium
transport along the tubule.
Emerging evidence from the last decade supports the concept
that the kidney is a calcium-sensing organ, with expression of
calcium-sensing receptors (CaSRs) along the nephron, particularly
in the TALH. The actions of kidney CaSRs are still not completely
understood, but some of the roles include counter-regulation of
parathyroid hormone (PTH) effects (by upregulation of the sodium
hydrogen antiporter-3 [NHE-3] and inhibition of phosphaturia)
and 1.25-vitamin D effects (by mitigation of the calcemic response),
inhibition of apical K1 recycling in the TALH with limitation
of paracellular calcium and magnesium transport, modulation
of claudin-14 (which regulates claudin 16 and 19), reduced
vasopressin-mediated trafficking and expression of aquaporin-2
(which explains the polyuria seen with hypercalcemia and modulates
the risk of kidney stones), and stimulation of renin release by the
juxtaglomerular cells, among others (12). CaSRs also cause an
increase in the sodium-chloride cotransporter activity in the distal
tubule (13). This effect contributes to the hypercalciuric effect of
the kidney CaSRs. Overall, these findings point to an integrated
action of kidney CaSRs in promoting urinary calcium excretion, a
likely counter-regulatory response to increased tubular calcium traf-
fic. CaSRs can be downregulated by PTH and phosphorus. Patients
4 The kidney is a calcium-sensing organ. When acti-
vated by increased calcium load, the kidney CaSR
promotes an integrated response that decreases
calcium reabsorption and thus increases calcium
excretion.
19
with autosomal dominant hypocalcemia type 1 (ADH1) present
hypocalcemia but variable calciuria. Therapy with calcium and vita-
min D may lead to hypercalciuria in these patients, which has been
linked to the risk of nephrocalcinosis and renal injury. By studying
mouse models and one ADH1 patient, van Megen et al. suggest cal-
ciuria is being modulated by renal CaSR response to calcemia (14).
Gain-of-function genetic variants of CaSRs are now known to cause
Bartter syndrome (15).
Calciotropic hormones regulate calcium excretion through com-
bined effects on bone, intestine, kidney, and parathyroid glands. PTH
release is triggered by low serum calcium level through the absence of
activation of CaSRs in the parathyroid cells. PTH exerts its multiple
effects in several cell types, leading to an integrated response that
releases calcium back to the circulation and restores normocalcemia.
In osteoblasts, PTH binds to its receptor, causing an increase in
RANKL (receptor activator of nuclear factor kB ligand) and a
decrease in osteoprotegerin expression, which collectively activate
osteoclasts by RANK-RANKL, resulting in an increase in bone
resorption. PTH also has noncellular effects and promotes calcium
buffering through increased expression of calcium-binding proteins,
osteocalcin, and osteonectin in the bone surface (16). In addition to
its bone effects, PTH increases tubular reabsorption of calcium by
activation of adenylyl cyclase systems in the proximal tubule and,
more importantly, through increased activity and expression of
TRPV5, increased expression of calbindin-D28k and reduced expres-
sion of Na/Cl cotransporter in the distal tubule. PTH also reduces the
availability of Na/Pi cotransporter in the proximal tubule and increases
the activity of 1a-hydroxylase. Despite the effect of PTH to augment
calcium tubular reabsorption, excess of PTH, such as seen in primary
hyperparathyroidism, leads to hypercalciuria, with increased risk of kid-
ney stones and nephrocalcinosis. This effect is a result of increased
calcium filtered load secondary to bone resorption and increased natri-
uresis secondary to downregulation of proteins related to sodium trans-
port, particularly NHE-3. FGF-23 and klotho have indirect effects on
calcium handling, by inhibiting the synthesis of PTH and 1,25-vitamin
D (the latter independently of the former). However, FGF-23 and
klotho have also been shown to regulate TRPV-5 independently of
PTH, thus promoting calcium reabsorption (17).
Whether 1,25-vitamin D has effects on tubular calcium trans-
port independent of its effect on calcium intestinal absorption in
humans is not clear. Several animal studies have shown that 1,25-
vitamin D enhances the expression of TRPV5, TRPV6, calbindins
and PMCA in distal tubules and collecting duct. Results from a
double knock-out model for TRPV5 and 1a-hydroxylase also sug-
gest that 1,25-vitamin D has direct tubular effects, increasing cal-
cium reabsorption (18). In addition, recent evidence suggests that
sclerostin, an osteocyte-derived hormone is another component of
bone metabolism. Sclerostin inhibits the Wnt-signaling pathway
and has antianabolic effects on osteoblasts. Mutations in its gene or
transcription factor are associated with increased bone mass (scleros-
teosis and van Buchem disease, respectively) (19). Sclerostin produc-
tion is inhibited by PTH, mechanical load, and estrogen among
others and stimulated by calcitonin and steroids. Experimental mod-
els have shown that sclerostin has direct and indirect effects on
1,25-vitamin D synthesis. Knock-out mice for sclerostin have
reduced urinary calcium excretion and fractional calcium excretion,
as well as evidence of increased 1,25-vitamin D synthesis, which was
reverted by sclerostin infusion (20).
20 Nephrology Self-Assessment Program - Vol 22, No 1, April 2023

Figure 1. Schematic representation of calcium transport along the renal tubule. Calcium (Ca11) is filtered at the glomerulus, and 65% of its
ultrafiltrable fraction is reabsorbed at the proximal tubule (PT). The remaining 20%, 10%, and 5% are handled in the thick ascending loop of
Henle (TALH), distal convoluted tubule (DCT), and connecting tubule, respectively. In the PT, Ca11 transport occurs mainly through a para-
cellular pathway due to a favorable electrochemical gradient secondary to sodium (Na) and to a lesser extent, due to transcellular transport. Clau-
dins (Cl) regulate paracellular transport in PT (Cl2) and TAL (Cl14 regulates Cl16 and 19). In the TALH, Ca11 transport is also paracellular,
dependent on the gradient created by active Na1 and Cl2 transport through Na1-K1-2Cl2 cotransporter in the luminal membrane, Na1/K1-
ATPase in the basolateral membrane and K1 recirculation through renal outer medullary potassium channel in the apical membrane. In the dis-
tal tubule, Ca21 reabsorption becomes an active transcellular process, involving the permeable ion channel (TRPV5) at the apical membrane,
intracellular binding to calbindins (CB28) and final extrusion to the circulation through Na1/Ca21 exchanger, plasma membrane Ca21-ATPase
(PMCA), and Na1-Ca21-K1 exchanger (NaCKX) localized at the basolateral membrane. There is no calcium reabsorption at the collecting
duct (CD), but apical calcium sensing receptor (CaSR) senses urinary calcium concentration, inhibiting water reabsorption (through the inhibi-
tion of aquaporin-2; AQP2) and stimulating urine acidification. Hormones that regulate calcium transport across the nephron are parathyroid
hormone, calcitriol, calcitonin, and estradiol. There is new evidence that sclerostin, Klotho, and FGF-23 also influence tubular calcium
reabsorption.

There are several other factors that regulate calcium transport,
which may be independent or mediated by calciotropic hormones.
A recent study evaluated the effects of high versus normal sodium
and low versus normal potassium diet on urinary calcium and
phosphate excretion in mice (21). The high-sodium diet increased
urinary calcium and phosphate excretion regardless of the dietary
potassium intake. However, serum calcium was lower with a high
sodium but low potassium diet suggesting an additional effect of
low-potassium diet in tubular calcium handling.
These abnormalities were accompanied by decreased calcium
reabsorption in the proximal tubule and TALH, likely mediated by
reduced expression of the sodium-proton exchanger (NHE3),
NKCC2, and claudin-16. In addition, the authors described an
increased calbindin-28k expression, suggesting the presence of a com-
pensatory response in the distal tubule that is not capable of revers-
ing the calcium urinary loss.
Hypercalciuria is a well-known epiphenomenon of acidemia.
Calcium phosphate is released from the skeleton as consequence of
Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
H1 buffering, causing an increase in the calcium (and phosphate)-
filtered load. On top of this short-term effect, studies have shown
that cell-mediated effects also occur with a reduction in osteoblast
activity and an increase in osteoclast activity, resulting in reduced
bone formation and increased resorption (22). In addition, particu-
larly in renal tubular acidosis type 1, other specific mechanisms may
be operative (urine pH, citrate concentration), and kidney stones
and nephrocalcinosis are common phenotypes in type 1 renal tubu-
lar acidosis. Imenez Silva et al. examined the effect of decreased
expression of protein-activated ovarian cancer G-protein (OGR1)
on acidosis-induced calcium excretion in mice (23). OGR1 is a pro-
tein normally expressed in the glomerulus, proximal tubule, and
endothelial cells and has been shown to reduce NHE-3 expression
in the proximal tubule. NH4Cl-induced metabolic acidosis increased
H1 excretion in both animals. However, whereas the wild-type ani-
mals demonstrated a positive association between H1 excretion and
calciuria with metabolic acidosis, the same effect was not observed
in knock-out mice, which showed higher expression of NHE-3,
TRPV5, and calbindin-D28k in comparison to the wild type. These
data suggest that OGR may detect low pH and modulate resulting
hypercalciuria.
There are sex differences in the urinary calcium and phosphate
handling. Androgen and estrogen receptors are expressed along the
nephron (24), and both experimental and human studies have
shown that urinary Ca21 excretion is higher in adult male compared
to adult females. This observation could be related to differences in
diet, higher osteoclastic activity in men, and higher calcium filtered
load (since men have higher absolute GFR values than women),
among others. In animal models, male mice present a reduced renal
expression of Ca21 transport proteins independently of calciotropic
hormones (25), and estrogens stimulate calcium reabsorption and
phosphate excretion (24). During childhood and particularly at
puberty, sex is also related to differences in bone mass gain and
development driven by sex steroids. A study evaluated the effects of
testosterone on tubular calcium handling according to aging and sex
(26). Testosterone upregulates expression of SMP-30 (renal senes-
cence marker protein-30, a calcium-binding protein present in renal
proximal tubular cells) and increases tubular calcium reabsorption in
mice, an effect mediated by increased plasma membrane calcium
ATPase-4 (PMCA4) expression. Conversely, aging was associated
with a reduction in testosterone and in SMP30 expression, as well as
an increase in urinary calcium excretion. Male rats presented higher
SMP30 expression in comparison to female rats of the same age,
and ovariectomy followed by testosterone replacement increased
SMP30 expression and reduced the hypercalciuric effect of ovariec-
tomy. This effect was reversed with the use of an androgen-receptor
inhibitor. All together, these findings suggest androgen-dependent
SMP30 expression may underlie sex differences in calcium handling
during puberty and bone loss in andropause.
Lactation is also related to important changes in renal and
intestinal calcium transport driven by the calcium transfer needed
for milk production. A recent animal study compared calcium and
magnesium excretion and protein expression in nonpregnant control
mice, lactating mice, or mice undergoing involution (27). There was
increased fractional calcium excretion and increased expression of
TRPV5 and calbindin-1 during lactation, with no change in the
expression of claudins. In addition, expression of renal 1a-
hydroxylase and 24-OHase were increased during lactation, along
21
with 1-25-vitamin D levels, which may explain the increased frac-
tional calcium excretion despite the enhanced paracellular calcium
reabsorption.
Adiponectin is an insulin-sensitizing adipokine known to be
reduced in obesity and associated with several beneficial effects,
including renoprotection in experimental models. On the other
hand, adiponectin has been associated with bone loss. Rutkowski
et al. evaluated mineral metabolism in adiponectin knock-out, wild-
type, and transgenic mice models (28). Overexpression of adiponec-
tin was related to lower bone mass, higher serum calcium, higher
calcium fractional excretion, lower FGF-23, with no changes in
PTH and 1.25 vitamin D, whereas the knock-out model presented
similar FGF-23 levels in comparison to the wild type but higher cir-
culating klotho levels. When challenged with a phosphate-enriched
diet (which results in increased phosphate and decreased calcium
fractional excretion due to reduced intestinal absorption), transgenic
mice showed higher calcium excretion, increased FGF-23, and frac-
tional phosphate excretion than the wild type. Knock-out mice
showed a smaller increase in PTH and lower FGF-23 levels. When
challenged with a calcium-enriched diet, all animals increased cal-
cium fractional excretion, but this effect was smaller in knock-out
and higher in transgenic mice. Overall, the findings from this study
suggest adiponectin modulates tubular transport of calcium and
phosphate, which is partially mediated by klotho expression. Levels
of adiponectin are known to be increased in CKD, and studies eval-
uating the effect of this adipokine in klotho expression in CKD-
MBD are needed.
Pathophysiology
Proteins involved in tubular calcium transport and intracellular cal-
cium trafficking are affected by a number of renal pathological con-
ditions, such as kidney stones, tubulopathies, and CKD, among
others.
Familial Hypomagnesemia with Hypercalciuria and
Nephrocalcinosis
Familial hypomagnesemia with hypercalciuria and nephrocalcinosis
is a monogenic disease caused by mutations in claudin-16 and in
claudin-19 (29,30). The calcium and magnesium wasting seen in
the disease is generally attributable to impairment in the paracellular
transport in the TALH. However, one specific mutation (missense
mutation c.908C.G, p.T303R) of claudin-16 is associated with a
calcium-specific abnormality, with no significant magnesium wast-
ing. Knowing that claudin-16 mRNA is expressed in the DCT,
Hou et al. hypothesized that this mutation is related to disturbance
of a specific function of claudin-16 in the DCT. Through a series of
experiments, the authors showed that phosphorylation of claudin-16
causes its expression in the apical membrane of DCT, along with an
increase in TRPV5 abundance and channel conductance (31).
Knock-down of claudin-16 expression in transgenic mouse kidney
caused delocalization of TRPV5 from the apical membrane of
DCT. In addition, the authors showed that a calcium-enriched diet
reduced the phosphorylation of claudin-16 through PTH-signaling.
Therefore, these results not only provide evidence for a combined
TALH and DCT defect in calcium reabsorption in certain mutations
of familial hypomagnesemia with hypercalciuria and nephrocalcinosis,
22
but also identified a novel regulatory effect of PTH in calcium tubu-
lar handling. Patients carrying CLDN19 mutations frequently exhibit
associated congenital ocular defects leading to variable visual impair-
ment (29,32).
Autosomal Dominant Tubulointerstitial Kidney Disease
A subtype of autosomal dominant tubulointerstitial kidney disease
(ADTKD) is caused by mutations in the transcription factor, hepa-
tocyte nuclear factor 1b (HNF1b) (33). HNF1b mutations are
associated with a phenotype including tubulointerstitial disease,
renal cysts, maturity-onset diabetes of the young, hypomagnesemia,
hypokalemia, and hypocalciuria. Kompatscher et al. evaluated the
effect of HNF1b in the TALH, showing that HNF1b causes an
activation of CaSRs, whereas a mutant protein abolished CaSR
expression (34). In addition, HNF1b knock-down in immortalized
kidney cell was associated with reduced CaSRs, reduction in
claudin-14, and upregulation of claudin-10, whereas kidney-specific
HNF1b knock-down was associated with important reduction in
CaSR expression, along with reduction in claudin-19 and claudin-
10b. These findings provide evidence for the mechanisms underly-
ing the hypocalciuric effects of ADTKD-HNF1b. ADTKD is also
observed in individuals with missense mutations in the SEC61A1
gene, and impaired protein transport of renin is observed, along
with reduction of the expression of calcium transporters. Using an
in vitro model of renal tubular cells, Sicking and colleagues observed
that administration of phenylbutyrate reversed the defective trans-
port of renin and calcium (35), opening an opportunity for pharma-
cological management of this genetic disease.
Renal Fibrosis
Interstitial fibrosis related to extracellular collagen deposition under-
lies progression of many kidney diseases. Proteins related to calcium
transport have recently been implicated in the development of renal
fibrosis. One study evaluated whether the CaSR was involved in col-
lagen expression of fibrosis induced by dietary adenine in mice (36).
The use of calcimimetic attenuated collagen deposition and abro-
gated the collagen upregulation induced by TGF-b1, whereas block-
ade of CaSRs enhanced collagen deposition induced by TGF-b1. In
another study, Orai-1 (calcium release-activated calcium channel
protein 1) knock-down prevented renal fibrosis and decreased
expression of proteins related to fibrosis in models of high-fat diet
and unilateral ureteral obstruction (37), with further experiments
suggesting a modulatory effect of Orai-1 over TGF-b1-induced
pathways. In addition, it has been hypothesized that sympathetic
activation has a role in CKD progression (38). The N-type Ca21
channel is expressed in nerves innervating the kidney, but its role is
not clear. Mishima et al., using a knock-out mouse for the N-type
Ca21 channel submitted to unilateral ureteral obstruction, showed
that (1) the N-type Ca21 channel is increased in the obstructed
fibrotic kidneys and (2) the knock-out mice presented a significantly
attenuated fibrotic response in comparison at least partly mediated
by reduction in renal sympathetic activation (39). More recently,
Piezo1, a mechanosensitive nonselective cation channel, was also
related to kidney fibrosis. Zhao et al. showed that Piezo1 protein
expression was markedly upregulated in human fibrotic kidneys. In
two different CKD models (unilateral ureter obstruction and folic
acid treatment), there was an increase in Piezo1 expression. They
Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
also showed that mechanical stretch, compression, stiffness or TGF-
b1 increased Piezo1, leading to profibrotic responses in cultured
tubular cells, which were prevented by inhibition or silence of
Piezo1. Importantly, the Piezo1-mediated calcium response was
dependent on extracellular calcium levels. When extracellular cal-
cium was chelated by EGTA, the profibrotic changes were signifi-
cantly attenuated (40).
Autosomal Dominant Polycystic Kidney Disease
Polycystin 2 has been characterized as a transient receptor potential
channel, playing a role in normal Ca21 signaling in the tubular cell
(41). It has been hypothesized that calcimimetics may play a thera-
peutic role in ADPKD. An in vitro study showed reduced basal rest-
ing Ca21 concentration and higher cAMP and mTOR levels in
PKD1-knock-down human proximal tubular cells in comparison to
wild-type cells. CaSR expression by these cells was confirmed and
selective CaSR activation with a calcimimetic reversed the abnormal-
ities described (42). In a second study using the same cell lines, the
authors showed that PKD1 knock-down cells had lower mitochon-
drial calcium levels and a severe reduction in ATP production in
comparison to the wild type, findings that were also reversed with a
calcimimetic (43). Mitochondrial TRPC3 upregulation has also
been shown in polycystin2 knock-down cells, causing an aggravation
of the mitochondrial abnormalities and cell proliferation (44). How-
ever, a previous in vivo study has shown that significant hypocalce-
mia prevented the use of calcimimetics in animal models of
ADPKD. A recent study, using a combination of small doses of a
calcimimetic (R-568) and a vasopressin V2 receptor antagonist (lixi-
vaptan) showed a reduction in cyst progression in two animal mod-
els of human ADPKD (45).
Diabetic Kidney Disease
Podocyte lesions and loss of podocytes are common features in dia-
betic kidney disease and calcium signaling disturbances have been
implicated in mediation of kidney diseases presenting podocytopa-
thy (46). Using the streptozotocin-induced diabetes model in
knock-out mice for NADPH oxidase 4, TRPC6, and double knock-
out for TRPC5 and TRPC6, Ilatovskaya et al. showed that
NADPH oxidase 4-deficient mice exhibited lower basal intracellular
calcium in podocytes and less damage, with blunting of the
angiotensin-induced calcium flux in glomeruli (47). H2O2 stimu-
lated calcium flux in wild-type animals, but this response was mini-
mized in TRPC6 knock-out models. The same group, in a more
recent study, identified that renal purinergic signaling is also
involved in diabetic kidney disease. They showed an increase in
ATP-mediated calcium influx in the podocytes, through the recep-
tors P2X4 and P2X7. This disruption in intracellular calcium
homeostasis contributes to podocytes dysfunction and aggravation
of diabetic kidney disease progression (48).
Chronic Kidney Disease
CKD is associated with a dramatic decrease in urinary calcium
excretion. However, the determinants of calciuria are not completely
understood, neither the effects of some medications commonly used
in this scenario, such as diuretics. Data from observational or retro-
spective studies have shown that furosemide increases calciuria,
whether thiazides have the opposite effect. Also, higher serum PTH
Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
is seen in furosemide users, although this effect could not be
completely explained by the increase in urinary calcium excretion.
Vasco et al. prospectively evaluated the effects of furosemide and
hydrochlorothiazide in a randomized trial in patients with stage 3
CKD for 1 year. At the follow-up, eGFR decreased, and PTH and
FGF-23 increased in both groups. However, furosemide was associ-
ated with a more significant increase in PTH, 1a,25-vitamin D,
procollagen type I N-propeptide (P1NP) and collagen type I
C-telopeptide (CTX), whereas hydrochlorothiazide attenuated the
PTH rise and caused a reduction in 1a,25-vitamin D and in these
bone turnover markers. This effect could not be explained by the
changes in calciuria, highlighting the different effects of these drugs
on vitamin D metabolism and bone remodeling (49).
Another question that arises is whether calcium supplementa-
tion in this population would increase calcium uptake by the skele-
ton or increase soft tissue deposition. Further understanding on this
matter is limited by technical difficulties in performing and assessing
calcium balance. One alternative is using the 44/42Ca ratio. The nat-
urally occurring isotopically light 42Ca is preferentially incorporated
into bone, whereas heavier 44Ca is excreted. The ratio increases
when bone formation exceeds resorption, so the higher the ratio, the
greater the skeleton positive balance. Shroff et al. have previously
shown that healthy children have a higher serum ratio than adults.
Specifically in CKD, they have shown that 44/42Ca ratio positively
correlated with serum calcium, 25-hydroxyvitamin D, alkaline phos-
phatase, and total bone mineral content measured by peripheral
quantitative computed tomography and inversely with PTH and
bone resorption markers. More impressive was the finding that in
children with CKD, the 44/42Ca ratio was 157% lower than that of
age-matched children and 29% lower than levels in elderly women
with osteoporosis, when adjusted for calcium containing medica-
tions and kidney function (50).
Renal Cell Cancer
CaSR has been implicated in the development of bone metastasis in
renal cell carcinoma and other cancers (51) and polymorphisms of
CaSR have been associated with increased risk of several cancers
(52). CaSR activates Rac and cell migration via the Gbg-PI3K-
mTORC2 pathway (53). Using a renal cell carcinoma cell line
transfected with CaSR, Frees et al. showed that calcium-treated cells
presented an increase in adhesion, production of extracellular pro-
teins, cell migration, and proliferation, and these effects were
abrogated by a CaSR antagonist (54). In addition, in vivo mice stud-
ies showed that bone metastasis was increased after injection of
CaSR-transfected cells, raising the hypothesis that targeting CaSRs
might be beneficial, at least in those with high CaSR expression.
Hypocalcemia
Hypocalcemia is commonly seen because of diseases related to cal-
cium transport or secondary to the use of drugs. We will review here
recent studies related to hypocalcemia, particularly related to drugs
that are increasingly being used in the CKD population.
Denosumab
Several case reports and studies have addressed the risk of hypocalce-
mia related to denosumab treatment for osteoporosis, metastatic
23
bone disease, or conditions such as immobilization. Denosumab (a
fully humanized monoclonal antibody against RANKL) inhibits
bone resorption, is metabolized by the liver and does not require
dose adjustment in CKD. It has been increasingly used in patients
with eGFR ,30 ml/min per 1.73 m2, in whom bisphosphonates
are not recommended or are contraindicated. However, denosumab
is associated with increased risk of hypocalcemia, and this risk may
be higher in those with low GFR, which is also associated with
lower calcium levels. In most patients, hypocalcemia is mild and
asymptomatic; however, symptomatic hypocalcemia occurs in small
percentage of patients (55).
Miyaoka et al. evaluated risk factors for low corrected calcium
levels after denosumab treatment in 77 patients with osteoporosis,
of whom 35% presented an eGFR of 30–59 ml/min per 1.73 m2
(56). The nadir of calcium levels occurred at day 7 after treatment.
Although none of the participants presented symptomatic hypocal-
cemia during the study, asymptomatic hypocalcemia (defined as a
corrected calcium level less than 8.5 mg/dl) occurred in 10.4%. Low
baseline corrected calcium, eGFR ,60 ml/min per 1.73 m2,
tartrate-resistant acid phosphatase-5b, bone alkaline phosphatase,
and pretreatment with antiresorptive agents were factors associated
with the absolute reduction in corrected calcium from baseline to
day 7. In a post hoc analysis of the FREEDOM trial (57), the safety
and efficacy of the use of denosumab in women with mild to mod-
erate CKD were evaluated during the trial phase and in the open-
label 7-year extension study. BMP gain over time and adverse effect
rates were similar among the CKD subgroups (normal, CKD G2,
G3a, and G3b).
In a series with 47 patients on dialysis, 25% presented acute
hypocalcemia following denosumab administration and tartrate-
resistant acid-phosphatase 5b was the best predictor of hypocalcemia,
along with bone alkaline phosphatase (58). Thongprayoon et al. per-
formed a meta-analysis evaluating the incidence of hypocalcemia in
patients with ESKD treated with denosumab (59). The authors
included six observational studies and 84 patients and describe a
pooled incidence of hypocalcemia of 42% (95% CI, 29 to 55), occur-
ring initially between 7 and 20 days and reaching a nadir between
2 weeks and 2 months after the first dose. There was a significant
increase in bone density parameters and reduction in alkaline phos-
phatase and PTH, with no significant changes in serum calcium or
phosphate after 3 months. The same group published another meta-
analysis restricted to kidney transplant, identifying five eligible studies
(one clinical trial and four observational studies) comprising 162 kid-
ney transplant patients (60), with the majority having an eGFR
.30 ml/min per 1.73 m2. During denosumab treatment, risk of
hypocalcemia was greater in the clinical trial and reported as small and
mild in observational studies (1.7%, 95% CI, 0.4 to 6.6). In addition,
after treatment, denosumab was related to increase in bone mineral
density and no change in Ca or PTH after treatment. Use of denosu-
mab is likely safer in patients presenting hypercalcemia pretreatment,
as suggested by a study evaluating the effects of denosumab in 14
hypercalcemic kidney transplant patients with osteoporosis (61).
In people with cancer, a retrospective study by Kanbayashi et al.
evaluated 327 patients with advanced disease and found that con-
comitant use of vonoprazan (a potassium-competitive acid blocker)
or dexamethasone, and pretreatment low serum calcium and hemo-
globin, as well as high alkaline phosphatase were significant predic-
tors for the development of denosumab-induced hypocalcemia (62).
24
In patients who are candidates to denosumab therapy, irrespec-
tively of the indication being for metastatic cancer or osteoporosis,
calcium and vitamin D should be corrected before denosumab
administration. Hypomagnesemia, when present, should also be
corrected. In those patients with CKD, is it recommended to check
calcium and PTH 1–2 weeks after administration and correcting
hypocalcemia, when present.
PD-1 Inhibitors
Programmed cell death protein 1 (PD-1) inhibitors are used in the
treatment of several tumors, such as melanoma, non–small cell lung
cancer, renal cell carcinoma, bladder and urothelial cancer, breast
cancer, among others. These drugs are associated with the risk of
urinary tract infections, electrolyte abnormalities and acute renal fail-
ure. A meta-analysis of 48 clinical trials including a total of 11,482
participants evaluated the risk of AKI and electrolyte abnormalities
in patients treated with PD-1 inhibitors (63). Of the 48 clinical
trials, 16 RCTs were used in the analysis assessing the risk of AKI
(n513) and electrolyte abnormalities (n55) and 39 clinical trials
were included in the analysis evaluating the incidence of electrolyte
abnormalities. The overall pooled relative risks of AKI and all elec-
trolyte abnormalities in patients treated with PD-1 inhibitors were
1.86 (95% CI, 0.95 to 3.64) and 1.67 (95% CI, 0.89 to 3.12),
respectively. In a subanalysis using patients treated with non-
nephrotoxic drugs as controls, the pooled RR of AKI in patients
treated with PD-1 inhibitors was 4.19 (95% CI, 1.57 to 11.18).
PD-1 inhibitors were associated with hypocalcemia, with a pooled
RR of 10.87 (95% CI, 1.40 to 84.16), and other specific electrolyte
disorders were not associated to treatment with PD-1 inhibitors.
The pooled estimated incidence rates of AKI and hypocalcemia in
patients treated with PD-1 inhibitors were 2.2% (95% CI, 1.5 to
3.0) and 1.0% (95% CI, 0.6 to 1.8), respectively. In addition, the
pooled estimated rate of interstitial nephritis was 16.6% (95% CI,
10.2 to 26.0). Several mechanisms have been hypothesized to medi-
ate the associations of PD-1 inhibitors and electrolyte disturbances
(64). Of, note, one study suggests that the incidence of hypocalce-
mia reported with PD inhibitors may be related to the reporting of
serum calcium not corrected for serum albumin (65).
Tyrosine Kinase Inhibitors
Hypocalcemia is a recognized potential side effect of this class of
drugs. According to a meta-analysis of seven studies, severe hypocal-
cemia is not uncommon, reaching almost 10% in patients that
received sorafenib (66). Although the exact mechanism is not
known, it seems to be more frequent in patients with thyroid cancer
and those with previous, but compensated hypoparathyroidism (67).
Parathyroidectomy
Hypocalcemia is a well-known severe complication after parathy-
roidectomy, particularly in severe hyperparathyroidism, with a need
for massive supplementation of calcium and vitamin D in the first
weeks following surgery (hungry bone syndrome) (68–70). A recent
meta-analysis done by Gao et al. compiling 2990 patients with
secondary hyperparathyroidism from 13 studies has shown that pre-
operative PTH, alkaline phosphatase and calcium were significant
predictors of hypocalcemia (71). Similar findings were described in
a single-center, retrospective study, which analyzed 1500 CKD
Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
patients that were submitted to parathyroidectomy between 2008
and 2019. Zhao et al. found that, beside the predictors previously
mentioned, the mass of excised parathyroid gland and age were also
associated with hypocalcemia, which was severe in 36.2% of the
patients. Curiously, hyperkalemia, a well-described, but not well-
explained, phenomenon was found in 24.6% of the patients (72).
Calcimimetics
Hypocalcemia is a common side effect of calcimimetics, and a recent
meta-analysis reported an increase in the risk of hypocalcemia by ten
times in people with CKD when compared to control (73). In this
same study, calcimimetic use was associated with improvement in
biochemical parameters, but not with all-cause or cardiovascular
mortality (73). It has been used also in the pediatric population
(74,75) with apparent safety and efficacy, although larger studies are
needed in this population. In addition, it is often used for manage-
ment of hyperparathyroidism post-transplant (76), although there is
controversy about the optimal treatment of this condition. A retro-
spective analysis of the EVOLVE study has shown that, among the
1938 patients randomized to cinacalcet, 1130 (58.3%) developed at
least one hypocalcemia episode during the first 16 weeks, and 356
(18.4%) were categorized as severe hypocalcemia (77). Risk factors
for hypocalcemia were high PTH and alkaline phosphatase, as well
as low serum calcium. Other studies have also identified high PTH
as a risk factor for calcimimetic-associated hypocalcemia (78,79).
This observation raises the question of whether hypocalcemia is
being triggered by a similar mechanism as seen in postparathyroi-
dectomy hungry bone syndrome, which should be treated with cal-
cium and calcitriol supplementation, or if it is predominantly caused
by reduced bone resorption related to more severe forms of hyper-
parathyroidism, therefore more likely to ultimately need parathy-
roidectomy (80).
Dialysate Calcium
The 2017 KDIGO Guideline on CKD-MBD suggests the use of a
dialysate calcium concentration between 1.25 and 1.50 mmol/L
(2.5 and 3.0 mEq/L) in CKD stage 5D. The rationale for this rec-
ommendation is to reduce the positive balance of calcium that often
occurs during years of dialysis treatment and likely reduce vascular
calcification and low turnover bone disease (81), although large
studies with hard clinical outcomes are still lacking. However, one
drawback of using low calcium dialysate is the increased risk of
hypocalcemia (82), which can result in increased risk of cardiac
arrythmias, sudden death, and higher PTH. Brunelli et al. have eval-
uated the consequences of the conversion to low calcium dialysate
(,2.5 mEqL) in a hemodialysis organization in the US reporting
greater rates of hospitalization for decompensated heart failure,
hypocalcemia, and intradialytic hypotension, with no differences in
all-cause mortality or total hospitalization rate (83). Hypocalcemia
has also been associated with increased risk of hypotension in
CRRT (84). The conversion to lower calcium dialysate was associ-
ated with lower serum calcium levels, increased serum phosphate
and PTH levels, and increased use of phosphate binders, vitamin D,
and calcimimetics (83). Similar biochemical and drug use trends
were reported elsewhere, whereas others have found 2.5 mEq/L-cal-
cium dialysate use was not associated with increased risk of hypocal-
cemia when compared to the use of 3 mEq/L dialysate (85). Further
Nephrology Self-Assessment Program - Vol 22, No 1, April 2023
studies are necessary and should assess not only the biochemical
parameters but also hard clinical outcomes.
Hypercalcemia
Hypercalcemia is a common reason for nephrology consult and is
most often related to underlying cancer, parathyroid disorders, and
drugs. Rarely, it can be caused by genetic conditions and loss-of-
function variants in CYP24A1 have been associated with
24-hydroxylase deficiency with concurrent hypercalcemia, nephro-
lithiasis, and nephrocalcinosis (86).
Malignancy
Usually, hypercalcemia is mediated by the production of PTH-
related peptide (PTHrP, which accounts for 80% of the cases) or
due to calcium release from bone by osteolytic metastases. Less than
1% of malignancy-related hypercalcemias are explained by increased
calcitriol or PTH. In the first case, there is some debate about which
is the source of excessive calcitriol synthesis, the tumor cells or adja-
cent macrophages. A recent published case report of a patient with
diffuse large B cell lymphoma and hypercalcemia documented the
increased expression of 1a-hydroxylase by lymphoma cells, confirm-
ing that these cells can produce the active form of vitamin D (87).
In the late, parathyroid carcinoma, which accounts for less than 1%
of primary hyperparathyroidism, is the source of PTH. A review of
1057 cases from the US National Cancer Database showed that,
although rare, this is a disease with high morbidity and mortality
(10-year survival rate of 57%), and in many cases related to the
degree of hypercalcemia (88). Finally, ectopic production of PTH,
such as by ovarian carcinoma or neuroendocrine tumors, is
described in anecdotical cases (89).
Fungal Infection
There are several case reports of pulmonary fungal infections (Pneu-
mocystic jirovecii and Histoplasma capsulatum) in immunosuppressed
patients, accompanied by hypercalcemia. These patients present
high serum levels of 1a,25-vitamin D and low PTH, a finding com-
patible with extra-renal synthesis of 1a,25-vitamin D (from the
granulomas). In an interesting report of hypercalcemia, the authors
described a case of an immunocompromised person affected by
invasive muscular microsporidial infection. The microsporidia was
surrounded by mononuclear inflammatory cells, mainly macro-
phages, likely the potential source of 1a,25 vitamin D (90).
Antibiotic Impregnated Calcium Sulfate Beads
There have been some reports of hypercalcemia after use of calcium
sulfate beads impregnated with antibiotics, which are occasionally
used for treatment of osteomyelitis. A retrospective analysis of an
orthopedic service in Switzerland, evaluated 215 patients who were
treated with antibiotic-loaded calcium sulfate beads. They found
symptomatic hypercalcemia in only two of them, and the increase in
serum calcium could also be explained by other factors, such as
immobilization and chronic kidney disease. However, serum cal-
cium was not systematically measured, and asymptomatic hypercal-
cemia could not be ruled out in many cases. Therefore, calcium
monitorization is recommended in those patients that will be
25
submitted to this procedure, particularly those with increased age,
CKD, and immobilization (91).
Silicone and Polymethylmethacrylate
Recently, hypercalcemia related to silicone and polymethylmetha-
crylate injections for cosmetic purposes has been described
(92,93). A systematic review by Tachamo et al. identified 23
patients with this condition. Polymethylmethacrylate induces an
inflammatory response with appearance of activated macrophages
in granulomas that produce 1-alpha-hydroxylase, increasing extra-
renal calcitriol production and leading to hypercalcemia, and fre-
quently, AKI (94). Although this procedure is not Food and Drug
Administration approved, it is commonly used in Latin America
and other countries (95).
Immobilization
Prolonged immobilization is another less common cause for hyper-
calcemia. However, its prevalence is likely increasing, considering
the expansion in number of patients and hospitalization days at
ICUs worldwide. This entity was described decades ago in patients
immobilized by traumatic spinal-cord injury (96) but is also seen in
critically ill patients. It is characterized by calcium release from the
skeleton and mediated by increased osteocyte synthesis of sclerostin,
which simultaneously inhibits bone formation and stimulates bone
resorption (97,98). The massive release of calcium from the skeleton
inhibits PTH, leading to the usual finding of suppressed PTH.
Those who have normal renal function can increase calcium excre-
tion and remain normocalcemic or show only very mild hypercalce-
mia. Conversely, when patients have CKD or AKI develops, the
reduced GFR impairs the ability to excrete calcium and hypercalce-
mia ensues. It is important to note that in patients who are on
continuous renal replacement therapy, particularly those using
citrate-anticoagulation, calcium removal may mitigate or abolish
the hypercalcemia, increasing the risk of underdiagnosis of the
immobilization syndrome (99,100). During the first wave of the
COVID-19 pandemic, several patients remained heavily sedated,
with neuromuscular blockage for weeks. Many of them also needed
continuous renal replacement therapy. An impressive bone loss was
documented in one study (101). A case report described a patient
who presented hypercalcemia only after renal function recovery,
emphasizing the need of a greater attention to bone loss in patients
with prolonged immobilization (102).
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