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Nephrocalcinosis: New insights into mechanisms and consequences

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Nephrol Dial Transplant (2009) 24: 2030–2035
doi: 10.1093/ndt/gfp115
Advance Access publication 18 March 2009
Editorial Review
Nephrocalcinosis: new insights into mechanisms and consequences
Benjamin A. Vervaet, Anja Verhulst, Patrick C. D’Haese and Marc E. De Broe
Department of Medicine, Laboratory of Pathophysiology, University of Antwerp, Antwerp, Belgium
Correspondence and offprint requests to: Patrick C. D’Haese; E-mail: Patrick.Dhaese@ua.ac.be
Keywords: crystal formation; crystal retention; nephrocalcinosis;
nephrolithiasis; Randall’s plaque
The most common form of renal stone disease, calcium
nephrolithiasis, is defined as the presentation of a macro-
scopic concrement of inorganic (calcium phosphate and/or
calcium phosphate) and organic material in the renal ca-
lyces and/or pelvis, either adhered to the papillae or pelvic
urothelium or not. In search of the mechanism underlying
calcium nephrolithiasis, in vitro and in vivo studies and ob-
servations in human biopsies have shown the presence of
two distinct types of renal microscopical crystal deposition
processes; one taking place within the tubular lumen (in-
tratubular nephrocalcinosis), and the other in the intersti-
tium (interstitial nephrocalcinosis). Recent observations,
however, strongly suggest that nephrocalcinosis and cal-
cium nephrolithiasis are to be considered two independent
pathologies and that nephrocalcinosis may cause calcium
nephrolithiasis only in particular conditions. In this review,
we discuss our current understanding of the mechanisms in-
volved in both types of nephrocalcinosis (intratubular and
interstitial), their possible consequences and their relation
to calcium nephrolithiasis.
Intratubular nephrocalcinosis
Mechanisms of intratubular nephrocalcinosis
Two key processes determine the development of intratubu-
lar nephrocalcinosis, crystal formation and crystal reten-
tion. Crystals (mainly calcium oxalate, CaOx, and calcium
phosphate, CaP) are thought to frequently form in the tubu-
lar fluid as a result of supersaturation mainly in the distal
nephron and can be considered a renal mechanism to ex-
crete an increased amount of waste per unit of volume
[1–4]. To ensure safe tubular crystal passage, the healthy
kidney on the one hand presents a non-crystal binding ep-
ithelium [5–9] and on the other hand is able to keep crystal
nucleation, growth and aggregation under control via uri-
nary micro- and macromolecular constituents such as cit-
rate, magnesium and proteins [10–13]. In addition, at the
physiological level, a high intratubular calcium concentra-


C The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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tion triggers a reduction in antidiuretic hormone-stimulated
water permeability of the collecting duct through the
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calcium-sensing receptor, leading to an increased urinary
volume and a reduced risk of supersaturation [14,15]. Fail-
ure or shortcomings of these defence/control mechanisms
result in crystal retention (= intratubular nephrocalcinosis),
either presenting as epithelial crystal adhesion, when crys-
tals smaller than the diameter of the tubular lumen adhere to
the tubular epithelium or as tubular crystal obstruction, in
the case of excessive crystal formation and/or aggregation
(see Figure 1). Whereas an obstruction is mainly a me-
chanical process determined by the diameter of the tubules
and the extent and rate of crystal formation/aggregation,
an adhesion is a subtle, complex cell biological pro-
cess in which a particular tubular epithelial phenotype
is responsible for firm crystal adhesion [5–8,16,17] (see
Figure 1).
Evidence is now available indicating that the luminal
surface of the tubular epithelium, under stress conditions,
expresses multiple crystal-binding molecules, which are
not present at the luminal membrane of intact differenti-
ated tubular epithelia. These molecules, such as sialic acid-
containing proteins and/or phospholipids, phosphatidyl
serine, nucleolin-related protein, annexin II, osteopontin
and hyaluronan, are expressed by dedifferentiated or regen-
erating cells [5,7,8,17–26]. The causal role of an aberrant
epithelial phenotype in crystal adhesion is corroborated
by consistent observations at three distinct research levels:
in vitro, in vivo and in clinical settings.
1. In vitro renal cell-lines as well as primary human ep-
ithelial cell cultures only bind crystals firmly in their
subconfluent phase, i.e. when cells are proliferating
and migrating, and lose this capacity once they be-
come confluent and fully polarized [6,8,17,24,27]. It
has to be noted that this is particularly true for epithelial
cells of distal origin, thought to be frequently exposed
to intraluminal crystals [28,29].
2. In rats treated with ethylene glycol (EG, a model of
hyperoxaluria/crystalluria) already after 1 day of treat-
ment, the numerous crystals present in the tubular fluid
were not adhering to the normal differentiated intact ep-
ithelium. After 4 days of treatment, however, crystals
Nephrol Dial Transplant (2009) 24: Editorial Review
Fig. 1. Schematic presentation of mechanisms involved in renal crystal handling and the development of nephrocalcinosis and nephrolithiasis. CaP,
calcium phosphate; CaOx, calcium oxalate; UrAc, uric acid; Br, brushite.
were found adhering to dedifferentiated/regenerating
cells [5]. Interestingly, during washout, shortly after
arrest of a 4-day EG-administration period, the num-
ber of regenerating tubular cells markedly increased,
as did crystal retention, while crystalluria decreased
to control values (Vervaet et al., unpublished results).
Additionally, in another study mild EG administration
did not result in nephrocalcinosis until a nephrotoxic
agent was injected intraperitoneally [30].
3. In a transplant protocol biopsy study of our group, all
patients showed tubular luminal expression of osteo-
pontin and hyaluronan at first biopsy 12 weeks post-
transplantation, while only 20% of these patients pre-
sented nephrocalcinosis. At second biopsy, 12 weeks
later, osteopontin and hyaluronan expression was still
present in all patients; however, it was associated with
100% nephrocalcinosis, evidencing luminal expres-
sion of these molecules to precede crystal adhesion
[16]. This early and maintained expression of particular
molecules may be related to recovery from ischaemia–
reperfusion and subsequent sustained renal stress by
exposure to potential nephrotoxic immunosuppressive
drugs. In the same study, it was found that preterm
infants, who are well known to be born with an im-
mature epithelium, also presented luminal osteopontin
and hyaluronan; however, nephrocalcinosis did not de-
velop until several days of life [16]. This delay most
likely is due to the time it takes for diet and/or medi-
cation protocols to induce crystalluria and subsequent
crystal adhesion [16,31,32].
2031
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In contrast, in some histological studies in rat and man,
intratubular crystals were observed adjacent to epithelial
cells that appeared morphologically normal [33,34]. These
observations may either suggest crystal adhesion to normal
differentiated cells or alternatively may represent a ‘snap-
shot’ of transient (non-adhesive) crystal–cell interactions.
Although it is not known whether these epithelia actually
present a normal apical membrane in terms of protein and
phospholipid composition, the latter possibility seems more
likely. Indeed, animal models of mild hyperoxaluria and
crystalluria (such as EG or minipump-infused oxalate) do
not immediately develop renal crystal retention by adhe-
sion or obstruction, but apparently require several days to
weeks of exposure [5,35]. This ‘incubation’ period, during
which nephrotoxic EG metabolites and/or transient toxic or
mechanical crystal–cell interactions may affect the tubular
epithelium, is in line with the need of a shift in the epithelial
phenotype prior to crystal adhesion [29,36,37].
In the case of severe injury, crystals may adhere to apop-
totic and/or necrotic cells (known to present altered mem-
brane surfaces) and even to denuded basement membranes
after cells have been lost from the epithelium [26,38,39].
Furthermore, besides crystal adhesion, nucleation of crys-
tals onto the tubular epithelium has been suggested to be a
potential mechanism underlying intratubular nephrocalci-
nosis [25]. In this process, crystallization starts at particular
sites on the epithelial surface instead of starting freely in the
tubular fluid. Remarkably, the composition of the cell sur-
face appears also to be a critical determinant in modulating
this process [25,40].
2032
Altogether, whereas excessive crystal formation/
aggregation may result in tubular obstruction and its dele-
terious consequences, crystal adhesion turns out to be a
consequence of epithelial phenotypical changes, which can
be induced by any renal insult/condition and, possibly, also
by passage of crystals/oxalate.
Consequences of intratubular nephrocalcinosis
Intratubular nephrocalcinosis is as harmful to renal function
as the number of tubules it functionally impairs. Whereas
the mechanism of tubular impairment is straightforward
for obstruction, it is harder to ascribe any direct deleterious
effect to crystal adhesion. Since both processes differ in
their nature, different ways of affecting renal function are to
be expected. While obstruction presents itself rather acutely,
adhesion most likely exerts chronic effects adding to the
severity of an already underlying pathology or condition.
Tubular obstruction acutely impairs tubular function by
mechanical blockage of tubular fluid flow, followed by
tubular atrophy, interstitial inflammation and interstitial fi-
brosis, and hence chronic renal damage/insufficiency de-
velops [41,42]. Histological evidence hereto was found in
pathologies with acute and/or excessive forms of crystal for-
mation and subsequent intratubular retention such as acute
phosphate nephropathy [43], primary hyperoxaluria, je-
junoileal bypass-induced enteric hyperoxaluria [33,44] and
several drug-induced crystal nephropathies (methotrexate,
acyclovir) [45,46]. Since the histopathology in these dif-
ferent types of crystal deposition shows important parallels
with classical (ureteral) obstructive nephropathy, the bulk of
the associated tubulo-interstitial changes most likely results
from obstruction itself rather than of a chemical (nephro-
toxic) effect/contribution of the different types of retained
crystals. With respect to nephrolithiasis, it has been ob-
served that in patients with primary hyperparathyroidism
and calcium phosphate stones, patients with brushite or
cystine stones and patients with distal renal tubular aci-
dosis, tubular obstruction presents itself as calcium phos-
phate (cystine in cystinuria) crystal plugging of the ducts
of Bellini with crystals protruding out of the papillary
slits/mouths into the pelvic lumen [47–50]. It is hypoth-
esized that these crystal plugs, besides inducing fibrosis,
tubular atrophy and even glomerular pathology [50], can
form the nidus or platform for stone formation in these
kidneys (see Figure 1).
In less severe/acute forms of nephrocalcinosis, as found
in transplant patients and preterm infants, the effect of mere
crystal adhesion might be more straightforward. Indeed, nu-
merous in vitro studies, mimicking these non-obstructive
crystal-cell interactions, investigate the epithelial reaction
to CaP and CaOx crystal contact (either apical or ba-
solateral) and report production of inflammatory media-
tors (MCP-1, PGE-2), reactive oxygen species (H2 O2 ) and
release of LDH, thereby marking crystal-induced injury
[21,29,51–53]. However, despite these reactions in vitro, up
to now a clinical detrimental effect of crystal–cell contact
or adhesion is not unequivocally proven in vivo. In kid-
ney transplant patients, Pinheiro et al. reported a 12-year
allograft survival rate of 75% in the absence of nephrocal-
cinosis, whereas in the presence of nephrocalcinosis, allo-
Nephrol Dial Transplant (2009) 24: Editorial Review
graft survival decreased to 48% [54]. Although these data
suggest an association between nephrocalcinosis and an in-
creased risk of allograft failure, it should be noted that half
of the allografts survive despite the presence of nephrocal-
cinosis. Also, in several prospective and retrospective stud-
ies, in which preterm infants with nephrocalcinosis were
compared with birth-weight- and postnatal (or gestational)
age-matched controls without nephrocalcinosis, no clear
evidence for an association between neonatal nephrocal-
cinosis and renal dysfunction in the long term was found
[55–58].
Overall, it is likely that the individual renal outcome de-
pends on numerous factors, such as the severity of the un-
derlying disorder and the extent, rate and duration of crystal
formation/adhesion on the one hand and the activity of re-
nal crystal clearing mechanisms on the other (see further
in the text) [34,59,60,61]. Possibly, adhered crystals may
affect normal tubular redifferentiation/regeneration ham-
pering restoration of a sufficient amount of functioning
tubules and, in addition, may further enlarge by growth
Downloaded from http://ndt.oxfordjournals.org/ by guest on December 27, 2015
and aggregation with other crystals leading to obstructive
tubulopathy.
Interstitial nephrocalcinosis
Mechanisms of interstitial nephrocalcinosis
The presence of crystals in the renal interstitium is defined
as interstitial nephrocalcinosis. Two independent mecha-
nisms may explain the appearance of these crystals in
the interstitium: translocation of intratubular crystals and
de novo interstitial crystal formation.
It has been hypothesized that translocation of crystals
can be established via transcytosis, a process during which
small intraluminal crystals are internalized within apical
vesicles (either receptor mediated or not) and translocated
transcellularly to the basolateral side where the crystals
are released into the interstitial extracellular environment
[62]. Although apical endocytosis of small crystals has been
described [28,59,60], to the best of our knowledge, there
is no evidence supporting the basolateral release of crys-
tals into the interstitium. Instead, these crystals most likely
disintegrate into lyosomes [60,61]. Recently, in an in vivo
study we described an alternative mechanism of transep-
ithelial crystal translocation, which also has been reported
under the term ‘exotubulosis’ by De Bruijn and co-workers
[34,63,64]. These studies demonstrated that intratubular ad-
hered crystals can be overgrown by tubular epithelial cells
adjacent to the crystal adhesion site. Within 2 weeks, these
proliferating and migrating cells cover the crystals and dif-
ferentiate into a new mature epithelium, with its basement
membrane on top of the crystals and its apical side directed
to the lumen, thereby restoring epithelial integrity of the
affected tubule (see Figure 1).
Both endocytosis and epithelial overgrowth do not seem
to be pathologic processes, and hence can be considered
defence mechanisms against renal calcification since crys-
tals disappear from the healthy kidney either in intracel-
lular vesicles (lysosomes) or via the extracellular intersti-
tium [34,60,61,65]. Currently, it is thought that crystals
disintegrate/dissolve due to a local decrease in pH and
Nephrol Dial Transplant (2009) 24: Editorial Review
exposure to proteases [34,61]. Deficiency or saturation
of these clearance mechanisms would reasonably result in
tubular and/or interstitial crystal accumulation. It is cur-
rently unknown, however, what happens to the ionic con-
stituents of the crystals after being dissolved. Are they im-
mediately cleared from the kidney or do they accumulate by
binding to interstitial macromolecular constituents (such as
hyaluronan) and, at a later stage, initiate interstitial super-
saturation and de novo crystal formation during the turnover
of these macromolecules?
Besides translocation, crystals can also be formed
de novo in the interstitium. It is not known, however,
whether this is merely due to a chemically driven su-
persaturation or whether cells are involved. Since up to
now, the presence of osteoblast-like cells has not been re-
ported in the parenchyma of calcified kidneys, no cell-
mediated pathological bone-forming process, similar to that
described for vascular calcification [66], can be suggested
in the kidney at the moment. Regardless of the mechanism,
de novo crystal formation is thought to be the process by
which the development of Randall’s plaque is initiated (see
Figure 1). In the clearest form of Randall’s plaque forma-
tion, as found in biopsies of patients with idiopathic calcium
oxalate nephrolithiasis (and some healthy controls), ultra-
structural studies did not reveal intratubular or intracellular
crystals. The smallest mineral deposits observed were scat-
tered nano-sized, laminated apatite-protein particles in the
basement membranes of the thin loops of Henle [33,67].
Although the specific mechanisms are still unclear, it is
known that these particles may extend into the medullary
interstitium and outgrow further towards the papillary sur-
face [67]. There, as an aggregated mineral syncytium, they
can either lie beneath the urothelium or be exposed to the
urinary environment after the loss of urothelial integrity
[33,67,68].
Various mineral types can be found in the intersti-
tium, e.g. calcium phosphate, calcium oxalate and adenine
[33,34]. Although translocation by epithelial overgrowth
gives a plausible explanation for the appearance of these
mineral deposits in the interstitium, de novo formation of
crystals other than calcium phosphate has to be considered.
However, up to now, no ‘non-calcium phosphate’ interstitial
crystal/particle outgrowth, histopathologically comparable
to classical Randall’s plaque formation, has been found
[47,48,50]. Also, current methodology does not allow for
the assessment of whether interstitial crystals originated in
the tubular lumen or were formed directly in the intersti-
tium. Therefore, it does not allow for the assessment of
whether or not in acute phosphate nephropathy, for exam-
ple, translocation is the main mechanism by which calcium
phosphate occasionally appears in the interstitium [34,43].
Identifying specific urinary or interstitial proteins incorpo-
rated in the crystals might provide clues on their site of
origin [69,70].
Consequences of interstitial nephrocalcinosis
Currently, no evidence has been provided showing that
mere interstitial nephrocalcinosis, whether de novo or ac-
quired via translocation, impairs renal function. Randall’s
plaque formation is not associated with inflammation, does
not damage epithelial cells and starts in basement mem-
2033
branes of morphologically normal epithelial cells of Henle’s
loop. Subsequently, when further interstitial crystal out-
growths lie in the vicinity of collecting ducts and ducts of
Bellini, these epithelia show no morphological abnormal-
ities [33]. Only when calcification completely surrounds
the thin loops of Henle, is an association with epithelial in-
jury found [33]. In addition, extensive calcification might
present itself as a physical interstitial barrier impairing
proper medullary/papillary function. Concentrating abil-
ity, however, seems not to be influenced since a correlation
between low urinary volume (and high urinary calcium)
and papillary plaque coverage was found [71]. Whether the
overall morphological absence of epithelial injury and in-
terstitial inflammation/fibrosis in the context of Randall’s
plaque formation is due to the crystal type (consistently be-
ing calcium phosphate) or to the site of origin is not known.
The main currently known clinically important feature
of Randall’s plaques is their proposed anchor function for
stones found attached to the papillae of patients with idio-
pathic calcium oxalate nephrolithiasis [33,72]. Moreover,
Downloaded from http://ndt.oxfordjournals.org/ by guest on December 27, 2015
ultrastructural studies corroborate the hypothesis that Ran-
dall’s plaques actually might serve as a platform for stone
development by progressive alternation of successive pro-
tein matrix deposition and crystal nucleation (see Figure 1)
[68]. This concept already was proposed by Alexander Ran-
dall in the late 1930s [73,74]. He observed calcium phos-
phate lesions (plaques) on the papillary surface and noticed
that renal stones were intimately attached to them. In ad-
dition, the mineral composition of the plaque (being cal-
cium phosphate) was different from that of the stones and
the plaques occurred in higher incidence than clinical renal
stones. Based on these observations, he concluded that renal
stones were originating as a slow deposition/crystallization
of urinary salts (calcium oxalate, calcium phosphate, uric
acid) upon a lesion of the renal papilla. These observations
have recently been confirmed and extended for patients
with idiopathic calcium oxalate nephrolithiasis [33,68,72].
In contrast to Randall’s plaques, initially appearing in-
nocuous, it is known that interstitial CaOx crystals can be
associated with inflammatory cells [34,75,76]. Whether this
is due to a higher reactivity of CaOx is not known. Although
it has been shown that fibroblasts can produce inflamma-
tory mediators upon contact with oxalate ions and calcium
oxalate crystals [53], it is of interest that interstitial crys-
tals and associated inflammation are always associated with
intraluminal crystal formation. The recruitment of inflam-
matory cells may therefore already be initiated by prior
luminal crystal–cell interactions inducing the production
of inflammatory mediators [21,29,51–53]. Altogether, it
can be suggested that interstitial inflammation can only be
found in disorders in which interstitial crystal deposition
is accompanied with intraluminal crystal formation and/or
handling.
Concluding paragraph
Nephrocalcinosis is a complex multifactorial histopathol-
ogy presenting itself via different mechanisms, each having
their own pathological course and mutually not exclusive.
Recent findings have made it possible to gain some new
insights into this complexity.
2034
A sufficient number of experimental and clinical ob-
servations strongly suggest that crystals do not adhere to
the normally differentiated tubular epithelium, but rather
attach to dedifferentiated/regenerating epithelial cells
[5,6,8,24,30]. We state that the shift of a normal tubular ep-
ithelial phenotype into a phenotype capable of binding crys-
tals can be induced by many unrelated insults/conditions.
Furthermore, if an epithelium binds crystals, kidneys can
react by several crystal clearing mechanisms involving
crystal degradation and dissolution, such as endocytosis and
epithelial overgrowth (‘exotubulosis’) [34,60,61,63,64].
However, when clearance mechanisms are surpassed or
compromised due to some underlying disease state or clini-
cal condition, intratubular crystals may accumulate and add
to the severity of that particular disorder. This might explain
why half of the renal allografts with nephrocalcinosis de-
teriorate more rapidly [54]. Most likely, these grafts are
already in a suboptimal state, favoring nephrocalcinosis,
and hence accelerating chronic allograft deterioration. In
this view, nephrocalcinosis may be considered a marker of
a less potent allograft.
Whereas intratubular nephrocalcinosis seems to develop
secondary to an underlying renal disorder/condition, the ini-
tiating event in interstitial nephrocalcinosis in the context
of Randall’s plaque formation does not seem to be associ-
ated with any kind of prior tubulo-interstitial morpholog-
ical aberration [33] or decreased renal function. However,
determining the mechanism initiating Randall’s plaque for-
mation is complicated by the lack of relevant experimental
models. Importantly, a recent study showed that stone for-
mation (one probable consequence of Randall’s plaques) is
associated with a 60% increased risk of developing chronic
kidney disease (Rule A et al. Abstract F-FC202 at the 41st
Annual Meeting of the American Society of Nephrology,
2008). Although this study does not show causation, or
does not include the type of stones or their possible initiating
mechanisms, it demonstrates the importance for continuous
efforts to unravel the exact mechanism of nephrolithiasis
(and by extension that of nephrocalcinosis), which, in the
long run, would allow for the establishment of preventive
strategies.
Conflict of interest statement. None declared.
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Received for publication: 26.12.08; Accepted in revised form: 24.2.09