Nephrol Dial Transplant (2009) 24: 20302035

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. DHaese and Marc E. De Broe
Department of Medicine, Laboratory of Pathophysiology, University of Antwerp, Antwerp, Belgium
Correspondence and offprint requests to: Patrick C. DHaese; E-mail: Patrick.Dhaese@ua.ac.be

Two key processes determine the development of intratubular nephrocalcinosis, crystal formation and crystal retention. Crystals (mainly calcium oxalate, CaOx, and calcium
phosphate, CaP) are thought to frequently form in the tubular fluid as a result of supersaturation mainly in the distal
nephron and can be considered a renal mechanism to excrete an increased amount of waste per unit of volume
[14]. To ensure safe tubular crystal passage, the healthy
kidney on the one hand presents a non-crystal binding epithelium [59] and on the other hand is able to keep crystal
nucleation, growth and aggregation under control via urinary micro- and macromolecular constituents such as citrate, magnesium and proteins [1013]. In addition, at the
physiological level, a high intratubular calcium concentra-

1. In vitro renal cell-lines as well as primary human epithelial cell cultures only bind crystals firmly in their
subconfluent phase, i.e. when cells are proliferating
and migrating, and lose this capacity once they become 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 treatment, the numerous crystals present in the tubular fluid
were not adhering to the normal differentiated intact epithelium. After 4 days of treatment, however, crystals

The most common form of renal stone disease, calcium

nephrolithiasis, is defined as the presentation of a macroscopic concrement of inorganic (calcium phosphate and/or
calcium phosphate) and organic material in the renal calyces 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 observations in human biopsies have shown the presence of
two distinct types of renal microscopical crystal deposition
processes; one taking place within the tubular lumen (intratubular nephrocalcinosis), and the other in the interstitium (interstitial nephrocalcinosis). Recent observations,
however, strongly suggest that nephrocalcinosis and calcium 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 involved in both types of nephrocalcinosis (intratubular and
interstitial), their possible consequences and their relation
to calcium nephrolithiasis.

Intratubular nephrocalcinosis

C The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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Mechanisms of intratubular nephrocalcinosis

tion triggers a reduction in antidiuretic hormone-stimulated

water permeability of the collecting duct through the
calcium-sensing receptor, leading to an increased urinary
volume and a reduced risk of supersaturation [14,15]. Failure or shortcomings of these defence/control mechanisms
result in crystal retention (= intratubular nephrocalcinosis),
either presenting as epithelial crystal adhesion, when crystals 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 mechanical 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 process in which a particular tubular epithelial phenotype
is responsible for firm crystal adhesion [58,16,17] (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 differentiated tubular epithelia. These molecules, such as sialic acidcontaining proteins and/or phospholipids, phosphatidyl
serine, nucleolin-related protein, annexin II, osteopontin
and hyaluronan, are expressed by dedifferentiated or regenerating cells [5,7,8,1726]. 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.

Keywords: crystal formation; crystal retention; nephrocalcinosis;

nephrolithiasis; Randalls plaque

Nephrol Dial Transplant (2009) 24: Editorial Review

2031

were found adhering to dedifferentiated/regenerating

cells [5]. Interestingly, during washout, shortly after
arrest of a 4-day EG-administration period, the number 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 osteopontin and hyaluronan at first biopsy 12 weeks posttransplantation, while only 20% of these patients presented 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 expression 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 immature epithelium, also presented luminal osteopontin
and hyaluronan; however, nephrocalcinosis did not develop until several days of life [16]. This delay most
likely is due to the time it takes for diet and/or medication protocols to induce crystalluria and subsequent
crystal adhesion [16,31,32].

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 snapshot of transient (non-adhesive) crystalcell 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 adhesion 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 crystalcell 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 apoptotic and/or necrotic cells (known to present altered membrane surfaces) and even to denuded basement membranes
after cells have been lost from the epithelium [26,38,39].
Furthermore, besides crystal adhesion, nucleation of crystals onto the tubular epithelium has been suggested to be a
potential mechanism underlying intratubular nephrocalcinosis [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 surface appears also to be a critical determinant in modulating
this process [25,40].

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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.

2032

Altogether, whereas excessive crystal formation/

aggregation may result in tubular obstruction and its deleterious 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

graft survival decreased to 48% [54]. Although these data

suggest an association between nephrocalcinosis and an increased risk of allograft failure, it should be noted that half
of the allografts survive despite the presence of nephrocalcinosis. Also, in several prospective and retrospective studies, 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 nephrocalcinosis and renal dysfunction in the long term was found
[5558].
Overall, it is likely that the individual renal outcome depends on numerous factors, such as the severity of the underlying disorder and the extent, rate and duration of crystal
formation/adhesion on the one hand and the activity of renal crystal clearing mechanisms on the other (see further
in the text) [34,59,60,61]. Possibly, adhered crystals may
affect normal tubular redifferentiation/regeneration hampering restoration of a sufficient amount of functioning
tubules and, in addition, may further enlarge by growth
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 mechanisms 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 crystals 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 transepithelial 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 adhered 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 differentiate 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 crystals disappear from the healthy kidney either in intracellular vesicles (lysosomes) or via the extracellular interstitium [34,60,61,65]. Currently, it is thought that crystals
disintegrate/dissolve due to a local decrease in pH and

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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 fibrosis, and hence chronic renal damage/insufficiency develops [41,42]. Histological evidence hereto was found in
pathologies with acute and/or excessive forms of crystal formation and subsequent intratubular retention such as acute
phosphate nephropathy [43], primary hyperoxaluria, jejunoileal bypass-induced enteric hyperoxaluria [33,44] and
several drug-induced crystal nephropathies (methotrexate,
acyclovir) [45,46]. Since the histopathology in these different 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 (nephrotoxic) effect/contribution of the different types of retained
crystals. With respect to nephrolithiasis, it has been observed that in patients with primary hyperparathyroidism
and calcium phosphate stones, patients with brushite or
cystine stones and patients with distal renal tubular acidosis, tubular obstruction presents itself as calcium phosphate (cystine in cystinuria) crystal plugging of the ducts
of Bellini with crystals protruding out of the papillary
slits/mouths into the pelvic lumen [4750]. It is hypothesized 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, numerous in vitro studies, mimicking these non-obstructive
crystal-cell interactions, investigate the epithelial reaction
to CaP and CaOx crystal contact (either apical or basolateral) and report production of inflammatory mediators (MCP-1, PGE-2), reactive oxygen species (H2 O2 ) and
release of LDH, thereby marking crystal-induced injury
[21,29,5153]. However, despite these reactions in vitro, up
to now a clinical detrimental effect of crystalcell contact
or adhesion is not unequivocally proven in vivo. In kidney transplant patients, Pinheiro et al. reported a 12-year
allograft survival rate of 75% in the absence of nephrocalcinosis, whereas in the presence of nephrocalcinosis, allo-

Nephrol Dial Transplant (2009) 24: Editorial Review

Nephrol Dial Transplant (2009) 24: Editorial Review

Consequences of interstitial nephrocalcinosis

Currently, no evidence has been provided showing that
mere interstitial nephrocalcinosis, whether de novo or acquired via translocation, impairs renal function. Randalls
plaque formation is not associated with inflammation, does
not damage epithelial cells and starts in basement mem-

branes of morphologically normal epithelial cells of Henles

loop. Subsequently, when further interstitial crystal outgrowths lie in the vicinity of collecting ducts and ducts of
Bellini, these epithelia show no morphological abnormalities [33]. Only when calcification completely surrounds
the thin loops of Henle, is an association with epithelial injury found [33]. In addition, extensive calcification might
present itself as a physical interstitial barrier impairing
proper medullary/papillary function. Concentrating ability, 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 interstitial inflammation/fibrosis in the context of Randalls
plaque formation is due to the crystal type (consistently being calcium phosphate) or to the site of origin is not known.
The main currently known clinically important feature
of Randalls plaques is their proposed anchor function for
stones found attached to the papillae of patients with idiopathic calcium oxalate nephrolithiasis [33,72]. Moreover,
ultrastructural studies corroborate the hypothesis that Randalls plaques actually might serve as a platform for stone
development by progressive alternation of successive protein matrix deposition and crystal nucleation (see Figure 1)
[68]. This concept already was proposed by Alexander Randall in the late 1930s [73,74]. He observed calcium phosphate lesions (plaques) on the papillary surface and noticed
that renal stones were intimately attached to them. In addition, the mineral composition of the plaque (being calcium 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 Randalls plaques, initially appearing innocuous, 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 inflammatory mediators upon contact with oxalate ions and calcium
oxalate crystals [53], it is of interest that interstitial crystals and associated inflammation are always associated with
intraluminal crystal formation. The recruitment of inflammatory cells may therefore already be initiated by prior
luminal crystalcell interactions inducing the production
of inflammatory mediators [21,29,5153]. 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 histopathology 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.

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exposure to proteases [34,61]. Deficiency or saturation

of these clearance mechanisms would reasonably result in
tubular and/or interstitial crystal accumulation. It is currently unknown, however, what happens to the ionic constituents of the crystals after being dissolved. Are they immediately cleared from the kidney or do they accumulate by
binding to interstitial macromolecular constituents (such as
hyaluronan) and, at a later stage, initiate interstitial supersaturation 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 supersaturation or whether cells are involved. Since up to
now, the presence of osteoblast-like cells has not been reported in the parenchyma of calcified kidneys, no cellmediated 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 Randalls plaque is initiated (see
Figure 1). In the clearest form of Randalls plaque formation, as found in biopsies of patients with idiopathic calcium
oxalate nephrolithiasis (and some healthy controls), ultrastructural studies did not reveal intratubular or intracellular
crystals. The smallest mineral deposits observed were scattered 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 surface [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 interstitium, 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 Randalls 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 interstitium. Therefore, it does not allow for the assessment of
whether or not in acute phosphate nephropathy, for example, translocation is the main mechanism by which calcium
phosphate occasionally appears in the interstitium [34,43].
Identifying specific urinary or interstitial proteins incorporated in the crystals might provide clues on their site of
origin [69,70].

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Conflict of interest statement. None declared.
24.

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A sufficient number of experimental and clinical observations 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 epithelial phenotype into a phenotype capable of binding crystals can be induced by many unrelated insults/conditions.
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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
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