Nephrol Dial Transplant (2009) 24: 3659–3668
doi: 10.1093/ndt/gfp418
Advance Access publication 29 August 2009
Crystalluric and tubular epithelial parameters during the onset of
intratubular nephrocalcinosis: illustration of the ‘fixed particle’
theory in vivo
Benjamin A. Vervaet, Patrick C. D’Haese, Marc E. De Broe and Anja Verhulst
Laboratory of Pathophysiology, Departments of Medicine and Pharmaceutical, Biomedical and Veterinary Sciences, University of
Antwerp, Antwerp, Belgium
Correspondence and offprint requests to: Patrick C. D’Haese; E-mail: [email protected]
Abstract
Background. The ‘fixed particle’ theory states that, besides
crystal formation in the tubular fluid, crystal adhesion to the
tubular epithelium is a prerequisite for the development of
intratubular nephrocalcinosis. It has been hypothesized that
the tubular epithelium, in order to bind crystals, needs to
be phenotypically altered. Whereas most evidence hereto
is provided by in vitro experiments, we set out to illustrate
this theory in vivo.
Methods. We simultaneously investigated the temporal
changes of nephrocalcinosis-associated parameters during and shortly after a 4-day ethylene glycol (EG)administration period in rats. We measured oxaluria, crystal formation, crystalluria, apoptosis, epithelial injury/
regeneration and luminal membrane expression of several
crystal-binding molecules [hyaluronan (HA), osteopontin
(OPN) and for the first time in vivo, annexin-2 (ANX2)
and nucleolin-related-protein (NRP) and one of their receptors (CD44, HA/OPN-receptor]. Clinically, renal biopsies
of preterm infants, transplant patients and acute phosphate
nephropathy patients were stained for ANX2, NRP, HA and
OPN.
Results. In the presence of a rather constant and persistent intratubular crystal formation, crystal retention gradually increased during EG-administration and markedly increased after arrest thereof, indicating that the development
of crystal adhesion requires more than just the presence of
crystals in the tubular fluid. All luminal membrane markers and a regenerating/dedifferentiated epithelium, unlike
apoptosis, to various extents were upregulated concurrently
and in association with crystal adhesion. However, both in
humans and rats, expression of luminal molecules was not
confined to crystal-containing tubules.
Conclusions. Altogether, these findings allow better insight
into the mechanisms underlying the ‘fixed particle’ theory
in vivo and indicate that an altered epithelial phenotype
with crystal-binding properties precedes crystal adhesion,
thereby corroborating the requirement of tubular epithelial
phenotypical changes in the development of intratubular
nephrocalcinosis.
Keywords: crystal adhesion; crystal-binding molecules; crystal
formation; nephrocalcinosis; regenerating/dedifferentiated tubular
epithelium
Introduction
Intratubular nephrocalcinosis can be defined as the histological observation of calcium oxalate or calcium phosphate crystals retained within the tubules of the kidney. As
to the mechanism of crystal retention, Finlayson and Reid
calculated that crystals cannot grow large enough during
their transit time through the tubules to be retained in the
tubules by their size (the ‘free particle’ theory). This led
to the hypothesis that crystals can only be retained in the
kidney by adhering to the tubular epithelium (the ‘fixed
particle’ theory) [1]. Although it has now been established
that crystal retention by size can occur in pathologies presenting extreme crystal formation and/or aggregation (i.e.
acute phosphate nephropathy and primary hyperoxaluria),
intratubular crystals (with diameters smaller than that of the
tubular lumen) are frequently found adhered to the distal
tubular epithelium, particularly in pathologies presenting
milder forms of nephrocalcinosis such as transplant patients and preterm infants [1–8].
As to the mechanism of crystal adhesion, numerous
in vitro studies have shown that a fully differentiated distal tubular epithelium does not bind crystals, whereas injured and regenerating epithelial cells do have affinity for
crystals [9–14]. Research on the molecular nature of the
crystal-binding phenotype has identified a growing list
of membrane-associated molecules with affinity for calcium crystals for which it is thought that their luminal expression is causative for crystal adhesion, including phosphatidylserine (PS), hyaluronan (HA), osteopontin (OPN),
C The Author 2009. Published by Oxford University Press [on behalf of ERA-EDTA]. All rights reserved.
For Permissions, please e-mail: [email protected]
3660
nucleolin-related-protein (NRP) and annexin-2 (ANX2)
[4,5,10–19].
Phosphatidylserine (PS) is a lipid membrane constituent
that normally resides in the inner leaflet of the double layer
of the plasma membrane. Upon cellular injury and during
apoptosis, however, this lipid flips to the apical leaflet [20].
Phosphatidylserine has been shown to bind crystals in injured cell cultures and some studies report crystal adhesion
to apoptotic cells [12,14,21,22].
Hyaluronan is a high molecular mass polysaccharide. Its
expression in the healthy kidney is primarily confined to
the interstitium of the inner medulla where it plays a role in
renal water handling [23]. In response to mitogens, stress
or mechanical injury tubular epithelial cells express HA
at their apical membrane, suggesting a supporting role in
epithelial proliferation/regeneration processes [24]. HA has
been identified as a major crystal-binding molecule since
crystal retention to HA-expressing proliferating/migrating
MDCK-I cells decreased substantially upon treatment with
Streptomyces hyaluronidase [15].
In contrast to HA, OPN, ANX2 and NRP are glycophosphoproteins. In the normal kidney, OPN is moderately expressed on the luminal membrane of several distal
tubules and abundantly present in the tubular fluid. OPN
has crystal-binding properties, is upregulated during injury/inflammation and thought to function as a chemokine
[25]. Also, since membrane-associated OPN, like HA, is upregulated during regeneration, it has been linked to tubular
crystal retention [11,16,26]. In this context, it is worth mentioning that HA and OPN have a mutual transmembrane cell
surface receptor, CD44, which plays a role in cell–matrix
interactions and signalling [27,28]. Urinary OPN, however,
is currently considered to play an inhibitory role in crystal
formation/retention [29,30].
Nucleolin-related protein has been identified as a
nucleus-cytoplasm shuttle protein [31–33]. In vitro data
by Sorokina et al. provided evidence that proliferating/
migrating cells express NRP on their apical surface
and demonstrated enhanced calcium oxalate monohydrate
(COM) crystal attachment [17].
Annexin-2 is involved in the structural organization and
dynamics of endocytic and secretory pathways [34]. In normal tissue of human tumour nephrectomies ANX2 is localized at the luminal membrane, in collecting ducts, thin and
(part of) thick loop of Henle and many distal convoluted
tubules [35]. Expression of ANX2, particularly cytoplasmic
at corticomedullary junction, is upregulated and selectively
associated with the dedifferentiation and regeneration period following acute renal failure [36]. In vitro ANX2 is also
present on renal epithelial cells and has been demonstrated
to bind COM crystals [18,37].
For these molecules, the number of studies investigating
their actual association with intratubular nephrocalcinosis
in vivo is rather limited [16,29,30]. Also, the ‘fixed particle’
theory is mainly based on in vitro observations. Therefore,
in order to evaluate this theory and to get more insight into
the actual epithelial phenotypical modifications during the
development of nephrocalcinosis in vivo, we simultaneously investigated the temporal changes of oxaluria, crystal formation, crystalluria, epithelial injury/regeneration,
apoptosis and the luminal membrane expression of HA,
B. A. Vervaet et al.
OPN, CD44, and for the first time in vivo, ANX2 and
NRP daily during and shortly after a 4-day ethylene glycoladministration period in rats. In addition, the expression
of HA, OPN and, for the first time in humans, ANX2 and
NRP was examined in biopsies of human pathologies or
conditions with a high prevalence of nephrocalcinosis, i.e.
preterm infants [7,38,39], transplant patients [6] and patients with acute phosphate nephropathy [40], thus presenting a clinically relevant counterpart for our experimental
study.
Materials and methods
Experimental design
Thirty-five male Wistar rats (250 g; Iffa-Credo) were divided into five
groups (n = 7 each). Groups 1–4 received EG-supplemented drinking
water (0.75% vol/vol) and were killed after 1, 2, 3 or 4 days. Group 5
received the EG-solution for 4 days and was killed 2 days after arrest of EGadministration. Twenty-four hours before killing, the animals were housed
in metabolic cages for urine collection and monitoring of fluid intake.
After sedation (Nembutal), a blood sample was collected, and kidneys
were excised. Sagittal slices of renal tissue were fixed in methacarn (60%
methanol, 30% chloroform, 10% acetic acid) or Dubosq–Brasil fixative
(47% ethanol, 11.7% H2 O, 23.5% formaldehyde, 17.6% acetic acid and
4 mM picric acid) for 4 h and embedded in paraffin (52◦ C; Kendall,
Mansfield, MA, USA). One additional sagittal slice was fixed in formol–
calcium fixative for 1.5 h and stored in liquid nitrogen. Urine and serum
samples were frozen at −20◦ C. Experiments were approved by the local
Ethical Committee for Animal Experiments of the Antwerp University.
Urine and serum biochemistry
Oxalate was determined by an enzymatic colorimetric assay (Sigma Diagnostics, Deisenhofen, Germany) in 5 ml urine samples acidified with
100 µl hydrochloric acid (1M). Serum creatinine concentration was analysed on a routine autoanalyser system (Vitros 750 XRC).
Crystalluria
The amount of urinary crystals was determined by centrifuging (1125 g)
3 ml urine for 10 minutes. The urinary pellet was resuspended in 500 µl
TSB and acidified with 100 µl 12% HCl, thereby dissolving crystals. The
pellet calcium content was determined by flame atomic absorption spectrometry (PerkinElmer, Norwalk, CT, USA) as a measure of crystalluria.
The ‘non-crystal-bound’ calcium was determined by measuring the concentration of calcium in the supernatant of 125 µl centrifuged urine. A
decrease in the supernatant calcium is a measure of crystal formation, since
crystal formation recruits free urinary calcium into a solid centrifugeable
crystal structure. Crystalluria was also evaluated microscopically.
Renal crystal content
Renal calcium deposits were visualized by von Kossa staining. Dubosq–
Brasil-fixed 4-µm tissue sections were incubated in 5% silver-nitrate (45
min), rinsed in water, incubated in 1% pyrogallic acid (3 min), again rinsed,
fixed in 5% sodium thiosulfate (1 min) and counterstained with haematoxylin/eosin. Per section (n = 1/rat), crystal retention was quantified by
counting the number of Von Kossa-positive tubules.
Tubular morphology
Renal tissue sections (4 µm) were stained for proliferating cell nuclear antigen (PCNA). For this, methacarn-fixed tissue sections were blocked with
normal horse serum and incubated with mouse anti-human PCNA (Dako,
Carpinteria, CA, USA). The sections were subsequently incubated with the
biotinylated horse anti-mouse antibody (Vector, Burlingame, CA, USA).
Finally, avidin-biotin peroxidase complex (Vector) and diaminobenzidine
were used to detect PCNA. Subsequently, the sections were stained with
periodic acid-schiff (PAS). Nuclei were counterstained with methylgreen.
Tubular injury/regeneration was evaluated by scoring 203 tubular crosssections per renal section [75 in cortex, 78 in Outer Stripe of Outer Medulla
(OSOM), 25 in Inner Stripe of Outer Medulla (ISOM) and 25 in Inner
Fixed particle theory in vivo
3661
Table 1. Scoring system for the evaluation of tubular morphology
(necropsy tissue, n = 25) were stained with ANX2, NRP, HA and OPN
for phenotypical evaluation of the tubules.
Score Morphology
0
1
2
3
4
5
Normal tubule
Tubule with luminal debris
Tubule with brush border injury/dilated tubule
Tubule with PCNA-positive cells
Tubule with flattened cells
Tubule with flattened PCNA-positive cells
Statistics
⎫
⎬
⎭
Injury
Regeneration
Medulla] by means of the semi-quantitative scoring system presented in
Table 1. Results were presented as the weighted mean percentage of tubules
with a particular score relative to the number of tubules scored per group.
Data were either presented as mean ± SD or as individual values and
median (range). Serum creatinine, oxaluria, urinary pellet, supernatant
calcium and the amount of crystal-containing tubules were analysed by
the Kruskal–Wallis test, followed by a Mann–Whitney U-test in combination with Bonferroni correction when more than two groups were compared. Frequencies of the morphological scores and luminal expression
of molecules were analysed by the chi-square-test in combination with
Bonferroni correction. Correlations were assessed by the non-parametric
Spearman ρ-correlation. P < 0.05 was considered significant. Statistics
were performed with SPSS 14.0.
Results
Luminal ANX2-, NRP-, HA-, OPN- and CD44-expression
Renal tissue sections were stained for ANX2, NRP, HA, OPN and CD44
[16]. The methacarn-fixed tissue sections were blocked with 1% BSA for
HA- and with normal horse serum for ANX2-, NRP-, OPN- and CD44staining and incubated with primary labels (biotinylated HA-bindingprotein, Seikagaku, Falmouth, MA, USA; rabbit anti-human ANX2antibody, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; goat
anti-human C23-(nucleolin)-antibody, Santa Cruz Biotechnology Inc.;
goat anti-human OPN-(OP199)-antibody, C.M. Giachelli, University of
Washington; or mouse anti-human CD44-antibody, Bender Medsystems,
Burlingame, CA, USA). For ANX2, NRP, OPN and CD44 sections were
subsequently incubated with secondary labels, biotinylated goat antirabbit, horse anti-goat and horse anti-mouse antibodies (Vector), respectively. Finally, avidin-biotin-peroxidase-complex and diaminobenzidine
were used to detect HA, OPN, and CD44. The sections were counterstained with methylgreen. No staining was observed when primary labels
were omitted.
Luminal ANX2-, NRP-, HA-, OPN- and CD44-expression was quantified in 200 randomly chosen tubules (100 in cortex, 50 in OSOM, 25 in
ISOM and 25 in inner medulla). Scoring results are presented as individual
values (i.e. weighted mean percentage of positive tubules over all renal
regions) and median (range) of those weighted means.
Luminal ANX2-, NRP-, HA-, OPN- and CD44-expression in
crystal-containing tubules
Out of each group, an animal with a sufficient amount of crystal-containing
tubules was selected for detailed evaluation on serial sections. The animals
presented 15 (Day 1), 250 (Day 2), 558 (Day 3), 918 (Day 4) and 1326 (Day
2 after arrest of EG-administration) crystal-containing tubules, respectively. Per animal, two series of serial sections were prepared. One section
out of each series was stained with Von Kossa for visualization of crystals and the remaining sections were stained for ANX2 and NRP and for
HA, OPN and CD44, respectively. For each series, 100 crystal-containing
tubules (with crystals adjacent to the epithelium) were randomly selected
on the Von Kossa-stained sections (25 in cortex, 25 in OSOM, 25 in ISOM
and 25 in inner medulla) and were scored for the presence or the absence
of luminal ANX2-, NRP-, HA-, OPN- and CD44-expression on the serial
sections. Crystal-containing tubules in which crystals were either overgrown by the tubular epithelium [41] or lying free in the lumen were not
included. For reason of clarity, scoring results of all renal regions and
experimental groups were combined and presented as the weighted mean
(±weighted SD).
Apoptosis
Out of each group, we prepared two serial frozen sections of one animal
with a sufficient amount of renal crystals. One section was stained with
Von Kossa for visualization of crystals, and the other by the TUNELmethod for visualization of apoptotic nuclei.
Clinical counterpart
Tissue sections of renal biopsies from patients with acute phosphate
nephropathy (n = 16), transplant patients (n = 5) and preterm infants
Urine and serum biochemistry
In the control group, urinary oxalate excretion was 0.77 mg/
24 h (range: 0.02–1.04 mg/24 h). Supplementation of
the drinking water with 0.75% EG induced hyperoxaluria
within 24 h (median: 2.25 mg/24 h; range: 1.83–2.57 mg/24
h) and, although to a lower extent, persisted until 2 days after
stopping EG-administration (Figure 1a). Serum creatinine
did not change significantly during the study, indicating that
renal function was preserved (median: 0.4 mg/dl; range:
0.1–2.9 mg/dl). Urine production and water consumption
increased slightly (but not significantly) compared with
controls during EG-administration (data not shown).
Crystalluria
Control animals did not show calcium oxalate crystals
in their urinary pellets (Figure 1c). Accordingly, the calcium content in pellets of centrifuged urine of controls
was low (median: 0.17 mg/24 h; range: 0.06–0.57 mg/
24 h) (Figure 1b). However, from 24 h after starting EGadministration until the end of the administration period,
the urinary pellets of treated animals contained calcium oxalate crystals (mainly bipyramidal calcium oxalate
dihydrate—COD), corroborated by a 3.5 to 9-fold increase
in the pellet calcium content, indicating crystalluria. A decrease in the calcium content of the supernatant of centrifuged urine within 24 h after start of EG-administration
from 5.8 mg/24 h (control, range: 1.40–8.43 mg/24 h) to
1.81 mg/24 h (range: 1.59–4.04 mg/24 h) confirmed crystal formation (Figure 1d). Within 48 h after arrest of EGadministration, calcium oxalate crystals were no longer observed by microscopy and the centrifugeable amount of
calcium fell within the range of control values.
Renal crystal content
Control rat kidneys showed no Von Kossa-positive
crystals in the tubules. One day after start of EGadministration, five crystal-containing tubules/sagittal renal section (range: 1–20) were found, increasing further
during EG-administration to 38 (range: 0–918) at Day 4
(Figure 2). Two days after arrest of EG-administration,
the median of crystal adhesion showed an almost 10-fold
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B. A. Vervaet et al.
Fig. 1. Urinary biochemistry showing hyperoxaluria (a), an increased calcium content in the pellet (b) and decreased calcium content in the supernatant
(d) of centrifuged urine corroborating crystalluria during EG-loading. (c) Urinary sediment inspected by bright field microscopy of a control rat (c1)
and a 0.75% ethylene glycol (EG)-treated rat (c2) at Day 1 after start of EG administration (500×). (c1) Control rats did not show crystals in their urine.
(c2) Sediment of treated rats showed abundant calcium oxalate crystals; both monohydrate (arrowheads) and dihydrate (arrows). Data are presented as
individual values (diamonds) and median (horizontal bars). a, b, c, d and e: P ≤ 0.05 versus control (Ctr) and Days 1, 2, 3 and 4, respectively, by the
Mann–Whitney U-test. No significant changes were observed versus Day 1, 2, 3 and 4, hence b, c, d and e are not displayed.
increase to 364 tubules/section (range: 2–1326). No intracellular crystals were observed throughout the study.
Tubular morphology
Fig. 2. Renal crystal content in function of time during and shortly after a
4-day EG-administration period. Quantification of the amount of crystalcontaining tubules on Von Kossa-stained sections (n = 1/animal) shows
the development of nephrocalcinosis. Data are presented as individual
values (diamonds) and median (horizontal bars). a, b, c, d and e: P ≤ 0.05
versus control (Ctr) and Days 1, 2, 3 and 4, respectively, by the Mann–
Whitney U-test. No significant changes were observed versus Day 1, 2, 3
and 4, hence b, c, d and e are not displayed. Also, although cortex, outer
stripe of outer medulla (OSOM), inner stripe of outer medulla (ISOM)
and inner medulla were scored separately, results of these compartments
were combined to avoid overloading data presentation.
Supplementation of drinking water with 0.75% EG induced moderate changes in tubular morphology without
overt necrosis. Tubular morphology after one day of EGadministration was comparable to controls (Figure 3) [16].
Morphological analysis of renal sections at Days 2 and
3 during EG-administration showed the appearance of injury and regeneration that extended from Day 2 on. These
events were evidenced by a slight rise in luminal debris, dilatation and proximal brush border injury on the
one hand and the appearance of flattened cells and increased PCNA-expression on the other hand. After arrest of
EG-administration, the number of regenerating tubules increased significantly, as did the numbers of injured/dilated
tubules (Figure 3). Overall, tubules with flattened and
PCNA-positive cells and injured/dilated tubules correlated
best with the renal crystal content (Table 2).
Since regenerating proximal (PT) and distal (DT)
tubules, with complete epithelial flattening, morphologically cannot be distinguished from each other, no distinction
was made between these two cell types in this study.
Fixed particle theory in vivo
3663
Fig. 3. (a) General evaluation of tubular morphology. (b) Scoring results of tubules with flattened phenotype, either with or without PCNA-positive
nuclei, are presented in more detail to show the relation with crystal retention in Figure 2. Data are presented as mean percentages of a particular
phenotype per group. a, b, c, d and e: P ≤ 0.05 versus control (Ctr) and Days 1, 2, 3 and 4, respectively, by the Mann–Whitney U-test. Y-axis in (a)
runs to 100%, but was truncated at 50%. Also, although cortex, outer stripe of outer medulla (OSOM), inner stripe of outer medulla (ISOM) and inner
medulla were scored separately, results of these compartments were combined to avoid overloading data presentation. The legend includes examples of
the different phenotypical scores, with black arrows pointing towards the respective phenotypical characteristics.
Luminal ANX2-, NRP-, HA-, OPN- and CD44-expression
in rats
In control animals, luminal expression of HA, CD44
and NRP was absent or very low (median <5% of
scored tubules), while ANX2 and OPN had a relatively
high baseline expression (median ca. 10%). During EGadministration, luminal expression of ANX2, NRP, HA,
OPN and CD44 increased at different degrees concomi-
tantly with crystal retention (Figure 4a–e). After arrest of
EG-administration, luminal HA-, CD44- and, to a certain
extent, ANX2-expression increased even further, as was
the amount of renal crystals, while expression of NRP and
OPN already had reached control levels at that time. Of the
proteins under study, HA- and CD44-expression correlated
best and significantly with the amount of renal crystals, r =
0.651 and 0.646, respectively (Table 2).
3664
B. A. Vervaet et al.
Fig. 4. (a–e) Luminal expression of ANX2, NUCL, HA, OPN and CD44. Data are presented as individual percentages of scored tubules (diamonds)
and median (horizontal bars). a, b, c, d and e: P < 0.05 versus control (Ctr) and Days 1, 2, 3 and 4, respectively, by the Mann–Whitney U-test.
Although cortex, outer stripe of outer medulla (OSOM), inner stripe of outer medulla (ISOM) and inner medulla were scored separately, results of these
compartments were combined to avoid overloading data presentation. (f ) Luminal expression of ANX2, NRP, HA, OPN and CD44 in crystal-containing
tubules. Weighted average (± weighted SD) of the combined scores of all renal regions and animals.
Table 2. Spearman’s ρ correlation of the number of crystal-containing tubules (Von Kossa) with tubular morphology and expression of luminal
molecules
Tubular morphology
Von Kossa
Luminal molecules
Von Kossa
Normal tubule
Tubule with luminal debris
Tubule with brush border injury/dilated tubule
Tubule with PCNA-positive cells
Tubule with flattened cells
Tubule with flattened PCNA-positive cells
−0.698∗
0.181
0.628∗
0.634∗
0.738∗
0.360∗
Annexine 2
Nucleolin
Hyaluronan
Osteopontin
CD44
0.489∗
0.205
0.651∗
0.514∗
0.646∗
∗ Significant
with P < 0.05.
Fixed particle theory in vivo
3665
Luminal ANX2-, NRP-, HA-, OPN- and CD44-expression
in crystal-containing tubules
For the two series of serial sections, 356 (ANX2, NRP)
and 347 (HA, OPN, CD44) crystal-containing tubules were
scored. ANX2, NRP, HA, OPN and CD44 were luminally
present in 40 ± 7%, 26 ± 11%, 33 ± 12%, 60 ± 12%,
and 44 ± 18% of crystal-containing tubules, respectively
(mean ± SD, Figure 4f). Taking overlapping expression
into account, it was found for ANX2 and NRP that 49 ±
9% of crystal-containing tubules did not present either of
these molecules, whereas for HA, OPN and CD44, this was
26 ± 13%. In addition, it was noticed that tubules without
intratubular crystals could also present luminal expression
of these molecules.
Apoptosis
Regardless of the duration of EG-administration, none or
only occasional apoptotic cells were present in the renal tissue. Therefore, no detailed quantification could be
performed.
Luminal ANX2-, NRP-, HA- and OPN-expression
in humans
In transplant patients, preterm infants and patients with
acute phosphate nephropathy ANX2, HA and OPN were
expressed on the luminal membrane at different degrees.
HA and OPN were clearly present, whereas ANX2 was
expressed to a lower extent. Overall, luminal NRP was
scarcely present in these pathologies, nevertheless an occasional tubule presenting luminal NRP-expression could be
identified (Figure 5). In contrast to the tissue of acute phosphate nephropathy patients, the tissue available for transplant patients and preterm infants did not show any crystal
deposits. In acute phosphate nephropathy patients, luminal
HA-, OPN-, ANX2- and NRP-expression could be found
in tubules with and without crystals.
Discussion
The ‘fixed particle’ theory, by which non-obstructive
nephrocalcinosis is currently explained, states that intratubular crystal retention occurs by adhesion of crystals to
the tubular epithelium [1]. Whereas in vitro studies provided
evidence for the potential involvement of certain crystalbinding molecules expressed on the luminal membrane of
phenotypically altered epithelial cells [11,12,15,17,18,24],
in vivo studies actually relating these findings to nephrocalcinosis are scarce [4,16,29,30,42]. In this in vivo study, for
the first time, data were presented in which relevant urinary
and epithelial parameters were investigated simultaneously
during the onset of (EG-induced) intratubular nephrocalcinosis.
The EG-model is widely accepted for inducing intratubular nephrocalcinosis. Although injurious to the kidney, a
low-dose (0.75% vol/vol) and short-term (4 days) EGadministration does not result in acute renal failure [16,43–
45]. Here, hyperoxaluria was present within the first 24 h
Fig. 5. Luminal expression of ANX2, NRP, OPN and HA in human
pathology with a high incidence of nephrocalcinosis. HA and OPN
were most abundantly present. ANX2 was present to a lower extent and
only an occasional tubule with NRP could be found. Arrow: luminal
expression.
3666
and was accompanied by a gradual decrease in the amount
of calcium in the supernatant of centrifuged urine, suggesting that calcium was being incorporated into a centrifugeable crystal-bound phase (= crystal formation). Indeed, crystalluria was confirmed both microscopically and
by the presence of an increased amount of calcium in
the urinary pellet. Remarkably, during the course of EGadministration, the decrease in supernatant calcium was
larger than the increase in pellet calcium, suggesting a reduced crystal excretion, which in turn was confirmed by
a concomitantly increasing number of crystal-containing
tubules.
As to the mechanism of crystal adhesion, three interesting observations were made. Firstly, whereas crystal
formation/excretion was already present at Day 1 of EGadministration, crystal retention only developed gradually,
illustrating that the kidney requires a certain incubation
period towards crystal adhesion. Similarly, Marengo et al.
found that crystal retention did not develop until 2 weeks of
continuous mini-pump infused oxalate exposure, whereas
crystalluria was already induced within 4 days [46]. The
fact that their observations were accompanied by an average hyperoxaluria almost double to ours (4.4 mg/24 h)
indicates that mere non-extreme hyperoxaluria/crystalluria
might not be that effective in inducing nephrocalcinosis
as compared to the EG-model. Since oxalate seems to be
injurious to the tubular epithelium only at supraphysiological concentrations [47] and EG, next to oxalate, is also
metabolized to nephrotoxic glycolate and glyoxylate, the
difference between these models might be explained by
their distinctive effects on the tubular epithelial phenotype.
Secondly, the fact that no unequivocal temporal order of
appearance between the increasing epithelial phenotypical changes and the concurrently increasing crystal adhesion could be deduced actually supports the involvement
of epithelial changes in the ‘fixed particle’ theory, since
conversion of a normal epithelium into a crystal-binding
one, in an environment that is loaded with crystals, would
immediately result in crystal adhesion. Thirdly, after arrest of EG-administration, the number of crystal-containing
tubules clearly increased and crystal excretion no longer
occurred, whereas crystal formation was still present and
comparable with (or even lower than) the level of formation
during EG-administration (i.e. slightly decreased hyperoxaluria and increased calcium content in urinary supernatant,
Figure 1). In other words, whereas maximal hyperoxaluria
(and thus crystal formation) was already reached during
EG-administration, markedly more crystals were retained
in the kidney after arrest of EG-administration. This again
clearly indicates that the development of crystal adhesion
requires more than just the mere presence of crystals in
the tubular fluid. Interestingly, morphological evaluation
revealed an increased presence of both injured and regenerating epithelia—with known crystal-binding affinity
[16]—after arrest of EG-administration (Figure 3). As injury increased by only 50% and regeneration increased by
more than 500% (Figure 3), the observed effects on crystal
retention and excretion presumably were due to the release
of the toxic EG-pressure on the kidney by which the latter
could fully unfold its natural regenerating ability. In line
with this, a rat study of Asselman et al., with continuous
B. A. Vervaet et al.
EG-administration for 4 and 8 days, did not show a marked
drop in crystal excretion, yet did show a persistent crystalluria and crystal retention [16], indicating that withdrawal
of EG apparently is necessary to induce the epithelial phenotypical shift favouring regeneration over injury as the
main crystal-binding phenotype.
Based on morphological data, however, drawing conclusions as to whether either injury or regeneration is the most
important determinant of crystal retention should be done
with caution. Although the morphological features applied
in our scoring system are well recognized, they most likely
do not allow a clear distinction between injured and regenerating epithelial cells on the molecular phenotypical level.
Moreover, it actually seems that injury and regeneration are
that intimately associated with each other that both processes share common overlapping phenotypical features.
For example, it is known that epithelial injury results in
cellular dedifferentiation, characterized by presentation of
a flattened cell morphology, lack of tight junctions and loss
of apical-basal polarity [48–50]. These features, however,
also are characteristic of differentiating or regenerating epithelial cells [51,52]. Therefore, at this moment, we prefer
to use the term dedifferentiated phenotype to address the
putative crystal-binding epithelium rather than specifically
referring to either an injured or regenerating phenotype.
As to the association of apoptosis with the development of nephrocalcinosis, TUNEL-staining was generally
negative. The negligible amount of apoptotic cells could
therefore not have been responsible for the vast number
of adhered crystals. However, the possibility of crystal adhesion to phosphatidylserine in particular is not excluded,
since injured (non-apoptotic) cells could still have presented
this phospholipid (not investigated in the present study)
at their luminal membrane [12,14]. The expression of all
other crystal-binding molecules under study, ANX2, NRP,
HA, OPN and CD44, increased at some point during EGadministration (Figure 4a–e). However, only the expression
pattern of HA, CD44 and, to a certain extent, ANX2 fitted best with the crystal adhesion pattern and showed an
increased expression after arrest of EG-administration. Of
these HA, and its receptor CD44, are the most likely candidates to be quantitatively involved in crystal adhesion
in vivo, since both molecules, in contrast to ANX2, are absent when no crystal retention occurs and present when it
does occur. Remarkably, however, all molecules presented
more luminal expression in crystal-containing tubules as
would be expected if the association with crystal adhesion
was based on coincidence alone, since the expression level
in crystal-containing tubules (Figure 4f ) was always higher
than the general expression over all tubules (Figure 4a–e).
Therefore, in addition to HA and CD44, an involvement of
OPN, ANX2 and NRP with crystal retention cannot be excluded. Furthermore, retention cannot be explained solely
by the molecules under study, suggesting the involvement
of either noninvestigated (such as phosphatidylserine and
sialic acid residues [19]) or new yet to be discovered crystalbinding molecules.
As demonstrated in the present study and by Verhulst
et al. [4], the luminal presence of several crystal binding molecules in humans with nephrocalcinosis of different aetiology suggests parallels in the crystal adhesion
Fixed particle theory in vivo
mechanism of rat and man. Here, the most important observation is that luminal HA-, OPN-, ANX2- and NRPexpression, in humans as well as in rats, is not confined to
crystal-containing tubules. This suggests, on the one hand,
that phenotypical alterations precede crystal adhesion and,
on the other hand, that these changes (characteristic of a
crystal-binding epithelium) are not necessarily due to intratubular crystal retention, however, can be induced by
multiple renal insults/conditions, such as epithelial immaturity, ischemia/reperfusion, inflammation and even passage
of intratubular crystals. Patients therefore yet would not
present an epithelial phenotype with affinity for crystals,
yet would not present nephrocalcinosis as long as crystals
are not formed. Furthermore, the discrepancy in ANX2 and
NRP between humans and rats might indicate that there is
either a basic difference between the EG-model and human
pathology or, alternatively, that there is no unique crystalbinding epithelium with a particular molecular composition. Although not proven by our current data, it is an intriguing concept that the composition and the individual
association of the molecules with crystal retention might
depend on the nature of the underlying disorder/insult. For
example, HA might be more important in nephrocalcinosis
associated with transplant patients and preterms that clearly
present renal epithelial regeneration [4], while ANX2 might
be more important in Dent’s disease since clc-5 deficiency
leads to a luminal upregulation of ANX2 [37]. Additionally, whether and to what extent the crystal type plays a role
in this is currently not known. However, within the current
context, the effect perhaps might not be that dramatic since
both calcium phosphate and calcium oxalate crystals can
be injurious to the tubular epithelium [53] and rapidly adhere to anionic sites on the surface of renal epithelial cells
[54,55], indicating comparable reaction patterns.
In conclusion, the data presented here allow better insight into the mechanisms underlying the ‘fixed particle’
theory in vivo. The first event during the onset of (low-dose
EG-induced) nephrocalcinosis is crystal formation in the
tubular fluid, yet without crystal retention. Next, the tubular epithelium gets injured and luminally expresses several crystal-binding molecules, generally associated with
dedifferentiated (injured/regenerating) cells. Here, the association of nephrocalcinosis with luminal HA, OPN and
CD44 is confirmed [16] and, for the first time in vivo, extended with ANX2 and NRP. Furthermore, crystal retention
gradually develops and, as evidenced immunohistochemically in rat and man, the luminal phenotypical changes can
also be found in tubules without adhered crystals. These
observations therefore indicate that epithelial phenotypical
alterations precede crystal adhesion and corroborate the requirement of a phenotypical shift in the development of
intratubular nephrocalcinosis.
Acknowledgements. We express our gratitude to Drs Markowitz
(Columbia College, New York), Mengel and Gwinner (Hannover Medical School, Germany) for the generous gift of the human kidney biopsy
samples. We also would like to thank Dr C.M. Giachelli (University of
Washington, Seattle, WA, USA) for the generous gift of the OP199 antiserum. We thank Simonne Dauwe, Geert Dams and Ludwig Lamberts for
excellent technical assistance, as well as the biochemistry laboratory of
the Antwerp University Hospital. A.V. is post-doctoral fellow of the Fund
for scientific Research Flanders (FWO).
3667
Conflict of interest statement. None declared.
References
1. Finlayson B, Reid F. The expectation of free and fixed particles in
urinary stone disease. Invest Urol 1978; 15: 442–448
2. Markowitz GS, Nasr SH, Klein P et al. Renal failure due to acute
nephrocalcinosis following oral sodium phosphate bowel cleansing.
Hum Pathol 2004; 35: 675–684
3. Kok DJ, Khan SR. Calcium oxalate nephrolithiasis, a free or fixed
particle disease. Kidney Int 1994; 46: 847–854
4. Verhulst A, Asselman M, De Naeyer S et al. Preconditioning of the
distal tubular epithelium of the human kidney precedes nephrocalcinosis. Kidney Int 2005; 68: 1643–1647
5. Khan SR, Shevock PN, Hackett RL. Acute hyperoxaluria, renal injury
and calcium oxalate urolithiasis. J Urol 1992; 147: 226–230
6. Nankivell BJ, Borrows RJ, Fung CL et al. The natural history of
chronic allograft nephropathy. N Engl J Med 2003; 349: 2326–2333
7. Schell-Feith EA, Kist-Van Holthe JE, Conneman N et al. Etiology of
nephrocalcinosis in preterm neonates: association of nutritional intake
and urinary parameters. Kidney Int 2000; 58: 2102–2110
8. Vervaet BA, Verhulst A, D’Haese PC et al. Nephrocalcinosis: new
insights into mechanisms and consequences. Nephrol Dial Transplant
2009; 24: 2030–2035
9. Verkoelen CF, Van Der Boom BG, Kok DJ et al. Cell type-specific
acquired protection from crystal adherence by renal tubule cells in
culture. Kidney Int 1999; 55: 1426–1433
10. Verkoelen CF, Van Der Boom BG, Houtsmuller AB et al. Increased
calcium oxalate monohydrate crystal binding to injured renal tubular
epithelial cells in culture. Am J Physiol 1998; 274: F958–F965
11. Verhulst A, Asselman M, Persy VP et al. Crystal retention capacity
of cells in the human nephron: involvement of CD44 and its ligands
hyaluronic acid and osteopontin in the transition of a crystal bindinginto a nonadherent epithelium. J Am Soc Nephrol 2003; 14: 107–115
12. Bigelow MW, Wiessner JH, Kleinman JG et al. Surface exposure
of phosphatidylserine increases calcium oxalate crystal attachment to
IMCD cells. Am J Physiol 1997; 272: F55–F62
13. Riese RJ, Mandel NS, Wiessner JH et al. Cell polarity and calcium oxalate crystal adherence to cultured collecting duct cells. Am J Physiol
1992; 262: F177–F184
14. Wiessner JH, Hasegawa AT, Hung LY et al. Mechanisms of calcium
oxalate crystal attachment to injured renal collecting duct cells. Kidney
Int 2001; 59: 637–644
15. Verkoelen CF, Van Der Boom BG, Romijn JC. Identification of
hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells. Kidney Int 2000; 58: 1045–1054
16. Asselman M, Verhulst A, De Broe ME et al. Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys. J Am Soc
Nephrol 2003; 14: 3155–3166
17. Sorokina EA, Wesson JA, Kleinman JG. An acidic peptide sequence
of nucleolin-related protein can mediate the attachment of calcium
oxalate to renal tubule cells. J Am Soc Nephrol 2004; 15: 2057–2065
18. Kumar V, Farell G, Deganello S et al. Annexin II is present on renal
epithelial cells and binds calcium oxalate monohydrate crystals. J Am
Soc Nephrol 2003; 14: 289–297
19. Asselman M, Verkoelen CF. Crystal-cell interaction in the pathogenesis of kidney stone disease. Curr Opin Urol 2002; 12: 271–276
20. Martin SJ, Reutelingsperger CP, McGahon AJ et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of
apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995; 182: 1545–1556
21. Sarica K, Erbagci A, Yagci F et al. Limitation of apoptotic changes
in renal tubular cell injury induced by hyperoxaluria. Urol Res 2004;
32: 271–277
22. Cao LC, Jonassen J, Honeyman TW et al. Oxalate-induced redistribution of phosphatidylserine in renal epithelial cells: implications for
kidney stone disease. Am J Nephrol 2001; 21: 69–77
3668
23. Hansell P, Goransson V, Odlind C et al. Hyaluronan content in the
kidney in different states of body hydration. Kidney Int 2000; 58:
2061–2068
24. Asselman M, Verhulst A, Van Ballegooijen ES et al. Hyaluronan is
apically secreted and expressed by proliferating or regenerating renal
tubular cells. Kidney Int 2005; 68: 71–83
25. Xie Y, Sakatsume M, Nishi S et al. Expression, roles, receptors, and
regulation of osteopontin in the kidney. Kidney Int 2001; 60: 1645–
1657
26. Yamate T, Kohri K, Umekawa T et al. Interaction between osteopontin
on madin darby canine kidney cell membrane and calcium oxalate
crystal. Urol Int 1999; 62: 81–86
27. Aruffo A, Stamenkovic I, Melnick M et al. CD44 is the principal cell
surface receptor for hyaluronate. Cell 1990; 61: 1303–1313
28. Weber GF, Ashkar S, Glimcher MJ et al. Receptor-ligand interaction
between CD44 and osteopontin (Eta-1). Science 1996; 271: 509–512
29. Wesson JA, Johnson RJ, Mazzali M et al. Osteopontin is a critical
inhibitor of calcium oxalate crystal formation and retention in renal
tubules. J Am Soc Nephrol 2003; 14: 139–147
30. Mo L, Liaw L, Evan AP et al. Renal calcinosis and stone formation
in mice lacking osteopontin, Tamm–Horsfall protein, or both. Am J
Physiol Renal Physiol 2007; 293: F1935–F1943
31. Borer RA, Lehner CF, Eppenberger HM et al. Major nucleolar proteins
shuttle between nucleus and cytoplasm. Cell 1989; 56: 379–390
32. Schmidt-Zachmann MS, Dargemont C, Kuhn LC et al. Nuclear export
of proteins: the role of nuclear retention. Cell 1993; 74: 493–504
33. Nigg EA. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 1997; 386: 779–787
34. Monastyrskaya K, Babiychuk EB, Hostettler A et al. Annexins as
intracellular calcium sensors. Cell Calcium 2007; 41: 207–219
35. Domoto T, Miyama Y, Suzuki H et al. Evaluation of S100A10, annexin II and B-FABP expression as markers for renal cell carcinoma.
Cancer Sci 2007; 98: 77–82
36. Cheng CW, Rifai A, Ka SM et al. Calcium-binding proteins annexin
A2 and S100A6 are sensors of tubular injury and recovery in acute
renal failure. Kidney Int 2005; 68: 2694–2703
37. Carr G, Simmons NL, Sayer JA. Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration
in collecting duct epithelial cells. Cell Mol Life Sci 2006; 63: 367–
377
38. Short A, Cooke RW. The incidence of renal calcification in preterm
infants. Arch Dis Child 1991; 66: 412–417
39. Saarela T, Vaarala A, Lanning P et al. Incidence, ultrasonic patterns
and resolution of nephrocalcinosis in very low birthweight infants.
Acta Paediatr 1999; 88: 655–660
40. Markowitz GS, Stokes MB, Radhakrishnan J et al. Acute phosphate
nephropathy following oral sodium phosphate bowel purgative: an
B. A. Vervaet et al.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
underrecognized cause of chronic renal failure. J Am Soc Nephrol
2005; 16: 3389–3396
Vervaet BA, Verhulst A, Dauwe SE et al. An active renal crystal
clearance mechanism in rat and man. Kidney Int 2009; 75: 41–51
Evan AP, Coe FL, Gillen D et al. Renal intratubular crystals and
hyaluronan staining occur in stone formers with bypass surgery but
not with idiopathic calcium oxalate stones. Anat Rec (Hoboken) 2008;
291: 325–334
Gambaro G, Valente ML, Zanetti E et al. Mild tubular damage induces calcium oxalate crystalluria in a model of subtle hyperoxaluria:
evidence that a second hit is necessary for renal lithogenesis. J Am
Soc Nephrol 2006; 17: 2213–2219
Green ML, Hatch M, Freel RW. Ethylene glycol induces hyperoxaluria
without metabolic acidosis in rats. Am J Physiol Renal Physiol 2005;
289: F536–F543
Yamaguchi S, Wiessner JH, Hasegawa AT et al. Study of a rat model
for calcium oxalate crystal formation without severe renal damage in
selected conditions. Int J Urol 2005; 12: 290–298
Marengo SR, Chen DH, MacLennan GT et al. Minipump induced
hyperoxaluria and crystal deposition in rats: a model for calcium
oxalate urolithiasis. J Urol 2004; 171: 1304–1308
Schepers MS, Van Ballegooijen ES, Bangma CH et al. Oxalate is toxic
to renal tubular cells only at supraphysiologic concentrations. Kidney
Int 2005; 68: 1660–1669
Hay ED. The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev Dyn 2005; 233:
706–720
Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J
Kidney Dis 1995; 26: 678–690
Bacallao R, Fine LG. Molecular events in the organization of renal
tubular epithelium: from nephrogenesis to regeneration. Am J Physiol
1989; 257: F913–F924
Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N Engl J
Med 1996; 334: 1448–1460
Bonventre JV. Dedifferentiation and proliferation of surviving epithelial cells in acute renal failure. J Am Soc Nephrol 2003; 14(Suppl 1):
S55–S61
Khan SR. Crystal-induced inflammation of the kidneys: results from
human studies, animal models, and tissue-culture studies. Clin Exp
Nephrol 2004; 8: 75–88
Lieske JC, Leonard R, Swift H et al. Adhesion of calcium oxalate
monohydrate crystals to anionic sites on the surface of renal epithelial
cells. Am J Physiol 1996; 270: F192–F199
Lieske JC, Norris R, Toback FG. Adhesion of hydroxyapatite crystals
to anionic sites on the surface of renal epithelial cells. Am J Physiol
1997; 273: F224–F233
Received for publication: 28.4.09; Accepted in revised form: 23.7.09