Original Paper
NEPHRON
Nephron
F. Grases
L. García-Ferragut
A. Costa-Bauzá
J.G. March
Laboratory ofUrochemistry,
Faculty of
Science, University ofBalearic Islands,
Palma de Mallorca, Spain
1996;73:561-568
Study of the Effects of Different
Substances on the Early Stages of
Papillary Stone Formation
..................................................................................................
Key Words
Abstract
Calcium oxalate crystallization
Heterogeneous nucleation
Inhibitors
Citrate
Phytate
Saponins
We have studied the early stages of calcium oxalate monohydrate (COM) crystallization, mainly heterogeneous nucleation, on fixed particles. Experiments
were performed using a flow system that closely simulates the human renal
conditions related to COM heterogeneous nucleation. The effects of citrate,
phytate, aescin and glycyrrhinic acid in the COM heterogeneous nucleation on
different solid surfaces as calcium phosphate, a mixture ofmucin and calcium
phosphate and wax were studied. The presence of citrate at normal urinary
concentrations (1.85.10-3 M) totally inhibited the development ofCOM crystals only on the most hydrophobic assayed material (wax). Saponins (aescin
and glycyrrhinic acid) only exhibited important inhibitory effects of COM
crystallization on the hydrophobic wax substrate but in no case the formation
of COM crystals was totally prevented. Phytate exerted the most remarkable
effects on the heterogeneous nucleation of COM crystals, thus, when it was
present at 1.43.10-7 M, it totally prevented the development of COM crystals
on the three assayed solid substrates.
Introduction
The actual knowledge of papillary renal stone formation demonstrates that the determining step in the formation of this kind of renal stone is the formation of the first
attached particles to the papillary urothelium. Nevertheless, the exact mechanism that such processes imply is still
not totally understood. Thus, it seems clear that the
antiadherent glycosaminoglycans layer plays an important role in preventing the adhesion of preformed particles to the urothelium and avoiding that particles form
directly on the urothelium [1]. Some authors claimed that
cell endocytosis of calcium oxalate monohydrate (COM)
crystals is responsible for such fixed particles formation
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[2]. Several studies show that preexistent attached particles of phosphates, uric acid, mucoprotein aggregates,
etc., act as powerful inductors of calcium oxalate crystal
formation [3-6]. It must be considered that it was clearly
demonstrated that calcium oxalate formation through homogeneous nucleation is very improbable in the conditions prevailing in the kidney [7]. Referring to the aspects
about the influence of diverse substances (crystallization
inhibitors) on the formation processes of such attached
microuroliths, few aspects are known. In this paper the
effects of citrate, phytate, aescin and glycyrrhinic acid
(two saponins) on the early stages of COM crystallization
have been studied, in conditions simulating those found
in the human kidney.
F. Grases
Laboratory ofUrochemistry
Faculty ofSciencc
University of Balearic lslands
E-07071 Palma de Mallorca (Spain)
Accepted:
August 8, 1995
Material and Methods
Synthetic Urine
The synthetic urine supersaturated with respect to calcium oxalate ([Ca2+] = 3.5.10-3 M [oxalate] = 3.0.10-4 M, supersaturation =
c/ceq = 19.2) was prepared immediately before use by mixing with a
tee-type mixing chamber equal volumes of solutions A and B. Solution A contained
1.93·1O-2M
Na2S04·IOH20,
5.92·10-3M
MgS04·7H20,
S.67·1O-2M NH4Cl, 0.163M KCl and [Ca2+] =
7.0.10-3 M. Solution B contained 1.54.10-2 M NaH2P04·2H20,
1.56.10-2 M Na2HP04·12H20,
0.223 M NaCl and [oxalate] =
6.0.10-4 M. pH of both solutions was adjusted to 5.50 to avoid calcium phosphates precipitation and 0.33 mI of H202 30% were added
per liter of artificial urine as disinfectant to prevent natural bacterium production and multiplication. It has been proved that such
hydrogen peroxide concentration did not significantly alter the stability ofthe oxalate ions. Chemicals of reagent-grade purity were dissolved in deionized and redistilled water.
Substrate Preparation
Wax substrate: 0.3 mI of molten wax are introduced in the sample
holder (fig. 1) and left to solidify.
Calcium phosphate substrate: 0.40 g of CaHP04· 2H20 (Panreac)
were used to prepare a pellet of 13 mm in diameter and approximately 1.6 mm high by compressing them with a hydraulic press (Graseby
Specac).
Calcium phosphate-mucin substrate: 0.392 g of CaHP04·2H20
and O.OOSg of mucin (from porcine stomach supplied by Sigma) (2 %
ofmucin) were used to prepare a pellet ofthe same size as the calcium
phosphate pellets by compressing them with a hydraulic press.
Simulation ofthe Early Stages ofCOM Stone Formation
In a temperature-controlled
(37 o C) chamber, 3 cylindrical flasks
of 3 cm diameter and 5 cm high were placed (fig. 1,2). The substrate
with its correspondent holder was situated in the flask. Synthetic
urine was introduced, fresh prepared, by a multichanel peristaltic
pump, with arate of 780 mUday into the bottom of the flasks overflowing constantly. The system was kept working during two different periods of time (3 and 6 h). When the experiment was finished
the substrate was removed from the system, rinsed with distilled
water and dried at room temperature in a desiccator. Number, type
and size of crystals developed on the substrate surface were evaluated
by Hitachi S-530 scanning electron microscopy. The images have
been processed by a segmentation method of image analysis providing an accurate location of the regions and regularity of its boundaries (fig. 3). This method is based on a mathematical model, which
minimizes a kind of functional energy associated to the image [S].
The image processing was performed with a program integrated in
the scientific software of image processing called 'Megawave' that
runs on a workstation IPCSun.
EjJects ofVarious Compounds
The effects of sodium citrate (supplied by Probus) in the concentration range 5.30.10-4 - 3.17.10-3 M, sodium phytate (supplied by
Sigma) in the concentration range 7.15.10-8 - 1.43.10-6 M, aescin
dihydrate (a saponin supplied by Fluka) in the concentration range
4.40.10-6 - 1.32.10-5 M and glycyrrhinic acid monoamonic salt (a
saponin supplied by Fluka) in the concentration range 2.3S· 10-5 S.93 .10-5 M were assayed by addition of different amounts ofthese
substances to artificial urine. Saponin derivatives are glycosids that
562
Nephron
1996;73:561-568
3 cm
Fig. 1. Simulation unit of calcium oxalate crystallization in flux
conditions. A = Artificial urine A solution channel; B = artificial
urine B solution channel; T = T-type mixing chamber of A and B
solutions; C = artificial urine container; D = waste collection funnel;
S = substrate holder detail.
appear as components of a great number of medical herbs with
claimed antilithiasic properties [11]. Comparison of the number of
COM crystals (determined as explained above) formed on a given
area (S.40·1O-3 mm2) ofthe pellet surface in presence and absence of
a particular additive permitted a direct evaluation of the inhibition
of COM heterogeneous nucleation:
% nucleation inhibition (In) =
Number of crystals in absence of admixtures Number of crystals in presence of admixture
Number of crystals in absence of admixtures
To assure the reproducibility
ment was repeated three times.
x 100
ofthe obtained results, each experi-
Calcium-Citrate Complexation
Due to the high concentration of citrate used and considering its
complexing capacity of calcium ions, in experiments in which the
action of citrate ions was evaluated, a supplement of calcium was
added to obtain the same calcium oxalate supersaturation value (S =
c/ceq = 19.2) that is found in the absence of citrate. It must be considered that a decrease in the supersaturation would imply a decrease in
the nucleation rate that could not be assigned to inhibitory effects.
The amount of added calcium ions was potentiometrically calculated
using a selective calcium electro de (Ingold) and a potentiometer (Crison 2002). Activity of free calcium ions must be the same in citrate
Grases/García- F erragut/Costa- Bauzá/
March
(C)
2
3a
3b
Fig. 2. Scheme of the parallel system used to study the crystallization of ca1cium oxalate. A = Artificial urine A
= controlled temperature chamber; D = urine collector; P = multichannel
solution; B = artificial urine B solution;
peristaltic pump; U = units of ca1cium oxalate crystallization.
Fig. 3. a Digitalized scanning electron microscope photography, and b the corresponding segmented one for
image-treating procedure.
e
Artificial Simulation ofStone Formation
Nephron 1996;73:561-568
563
a
b
Fig. 4. Scanning electron microscope images of COM crystals
developed on three different substrates during a 6-hour period at
37 o C and in the absence of inhibitors. a Calcium phosphate substrate. b Mixture of calcium phosphate and mucin substrate. e Wax
substrate.
presence and absence, consequently when citrate was present an
amount of calcium that fulfilled such conditions was added in each
case. Thus, in presence of citrate 0.53.10-3 M, total calcium concentration was 3.65· 10-3 M, in presence of citrate 1.85· 10-3 M, total
calcium concentration was 4.00.10-3 M and in presence of citrate
3.17.10-3 M, total calcium concentration was 4.40.10-3 M.
When using phytate, due to the low used levels, the decrease in
the free calcium concentration was practically negligible, as it was
potentiometrical1y observed and, consequently, in this case it was not
necessary to add a calcium supplement.
Results
The COM crystals formed by heterogeneous nuc!eation on the three assayed substrates are shown in figure 4.
It is interesting to observe that the use of a mixture of
calcium phosphate and mucin as substrate induced a
decrease of the size of the crystals, also being their morphology changed. Thus, when mucin was absent in the
substrate, crystals were polyhedral whereas in its presence, plate hexagonal crystals were observed.
564
Ncphron 1996;73:561-568
e
The effects of citrate in the COM heterogeneous nucleation on calcium phosphate, a mixture of mucin and
calcium phosphate, and wax, are shown in figure 5. When
citrate was present, a total absence of COM crystals was
observed in the citrate concentration range 1.85.10-3 3.17.10-3 M, but only on wax. It is noteworthy that citrate
practically caused no effects in the COM heterogeneous
nucleation on the three assayed surfaces at a 5.24.10-4 M
concentration in the assayed conditions.
The effects of aescin in the COM heterogeneous nucleation on the three studied substrates are shown in figure 6. The total absence of COM crystals was observed
only for the highest assayed concentration (1.32.10-5 M)
and exclusively when the used substrate was wax. Aescin
practically caused no effects in the COM heterogeneous
nucleation on mucin-calcium phosphate surfaces for all
studied concentrations and only slight inhibitory effects
on phosphate surfaces.
The effects of glycyrrhinic acid in the COM heterogeeous nucleation on the three assayed substrates are pre-
Grases/García- F erragut/Costa- Bauzá/
March
In ('Yo)
In ('Yo)
100
o
100
(3)
o-
80
80
60
60
40
40
20
20
a
o
o
2
[Citrate]
X
3
(3)
(2)
o
4
0.4
0.6
0.8
[Aescin]
103 (M)
In ('Yo)
In ('Yo)
100
100
80
80
60
60
40
40
20
20
1.0
X
1.2
I
1.4
1.2
I
1.4
105 (M)
b
o
o
2
[Citrate]
X
3
4
0.6
103 (M)
0.8
[Aescin]
1.0
X
a
b
105 (M)
Fig. 5. Inhibitory capacity (In) of citrate on the calcium oxalate
crystallization in the studied conditions at (a) 3 h, (b) 6 h, on (1) calcium phosphate substrate, (2) mixture of calcium phosphate and
mucin substrate, and (3) wax substrate.
Fig. 6. Inhibitory capacity (In) of aescin on the calcium oxalate
crystallization in the studied conditions at (a) 3 h, (b) 6 h, on (1) calcium phosphate substrate, (2) mixture of calcium phosphate and
mucin substrate, and (3) wax substrate.
sented in figure 7. As can be seen, at short times and when
wax was used as substrate, the glycyrrhinic acid exhibited
an important inhibitory effect in the COM heterogeneous
nucleation, thus in the range 5.95.10-5 - 8.93.10-5 Mthe
total absence of COM crystals was observed. It is interesting to notice as only slight inhibitory effects on calcium
phosphate and mucin-calcium phosphate surfaces for al!
studied concentrations were detected.
The effects of phytate in the COM heterogeneous
nucleation on the three studied substrates are presented in
figure 8. As is shown, when phytate was present, a total
absence of COM crystals was observed in the range
1.43.10-7 - 1.43.10-6 M on wax, 7.15.10-7 - 1.43·
10-6 M on calcium phosphate and 1.43.10-6 M on the
mixture mucin-calcium phosphate (3 h). In fact, phytate
provoked inhibitory effects in the COM heterogeneous
nucleation practical!y on al! the surfaces and in al! the
assayed concentrations except in the lowest one (7.15·
10-8 M), in which slight or no effects were detected.
The results shown in this paper, according to others
previously presented [3-6], clearly demonstrated that
when a nonprotected nonrenewed solid surface was in
contact with normal synthetic urine, sooner or latter calcium oxalate deposits develop on it through heterogeneous nucleation processes. It is also interesting to observe how the COM heterogeneous nucleation capacity of
Artificial Simulation ofStone Formation
Nephron 1996;73:561-568
Discussion and Conclusions
565
In (%)
100
o-
o
In
o
(%)
100
(3)
(3)
80
80
60
60
40
40
20
20
o
I
10
468
2
a
a
o
o
5
[Glycyrrhinic acid] x 105 (M)
In
10
15
20
15
20
[Phytate] x 107 (M)
(%)
120
(%)
100
In
(3)
100
O-
o-
-----o
80
80
60
60
40
40
20
20
(2)
, b
o
2
468
[Glycyrrhinic acid]
10
X
105 (M)
o
,
o
5
10
[Phytate]
X
b
107 (M)
Fig. 7. Inhibitory capacity (In) of glycyrrhinic acid on the calcium
oxalate crystallization in the studied conditions at (a) 3 h, (b) 6 h, on
(1) calcium phosphate substrate, (2) mixture of calcium phosphate
and mucin substrate, and (3) wax substrate.
Fig. 8. Inhibitory capacity (In) of phytate on the calcium oxalate
crystallization in the studied conditions at (a) 3 h, (b) 6 h, on (1) calcium phosphate substrate, (2) mixture of calcium phosphate and
mucin substrate, and (3) wax substrate.
calcium phosphate is notably higher than the capacity of a
more hydrophobic substrate such as wax. According to
these results, the activity on the nuc1eation and growth of
COM of the most polar surfaces was greater than that
caused by non polar surfaces, thereupon being in good
accordance with the finding of other authors [9]. It is also
c1early seen that the capacity of a substance to inhibit heterogeneous nuc1eation on a substrate also greatly depends
on the nature of such substrate.
The study of the effects of citrate on COM heterogeneous nuc1eation demonstrated that, when calcium phosphate acted as heterogeneous nuc1eant even at the higher
assayed citrate concentrations (3.17.10-3 M) and at
shorter times (3 h), the formation of COM crystals was
not totally prevented although it was notably reduced (see
fig. 5a). This indicates the high capacity of calcium phosphate salts to promote the formation of calcium oxalate
deposits on those crystals. Consequently, the important
risk factor that the presence of calcium phosphate adhered deposits to the urothelium implies, conceming the
development of oxalocalcic uroliths, is demonstrated. It is
interesting to observe how the presence ofmucin (a glycoprotein) notably reduced the development of COM crystals on the calcium phosphate pellets, probably due to its
continuous flow from the pellet surface to the solution.
This demonstrated the importance of the existence of a
continuously renewed glycosaminoglycan layer covering
the urothelium and protecting it against urolith development. The presence of citrate at normal urinary concentrations (1.85.10-3 M) or higher concentrations (3.17·
566
Nephron 1996;73:561-568
Grases/García- F erragut/Costa- Bauzá/
March
10-3 M) totally inhibited the development of COM crystals on the more hydrophobic assayed material (wax), this
showing its high inhibitory efficacy on the heterogeneous
nucleation on certain types of hydrophobic materials.
Nevertheless, it must be pointed out that when lower concentrations were assayed (5.29.10-4 M) these were not
high enough to avoid the deve10pment of COM crystals
on the wax surface (see fig. 5), likewise indicating that
hypocitraturia really implies an important risk factor to
develop renal stones.
Saponins are surface-active naturally-occurring substances normally found in plants. Due to their surface
activity, they can be used as stabilizers of colloidal suspensions [10], and hence, aescin (a saponin) has been proposed to avoid secondary aggregation (formation of aggregates as a consequence of crysta1-crystal collisions) in uric
acid urolithiasis. Because the presence of saponins in
practically all herb infusions to which some antilithiasic
properties were assigned [11], we decided to study the
effects of two saponins (aescin and glycyrrhinic acid), at
therapeutic quantities, on the heterogeneous nucleation
and development ofCOM crystals. The concentrations of
saponins in the experimentalliquids were chosen considering oral doses within the therapeutically recommended
margins, supposing a diuresis of 1.5 liters124 h and considering an excretion of the intact saponin between 1 and
10% of the ingested amount. As can be observed in figures 6 and 7, both saponins exhibited important inhibitory effects of COM crystallization on the hydrophobic wax
substrate, but the effects on calcium phosphate substrate
were the weakest in both cases. It is interesting to observe
that when COM crystals were present, these saponins did
not significantly affect the formation of COM agglomerates on the pellet surface. The presence of some aggregates
on the pellet surface, despite the absence of significant
crystalluria, demonstrates that such aggregates were
formed through primary aggregation. Primary aggregation consists of a type of crystal malgrowth that takes
place on the surface of a1ready developed crystals, the socalled parent crystals [12]. This process results in concretions consisting of intergrown crystals with a complex
crystal arrangement as observed in the core of COM calculi [13]. From these facts it can be concluded that saponins can produce important inhibitory effects in the heterogeneous nucleation on hydrophobic materials, but no
important action must be expected on heterogeneous
nucleation on calcium phosphates or in primary aggregation.
Phytate exerted the most remarkable effects on the heterogeneous nucleation of COM crystals. This high inhibi-
Artificial Simulation ofStone Formation
tory capacity of calcium oxalate crystallization caused by
phytate is in good agreement with previously reported
papers in which the effects on crystal growth [14] and
crystal nucleation [15] were studied. Thus, as can be seen
in figure 8, the presence of very low concentrations (like
1.43.10-7 M) caused a total inhibition of COM crystals
development on the wax substrate and caused important
inhibitory effects of the heterogeneous nucleation on calcium phosphate. It is interesting to observe that when
phytate was present at 1.43.10-7 M, it totally prevented
the development of COM crystals on the three assayed
substrates used to provoke heterogeneous nucleation of
such crystals.
It is interesting to compare the effects of the assayed
inhibitors found in this paper with those obtained previously in similar conditions but using a system in which
the artificial urine was in static conditions [5]. Thus, the
inhibitory effects were weaker, although similar in nature,
in static conditions, yielding that the urodynamics are
also important to determine the activity of a given inhibitoro This fact demonstrates that the presence of retention
zones with scarce urodynamic efficacy in the kidney
implies an important lithiasic risk factor. Thus, these
zones would be specially susceptible to form calculi also
due to the reduced action of inhibitors.
Acknowledgement
Financial support by Dirección General de Investigación Científica y Técnica (grant PB 92-0249) is gratefully acknowledged.
Nephron 1996;73:561-568
567
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