Nephron
Editors: G.M. Berlyne, Brooklyn, N.Y.;
S. Giovannetti, Pisa
Original Paper
Reprint
Publisher: S.Karger AG, Basel
Printed in Switzerland
Nephron 1993;65:77-81
l •••••••••••••••••••••••••••••••••••••••••••••••••••••••
F. Grasesa
A. Costa-Bauzáa
J. G. Marcha
O. Sohnelb
a
b
Department of Chemistry,
University of the Balearic Islands,
Palma de Mallorca, Spain;
Institute ofTechnology,
Department of Inorganic Processes,
Pardubice, Czechoslovakia
Artificial Simulation of Renal Stone
Formation
Influence of Some Urinary Components
..............................................
.••••.......•••••.......•••••.......•••••........••••.........••••........•••••.....••••••......
Key Words
Abstract
Calcium oxalate
Citrate
Pyrophosphate
Glycosaminoglycans
The effect of natural admixtures occurring in human urine (citrate, pyrophosphate and glycosaminoglycans) on the precipitation of stone-forming compounds was studied. Experiments were carried out under conditions c10sely
simulating the early stages of renal stone formation. Among the studied admixtures, citrate was determined as the most effective substance preventing the
phosphate partic1e formation. Indeed, in the presence of citrate, some calcium
oxalate monohydrate crystals were found. Pyrophosphate induced the formation of calcium oxalate dihydrate crystals. Phosphate crystals appeared at pH 6
and never at pH 5. The easy formation of phosphate partic1es supports the
hypothesis that these crystals represent a very important heterogeneous nuc1eusinitiating oxalocalcic calculus formation in the kidney. Reported results also
indicated uric acid as a significant heterogeneous nuc1eus of calcium oxalate
monohydrate crystals at urinary pH equal or lower than 5 and the important role
of bacteria in increasing the organic detritus deposited on the solid surfaces.
Introduction
The formation of a heterogeneous nuc1eus inside the
upper urinary tract, which later serves as a substrate for
oxalocalcic stone formation, represents the principal step in
this kind of calculogenesis. However, the majority of
studies conceming oxalocalcic urolithiasis was devoted to
crystal growth [1-4] and secondary agglomeration of calcium oxalate crystals [5-8], though both processes are of
minor significance at early stages of stone formation. Basic
processes of oxalocalcic stone formation, i.e. nuc1eation
and primary agglomeration of calcium oxalate, received
substantially lower attention [9].Moreover, previous studies
dealing with nuc1eation and primary agglomeration were
mostIy conducted under experimental conditions dissimilar to those prevailing in the kidney. Therefore, these results
Accepted:
October 20, 1992
relevant to urolithiasis can be regarded as questionable, at
least to some extent.
This contribution deals with the effect exerted by admixtures occurring in human urine on the precipitation of
stone-forming compounds from synthetic urine. Experiments were performed under conditions c10selysimulating
the early stages of stone formation. The studied admixtures,
citrate, pyrophosphate and glycosaminoglycans (GAGs)
pro mote or inhibit stone formation.
Material and Methods
In all experiments, the synthetic mine used was prepared by
mixing equal volumes ofsolutions A and B, the composition ofwhich
is given in table 1.Both solutions contained HzOz (0.066%), if not stated
otherwise, as a disinfectant preventing natural bacterium production
F. Grases
Department of Chemistry
University of the Balearic Islands
E-0707! Palma de Mallorca (Spain)
© 1993 S. Karger AG, Basel
0028-2766/93/
0651-0077$2.75/0
Table 1. Composition
of the artificial urine used
Solution A, gil
Solution B, gil
11.02
1.46
4.64
12.13
2.65 NaH2P04·2 HP
18.82 Na2HP04 ·12 H20
13.05 NaCI
Na2S04·10 H20
MgS04·7 H20
NH4CI
KCI
0.076 Na2C204
1.04 CaCI2·3.5 H20
(0.12 chondroitin sulfate)
(0.5 uric acid)
(1.00 C6Hs07Na3·2 HP)
(0.0375 Na4pp7'1O H20)
Optional admixtures are given in parantheses.
2a
•
•
4
• ¡-1
•
Ó
Ó
2
•eJ--- •
4
Solution B
____5
~3
2b
Fig.2. Phosphate (a) and COM crystals (b) developed
substrate when inhibitor substances were absent (pH = 6).
on the
Waste
Fig.l. Scheme of the experimental device. I = Isolated and thermostatic box; 2 = tee-mixing chamber; 3 = changeable spherical
substrate; 4 = coil; 5 = container; 6 = pure water.
and multiplication. It has been proved that such hydrogen peroxide
concentrations did not significantly alter the stability of the oxalate
ions. A required quantity of admixture was dissolved in solution A
and/or B prior to the experiment. The solutions were mixed in a
tee-type mixing chamber. Prepared synthetic urine was delivered
dropwise with arate of 350 mI per day to the apex of a ball serving as a
substrate for the solid precipitation from the urine. The ball, approximately 7 mm in diamater, composed of inorganic material with porous
structure, was ofvolcanic origin (complex silicate: 12%Si, 55% Ca, 14%
Fe, 6% Al). In some experiments, the volcanic ball was covered with a
hydrophobic plastic material (Parafilm; American Can Co.) The dropping rate was fast enough to ensure continuous renewal of the liquid
layer covering the ball surface. AlI experiments were performed at
37°C. A schematic diagram ofthe used experimental device is shown
in the figure l. Equal volumetricflows ofsolutions A and B were mixed
78
in the tee-type mixing chamber. A flow rate of 0.12 mI min-1 of each
solution was maintained by a multichannel peristaltic pump. A 5-mmlong changeable cylindrical tube of I-mm inner diameter delivered
mixed solutions (artificial urine) dropwise to the apex of the ball.
Each experiment was conducted continuously of 96 h. Then the
ball was removed from the equipment, rinsed with water and dried at
room temperature in a desiccator. Crystals developed on the ball
surface were observed by a Hitachi S-530 scanning electron microscope equipped with the EDAX analytical device. AlI observations
were repeated three times to ensure the reproducibility ofthe results.
Results
Crystals which developed on the ball surface were identified according to their habit and, in disputable cases, by
EDAX analysis of the constituting element. Calcium oxalate monohydrate (COM) formed typical plate-like crystals
(fig. 2) and calcium oxalate dihydrate (COD) crystallized as
Grases/ Costa -Bauzá/March/S6hnel
Artificial Oxalocalcic Renal Stone
Generation
3b
3a
3c
Fig.3. a COM crystals formed in the presence of 320 mg/l of
citrate. b Phosphate and COM crystals formed in the presence of
60 mg/I of GAGs. e COD and phosphate crystals formed in the
presence of7.5 mg/l ofpyrophosphate. d COM crystals formed in the
presence of320 mg/l of citrate and 60 mg/l ofGAGs. AlI experiments
were performed at pH = 6 and in the presence of disinfectant (HzOz).
pyramids (fig. 3). Two particle types ofhabit differing from
COM and COD, were detected: those composed ofCa and
P represented calcium phosphates (fig. 2, 4) and those
without such elements corresponded to uric acid (fig. 5).
A large quantity of phoshate particles together with a
limited amount of COM crystals formed on the substrate
surface at experiments performed with synthetic urine containing H202 at pH 6 and 3rC (fig. 2). Considering the
shape and the pH values where the calcium phosphate
crystals were formed, it was assumed that they consisted of
brushite. Small islands ofCOM crystals and no phosphate
particles were observed during similar experiments carried
out in the presence of 320 ppm of citrate or a mixture of 320
ppm of citrate and 60 ppm GAGs (fig. 3). Addition of 60
ppm GAGs to synthetic urine caused the formation of only
a small quantity of phosphate crystals, whereas addition of
7.5 ppm pyrophosphate induced precipitation of a substan-
tital amount of phosphate particles together with COD
pyramids. However, in experiments carried out in the presence of citrate and pyrophosphate, only a limited quantity
of phosphate particles and no oxalate crystals were detected. (fig. 3). The results are summarized in table 2.
Large quantities of uric acid and COM crystals were
observed in experiments performed with synthetic urine
containing 250 ppm of uric acid and 320 ppm of citrate at
pH 5 (fig. 5). It must be emphasized that no phosphate
particles were observed in this case, contrary to experiments
carried out with synthetic urine at pH 6.
In experiments with synthetic urine containing no disinfectant at pH 6, large amounts of organic material including
fibers of organic origin accompanied the formed phosphate
particles and COM crystals, (fig. 6).
Finally, COM crystals and spherulitic calcium phosphate particles, probably apatite, were formed on the sub-
79
Fig.4. COM and spherulitic calcium phosphate crystals developed on the substrate covered by plastic material at pH 6.7 and in the
presence of 320 mg/I of citrate.
Fig.5. COM and uric acid crystals obtained when 250 mg/I ofunc
acid were present in the synthetic urine at pH = 5 and in the presence
of disinfectant (H202).
Table 2. Summary of experimental results with synthetic urine
at pH = 6 in the presence of admixtures, quoted as a number of islands of respecti ve crystals on the hall surface
>S
>SOO
COD
I OO
2COM
O
34
>Sphosphate
O
Crystals
observed
ent
The values shown are average s of three replicates fm each measurement.
Fig.6. Phosphate crystals and organic fibers obtained in the presence of bacteria.
strate surface covered by the plastic material, at experiments performed with synthetic urine containing H202 and
citrate at pH 6.7 and 37°C, as can be seen in figure 4.
Discussion
Crystals of stone-forming compounds develop on the
surface of a solid in contact with urine supersaturated with
respect of these salts. However, the presence of some urinary components, mainly citrate, GAGs and pyrophosphate, can even in minute quantities substantially modity
the formation and development of solid form urine. Results
80
presented in this contribution indicate that considering the
amounts present in real urine, the citrate acted as the most
effective substance preventing phosphate particle formation among studied admixtures. Indeed, in the presence of
citrate, no phosphate particles could be detected (fig. 2, 3,
table 2) though some COM crystals were found. This effect
must be attributed to two facts: the decrease in supersaturation of calcium salts caused by the complexing capacity of
citrate and the well-known inhibitory capacity of this substance. The comparison of the effects of GAGs and pyrophosphate showed that the latter is less effective in preventing phosphate particle formation. Moreover, pyrophosphate induced the formation of COD crystals. Thus, the
Grases/ Costa -Bauzá/March/Sohnel
Artificial Oxalocalcic Renal Stone
Generation
hindrance capacity with respect to phosphate formation of
the studied admixtures increases in succession pyrophosphate < GAGs < citrate.
Phosphate crystals form at experiments performed at pH
6, i.e. under conditions considered as physiologically normal in urine, and never at pH 5. This result emphasizes the
susceptibility of urine to easily form phosphate partic1es
and lends support to the hypothesis that phosphate crystals
represent a very important heterogeneous nuc1eus-initiating oxalocalcic calculus formation in the kidney.
Reported results indicate that at urinary pH equal or
lower than 5, uric acid crystallizes in appreciable amounts
and citrate exerts litde inhibitory effect on the process (Fig.
5). As can be seen, uric acid represents also an effective
heterogeneous nuc1eus of COM crystals. These findings are
in good agreement with the idea regarding uric acid as
heterogeneous nuc1eus for calcium oxalate [10,11].Also, the
well-known fact that inhibitors ofuric acid synthesis, such
as allopurinol, occasionally exhibit an effective therapeutic
action on oxalocalcic urolithiasis, corroborates the suggested idea.
The important role that bacteria can play in oxalocalcic
calculogenesis must be recognized. The presence of bacteria resulted in a notable increase in organic detritus de-
posited on the solid surface, some ofit in a fibrous form (fig.
6). This material can serve as a substrate for heterogeneous
nuc1eation of calcium oxalate crystals [12]facilitating thus
the stone formation and developmenl.
Finally, the formation of COM and spherulitic calcium
phosphate when using a solid substrate of very different
nature from the inorganic ball, such as plastic material,
c1early demonstrated that when a solid substrate is in contact with urine in the kidney, calcium salts will precipitate
on its surface and sooner or later develop into a full size
renal calculus. Active admixtures, such as citrate, slow
down the rate of this development, but do not bring the
process to a complete hall. This also demonstrated the
importance of the presence of the continuously renewed
antiadherent layer of GAGs that cover the inner walls of the
kidney. It is evident that such continuous renewing processes avoid the formation of growing scales.
Acknowledgement
The financial support of the 'Dirección General de Investigación
Científica y Técnica' (grant PB 89-0423) is gratefully acknowledged.
..................................................................................................................................................
References
Gardner GL: Nucleation and crystal growth of
calcium oxalate trihydrate. J Crystal Growth
1975;30: 158-168.
2 Nancollas GH, Gardner GL: Kinetics of crystal
growth of calcium oxalate monohydrate. J Crystal Growth 1974;21:267-276.
3 Werness PG, Duckworth SC, Smith LH: Calcium oxalate dihydrate crystal growth. Invest
UroI1979;17:230-233.
4 Gardner GL: Effect of pyrophosphate
and
phosphonate anions on the crystal growth kinetics of calcium oxalate hydrates. J Phys Chem
1978;82:664-670.
5 Hartel RWE, Gottung BE, Randolph AD,
Drach GW: Mechanisms and kinetic modelling
of calcium oxalate crystal aggregation in a urinelike liguor. I. Mechanism. AICHE J 1986;32:
1176-1180.
6 Ryall RG, Ryall RL, Marshall VR: A computer
model for the determination
of extents of
growth and aggregation of crystals from changes
in their size distribution. J Crystal Growth
1986;76:290-298.
7 Ryall RG, Ryall RL, Marshall VR: Interpretation of particle growth and aggregation pattern
obtained from the Coulter-counter. A simple
theoretical model. Invest Uro11981; 18:396-400.
8 Hartel RW, Randolph AD: Mechanisms and
kinetic modeling of calcium oxalate crystal aggregation in a urinelike liguor. n. Kinetic modeling. AICHE J 1986;32:1186-1189.
9 Grases F, Masárová L, Sohnel 0, Costa-Bauzá
A: Agglomeration of calcium oxalate monohydrate in synthetic urine. Br J Urol 1992;70:
240-246.
10 Mandel NS, Mandel GS: Epitaxis between
stone-forming crystals at the atomic level; in
Smith LH, Robertson WG, Finlayson B (eds):
Urolithiasis: Clinical and Basic Research. New
York, Plenum Press, 1981.
I 1 Grases F, Costa- Bauzá A, March JG, Masárová
L: Glycosaminoglycans,
uric acid and calcium
oxalate urolithiasis. Urol Rest 1991;19:375-380.
12 Grases F, Costa-Bauzá A: Study of factors affecting calcium oxalate crystalline aggregation.
Br J UroI1990;66:240-244.
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