Original Paper
Nephron 1993:63: 176-182
O. SÓlznel
F. Grases
University of the Balearic Islands,
Department of Chemistry,
Palma de Mallorca, Spain
Fine Structure of
Calcium Oxalate Monohydrate
Renal Calculi
................................................................................................
Key Words
Calcium oxalate monohydrate
Papillary calculi
Structure
Formation mechanism
Abstract
Fine structure, location and size of the core of 12 calcium oxalate monohydrate
(COM) papillar calculi from different 'idiopathic' stone-formers were studied by
an optical and scanning electro n microscope equipped with the EDAX analytical device. Each individual core exhibited a unique overall structure composed
of loosely arranged twined and intergrown crystals of plate-like and/or columnar shape and particles of'rosette' structure with considerable void space among
crystals in some cases or compact structure in others. Crystals were covered by a
thin layer or organic material mostly invisible to the microscope. Sometimes
debris of organic origin in a core was observed. A substantial amount of organic
matrix appeared at the core boundary, often in the form of amorphous plates.
The outer striated layer of COM stone consisting of tightly packed columnar
crystals originated on this matrix. The stone core was located near the stone
surface that was attached to the kidney wall and contained foreign particles that
act as heterogeneous nucleants of calcium oxalate crystals.
Introduction
Based on reconsidering the contemporary knowledge on
urolithiasis and available results of in vitro experiments
from the viewpoint of theory of solution crystallization the
following mechanism of calcium oxalate monohydrate
(COM) stone generation can be suggested: Several crystals
ofCOM or an appropriate foreign substance attached to the
kidney wall at sites with damaged anti-adherent glycosaminoglycans protective layer represent a stone nucleus. COM
crystals increase in size by regular crystalline growth and
multiply mainly by primary agglomeration, i.e. by an aberrant growth of parent crystals taking place on surface imperfections and/or crystal tips. Particles of foreign substances serve as heterogeneous nuclei for COM crystal forma-
Accepted:
March 11.1992
tion that further develop as already described. The resulting
concretion consisting of loosely arranged intergrown and
twined crystals represents a stone coreo At a certain stage of
development this core becomes covered by a layer of the
organic matrix that brings the core growth to a complete
hall. New crystals nucleated later on the organic matrix
layer develop by further growth into the outer striated layer
ofthe COM calculus.
The mechanism of core development, particularly the
role of primary agglomeration, has already been verified by
studying the CO M calculi core structure [1,2], by comparing
the core structure with configurations appearing in precipitating experiments performed in vitro [3-5]. Although nucleation of calcium oxalate crystals on organic substrate
(uromucoid particles) has also been observed in in vitro
Prof. F. Grases
Departmem
University
of Chemislry.
of the Balearic
Faculty
of Sciences
Islands
© 1993S. Karger AG. Basel
0028-2766/93/
063>017652.75/0
E-D7071 Palma de Mallorca (Spain)
I
Stone-former 4
mg/I
Stone-former
398271
336.8
\.4 20.3
Creatinine
18.7
33,4
53.8
5\.5
27.6
45.6
35.9
35.5
19.5
18.6
20.3
17.9
100
3\.2
87.0
35.8
\.9
Uric
81.4
acid
98
l\.O
17.1
9.0
66.1
97
IIA
9.5
52.1
-D.8
62.8
93
110.9
22,4
\.5
9.2
58.5
Phosphorus
:'Ig
Stone-former
Table 1.
of serum biochemical Jata
experiments
[6], 101
direct
evidence that such mechanism par- Stone-former
6 Summary
Ca
The study was repeated
3 times for each stone-former.
useful information
on the stone formation mechanism,
Methods and Results
The fine structure of 12 spontaneously
passed renal
COM calculi from 8 'idiopathic' stone-formers was studied.
Stones were first dried in a vacuum dessicator. The outer
morphology of each stone was observed in an optical stereoscopic microscope in arder to determine the stone surface
which adhered to the kidney. Then the stone was broken by
a sharp spatula perpendicularly
to this surface and the
location and size of the core examined by an optical microscope. Stone fragments were covered by a layer of gold and
observed in the scanning electron microscope equipped
with the EDAX analytical device. Calculi for which the
surface of adherence was difficult to identify with certainty
were excluded from this study. The main urine and serum
biochemical parameters of the patients whose stones were
studied are shown in table 1 and 2.
The studied stones, 2-5 mm in diameter, contained a
core varying in size between approximately 0.5 and 2 mm
Creatinine
Urie5.18
aeid
2.9
194
8.5336
72
32.4
349
1.7
52.0
02
933
16.9
246
249
2.l
44
23.7
202
371
156
323
5.29
470
23.2
\.6
75.9
453
516
\.597
887
295
22.8
1.5
1.765
1165
25
497
53.7
214
8.7
54
27l.1
320
\.9
346
Citrate
Diuresis
Oxalate
308
410
4.85
13.3
498
430
4.97
2.2
4.25
76
25.7
310
652
334
81l
5.51
20.8
447
5,42
670
423
1.740
1.070
62.9
693
5.12
5.98
2.7
Table22.2
2.Ca
Sum1566
.070
mgII
mg/1
mg!\
mg/1
mg/1mgII
mg!\
Phosphorus
Mg
Stone-former 1
mary of urinary bio-
pH
(table 3). The stone/core
diameter ratio varied between
approximately
2 and 5. The core was formed by loosely
arranged intergrown and twined crystals of plate-like and
columnar shape that is typical for COM (fig. 1). Particles
with 'rosette' structure (fig. 2) were observed in every studied core, but frequency of their occurrence varied in individual cases. In some cases, a considerable void space
among crystals constituting the core created an impression
at observation under high magnification observation that
the stone was a partially hollow object (fig. 3). At low
magnification,
however, the fractured surface of a stone
core appears mostly compact with occasional occurrence of
cávities, though in several cases the core appeared significantly porous. Each core, even of stones produced by the
same stone-former, exhibits a unique overall fine structure,
i.e. unique arrangement
of crystals, dissimilar to other
cores. However, crystals constituting the core display in
liters
The study was repeated 3 times for each stone-former. Urine accumulated oyer a 24-hour periodo following free
die!. pH corresponds to urine aceumulated oyer a 2-hour periodo following an oyemight fas!.
l77
-
void
yoid
')4
')oid
mm
in matrix
0.6
].4
2.4
ycompactness
2.2
0.9
1.5
diameter
Stane/core
diameter
thecores
core
several
2.5
3.5
5ratio
0.5
0.8
11.1
1.2
vyoid
void
updetected
ric
acid
232.5
Core
Core
the
core
51.5
2.5
3.5
Table 3. Main
3.4
phosphates
compac!
sourrounding
compact
hosphates
present
Organic
phosphates
Foreign
particles
Stane-former ].
Stone-forlller
7.
8.
9.
Stone-former
5.
3.
characteristics
4.
6a.
7.3b.
8) 4b)
l.the
2.
(fig.4a)
1b.
2. 5.01'6.
(fig.la.9)
(fig.
(fig.6b)
3a)
]0)
diameter
Stone
mm
a
50 IJm
20IJm
Fig.1. Fine structure 01' a COM calculus coreo Imergrown and twined plate-like (a) and
columnar (b) crystals forllling the coreo
178
Sohne]/Grases
Structure 01' Calcium Oxalate Monohydrate
Renal Calculi
200 IJm
Fig.2. Partic1es with 'rosette' structure present in the stone coreo
each case the identical and typical features described above.
Foreign particIes such as phosphates, that can act as heterogenous nucIei of calcium oxalate, were found in each
calculus (fig. 4).
The surface of the majority of crystals constituting the
core was covered by a thin layer of an organic material
invisible even to the electron microscope. Its presence was
determined by EDAX analysis ofthe same site performed
at low (4 kV) and high (15 kV) voltages. The low-voltage
analysis that examines only the surface layers detected no
calcium or other metallic elements, whereas the high-voltage analysis penetrating deep into the object showed calcium as a principal component ofthe crystal. COM crystals
grown in a system containing no organic material showed
calcium both at low- and high-voltage analysis. Hence, an
organic layer must cover the crystral surface. Debris of
evidently organic origin can occasionally be observed on
the surface of some crystals constituting the core (fig. 5).
3b
100 IJm
Fig.3. a Hollow appearance ofthe stone coreo b Compact appearance of the stone coreo
Fig.4. Plate-like and spherulitic crystals of phosphate (determined
by EDAX).
179
50
¡.1m
Fig.5. Oebris of organic origin indicated by arrow on the surface
of crystals fonning the coreo
100 IJm
Fig.7. Columnar crystals fonning the outer striated layer of a
COM calculus originating on the matrix layer.
I
6a
/
50
200 IJm
¡.1m
Fig.8. The lower end of the striated layer.
At the core boundary a layer of organic origin, often as
large amorphous plate-like particles, can always be observed (fig. 6). This ¡ayer is composed of only organic
material since no calcium or other metallic elements were
6b
50
¡.1m
Fig.6. The organic matrix in the fonn of plates occurring at the
core boundary.
180
Sohnel/Grases
determined by the high-voltage EDAX analysis. In fact, a
hole was usually burnt at the analyzed spot. It is on this
organic matrix that the columnar crystals forming the outer
striated layer ofCOM stone originate (fig. 7). The columnar
crystals are evidently attached by their lower ends to the
matrix layer. Tightly arranged columnar crystals forming
the striated layer can be seen in figure 8 where the sone
fracturing incidentally discloses the lower end of the layer.
Structure of Calcium Oxalate Monohydrate
Renal Calculi
I
.
.'
Each such core then served as a source of new striated layer
formation, so the whole stone was composed of several
smaller stones firmly connected together.
The core of papillar COM stones was located in close
vicinity to the surface where the stone adhered to the kidney
wall. A compact layer, similar to the outer striated layer,
developed around the core also towards the kidney wall.
This layer was considerably thinner (sometimes only fractions of a millimeter) than the striated layer in the direction
perpendicular to the kidney wall that usually reached a few
millimetres (fig. 10).
The main characteristics of each one of the twelve studied renal stones are summarized in table 1.
100 ~m
Fig.9. The layer of the organic matrix (indicated by an arrow)
covering the sto ne.
Fig.10. Cross-section of a COM sto ne. The surface of attachment
lo the kidney wall is indicated by arrow.
In one case the whole stone of 1.5mm in size consisted of
loosely arranged aggregated crystals covered by a layer of
an organic material (fig. 9). The stone interior was highly
porous. The organic layer surface was divided by cracks
into separate plates of identical appearance to the particles
of the matrix at the boundary of a core inside the stone
(fig. 6). This whole stone represented, in fact, the stone coreo
The largest caIculus studied, around 5 mm in size, exhibited several regions with loose arrangements of crystals
that can be recognised as additional cores. The initial core
gave rise to the striated layer on which these additional
cores originated at a certain stage of stone development.
Discussion and Conclusions
The performed study of the COM caIculi structure unequivocally showed that the stone core consists of loosely
arranged intergrown and twined crystals. This structure
closely resembles the structure of artificially grown COM
concretions and stones that developed by the mechanism of
primary agglomeration. This observation confirms the crucial role of primary agglomeration in the generation of the
real stone nucleus. The stone core can exhibit a compact
structure with occasional occurrence of cavities or a hollow
feature with a considerable void space among the crystals.
80th structures can be observed in different caIculi belonging to the same stone-former. This seems to indicate that the
Cdre compactness depends on the number and location of
the heterogeneous nuclei that are responsible for starting
the caIculus growth.
The location of a papilIar caIculus core near the surface
of stone attachment to the kidney wall confirms that the
stone nucleus is formed by crystals adhered to this wall.
Additional cores formed on the striated outer layer originating on the initial core could be distinguished in larger
stones. Each core gave birth to a 'new caIculus' that was
firmly attached to the initial caIculus. There are indications
that each larger calculus consists of a number of smaller
stones formed by such a mechanism. However, the reason
of additional core formation is not clear for the present.
Crystals forming the stone core are covered by a thin
layer of organic material. Oebris of organic origin can
occasionally be observed inside the coreoHowever, massive
presence of the organic matrix inside the core was not
detected. This fact seems to indicate that the stone core
develops rather quickly.
The core when reaching a certain size becomes covered
by a relatively thick layer of organic matrix. The matrix can
181
be invariably observed at the boundary of both initial and
additional cores. Why this layer develops at a certain stage
of stone generation remains to be clarified. The different
stone/ core diameter ratios can be attributed to different
calculus location in the kidney and to different times
elapsed before calculus expulsion.
The outer striated layer composed of columnar crystals
originates on the layer of organic matrix covering the calculus coreo This layer extends several millimeters into the
inner space ofthe kidney but only a fraction ofthis distance
in the direction towards the kidney wall in the case of the
initial coreo This implies that urine al so has access, although
severely restricted, to the calculus base. Crystals forming
the striated layer apparently grow by a slow regular crystalline growth. Considering the COM crystal growth rate
3.3 x 10-5 mol min-1 m-2 [11], i.e. approximately 3.6 x 10-11 m
S-I, indicates that development
of a layer 3 mm thick
would require around 960 days, i.e. 2.7 years. Therefore,
considerable time is required for a calculus to reach a size
of several millimetres in diameter when it will usually be
spontaneously
released from the kidney wall and washed
away from the upper urinay tract. It is interesting to note
that the estimated time of a papillar stone development
coincides with the average period of stone formation by
recurrent stone-formers [12].
The constant presence of heterogeneous
nucleants in
the core supports the hypothesis that preventing formation of stone nucleus by suppression
of ¡he solid-phase
nucleation in the kidney, would eliminate this kind of
urolithiasis.
Moreover,
features observed in the renal
stones, particularly the core location, the prevailing mechanism of core development
and the role of the organic
matrix in the striated layer formation, correspond with the
principal aspects of the mechanism of stone generation
proposed in the 'lntroduction'
and thus represent a sound
confirmation
of this mechanism validity.
Acknowledgement
Financial assistance from the Spanish Dirección General de Investigación Cientifica y Técnica, grant No. SAB 91-0040 and PB 89-0423,
is gratefulIy acknowledged .
...................................................................................
.
References
Seyfart H-H, Hahne s: Microscopic examinations of urinary calculi. Jena Rev 1978;23:
182-187.
2 Kim KH, Johnson FB: Calcium oxalate crystal
growth in human urinary stones. Scanning Electron Microsc 1981:iii: 147-154.
3 Grases F, Millan A. S6hnel O: Role of agglomeration in calcium oxalate monohydrate uroliths
development. Nephron 1992:61: 145-150.
4 Grases F, Masárová L. S6hnel 0, Costa-Bauzá
A: Agglomeration of calcium oxalate monohydrate in synthetic urine. Sr J Uro11992; in press.
5 Millan A, Grases F, S6hnel 0, Krivánková 1:
Semi-batch precipitation of calcium oxalate
monohydrate.
Cryst Res Technol 1992:27:
31-39.
182
6 Grases F, Costa-Sauzá
A: Study of factors af-
fecting calcium oxalate crystalline aggregation.
Br J UroI1990;66:240-244;
7 Gibson RI: Descriptive human pathological
mineralogy. Am Mineralogist 1974:59:11771182.
8 Iwata H, Nishio S, Wakatsuki A, Odi K, Takeuchi M: Architecture of calcium oxalate monohydrate urinary calculi.J Uro11985: 133:334-338.
9 Meyer AS, Finlayson S, DuBois L: Direct observation of urinary stone ultrastructure. Br J Urol
1971;43: 154-163.
S6hnel/Grases
10 Murphy BT, Pyrah LN: The composition, structure and mechanisms of the formation of urinary calculi. Br J UroI1962:34:129-159.
11 Singh RP, Gaur SS, White DJ, Nancollas GH:
Surface effects in the crystal growth of calcium
oxalate monohydrate. J Colloid Interface Sci
1987: li8 :379-386.
12 Ljunghall S, Danielson BG: A prospective study
of renal stone recurrences. Sr J Urol 1984:56:
122-124.
Structure of Calcium Oxalate Monohydrate
Renal Calculi
I