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MCGILL UNIVERSITY
CALCIUM OXALATE CRYSTAL FORMATION INHUMAN URINE AND IDENTIFICATION OF
MINERAL-BINDING PROTEINS
Dy
QUYNH DUNG SARAH NGUYEN
A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES ANDRESEARCH IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE
DEGREE OF MASTER OF SCIENCE
FACULTY OF DENTISTRY
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TABLE OF CONTENTS
List of Abbreviations .IV
List of Figures VII
Abstract and Résumé .IX
Introduction and Literature Review 1Kidney stones 2Kidney stone composition 2Kidney stone formation 3Crystallization 4
Supersaturation 4Nucleation 4Growth 6Aggregation 6Calcium·oxalate crystals 7
Kidney stone matrix 9Urinary proteins 11
Osteopontin 12Urinary Prothrombin Fragment 1 13Albumin 14Tamm-Horsfall Protein 15
Urine from stone formers versus non-stone formers 17Urine ofmales versus females 18
Rationale and objectives ofthis research project 20
Materials and Methods 22The precipitation ofcalcium oxalate (CO) crystals from human urine 23
Part 1: The effects ofurine manipulation 23Osteopontin: Further characterization 26
Thrombin digestion 26Osteopontin associated with calcium oxalatecrystals versus calcium phosphate crystalsprecipitatedfrom male urine 27
Part II: Gender differences 27The precipitation ofcalcium oxalate (CO) crystals from rat urine 28SnS-PAGE ofcrystal associated proteins 29Gel staining methods 29
Double staining: Stains-Ali / Ag nitrate 29Si/ver Staining 30
Western blotting 31Light and fluorescence microscopy 34Scanning Electron Microscopy (SEM) .35
II
Immunohistochemistry ofkidney stones .35Paraffin embedding 36Hematoxylin and eosin staining 36Immunohistochemicallocalization ofmatrix proteins .36
Preparation ofpure inorganic calcium oxalate dihydrate (Weddelite)crystals 37Hydroxyapatite (HAP) beads 38Poly-L-Aspartic acid (poly-Asp/PA) .38
Fluorescein isothiocyanate labeling ofpoly-Asp .38Inhibition ofcalcium oxalate dihydrate growth 39Competitive peptide/protein binding assays .40
Results , 42Precipitation ofcalcium oxalate crystals from urine .43Immunohistochemical staining ofcalcium oxalate kidney stones for
osteopontin ; 65Calcium oxalate crystals 68Hydroxyapatite beads ,.78
Discussion 86Precipitation ofcalcium oxalate crystals from urine 87Immunohistochemical staining ofcalcium oxalate kidney stones for
osteopontin 96The use ofsynthetic calcium oxalate crystals for peptide/protein-bindinganalysis 96The use ofBioRad hydroxyapatite ceramide beads for peptide/protein-bindinganalysis 101
Conclusions and summary " 104
References 108
Acknowledgements 118
III
LIST OF ABBREVIATIONS
oc degrees Celsius
Abs absorbance
Ag silver
APS ammonium persulfate
BSA bovine serum albumin
Ca calcium
CaCh calcium cWoride
CaP calcium phosphate
CO calcium oxalate
COD calcium oxalate dihydrate
COM calcium oxalate monohydrate
COT calcium oxalate trihydrate
CMP crystal rnatrix protein
ddH20 double deionized water
EDS Electron Dispersive Spectroscopy
EDTA ethylenediaminetetracetic acid
FACS Fluorescence Activated Cell Sorter
FITC fluoresceinisothiocyanate
FLM fluorescence light microscopy
g gram
GAG glycosaminoglycan
Glu glutamic acid
IV
H&E hematoxylin and eosin
HAP hydroxyapatite
HCL hydrochloric aeid
HRP horseradish peroxidase
HSA human serum albumin
KCL potassium chloride
kDa kilodalton
KH2P04 potassium dihydrogen orthophosphate
1 liter
LM light microscopy
M moles per liter
MEB microscopie éléctronique à balayage
mg milligrams
mM millimoles per liter
MW molecular weight
NaCI sodium chloride
Na2HP04 sodium phosphate dibasic, anhydrous
NaN3 sodium azide
NaOx sodium oxalate
NaP sodium phosphate
OC oxalate de calcium
OPN osteopontin
PBS phosphate buffer saline
v
PMSF phenylmethylsufanylflouride
poly-Asp/PA poly-L-aspartic acid
RBC red blood cell
RGD arginine-glycine-aspartate (Arg-Gly-Asp)
RNA ribonuc1eic acid
rpm
SDS
SDS-PAGE
SEM
SS
TEMED
THP
Tween20
J.tg
J.tl
UMM
UPTFI
V
w/v
revolutions per minute
sodium dodecyl sulfate
sodium dodecyl sulfate polyacrylamide gel electrophoresis
scanning electron microscopy
supersaturation
N,N,N',Nil-tetramethylethylenediamine
Tamm-Horsfall protein
polyoxyethylene sorbitan monolaurate
microgram, IxlO-6
microliter
urinary macromolecules
urinary prothrombin fragment 1
volt
weight per volume
VI
LIST OF FIGURES
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Mode! for the initial intratubular events in the formation and aggregation
ofcalcium oxalate and calcium phosphate crystals leading to kidney stone
formation (page 5)
Important factors for the development ofa calcium stone (page 7)
SDS-PAGE ofmale urine samples (page 48)
Western blots ofmale urine samples (page 49)
SEM ofcrystals from male urine (page 50)
SDS-PAGE offemale urine samples (page 51)
SDS-PAGE ofCMP digested with thrornbin (page 52)
Western blot ofCMP frorn CO and CaP crystals (page 53)
SEM and X-ray rnicroanalysis ofCO and CaP crystals (page 54)
SDS-PAGE ofmale and female urine samples (page 55)
Western blots ofmale and female urine samples (page 56)
Western blots ofmale and female concentrated urine samples (page 57)
SDS-PAGE ofmale and female CMP samples (page 58)
Western blots ofmale and female CMP samples (page 59)
SDS-PAGE ofthe supernatants obtained frorn male and female urine
samples (page 60)
Western blots ofthe supernatants obtained frorn male and female urine
samples (page 61)
SEM ofcrystals frorn male and female urine (page 62)
VII
Figure 18.
Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.
SDS-PAGE and Western blots ofrat urine samples (page 63)
SEM ofcrystals from rat urine (page 64)
LM ofH&E-stained kidney stone sections (page 66)
LM ofkidney stone sections stained for OPN (page 67)
LM ofsynthetic COD crystals (page 71)
SEM ofsynthetic COD crystals (page 72)
SDS-PAGE ofpoly-Asp (page 73)
LM ofCOD grown with poly-Asp (page 74)
SEM ofCOD grown with poly-Asp (page 75)
LM ofCOD grown with HSA (page 76)
SEM ofCOD grown with HSA (page 77)
LM ofHAP beads (page 80)
SEM ofHAP beads (page 81)
FLM ofHAP beads and poly-Asp (page 82)
FACS analysis ofHAP beads and poly-Asp (page 83)
FLM ofHAP beads and HSA (page 84)
FACS analysis ofHAP beads and HSA (page 85)
VIII
ABSTRACT AND RÉSUMÉ
IX
ABSTRACT
Urolithiasis occurs in 20% of males and 5-10% offemales, and 75% ofkidney stones contain
calcium oxalate (CO) mineraI. To analyze mineral-binding proteins and to make gender
comparisons, using the model of Doyle et al. (Clin Chem, 37: 1589-1594, 1991), CO crystals
were generated in whole and centrifuged urine samples and then washed with water or sodium
hydroxide. Crystals and mineral-binding proteins were analyzed by SDS-PAGE, Western
blotting and electron microscopy (SEM). Regardless ofurine or crystal treatment, osteopontin
and UPTF 1 proteins were consistently present in the samples, whereas THP and albumin were
partially removed. SEM showed larger crystals precipitated from female than from male urine.
Western blotting demonstrated more albumin bound to crystals from females. In other
experiments, CO crystals were grown in the presence ofpoly-L-aspartic acid (PA) and albumin.
SEM demonstrated that these proteins affected CO crystallization. Competitive protein-binding
assays and fluorescence activated cell sorter analysis after binding of PA and albumin to
hydroxyapatite indicated that PA binds hydroxyapatite with a stronger affinity than albumin.
RÉSUMÉ
La lithiase rénale se produit chez 20% des hommes et 5-10% des femmes et 75% des pierres
rénales contiennent de l'oxalate de calcium (OC) comme minéral principal. En utilisant le modèle
de Doyle et al. (Clin Chem, 37: 1589-1594, 1991) pour analyser les protéines et pour comparer
les deux sexes, des cristaux OC ont été précipité de l'urine centrifugé et non- centrifugé. Les
protéines liées aux cristaux ont été analysé par l'éléctrophorèse (SDS-PAGE), Western et par la
microscopie électronique à balayage (MEB). Les protéines d'ostéopontine et la prothombine
urinaire (fragment 1) n'ont pas été influencé, tandis que la protéine de Tamm-Horsfall et
l'albumine ont été éliminé des échantillons d'urine traité. Le MEB a démontré que des plus gros
cristaux ont précipité de l'urine des femmes que de l'urine des hommes, et les Westerns ont
démontré qu'il y a plus d'albumine liée aux cristaux des femmes. Dans d'autres expériences, la
croissance des cristaux OC en présence de l'acide Poly-L-Aspartique (PA) et de l'albumine,
analysée par MEB, a démontré un effet marquant sur la cristallisation de OC par ces protéines.
Les analyses de FACS de la liaison de PA et d'albumine à l'apathite ont indiqué que la PA se lie à
l'apathite avec une affinité plus importante que l'albumine.
x
INTRODUCTION AND
LITERATURE REVIEW
INTRODUCTION AND LITERATURE REVIEW
Kidney stones
Kidney stone disease affects approximately 10-12% of individuals in the
industrialized world [4, 91]. Clinical protocols exist to identi:fy the risk factors leading to
renal stone formation, however many cases remain idiopathic, with no identifiable
biochemical or anatomical markers [92]. The risk ofrecurrence is high, ranging from 60
70% after 10 years, thus placing increased importance on prevention ofsecondary stone
formation [4, 91, 93].
Increased incidence ofurolithiasis is associated with male gender (a group
comprising two-thirds ofstone formers), increasing age (up until the age of65), low urine
volume, hereditary factors and disorders, as weIl as other kidney disorders and
geographic factors. Other fàctors which can influence the rate of stone disease are
dietary intake, hypercalciuria, hyperuricosuria, hyperoxaluria, hypocitraturia, and
acidosis [34]. The formation ofcalcium oxalate stones, in particular, can he caused by
primary hyperthyroidism, idiopathic hypercalciuria, low urine citrate level,
hyperoxaluria, or hyperuricosuria, and in the case ofcalcium phosphate stones, renal
tubular acidosis [21].
Kidney stone composition
About 80% ofrenal stones are composed ofcalcium oxalate (CO) and calcium
phosphate (CaP) [34,90] and about 70% ofthese stones are calcium oxalate [87]. The
2
remaining stones are composed ofuric acid or mixed urie acid and calcium (10%),
struvite stones (10%), and cystine stones (l%) [34]. Approximately two-thirds ofkidney
stones contain more than one type ofcrysta~ and CO mixed with CaP is the most
common combination encountered [45]. Calcium oxalate kidney stone disease is a
common clinical problem occurring at an annual rate of 1 in 1,000 people. There is as
yet no completely corrective therapy for idiopathie calcium oxalate urolithiasis [69]. As
most other stone types have medical conditions associated with them and only 10% of
CO stones have an identifiable pathology, the remainder ofthe discussion on kidney
stones will he limited to the pathophysiology ofidiopathie calcium oxalate stone formers
[21,34].
Kidney stone formation
There are three main contributing issues relevant to kidney stone formation:
supersaturation ofurine with respect to ions (such as calcium and oxalate), crystal
nucleation, growth and aggregation, and the presence of inhibitors and promoters of
crystallization [83]. Stone formation is a complex process, involving the formation ofa
crystalline (minerai) and a non-crystalline (organic) phase [34, 90]. The crystalline phase
will he further discussed in the section entitled "Crystallization" and the non-crystalline
phase in the section entitled "Kidney Stone Matrix".
3
Crystallization
Supersaturation
One ofthe important factors for CO crystallization is supersaturation (SS) ofthe
urine with respect to calcium and oxalate. This is defined by Asplin et al. (1997) as the
ratio ofthe concentration ofthe dissolved salt divided by the solubility ofthat salt in
urine, at body temperature. As the SS increases, a level is attained at which a solid phase
formation is possible, and this is referred to as the upper limit ofthe metastable range.
Below this point, supersaturation allows the growth ofpreformed crystals but not their de
novo formation. The typical concentrations in human urine are 2-8 mM for calcium and
0.2-0.5 mM for oxalate [2]. Normal human urine is in significant excess ofcalcium ions
with a Ca:Ox ratio in the range of6:1 to 10:1. An even wider range is seen in the urine of
stone patients [86].
Nucleation
The interaction between mineraI phases and macromolecules in vivo is a complex
phenomenon, influenced by physiological factors such as inorganic ion concentration and
pH [18]. Although human urine is generally insufficiently supersaturated with respect to
calcium oxalate to induce nuc1eation ofCO crystals, the dissolved calcium oxalate can
form nuc1ei when its supersaturation reaches 7-11 times its solubility [21, 45]. This type
offormation is known as homogeneous nuc1eation. More commonly, nuc1ei are formed
on existing surfaces in a process referred to as heterogeneous nucleation. Surfaces in the
kidney that can serve as sites for heterogeneous nuc1eation are epitheliallinings, cellular
debris, urinary casts (such as aggregations ofTamm-Horsfall protein [THP]) and other
4
crystals. Anything that increases the rate offormation ofheterogeneous nuclei in tubular
fluid or urine would lower the supersaturation at which crystals :fust form.
Hyperuricosuria, which promotes the formation ofCO crystallization for example, may
have an effect by producing urate or uric acid seeds that could serve as sites for
heterogeneous nucleation [21,37,88].
Another type ofmineral that could serve as sites ofCO nucleation is CaP. Studies
have shown that more than 70% ofoxalate-rich stones had CaP within or near their
central core [38]. Calcium phosphate crystals that remain in the nephron or in the renal
collecting ducts could thereby act as possible promoters ofCO nucleation. Under normal
conditions in the kidney, the pH is around 6.75 in the proximal tubule and 6.45 in the
distal tubule [20, 38, 45]. Calcium phosphate crystals would most likely form under
these conditions (at a pH higher than 6.2), whereas CO would form in the collecting duct
(at a pH between 5.0-6.2) [20, 38, 45]. Theoretically, CaP formed in the nephron could
partly or completely dissolve in the collecting duct when the pH is low. This would
cause an increased local concentration ofcalcium and thus an increased supersaturation
with respect to CO, promoting CO precipitation on preformed CaP crystals [39].
Proximal tubule Distal tubule DiSlllltubule Collecting-ductLoop ofHenle prox part distpart
..... .. Cal + Caz>
••• ca2••caz
, •--. •• Caz'Caz,
Caz, Cah
pH 6<75 -1.4 6.45 MS 5<5-6.1
• CaP crystals t2J CaOx crystals
Figure 1. Model for the initial intratubular events in the formation and aggregation of
calcium oxalate and calcium phosphate crystals leading to kidney stone formation [39].
5
Growth
Nucleation is only the first step in the process ofstone formation. Growth and
aggregation ofthese microscopie nuclei into masses that eventually hecome large enough
to he occluded within the kidney proper are aIso essential steps in the process ofstone
formation. Within the five to seven minutes required for urine to pass through the
nephrons, mineraI nuclei do not attain a size large enough to he retained and block the
lumens ofthe kidney tubules [21]. Since the rate ofCO crystal growth is approxiInately
2 Jlm per minute in an uninhibited environment, the probability ofa single crystal
attaining a large enough size by growth aIone is extremely low [37].
Aggregation
The process ofaggregation involves the formation ofa new, larger mass by
adhesion ofexisting particles in an energetically favorable process, and hence occurs
naturally. The rate ofaggregation is controlled by factors such as Van der Waals forces
and the viscous properties ofsurrounding molecules, and can he influenced by the
saturation level ofthe urine. Aggregation is a quick process whereby the formation of
larger particles occurs within seconds, and therefore is considered to he more critical than
nucleation and growth ofcrystaIs in the process ofstone formation [37].
6
CaP supersamrationUMM
CaP supersatUration
UMM
LowpH
CaOx supersatlffiltionUMM
UMM
CaOx supersaturationUMM
CaOx supersaturatiQnUMM
caOxCaP stolle formation
Citrate
UMMCitrate
MagnesiumPyrophosphate
UMM
HigbpH
UMMCitrate
Pyrophosphate
UMM
UMMCitrate
Pyrophosphate
UMMCitrate
Pyrophosphate
Figure 2. Important factors for the development ofa calcium stone (UMM=urinary
macromolecules) [39].
Calcium oxalate crystals
Calcium oxalate crystals exist in three forms depending on their hydration
state: calcium oxalate monohydrate (COM), dihydrate (COD) and trihydrate (COT).
Although COT has not been reported to be a component in kidney stones, some believe
7
that it may he an important precursor in their formation [16]. As the existence ofCOT in
urine and stones has yet to he confirmed, the remainder ofthe discussion ofCO crystals
will focus on COM and COD. The frequency ofCOM is approximately twice that of
COD, although many stones contain both crystal types [27, 59]. COM is usually found in
stone formers' urine but seldom in healthy urine, and asymptomatic crystals are usually
COD [17].
The formation ofCOD crystals has heen reported to he an important factor in
kidney stone formation. COD crystals have a higher positive charge than COM crystals
due to the fact that they display more calcium ions per unit ofcell and therefore present
more repulsive charges hetween crystals, which would in turn decrease the formation of
aggregates. As mentioned above, aggregation is one of the most important steps in the
formation of stones, therefore COD crystallization could decrease stone formation.
Furthermore, as COD crystals have a lower negative charge than COM crystals, they
would he less prone to adhere to cell surfaces and therefore contribute to a lesser degree
to the retention in the collecting ducts [17]. This theory is supported by the fact that
COM crystals are large cationic particles, presenting calcium ions at their surface that
would have a stronger affinity for anionic molecules on rerral epithelial cell membranes
than would COD crystals [84, 87]. Studies have shown that approximately 50% more
COM crystals appears to bind to inner medullary collecting duct cells than COD for a
given amount ofadded crystals [87].
It has been proposed that normal human urine contains factors that can influence
CO crystal structure in the direction ofCOD. The presence ofurinary macromolecular
inhibitors ofcrystal growth can cause preferential crystallization ofCOD from
8
supersaturated solutions ofcalcium chloride and sodium oxalate, rather than COM [87].
Sorne macromolecules have been shown to influence CO crystallization in favor ofCOD,
specifically RNA, heparin and poly-aspartic acid. These also are known to inhibit CO
crystallization. Other macromolecules reported to have an effect on crystal structure are
nephrocalcin, osteopontin (OPN) and urinary prothrombin fragment 1 (UPTFl), which
are also protein inhibitors ofCO nuc1eation and growth. These proteins contain
polyanionic regions and a net negative charge [27, 87]. Albumin has also been found to
have an affect on crystal structure [17].
Kidney stone matrix
The matrix ofkidney stones is a heterogeneous mixture oforganic molecules
originating from serum, kidney and urine. It is composed ofa combination ofproteins,
carbohydrates, nucleic acids and glycosaminoglycans (GAG). The breakdown ofthe
components is as follows: 64% protein, 9% non-amino sugars, 5% glucosamine, 10%
bound water and 12% organic ash, all ofwhich are present from the center to the surface
ofthe stone [26, 65]. Sources ofthe matrix inc1ude the adsorption and binding ofurinary
macromolecules, the incorporation ofexfoliated epithelial cells and their degradation
products, and blood [46]. It has been suggested that the composition ofthe matrix is
constant regardless of the crystalline component ofthe stone, while others believe that
various types of stones have distinct organic compositions [65, 94, 95]. Two to three
percent ofthe total dry weight ofa stone is matrix and it is distributed throughout the
stone, occupying a larger volume than is represented by its weight [46].
9
Matrix inclusion can be divided into two distinct steps tOOt ultimately lead to
different types ofmatrices. The fust step includes nucleation and growth ofcrystals in
the renal tubules tOOt would bind urinary molecules present depending on crystal-surface
affinity. When the crystals are retained in the kidney tubule, they then induce the second
type ofmatrix involving contributions from the renal epithelium. This process is
considered as being traumatic and leads to a significantly altered composition ofurinary
macromolecules. In such a case, the crystals would bind molecules based on their
mineraI type and mineral-binding affinities, thus resulting in the deposition ofchemically
distinct matrices [26].
The association oforganic matrix with the mineraI component is believed to occur
very early on in stone formation [5]. Sorne believe tOOt the stone matrix is present only
passive1y because these components are present in the urine at the time when the stones
form. Others speculate that the organic matrix plays a more significant, active role in
stone formation, contributing to steps such as crystal nucleation, crystal growth and the
overall composition ofthe stones [46]. The mechanism in which proteins adsorb to
growing crystals depends on the properties ofthe protein surfaces and crystal faces
involved in the crystal interactions and those exposed to the surrounding urine.
Theoretically, macromolecules with a very high negative charge and strong aflinities for
urinary crystals would become irreversibly bound and remain as components of the
matrix [19].
Considerable controversy has accompanied discussions of the organic matrix and
the way in which it said to associate with the mineraI content ofa stone. Terms such as
"incorporation" and "inclusion" have widespread use in the literature on protein
10
association in kidney stones, however these tenns perhaps erroneously insinuate that
macromolecules as large as THP, with a monomeric molecular weight of85 kDa, are
literally included/occluded into the atomic lattice ofa crystal. In tenns ofprotein binding
to calcium oxalate, it is thought that organic materials bind to the crystals ionically or by
Van der Waals forces. Another theory is that the proteins are adhesive, binding crystals
in the preliminary stages ofgrowth, but then are displaced during the other steps ofstone
formation. Only tightly bound proteins would be expected to remain between the crystals
and therefore would become part of the stone as a whole [5]. This is the other
controversial aspect: are proteins present or absent from crystal surfaces? Studies have
shown that in sorne sections ofCO stones, crystal surfaces are coated with altemating
electron-dense and light fibrils, and that other surfaces are covered with a more
amorphous granular material, which would indicate the presence ofprotein [12, 61].
Others have shown that crystal surfaces are smooth and devoid ofproteinaceous material
[65].
Urinary Proteins
Urine contains over 200 proteins, and a number ofthese proteins are suspected to
play a role in the processes involved in kidney stone formation. Ofthese proteins, a few
have been noted as potential inhibitors ofcrystallization, most notably osteopontin,
urinary prothrombin fragment 1, albumin and Tamm-Horsfall protein [3,5,26,36,60,
64,65,88].
11
Osteopontin
Osteopontin, also known as uropontin, was initially discovered in bone matrix
[13]. It has been associated with mineralization processes, including kidney stones, as a
potent inhibitor ofCO crystal nucleation, growth and aggregation [1, 78] and as an
inhibitor ofnucleation and growth ofCaP [1, 70, 82]. Osteopontin is considered to be an
important protein in calcified tissues due to its strong calcium-binding and its cell
adhesion activity via the RGD tripeptide that binds to integrins [68].
Osteopontin is a 44 kDa, highly acidic glycosylated phosphoprotein which is
secreted in the kidneys by the thin and thick ascending limbs ofthe loop ofHenle and by
the papillary surface epithelium ofthe renal calyces [44, 82]. It is produced by many
types ofepithelial cells and can be found in normal plasma and in various bodily fluids
such as bile, urine and milk [13, 70].
Human OPN is encoded by a single gene containing seven exons and six introns
[31]. Three different splice variants ofthis protein have been identified as weIl as
numerous isofonns due to substantial post-translational modifications such as
glycosylation, phosphorylation and sulfation [23, 24, 51]. The protein contains 42 serine
and 14 threonine residues which are highly conserved and suitably located for
phosphorylation [41, 70]. Phosphorylation of the protein may be important with respect
to its inhibitory activity, as dephosphorylated fonns ofOPN have been demonstrated to
be less effective inlnbitors ofCaP nucleation and growth [88].
Human OPN is abundant in acidic amino acids with 48 aspartic acid and 27
glutamic acid residues along its 298 amino acid peptide sequence. The majority ofthe
aspartic acid residues are found at the N-terminal end of the protein [41]. Found to be
12
incorporated into the urinary stone matrix [61], the 15-20% aspartic acid residue content
ofOPN is thought to be important in mineraI binding [28, 70, 86]. This protein is also
high in sialic aeid [78 ,70] which plays an indirect role in crystal binding by forming a
bridge between transiently expressed crystal binding molecules and cell surfaces [84].
Osteopontin is effieiently cleaved by thrombin, splitting the protein into
approximately equal-sized OOlves [23]. The thrombin cleavage site is highly conserved,
which suggests tOOt the cleavage ofOPN by thrombin plays an important role in
physiological processes. The activity ofthe cell-binding domain, the arginine-glycine
aspartate (RGD) sequence, is modulated by thrombin cleavage [24, 51] and this results in
a 2-3 fold increase in the chemotactic activity of OPN [31] perhaps by exposing a cryptic
adhesion sequence [51].
Urinary OPN concentration varies inversely with urinary volume [51, 96] and has
been detected to be excreted at a rate of3 mg/l [66, 88]. The urinary excretion levels of
OPN in stone formers has been shown to be less than in non-stone formers, possibly
attributable to a lower rate of synthesis or by incorporation into stones [50, 66, 89]. OPN
is considered to he a strong inhibitor ofall aspects ofCO crystallization in vitro at
physiologically relevant concentrations [1, 41].
Urinary Prothrombin Fragment 1
Prothrombin is the parent molecule ofurinary prothrombin fragment 1 (UPTFl)
whose function is primarily associated with blood coagulation [74]. Prothrombin and its
fragments can be found in four differynt forms: intact prothrombin, prothrombin fragment
1 (UPTFl), prothrombin fragment 2, and prothrombin fragment 1+2 [36]. Prothrombin
13
contaîns ten y-carboxy-glutamic acid residues in its N-terminal domain, amino acid
residues that have a high affinity for calcium ions. Fragments containing this domain, the
FI +2 and FI, are released during the coagulation process [80, 88].
Urinary prothrombin fragment 1 has been detected in CO and CaP crystal
preparations freshly precipitated from urine, and its reported molecular weight is about
31 kDa [80, 88]. Excretion ofUPTFI by non-stone formers has been reported to occur at
a rate ofOA mg/day [9]. The prothrombin gene is expressed in the kidney, therefore it is
speculated that the protein has activation pathways and functions other than solely in the
coagulation cascade. Also, immunohistochemical studies have located UPTFI in the
more lithogenic regions of the human kidney [35], more specifica11y in the distal
convoluted tubules and in the thick ascending loop ofHenle [44].
Studies on UPTFI have shown a strong inhibitory activity on CO crystal growth
or aggregation at physiological concentrations, and therefore, its role in urine is presumed
to be the prevention of stone formation [66]. A positive correlation has been found
between UPTFI and OPN levels in that decreased excretion ofUPTFI correlates with a
similar decrease in OPN excretion. This may be an important factor in the urinary
inhibition ofCO crystallization [66].
Albumin
Albumin is one ofthe most abundant proteins in urine, excreted at a rate of
approximately 2.5 mg/day in stone formers and in non-stone formers. Although it has
been detected as a component ofthe stone matrix, the process ofits inc1usion is not yet
known. Albumin is a powerful nuc1eator ofCO crystals in vitro, with the polymerie
14
fonns being more active than monomers. The nucleation ofmineraI by albumin
apparently Ieads to the exclusive formation ofCOD crystaIs, whereas COM crystals are
formed in its absence. As mentioned above, COD crystallization would serve to decrease
stone formation [17].
Albumin has also been speculated to be involved in the aggregation process of
CO crystals, since it was shown that albumin reduces the size ofcrystal aggregates in a
concentration-dependent manner. Calcium oxalate dihydrate crystals containing albumin
aggregate onto COM crystals, or free albumin could absorb onto the surface ofcrystals
that have aIready formed [17].
Another mechanism by which albumin could serve in preventing stone formation
is by inducing the formation ofnumerous crystals that would remain small enough to be
easily eliminated. Furthermore, albumin would favor the formation ofCOD, which is
less susceptible to retention in the urinary system [17].
Tamm-Horsfall Protein
Tamm-Horsfall protein (THP), also known as uromodulin, is the most abundant
protein in urine, with a urinary excretion of20-200 mg/day [83, 88]. THP bas a
molecular weight of 80-85 kDa, but tends to self-aggregate into macromolecules as large
as 23 million daltons, especially under conditions ofelevated concentrations ofcations
such as sodium, calcium, magnesium, and hydrogen (i.e., under conditions ofhigh ionic
strength and low pH) [65, 83] as weIl as at high concentrations ofTHP itself[88].
THP is made by renal epithelial cells in the thick ascending limb ofHenle [21]
[44] where it may possibly act to render the nephron wall impermeable to water [83]. It
15
is also produced in distal tubules in the form ofsubunits [65]. THP can be found on
individual as weil as inter-crystalline surfaces where it serves to connect crystals inside
renal tubules [44].
THP is 616 amino acids in length [41] and contains an Arg-Gly-Asp (RGD)
integrin-binding motif, which functions in cell-matrix interactions [88]. THP also
contains 25-30% carbohydrate (12% hexose, and 11% hexosamine), ofwhich up to 50%
is sialylated [65, 83].
The role ofTHP in calcium stone formation is a controversial one. It has been
reported to both promote [41, 72] and inhibit [1,49] CO crystallization in vitro [83].
Although some confirm the presence ofTHP in the stone matrix [88], other studies have
obtained contradictory results [26]. It has been found to inhibit crystallization by coating
crystals and thus preventing additional crystal growth and/or aggregation [21, 83].
However, the tendency towards self-aggregation and polymerization would lead to
promotion by forming a mesh-like structure to which crystals could adhere, thus initiating
crystal growth and/or aggregation [83,88]. The degree ofaggregation appears to dictate
whether THP will act to retard or promote crystallization [88].
Differences in THP isolated from stone formers and non-stone formers have been
described. It is speculated that processing steps involved in handling urine samples could
play a role by disrupting carbohydrate residues and that the THP from stone formers may
be more susceptible to those effects than THP from non-stone formers [83]. Other
studies have shown a decrease in excretion in stone formers, an observation suggesting an
important role in CO crystallization in vivo [71, 81].
16
Urine from stone formers versus non-stone formers
The urine from stone formers and non-stone formers is common1y supersaturated
with respect to calcium oxalate. Crystals produced in urine from stone-formers and non
stone formers are morphologica11y identical [73]. Calcium oxalate crystals are frequently
found in the urine ofstone-formers and ofnon-stone formers. Therefore, the formation
ofa kidney stone is most probably the result ofcrystal retention in the kidney on the basis
ofcrystal size or shape, aggregation or adherence to the kidney tubule epithelium [85].
Although both CO and CaP have heen found in the urine of stone formers and non-stone
formers, mixed co-caP crystal formation is more common1y found in stone-forming
patients [45].
Genera11y, stone formers excrete more crystals as either small crystals similar to
those in non-stone formers, or large crystals [85, 88]. This is not entirely due to the fact
that the supersaturation in the stone formers' urine is greater; when a small amount of
oxalate is added to non-stone formers urine in order to increase the saturation to that of
the stone formers, no increase in crystal size is detected [88].
It appears that there is a highly selective process, and not a random absorption of
macromolecules onto the surface ofgrowing crystals, tOOt is required for the occurrence
ofstone formation. An important difference hetween stone formers and non-stone
formers is the production or deposition ofthis required material in the stone formers [64].
This required material affects crystallization processes and hence can he referred to as an
inhibitor or a promoter ofcrystallization. It is presumed tOOt kidney stone formation is
due to the absence or modification ofurinary inhibitors [73]. Since the crystal matrix OOs
he shown to he an inhibitor ofcrystallization, this would suggest tOOt the difference
17
between stone formers and non-stone formers lies in the composition and properties of
the matrix [7, 25, 27].
Studies have found modifications with respect to OPN, UPTF1, and THP levels or
activity in stone-formers as compared to non-stone formers. On average, urinary OPN
and UPTFI concentrations were found to he lower for stone-formers than non-stone
formers [67]. This finding are suggestive oftwo possibilities: 1) these proteins exist in
the urine ofstone-formers at a lower concentration than in non-stone formers, and/or 2)
attributable to the fact that stone formers produce larger crystals in a higher quantity than
non-stone formers, these proteins are detected at a lower level due to their increased
inclusion into the crystal matrix.
THP isolated from the urine ofrecurrent stone formers has a decreased capacity to
inhibit crystal aggregation. The higher calcium concentrations found in stone-formers'
urine promotes THP self-aggregation, which in turn translates into increased crystal
aggregation. Viscosity measurement studies suggest that the degree of self-aggregation is
inversely proportional to the inhibitory activity ofTHP, and can be reversed by the
addition ofcitrate [88].
Urine of males versus females
It has been suggested that the predominance ofstone formation in males can he
explained by sex-dependent differences in concentrations and/or activity ofurinary
inhibitors or promoters, which in turn maybe regulated by sex hormones [14, 75]. The
well documented disparity in the rate ofCO lithogenesis between men and women being
approximately 2-3:1 (male:female ratio) [14,43] suggests that sex hormones likely play a
18
role in stone formation, not only by influencing the urinary conditions which increase the
probability ofCO precipitation, but aiso by affecting the likelihood that these crystais
will be retained in the urinary collecting system. This is supported by studies which
show that higher Ievels ofestrogen activity during the reproductive years offemales
protect women from kidney stone formation [77]; by corollary, administration of
testosterone in rats promotes CO stone formation [43,55]. These studies aiso predict a
Iesser role for female sex hormones. Estrogen appears to inhibit the excretion ofurinary
calcium and it has been shown that female sex hormone supplementation inhibits the
excretion ofurinary oxalate [43].
The urine from females is less likely to undergo precipitation ofCO than that
from males, possibly attributable to the lower urinary concentrations ofcalcium (Ca).
Although this might be considered sufficient evidence to explain the difference between
stone formation rates in men and women, as precipitation of insoluble crystals is the fust,
and absolute, requirement for stone development, the nucleation ofcrystals is, as
mentioned above, not sufficient to cause CO stone formation [14].
It has been demonstrated that female urine produces significantly larger volumes
ofcrystals as weIl as Iarger individuai crystalline particles following induction of
crystallization, although the overall mass ofthe particles produced are the same in both
genders. The formation oflarger crystals could result from greater crystal growth, which
might occur in higher concentrations ofcalcium and oxalate. Although urinary
concentrations ofcalcium are generally lower in the females, their endogenous oxalate
levels are slightly higher. This is pertinent because the concentration ofoxalate is about
15 times more influential on crystallization than are urinary calcium levels. Another
19
explanation for the greater crystal volume could be an enhanced inclusion oforganic
matrix into the crystalline structure, which would increase the volume, but not the mass
ofthe precipitated crystals [14].
Interestingly, crystals deposited from the urine ofmen, though smaller than those
from the urine ofwomen, appear to he more highly aggregated. Although reduced
aggregation ofthe crystals in female urine suggests that they might he less likely to he
retained in the kidney, this would he at least partially countered by the formation of larger
crystals, occupying a greater total volume. Both men and women produce particles
averaging 7.1 ~m in size, and therefore crystals precipitated from both genders would be
equally prone to retention in the urinary tract. This would indicate that the greater
tendency for men to form CO stones may he linked to their increased predisposition to
nucleate CO crystals, rather than to the formation oflarger crystalline particles more
likely to he retained in the renal collecting system [14].
Rationale and objectives of this research project
Using an established crystal precipitation methodology for urine, tbis project
attempts to clarifY sorne discrepancies found in the literature with respect to the
processing ofurine samples and the protein content ofprecipitated calcium oxalate
crystals. In particular, it focuses on the association ofosteopontin, urinary prothrombin
fragment 1, albumin and Tamm-Horsfall protein with the precipitated mineral phase.
Experiments have also been performed in an effort to find differences hetween the urine
and crystals precipitated from the urine ofmale and female non-stone formers.
20
As a second part to this thesis, the mechanism ofprotein binding to calcium
oxalate crystals has been preliminarily explored. Given the high content ofaspartic acid
and the presence ofa contiguous stretch ofpoly-Asp in OPN, peptide/protein-binding
studies have been performed using poly-Asp and albumin (for comparative purposes).
Binding ofthese peptide/proteins to hydroxyapatite, a putative precursor phase to kidney
stone formation, has been studied in order to better understand protein-mineral
interactions in general, and to model what may be the earliest stages ofurolithiasis.
Future experiments are planned to examine peptide/protein interactions with calcium
oxalate.
21
MATERIALS AND METHODS
MATERIALS AND METRODS
The precipitation of calcium oxalate (CO) crystals from human urine
Part J: The effects ofurine manipulation
The metastable limit of the urine with respect to oxalate was determined as
described by Ryall et al. [73]. Briefly, sodium oxalate (NaOx, Fisher Chemicals) was
added to aliquots ofmale urine by titration and incubated for 30 minutes at 37°C in a
shaking incubator (New Brunswick Co. Inc. Series 25 Incubator Shaker) [73]. The
presence of crystals in the solution was verified by light microscopy. The metastable
limit was determined to be slightly below 4 mM NaOx (final concentration), and
therefore this was the concentration used in the experiments involving the precipitation of
calcium oxalate crystals from the urine.
Doyle et al. [26], originally described the protocol for the precipitation of CO
crystals from urine. A modified version of this protocol was used. Briefly, fresh
moming urine (200-500 ml volume) was collected without added anti-bacterial agents
from 6 males between the ages of23 and 45 years with no history ofkidney stones. Each
sample was treated individually. The pH ofeach sample was taken and urine analysis
was performed using ChemStrip 5L (Roche) for the presence ofleukocytes, glucose,
ketones, or red blood cells (RBCs) in the sample. Samples with positive leukocytes, or
RBCs would have been rejected in this study, but none had abnormal detectable
concentrations, and thus all samples were used. Attempts were made to quantify the
urinary protein concentrations by the Micro BCA Protein Assay Reagent Kit (Pierce) as
well as by the mini-Lowry method. As neither ofthese assays provided accurate or
23
reproducible readings due to interfering substances commonly found in urine [97, 98], we
opted to normalize the samples by their relative urine/creatinine levels. One-ml aliquots
of each sample were taken for urine/creatinine analysis, which was performed at the
chemistry laboratory of the Montreal General Hospital. The remainder of the samples
was kept at 4°C until processed, usually within a few hours of collection.
In order to address the effect ofcentrifugation, the samples were halved and each
half was placed in a 500 ml centrifuge bottle. One half of the sample was centrifuged at
8,200 rpm for 30 minutes at 22°C in a Beckman J2-MC centrifuge fitted with a J2-14
rotor. An aliquot of the supematant (SN) and the pellet were taken and stored at 4°C
until further analysis. The uncentrifuged and the centrifuged (SN remaining after initial
centrifugation) samples were placed in a shaking incubator at 37°C. A 200 mM solution
of sodium oxalate (NaOx) was added to both samples to get a final concentration of4
mM ofNaOx at intervals of one hour for a total of 3 hours. The volume ofNaOx
solution added was corrected in order to keep the final concentration of oxalate at the
metastable limit.
To collect the crystals, the samples were centrifuged at 8,200 rpm for 30 minutes
at 22°C. The crystals were harvested by filtration through a hydrophilic polypropylene
membrane (0.2-0.8 Ilm pore size, Gelman Sciences) and washed for six cycles with 40 ml
of double-distilled water in 50 ml tubes (Corning). Samples that were too viscous to f10w
through the membranes were passed through a prefilter (Fisher), and then reapplied to the
membrane.
In order to observe the effect of different washing conditions on the crystals, the
crystals obtained from centrifuged and uncentrifuged urine were halved. Each halfwas
24
washed either for 2 more cycles with ddH20 or for 3 cycles with 40 ml of O. 1 M sodium
hydroxide (NaOH) (BDH Chemicals).
An aliquot of the crystals was taken for each of the 4 conditions: 1) uncentrifuged
urine/crystals washed with ddH20, 2) uncentrifuged urine/crystals washed with NaOH, 3)
centrifuged urine/crystals washed with ddH20, 4) centrifuged urine/crystals washed with
NaOH. These crystals were placed in 1.5ml microcentrifuge tubes (Fisher) and washed 2
more times with ddH20, in order to remove the NaOH, by spinning for 30 seconds at
13,000 rpm using a Beckman Microfuge Il centrifuge. The crystals were resuspended in
500 J11 of ddH20 and were stored at 4°C for future scanning electron microscopy (SEM)
studies (see below).
To obtain/release crystal-bound proteins, the remaining crystals were weighed and
placed in 15 ml centrifuge tubes (Corning) for demineralization with the appropriate
volume of0.25 M ethylene-diamine-tetracetic acid (EDTA) (BDH Chemicals) at pH S.O
to obtain 7 ml EDTA per 30 mg of crystals for a period of 3 days at 4°C with gentle
inversion [26]. By this demineralization method, the matrix proteins were eluted into the
solution. The EDTA was removed by dialyzing against ddH20 using cellulose
membranes with a 12 kDa molecular weight cutoff (Sigma), which were prepared by
boiling for 30 minutes in ddH20 to remove the glycerol, and then rinsed thorougWy with
ddH20 before loading the samples. Dialysis was done over 24 hours at 4°C with 3 water
changes and agitation using a magnetic stirrer. The extracts were collected in 15 ml
centrifuge tubes (Corning) and frozen at SO°C (30 minutes to one hour) then dried under
vacuum (Vitris Sentry-Freezemobile 12EL) for 24-72 hours and reconstituted in the
appropriate volume of ddH20 to concentrate the samples 5-fold. Ail samples, including
25
the original urine samples, supematants (obtained from centrifuged samples as well as
those taken after collection of crystals), crystal extracts and pellets (obtained from
centrifuged samples) were stored at -20°C (ifprocessed within one day) or -SO°C (if
processed after more than one day).
To determine the effect of storage temperature, protease inhibitors, type, and
temperature ofurine collection on urinary proteins, a 24-hour urine sample was collected
from one female control (age 26). Halfofthe urine was stored at 4°C and the other half
at room temperature. The following mixture ofprotease inhibitors were added to half of
each of the samples: 5 ~g/mlleupeptin (Sigma), 100 ~g/ml benzamidine (Sigma) and 0.1
mM phenylmethylsulfonyl fluoride (PMSF) (Sigma). Halfofthese samples were stored
at 4°C, and the other half at -80°C, for 4 days before processing. On the fifth day, a fresh
morning sample ofurine was taken from the subject and the same protease inhibitors
were added to half of the sample. The samples were vortexed and processed within one
hour.
Osteopontin: Further characterization
Thrombin digestion
Digestion ofOPN with thrombin generates two fragments ofapproximately 30
kDa, and this was used to aid in the identification of OPN in polyacrylamide gels. One
hundred ng of sample were digested with thrombin (Boehringer Mannheim) using 0.01
units ofenzyme per ~g ofprotein. The enzyme and proteins were placed in 1.5 ml
microcentrifuge tubes with 100 mM Tris-HCl, at pH 8.0 with 150 mM NaCI, and the
reaction took place at room temperature overnight [40, 76]. Urinary OPN (50 ng) was
26
used as a positive control and HSA (500 ng) was used as a negative control. The samples
were prepared and loaded onto a 12% polyacrylamide gel as above. The gel was stained
using the double staining method with Stains-AlliAg nitrate.
Osteopontin associated with calcium oxalate crystals versus calcium phosphate crystals
precipitatedfrom male urine
CO crystals were precipitated from male urine as stated above. In other
experiments, CaP crystals were precipitated as follows: after determination of the
metastable limit ofurine with respect to phosphate, a 0.1 M sodium phosphate (NaP)
solution was added to male control urine as described in Atmani et al. [3] to obtain 15
mlIl NaP. The remaining steps to collect and wash the crystals were identical to the
procedures used for CO crystals. Samples were taken for SEM and for further analysis
by Western blotting using antibodies against OPN.
Part II: Gender differences
From the results obtained in Part 1, all remaining crystal precipitation experiments
from urine were performed in whole urine (uncentrifuged) and the crystals obtained were
washed for 8 cycles with ddH20 only. Fresh morning urine (200-500 ml volumes) was
collected from 7 males and 15 females between the ages of20 and 45 years with no
history ofkidney stones. The female samples were collected in bottles treated with
thymol, an anti-bacterial agent. Again, each sample was treated individually and aliquots
were taken for pH and urine/creatinine analysis. None of the samples presented abnormal
urine analysis results. Crystals precipitated from the urine offemales were first passed
27
through a prefilter (Fisher) in order to remove the thymol, and then washed for 8 cycles
with ddH20. Aliquots ofthe crystals were taken for SEM and stored at 4°C in ddH20
until analyzed. Crystal extracts were halved and processed as follows: one half was
dialyzed and lyophilized as described above, the other half was concentrated and desalted
using a Centricon Plus-20 (10,000 MW cutoff, Millipore) with a Beckman J6-MC
centrifuge fitted with a JS-4.2SM rotor spun at 4,000 rpm for 25 minute cycles at 4°C.
Once the concentration was complete, ddH20 was added for 2 cycles in order to desalt
the extracts. The extracts were concentrated s-fold.
Male and female urine samples were concentrated using YM-10 centrifugaI filter
devices (10,000 MW cutoff, Millipore) in aDamonlIEC Division CRU-sOOO centrifuge
at 4,200 rpm for 4s-minute cycles at 4°C until concentrated 10-fold. These samples were
stored at -20°C until analyzed by Western blotting for the presence ofOPN.
The precipitation of calcium oxalate (CO) crystals from rat urine
Urine was extracted from the bladder of3 adult male Sprague Dawley rats (350
400 g, Charles River Laboratory) with a syringe (Becton Dickinson) and placed into 1.5
ml microcentrifuge tubes (Fisher). CaOx crystals were precipitated in one of the samples
using a volume of 500 JlI with the addition of a 200 mM NaOx solution for a 10 mM final
oxalate concentration. The crystals were collected by centrifugation in a Beckman
Microfuge Il operating at 13,000 rpm for 15 minutes at room temperature. The
supernatant was recuperated in a 1.5 ml-microcentrifuge tube (Fisher) and the pellet
(containing the CaOx crystals) was washed 6 times with 500 JlI of ddH20. An aliquot
was taken for SEM analysis and was stored at 4°C until processed. The remaining
28
crystals were demineralized with 1 ml of250 mM EDTA (pH 8.0) at 4°C for 3 days. The
extract was desalted (with ddH20) using Centricon YM-lO centrifugaI filter devices
(10,000 MW cutoff, Millipore) in a DamonlIEC Division CRU-5000 centrifuge at 4,200
rpm for two, 15-minute cycles at 4°C. The extract was collected in two 1.5 ml
microcentrifuge tubes (Fisher) and stored at -20°C until processed.
SnS-PAGE of crystal-associated proteins
Samples (20 III total volume) were mixed with 5 III of5X SDS-PAGE sample
buffer [0.5 M Tris-HCl (Bio-Rad) pH 6.8, 10% SDS (w/v, Bio-Rad), 50% glycerol
(Sigma), 0.0005% bromophenol blue (w/v, Sigma), 10% 2-mercaptoethanol (Fisher)] and
boiled for 5 minutes. Polyacrylamide gels were prepared according to the protocol for
the Mini-Protean III Cell gel apparatus from Bio-Rad. Unless otherwise stated, 12%
separation gels were prepared [12% AcrylamidelBis (Bio-Rad), 33.5% ddH20, 0.1%
SDS, 0.375 M Tris-HCl pH 8.8,0.0006% TEMED (Bio-Rad), 0.05% APS] and allowed
to polymerize. After 1 hour, a 4% stacking gel [4.5% AcrylamidelBis, 58.4% ddH20,
0.125 M Tris-HCl pH 6.8, 0.1% SDS, 0.0008% TEMED, 0.05% APS] was poured above
the separation gel and 1 mm-thick, 10-well combs were inserted into the gel. After
polymerization was complete (usually between 15-60 minutes) samples were loaded and
migration proceeded in electrophoresis running buffer at 200 V until the bromophenol
blue marker exited the gel (usually between 45-75 minutes) [54).
Gel Staining Methods
Double Staining: Stains-AIVAg nitrate
29
,.,,-.
In order to optimize the visualization ofacidic proteins such as OPN, the
following protocol, first described by Goldberg et al. [32] was used [32, 78]. Following
electrophoresis, polyacrylamide gels were washed in 25% isopropanol (Fisher) in order to
remove the sns. The solution was changed every 15 minutes for 90-120 minutes. Using
a light-sensitive container, the gels were placed in a generous volume of Stains-AIl
solution [30 JlM Tris, 7.5% formamide (Fisher), 25% isopropanol, 0.025% Stains-AIl
(ICN)] and stained overnight at room temperature. The stain was removed and the gels
were washed with 25% isopropanol until the background was clear. Gels were quickly
rinsed 3 times with ddH20 and incubated in a light-sensitive container with a freshly
prepared 12 mM silver nitrate (AgN03,Fisher) solution for 30 minutes at room
temperature then rapidly rinsed 3 times with ddH20. The gels were incubated briefly in
freshly prepared developing solution [0.28 M sodium carbonate anhydrous (Fisher),
0.15% of37% stock formaldehyde (Fisher)] until the staining was complete. The
staining reaction was terminated with 10% acetic acid (CH3COOH) (Fisher).
Si/ver Staining
The protocol to stain polyacrylamide gels with silver nitrate was described by
Blum et al. [10]. Following electrophoresis, gels were fixed [30% ethanol (EtOH)
(Fisher), 10% AcOH] for 90 minutes (3 changes/ 30 minutes). After rinsing for 10
minutes in 20% EtOH and 10 minutes in dH20, the gels were sensitized for 1 minute in
0.02% sodium thiosulfate (Na2S203 /STS). The gels were then rinsed 3 times for 30
seconds in ddH20 and were incubated for 30 minutes in a freshly prepared silver nitrate
solution [0.2% AgN03, 0.05% formaldehyde]. Following five, lO-second washings with
30
ddH20, the gels were incubated for 5-15 minutes in freshly prepared developing solution
[0.009% formaldehyde, 0.001 % STS, 3% potassium carbonate (K2C03)]. The reaction
was terminated by incubating for 30 minutes with 5% Tris/ 2.5% AcOH. The gels were
rinsed and stored in ddH20.
Western blotting
Western blot analysis was performed using the Mini Trans-Blot Transfer Cell
system from Bio-Rad [15]. For the samples obtained from the six initial male controls,
Immobilon-P transfer membranes (Millipore) were soaked in 100% methanol (Fisher) for
30 seconds and then incubated in transfer buffer [1.5% glycine, 0.3% Tris, 20%
methanol] until ready to use. Polyacrylamide gels were soaked in transfer buffer for 10
minutes and were placed against membranes and inserted into gel holder cassettes for the
electro-transfer. Fiber pads and filter paper (Schleicher & Schuell) were placed on either
side ofthe gels and the membranes. A Bio-Ice cooling unit, frozen at -20°C, a magnetic
stirrer and transfer buffer were added to the Transfer Cell and the whole system was
placed on a stirring plate (Coming) at 4°C overnight at a constant voltage of30 V.
Following the transfer, membranes were rinsed in ddH20 for 2 minutes and then
incubated in Ponceau Red [0.2% Ponceau S (Sigma), 3.5% TCA (Fisher)] for five
minutes in order to mark the proteins tOOt were used as standards (Low Molecular Weight
range, Broad Molecular Weight or Prestained Broad Molecular Weight, Bio-Rad).
Membranes were washed with ddH20 to destain the Ponceau red background and the
membranes were rinsed in PBS [0.02% KCI (Fisher), 0.14% Na2HP04 (lT.Baker),
0.024% KH2P04 (BDH Chemicals), 0.85% NaCI (Fisher), 0.1% Tween 20 (ICN)] for 2
31
minutes. Membranes were incubated in a generous volume ofblocking buffer [3% BSA
(ICN) in PBS] for one hour at room temperature on an orbital shaker (Bellco
Biotechnology) set on the lowest speed. The resolved proteins were immunodetected
using the following primary antibodies: 1) monoclonal mouse anti-human OPN Mb53
antibody [8] (a gift from Dr. Ann Chambers, University ofWestern Ontario), 2)
polyclonal OPN LF-124 (anti-N-terminal) or LF-123 (anti-C-terminal) antibodies [29] (a
gift from Dr. Larry Fisher, National Institutes ofHealth, Bethesda, Maryland), 3)
monoclonal mouse anti-human urinary prothrombin fragment 1 (UPTFl) B19-1 antibody
(a gift from Dr. Rosemary L. Ryall, Australia), 4) polyclonal rabbit anti-human serum
albumin (Sigma), 5) polyclonal rabbit anti-human Tamm-Horsfall protein (a gift from Dr.
Nakagawa, University ofChicago). The antibodies were gently vortexed in a 3%
BSAlPBS solution in the following dilutions: 1) anti-OPN Mb531 1:5,000, 2) LF-1241
1:2,000, LF-1231 1:10,000, 3) anti-UPTFI B19-11 1:2,000,4) anti-HSA/ 1:5,000, 5) anti
THPI 1:5,000, and incubated, gently agitating, with the membranes for two hours at room
temperature.
Membranes were washed vigorously 3 times for 10 minutes in blocking buffer.
Secondary antibodies used were either goat anti-mouse-HRPI 1:2,000 (Dupont) or goat
anti-rabbitl 1:2,000 (Pierce) for the monoclonal or polyclonal antibodies, respectively.
These were added to PBS and incubated with the membranes, gently shaking, for one
hour at room temperature. The membranes were washed vigorously 3 times for 10
minutes with PBS and the proteins were visualized by chemiluminescence with the ECL
Western blotting detection system (Amersham Pharmacia Biotech) using Super RX Fuji
medical x-ray films (Amersham).
32
The remaining samples (obtained from the 7 other male controls and the 15
female controls) were immunodetected using Immun-Blot PVDF membranes (Bio-Rad),
which were soaked in 100% methanol (Fisher) for 30 seconds and then incubated in
transfer buffer [192 M glycine, 25 mM Tris, 20% methanol]. Polyacrylamide gels were
soaked in transfer buffer for 10 minutes and were placed against membranes and inserted
into gel holder cassettes for the electro-transfer. Fiber pads and filter paper (Bio-Rad)
were placed on either side ofthe gels and the membranes. A Bio-Ice cooling unit, frozen
at -20°C, a magnetic stirrer and transfer buffer were added to the Transfer Cell and the
whole system was placed on a stirring plate (Fisher) at room temperature for 1 hour at a
voltage of 100 V.
Following the transfer, membranes were rinsed in ddH20 for 2 minutes and
blocked with 2% BSA (Sigma) in TBS [50 mM Tris pH 7.4, 0.15 M NaCI (Fisher), 0.1%
Tween 20 (Fisher)] for one hour at room temperature on an orbital shaker (Fisher) set on
the lowest speed. The resolved proteins were immunodetected using the following
primary antibodies: 1) monoclonal mouse anti-human OPN Mb53 antibody (a gift from
Dr. Ann Chambers, University ofWestem Ontario), 2) polyclonal OPN LF-124 (anti-N
terminal) or LF-123 (anti-C-terminal) antibodies (a gift from Dr. Larry Fisher, National
Institutes ofHealth, Bethesda, Maryland), 3) monoclonal mouse anti-human urinary
prothrombin fragment 1 (UPTF1) B19-1 antibody (a gift from Dr. Rosemary L. Ryall,
Australia), 4) polyclonal rabbit anti-human serum albumin, 5) polyclonal rabbit anti
human Tamm-Horsfall protein (a gift from Dr. Nakagawa, University ofChicago). The
antibodies were gendy vortexed with TBS in the following dilutions: 1) anti-OPN Mb53/
1:2,500,2) LF-124/ 1:2,500, LF-123/ 1:1,000, 3) anti-UPTFI BI9-l/ 1:2,500,4) anti-
33
HSA/ 1:2,500, 5) anti-THP/ 1:2,500, and incubated with the membranes, gentlyagitating,
for 2 hours at room temperature.
Membranes were washed vigorously 5 times for 5 minutes in TBS. Secondary
antibodies used were either anti-mouse-alkaline phosphatase conjugate/ 1:3,000
(Cedarlane Labs) or anti-rabbit-alkaline phosphatase conjugate/ 1:3,000 (Cedarlane Labs)
for the monoclonal or polyclonal antibodies, respectively. These were added to TBS and
incubated with the membranes, gently shaking, for one hour at room temperature. The
membranes were washed vigorously 5 times for 5 minutes with TBS and the proteins
were visualized using Fast 5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium
tablets (Sigma) which were dissolved in 10 ml ddH20. The membranes were removed
from solution and the reaction stopped with 10 mM EDTA in 50 mM Tris-HCI pH 7.4.
After 15 minutes, the membranes were washed for 30 seconds with ddH20 and allowed
to dry in air on Kimwipes EX-L Delicate Task Wipers (Kimberly Clark).
Light and fluorescence microscopy
Crystal suspensions to be observed under light or fluorescence microscopy were
placed on precleaned microscope slides (25x75x1 mm, Fisherbrand) under a microscope
coyer slip (22x22 mm, Fisherbrand). The samples were visualized immediately and the
images captured with a Sony 3CCD color video camera attached to a Leica (Leitz) DM
RBE microscope and using Northem Eclipse imaging software (version 6.0, Empix
Imaging).
34
Scanning electron microscopy (SEM)
Samples destined for SEM were prepared as follows: one drop (between 1-5 J.ll)
ofwell-vortexed crystal suspension was mounted on aluminum stubs or carbon planchets.
the samples were dried in air and sputter-coated with gold or palladium for 5 minutes in
a Hummer VI Sputtering System (Anatech Ltd). The coated samples were examined
using a JEOL JSM-840A SEM operating at an acce1erating voltage of 15 keVand
visualized using the EDAX 840 Imaging program.
Immunohistochemistry of kidney stones
Kidney stones were obtained by surgical removal from two female patients (age
66 and 69) at the Royal Victoria Hospital Kidney Stone Clïnic. These were characterized
as calcium oxalate-containing stones by x-ray diffraction. The stones were washed 8
times with tap water and placed in separate 20 ml glass scintillation vials (Kimble). The
stones were fixed in 1% glutaraldehyde and 1% paraformaldehyde, with the fixative
being changed daily, for one week at room temperature on a rotator. The fixative was
removed and the samples were rinsed with ddH20. Wash buffer [0.1 M sodium
cacodylate (J.T. Baker, 5% sucrose (J.T. Baker) pH 7.3] was added and the samples were
left to incubate for 3 days at 4°C. The stones were decalcified in 4.13% EDTN1%
glutaraldehyde (20 mVvial) on the rotary shaker at 4°C. The solution was changed daily
until the samples were demineralized (between 12-26 days), and then they were placed in
wash buffer until further processing.
35
Paraffin embedding
The kidney stone samples were embedded in paraffin at the pathology laboratory
ofthe Montreal General Hospital. Briefly, the samples were placed into cassettes
(Simport Plastics Ud) soaked in 0.1 M PBS. Samples were dehydrated in a Fisher
Histomatic Tissue Processor (modelI66) followed by paraffin embedding in a Shandon
Embedding Center. Tissue blocks were trimmed and placed in a rotary microtome and
eut into 1 ~m-thick sections. The sections were floated onto glass slides, dried and stored
until further processing.
Hematoxylin and eosin staining
Hematoxylin and eosin staining was also performed at the pathology laboratory of
the Montreal General Hospital in a Tissue Tek SCA automatic H&E processor (Sakura).
Immunohistochemicallocalization ofmatrix proteins
Kidney stone sections were immunostained for the presence ofOPN using LF
123 polyclonal antibodies. Deparaffinized sections were treated with 1% bovine
testicular hyaluronidase (Sigma) at 37°C for 30 minutes. LF-123 was diluted to 1:200 in
5% normal goat serum/0.2% BSA in TBS with 0.01% Tween-20 (TBST) (50 mM Tris
HCI, 150 mM NaCI, 0.01% Tween-20, pH 7.6). Sections were washed for 3 cycles of5
minutes each with TBST. Biotinylated goat anti-rabbit IgG (Caltag Laboratories)
antibodies were allowed to incubate at a dilution of 1:200 in the same buffer for 45
minutes at room temperature. The sections were washed for 3 cycles of 5 minutes each
with TBST and treated with the Vectastain ABC-AP kit (Vector Laboratories) for an
36
additional45 minutes at room temperature. Color development was achieved with
treatment with Fast Red TR/Naphthol AS-MX phosphate (Sigma) containing ImM
levamisole to inhibit endogenous alkaline phosphatases. After 10 minutes, sections were
counterstained with methyl green (Vector Laboratories) and mounted in Kaiser's glycerol
jelly.
Preparation of pure inorganic calcium oxalate dihydrate (Weddelite) crystals
The precise conditions necessary for the growth ofuncontaminated con crystals
was described by Lepage and Tawashi [56]. AlI solutions were filtered using Whatman
(qualitative 4) filter papers prior to use. Three milliliters of0.005 M NaOx (room
temperature) was added to 5 ml of 1 M calcium chloride solution (Fisher) (at 4°C) in
polystyrene 15 ml centrifuge tubes (Fisherbrand). The NaOx solution was added to the
center ofthe air-liquid interface ofthe calcium chloride solution using a pipette. The
mixture was left without agitation for 24 hours at 4°C. The con crystals that formed
were collected by centrifuging at 6,000 rpm for 5 minutes at 4°C. con crystals are
stable in air at 4°C for 2 weeks; however, when these crystals are kept in normal saline
(Ca2C204) at 37°C, theyare stable for only 24 hours. This is due to the graduaI
conversion to the monohydrate form, which is stable in normal saline at 37°C [56]. As
CO is insoluble in alcohol [6], the crystals were washed 3 times with ddH20 and
dehydrated sequentially (30%, 50%, 70%, 100% EtOH) in EtOH. The 100% EtOH
solution was changed 3 times to ensure that the dehYdration was complete. The crystals
were kept at 4°C and rehYdrated sequentially as needed.
37
To determine the effects ofNaOH washing on the crystals, the crystals were
washed 3 times with 0.1 M NaOH and dehydrated as described above. A sample was
taken for analysis by SEM.
Hydroxyapatite (HAP) beads
Macro-Prep ceramic hydroxyapatite type II beads were purchased from BioRad
and 0.01 g was suspended in 600 ~l of50 mM Tris-HCl (pH 7.4) and washed 3 times,
allowing the beads to settle between washings for 5-10 minutes at room temperature.
Hemocytometer counting (0.100 mm deep Neubauer lmproved Bright-line, La Fontaine)
demonstrated that 100 ~l ofthis suspension contained approximately lxl06 beads. The
beads were analyzed by LM and SEM.
Poly-L-Aspartic acid (poly-Asp)
Poly-Asp has been used as a convenient and cost-efficient model for modeling the
poly-Asp stretch found in OPN [86]. Synthetic aspartic acid polymers (MW range 5,000
15,000 daltons) were purchased from Sigma and were analyzed by SDS-PAGE stained
by Stains-AlI or Ag stain on 18-20% gels.
Fluorescein isothiocyanate labeling ofpoly-Asp
Using the Fluorotag FlTC conjugation kit (Sigma), poly-Asp was coupled to
FITC in a 10:1 (label to peptide) ratio, determined to be the optimallabeling conditions,
as per the protocol included with the kit. Briefly, the peptide was dissolved in a 0.1 M
sodium carbonate-bicarbonate buffer (pH 9.0) at 5 mg/ml, and 1.0 ml ofthis solution was
38
placed into a 5 ml glass scintillation vial (Kimble) covered with aluminum foil to block
light entry, and agitated with a magnetic stir bar. Two milligrams offluorescein
isothiocyanate, Isomer 1 was reconstituted in 2 ml of0.1 M sodium carbonate-bicarbonate
buffer (pH 9.0) and vortexed until dissolved. A 10:1 dilution ofFITC was prepared in
the same buffer and 250 ml ofthis solution was added dropwise to the peptide while
stirring. The reaction was incubated at room temperature with gentle stirring for 2 hours.
The labeled peptide was isolated using Sephadex G-25 column chromatography. The
columns were equilibrated with 30 ml PBS solution before adding the reaction mixture to
the top ofthe column gel bed. The'fractions were obtained by eluting the column with 10
ml PBS, collecting 1.0 ml fractions in 1.5 ml microcentrifuge tubes. The columns were
regenerated by washing with 50 ml PBS. Ten milliliters ofPBS containing 0.05%
sodium azide was added to the columns for prolonged storage at 4°C. The fractions were
pooled and concentrated using Centricon YM-3 centrifugaI filter devices (3,000 MW
cutoff, Millipore) in a DamonlIEC Division CRU-5000 centrifuge operating at 4,200 rpm
for 45-minute cycles at 4°C until complete. A 0.1% (w/v) sodium azide solution was
added to the labeled peptide for storage at 4°C, and protected from light.
Inhibition of calcium oxalate dihydrate growth
To determine the effect ofproteins and peptides on the growth ofCOD crystals,
HSA (Sigma) and poly-Asp (Sigma) were added to the NaOx solution prior to
crystallization. The effects ofHSA were determined in the range of 1-200 Jlg/ml [17].
An 80 Jlg/ml HSA solution was prepared in ddH20 and the appropriate volume was
added to 3 ml ofa 0.005 M NaOx solution to give a final concentration of 1, 5, 10, 20,
39
SO, 100, or 20 Jlg/ml HSA. The effect ofpoly-Asp on CO crystallization was studied in
the range ofO.OS-2 JlM [86]. A S.92 mg/ml stock solution ofpoly-Asp was prepared in
ddH20 and the appropriate volume ofthis solution was added to 3 ml ofO.OOS M NaOx
to obtain O.OS, 0.1, 0.2S, O.S, l, or 2 JlM final concentration ofpoly-Asp. The solutions
were added to S ml of 1 M CaCh as described above and were allowed to sit unperturbed
for 24 hours at 4°C. The crystals were collected by centrifugation and were washed once
with ddH20 and subsequently dehydrated as previously described. The crystals were
analyzed by LM and SEM.
Competitive peptide/protein binding assays
The competitive binding ofunlabeled protein versus FITC-labeled poly-Asp to
HAP beads was deterrnined for HSA and poly-Asp. The arnount of225 Jlg labeled
peptide was considered to be the minimum arnount ofpeptide required to produce a
strong enough fluorescence signal as detected by the fluorescence microscope. The
labeled peptide was competed offwith l, 10, 100,200 and SOO tirnes unlabeled poly-Asp
[33] or HSA. One hundred microliter aliquots (lx106 beads) ofHAP beads were placed
in 1.5 ml microcentrifuge tubes and allowed to settle. The supernatant was removed and
an appropriate volume ofSO mM Tris-HCl (pH 7.4) was added. A 250 mg/ml solution of
HSA or poly-Asp was prepared in 50 mM Tris-HCl (pH 7.4). Labeled and unlabeled
proteins were rnixed in 0.6 ml microcentrifuge tubes prior to addition to the beads with a
final reaction volume ofSOO Jll. The reaction tirne was 2 hours at room temperature on a
rotator with the solution protected from light. The beads were allowed to settle and the
40
supematant containing unbound protein was removed. The beads were washed 3 times
with 500 JlI of50 mM Tris-HCI (pH 7.4) and were resuspended in the same volume.
To determine autofluorescence ofthe beads, one tube was prepared containing the beads
alone with 500 JlI of50 mM Tris-HCI (pH 7.4). The autofluorescence ofunlabeled
protein was assessed by binding IX (225 Ilg) protein with the beads. Maximum
fluorescence was determined by binding 225 Ilg ofFITC-labeled peptide to the beads
atone. The effects ofthe addition ofunlabeled protein were observed by fluorescence
microscopy and by Fluorescence Activated Cell Sorting (FACS) (Becton Dickenson
FacScan).
41
RESULTS
Results: Precipitation of calcium oxalate crystals from urine
The effects ofurine manipulation
The effects ofurine treatment on the proteins that bind to crystals are shown in
Figures 3-5. These experiments were repeated on six individually treated male control
urines and the results depicted in these figures are typical ofthe tendencies observed in
aIl the samples. The electrophoretic mobility ofuncentrifuged urine, also referred to as
whole urine, can be seen in Figure 3 (lane 1). Using the double staining method of
Stains-AlI and Ag nitrate, four banding regions can he identified at 95,67,40 and 18
kDa. Western blotting confirmed the abundance ofHSA and THP in urine, whereas OPN
and UPTFI are present in concentrations barely detectable by this method (Figure 4).
Centrifugation of the urine appears to have an effect at electrophoretic mobilities
of95 and 67 kDa, as visualized by Stains-All/Ag nitrate staining ofSnS-PAGE (lane 9).
The Western blots confirm that centrifugation removes most ofthe THP (95 kDa) as weIl
as sorne of the albumin (67 kDa) from the urine, as seen in the pellet obtained after
centrifugation of the urine (lane 9). It does not appear to have a significant effect on the
CMPs, as the bands obtained from uncentrifuged urine are identical to those from
centrifuged urine (lanes 2 and 6). The CMPs obtained from whole urine show an
electrophoretic banding pattern in three regions, between 67-43 kDa, 31 kDa and 18 kDa,
as stained by Stains-AWAg nitrate. By Western blotting, OPN and UPTFI are much
more abundant CMPs than HSA and THP, which are bare1y detectable by this method.
The solution remaining after the crystals are collected (supernatant) contains the proteins
that are not bound to the crystals, and this seems to include the majority ofthe urinary
proteins (lanes 4 and 8).
43
Washing the crystals with 0.1 M NaOH instead ofddH20 removed the crystal
bound THP and most ofthe HSA as shown by Western blotting (Figure 4, lanes 2,3,6
and 7). There was no significant effect on the crystal-bound OPN and UPTFI.
Scanning electron micrographs ofthe crystals obtained from uncentrifuged urine
washed with ddH20 and NaOH, as well as those obtained from centrifuged urine washed
with ddH20 and NaOH, are depicted in Figure 5. AU four conditions yielded both types
ofCO crysta1s: COM and COD. There was no significant difference in size or
morphology of the crystals, suggesting that there is little or no effect ofcentrifugation or
washing conditions on the crystals precipitated from urine.
The effects ofurine collection and storage temperature as well as the addition of
protease inhibitors is shown in Figure 6 as visualized by Ag staining of SDS-PAGE. No
significant differences were detected in any of the 5-day-old urine samples compared to a
freshly voided sample from the same female control subject. The storage ofurine at
room temperature versus at 4°C did not appear to have an effect on the proteins (lanes 3-6
versus lanes 7-10), nor did storage temperature - with or without added protease
inhibitors.
Osteopontin: Further characterization
Thrombin digestion
To veri:fy that the bands with electrophoretic mobilities around 67 kDa seen in
CMP preparations were indeed OPN and not HSA, H8A, a CMP sample, as well as
urinary OPN were treated with thrombin and visualized by 8D8-PAGE and doubly
stained with Stains-AU and Ag nitrate (Figure 7). After digestion with thrombin, H8A
44
(lanes 1 and 4) remained at 67 kDa, whereas in the CMP samp1e, the bands found
between 43-67 kDa were rep1aced by bands at 31 kDa (lanes 2 and 5), indicating the
c1eavage ofthe mo1ecu1e as seen in the urinary OPN samp1e. A faint band was present at
67 kDa in the thrombin-digested CMP samp1e that either wou1d suggest the presence of
a1bumin or undigested OPN.
Osteopontin associated with calcium oxalate crystals versus calcium phosphate
crystals
The pattern ofbinding ofthe different OPN isoforms to CO crystals compared to
calcium phosphate (CaP) crysta1s is shown in Figure 8. The resu1ts obtained by Western
b10tting using anti-OPN antibodies suggests that the OPN isoforms were equa11y
incorporated into the CO crystals, whereas the higher and lower mo1ecu1ar-weight forms
ofOPN were preferentia11y bound to the CaP crystals. Figure 9 shows the scanning
e1ectron micrographs ofthe different types ofcrysta1s obtained in these experiments.
Gender differences
The resu1ts obtained from 9 male and 9 female control urines are shown in
Figures 10-12. The e1ectrophoretic banding patterns ofthe urinary proteins visualized by
Ag stain are shown in Figure 10. AlI samp1es were normalized for protein content using
urine/creatinine values. A significant amount of intra-gender variability in the patterns
was observed, a1though there appeared to be no significant qualitative inter-gender
differences. Western b10tting for OPN, UPTFl, HSA and THP (Figure Il) a1so
demonstrated similar patterns for these proteins in male and female samp1es, although
45
intra-gender variability appeared to he present for H8A only. As mentioned above, THP
and H8A are the most abundant ofthese proteins in urine. Western blotting using
monoclonal anti-OPN antibodies and the N- and C-terminal antibodies to OPN on urine
concentrated lO-fold confirmed the presence ofOPN in the urine (Figure 12). Using
these antibodies, the results obtained demonstrate a large variability hetween individuals
for this protein.
Figure 13 shows the electrophoretic banding patterns ofthe CMPs precipitated
from male (a) and female (b) urine. The intra-gender variability was greater in the
patterns observed in the female samples than in the male samples. On average, there
appeared to he qualitatively more proteins bound to the crystals precipitated from female
urines than male urines. Western blotting ofthe same samples using antibodies against
OPN, UPTFl, H8A and THP showed an increased binding ofH8A and THP to the
crystals from female urine versus those from male urine (Figure 14). There also appeared
to he different forms ofH8A binding to the crystals from female urine than from male
urine (Figure 14 c).
The electrophoretic mobility patterns ofthe proteins remaining after the crystals
were collected are depicted in Figure 15. There were no significant differences hetween
the genders shown either by 8D8-PAGE stained with Ag nitrate or by Western blotting
for OPN, UPTFl, H8A and THP. The presence ofH8A and THP in the supernatant
suggests that these two proteins are not completely bound to the crystals in both males
and females (Figure 16).
8canning electron microscopy demonstrated the presence ofboth COM and COD
crystals precipitated from the urine ofmales and females (Figure 17). On average, the
46
.--crystals precipitated from female samples appeared to he significantly larger than those
obtained from male urines, although the relative size between COM and COD within
each gender remained the same. The two types ofcrystals, COM and COD, were
consistently found in both genders, although some samples contained more COD than
COM.
Precipitation ofco crystals from rat urine
Previous studies in this laboratory have worked with a rat model ofkidney stone
formation, and for comparative purposes, we thus decided to look there also. Figures 18
and 19 show experimental results obtained from male rat urine samples. The SDS-PAGE
profiles ofurines from three rats are shown in lanes 1-3. These samples, compared to
human urine, contained qualitatively more protein. Comparatively, there were also a
greater number oflower molecular-weight proteins in human urine than in rat urine. The
CMP sample and supernatant were obtained from the urine sampIe in lane 3. From these
results, few proteins were incorporated into the crystals. The Western blots using the
anti-N- and anti-C-terminal ends ofOPN showed bands in the urine samples between 43
67 kDa and barely detectable band at 43 kDa in the CMP samples. Scanning electron
microscopy ofthe crystals precipitated from the rat urine showed both COM and COD
crystals. The COD crystals commonly showed a radiating stellate structure, a
morphology not typically seen in samples precipitated from human urine. The crystals
were approximately the same size as those seen in human female samples.
47
Figure 3 SnS-PAGE OF MALE URINE SAMPLES
1 2 3 4 5 6 7 8 9
95 kDa
67 kDa
43 kDa-
31 kDa-
18 kDa-
Typical sns-pAGE of male urine samples doubly stained with StainsAli and silver nitrate.
Legend:lane 1: whole urine sample (uncentrifuged) [other lanes obtained from same sample]lane 2: proteins from crystals precipitated from uncentrifuged urine washed with ddH20lane 3: proteins from crystals precipitated from uncentrifuged urine washed with NaOHlane 4: supematant remaining after crystals precipitated from uncentrifuged urinelane 5: centrifuged urine samplelane 6: proteins from crystals precipitated from centrifuged urine washed with ddH20lane 7: proteins from crystals precipitated from centrifuged urine washed with NaOHlane 8: supematant remaining after crystals precipitated from centrifuged urinelane 9: pellet obtained from the centrifuged urine sample
48
Figure 4
67 kDa-
a) 43 kDa-
WESTERN BLOTS OF MALEURINE SAMPLES
123456789
.....b) 31 kDa- ,. -c) 67 kDa-.,/'f,
d) 95kDa- ••
Western blotting of multiple typical male urine samplesa) monoclonal anti-OPN antibody, b) monoclonal anti-UPTFI antibody, c) polyclonalanti-HSA antibody, d) polyclonal anti-THP antibody.
Legend:lane 1: whole urine sample (uncentrifuged) [other lanes obtained from same sample]lane 2: proteins from crystals precipitated from uncentrifuged urine washed with ddH20lane 3: proteins from crystals precipitated from uncentrifuged urine washed with NaOHtane 4: supematant remaining after crystals precipitated from uncentrifuged urinelane 5: centrifuged urine samplela:ne 6: proteins from crystals precipitated from centrifuged urine washed with ddH20lane 7: proteins from crystals precipitated from centrifuged urine washed with NaOHlane 8: supematant remaining after crystals precipitated from centrifuged urinelane 9: pellet obtained from the centrifuged urine sample
49
Figure 5
a)-- 5JU1l
c)-- 5JU1l
SEM OF CRYSTALS FROMMALE URINE
b)-- 5JU1l
d)
Scanning electron micrographs of crystals obtained from a typical maleurine sample.a) crystals precipitated from uncentrifuged urine washed with ddH20, b) crystalsprecipitated from uncentrifuged urine washed with 0.1 M NaOH, c) crystals precipitatedfrom centrifuged urine washed with ddH20, d) crystals precipitated from centrifugedurine washed with 0.1 M NaOH.
50
Figure 6 SnS-PAGE OF FEMALE URINESAMPLES
1 2 3 4 5 6 7 8 9 10
95 kDa
66 kDa43 kDa-
31 kDa-
..-_..._-----"",.,."'" Î
sns-pAGE of whole female urine samples stained with silver nitrate:
Legend:1ane 1: fresh void with protease inhibitor added1ane 2: fresh void without protease inhibitor added1ane 3: urine collected at room temperature, stored at 4°C with protease inhibitor added1ane 4: urine collected at room temperature, stored at 4°C without protease inhibitor1ane 5: urine collected at room temperature, stored at -8üoC with protease inhibitor added1ane 6: urine collected at room temperature, stored at -8üoC without protease inhibitor1ane 7: urine collected at 4°C, stored at 4°C with protease inhibitor added1ane 8: urine collected at 4°C, stored at 4°C without protease inhibitor added1ane 9: urine collected at 4°C, stored at -8üoC with protease inhibitor added1ane 10: urine collected at 4°C, stored at -8üoC without protease inhibitor added
51
Figure 7 SDS-PAGE OF CMP DIGESTEDWITH THROMBIN
1 2 3 4 5 6
67 kDa-
43 kDa-
31 kDa-
sns-pAGE profile of CMP digested with thrombin, doubly stained withStains-Ali and silver nitrate. Asterisks (*) indicate thrombin-digestedproducts of OPN.
Legend:lane 1: HSA (negative control) (500 Ilg)lane 2: urinary OPN (50 ng)lane 3: CMP washed with 0.1 MNaOH (100 ng)lane 4: digested HSA (500 Ilg)lane 5: digested OPN (50 ng)lane 6: digested CMP washed with 0.1 M NaOH (100 ng)
52
Figure 8 WESTERN BLOT OF CMP FROM COAND CAP CRYSTALS
1 2CO
3 4CaP
Western blotting of CMP precipitated from male urine usingmonoclonal anti-OPN antibody.
Legend:lane 1: male urine samplelane 2: CMP obtained from CO crystals precipitated from male urine (using NaOx)lane 3: male urine samplelane 4: CMP obtained from CaP crystals precipitated from male urine (using NaP)
53
Figure 9 SEM AND X-RAY MICROANALYSIS OFCO AND CAP CRYSTALS
a)
SEM
Au
EDS---
c.
c.
b)--- 20Jlm
c.
Scanning electron micrographs of crystals precipitated from male urineusing a) NaOx, producing CO crystals and b) NaP, producing CaPcrystals, and the electron dispersive spectroscopy (EDS) analysesshowing the elemental composition of the minerai types. On the EDSfigures, the x-axis depicts the x-ray energy release profile and the y-axisthe number of counts.
54
Figure 10 SnS-PAGE OFMALEANDFEMALEURINE SAMPLES
a) MALE
95 kDa
67 kDa-
43 kDa
31 kDa-
18 kDa-
b)FEMALE
31 kDa-
95 kDa- 'III 1 ... 1 Il. JI' 1
:::::~I ~ .i ....'"
sns-pAGE stained with silver nitrate of representative male and femalewhole urine samples: a) 9 male urine samples and b) 9 female urinesamples.
55
Figure Il WESTERN BLOTS OF MALE AND FEMALEURINE SAMPLES
MALE URINE
a)OPN
+ control
67 kDa-, ...
43 kDa-
b) UPTFl
+ control
31 kDa- ..
c)HSA
67 kDa- .... 1fIlI!....,.....-'.---.. " ..c~~~.. _
FEMALE URINE
+ control
+ control
•
................::-:--I!~ .......~-, ~
d)THP
97 kDa- ---- - .....---_.._-..-.
Western blotting of samples obtained from 9 male and 9 female urinesdepicted in Figure 10 using: a) anti-OPN, b) anti-UPTF1, c) anti-HSA,d) anti-THP antibodies. CMP samples were used as positive controls in the antiOPN and anti-UPTFI blots (first lane in each blot).
56
Figure 12 WESTERN BLOTS OF MALE AND FEMALECONCENTRATED URINE SAMPLES
a)OPN
MALECONCENTRATEDURINE
FEMALE CONCNETRATEDURINE
67 kDa- ......
b) LF-123
.......•... ~67 kDa-III.I_
c) LF-124
..:0
-
67 kDa-~".~:t' !'r!l!Id
1
Western blotting of 9 concentrated male and female urine samplesdepicted in Figure 10 using a) anti-OPN, b) LF-123, and c) LF-124antibodies.
57
Figure 13 SnS-PAGE oFMALEANDFEMALECMPSAMPLES
a) MALE
95 kDa
65 kDa-
31 kDa-
18 kDa-
b)FEMALE
.. .... ...-
97 kDa
67 kDa-
43 kDa-
31 kDa-
SOS-PAGE stained with silver nitrate of CMP from crystalsprecipitated from male and female urine samples depicted in Figure 10:a) crystal matrix proteins from 9 male urine samples, b) crystal matrixproteins from 9 female urine samples.
58
Figure 14 WESTERN BLOTS OF MALE AND FEMALECMPSAMPLES
MALECMP FEMALECMP
a)OPN
67 kDa
43 kDa- _1 li --b) UPTFI
31 kDa-
c)HSA
--..- -...... _.-
67 kDa- · ,. - -.," r:1;~.\.'I· '
'·1·•·····.···.···-. • é. 1•...•..
l",'".. ,..
< 1;'.:' ;
d)THP
97kDa- _ ..-".~----"-;Z ~....-
Western blotting of CMP samples precipitated from 9 male and 9female urines depicted in Figure 10 using a) anti-OPN, b) anti-UPTF1,c) anti-HSA, d) anti-THP antibodies.
59
Figure 15 SnS-PAGE OF THE SUPERNATANTSOBTAINED FROM MALE AND FEMALE
URINE SAMPLES
a) MALE
95 kDa
67 kDa-
43 kDa-
31 kDa-
b)FEMALE
65 kDa-
43 kDa-
31 kDa-
t'",;., •. ,.""",•.",.'t,
11
sns-pAGE stained with silver nitrate of the supernatant remainingaCter precipitation of crystals from male and female urine samplesdepicted in Figure 10: a) 9 supernatants from male urine samples, b) 9supernatants from female urine samples.
60
Figure 16 WESTERN BLOTS OF THE SUPERNATANTSOBTAINED FROM MALE AND FEMALE
URINE SAMPLES
MALE SN
a)OPN+control
67 kDa-.iI
h) UPTF1+control
31 kDa- ..",.,
c)HSA
+control
+control
FEMALESN
67 kDa- _ _- -d)THP
97 kDa- -Western blotting of SN samples obtained aCter collection of crystalsfrom male and female urines depicted in Figure 10 using a) anti-OPN,b) anti-UPTF1, c) anti-HSA, d) anti-THP. CMP samples were used as positivecontraIs in the anti-OPN and anti-UPTFI (first lane in each blot).
61
Figure 17 SEM OF CRYSTALS FROM MALE ANDFEMALE URINE
MALE
-- 10JUll
FEMALE
10 JUIl
Scanning electron micrographs of crystals obtained from typical maleand female urine samples. Crystals precipitated from male samples are shown inpanels to the left and female samples to the right.
62
Figure 18 SnS-PAGE AND WESTERN BLOTS OFRAT URINE SAMPLES
--.....--""*'~
95 kDa
65 kDa-"
31 kDa-
18 kDa-
1 2 3 4 5
11 2
",.,95 kDa-' ,
';i~;i:::~~t~';
67 kDa-' ~.
43 kDa-~_
a)
4 5 1 2 3 4 5
95 kDa-t"-I" 1·t-'
b)'
SnS-PAGE profile oC rat urine stained with silver nitrate and Westernblotting using a) LF-123 and b) LF-124 polyclonal antibodies.
Legend:lane 1: rat male urine (from rat #1)lane 2: rat male urine (from rat #3)lane 3: rat male urine (from rat #2)lane 4: CMP from rat #2lane 5: SN from rat #2
63
Figure 19 SEM OF CRYSTALS FROM RAT URINE
- S",m -- lO",m
-- 5J1m -- lO",m
Scanning electron micrographs of crystals precipitated from rat urine.Top two panels show star-shaped COD crystals. Middle panels show mixed CaM andCOD. Bottom two panels show typical CaM crystals.
64
Results: Immunohistochemical staining ofhuman, calcium oxalate kidney
stones for osteopontin
Figure 20 shows a typical hematoxylin and eosin stained section ofdecalcified
paraffin embedded calcium oxalate kidney stone sections obtained from two female
patients. Figure 21 shows the immunohistochemicallocalization ofOPN by LF-123
antibodies. In both figures, layers/lamellae ofconcentrically deposited organic matrix
surround apparent niduses ofcalcification. OPN most often localizes intensely to the
lamellae (Figure 21). Such a layered appearance to the stones likely reflects alternating
cycles ofcrystal growth/deposition and matrix protein adsorption at the stone surface.
65
Figure 20 LM OF H&E-8TAINED KIDNEYSTONE SECTIONS
Light micrographs of human, calcium oxalate kidney stone sectionsstained with hematoxylin and eosin.
66
Figure 21 LM OF KIDNEY STONE SECTIONSSTAINED FOR OPN
Light micrographs of human, calcium oxalate kidney stone sectionsstained for OPN (antibody LF-123).
67
Results: Calcium oxalate crystals
Growth of inorganic COD crystals
Synthetic calcium oxalate dihydrate crystals grown in the absence oforganic
material produced crystals ranging in size from 1-15 J.lm. The shape of the crystals was
variable, with the majority ofthe crystals being ofthe typical bipyramidal morphology,
with the remaining crystals having rounded edges or star-like growth patterns. Figure 22
depicts the crystals as seen by light microscopy. Scanning electron microscopy ofthe
crystals is shown in Figure 23. The effect of0.1 M NaOH washing ofthe crystals is
depicted in Figure 23b, and these results suggest that this washing condition has no
significant effect on the crystals.
Crystal growth assay in the presence ofpeptide!protein
Commercially available poly-L-Asp was used to model the poly-Asp domain
found in OPN. In order to verify the molecular weight, 2 different acrylamide
concentrations and 2 staining methods were used. Figure 24 shows the peptide with a
maximum MW ofapproximately 15,000 daltons, as visualized by Ag nitrate (20% gel)
and Stains-AlI staining (18% gel) after SDS-PAGE.
Crystals were grown in the presence of0.05-2 J.lM PA to determine the effects of
the peptide on crystal growth. Results obtained are shown in Figures 25 and 26.
Compared to the crystals grown without added peptide (a), 0.05 J.lM PA had little or no
efi'ect, yielding crystals with approximately the same size or sma11er than the control
crystals (b). At a concentration of0.1 J.lM PA (c), the crystals obtained were still
approximately the same size as the control, however a more elongated variant of the
COD crystals was also present, a feature not present in the control samples. This
68
particular crystal shape was the oruy type formed at 0.25 JlM PA (d), the majority of
them being smaller than 5 Jlm. At 0.5 JlM PA (e), the crystals formed were elongated,
with the width being smaller towards the center than at the ends ofthe crystals, thus
distinguishing them from more typical, plate-like CaM crystals. The crystals produced
in the presence of 1 JlM PA (f) and 2 JlM [(g) and (h)] were thin and e1ongated. At 1 JlM
and 2 JlM PA concentrations, by visual inspection ofthe pellet obtained after
centrifugation ofthe crystals during collection, there was a marked reduction in the
quantity ofcrystals produced in the presence ofthis inhibitor. These results suggest a
significant effect ofPA on CO crystal growth at concentrations greater than 0.1 JlM.
The crystals obtained in the growth assays in the presence of 1-200 Jlg/ml HSA
are presented here by light (Figure 27) and scanning e1ectron (Figure 28) microscopy.
Panel (a) depicts crystals grown without any added protein. At 1 and 5 Jlg/ml HSA
[panels (b) and (c)], the crystals produced were as large, or even larger, than the control
crystals (>5 Jlm). At 10 Jlg/ml HSA (d), the average size ofthe crystals obtained
decreased, and this size change remained steady over 20 and 50 Jlg/ml HSA [panels (e)
and (f)]. At a concentration of 100 Jlg/ml (g), the number ofsmall crystals «2.5 Jlm)
increased with a fraction ofthe crystals remaining at their original size. The highest
protein concentration ofHSA tested was 200 Jlg/ml (h), and aIl of the crystals produced
at this concentration were smaller than 2.5 Jlm. These results demonstrate that in the
presence ofHSA, COD crystal growth is affected at concentrations as low as 10 Jlg/ml.
At higher concentrations (>100 Jlg/ml) ofHSA, growth of the crystals appears to be
secondary to nucleation ofnew crystals, yie1ding very numerous smaller crystals, aIl of
the COD morphology. There was no apparent difference in the overall amount of
69
crystals formed in the presence ofHSA by visual assessment ofthe pellets obtained after
centrifugation for the collection ofthe crystals, however, no attempt was made to
quantify the number ofcrystals produced.
70
Figure 22 LM OF SYNTHETIC COD CRYSTALS
- lOJ.lm
o
c o 0
Light micrographs of inorganic COD crystals synthesized in solution bythe addition of CaCh and NaOx (100: 1 Ca: Ox).
71
Figure 23 SEM OF SYNTHETIC COD CRYSTALS
a)
b)
- 21Jl1l
Scanning electron micrograph of synthetic COD crystals: a) washedwith ddH20 and b) washed with 0.1 M NaOH.
72
Figure 24 SnS-PAGE OFPOLy-ASp
•14 kDa-1
a)
1 2 3
b)
1 2 3
SnS-PAGE profile ofPoly-L-Aspartic acid stained with a) silver nitrate(20% gel) and with b) Stains-Ali (180/0 gel).
Legend:lane 1: 20 Ilg Poly-L-Aspartic acidlane 2: 50 Ilg Poly-L-Aspartic acidlane 3: 100 Ilg Poly-L-Aspartic acid
73
Figure 25 LM OF COD GROWN WITH POLy-Asp
tÔDtrol
(
a) b)
0.05 JlM
0.1 JlM 0.25 JlM
c) d)
0.5 JlM 1 JlM
e) t)
g) h)
Light micrographs of COD crystals grown in the presence of Poly-LAspartic acid (PA).a) no PA added (control), b) 0.05 /lM PA, c) 0.1 /lM PA, d) 0.25 /lM PA, e) 0.5 /lM PA,f) 1 /lM PA, g), h) 2 /lM PA.
74
Figure 26 SEM OF COD GROWNWITHPOLy-Asp
a) b)
c) . .
e) . ..
d) . "
f)
g) h)
Scanning electron micrographs of COD crystals grown in thepresence of Poly-L-Aspartic acid (PA).a) no PA added (control), b) 0.05 /lM PA, c) 0.1 /lM PA, d) 0.25 /lM PA, e) 0.5/lM PA, t) 1 /lM PA, g), h) 2 /lM PA.
75
Figure 27 LM OF COD GROWN WITH H8A
Ûntrol
a) b)
()J
IJ.lWml
5 J.1Wml 10 J.1~/ml
c) d)
20 J.lWml 50 J.lWml
e) Q
10Q.J1Wlb.l 200 J.l~/ml
g) h)
Light micrographs of COD crystals grown in the presence of albumin.a) no HSA added (control), b) 1 Ilg/ml HSA, c) 5 Ilg/ml HSA, d) 10 Ilg/ml HSA, e) 20Ilg/ml HSA, f) 50 Ilg/ml HSA, g) 100 ug/ml HSA h) 200 Ilg/ml HSA.
76
Figure 28 SEM OF COD GROWN WITH HSA
a) b)
c) d) - ..
e)
g)
t)
h)
Scanning electron micrographs of COD crystals grown in the presenceofalhumin.a) no HSA added (control), b) 1 ~glm1 HSA, c) 5 ~glml HSA, d) 10 ~glm1 HSA, e) 20~glm1 HSA, f) 50 ~glml HSA, g) 100 ~glml HSA h) 200 ~glml HSA.
77
Results: Hydroxyapatite (HAP) beads
Given the variability in size, number and shape ofthe synthetic CO crystals, and
also because we were interested in exploring protein interactions with the CaP phase of
kidney stones and other mineralized tissues, commercially available HAP beads were
used in competitive binding assays involving FITC-Iabeled PA and protein. Although
there was sorne variability in the size ofthe beads, the particulate population was
significantly more homogeneous than the COD crystals. The beads were larger than
COD, averaging 20 /lm in diameter. Light micrographs ofthe beads are shown in Figure
29, and scanning e1ectron micrographs ofthe same beads are depicted in Figure 30. The
surface ofthe beads, as seen in Figure 30a, was porous, and the porosity extended weIl
into the interior as observed after crushing ofthe beads in a microcentrifuge tube using a
pipette tip (Figure 30b).
Competitive peptidelprotein-binding assays
In order to study the binding characteristics ofFITC-labeled PA to HAP beads,
competitive binding studies were performed in the presence of IX, 10X, 100X, 200X and
500X unlabe1ed PA and HSA. The results for each protein were analyzed by
fluorescence microscopy and by FACS. Fluorescence micrographs (Figure 31) are
shown in the cases where the fluorescence was detectable by our imaging system.
Competition assays between labeled and unlabeled PA demonstrated that aIl of the
labeled peptide could he displaced by excess (200X) unlabeled PA (Figure 32g). This
indicates that the extra FITC group bound to the labeled PA does not have a significant
effect on its binding affinity to HAP. In these experiments, compared to the maximal
78
fluorescence as seen in the control samples (c), there was a graduaI decrease in the
fluorescence in the presence of increasing amounts ofexcess unlabeled PA.
The results obtained in the competition assays with HSA are shown in Figures 33
and 34. Differences in fluorescent labeling ofthe beads with FITC-Iabeled PA suggest
that HSA binds to HAP with less affinity than PA. The maximum excess HSA tested
(SOOX) inhibited the binding oflabeled PA to approximately the same degree as IX of
unlabeled PA, representing a SOO-fold difference in binding affinity ofHSA compared to
PA. There was a graduaI decrease in fluorescence accompanied by an increasing amount
ofexcess HSA, indicating a slight inhibition in the binding oflabeled PA to the HAP
beads by HSA. From the results obtained here, it is unlikely that any amount ofexcess
HSA would completely inhibit the binding ofPA to HAP.
79
Figure 29
-- 20/.lm
LM OF HAP BEADS
o
Light micrographs of BioRad hydroxyapatite ceramide type II beads.
80
Figure 30 SEM OF HAP BEADS
a)- lOpm
b)_ 2JU1l
-- 5JU1l
Scanning electron micrographs of BioRad hydroxyapatite beads: a)whole beads, b) crushed beads demonstrating the porosity of the surfaceof the beads.
81
Figure 31
a)
FLM OF HAP BEADSAND POLy-ASp
b)
1)
o 0
("'j
o control
2)
IX PA
Fluorescence light micrographs of BioRad HAP beads: competitionassay using FITC-Iabeled Poly-L-Asp and unlabeled Poly-L-Asp.Panell: FITC-Iabeled PA binding to beads alone, panel 2: IX competition withunlabeled peptide. a) fluorescence, b) bright field.
82
Figure 32 FACS ANALYSISOF HAPBEADS AND POLy-ASp
OC!N......
b) a) e) d) c)
~(I.J
ëQ;l>w
~,10 4
FL1-H
Histogram of FACS analysis: Fluorescence obtained after thecompetitive binding of FITC-labeled and unlabeled Poly-L-Asparticacid to BioRad HAP ceramide beads. The y-axis depicts the number ofevents that have corresponding relative fluorescence energy on the xaxis.
Legend:a) autof1uorescence (HAP beads alone)b) IX Poly-L-Aspc) FITC-Iabe1ed Poly-L-Aspd) IX Poly-L-Asp and FITC labe1ed-Poly-L-Aspe) IOX Poly-L-Asp and FITC labeled-Poly-L-Aspf) IOOX Poly-L-Asp and FITC labeled-Poly-L-Aspg) 200X Poly-L-Asp and FITC labeled-Poly-L-Asph) 500X Poly-L-Asp and FITC labeled Poly-L-Asp
83
Figure 33
1)
FLM OF HAP BEADS AND HSAa) b)
control
2)
3)
4)
lXHSA
lOXHSA
lOOXHSA
Fluorescence light micrographs of BioRad HAP beads: competitionassay using USA and FITC-labeled Poly-L-Asp.Panell: FITC-Iabe1ed PA binding to beads alone, panel 2: FITC-Iabeled PA plus IXHSA competition, panel 3: FITC-Iabeled PA plus IOX HSA, panel 4: FITC-Iabeled PAplus IOOX competition with HSA. a) fluorescence, b) bright field
84
Figure 34
coN.....
FACS ANALYSIS OF HAPBEADS AND HSA
a)
FL1-H
Histogram of FACS analysis: Fluorescence obtained aCter thecompetitive binding of FITC-Iabeled Poly-L-Aspartic acid and albuminto BioRad HAP ceramide beads. The y-axis depicts the number ofevents that have corresponding relative fluorescence energy on the xaxis.
Legend:a) autofluorescence (HAP ceramide beads)b) IXHSAc) FITC labeled Poly-L-Aspd) IX HSA and FITC labeled-Poly-L-Aspe) IOX HSA and FITC labeled-Poly-L-Aspf) lOOX HSA and FITC labeled-Poly-L-Aspg) 200X HSA and FITC labeled-Poly-L-Asph) 500X HSA and FITC labeled-Poly-L-Asp
10 4
DISCUSSION
DISCUSSION
The precipitation of calcium oxalate crystals (CO) from human urine
The study ofthe numerous factors known to contribute to idiopathic kidney stone
disease remains a perplexing one. Although great progress has been made in recent years
to identify possible pathogenic components which could play a role in urolithiasis, the
multifactorial nature ofthis disease suggests that a combination of factors are likely
responsible. Physiological, internaI factors such as urine chemistry and type and
abundance of urinary proteins, and in particular the proteins osteopontin, urinary
prothrombin fragment 1, albumin and Tamm-HorsfaIl protein, have aIl received much
attention in the field as candidates for regulating urolithiasis. Other factors that likely
contribute to kidney stone disease are fluid intake and diet, which are often required to be
modified in stone-formers after diagnosis ofthe disease in order to alter the urinary
concentrations ofcertain molecules known to affect crystallization [22].
The presence and concentrations of ions are major determinants of
nephrolithiasis. As human urine is supersaturated with respect to calcium and oxalate,
these ions combine to produce a near insoluble salt in the form ofcrystals that are
destined, in the non-stone former, to he excreted [63]. There are approximately 7,200
crystals present per milliliter ofurine in the non-stone former, which adds up to a daily
excretion of lxl07 crystals [52]. Not only do these ions contribute to the actual formation
ofcrystals, but it has been shown that oxalate Can cause proliferation of, and or injury to,
renal epithelial ceIls [48, 52]. This would likely result in an increase in crystal retention
87
and aggregation, as exposed regions ofdamaged cell membranes would serve as sites for
crystal attachment [52].
Factors present in urine that serve as inhibitors ofthese crystallization processes
likely regulate crystal growth during the process ofstone formation. Macromolecules
that have been identified as inhibitors ofCO crystallization include OPN, UPTF1, HSA,
THP, nephrocaIcin, uronic acid-rich protein, GAGs and lithostathine [88], and we
speculate that these molecules are either absent or aberrant in the urine of stone formers.
The physiological response to mineraIs in the body is to coat with protein, which
usually is believed to act in an inhibitory manner [61]. Most biological fluid systems are
complex and involve multiple organic components, which act not only by themselves as
monomer molecules, but often have the propensity to form large macromolecular
assemblies that may have similar or different functions.
Macromolecules can act to inhibit crystallization in numerous ways. Initially,
these proteins could prevent stone development by binding ions and by forming small
mineraI nuclei. Although the promotion ofnucleation as a means ofdecreasing stone
formation seems counterintuitive, the binding ofcalcium and oxalate by proteins would
lower their relative urinary supersaturation, thus decreasing their availability for the
growth ofpreformed larger nuclei [17]. In the latter case, the deposition ofnew mineraI
on pre-existing nuclei would increase their size, thus increasing the potential to remain in
the renal tubule. In the non-pathogenic scenario, the newly formed nuclei would be
washed away in the tubular fluid for eventuaI excretion in the urine. In the case of stone
formers, however, these nuclei remain in the kidney, serving as potential sites for new
mineraI deposition and/or aggregation ofother crystals. Inhibitory proteins could
88
potentially act at this stage by coating the crystal surfaces, thus decreasing the sites
available for aggregation or cell attachment. If the cell surface anions responsible for
mineraI binding, such as sialic acid, GAG-containing proteins and groups ofanionic
amino acids (such as glutamic and aspartic acid) succeed in anchoring the crystal to the
renal epithelium, two outcomes are possible: 1) the crystals remain on the cell surface
where they can potentially initiate stone formation, or 2) they are internalized via
membrane-lined vacuoles ofthe phagocytic/endosomal system to be dissolved within the
cell or carried into the interstitium [57]. The precise factors regulating these processes
are currently unknown, however, it is clear that proteins play a definite role in the
mechanisms leading to, or preventing, kidney stone formation.
The characterization ofproteins regulating crystallization may be key to
understanding the pathogenic mechanisms leading to stone formation. In this study, we
confirmed the association ofOPN, UPTF1, HSA and THP with CO crystals precipitated
from the urine ofnon-stone formers. Particular interest was centered on identifYing
difIerences between male and female non-stone formers, in the hopes ofdiscerning the
potential factor(s) leading to the higher incidence ofkidney stones in males. Although
several other reports have compared genders [3, 7, 14], this is the first study to do so in a
multiple, yet individual, manner, identifYing OPN, UPTF1, HSA and THP in the urine,
and crystal matrix, as well as looking at the morphology ofthe crystals obtained from the
same subjects. The role ofOPN and HSA in the mechanisms involved in crystallization
was further elucidated, confirming their importance in the modulation ofcrystal growth.
This study also provided insight into the mineral-binding behavior ofthese two proteins.
Prior to proceeding with the study, however, it was essential to establish the particular
89
effects that urine handIing/processing has on urinary proteins. A priori, it was decided
that the particular urine processing conditions that had the least affect on the proteins
thought to be involved in urolithiasis would therefore he used throughout the remainder
of this study.
The effects ofurine manipulation
In the past decade, there has been much controversy over the effects ofurine
manipulation on urinary proteins [5, 47, 47, 60]. Factors such as the centrifugation of
urine as weIl as the washing conditions of the crystals precipitated from urine yielded
inconsistent results in the literature; therefore, we sought to determine the effects ofthese
manipulations in a controlled and reproducible manner. This is the first systematic
comparison ofthe different urine preparation conditions on multiple samples from both
genders. The urine samples from six male non-stone formers were halved and then
centrifuged, or left as whole samples. Four banding regions were identified in the urine
at 95, 67, 40 and 18 kDa in aIl the samples processed, and only the 18 kDa region was
unaccounted for with the antibodies used in this study. Due to the similar electrophoretic
mobilities, we speculate that this band likely represents nephrocalcin [18,88], however,
further studies would he required in order to verify this statement. The remaining protein
bands are similar to those found previously in human urine [3, 5, 26, 60, 64], confirming
the abundance ofHSA and THP, and the much lower levels ofOPN and UPTFI in the
urine.
The effects ofurine centrifugation are to remove most of the THP as weIl as
partially removing HSA from the urine. The removal ofthese proteins by centrifugation
90
is most likely due to the potential formation ofaggregated forms, which would tend to
make them settle due to their higher molecular weight. Centrifugation does not have a
significant effect on crystal-bound proteins, as the banding pattern ofCMP obtained from
uncentrifuged urine is identical to those from centrifuged urine. The CMP samples
obtained from whole urine OOd an electrophoretic banding pattern in three regions,
between 67-43 kDa, 31 kDa and 18 kDa, similar to those found previously for CMPs
using similar methods and obtained from human urine [3, 5, 26, 60, 64]. By Western
blotting, OPN and UPTFI were found to be much more abundant than HSA and THP,
although these proteins were nevertheless found to be part ofthe matrix. Unlike previous
studies [3, 26, 79] showing a greater binding ofUPTFl than OPN to the crystals
precipitated from urine, the results obtained in this study did not confirm those findings;
we detected OPN at levels similar to UPTFI in aIl our samples. Although the levels of
HSA and THP detected in the matrix ofcrystals were slightly variable, these two proteins
are consistent components ofthe crystal matrix in the majority ofthe samples used in this
study. This suggests that the lack ofthese proteins in the matrices ofcrystals in other
studies [3, 26, 64, 65] is perOOps attributable to differences in the levels ofthese proteins
on an individual basis, or perhaps is attributable to the method ofurine processing used in
those studies.
Washing ofCO crystals with NaOH instead ofddH20 removed the crystal-bound
THP and most of the HSA; however, no significant effect on the crystal-bound OPN and
UPTFI was observed. It is has been noted tOOt centrifugation and filtration ofthe urine
prior to processing causes an almost complete loss ofTamm-Horsfall protein and sorne
reduction in the concentration ofalbumin and other urinary proteins. As these are the
91
two most abundant proteins found in urine and they are removed by NaOH washing, it
has been speculated that these proteins are non-selectively bound to the crystals [64].
In our work, both types ofCO crystals, COM and COD, were seen in all four
conditions ofcrystal preparation. There was no difference in size or morphology of the
crystals obtained in our samples, suggesting that there is little or no effect of
centrifugation or washing conditions on the crystals precipitated from urine.
To verny the effects ofurine storage conditions on urinary proteins, the effect of
the temperature at which urine samples are collected and stored as well as the effects of
protease inhibitors were compared to freshly voided samples. No significant differences
were detected in any ofthe five-day-old urine samples compared to a freshly voided
sample from the same female control subject. The storage ofurine at room temperature
versus at 4°C did not appear to have an effect on the proteins, nor did storage temperature
with or without added protease inhibitors. This indicated that the urinary proteins remain
unaffected by temperature within the time frame and by the detection methods used in
this study, although further confirmation by Western blotting with the antibodies used in
this study would be required.
Osteopontin: Further characterization
Thrombin digestion
Osteopontin, a protein accumulating in significant amounts with the precipitation
ofcalcium oxalate in human urine, is susceptible to proteolytic cleavage by thrombin - a
physiological processing step shown to be ofphysiological relevance in terms ofcell
adhesion [30], but ofunknown significance in terms ofits effects on crystallization.
92
Importantly, the cleaved forms ofOPN can escape detection by antibodies possibly due
to epitopes at the cleavage site or an alteration in conformation ofthe OPN molecule
itselfby proteolytic cleavage; the precise function ofthese processed/cleaved forms has
yet to be determined [51]. To verify that the bands with electrophoretic mobilities around
67 kDa seen in CMP preparations were indeed OPN and not HSA, HSA, a CMP sample,
as weIl as urinary OPN were aIl treated with thrombin. After digestion with thrombin,
HSA remained at 67 kDa whereas in the CMP sample, the bands found between 43-67
kDa are replaced by bands at 31 kDa indicating the cleavage of the molecule as seen in
the urinary OPN sample. A faint band present at 67 kDa in the thrombin-digested CMP
sample either would suggest the presence ofalbumin or undigested OPN. This indicates
that the majority ofthe bands found in the CMP preparation represent the intact form of
OPN, thus confirming its abundance in the matrix, while the remaining bands belong to
HSA, thus confirming the presence ofHSA in CO crystals.
Osteopontin in calcium oxalate crystals versus calcium phosphate crystals
The results obtained by Western blotting using anti-OPN antibodies suggest that
the OPN isoforms are equally bound/incorporated into the CO crystals, whereas CaP
crystals preferentially bind the higher and lower molecular-weight forms ofOPN. The
phosphorylation ofthis protein has been previously shown to have an effect on its
inhibitory activity for crystal growth in both CO and CaP [88], however, the specifie
effects ofpost-translational modifications on mineral binding are not yet known. Since
most ofthe urinary isoforms ofOPN are found in the so-called crystal matrix, with all of
them binding to the mineral phase, we believe that at least a portion of the binding sites
93
involved in crystal-protein interactions are likely related to the primary amino acid
sequence of the molecule.
Gender differences
In this study, a significant amount of intra-gender variability in the patterns was
observed, though there were no significant qualitative inter-gender differences with
respect to urine. Similar patterns for OPN, UPTFI and THP were noted in the male and
female samples, though intra-gender variability appeared to he present for HSA orny.
The presence ofOPN in the urine was confirmed in samples concentrated ten-fold,
demonstrating a large variability between individuals for this protein as detected by the
antibodies used.
Overall, the intra-gender variability was greater in the patterns observed in the
female samples than in the male samples. There are qualitatively more proteins bound to
the CMP precipitated from female urines than male urines with an increased crystal
binding ofHSA and THP in females, once again, confirming their existence in the matrix
ofcrystals precipitated from human urine. The presence ofthese proteins in the
supernatant, however, demonstrates that HSA and THP are not completely bound to the
crystals, suggesting the non-selective binding ofthese proteins to CO crystals.
Numerous different forms ofHSA were identified in the crystals obtained from
female urine that were absent from those obtained from male urine. This may be one of
the key differences hetween the genders. We suspect tOOt albumin modulates the
preferential formation ofCOD over COM crystals, which would lead to the decreased
formation ofaggregates hecause of the greater repulsive charges between COD crystals
94
[14]. Further studies are required, however, to determine the specific role that albumin
plays in the greater inhibitory capacity of female urine on stone formation. Although the
formation ofCOD over COM crystals would he beneficial, this would be counteracted by
the fact that larger crystals were identified in samples obtained from female urine. The
tendency for females to form larger crystals has heen documented [14], however, we
suspect that this is due to factors other than HSA, as supported by previous [14] as weIl
as CUITent evidence (in this study) that HSA acts to promote nucleation leading to
crystallization of smaller particles.
Precipitation ofco crystals from rat urine
In looking at another mammalian species for comparative purposes, the samples
precipitated from rat urine, compared to human urine, appeared to contain qualitatively
more protein, and in particular, low molecular-weight proteins. From the results obtained
in this study, there did not appear to he many proteins incorporated into the crystals
precipitated from rat urine. Using the antibodies against the anti-N-, and anti-C-terminal
ends ofOPN, bands were detected in the rat urine samples hetween 43-67 kDa and only a
very faint band, ifany at aIl, at 43 kDa in the CMP preparation. Crystals precipitated
from rat urine were in the form ofhoth COM and COD crystals, with the latter being
rather "steIlate" in form. This crystal morphology was not typically seen in samples
precipitated from human urine, though the crystals were approximately the same overall
size as those seen in human female samples. The difference in crystal morphology
observed is most likely due to the difference in the CMP bound to human crystals versus
those in rats, thus influencing crystal structure.
95
Immunohistochemical staining of calcium oxalate kidney stones for osteopontin
These results, obtained by hematoxylin and eosin staining of the calcium. oxalate
stones, demonstrated the layered arrangement ofmatrix in calcium. oxalate stones. The
concentric lamellar immunohistochemical staining pattern ofOPN confirms that the
protein is most like1y deposited in layers with the mineraI phase ofthe stones. Both the
concentric lamellae ofmatrix and the interlamellar substances (radial striations) label
intensely for OPN, and these findings are consistent with previous studies on kidney
stones [44, 61].
The use of synthetic calcium oxalate crystals for peptide/protein-binding analysis
Growth ofinorganic COD crysta/s
Calcium. oxalate dihydrate crystals grown in the absence oforganic material
yielded crystals ranging in size from 1-15 Jlm, as typically observed in previous studies
[86]. The shape ofthe crystals was variable, with the majority ofthe crystals being ofthe
typical bipyramidal morphology characteristic ofCOD, and the remaining crystals having
rounded edges or anvil-shaped morphologies characteristic of COM. Although it had
previously been shown that crystal-washing conditions have an effect on the crystal
surface by the removal ofprotein as small etched pits and cavities at the crystal surfaces
[58], SEM analysis ofcrystals obtained from urine washed with NaOH in this study,
however, did not show any evidence ofsuch protein removal. Surfaces of the crystals
obtained were smooth, even after the same treatment with 0.1 M NaOH used by Ryall et
96
al. [58]. To determine whether this washing condition directly affected the mineraI phase
ofthe crystal surface, synthetic CO crystals grown in solution devoid ofproteins were
washed using the same concentration ofNaOH. We were unable to reproduce the
etching and pitting observed on the surface ofthe crystals grown in the presence or
absence ofprotein, however, as there was no effect observed on the surface ofthe
crystals grown inorganically, this suggests that this particular washing condition does not
directly affect the mineraI phase ofthe crystal.
Crystal growth assay in the presence ofprotein
As other studies generally utilized systems that yielded COM crystals, we opted
to use one that would preferentially yield COD crystals [1, 14], giving crystals ofwell
defined geometries and clearly identifiable crystallographic faces for future imaging
studies on the interactions ofproteins with crystal surfaces. To begin these studies,
crystals were grown in the presence ofthe peptide PA, a well-characterlzed modulator of
crystal growth that represents an amino acid domain common to many acidic mineralized
tissue proteins, to determine the effects ofthis peptide on crystal growth in our system.
In comparison to the crystals grown without added peptide, low concentrations ofPA had
little or no effect on crystal growth, yielding crystals with approximately the same size or
smaller than the control crystals. Differences were observed starting at a concentration of
0.1 JlM PA and greater, where a more elongated version ofthe COD crystal were seen,
unlike in the control samples. This particular crystal shape was the only type formed at
0.25 JlM PA, with the majority ofthem being smaller than 5 Jlm. At increasing
concentrations ofPA, the crystals formed were more elongated, the width being smaller
97
towards the center than at the lengths ofthe crystals, distinguishing them from the typical
plate-like COM crystals. As these crystals were grown in a controlled, chemically
defined in vitro system, it is appropriate to assume that these are CO crystals. Although
this particular shape ofcrystal has not yet been reported in the literature, a similar, yet
less elongated dumbbell morphology has been referred to by McKee et al. [61], to
describe crystal ghosts representing the organic component associated with the mineraI
phase ofsmall crystalline particles as weIl as larger kidney stones. Such crystal forms
imply inhibition ofcrystal growth in the central regions ofthe elongated forms,
presumably from the direct inhibitory binding ofPA at these sites. The particular
significance ofthis crystal morphology is unknown, however it can be speculated that
such rounded edges would he beneficial in decreasing intratubular crystal-attachment by
reducing renal epithelial cell damage potentially caused by the rough edges ofa typical
bipyramidal-shaped COD crystal. Crystal-crystal associations, however, would most
likely remain the same, as the rounded ends ofthe crystals would have the same
propensity to interlock molecularly with the central regions ofan adjacent crystal of the
same morphology, thus joining and stacking them in the same manner as in the original
crystal forms.
By visual inspection, the size ofthe crystal-containing pellet obtained after
centrifuging the crystals during collection was markedly reduced by the addition ofthe
higher concentrations ofPA, suggesting that it acts not only on crystal growth but also to
inhibit the in vitro nucleation ofCO crystals.
To test the effects ofa urinary protein on crystal growth in our system, HSA was
used, and at relatively low concentrations ofHSA, the crystals produced were as large or
98
even larger than the control crystals (>5 fJm). This would he expected, as HSA was
previously shown to he a promoter of the nucleation ofCO crystals [17]; H8A binding to
the mineraI would increase the relative size ofthe crystal compared to the control crystaIs
grown without added protein. At 10 fJg/ml ofH8A, the average size of the crystaIs
obtained decreased, and this size change remained steady over 20 and 50 fJg/ml ofadded
HSA. At a concentration of 100 fJg/ml, the number ofsmall crystals (<2.5fJm) increased
with a fraction ofthe crystals remaining at their original size. The highest concentration
ofHSA tested was 200 fJg/ml, and aIl ofthe crystals produced were smaller than 2.5 fJm.
These results demonstrate that in the presence ofHSA, COD crystal growth is affected at
concentrations as low as 10 fJg/ml. At higher concentrations (>100 fJg/ml) ofHSA,
growth ofthe crystals appears to he secondary to nucleation ofnew crystals, yielding
very numerous smaller crystals, all of COD morphology. There was no apparent
difference in the overall amount ofcrystaIs formed in the presence ofHSA by visual
assessment ofthe pellets obtained after centrifugation for the collection of the crystals.
This is expected, as increasing concentrations ofadded HSA yielded smaller yet more
numerous crystals, whereas lower concentrations ofadded HSA yielded larger less
numerous crystals, thus producing the same overall particulate volume. The precise
quantification ofthe numher and size ofcrystals produced was not possible due to
constraints ofthe instruments available to us.
The results obtained in this study on the effects ofH8A on crystal structure are
consistent with previous findings [17]. The increased formation ofCOD crystals over
COM crystals in the presence ofH8A is attributed not to the chemical transformation of
COM crystals into COD, but to the inhibition ofCOM crystallization leading to a
99
relatively higher frequency ofCOD crystals [17]. As previously mentioned, the
formation ofsmaller crystals in the presence ofH8A is significant in decreasing the rate
ofstone formation, as smaller crystals would be less prone to occluding the tubular
lumen. Even ifpolymerized forms ofH8A have been shown to promote aggregation
[17], this would be counterbalanced by the formation ofsmaller crystals.
The paradoxical nature ofthe action ofthis protein, as well as others, on
crystallization underlines the notion that not one, but many factors acting in concert, are
responsible for determining whether a single crystal will develop into a stone. Protein
mineraI interactions have been previously studied [11, 17,42,53,62,86], demonstrating
several possible mechanisms for the regulation ofcrystallization by proteins. Many of
these protein-mineral interactions are higWy complex, involving specific groups on the
proteins, such as the carboxylate groups on OPN [42], as well as the secondary structure
ofthe protein, such as the repetitive homologous helical domains ofH8A [17]. A protein
with the required repetitive structure could act as a nucleator by providing a template for
the appropriate positioning ofions to form the :tirst crystallattice. Other proteins could
then act on crystallization by favoring new mineral deposition on one crystal plane over
another, or by blocking the sites ofnew mineraI deposition altogether. This particular
inhibition could result in a decreased rate ofgrowth on crystal surfaces that would
normally grow rapidly, conversely promoting growth on surfaces that would not usually
grow - thus modulating crystal structure. Aggregation and retention ofcrystals are
potentially affected in the same manner; the sites for crystal-cell or crystal-crystal
associations would be exposed or blocked depending on the nature ofthe
macromolecules present in the surrounding fluid.
100
The use ofBioRad hydroxyapatite (HAP) ceramide beads for peptide/protein
binding analysis
As calcium phosphate is predicted to play an important role as a nuc1eator of
calcium oxalate, and it is conveniently conunercially available in well-characterized
forms, it was used to simulate the CaP mineraI component in protein-binding assays. The
homogeneity of the bead population was also an advantage compared to the synthetic
COD crystals, as the size and shape ofthe latter crystals produced in this study were
highly variable. The HAP beads are larger than the COD crystals that we prepared,
averaging 20 Ilm in diameter, and demonstrated significant porosity. Thus while having
a smaller surface area per unit weight, the high level ofporosity permitted significant
additional binding ofour reagents used in the protein-binding studies - a feature
advantageous for fluorescent imaging ofthe beads and for quantitative studies (see
below).
Competitive protein-binding assays
In order to study the binding characteristics ofFITC-labeled Poly-L-Aspartic acid
(PA) on HAP beads, competitive binding studies were performed in the presence of IX,
10X, 100X, 200X and 500X unlabeled PA and albumin (HSA). The results for each
protein were analyzed by fluorescence light microscopy and by FACS. Competition
assays between labeled and unlabeled PA demonstrated that virtually all ofthe labeled
peptide could be displaced by excess (200X) unlabeled PA. This verified that the
addition ofan FITC group bound to the PA does not have a significant effect on its
101
binding affinity to HAP, as previously shown [33]. There was a graduaI decrease in the
fluorescence compared to the maximal fluorescence as seen in the control samples by the
different amounts ofexcess PA added, again confirming the findings ofprevious studies
ofPA binding to HAP [33], and showing that fluorescently labeled PA can be used to
model the interactions ofthis primary amino acid sequence (as is found in OPN) with
mineraI.
In terros ofthe specificity ofbinding, the results ofthis study show tOOt the
binding ofPA to HAP is highly specific, since very high concentrations (SOO-foId) of
HSA were required to inhibit FITC-Iabeled PA binding to the same degree as I-fold
unlabeled PA. There was a graduaI decrease in fluorescence ofthe beads with increasing
amounts ofHSA, indicating a slight inhibition in the binding oflabeled PA to the HAP
beads, likely representing nonspecific interactions ofFITC-labeled PA with the beads. It
is unlikely that anyamount ofexcess HSA would compIete1y inhibit the binding ofPA to
HAP indicating tOOt aithough HSA has been shown to have sorne eifects on
crystallization, its ability to bind to CaP is much less than that ofPA. The results from
this and other studies point to HSA OOving roles in urine more important than binding to
minerai surfaces.
Future studies
It wouid be informative to repeat the first part ofthis study using samples from
stone patients in order to assess the differences ifany, between urine samples obtained
from stone-formers and the results observed in this study for the urine ofnon-stone
formers. Further studies characterizing the urinary albumin from males and females may
102
also elucidate ifthe differences between the genders is in part due to albumin. With
respect to protein-crystal interactions, detailed studies ofCaP crystals should result in a
better understanding ofthe mineral-binding properties ofOPN, as well as UPTFl, HSA
and THP.
Although our data is consistent with the PA region ofOPN playing an important
role in binding and modulating calcium-based minerais in urine, additional studies are
required using intact, whole OPN in this and other crystal growth mode! systems. It
would also likely be informative to perform these experiments using UPTFI and THP.
Using a different system ofinorganic COD production might yield a crystal population
stable and homogeneous enough to repeat the protein-binding experiments in order to
compare binding capacities ofboth types ofminerals studied here.
103
CONCLUSIONS AND SUMMARY
CONCLUSIONS AND SUMMARY
The effect ofurine manipulation, such as centrifugation, was verified by sns
PAGE, Western blotting and SEM ofthe crystals precipitated from these urines.
Centrifugation has an effect on Tamm-Horsfall protein and albumin, as these proteins
were found in the pellet remaining after centrifugation. The washing conditions of
crystals obtained from human urine (by NaOH) had a significant effect on proteins that
are non-selectively bound to the crystals precipitated. We were able to identify these
proteins as THP and HSA. There was no effect ofthese urinary manipulations on the
types ofcrystals obtained from the urines, as both COM and COD crystals were found in
samples from uncentrifuged as weIl as centrifuged urines washed in both ddH20 and
NaOH.
From the results obtained in this study, the incorporation ofproteins into crystals
precipitated from urine is a selective process. OPN and UPTFl, which are abundantly
bound to the crystals, are usually found in urine at very low concentrations, whereas two
ofthe most abundant proteins in urine, THP and HSA are only found in scant amounts in
the crystals. Ifthe inclusion process were non-selective, one would expect the opposite
tendency to he true.
Conditions ofurine collection as weIl as storage temperatures do not appear to
have effect on urinary proteins. The addition ofprotease inhibitors does not have an
added effect on the proteins, indicating that there was no protein degradation due to
proteases in the urine. This was tested in female urine, known to he high in protease
105
activity. Further confirmation, by Western blotting, using the antibodies studied here are
required in order to confirm these findings.
As both OPN and HSA have electrophoretic mobilities ofapproximately 67 kDa,
the identification ofOPN bands in the CMP was achieved by treatment with thrombin, as
OPN contains a thrombin c1eavage site that splits the molecule almost into equal halves.
This demonstrated that the majority ofthe intensity ofthe bands in this region was OPN,
as only a very faint band remained at 67 kDa after treatment with thrombin, indicating the
presence ofHSA.
Studies in precipitated CaP crystals demonstrated a different incorporation pattern
ofOPN into this crystal type. By Western blotting, we showed that the different isoforms
ofOPN in urine are incorporated equa11y into CO crystals. Different isoforms OPN are
preferentially bound to CaP, suggesting that post-translational modifications ofthe
protein are important in the binding to this type ofcrystal.
With respect to the differences between genders, there are no significant
differences between the urines from male and female control subjects, however, the
crystals precipitated from female urine contain qualitative1y more protein than those
precipitated from male urine. There seems to be more albumin as well as THP bound to
crystals from female urine. By SEM, crystals precipitated from female urine were, on
average, larger than those precipitated from male urine. Both genders produced COM
and COD crystals.
Rat urine is abundant in proteins as seen by SDS-PAGE. The OPN bound to the
crystals precipitated from rat urine contain the same bands between 43-67 kDa as found
in human samples. The crystals are COM and COD and are approximately the same size
106
as those precipitated from human urine. The COD crystals are consistently of the
stellate-shaped morphology. There is no difference in the shape ofthe COM crystals
compared to human COM crystals.
Staining ofkidney stone sections obtained from two female patients confirmed
the findings ofother studies that showed that the mineral is deposited in lamellae between
layers ofmatrix. The presence ofOPN was also confirmed in these layers.
Inorganic COD have the same morphology as COD precipitated from urine as
shown by SEM. Poly-Asp decreases crystal production and appears to favor the
formation ofa modified form ofCOD in a calcium to oxalate ratio which would normally
produce bipyramidal-shaped COD crystals. The significance ofthis finding is not yet
known. Albumin appears to have an effect on the nucleation ofCOD crystals, producing
numerous small COD crystals. This finding is important because COD is the form of
crystals found most commonly in non-stone formers.
Protein binding to HAP beads was performed using PA and H8A. The binding of
FITC-Iabeled PA was inhibited by excess unlabeled PA but not so efficiently by H8A.
This finding indicates that PA binds to HAP to a greater degree than H8A.
In summary, the results obtained here demonstrate that the model used to
characterize urine in this study is a valid one, producing consistent findings. We have
shown that the process ofincorporation ofproteins into calcium oxalate is a selective one
and that differences exist between the crystals precipitated from the urines ofmales and
females. Growth ofcalcium oxalate crystals in the presence ofpeptides/proteins
indicates that proteins influence crystal structure. Protein-mineral binding studies
demonstrated that different proteins bind to mineraI with different affinities.
107
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ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
.:. 1 would like to thank Dr. Marc D. McKee and Dr. Denise Arsenault for giving me the
tremendous opportunity to do this work. Thank-you for your guidance and for
believing in me.
•:. Roxana Atanasiu, thank-you for teaching and guiding me through the fust part ofmy
Masters as well as for your contribution to Figure 8 (sample preparation and WB).
•:. Douglas Vandor, you are an inspiration to do bigger and better things.
•:. Thank-you Isabelle Turgeon for YOur friendship and advice, for ordering all ofthose
things 1needed 1ast minute, and for proofreading my abstracts.
•:. Caroline Tanguay: thank-you for your help, for all those hours oftalking, looking at
pictures, your continuous support and for proofreading my abstracts.
•:. Mari Kaartinen, thank-you for all your help, your insights and your company in and
outside the office. 1will always he anti-chicken.
•:. Thank-you SherifEI-Madaawy, for your help with the kidney stone sections.
•:. To Helen Campbell, many thanks for your assistance and for teaching me to use the
SEM and X-ray microanalysis.
•:. Jaime Sanchez-Dardon: thank-you for your assistance in using the FACscan.
•:. 1would also like to thank Dr. C.E. Smith for keeping me up-to-date with the literature
and for his insight on anomalous gels and dentistry.
•:. Dad, Mom, Grandma, Golda and Tommy, thanks for heing so patient with me; 1do
not know where 1 would be without you. 1 love you aU very much.
•:. Thank-you John W. Graham for providing excellent conversation, heing more than
pleasant company and for proofreading my thesis.
•:. And last but not least, thanks to aU my friends, especiaUy Eva Lee, Helen Fong,
Kristen Itagawa, Patricia Tellis, Steve Villeneuve, and Toshiro Nguyen for being
there for me throughout my thesis and making life that much sweeter.
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