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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