JARKKO JOKIHAARA
Chronic Renal Insufficiency and Bone
ACADEMIC DISSERTATION
To be presented, with the permission of
the Faculty of Medicine of the University of Tampere,
for public discussion in the small auditorium of Building K,
Medical School of the University of Tampere,
Teiskontie 35, Tampere, on August 24th, 2007, at 12 o’clock.
U N I V E R S I T Y O F TA M P E R E
ACADEMIC DISSERTATION
University of Tampere, Medical School
Tampere University Hospital, Department of Surgery
UKK Institute
Finnish Graduate School for Musculoskeletal Diseases
Finland
Supervised by
Docent Teppo Järvinen
University of Tampere
Professor Ilkka Pörsti
University of Tampere
Reviewed by
Professor Sulin Cheng
University of Jyväskylä
Docent Risto Ikäheimo
University of Oulu
Distribution
Bookshop TAJU
P.O. Box 617
33014 University of Tampere
Finland
Tel. +358 3 3551 6055
Fax +358 3 3551 7685
[email protected]
www.uta.fi/taju
http://granum.uta.fi
Cover design by
Juha Siro
Acta Universitatis Tamperensis 1240
ISBN 978-951-44-7002-8 (print)
ISSN 1455-1616
Tampereen Yliopistopaino Oy – Juvenes Print
Tampere 2007
Acta Electronica Universitatis Tamperensis 633
ISBN 978-951-44-7003-5 (pdf )
ISSN 1456-954X
http://acta.uta.fi
CONTENTS
CONTENTS............................................................................................... 3
LIST OF ORIGINAL PUBLICATIONS .................................................... 6
ABBREVIATIONS.................................................................................... 7
ABSTRACT............................................................................................... 8
INTRODUCTION...................................................................................... 9
REVIEW OF THE LITERATURE........................................................... 11
1
Bone biology.............................................................................................11
1.1 Bone anatomy ..................................................................................11
1.2 Function of bone ..............................................................................12
1.2.1 Locomotive function ............................................................12
1.2.2 Protective and supportive function........................................13
1.2.3 Metabolic function ...............................................................13
1.3 Bone turnover ..................................................................................14
1.4 Functional bone adaptation...............................................................16
2
Characterization of bone ...........................................................................16
2.1 Material ...........................................................................................17
2.2 Mass and geometry ..........................................................................17
2.3 Biomechanics...................................................................................18
3
Chronic kidney disease-bone and mineral disorder ....................................19
3.1 Physiology of mineral metabolism ...................................................19
3.2 Definition of chronic kidney disease-bone and mineral disorder.......20
3.3 Epidemiology of chronic kidney disease-bone and mineral
disorder ............................................................................................21
3.4 Pathogenesis of chronic kidney disease-bone and mineral
disorder ............................................................................................22
3.5 Effects of chronic kidney disease-bone and mineral disorder on
bone .................................................................................................23
3
3.6
3.7
3.5.1 Experimental chronic kidney disease-bone and mineral
disorder................................................................................ 23
3.5.2 Clinical kidney disease-bone and mineral disorder ............... 25
Pharmacological treatments for chronic kidney disease-bone
and mineral disorder ........................................................................ 26
3.6.1 Calcium-based phosphate binders ........................................ 26
3.6.2 Sevelamer ............................................................................ 27
3.6.3 Calcitriol and paricalcitol..................................................... 27
3.6.4 Bisphosphonates .................................................................. 28
Evidence for the efficacy of the prevention and treatment of
chronic kidney disease-bone and mineral disorder ........................... 28
AIMS OF THE STUDY ........................................................................... 30
MATERIALS AND METHODS.............................................................. 31
1
Animals.................................................................................................... 31
2
Nephrectomy............................................................................................ 31
3
Pharmacological treatments ...................................................................... 32
3.1 Phosphate binding therapies ............................................................ 32
3.1.1 Calcium salts ....................................................................... 32
3.1.2 Sevelamer ............................................................................ 32
3.2 Paricalcitol ...................................................................................... 32
3.3 Pamidronate .................................................................................... 34
4
Samples.................................................................................................... 34
4.1 Blood .............................................................................................. 34
4.2 Kidney ............................................................................................ 34
4.3 Bone................................................................................................ 34
4.3.1 Peripheral quantitative computed tomography...................... 35
4.3.2 Geometrical measurements .................................................. 36
4.3.3 Dual-energy X-ray absorptiometry....................................... 37
4.3.4 Bone histomorphometry....................................................... 37
4.3.5 Biomechanical testing .......................................................... 37
5
Statistical analysis .................................................................................... 39
RESULTS ................................................................................................ 40
4
1
The effects of experimental chronic renal insufficiency on bone
structure and strength................................................................................ 41
2
The effects of the treatment of hyperphosphatemia on bone...................... 43
3
The effects of paricalcitol treatment on bone............................................. 45
4
The effects of pamidronate administration on bone in experimental
chronic renal insufficiency ........................................................................47
DISCUSSION .......................................................................................... 50
SUMMARY AND CONCLUSIONS ....................................................... 56
ACKNOWLEDGEMENTS...................................................................... 57
REFERENCES......................................................................................... 59
ORIGINAL COMMUNICATIONS ......................................................... 71
5
LIST OF ORIGINAL PUBLICATIONS
The thesis is based upon the following original publications, referred to in the
text by roman numerals (I-IV):
I
Järvinen TLN, Sievänen H, Jokihaara J, Einhorn TA (2005):
Revival of bone strength: The bottom line. J Bone Miner Res
20:717-720.
II
Jokihaara J, Järvinen TLN, Jolma P, Kööbi P, Kalliovalkama J,
Tuukkanen J, Saha H, Sievänen H, Kannus P, Pörsti I (2006):
Renal insufficiency-induced bone loss is associated with an
increase in bone size and preservation of strength in rat proximal
femur. Bone 39:353-360.
III
Jokihaara J, Pörsti I, Pajamäki I, Vuohelainen T, Jolma P, Kööbi P,
Kalliovalkama J, Niemelä O, Kannus P, Sievänen H, Järvinen TLN
(2006): Paricalcitol [19-Nor-1,25-(OH)2D2] in the treatment of
experimental renal bone disease. J Bone Miner Res 21:745-751.
IV
Jokihaara J, Pörsti I, Kööbi P, Jolma P, Mustonen J, Saha H,
Sievänen H, Kannus P, Iwaniec U, Turner RT, Järvinen TLN:
Pamidronate in experimental renal osteodystrophy. Manuscript
submitted.
Original communications I and III are reproduced with permission of the American
Society for Bone and Mineral Research. Original communication II is reproduced with
permission of Elsevier.
6
ABBREVIATIONS
aBMD
ANOVA
AP
APD
BMC
cBMD
cCSA
CKD
CKD-MBD
CRI
CSMI
CVrms
CWT
DXA
ML
NTX
OPG
PAR
PTH
pQCT
RANK
RANK-L
ROD
SD
SEV
Sham
TBMC
tCSA
vBMD
Areal bone mineral density
Analysis of variance
Anteroposterior direction
Pamidronate
Bone mineral content
Cortical volumetric bone mineral density
Cortical bone cross-sectional area
Chronic kidney disease
Chronic kidney disease-mineral and bone disorder
Chronic renal insufficiency
Cross-sectional moment of inertia
Average root-mean-square coefficient of variation
Cortical wall thickness
Dual energy X-ray absorptiometry
Mediolateral direction
Subtotal surgical 5/6 nephrectomy
Osteoprotegerin
Paricalcitol
Parathyroid hormone
Peripheral quantitative computed tomography
Receptor activator for nuclear factor B
Receptor activator for nuclear factor B -ligand
Renal osteodystrophy
Standard deviation
Sevelamer hydrochloride
Surgical sham-operation
Total bone mineral content of a whole bone
Total bone cross-sectional area
Total volumetric bone mineral density
7
ABSTRACT
The rationale for this thesis was to study the effects of chronic renal
insufficiency (CRI) on bone structure. CRI has been associated with skeletal
lesions and this related bone disorder has been termed renal osteodystrophy or
more extensively as chronic kidney disease-bone and mineral disorder (CKDBMD). The definition and evaluation of renal osteodystrophy has predominantly
focused on changes in the trabecular bone and circulating systemic biochemical
markers of bone turnover. Accordingly, one objective of this study was to assess
the changes in the cortical bone with peripheral quantitative tomography, and
particularly, in the bone structural strength with mechanical testing. In this study,
experimental CRI was shown to be associated with decreased volumetric cortical
bone mineral density and that along with the progression of CRI bone strength
may eventually deteriorate. Other main objective of this study was to assess the
efficacy of four different pharmacological treatments (calcium carbonate,
sevelamer, paricalcitol, pamidronate) on bone in CRI. These were chosen to
represent clinical treatment options for the consequences of impaired renal
function. It is hypothesized that the general treatment of renal condition is
beneficial also to the bones, but there is also some concern about the possible
adverse skeletal side effects. In this study, calcium carbonate, sevelamer, and
paricalcitol treatments were observed to alleviate the CRI-induced detrimental
changes in bone. Calcium salts and sevelamer are phosphate binding agents are
clinically used to alleviate hyperphosphatemia in CRI. Sevelamer treatment was
associated with positive effects on bone, and although calcium carbonate
treatment simultaneously suppressed parathyroid hormone secretion, no
detrimental effects on bone strength were observed. Similarly, the amelioration
of secondary hyperparathyroidism with vitamin D analogue paricalcitol was
beneficial also on bone. In addition, it was shown that pamidronate treatment
was associated with increased bone mineral content in CRI. Altogether these
findings suggest that experimental CRI exerts detrimental effects on bone, but
however, these above mentioned treatments were shown to be effective in
preventing these changes.
8
INTRODUCTION
The main function of the skeleton is locomotion and appropriate mechanical
competence is crucial for bones to function properly. Bones must be strong
enough to bear loading without fracturing, but simultaneously bone mass should
be reasonable to achieve overall energy efficiency. It seems evident that each
bone has adapted a precise three-dimensional structure that is an optimum in the
light of these requirements. Sufficiently high mineral content of bone tissue is
needed to achieve the appropriate structural properties. Minerals are equally
essential in numerous vital biochemical processes in the body and systemic
concentrations and metabolism of minerals, notably calcium and phosphorous,
are therefore controlled delicately. Kidneys and bones are among the organs that
are critical to this intricate regulation system. However, if body is subjected to
metabolic distress, such as the general disorder of mineral metabolism associated
with chronic kidney disease, the homeostatic control system may be influenced
by inappropriate factors. Thus, as a consequence of impaired renal function the
properties of bone may be affected so that the bone becomes inadequate for
competent mechanical function. Particularly, increased bone fragility is a
detrimental consequence of unsatisfactory bone structure as it may predispose to
bone fractures.
Chronic impairment of renal function, chronic renal insufficiency (CRI), has
been recognized as a growing public health problem and prevalence of CRI is
constantly increasing worldwide. This may be explicable in terms of increased
prevalence of risk factors that have been associated with CRI, including diabetes
mellitus and hypertension. The pathogenesis of CRI originates in gradual
decrease in glomerular filtration and loss of metabolically active kidney tissue.
In other words, the capacity of kidneys to excrete substances and to produce
active vitamin D (calcitriol) becomes deficient and results in disturbances in the
homeostasis of phosphorous and calcium, particularly hyperphosphatemia and
hypocalcemia, and low levels of calcitriol. Circulating serum calcium and
phosphorous concentrations are regulated to a large extent by parathyroid
hormone (PTH) and calcitriol, which act on three target organs: kidney, intestine,
and bone. Increased PTH levels are needed to maintain physiological calcium
and phosphorous concentrations in CRI and this leads to the development of
secondary hyperparathyroidism. Mild CRI is generally asymptomatic, but if the
condition is left untreated, the disturbances of mineral metabolism may
eventually result in cardiovascular calcifications, skeletal lesions, and other
detrimental complications. This condition is extensively termed as chronic
kidney disease-bone and mineral disorder (CKD-BMD), and the bone lesions
9
have traditionally been specified as renal osteodystrophy. CKD-BMD is widely
considered the most complex and least predictable form of metabolic bone
diseases. Advanced CRI has been associated with increased bone fragility,
whereby the skeletal changes in CKD-BMD have attracted considerable
scientific interest. However, information on changes in the bone structure and
mechanical competence of bones is scarce.
The rationale for this series of experiments was to utilize a structurally
oriented approach to characterize the effects of CRI on bone. Mechanical
competence is a fundamental feature of bone and therefore characterization of
the bone structural strength and its determinants is warranted, and indispensable,
in the evaluation of bone. One objective of the clinical treatment of patients with
CRI is to prevent and overcome the consequences of the underlying
hyperphosphatemia, hypocalcemia, low calcitriol levels, and the associated
secondary hyperparathyroidism. There are distinct pharmacological treatment
options available, and because of the complexity of the consequences of CRI,
there are a myriad of mechanisms and possible treatment outcomes to consider.
In general, the cornerstones of the pharmacological management of disturbances
in mineral metabolism are alleviation of hyperphosphatemia and inhibition of
excessive parathyroid function. In this thesis, the treatments were chosen to
represent the current clinical pharmacological treatment options for the general
management of CRI and the rationale was to evaluate the influence of these
widely used interventions on bone structure. Specifically, the objectives were to
evaluate the effects of treatment of hyperphosphatemia with calcium salts and
sevelamer, and the effects of vitamin D analogue paricalcitol on bone.
Additionally, the effects of bisphosphonate treatment on bone in CRI were also
explored; bisphosphonates are shown to be potent inhibitors of bone resorption,
but however, the net effect may not be desirable in CKD-BMD. Furthermore,
outcome of any bone affecting drug treatment is somewhat unpredictable in
CKD-BMD.
10
REVIEW OF THE LITERATURE
1
Bone biology
1.1 Bone anatomy
Bones are the hard and rigid constituents of skeleton. In each bone, the solid
bone tissue is enclosed between outer periosteal envelope and inner endosteal
envelope (Figure 1). Inside the bone endosteal perimeter demarcates the solid
portion and the marrow cavity (medullary canal which encompasses the bone
marrow). The texture of the solid portion is either dense cortical bone or
honeycomb-like trabecular bone. Periosteal envelope includes mainly cortical
bone, and articular cartilage in joints, while endosteal perimeter is considered to
include trabecular, endocortical, and intracortical subdivisions which are in
continuity. The solid bone tissue consists of bone cells and extracellular matrix,
which comprises of mineral compound that resembles hydroxyapatite.
The shape of bones varies greatly according to the specific function of each
given bone. The overall shape of each bone may be grossly described as long
(femur, tibia, humerus) or flat (skull, ilium, scapula). The proportion of cortical
to trabecular bone is distinct in different bones and also in different parts within
a given bone: shaft (diaphysis) of a long bone resembles a cylinder made of
cortical bone, while in the ends of a long bone (metaphysis and epiphysis), and
in flat bones overall, a thinner cortical shell is filled with a spongy mesh of
trabecular bone.
Figure 1. Schematic structure of a long bone.
11
1.2 Function of bone
The purpose of the skeleton is to contribute to the survival of the organism in the
wild. Bones have locomotive, protective, and metabolic function, and like all
organs, tissue builds up functional structures that are optimum to carry out these
functions (Einhorn 1992). Each bone presents a species-specific size, shape and
internal structure to outcome the evolutionary driven genetic adaptation in the
population and this is the starting point for physiological bone adaptation in the
individual during its lifespan (Currey 2003a, Currey 2003b).
1.2.1 Locomotive function
The primary function of bones is locomotion (Einhorn 1992). Bones build up
skeleton, which may be considered as a mechanism that consists of supports,
levers, joints, and sites for muscle attachment that are needed for movement.
Skeleton also bears the body. Bone strength and stiffness are of greatest
importance in the locomotive function of bones (Einhorn 1992, Currey 2003b,
Currey 2004). Bones must be able to resist bending and compression to perform
as levers in musculoskeletal system (Currey 2004). Equally, bones must resist
the forces applied to them by muscle contraction and gravity without breaking,
and only adequately strong bones make movement possible. The precise threedimensional structure and composition of each bone is critical to its ability to
function effectively in locomotion (Einhorn 1992, Burr 1997, Frost 1997, Parfitt
1998, Currey 2001, van der Meulen et al. 2001, Turner 2002, Currey 2003a,
Currey 2003b, Currey 2004). When considering the attributes of the other
constituents of skeleton; cartilage, tendons and ligaments are less rigid but
appropriate in toughness, tensile strength, and flexibility thus providing for
particular features such as bone adhesion and joint movement.
Cortical bone is strong and stiff because of the high mineral content and
combination of dense cortical bone and hollow marrow cavity provides for
combination of strength and low weight (Einhorn 1992, Turner and Burr 1993,
Currey 2001, van der Meulen et al. 2001). Cortical bone is superior to trabecular
bone in terms of whole bone strength and load bearing in the skeleton is mostly
carried out by cortical bone (Augat et al. 1996, Bell et al. 1999, Haidekker et al.
1999, Crabtree et al. 2001, Muller et al. 2003, Pistoia et al. 2003). For example,
the thickness of the cortical shell has been shown to be as small as 0.3–0.4 mm
in the vertebral body, but still the biomechanical role of this thin cortical shell
can be substantial, being about 45% at the midtransverse section (Eswaran et al.
2006).
From the evolutionary point of view, the skeleton is subjected to an obvious
need for a mechanism to attain perfect temporospatial specificity for metabolic
needs of systemic hormones without compromising the mechanical competence
of locomotive apparatus, the bottom line in terms of viability of vertebrates.
While hormones affect the bone mineral mass, the ultimate currency in the
12
economy of survival is the metabolic energy efficiency (Currey 2003b). Bones
being twice as dense as other tissues, bone mass has a profound effect on body
mass, which in turn governs metabolic energy requirements. Accordingly, it
appears that basic physical principles have simply resulted in evolutionary
pressures to specifically reduce and optimize bone mass required to function in a
given evolutionary niche.
1.2.2 Protective and supportive function
In addition to locomotive function, strength and stiffness of bone is also utilized
to form protective and supportive tissues. Skeleton defines the general shape of
the body and acts as a supporting framework for all other systems of the body. In
addition, skeleton forms support for body cavities in which internal structures are
located, and equally, skeleton provides mechanical protection for certain organs
and vital structures. For example, the vertebral column protects the spinal cord,
the skull protects the brain, ribs protect the heart and lungs, and long bone
diaphysis encompasses hematopoietic red bone marrow (Moore and Agur 2007).
1.2.3 Metabolic function
The overall shape or precise distribution and orientation of bone are of less
importance to the successful execution of the metabolic functions, particularly
the former, whereas the existence of metabolically active surfaces is obviously
more critical. In the whole skeleton, cortical bone represents about 75% of the
volume and mass, but only 25% of the surface area (Parfitt 1998). Thus, the
importance of trabecular bone becomes readily apparent concerning the mineral
transfer function. Because of the honeycomb-like texture, the trabecular bone
possesses a large surface area with close proximity to circulation and bone
marrow. Bone tissue is a reserve of ions, particularly calcium and phosphate, and
99% of total body calcium is deposited in bone (Moe 2005). Systemic control of
calcium and phosphorous metabolism is a complex and closely regulated process
involving the kidneys, intestine, parathyroid glands, and the skeleton (Figure 2).
In addition to mineral metabolism, skeleton is also a source of hematopoietic
cells, cytokines and growth factors.
13
Figure 2. A simplified diagram of the regulation of calcium and phosphorous balance.
1.3 Bone turnover
Bone remodeling is a coupled multi-step process of bone resorption and
formation at discrete locations in the skeleton. The rate of bone remodeling is
referred to as bone turnover. In bone physiology, turnover means replacement
referring to proportional volume replacement per time unit, usually expressed as
percent/year (Parfitt 2002). The primary objective of bone remodeling is to
prevent bone becoming too old, remodeling makes less direct contribution to
mineral homeostasis (Parfitt 2003). In human adult skeleton, bone resorption and
formation is continuously occurring at about 1–2 million microscopic sites
(Rodan and Martin 2000). Bone turnover is mainly affected by mechanical
stimuli, hormones, cytokines, and growth factors that influence the recruitment,
differentiation and activity of osteoclasts, osteoblasts and osteocytes.
During the formation phase of remodeling, the collagenous structure of the
bone becomes calcified in a process called mineralization. The density of
unmineralized bone is lower and it increases as all free matrix water is being
replaced by mineral. Thus, the amount of bone mineral in a particular volume is
decreased if bone turnover is high and the mean age of bone is low, or bone
mineralization is impaired (Parfitt 2003). Reciprocally, the degree of
mineralization is increased if bone turnover is low and mean bone age is high,
however, complete mineralization is normally prevented presumably by some
property of living osteocytes (Parfitt 2003). Formation rate is frequently in
balance with that of the resorption rate, but not always and the imbalance can
affect bone volume (Parfitt 1998, Salusky and Goodman 2001, Parfitt 2003).
The instrument of remodeling is a multicellular unit which consists of
osteoclasts, osteoblasts, and precursors of those (Figure 3). Osteoclasts are bone
14
resorbing cells that are derived from the circulating progenitor cells. Activation
of osteoclasts is responsible for bone resorption and that takes place for about 3
weeks per microscopic site (Rodan and Martin 2000). During bone resorption
osteoclasts endocytose the degradation products of bone matrix, which are then
transported through the resorbing osteoclast, and thus osteoclasts can
simultaneously remove large amounts of bone matrix and penetrate into bone
(Salo et al. 1997). Osteoblasts, the bone forming cells, are derived from the
marrow stromal cells and some of them eventually become osteocytes or lining
cells. Once the osteoclasts have resorbed bone, the osteoblasts fill the resorption
pit with unmineralized matrix, the osteoid. To complete the remodeling cycle,
the osteoid is finally mineralized. The entire rebuild process takes about 3–4
months (Rodan and Martin 2000).
Figure 3. Basic multicellular unit of bone remodeling and a simplified diagram of the
regulation of osteoclast activation. OB, osteoblast; SC, stromal cell; OC, osteoclast;
OPG, osteoprotegerin; RANK, receptor activator for nuclear factor B; RANK-L,
receptor activator for nuclear activator B -ligand.
Osteoblasts and its precursors (marrow stromal cells) regulate osteoclast
activity via osteoprotegerin / receptor activator for nuclear factor B -ligand
(OPG/RANK-L) system (Suda et al. 1999, Boyle et al. 2003, Harada and Rodan
2003, Martin and Sims 2005). Receptor activator for nuclear factor B (RANK)
is located on osteoclasts precursor cells, whereas osteoblasts and stromal cells
express RANK-L, which activates the development and controls the activity of
mature osteoclasts (Figure 3) (Boyle et al. 2003). The interactions of RANKL
and RANK are also controlled by OPG: soluble OPG acts as a decoy receptor
and binds also to RANK-L and subsequently blocks the binding of RANK to
RANK-L, and thus decreases the activation of the osteoclasts (Suda et al. 1999,
Boyle et al. 2003). This OPG/RANK-L system is regulated by a large number of
different hormones, growth factors and cytokines that are known to have an
effect on bone turnover (Suda et al. 1999, Boyle et al. 2003, Harada and Rodan
15
2003). Parathyroid hormone (PTH) is among the best known factors that affect
bone formation and resorption (Boyle et al. 2003, Harada and Rodan 2003).
1.4 Functional bone adaptation
Differences between corresponding individual bones may be classified as
outcome of long-term (evolutionary) or short-term (during a single lifetime)
adaptation (Currey 2003a). Different species have distinct bones because of
evolutionary adaptation driven by natural selection. During individual’s lifetime,
bones are adapted to the prevailing predominant functional requirements (Currey
2003b) and this phenomenon is referred as functional bone adaptation. In theory,
optimal locomotive structure is light for speed and energy efficiency but still
strong for load bearing. These conflicting properties are balanced in bone;
functional adaptation seems to optimize appropriate structures of minimal weight
and maximal strength (Einhorn 1992, Turner and Burr 1993, van der Meulen et
al. 2001, Currey 2003a, Currey 2003b).
The concept of bone functional adaptation dates back to the writings by
Julius Wolff (1892), which introduced the premise that biological processes
could be regulated by mechanical loading. Mechanical forces can modulate the
incessant regulation of bone turnover at specific desired sites within bone (Frost
2003). Loading-induced strains in bone are associated with corresponding
changes in bone structure which seems to gradually optimize the bone structure
for the prevailing loading environment (Frost 2003). Bones are also able to some
extent counteract the deleterious effect on some material property by an
alteration in the architecture of the whole bone (Currey 2004).
Functional bone adaptation may influence bone strength by changing the
absolute bone mass and the distribution of the given bone mass (Turner 2002,
Currey 2003a, Currey 2003b, Currey 2004, Ruff et al. 2006). Larger bones can
carry more loads and optimized distribution minimizes the mass needed to
accomplish structures for the predominant mechanical demands. In addition to
these structural changes of adding and redistributing of bone mass, bone strength
is also influenced by material properties of bone tissue (Turner 2002).
2
Characterization of bone
The characteristics of bone can be described at different levels: tissue level
(material) properties are defined as qualities which are independent of bone size
or structure, while characteristics of whole bone or an intact anatomical unit may
be considered as organ level structural properties (morphology) (Einhorn 1992,
Currey 2001, van der Meulen et al. 2001).
16
2.1 Material
The solid bone tissue is characterized by physiologically mineralized organic
matrix and water. In adult human bone, mineral accounts for 60% to 70% and
organic matrix for 30% to 40% of the dry weight (Standring 2005). In living
bone, the matrix is moderately hydrated and water accounts for 10% to 20% of
the mass. The organic matrix consists predominantly of type 1 collagen and bone
mineral resembles geological hydroxyapatite (Ca10(PO4)6(OH)2). The matrix
mineral accumulates at the expense of water so that the tissue volume does not
change along the mineralization process.
Mineral density of bone is a measure of mass per unit volume. The definition
of apparent mineral density must be distinguished from material density (true
bone density). Apparent density of bone equals its total mass divided by its total
volume (volumetric bone mineral density, vBMD), whereas in determination of
true bone material density only a certain volume of solid cortical bone is
included in the analysis (cortical volumetric bone mineral density, cBMD).
Computed analysis methods have enabled the attainment of apparent volumetric
bone density for any given precise volume of interest, defined anatomically or
arbitrarily, since the mass encompassed by this given particular volume can be
unambiguously determined. Nevertheless, the determination of true material
density for the whole bone is impracticable, because the outer edges of a bone
encompass cortical and trabecular bone compartments, and bone marrow, and all
these constituents have different tissue densities.
Furthermore, true bone density is the density of bone material when it is
considered as uniform material over the particular volume, i.e. material density.
The volume to which the mass is referred should therefore be defined to include
only bone tissue, and marrow cavity as well as the cavities within trabecular
bone need to be excluded. For greatest accuracy, the bone volume should even
exclude osteocyte lacunae and canaliculae within cortical bone, but in practice
this refinement is usually disregarded. Accordingly, the usual interpretation of
increased cortical bone porosity is that bone tissue density is decreased (Parfitt
1998).
2.2 Mass and geometry
The bulk of bone is usually referred as bone mass, which is influenced by
absolute size of the whole bone and properties of bone tissue and structure
within, and it is usually defined as bone mineral content (BMC). Each bone has
specific spatial dimensions and mass distribution which define its geometrical
attributes. Periosteal bone formation defines the total cross-sectional area
(tCSA), which includes cortical bone, trabecular bone and marrow area.
Endocortical bone formation and resorption define cortical wall thickness (CWT)
and cortical bone cross-sectional area (cCSA). However, the separation between
cortical and trabecular bone is not always definite as the shift is a continuum.
17
The marrow area in diaphysis of a long bone is defined as the area surrounded by
endosteal surface. The outside diameter of bone (width, thickness) is defined as
the distance between the opposite periosteal surfaces, and accordingly, the inside
diameter of bone marrow cavity (inside width, inside thickness) is defined as the
diameter between the opposite endosteal surfaces.
2.3 Biomechanics
Whole bone strength equals to the ultimate force the bone can sustain without a
fracture. Bone fractures occur when applied load exceeds the bone strength, and
accordingly, bone strength may also be defined as the breaking load (unit
newton, N) needed to break the bone structure. Whole bone strength is
influenced by the tissue level material properties and organ level structural
properties of bone. (Einhorn 1992, Turner and Burr 1993, Currey 2001, van der
Meulen et al. 2001, Turner 2002)
The relationship between the load applied to structure (unit newton, N) and
deformation (unit millimeter, mm) in response to the load is called a loaddeformation curve (Figure 4). The load-deformation curve can be divided into
the elastic deformation region and plastic deformation region; elastic
deformation is reversible, while plastic deformation refers to permanent
structural damage (Turner and Burr 1993).
Figure 4. A typical load-deformation curve of bone mechanical testing until
failure.
18
Mechanical competence of whole bone represents the net influence of all
factors acting at lower physical scales (i.e. material properties, mineral mass or
geometry) when considering bone as a hierarchical structure (Hernandez and
Keaveny 2006). Determination of bone strength by structural testing measures
both structural and material properties of bone (Currey 2001, van der Meulen et
al. 2001, Turner 2002, Hernandez and Keaveny 2006). Furthermore, it has been
argued that conclusions regarding bone mechanical function based solely on
properties of bone material, mineral content or geometry are inappropriate and
likely to be misleading (van der Meulen et al. 2001, Turner 2002).
3
Chronic kidney disease-bone and mineral disorder
3.1 Physiology of mineral metabolism
Physiological extracellular calcium and phosphate concentrations are essential to
life and the deliberate mineral homeostasis is regulated by controlling the serum
concentrations of the corresponding ions. Kidneys excrete water and electrolytes,
as well as various other substances, in order to control the appropriate
concentrations of these in body fluids. Calcitriol and PTH are the principal
hormones mediating the regulation of calcium and phosphorous concentration
through actions on kidney, bone and intestine (Figure 2).
There is approximately 1 000 g of calcium and 600 g of phosphorous in
human body stores. Ninety-nine percent of the calcium is in bone, 0.9% is
intracellular and 0.1% in extracellular space. For phosphorous, the distribution is
85% in bone, 14% in intracellular, and 1% in extracellular space. The average
daily intake is from 0.05% to 0.10% (from 0.5 g to 1 g) for calcium and from
0.15% to 0.23% (from 0.9 g to 1.4 g) for phosphorous when calculated as
percentage of the total reserve in body (absolute amounts in parenthesis). The
absorption of calcium and phosphorous in intestine is both active and passive,
and one third of the absorbed excess calcium and two thirds of excess
phosphorous is excreted in urine by the kidneys and the rest in stool (Moe 2005).
Calcitriol acts on bone to increase mineralization and enhance osteoclast
activity, and there are also other minor effects. Calcitriol acts also on parathyroid
gland to inhibit PTH secretion, and on intestine to increase calcium and
phosphorous absorption. For the present, the action of calcitriol on kidney
calcium and/or phosphorous uptake/excretion is controversial. The sources of
calcitriol are dietary ergocalciferol and conversion of 7-dehydrocholesterol to
cholecalciferol. These precursors are hydroxylated in liver to 25hydroxycholecalciferol, which is then further hydroxylated by 1- -hydroxylase
to calcitriol (1,25-dihydroxycholecalciferol) in the proximal nephron of kidney
(Fraser and Kodicek 1970). The synthesis is regulated by altering 1- -
19
hydroxylase activity, which is increased by low phosphorous, low calcium, low
calcitriol, and increased by PTH.
PTH increases calcium reabsorption and decreases phosphorous reabsorption
from the kidney and increases bone resorption and formation by multiple
mechanisms. PTH is released from parathyroid glands in response to lowering of
Ca2++ concentration (hypocalcemia)
via Ca2++ sensing receptor.
Hyperphosphatemia stimulates PTH secretion (Fine et al. 1993). Calcitriol
inhibits PTH secretion and PTH enhances the synthesis of calcitriol, and thus
PTH indirectly increases intestinal absorption of calcium, thereby completing the
regulatory feedback loop. Active PTH is cleaved from pre-pro PTH to pro PTH
in parathyroid gland, and then secreted and catabolized to active N-terminal 1-84
PTH and less active C-terminal 7-84 fragments. Kidneys are responsible for the
plasma clearance and subsequent degradation of 1-84 PTH and a variety of the
truncated derivatives of PTH (Freitag et al. 1978).
Phosphatonins are a group of substances (e.g. fibroblast growth factor 23,
frizzled related protein 4, and matrix extracellular phosphoglycoprotein) that
appear to be involved in regulation of serum phosphorous levels in certain
diseases, such as tumor-induced osteomalacia, X-linked hypophosphatemic
rickets, and autosomal dominant hypophosphatemic rickets, but for the present,
the role of phosphatonins in normal physiology remains unresolved.
3.2 Definition of chronic kidney disease-bone and mineral disorder
Chronic kidney disease (CKD) is defined as long-term kidney damage and it is
commonly associated with deterioration of kidney function. CKD may be staged
according to severity of functional impairment, but in literature on experimental
studies this staging is often omitted. Accordingly, in experimental studies
deteriorated kidney function is widely referred as CRI regardless of the severity
of the impairment. The use of the terms renal failure and end-stage renal disease
are somewhat problematic in experimental settings due to their clinical
definitions. Therefore, throughout the results and discussion sections of this
thesis, CRI of the utmost severity is referred as advanced CRI.
The complex and multifactorial skeletal changes associated to CKD have
been traditionally described as renal osteodystrophy (Stanbury and Lumb 1962,
Stanbury et al. 1969, Parfitt 1972, Parkinson et al. 1979, Hruska and Teitelbaum
1995, Martin et al. 2004), although other terms have also been used, such as
renal bone disease and uremic bone disease. In recent years, organizations called
Kidney Disease: Improving Outcomes Quality Initiative (K/DOQI) and Kidney
Disease Improving Global Outcomes (KDIGO) have defined renal
osteodystrophy (ROD) as a constellation of bone disorders present or
exacerbated by CKD that lead to bone fragility and fractures, abnormal mineral
metabolism, and extraskeletal manifestations (Massry et al. 2003, Moe and
Drueke 2004, Moe et al. 2006), or concisely as an alteration of bone morphology
in patients with CKD (Moe et al. 2006). In addition, the recent KDIGO position
20
statement also introduced a broad systemic entity or syndrome called chronic
kidney disease-mineral and bone disorder (CKD-MBD) which covers all the
clinical, biochemical, and imaging abnormalities due to CKD manifested by the
following: abnormalities of calcium, phosphorous, PTH, or vitamin D
metabolism; abnormalities in bone turnover, mineralization, volume, linear
growth, or strength; and vascular or other soft tissue calcification (Moe et al.
2006). The term osteoporosis is not recommended when describing skeletal
complications that are associated with CKD (Cunningham et al. 2004, Moe et al.
2006).
3.3 Epidemiology of chronic kidney disease-bone and mineral
disorder
First notes on bone abnormalities associated with renal diseases date back to 19 th
century and the first epidemiological reports were published during the 1970s
and 1980s when fractures in significant number were associated to the use of
aluminum-containing dialysis fluids (Parfitt et al. 1972, Ward et al. 1978,
Parkinson et al. 1979). The identification of aluminum-related osteomalacia led
to changes in composition of dialysis fluids and thereafter the clinical entity of
aluminum-related osteodystrophy has practically vanished.
Prevalence of impaired renal function in general adult population is estimated
to be approximately 11% (Chadban et al. 2003, Coresh et al. 2003). Rix et al.
(1999) have analyzed biochemical markers of bone turnover together with bone
mineral density data in mild-to-moderate CRI patients and concluded that
skeletal changes seem to initiate early in the course of CKD. In unselected
population of predialysis CRI patients, abnormal bone histology was found in
68% of the patients with severe impairment of renal function (Spasovski et al.
2003). Among dialysis patients, 46% have been reported to display gross
histological bone abnormalities (Barreto et al. 2006).
Epidemiological studies on U.S. dialysis population report approximately
four-fold increases in the incidence of hip fractures in comparison to agematched population (Alem et al. 2000, Stehman-Breen et al. 2000). In other
populations on dialysis, the prevalence of vertebral fracture has been reported to
be as high as 21% (Atsumi et al. 1999) and even higher incidence of hip
fractures has been reported (Coco and Rush 2000). Further, there is also
epidemiological data that older women with less severe (moderate) renal
dysfunction are at increased risk of hip fracture (Ensrud et al. 2007). However, in
this context, one should recall that that CKD-BMD is usually asymptomatic and
the clinical complications appear late in the course of CKD (Llach and
Fernandez 2003).
21
3.4 Pathogenesis of chronic kidney disease-bone and mineral
disorder
Disturbances in mineral metabolism and the resulting bone changes are common
complications of CKD. The major factors in the pathogenesis of CKD-BMD
include renal insufficiency-associated secondary hyperparathyroidism with the
associated phosphorous retention (hyperphosphatemia) as renal glomerular
filtration rate falls and a decrease in calcitriol levels as metabolically active renal
mass is reduced (Figure 5). As a consequence, the serum levels of calcium are
affected (Slatopolsky et al. 1971, Goodman 2001, Llach and Velasquez Forero
2001, Martin and Gonzalez 2001a). In incipient CRI, the plasma levels of PTH
increase in an obvious attempt to maintain physiological calcium levels and to
overcome hyperphosphatemia, but elevated PTH levels may also be observed in
absence of hypocalcemia (Lopez-Hilker et al. 1986). Due to this response, the
early stage of CKD may be present with elevated PTH levels and normal serum
phosphate, calcium and calcitriol levels (Fukagawa et al. 1991, Fajtova et al.
1995, Martinez et al. 1997). But concurrently, particularly the incessant
incidence of high phosphorous levels causes activation and progressive growth
of parathyroid glands resulting in a process called secondary
hyperparathyroidism (Slatopolsky and Delmez 1994, Naveh-Many et al. 1995,
Slatopolsky et al. 1996). In addition, abnormal regulation of calcitriol
metabolism is also observed with the secondary hyperparathyroidism (Fukuda et
al. 1993). Nevertheless, if the renal insufficiency persists and advances, serum
phosphorous and calcium levels cannot be maintained within an optimal range
despite the development of secondary hyperparathyroidism.
Figure 5. Some of the crucial factors that contribute to the development of secondary
hyperparathyroidism in CRI.
In clinical CKD-BMD, persistent high PTH may result in representative
histological characteristics of high bone turnover, and in contrast, prevailing low
PTH levels are associated with histological features of low bone turnover
22
(Salusky and Goodman 2001, Llach and Fernandez 2003, Lehmann et al. 2005).
In general, the histological representation of ROD based on determination of
bone turnover and mineralization varies greatly from such entities as osteitis
fibrosa, osteomalacia, adynamic bone disease, to mixed uremic bone disease.
However, it needs to be noticed that in the clinical setting the histological
features of ROD are often obscured by the simultaneous existence of diabetes
mellitus, immobilization, glucocorticoid-related osteoporosis, and changes
induced by prior parathyroid surgery (Salusky and Goodman 2001), which are
common in CRI patient population. Further, the histological features may also be
influenced by such factors as the race, sex, age, type of kidney disease, nutrition,
and menopausal status (Martin et al. 2004).
In addition to the determination of PTH levels, several circulating
biochemical markers of bone formation and resorption have also been used in the
clinical characterization of bone turnover related to CRI: total or bone-specific
alkaline phosphatase, osteocalcin, and carboxy-terminal propeptide of type I
collagen as indicators of bone formation, and, tartrate-resistant acid phosphatase,
cross-linked C-telopeptide of type I collagen, and N-telopeptide of collagen
cross-links as indicators of bone resorption, but their significance and
applicability are poor or remain to be established (Urena et al. 1996, Coen et al.
1998, Rix et al. 1999, Urena and De Vernejoul 1999, Salusky and Goodman
2001, Moe et al. 2006). Particularly in CRI, the measures of several of these
biomarkers are unreliable, because their elimination depends on kidney function
and the markers accumulate in serum if there is dysfunction of glomerular
filtration and excretion (Martin et al. 2004, Moe et al. 2006).
3.5 Effects of chronic kidney disease-bone and mineral disorder on
bone
3.5.1 Experimental chronic kidney disease-bone and mineral disorder
Subtotally nephrectomized rat has been widely used as an experimental model of
CRI and it has been shown to resemble the characteristics of CKD-BMD in
biochemistry and bone histology (Kaye 1974, Jablonski et al. 1993, Jablonski et
al. 1994, Slatopolsky et al. 1996, Turner et al. 1996, Miller et al. 1998, Sanchez
et al. 1998, Geng et al. 2000, Sanchez et al. 2000, Freesmeyer et al. 2001,
Sanchez and He 2003, Slatopolsky et al. 2003, Sanchez et al. 2004). More
recently, adenine-containing diet has been introduced as a novel means to impair
renal function (Katsumata et al. 2003, Nagano et al. 2006), but data on the
effects of this model of CKD-BMD is scarce.
The most common forms of ROD are likely to be largely attributable to
variations in the plasma levels of PTH. Continuous PTH excess seems to induce
an overall catabolic effect on the cortical bone in ROD, the suggested
mechanism being an increase in the porosity due to an increase in bone turnover
23
(Parfitt 1996, Miller et al. 1998, Parfitt 1998, Schober et al. 1998, Slatopolsky et
al. 2003, Jamal et al. 2006a). Quite intriguingly, the effect of PTH excess on
trabecular bone is generally anabolic in ROD, but there seems to be a myriad of
possible mechanisms (Miller et al. 1998, Parfitt 1998, Schober et al. 1998,
Slatopolsky et al. 2003). Furthermore, Miller et al. (1998) have also suggested
that slightly elevated PTH levels may result in decreased amount of trabecular
bone.
Sanchez et al. have studied the effects of CRI on bone growth in weanling
rats (Sanchez et al. 1998, Sanchez et al. 2000, Sanchez and He 2003, Sanchez et
al. 2004). The findings are somewhat controversial, as the authors have shown
data that CRI restricts longitudinal bone growth in one experiment (Sanchez et
al. 2004), whereas another experiment failed to show this effect (Sanchez et al.
2000). Others have shown no change in bone length in the nephrectomized
(NTX) rats (Kaye 1974). All in all, these studies suggest that when evaluating
the effect of CRI on bone longitudinal growth, the changes in body weight need
to be included in the analysis. Reduced bone longitudinal growth has been
associated with reduced weight gain (Sanchez et al. 2004), whereas in most of
the studies NTX has not been associated with either reduced gain in bone length
or body weight (Kaye 1974, Sanchez et al. 2000). However, in general NTX
does not seem to influence body weight in pair fed (Russell and Avioli 1972,
Kaye 1974) or in ad libitum fed rats (Kaye 1974, Slatopolsky et al. 1996, Miller
et al. 1998, Sanchez et al. 1998, Sanchez et al. 2000, Freesmeyer et al. 2001,
Sanchez and He 2003).
Abnormal mineralization is observed in subtotally nephrectomized rats after
2 weeks (Russell and Avioli 1972). Turner et al. (1996) have estimated bone
mineral density 6 months after a NTX operation from a section of rat femoral
midshaft by dividing wet weight by volume and concluded that advanced CRI is
associated with a decrease in bone density. In a 12-week experiment, Miller et al.
(1998) determined BMC and bone width by single-photon absorptiometry at 10
equidistant sites in femur and calculated bone mineral density as the BMC
divided by bone width (i.e. areal bone mineral density, aBMD). They reported
that aBMD is uniformly decreased in mild ROD and unchanged in moderate
disease, while in severe ROD, aBMD is decreased only in proximal femur and
increased in distal femur (Miller et al. 1998). However, they did not provide data
on actual changes in BMC or bone width at these sites. In the
histomorphometrical determination of rat tibial metaphysis the trabecular bone
volume per total volume is increased and in tibial shaft cortical bone porosity
ratio is increased (Miller et al. 1998, Slatopolsky et al. 2003). In rat lumbar
vertebrae CRI is associated with increased bone volume per tissue volume
(BV/TV) and osteoid thickness (Turner et al. 1996).
The changes in the gross bone geometry of NTX rats include a decrease in
outer diameters of femur (Jablonski et al. 1993); or no change in the outer
diameter, an increase in inner diameter, and a decrease in CWT (Jablonski et al.
1994). Miller et al. (1998) observed no change in tCSA, cCSA, or marrow area.
More recently, Slatopolsky et al. (2003) have reported a slight increase in tCSA
24
with no change in cCSA, marrow area or CWT after 8 weeks of CRI, and an
additional decrease in marrow area and an increase in CWT after 16 weeks of
CRI.
There is sparse data on the effects of CRI on whole bone strength. The group
of Jablonski has reported that bone strength is maintained during 28 or 33 weeks
of CRI (Jablonski et al. 1993, Jablonski et al. 1995), but decreased bone strength
was reported after 38 weeks of CRI (Jablonski et al. 1993). After 6 months of
advanced CRI, Turner et al. (1996) have determined breaking load by three-point
bending in femoral midshaft and by compression in lumbar vertebrae, and
observed a decrease in breaking strength in both locations.
3.5.2 Clinical kidney disease-bone and mineral disorder
Bone measurements with dual energy X-ray absorptiometry (DXA) in patients
with CKD have provided data on changes in bone mineral density along the
course of the disease and most of the studies conclude that reduced renal
function is associated with decreased bone mineral density (Bianchi et al. 1992,
Gabay et al. 1993, Rix et al. 1999, Hsu et al. 2002, Klawansky et al. 2003, Lobao
et al. 2004). Furthermore, when patients are classified according to the
impairment of renal function, there seems to be a close relationship between
renal function and bone mineral density (Bianchi et al. 1992, Rix et al. 1999, Hsu
et al. 2002). These studies also suggest that deleterious processes affecting bone
mineral properties commence early in the course of CRI. However, although a
large general population sample (National Health and Nutrition Examination
Survey of USA, NHANES III) did indeed confirm that renal function correlates
with bone mineral density, it was also shown that when the data was adjusted for
age and weight, the negative association between renal function and bone
mineral density extinguishes (Hsu et al. 2002). On the contrary, Klawasky et al.
(2003) have analyzed the same data and concluded that decreased renal function
was associated with reduced bone mineral density, but the adjustments for age
and weight were absent.
The most consistent finding regarding end-stage renal disease patients in
dialysis is a decrease in the DXA-derived bone mineral density, but similarly to
predialysis patients, the relationship was affected by age, gender or body mass
index (BMI) (Bianchi et al. 1992, Gabay et al. 1993, Russo et al. 1998, Atsumi et
al. 1999, Taal et al. 1999, Jamal et al. 2006a). In a detailed evaluation of bone
loss in patients on dialysis by single-photon absorptiometry, Schober et al.
(1998) found that particularly cortical bone volume is reduced and this is due to
an increase in cortical porosity and a decrease in CWT. Russo et al. (1998) used
computed tomography to assess the cortical compartment showed that cBMD
and cCSA were reduced in long bone diaphysis. Recent data from Jamal et al.
(2006a) corroborated these changes observed in cortical bone, and furthermore,
they suggest that after adjusting for age, weight and sex, a decrease in cBMD,
cCSA and CWT are associated with an increase in fracture risk in dialysis
25
patients. The changes associated with ROD in bone sites which are rich in
trabecular bone seem to be more complex (Schober et al. 1998); Russo et al.
(1998) reported no differences in trabecular vBMD while Jamal et al. (2006a)
observed a decrease in vBMD. However, the separation between cortical and
trabecular bone was defined as a fixed percentage of total area in both studies
and this may generate inaccuracy particularly in patients with ROD, as the
disease is associated with changes in the mineralization of the endosteal surface.
3.6 Pharmacological treatments for chronic kidney disease-bone
and mineral disorder
3.6.1 Calcium-based phosphate binders
Data from experimental studies show that excess intake of dietary phosphorous
is associated with an increase in plasma levels of phosphorous and growth of the
parathyroid glands in CRI rats (Kaye 1974). In vivo and in vitro data has also
shown that phosphorous has a direct stimulatory effect on PTH secretion and
parathyroid cell proliferation, and that restriction of dietary phosphate prevents
the development of these changes independently of changes in calcium and
calcitriol levels (Kilav et al. 1995, Naveh-Many et al. 1995, Almaden et al. 1996,
Denda et al. 1996, Slatopolsky et al. 1996). Reduced phosphate intake has been
associated with an increase in calcitriol levels in healthy adults (Portale et al.
1986) and amelioration of secondary hyperparathyroidism in CRI patients
(Martinez et al. 1997).
The mechanism of action of the phosphate-binding agents is to form nonabsorbable compounds with phosphorous in the intestine lumen. Calcium salts
(carbonate or acetate) are the most widely used phosphate binders and have been
shown to be effective in lowering serum phosphorous and PTH levels in
experimental and clinical studies with different degrees of CRI (Kaye 1974,
Slatopolsky et al. 1986, Johnson et al. 2002, Coladonato 2005, Salusky 2006).
The use of phosphate-binders is well established in the clinical treatment of
CKD-BMD.
However, calcium salts contain a high proportion of elemental calcium and
from 20% to 30% of that amount is absorbed from the intestine. The resulting
increase in the calcium intake may affect bone metabolism in CKD (Salusky and
Goodman 2001). Particularly, there is a great concern that the use of calcium
salts as phosphate binders together with long-term imbalance between calcium
and phosphorous may contribute to the development and progression of
cardiovascular calcification in CKD patients (Goodman et al. 2000, Llach and
Fernandez 2003, Goldsmith et al. 2004, London et al. 2004).
26
3.6.2 Sevelamer
Sevelamer hydrochloride is a specifically designed hydrogel of cross-linked
poly-allylamine which is not absorbed and does not contain calcium or
aluminum (Burke et al. 1997, Plone et al. 2002). Sevelamer acts as a phosphate
binder in the intestine and the treatment has been associated with reduced
phosphorous and PTH levels in experimental CRI (Rosenbaum et al. 1997,
Nagano et al. 2001, Cozzolino et al. 2002, Cozzolino et al. 2003, Katsumata et
al. 2003, Nagano et al. 2003a, Nagano et al. 2003b). The efficacy of the
compound in phosphate binding seems fairly similar to calcium salts, but the use
of sevelamer is not associated with the above noted potentially harmful elevation
in calcium levels (Cozzolino et al. 2002, Cozzolino et al. 2003). These
experimental findings are in agreement with the data from clinical studies and
sevelamer has been considered a potentially valuable treatment option for
patients with CRI. (Chertow et al. 1997, Bleyer et al. 1999, Chertow et al. 1999a,
Chertow et al. 1999b, Slatopolsky et al. 1999, Chertow et al. 2002, Qunibi et al.
2004, Asmus et al. 2005, Raggi et al. 2005, Salusky 2006).
3.6.3 Calcitriol and paricalcitol
Calcitriol controls the growth of the parathyroid gland and inhibits the synthesis
and secretion of PTH in parathyroid cells (Llach and Velasquez Forero 2001,
Martin and Gonzalez 2001a) and therefore calcitriol has been used in the
treatment of secondary hyperparathyroidism. However, calcitriol also increases
the intestinal absorption of calcium and phosphorous and therefore inappropriate
treatment may contribute to the development of toxic hypercalcemia and
hyperphosphatemia (Llach and Velasquez Forero 2001, Llach and Fernandez
2003). Long-term calcitriol therapy may also contribute to the suppression of
bone turnover in CRI (Goodman et al. 1994, Salusky and Goodman 2001, Llach
and Fernandez 2003).
Certain synthetic analogs of calcitriol have been developed with an
expectation of exhibiting a similar suppressive activity on the parathyroid but
lesser potential to induce hypercalcemia and hyperphosphatemia than native
calcitriol. Currently the most salient such compounds are paricalcitol (19-nor1,25-(OH)2D2), 22-OCT (22-oxa-1,25-(OH)2D3,), alfacalcidiol (1- -OH-D3), and
doxercalciferol (1- -OH-D2). Paricalcitol has been shown to effectively suppress
CKD related increase in plasma PTH with less pronounced effects on serum
calcium and phosphorous levels than calcitriol in experimental (Brown et al.
1990, Slatopolsky et al. 1995, Takahashi et al. 1997, Brown et al. 2002,
Slatopolsky et al. 2002, Slatopolsky et al. 2003) and clinical studies (Llach et al.
1998, Martin et al. 1998a, Martin et al. 1998b, Martin and Gonzalez 2001b,
Sprague et al. 2001, Sprague et al. 2003). It has also been suggested that there
may be a general survival advantage when paricalcitol is compared to calcitriol
treatment (Drueke and McCarron 2003, Teng et al. 2003).
27
3.6.4 Bisphosphonates
Bisphosphonates are inhibitors of bone resorption. The compound concentrates
in bone wherein they are taken up by osteoclasts, and subsequently, acts by
inhibiting osteoclast activity. Bisphosphonate treatment has been shown to result
in reduced bone resorption and positive net bone balance (Rodan and Fleisch
1996, Fleisch 1998). Alendronate and risendronate are well proven agents in the
prevention and treatment of postmenopausal osteoporosis (Chavassieux et al.
1997, Cranney et al. 2002), but data on the fracture-prevention efficacy of
pamidronate is scarce.
Bisphosphonates seem to reduce bone turnover regardless of its cause, and
accordingly, bisphosphonates may be considered as potential treatment options
for CKD-BMD. Accordingly, it has been speculated that bisphosphonates could
be particularly favorable in the case of high bone turnover ROD and the
associated increased cortical porosity. However, although bisphosphonates are
widely used in the treatment of postmenopausal osteoporosis, it has been
speculated whether bisphosphonate treatment is associated with impaired
microdamage repair (Burr et al. 1997, Mashiba et al. 2000, Fan and Cunningham
2001) or with inappropriate increase in mineralization causing increased
brittleness (Meunier and Boivin 1997, Currey 2004). Reduction of bone turnover
may be a therapeutic objective in ROD, but currently the use of bisphosphonates
in ROD is not recommended (Rodan and Martin 2000, Fan and Cunningham
2001, Turner 2002, Klawansky et al. 2003, Massry et al. 2003, Langman et al.
2005).
Pamidronate (3-amino-1-hydroxypropylidene-1,1-biphosphonate) contains
nitrogen in the side chain and it is called an amino-substituted second-generation
bisphosphonate. The alteration in the side chain substitution influences the
affectivity of the bisphosphonate. The structure of bisphosphonates resembles
inorganic pyrophosphate but instead of the oxygen bridge (P-O-P) there are two
C-P bonds on the same carbon and two additional lateral side chains (P-C-P).
This structure is resistant to enzymatic hydrolysis and therefore bisphosphonates
are excreted unaltered by an active process in the kidneys (Rodan and Fleisch
1996).
3.7 Evidence for the efficacy of the prevention and treatment of
chronic kidney disease-bone and mineral disorder
An effective treatment for bone fragility should improve the biomechanical
properties of bone as organ, i.e. a combination of positive effects on the various
particular of bone structural strength (material properties, mineral mass,
geometry, etc.) that result in an increase in bone mechanical competence (Turner
2002).
In adenine-induced model of CRI in rats, Katsumata et al. (2003) evaluated
the effects of 4-week sevelamer treatment on bone histology and found that
28
cortical bone porosity was decreased. Fifty-two week treatment with calcium
salts has been associated with reduction in thoracic trabecular and cortical bone
attenuation evaluated by computed tomography as an index of bone mineral
density in dialysis patients (Raggi et al. 2005). Raggi et al. (2005) also compared
the effects of the treatment with calcium salts to a treatment with sevelamer, and
they concluded that the sevelamer treatment is associated with more beneficial
effect on both cortical and trabecular bone mineral amount than the treatment
with calcium salts. Asmus et al. (2005) reported that calcium salt administration
was associated with decreased trabecular vBMD while sevelamer treatment
prevented the change in a two-year follow up study comparing the effects of
sevelamer and calcium administration in dialysis patients. However, in this
study, no difference was observed in cBMD between the groups (Asmus et al.
2005). On the other hand, Salusky et al. (2005) analyzed bone histology in
dialysis patients treated with either sevelamer or calcium salts and concluded that
both treatments are equally efficient in controlling ROD. However, there is no
data on the effects of sevelamer treatment on any indices of bone strength in CRI
Paricalcitol has been shown to be approximately ten times less effective in
mobilizing calcium and phosphorous from bone than calcitriol (Finch et al.
1999). Finch et al. (2001) also suggested that it is unlikely that paricalcitol
treatment would have a deleterious effect on bone. Subsequently, it was also
shown that paricalcitol treatment efficiently reduced cortical porosity and
ameliorated trabecular bone pathology that is associated with ROD (Slatopolsky
et al. 2003). Calcitriol treatment has been shown to enhance bone mechanical
properties in CRI (Jablonski et al. 1995), but there is no data on the effects of
paricalcitol treatment on bone strength.
In moderate experimental CRI, ibandronate (bisphosphonate) treatment was
associated with decreased bone turnover and erosion with increased bone volume
(Geng et al. 2000). Data on the efficacy of bisphosphonates in patients with
predialysis CKD or in dialysis patients is scarce, presumably because
bisphosphonate treatment is not used in CKD patients. However, Torregrosa et
al. (2003) have shown that pamidronate treatment is associated with an increase
in aBMD in lumbar spine and proximal femur in dialysis patients, although the
study was devoid of a control group. There is no data on the effects of
bisphosphonate treatment on any indices of bone strength in CRI. As a remark,
bisphosphonate treatment has been shown to result in a positive effect on bone
mineral density when used in renal transplant patients (Fan et al. 2000, Grotz et
al. 2001, Coco et al. 2003, Fan et al. 2003, Haas et al. 2003). It has, however,
been suggested that in this context the positive effect of bisphosphonate
treatment is not long-lasting (Schwarz et al. 2004).
29
AIMS OF THE STUDY
Since the principal task of bones is to allow locomotion and bear incident loads
without breaking (Einhorn 1992, Parfitt 1998, van der Meulen et al. 2001), an
organ level approach was established to assess the skeletal changes associated
with CRI. Equally, the objective was to evaluate the effects of certain
pharmacological treatments on bone structure in CKD-BMD. Accordingly, the
specific objectives of the individual studies were the following:
1.
To characterize the changes in bone structure and mechanical
strength attributable to experimental CRI.
2.
To determine the effects of calcium carbonate and sevelamer
treatments on bone in the management of CRI associated
hyperphosphatemia.
3.
To evaluate the efficacy of vitamin D analogue paricalcitol
treatment on bone in CRI.
4.
To explore the effects of
administration on bone in CRI.
30
bisphosphonate
pamidronate
MATERIALS AND METHODS
1
Animals
Male rats of Sprague-Dawley strain were used in the experiments of this thesis.
The specific objective of this series of experiments was to assess the effects of
the disease per se (CRI) and the potential agents used to counteract these
deleterious effects on bone structure and strength. Being obvious that human
experimentation cannot generate a suitable sample for such analysis, it is readily
apparent that an appropriately designed animal experiment is reasoned and
justified approach (Marcus et al. 1996). All the experimental study designs were
approved by the Animal Experimentation Committee of the University of
Tampere, Finland, and the Provincial Government of Western Finland
Department of Social Affairs and Health, Finland. The studies conformed to the
NIH Guide for the Care and Use of Laboratory Animals.
Animals were housed two per cage in an animal laboratory at 22ºC
temperature with light cycle of 12 hours and fed standard laboratory food pellets
containing 0.9% calcium, 0.8% phosphate, 0.27% sodium, 0.2% magnesium,
0.6% potassium, 1 500 IU/kg 1,25(OH)2D3, and 12 550 kJ/kg energy (Lactamin;
AnalyCen, Lindköping, Sweden), except the in treatment groups of the study II
(see below), and in studies III and IV in which the calcium content of the diet
was 0.3% for all groups during the treatment period.
2
Nephrectomy
Animals were operated at the age of 8 weeks in all these experimental studies.
Operations were performed under 75 mg/kg ketamine (Parker-Davis
Scandinavia, Solna, Sweden) and 2.5 mg/kg diatzepam (Orion Pharma, Espoo,
Finland) anesthesia that was given intraperitoneally. Rats were randomly
subjected to either surgical bilateral kidney decapsulation (Sham) or subtotal 5/6
nephrectomy surgery, except for study IV, in which the rats were subjected to
unilateral nephrectomy instead of kidney decapsulation. In the 5/6 nephrectomy
operation, the entire right kidney was removed, along with the upper and lower
poles of the left kidney (Jolma et al. 2003, Pörsti et al. 2004). In the
decapsulation, the kidney capsule was surgically removed but kidneys were left
otherwise intact. The unilateral nephrectomy was performed as a Sham-operation
31
in study IV to create equal inflammatory conditions in all groups and
accordingly, to be able to eliminate the potential confounding influence of
surgical trauma on the renal function and drug pharmacodynamics. In unilateral
nephrectomy right kidney and small sections of the upper and lower poles of the
left kidney were removed. It was confirmed that unilateral nephrectomy did not
result in CRI. The surgical operations were followed by 4 week (II), 14 week
(additional separate assessment of effects of sevelamer treatment), 15 week (III),
and 14 week (IV) periods of disease progression.
3
Pharmacological treatments
3.1 Phosphate binding therapies
3.1.1 Calcium salts
In the study II, after the disease progression the animals were allocated to 3.0%
calcium diet (Lactamin; AnalyCen, Lindköping, Sweden) or continued on the
previous 0.3% calcium diet for the subsequent 8 weeks. The extra calcium was
supplied as calcium carbonate salt and otherwise the chows were identical to the
control groups (Figure 6).
3.1.2 Sevelamer
The effects of sevelamer treatment were assessed in an additional experiment
(Figure 6). Accordingly, after the disease progression the animals were allocated
to untreated and sevelamer treated, and the latter groups received diet containing
3% sevelamer (SEV; sevelamer hydrocholoride; Genzyme, Cambridge, MA,
USA) for 9 weeks.
3.2 Paricalcitol
In the study III, animals in the treatment group were given intraperitoneal
injections of paricalcitol (PAR; 19-nor-1,25-(OH)2D2; Abbott Laboratories, IL,
USA) 100 ng per rat three times a week for 12 weeks (Figure 6).
32
Figure 6. Flowchart of the individual studies. AS, additional study assessing the effects
of sevelamer and CRI.
33
3.3 Pamidronate
In the study IV, pamidronate (APD; 3-amino-1-hydroxypropylidene-1,1biphosphonate;
Novartis,
Switzerland)
was
administered
3 mg/kg
subcutaneously once a week for 8 weeks (Figure 6). The drug dose was chosen
on the basis of previous publications on APD treatment in rat, in which the
weekly dose has ranged between 0.5-10.5 mg/kg (Bourrin et al. 2002, Mayahara
and Sasaki 2003, Mekraldi et al. 2005).
4
Samples
4.1 Blood
At sacrifice, the carotid artery was cannulated and blood samples for creatinine,
urea, phosphate, and PTH were drawn into chilled tubes (II-IV). The tubes
contained ethylenediaminetetraacetic acid (EDTA) or heparin as anticoagulants,
as appropriate, after which the samples were centrifuged, and the plasma stored
at -70ºC until analysis. Plasma creatinine was measured by the colorimetric assay
according to Jaffe, urea by colorimetric enzymatic dry chemistry, and phosphate
by colorimetric dry chemistry (Vitros 950 analyzer, Johnson & Johnson Clinical
Diagnostics, NY, USA). PTH was measured using immunometric assay
(Immutopics, San Clemente, California, USA).
4.2 Kidney
Five-micrometer thick sections of aortic artery and kidney were stained with the
von Kossa method and processed for light microscope evaluation (II-IV). An
expert who was blinded to the experiments qualified all histology at x200
magnification. The calcification was counted from 10 random sections. Each
field was divided into 100 grids, and each grid containing foci of calcification
denoted one score. Total scores of each field were counted. The index of
calcification was determined for each rat by the mean score of the calculated 10
fields (Pörsti et al. 2004).
4.3 Bone
At sacrifice, both hindlimbs were excised and all the surrounding skin, muscle
and soft tissue were carefully excised to collect both femora. The femora were
then wrapped in saline soaked gauze bandage and stored at -20ºC in small, sealed
freezer bags. At the day of measurement, the bones were slowly thawed at room
34
temperature at least 15 h before actual mechanical testing and kept wrapped in
the saline-soaked gauzes except during measurements. For each rat, all
measurements were performed successively in the same order. For histological
determination, certain femora were placed in 70% ethanol solution after
excision.
4.3.1 Peripheral quantitative computed tomography
The cross-sections of the femoral necks, femoral midshaft and distal femur were
scanned with a commercial peripheral quantitative computed tomography
(pQCT) system (Stratec XCT Research M with Software version 5.40B; Stratec
Medizintechnik, Birkenfield, Germany) (Figure 7).
Figure 7. A longitudinal slice of rat proximal femur (left) and corresponding
representative (magnified) cross-sectional slices of femoral midshaft (bottom right) and
femoral neck (top right).
35
For femoral neck analysis, the femur was inserted into a special plastic tube
with femoral neck in axial direction. The scan line was adjusted to midneck
using scout view option of the software and one cross-sectional image was
scanned with a voxel size of 0.070 x 0.070 x 0.5 mm3 and scan speed 3.0 mm/s.
Total cross-sectional area (tCSA), total bone mineral content (BMC), and total
volumetric bone mineral density (vBMD) were recorded as given by the pQCT
software. Each bone was measured twice with repositioning of the sample
between the measurements, and the average of the measurements was used as the
variable. In our laboratory, the average (root-mean-square) coefficient of
variation (CVrms) were 3.9% for tCSA and 2.1% for vBMD, respectively
(Pajamäki et al. 2003).
For femoral midshaft and distal femur analysis, the femur was inserted into a
specially constructed plastic tube. One cross-sectional slice from each bone was
scanned at midshaft (at 50% of the measured femur length) and distal femur
(10% of the femur length, measured from the distal end) with a voxel size, 0.070
x 0.070 x 0.5 mm3 and scan speed 3.0 mm/s. The tCSA, cortical cross-sectional
area (cCSA), cortical volumetric bone mineral density (cBMD), and BMC of the
femoral midshaft, and tCSA and BMC of the distal femur were recorded as given
by the pQCT software. In our laboratory, the CVrms in the femoral midshaft were
0.9% for the tCSA, 1.5% for the cCSA, and 0.6% for the cBMD (Pajamäki et al.
2003).
4.3.2 Geometrical measurements
A digimatic caliper (Mitutoyo 500, Andover, United Kingdom) and pQCT image
analysis, both providing a resolution of 0.01 mm, were used to determine the
bone dimensions and geometry. The length of the femur was measured from the
tip of the greater trochanter to the intercondylar notch. The outside diameters of
the femoral shaft were measured in the mediolateral (MLOUTSIDE) and
anteroposterior (APOUTISDE) directions. Likewise, the orthogonal inside diameters
(MLINSIDE, APINSIDE) of the medullary canal of the femoral shaft were determined
at the break line after three-point bending of the shaft (described in the
subsequent text). Additionally, the femoral shaft was considered a hollow elliptic
shaped structure, and following geometric indices were determined: the average
cortical wall thickness as given by the pQCT software; cortical wall thickness
(CWT) in ML (CWTML) and AP (CWT AP) directions as CWTML = (MLOUTSIDEMLINSIDE)/2 and CWTAP = (APOUTSIDE-APINSIDE)/2; principal cross-sectional
moments of inertia (CSMI MAX and CSMIMIN) and the polar section modulus
(SSIPOLAR) according to common engineering principles. In our laboratory, CVrms
for repeated measurements of femur dimensions range from 0.2% (femur length)
to 4.0% (midshaft inside width) (Järvinen et al. 1998a, Järvinen et al. 1998b).
36
4.3.3 Dual-energy X-ray absorptiometry
The total bone mineral content of the whole femur (TBMC) was measured with
dual-energy X-ray absorptiometry (DXA) (II, IV). All measurements were done
using a standard DXA scanner (Norland XR-26; Norland Corporation, Fort
Atkinson, Wisconsin, USA) with additional scanner modification for small
animal measurements (Sievänen et al. 1994a). The standard general scan option
(version 2.2.2) was applied for scanning and the scan data was analyzed with the
research scan option (version 2.5.2) Pixel spacing was 0.5 x 0.5 mm and scan
speed 10 mm/s. For scanning, the femur was placed at a constant location of the
scan table on its posterior surface. The baseline point was located on the soft
tissue equivalent. The scanner was calibrated daily according to manufacturer’s
recommendations, and the scanner performance was controlled by the quality
assurance protocol of our laboratory (Sievänen et al. 1994b). In our laboratory,
the precision (CVrms) for repeated measurements of TBMC of rat femur is 1.6%
(Järvinen et al. 1998a, Järvinen et al. 1998b).
4.3.4 Bone histomorphometry
The histomorphometric measurements were performed using the OsteoMeasure
Analysis System (OsteoMetrics, Atlanta GA, USA) (IV). The distal femoral
metaphysis was dehydrated and embedded without demineralization. The tissue
was sectioned at 5 microns and the sections stained with toluidine blue. An area
of 7.5 mm2, immediately distal to the primary spongiosa and excluding the
cortical bone margins, was analyzed to obtain the following static bone
measurements and calculated values as described (Turner et al. 1998): bone
volume normalized to tissue volume (BV/TV), trabecular number, trabecular
thickness, and trabecular separation (Turner et al. 1998).
4.3.5 Biomechanical testing
Femora were mechanically tested using a Lloyd material testing device (LR5K,
J.J. Lloyd Instruments, Southampton, United Kingdom). Anteroposterior threepoint bending, mediolateral three-point bending, and compression of femoral
neck were performed (Sogaard et al. 1994, Järvinen et al. 1998a, Järvinen et al.
1998b, Leppänen et al. 2006).
For the anteroposterior three-point bending, the femur was placed on its
posterior surface on the supports of the bending apparatus (Figure 8A). For each
bone, these supports were placed individually just distal to the trochanter minor
and the other just proximal to the condyles of the femur. Before the actual
testing, a small stabilizing preload was applied on the superior (anterior) surface
of the femur at a rate of 0.1 mm/s using a crossbar fixture (a plate with rounded
edges of 10 mm diameter). The load was then applied at a rate of 1.0 mm/s until
37
the failure of the specimen, and the breaking load and energy absorption were
determined.
For the mediolateral three-point bending, the femur was placed on its lateral
surface on the lower supports of the bending apparatus (Figure 8B). For each
bone, these supports were placed individually so that one was under the
trochanter major and the other under the distal femur. To prevent the otherwise
unavoidable twisting of the bone to the anteroposterior position during loading,
the intercondylar fossa of femur was gently pressed between the blades of blunt
pliers tightly attached to the bending apparatus. The preloading and actual
loading were carried out as described above (Leppänen et al. 2006).
Figure 8. (A) Three-point bending of the rat femoral midshaft in anteroposterior
direction; (B) three-point bending of the femoral midshaft in mediolateral direction; and
(C) compression test of the femoral neck.
After the three-point bending of the femoral shaft, the proximal part of each
specimen was collected and femoral neck was subjected to compression test
using the materials testing machine. In the femoral neck compression test, the
proximal half of each femur was mounted in a specially constructed fixation
device (Sogaard et al. 1994). The specimen was then placed under the materials
testing device, and the vertical load was applied to the top of the femoral head
using a brass crossbar until failure of the femoral neck (Figure 8C). The
38
preloading and actual loading were carried out as described above. The breaking
load was determined from a load-deformation curve. In our laboratory the CVrms
of the breaking load for anteroposterior three-point bending, mediolateral threepoint bending and femoral neck compression are 5.0%, 3.8%, and 7.6%,
respectively (Järvinen et al. 1998a, Järvinen et al. 1998b, Leppänen et al. 2006).
5
Statistical analysis
All measurements and analysis were blinded to the group assignments. The
descriptive variables were reported as mean and standard deviations, and
measurement results were reported as mean and standard deviations, standard
error of mean or 95% confidence intervals. Two-way analysis of variance
(ANOVA) was used to determine the effects of CRI and the effects of calcium
salt, sevelamer, and pamidronate treatment. One-way ANOVA was used to
determine the effect of paricalcitol treatment in CRI. Differences were
considered significant when P < 0.05. The predominant stresses in the long
bones of the lower extremities stem from weight bearing, concomitant bending,
and torsional loading produced by the muscles attached to long bones (Burr
1997, Frost 1997). Accordingly, to eliminate the inherent bias arising from
comparisons between study groups that may differ in body weight and size (e.g.
uremic versus control), data pertaining to bone mechanical competence was
equalized to in terms of the animal’s apparent loading environment by using the
body weight and femoral length of each rat as covariates in the analysis (Järvinen
et al. 2003a, Järvinen et al. 2003b, Pajamäki et al. 2003).
39
RESULTS
The surgical subtotal nephrectomy was found effective in inducing a disease
state mimicking that of typical CRI in humans, characterized by elevated plasma
creatinine, urea, phosphorous and PTH levels. These values are generally used as
indices of impairment of renal function and to approximate the prevailing
metabolic disorder, and accordingly, these values may be considered to reflect
the severity of CRI. In the histological analyses of kidney tissue, the CRI rats
showed moderate increases in glomerulosclerosis, interstitial damage and kidney
calcifications when compared with the Sham rats. Thus, the morphology of the
kidneys confirmed the CRI which was indicated by changes in serum chemistry.
The determination of whole bone strength effectively covers virtually all of the
individual variability that may be instantaneously, sporadically or permanently
present in structural particulars (size and shape of the bone, cortical thickness
and specific cortical geometry, trabecular architecture, etc.), providing the
ultimate assay on bone functional capacity (I). Accordingly, an organ-level
assessment was reasoned approach to explore the effects of experimental CRI on
bone (I).
The thesis includes data that has not yet been published, since a discrete
additional experimental study was carried out to assess the effects of
hyperphosphatemia treatment with sevelamer administration. To make the results
from the individual studies commensurate with each other, the severity of the
renal condition must be addressed, and accordingly, indices of renal function
were determined by serum biochemistry in each study (Figure 9). When these
characteristics are combined with the mortality data (Figure 6) over the course of
the disease, it is possible to approximate the severity of CRI in each study.
Accordingly, studies II and IV may be considered to represent mild-to-moderate
CRI, whereas study III and the additional study on effects of sevelamer treatment
are models of severely advanced CRI.
40
Figure 9. Characteristics of serum biochemistry in each study (roman numerals, II-IV;
AS, additional study assessing the effects of sevelamer and CRI). Plasma levels of (A)
creatinine; (B) urea; (C) phosphate; and (D) PTH. Bars represent the mean in untreated
CRI group of each study. The dashed line represents the mean value in Sham and solid
lines limit the range of standard deviation.
1
The effects of experimental chronic renal
insufficiency on bone structure and strength
In this structurally oriented (I) series of experiments the CRI-induced changes on
bone were assessed and CRI was associated with deterioration of bone in all
experiments. Already mild-to-moderate CRI induced a significant decrease in the
BMC of the whole femur, a deficit that ranged from -4.8% (data from the
additional study on effects of sevelamer treatment) to -6.2% (II). In the
evaluation of the trabecular structure in the distal femoral metaphysis, the
present model of CRI resulted in the characteristic histomorphometric features of
ROD, including increased bone turnover and peritrabecular fibrosis (IV). In the
site-specific analysis of the femur, it was possible to depict the extent to which
the vBMD or cBMD, cross-sectional geometry, BMC, and breaking load of the
different regions of rat femur were affected by the disease in all experiments.
CRI had the most pronounced effect on vBMD. In the femoral neck the
changes ranged from -6.5% to -11% (Figures 10A, 11C, and 12A) and in mildto-moderate CRI a simultaneous increase in tCSA was observed (Figure 10B).
To further examine the relationship between the renal function and bone
geometry of the femoral neck, additional 25 Sham and 7 subtotally
nephrectomized animals (NTX) were pooled with the data from study II. The
added Sham and NTX animals showed similar changes in plasma creatinine and
urea to the previous results. In the pooled analyses, the NTX rats showed
corresponding increases of plasma creatinine and urea as above, and reduced
41
bone mineral density and increased cross-sectional area in the femoral neck
(Figures 13A and 13B). However, no differences were observed in the breaking
load, while vBMD of the femoral neck showed a significant inverse correlation
with tCSA in both Sham and NTX rats (Figures 13C and 13D). The mechanical
strength of femoral neck was maintained in mild-to-moderate CRI (Figure 10D)
(II). However, in groups with more advanced CRI a decrease in breaking load up
to -16% was observed (III, and data from the additional study on effects of
sevelamer treatment) (Figures 11F and 12D).
Figure 10. The effects of CRI and calcium treatment (Ca) in femoral neck: (A) vBMD;
(B) tCSA; (C) BMC; and (D) breaking load. Bars represent the mean + SD. Significant
differences are indicated: a P < 0.01 CRI-groups vs. Sham-groups; b P < 0.05 CRI vs.
Sham; c P < 0.001 CRI+Ca vs. Sham+Ca; d P < 0.05 CRI groups vs. Sham-groups.
The results from the analysis of femoral midshaft showed a decrease in
cBMD ranging from -1.2% to -6.6% (Figures 14A and 15A). No CRI-induced
differences were observed in tCSA or cCSA between the study groups (Figures
14B, 14C, 15B, 15C, and 16), and CRI was associated with a decrease in BMC
(Figures 14D and 15E). However, a localized thinning of the cortex in the medial
and lateral aspects of the femoral midshaft was evidenced in the determination of
CWT in association with severe CRI (data from the additional study on effects of
sevelamer treatment) (Figure 14F). Bone strength was evidently maintained in
mild-to-moderate CRI (Figure 15F), whereas the more advanced CRI was
associated with a reduction in the breaking load of the femoral midshaft (Figures
42
14G and 14H). In distal femur, mild-to-moderate CRI induced histological
features of ROD, but no changes were detected in amount of bone, or in the
trabecular thickness or number.
Figure 11. The effects of CRI and sevelamer treatment (Sev) in femoral neck: (A)
width in mediolateral direction; (B) thickness in cephalocaudal direction; (C) vBMD;
(D) tCSA; (E) BMC; and (F) breaking load. Bars represent the mean + SD. Significant
differences are indicated: a P < 0.05 vs. Sham, b P < 0.05 vs. CRI, c P < 0.05 CRI-groups
vs. Sham-groups.
2
The effects of the treatment of hyperphosphatemia
on bone
CRI results in a progressive hyperphosphatemia (Figure 9C), a condition that is
associated with the above described detrimental effects on bone. Accordingly,
the effects of two different dietary phosphorous binding agents, calcium
carbonate and sevelamer hydrochloride, on bone structure were investigated in
43
two separate studies (II, and data from the additional study on the effects of
sevelamer treatment). In study II, the administration of calcium salts in
experimental CRI was efficient in reducing phosphorous levels and resulted in
anticipated suppression of PTH levels. In terms of bone mineral content, calcium
administration revoked the CRI-induced decrease in BMC of the whole femur. In
this study calcium administration was not associated with any changes in the
site-specific analysis of vBMD, tCSA or BMC in the femoral neck (Figures 10AC) or femoral midshaft, and consistently, no differences were observed in the
evaluation of morphological indices (CSMIMAX, CSMIMIN and SSIPOLAR) of the
femoral midshaft. Moreover, the effects of calcium administration did not induce
any changes in breaking load of the femoral neck (Figure 10D) or femoral
midshaft.
To eliminate the possible effects of increased dietary calcium, the effects of
sevelamer treatment in advanced CRI were evaluated in an additional
experiment. In this study the effects of sevelamer treatment on bone were clearly
evident, although elevated phosphorous (hyperphosphatemia) and PTH levels
were observed in the end of the study. Sevelamer administration attenuated the
CRI-induced decrease in vBMD of the femoral neck (Figure 11C) and in cBMD
of the femoral midshaft (Figure 14C). In addition, sevelamer administration was
also associated with an increased amount of cortical bone in femoral midshaft
(Figures 14C-F). As a result, we observed that sevelamer treatment ameliorated
the deterioration of bone strength in CRI (Figures 11F, 14G and 14H).
Figure 12. The effects of CRI and pamidronate (PAR) treatment in femoral neck: (A)
vBMD; (B) tCSA; (C) BMC; and (D) breaking load. Bars represent the mean + SD.
Significant differences are indicated: a P < 0.05 vs. Sham, b P < 0.05 vs. CRI.
44
Figure 13. Scatter plots of (A) vBMD and (B) tCSA in relation to plasma urea (dashed
line at 7.5 mmol/l denotes highest urea concentration of normal (Sham) rats), and scatter
plots and correlations of vBMD and tCSA in (C) 34 Sham rats and (D) 17 5/6
nephrectomized (NTX) rats.
3
The effects of paricalcitol treatment on bone
In study III, paricalcitol treatment significantly restrained the CRI-induced
increase in PTH levels without any effect on calcium or phosphorous levels. CRI
is associated with decreased calcitriol levels, and as anticipated, paricalcitol
administration suppressed calcitriol levels further. In the bone analysis,
paricalcitol treatment was associated with a beneficial effect on CRI-induced
decreases in vBMD of the femoral neck (Figure 12A) and in cBMD of the
femoral midshaft. Most importantly, paricalcitol treatment completely prevented
the CRI associated decrease in breaking load in the femoral neck (Figure 12D).
45
Figure 14. The effects of CRI and sevelamer treatment (Sev) in femoral midshaft: (A)
cBMD; (B) tCSA; (C) cCSA; (D) BMC; (E) cortical thickness in anteroposterior
direction; (F) cortical thickness in mediolateral direction; (G) breaking load in
anteroposterior and (H) mediolateral direction. Bars represent the mean + SD.
Significant differences are indicated: a P < 0.05 vs. Sham, b P < 0.05 vs. CRI, c P < 0.05
sevelamer treatment groups vs. untreated groups.
46
4
The effects of pamidronate administration on bone in
experimental chronic renal insufficiency
According to the general mechanisms of the action of bisphosphonates, the
effects of pamidronate treatment on bone were evident although the general
characteristics of CRI in serum biochemistry (creatinine, urea, phosphate and
PTH) were not affected (IV). Pamidronate treatment resulted in an increase in
total BMC of whole femur, an effect that was observed in both Sham and CRI
groups, but intriguingly, the treatment effect was substantially greater in the CRI
group. In histological analysis of distal femoral metaphysis, pamidronate
treatment was shown to result in an increase in relative bone volume (BV/TV) in
both the CRI and Sham groups. This increase in BV/TV is most likely
attributable to increases in trabecular thickness and number, and consequently, a
decrease in trabecular separation. However, also relative volume of osteoid was
increased in association with pamidronate treatment (histomorphometrical data
not shown).
In the structural analysis of the femoral midshaft, the most pronounced effect
of pamidronate treatment was an increase in the amount of cortical bone
particularly in the CRI group (Figures 13C and 13D). Increased cCSA together
with decreased tCSA and reduced inside diameters of the marrow cavity
(marrow area) indicate that the net bone gain was located on the endosteal
surface (Figure 16). As a result, an increase in BMC was observed but no
differences were detected in the cBMD between the treated and untreated groups
(Figures 15A and 15E). In mild-to-moderate CRI, pamidronate treatment was not
associated with changes in bone strength (Figure 15F).
47
Figure 15. The effects of CRI and pamidronate administration (APD) in femoral
midshaft: (A) cBMD; (B) tCSA; (C) cCSA; (D) average cortical thickness; (E) tBMC;
(F) breaking load in anteroposterior direction. Bars represent the mean + SD. Significant
differences are indicated: a P > 0.01 CRI-groups vs. Sham-groups; b P < 0.05 CRI vs.
Sham; c P < 0.05 pamidronate treatment groups vs. untreated groups; d P < 0.05
CRI+APD vs. CRI; e P < 0.05 for interaction between CRI and pamidronate treatment.
48
Figure 16. The effects of CRI and pamidronate administration (APD) on marrow cavity
(medullary canal) dimensions in femoral midshaft. Cross-section of bone is depicted
white (in Sham) or grey (in CRI), and effect of APD treatment is black (in both). APD
treatment effect P < 0.05 in both mediolateral (vertically in picture) and anteroposterior
directions (horizontally in picture); NS, statistically non-significant difference.
49
DISCUSSION
The rationale for this thesis was prompted by the fact that traditionally the
research on ROD has been mostly focused on non-mechanical functions of bones
and on material properties of trabecular bone, although the increased
susceptibility to fractures is the most important clinical manifestation of all
metabolic bone disorders (Parfitt 1998). Accordingly, albeit the increased risk of
fractures is recognized as a skeletal complication of CRI, the assessment of the
mechanical properties of bone structure has not been properly included in the
definitions of ROD and CKD-MBD (Massry et al. 2003, Moe et al. 2006,
Sprague 2007). Furthermore, analysis of cortical bone has been almost
completely ignored from the context of ROD despite the fact that cortical bone is
the main determinant of bone strength (Ferretti et al. 1995, Augat et al. 1996,
Parfitt 1998, Massry et al. 2003, Moe et al. 2006). Inspired by this apparent
discrepancy, an organ level structural approach was established to evaluate the
consequences of experimental CRI on bone (I). Accordingly, the prime focus of
the analysis in this thesis was on the evaluation of mechanical competence of
bone (I).
The first main objective of the thesis was to assess the effects of CRI on bone
structure, and broaden the knowledge of changes in bone strength and its main
determinants in CRI (I). This present study confirms that CRI is associated with
detrimental effects on bone and suggest that the mechanical competence of bone
is maintained despite evident ROD associated with mild-to-moderate CRI. The
present model of experimental mild-to-moderate CRI induced a loss of cortical
bone, but there were no differences in mechanical testing of bone strength in
femoral midshaft or femoral neck (II, IV). However, in severely advanced CRI
(data from the additional study on effects of sevelamer treatment), the
mechanical competence of bone was eventually deteriorated. These findings are
in agreement with current understanding of the CKD-BMD, and additionally, the
results suggest that bones possess quite a substantial capacity to resist the
detrimental effects associated with CRI before actual structural decay occurs.
The second main objective of the study was to evaluate the skeletal effects of
three pharmacological treatments that are currently used in the clinical
management of CRI. The treatments were chosen to represent the cornerstones
of the general pharmacological treatment of CRI (Elder 2002, Emmett 2004,
Martin et al. 2004, Friedman 2005): alleviation of hyperphosphatemia with two
distinct phosphate binding agents, calcium carbonate (II) and sevelamer (data
from the additional study), and amelioration of secondary hyperparathyroidism
with vitamin D analogue paricalcitol (III). Concerning the efficacy of the
50
treatment with calcium salts in prevention of the deleterious effects of CRI, the
high calcium diet was found to increase the total BMC of the whole femur,
although the only statistically significant effect of treatment was observed in the
vBMD of the femoral neck (II). Nevertheless, there is a concern of so called
adynamic bone in association with administration of therapeutic doses of
calcium salts, because high calcium intake may oversuppress PTH levels, and
that may have adverse consequences on bone (Salusky and Goodman 2001).
Although a significant suppression of PTH levels below the physiological range
was observed, calcium salt administration treatment was not associated with any
differences in bone strength in Sham or CRI groups (II). Similarly to the calcium
salts, sevelamer also binds phosphate in the intestine, but unlike the calcium
salts, sevelamer is not absorbed and therefore it is not supposed to have any
direct effect on PTH levels (Plone et al. 2002). In the present study, the treatment
with sevelamer was shown to prevent CRI-induced decreases in cBMD, BMC,
and bone strength in the femoral midshaft, and the beneficial effect of sevelamer
treatment was also evident in the femoral neck (data from additional study on the
effects of sevelamer treatment). The known mechanism of sevelamer is to act as
a phosphate binder, and as no differences were detected in the serum
phosphorous levels between the treated and untreated groups at the end of the
study, the results of the present study suggest that the beneficial effect of
sevelamer was attributable to its action along the course of CRI. In previous
experimental studies, sevelamer has been shown to decrease serum phosphorous
(Rosenbaum et al. 1997, Nagano et al. 2001, Cozzolino et al. 2002, Cozzolino et
al. 2003, Katsumata et al. 2003, Nagano et al. 2003a, Nagano et al. 2003b), and
the lack of effect in the end of the study may be explained by the very severely
advanced CRI, in which the hyperphosphatemia had become too grave to
overcome. Combining the similar findings of calcium carbonate and sevelamer
treatments, it may be concluded that the control of hyperphosphatemia is
relevant also in terms of prevention of the bone lesions and the use of dietary
calcium carbonate or sevelamer as a phosphate binding agents seem viable
treatment options. However, the comparison between the efficacy of calcium
carbonate and sevelamer treatment was not possible, because the severity of the
CRI and ROD was different in study II and in the additional study on the effects
of sevelamer treatment.
Apart from the prevention of phosphate absorption, the results of study III
show that amelioration of secondary hyperparathyroidism with paricalcitol
decreased PTH levels and prevented the CRI-induced decrease in cBMD of the
femoral midshaft and vBMD of the femoral neck. These findings complement
the previously published results on the effect of paricalcitol on bone histology
(Slatopolsky et al. 2003). The results of the present study further show that the
treatment prevented the CRI-induced loss on bone strength in the femoral neck.
When considering together the effects of controlling hyperphosphatemia by
hindering phosphate absorption and the effects of inhibition of parathyroid
function by vitamin D analogue, the results not only highlight the significance of
high phosphorous levels in the development of bone abnormalities, but also
51
pinpoint that PTH is the essential mediator of these effects in ROD. In essence, it
may be concluded that the general management of the hyperphosphatemia and
secondary hyperparathyroidism with these interventions was proven to be
beneficial to bone in present experimental model of CRI.
When considering the effects of pamidronate treatment on bone in CRI, it
needs be pointed out that bisphosphonates are not widely used, or even currently
recommended, treatment options of ROD and thus the treatment does not
represent clinical management of CRI. However, the use of bisphosphonates in
CRI population has been considered (Rodan and Martin 2000, Fan and
Cunningham 2001, Turner 2002, Klawansky et al. 2003, Massry et al. 2003,
Langman et al. 2005). In study IV, pamidronate treatment was associated with an
increase in BMC and cCSA, and the effects were particularly evident in CRI
group. This interaction suggests that CRI may augment the effect of
bisphosphonate treatment on bone. Detailed analysis of the cross-sectional
geometry of femoral midshaft showed that the amount of endosteal bone was
increased after pamidronate treatment (IV). When combined with the findings of
increased BMC and BV/TV in the distal femur, it may be suggested that the
effect of pamidronate was pronounced in bone surfaces that are metabolically
most active. However, there were no differences between study groups in
breaking load of the femoral midshaft and the plausible explanation for this is
that the apposition of bone on endosteal surface has only a minor influence on
the mechanical strength of the structure (I, IV) (Turner and Burr 1993, Turner
2002).
In addition to increased cortical porosity, another important factor for
impaired cortical bone structure in ROD is generalized cortical thinning. An
apparent mechanism for thinner cortices is that the increase in net endocortical
resorption exceeds the slow rate of net periosteal apposition (Parfitt 2003). It has
been suggested that ROD represents a spectrum of disorders of bone turnover
that range from high- to low-turnover skeletal lesions (Hruska and Teitelbaum
1995, Salusky and Goodman 1995) and the complex outcome is reflected in the
wide scatter of bone abnormalities which have been associated with ROD
according to the current literature. However, it has also been discussed that the
decrease in cortical thickness is the major determinant of bone strength
regardless of subsequent development and regardless of the histological
classification or the disorder of bone remodeling that may be present at the time
the fracture occurs (Parfitt 1998, Schober et al. 1998).
In general, there are numerous studies which have reported associations
between bone mineral density values and bone fragility or even with incidence
bone fractures. This has been adapted also to the current literature on ROD, but
the use of the term “bone density” has been somewhat vague and confusing
(Sievänen 2000). Firstly, the distinction between the different definitions of bone
density has not been explicit. DXA is a widely used technique to assess BMC
and bone density, and thus derived measures of density may be broadly termed
as areal density values. In this thesis, the bone density results refer uniformly to
actual volumetric cBMD or vBMD values which are determined by pQCT and
52
accordingly defined as mass per unit volume (mg/cm3), not per unit area
(mg/cm2). Secondly, especially in ROD, the interpretation of numerical changes
in areal density values is uncertain, because the calculations are based on
assumptions that changes in bone three-dimensional structure and material
density are known or predictable. In ROD, these underlying assumptions may no
longer hold since the known effects are divergent on cortical and trabecular
bone, and the overall pattern of changes on bone structure is complex and
unpredictable for the present (Parfitt 1998). Change in such derived areal density
value may thus arise from any undefined change(s) in cortical or trabecular bone
tissue properties, or from geometrical alteration, and the eventual mechanical
significance of the occurrence might not correlate at all with the numerical value
(Parfitt 1998, van der Meulen et al. 2001, Martin et al. 2004). On the other hand,
the ostensibly unchanged areal density value may conceal some distinctive
changes resulting from possible redistribution of bone mineral between cortical
and trabecular compartments. Therefore, particularly in ROD, some changes in
bone may have influence on bone strength (I) although those changes are not
reflected by DXA assessment of bone (van der Meulen et al. 2001, Elder 2002,
Turner 2002). Nevertheless, the K/DOQI guidelines recommend the use of DXA
in CKD patients for the evaluation of the risk factors for fractures (Massry et al.
2003), although the role of DXA measurements in assessment of ROD is not
well established (Martin et al. 2004).
In previous experimental studies the group of Jablonski et al. have shown that
the material properties of bones were quite well preserved even 240 days after
5/6 nephrectomy (Jablonski et al. 1993, Jablonski et al. 1994, Jablonski et al.
1995). Further, Turner et al. (1996) have shown that in rats with advanced CRI
the bone strength is deteriorated. Thus the finding of reduced bone mineral
density with decreased bone strength in response to advanced CRI in the present
study is in accordance with previous experimental findings. These are also in
agreement with the clinical data on incidence of fractures in patients with predialysis CRI (Ensrud et al. 2007) and in end-stage renal disease patients (Atsumi
et al. 1999, Alem et al. 2000, Coco and Rush 2000, Stehman-Breen et al. 2000,
Jamal et al. 2006a).
However, although the finding of decreased bone strength may be associated
with increased incidence of fractures, the relationship may not be as
straightforward as it appears. It should be recalled that that bone mineral density
as itself is only a modest risk factor of fractures - some 85% of the contribution
to the rise in fracture risk with age is unrelated to bone mineral density (Wilkin
and Devendra 2001). According to several well-designed clinical studies on risk
factors of fractures among elderly people (Hayes et al. 1993, Nevitt and
Cummings 1993, Schwartz et al. 1998, Parkkari et al. 1999, Carter et al. 2000,
Palvanen et al. 2000, Wei et al. 2001, Lee et al. 2002, Robinovitch et al. 2003),
falling with its determinants – not the decrease in bone mineral density – has
been shown to be the strongest single risk factor for a fracture. When a person
falls, the type and severity of falling are crucial in determining whether or not a
53
fracture occurs (Hayes et al. 1993, Parkkari et al. 1999, Carter et al. 2000,
Palvanen et al. 2000, Wei et al. 2001, Robinovitch et al. 2003).
Therefore, although reduced bone mineral density and elevated levels of
PTH, and both bone formation and resorption, have been detected already at an
early stage of CRI, the most likely explanation for increased fracture rates in
association with CRI is the more frequent occurrence of falls among CRI
patients (Roberts et al. 2003, Cook and Jassal 2005, Desmet et al. 2005). The
etiology of falls is multifactorial (Kannus et al. 2002, Tinetti 2003, Kannus et al.
2005) and it is plausible that prevalence of some general risk factors of falling is
higher among the CRI population. These risk factors may include general frailty
with other chronic diseases, gait disabilities, peripheral neuropathy, impaired
muscle strength, peripheral vascular disease, and vision impairment (StehmanBreen 2004, Jamal et al. 2006b). According to Desmet et al. (2005), particularly
diabetes, polypharmacy and failed walking test are independent risk factors for
falling in dialysis population. And further, epidemiological studies showing
increased fracture risk in dialysis population show also that body mass index
(BMI), age, gender, race and presence of peripheral vascular disease are
independently associated with fracture risk, while PTH, phosphate and calcium
levels do not predict fracture risk (Alem et al. 2000, Stehman-Breen et al. 2000,
Jamal et al. 2006b).
Assessment of bone biopsy is a widely used method to characterize ROD in
clinical and experimental studies. The histomorphometrical data from study IV
showed gross changes in association with pamidronate treatment. According to
the basic principles of bone biology, the outcome of these histological changes,
regardless of the cause, should ultimately translate into changes in bone
structure, and possibly, but not necessarily, altered mechanical competence of
bone (I). In the data from study IV, the histomorphometrical findings were
translated into changes in BMC, but however, no differences were observed
between the study groups in strength of femoral midshaft. Thus, this data
supports the claims (van der Meulen et al. 2001) that although the
characterization of the changes in bone histomorphometry provides information
on bone turnover, but the interpretation of these findings in terms of bone
strength is inappropriate if the outcome, the strength of whole bone as a
structure, remains unknown (I). Geometrical variables and mineral properties
may be considered as determinants or surrogate measures of bone strength (I),
but the relationship between whole bone strength and these underlying
determinants is complex (Ruff et al. 2006). Furthermore, particularly in ROD,
the relationship between bone strength and changes in bone geometry and
mineral properties is unclear, because the changes in bone tissue properties are
diverse and unpredictable (Parfitt 1998). Determination of bone material texture
properties (e.g. histomorphometrical variables) and structural properties (e.g.,
geometry or cortical wall thickness) are essential elements of the characterization
of ROD, but when assessing the efficiency of certain intervention on bone
structure in ROD, the chances should be evaluated with data on to the total
outcome, which is the strength of the bone structure (I) (Turner 2002).
54
Kidney Disease: Improving Global Outcomes (KDIGO) has stated that in
CKD the contribution of bone mass to bone strength is of uncertain value and
emphasized the assessment of bone strength (Moe et al. 2006). It is also possible
that bone mineral density values altogether may be quite misleading in ROD, in
addition to the above mentioned matter, if bone resorption is being replaced with
newly formed bone which has defective mechanical properties because of
inappropriate microstructure (Parfitt 2003). These statements further emphasize
the importance of mechanical testing of bone structure in ROD. Along these
lines, Hernandes and Keaveny (2006) have recently stated that fracture is a
mechanical event and necessitates that any relevant change in bone structure
must change bone biomechanical performance. In this study, mechanical testing
of a whole bone was used to determine directly and unambiguously the structural
strength of bone as an organ, and as van der Meulen et al. (2001) have discussed,
there is no alternative to mechanical testing of whole bones in terms of bone
strength (I).
55
SUMMARY AND CONCLUSIONS
1.
Experimental CRI was associated with gradual deterioration of
bone structure and the most evident detrimental changes were
observed in the measures of cBMD in the femoral midshaft and
vBMD in the femoral neck. The mechanical strength of bone
structure was maintained in mild-to-moderate CRI (II, IV), but
advanced CRI was associated with detrimental changes in bone
strength (data from the additional study on effects of sevelamer
treatment).
2.
The alleviation of CRI-induced hyperphosphatemia through the use
of calcium carbonate (II) and sevelamer (data from the additional
study on effects of sevelamer treatment) were found to be effective
in the prevention of deleterious changes associated with CRI in
femoral midshaft and in femoral neck.
3.
Administration of vitamin D analogue paricalcitol was observed to
prevent the loss of bone mineral and bone strength in femoral neck
in CRI (III).
4.
Bisphosphonate pamidronate was found to increase BMC in
endosteal surface of femoral midshaft and in distal femur,
particularly in association with CRI, while no differences were
observed in bone strength (IV).
56
ACKNOWLEDGEMENTS
This study was carried out at the Department of Surgery, Medical School,
University of Tampere, Tampere University Hospital, and UKK-institute,
Tampere, Finland, from 2000 to 2007.
Firstly, I want to thank Markku Järvinen, M.D., Professor and Head of the
Department of Surgery, University of Tampere, for his standing efforts to
establish the most favorable studying and working environment which I have
enjoyed over the years.
I have been privileged to work under the guidance of brilliant supervisors
who have vast knowledge combined with motivating attitude. I express my
professional and personal gratitude to Docent Teppo Järvinen, M.D., mvp, for
the paramount contribution. I have enjoyed outstanding guidance, thoughtful
support, and attentively provided prime facilities, which have all been of vital
importance in this work. Similarly, I am grateful to Professor Ilkka Pörsti, M.D.,
for the major efforts he has put into this study. I have had the benefit of
educative instruction and constructive advice. The making of thesis has been a
rewarding learning experience.
I have felt honored to work with Harri Sievänen, D.Sc., and Pekka Kannus,
M.D., whose comprehensive expertise has provided for many enlightening
insights and whose crucial contribution to the different manuscripts has been of
great value.
There are many friends and colleagues who have been obliging and
invaluable in many occasions. Particularly, I offer my thanks to Pasi Jolma,
M.D., for the lucid and fundamental wit. Tero Järvinen, M.D., Janne Nurmi,
D.V.M., and Peeter Kööbi, M.D. have vitally pitched in to assist me in various
tasks. Over and above, it gives me a great pleasure to thank beyond measure Ilari
Pajamäki, M.D., and Olli Leppänen, B.M., jolly members of the gentleman’s
social club, for their committed and instrumental exertions by the workbench.
Professor Jukka Mustonen, M.D., is warmly thanked for commenting various
manuscripts. I wish to thank the collaborators of the original publications Jarkko
Kalliovalkama, M.D., Onni Niemelä, M.D., Heikki Saha, M.D., Juha
Tuukkanen, Ph.D., Tuomo Vuohelainen, M.D., Russell T. Turner, Ph.D., Urszula
Iwaniec, Ph.D., and Thomas A. Einhorn, M.D. I’m appreciative of broadening
views and discussions of Saija Kontulainen, Ph.D., Ari Heinonen, Ph.D., Kirsti
Uusi-Rasi, Ph.D., and Riku Nikander, M.Sc. Here and now, I express my thanks
to Docent Risto Ikäheimo, M.D., and Professor Sulin Cheng, Ph.D., for their
comments and support of my work. Further, it is an honor to have Dr. Frank
57
Rauch, M.D., McGill University, Canada, as my official opponent in the
dissertation.
I’m grateful to the Professor Heather McKay, Ph.D., and Karim Khan, M.D.,
for providing me everything I needed on the home straight of this study and also
for the forthcoming opportunities.
Some people have an eminent influence on others’slogging. Over the course
of this study and beyond, I have particularly appreciated the genuine presences
of Juuso Ikonen and Olli Komulainen. Moreover, others not mentioned in this
context are not forgotten but thanked elsewhere. I thank my parents and my
brother Jussi for their support.
Finally, I try to put into words my deepest of gratitude to Heta, the love and
light of my life, for her unfailing understanding and encouragement.
This study has been supported by grants from Competitive research funding of
the Pirkanmaa Hospital District, Finnish Graduate School for Musculoskeletal
Diseases, The Finnish Kidney Foundation, The Finnish Medical Foundation, and
The Pirkanmaa Regional Fund of the Finnish Cultural Foundation.
58
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