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\begin{document}

\title{EVALUATION OF RECEPTOR ACTIVATOR OF NUCLEAR FACTOR KAPPA B L\selectlanguage{polish}İ\selectlanguage{english}GAND
(RANKL) AND OSTEOPROTEGERIN (OPG) LEVELS IN PATIENTS DIAGNOSED WITH
BRONCHIECTASIS AND OSTEOPOROSIS\selectlanguage{english}}



\author[1]{Kemal Güleç}%
\author[1]{Sevgi Pekcan}%
\author[1]{Beray Selver Eklioglu}%
\author[1]{Asli Yilmaz}%
\author[2]{Gokcen Unal}%
\author[1]{Sevil Kurban}%
\affil[1]{Necmettin Erbakan University Meram Medical Faculty Hospital}%
\affil[2]{Necmettin Erbakan Universitesi Meram Tip Fakultesi Hastanesi}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Objective: Bronchiectasis is a condition characterized by irreversible
abnormal dilation and anatomic breakdown of the bronchial tree. In our
study we aimed to understand the causes of osteoporosis at molecular
levels and to investigatethe usefulness of RANKL and OPG levels as
markers in early diagnosis and follow-up of osteoporosis, RANKL
inhibitors in the treatment of osteoporosis. Materials and Methods: 30
noncystic fibrosis bronchiectasis patients were diagnosed as
osteoporosis with DEXA and applied to the Pediatrics Chest Diseases
Department of Meram Medical Faculty of Necmettin Erbakan University
between June 2015 and June 2016 were included in our study. BMD values
were determined by DEXA and QUS in the patient group and only QUS in the
control group. Results: In the patient group 56.6\% (n: 17) were male,
43.4\% (n: 13) were. There was no statistically significant difference
between RANKL, OPG, RANKL / OPG ratios, BALP, OST, NTX values between
the patient and control group (p\textgreater{} 0.05). The median serum
Ca level was 9.51 (IQC: 0.74) in the patient group and 9.75 (IQC: 0.47)
in the control group, there was a statistically significant difference
between them (p = 0.003). There was a strong positive correlation
between QUS z scores and DEXA z scores in the patient group (p = 0.008).
Conclusion: In conclusion, we could not pinpoint the role of RANK /
RANKL / OPG in the pathogenesis of osteoporosis in patients with
noncystic fibrosis bronchiectasis. However, we think it is appropriate
to conduct studies on a wider series in this topic.%
\end{abstract}%



\sloppy


\textbf{EVALUATION OF RECEPTOR ACTIVATOR NUCLEAR KAPPA B LIGAND (RANKL)
AND OSTEOPROTEGERIN (OPG) LEVELS IN PATIENTS WITH OSTEOPOROSIS AND
BRONCHIECTASIS}

\textbf{INTRODUCTION}

Bronchiectasis is a condition characterized by irreversible abnormal
dilation and anatomic breakdown of the bronchial tree. Although the
frequency pf bronchiectasis has been declining due to the improvement in
nutrition and sanitation circumstances, increased rate of vaccination
and early and frequent use of antibiotics, it still causes a serious
health problem in developing countries (1,2). Pifferi et al. reported
the frequency of bronchiectasis as 1/6000 in general pediatric
population in their study in 2004 (3).

The most common cause of bronchiectasis in developed countries is Cystic
Fibrosis (CF). Bronchiectatic patients with CF and noncystic fibrosis
have many common features. Chronic and recurrent lung infections,
chronic inflammation, inhaled corticosteroid use in inactivity and acute
exacerbations, bronchial hyperreactivity and nutritional problems are
the characteristic features of both groups of diseases (4).

Osteoporosis and osteopenia were reported to be more frequent in
patients diagnosed with CF and noncystic fibrosis bronchiectasis
compared to normal population and the frequency was reported to increase
with increasing age (4-5).

Osteoporosis is a systemic bone disease characterized with decreased
bone mineral density (BMD) and impairment of inner architecture. The
amount of mineralization is normal in all forms of osteoporosis, while
decreased bone volume in especially trabecular bone and decreased
trabecular bone cycle is noticed. Osteoporosis is evaluated using BMD,
dual-energy X-ray absorptiometry (DEXA), quantitative computed
tomography (QCT) and quantitative ultrasonography (QUS). Osteoporosis is
frequently encountered in chronic diseases, especially with long
hospital stay, inadequate vitamin D intake and drug use. Vitamin K and D
deficiency in cystic fibrosis is thought to be associated with
osteoporosis (6).

Many mechanisms are responsible in the development of osteoporosis. Bone
resorption secondary to the changes in RANK-RANKL-OPG system is in the
forefront recently (7). OPG is a member of the receptor TNFR super
family, and is also known as TNFRS11B. Another name of it is
osteoclastogenesis inhibitory factor (OCIF). Original OPG molecule is a
polypeptide composed of 401 amino acids, and the mature protein composed
of 380 amino acids is formed by separation of the 21-amino acid
propeptide part.

The association between osteoporosis and RANKL/OPG levels has been
demonstrated in the literature in patients with CF in whom
bronchiectasis is common. However, no study has been published
evaluating the association between osteoporosis and RANKL/OPG levels in
patients with noncystic fibrosis bronchiectasis. The aim of this study
was to measure the RANKL and OPG levels in patients with noncystic
fibrosis bronchiectasis and with osteoporosis. And to evaluate the
applicability of using RANKL and OPG levels as markers in the early
diagnosis of osteoporosis and its follow-up.

\textbf{MATERIAL AND METHOD}

Patients between 5-18 years of age and applied to the outpatient clinics
of Pediatric Pulmonary Diseases with the diagnosis of non-cystic
fibrosis bronchiectasis and osteoporosis were included in the study.
Patients presented to the outpatient clinics of pediatric for routine
follow-up with no chronic disease were included as the control group.
DEXA measurement was performed in order to evaluate the bone mass of
patients with bronchiectasis.

BMD value measured by DEXA is expressed by Z and T scores. Comparison of
the measured bone mass with reference values according to age and gender
and definition of it as standard deviation is the Z score. The standard
deviation of comparison of the bone mass with the mean bone mass of the
young adult reference population is the T score. BMD measured in
children is evaluated as Z score. The length of the measured bone in
addition to the height, pubertal phase, skeletal maturation, race and
body composition is considered in order to correctly interpret the data
obtained by DEXA method (8). Another method of evaluation in children is
QUS (9).

Ethics board approval was obtained from the Ethics Board Committee dated
January 8, 2016, and numbered 2016/399. An informed consent was obtained
from all patients included in the study after explaining the aim and
extent of the project in detail to each of them.

\subsection*{Collection and Storage of Samples and Study
Method}

{\label{collection-and-storage-of-samples-and-study-method}}

Blood samples obtained were centrifuged at a cooling centrifugation
device with the trademark Hettich Rotina 46R (\emph{Hettich}Zentrifugen,
\emph{Tuttlingen} , \emph{Germany)} at 4000 cycles/minute for 10 minutes
and the sera were separated. Serum samples were stored in New Brunswick
U570 (New Brunswick Scientific, New Jersey, ABD) refrigerator at -80\selectlanguage{ngerman}°C.
Enzyme Linked Immunosorbent Assay (ELISA) method was used in the serum
samples using human RANKL (Biovendor Research and Diagnostic Products
Karasek, The Czech Republic), OPG Biovendor Research and Diagnostic
Products Karasek, The Czech Republic, bALP (Quidel Corporation, San
Diego, USA), OST (Biovendor Research and Diagnostic Products Karasek,
The Czech Republic), NTX (Alere Scarborough, Scarborough, USA) kits.

The results were calculated according to the absorbance concentration
calibration graphics using Biotek ELX 50 microplate washer
(\emph{BioTek} Instruments, Vermont, USA) and Bio-rad Microplate
absorbance reader xMark (Bio-rad Laboratories, \emph{California, USA} )
system.

\textbf{STATISTICAL ANALYSIS}

All analyzes of the study were performed using SPSS 20.0 package
program. Nominal scale (categorical) variables were presented using
frequency and percentage; proportional scale (numerical) variables with
normal distribution using mean±SS and with non normal distribution using
median (interquartile range) in tables and graphics. All the
proportional scale variables were checked for whether they were normally
distributed or not using Kolmogorov-Smirnov and Shapiro-Wilk analyzes.
Age, bALP, Bone US BMD and Mg values were found to be normally
distributed in the groups (p\textgreater{}0,05), and the remaining
values were found to be non-normally distributed with a high skewness
value. Therefore, Student t-test was used in independent two-group
comparison for parameters of Age, bALP, Bone US BMD and Mg since they
were parametric variables and Mann-Whitney U test was preferred for
nonparametric variables. Monte Carlo corrected chi-square analysis was
used to determine the significance of the association between
categorical variables. Pearson correlation coefficient was used when
both parameters were normally distributed to establish the correlation
between the numerical variables. Spearman's Rho correlation coefficient
was used when any of the parameters was non normally distributed. Type-1
error level was accepted as 5 \% in all the analyzes and
p\textless{}0,05 was accepted as statistically significant.

\textbf{RESULTS}

Among the 30 patients included in the study, 56,6 \% (n:17) were boys
and 43,3\% (n:13) were girls with a mean age of 13,3±3,8 (5-18) years.
Among the 40 patients included as the control group, 35 \% (n:14) were
boys and 65\% (n:26) were girls with a mean age of 11,8±3,5 (5-17)
years. No significant differences were found in age and gender between
the patient and control groups (p\textgreater{}0,05).

When the underlying cause was evaluated in the study group, 13 was found
to be immune deficiency, 9 PSD, 4 infection, 3 idiopathic and 1 as Mac
Leod Syndrome.

Laboratory values of the patient and control groups is given in Table
1-2-3.

Correlations are given in Table 4-5.

\textbf{DISCUSSION}

Bronchiectasis is increasingly being diagnosed with the introduction of
high-resolution computed tomography. Primary cause of noncystic fibrosis
bronchiectasis is postinfectious causes worldwide; however, this has
changed in some regions with the efficient use of antibiotics and
decreased prevalence of tuberculosis and childhood pneumonia. The
etiology pf bronchiectasis could be detected in 40-63\% of the cases in
different studies performed (10-11).

Studies have been published in the literature suggesting the effect of
RANK/RANKL/OPG system on the development of osteoporosis in patients
with cystic fibrosis. No study has been published that demonstrates the
association of osteoporosis and RANK/RANKL/OPG system in patients with
noncystic fibrosis bronchiectasis.

OPG is a member of the receptor TNFR super family and is also known as
TNFRS11B. It is released extracellularly as a soluble glycoprotein and
functions as a trap receptor for RANKL (12). Osteoporosis was developed
in rats with OPG failure (13). The osteoprotective role of OPG was
confirmed with demonstration of a 100-kb homozygous deletion in the OPG
gene (chromosome 8q24.2) in juvenile Paget's disease and deletion in the
third exon of OPG in idiopathic hyperphosphatasia (14-15). RANKL gene is
localized in the chromosome 13q14 and in the exon 6. It is a peptide
formed of 317 amino acids composed of two soluble forms such as membrane
bound cellular form in 40-45 Da and biologically active form in 32 kDa.
RANKL is a key regulator in osteoclastogenesis and osteopetrosis is
developed in rats without RANKL due to osteoclast deficiency. Presence
of RANKL and M-CSF in the environment is required and sufficient for the
conversion of osteoclast precursors to mature osteoclasts (12-16). RANKL
receptor has been defined as RANK. RANK is a transmembrane protein
composed of a total of 616 amino acids including a short transmembrane
of 21 amino acids and large cytoplasmic parts (12,16,17).

Osteoclast precursors convert into a multinuclear cell with the effect
of many factors such as cytokines, hormones and growth factors. These
cells differentiate into active osteoclasts in the presence of MCSF and
RANKL. Osteoclasts, once differentiated, start to destruct the bone
surface to produce lacunae. OPG, the trap receptor of the RANKL inhibits
the continuous binding od RANKL to RANK and causes apoptosis of the
osteoclasts. Subsequently, preosteoblasts convert to osteoblasts for new
bone formation (18). RANKL was defined by four independent groups and
was named as TRANCE, ODF, osteoprotegerin ligand (OPGL) and TNFSF11.

RANKL receptor has been defined as RANK. Other names of it are
TNF-related activation-induced cytokine receptor (TRANCE-R) or
osteoclast differentiation and activation receptor (ODAR). RANK is a
transmembrane protein with a total of 616 amino acids with the
extracellular part composed of a 28-amino acid signal peptide, a short
transmembrane, and large cytoplasmic parts. Osteoporosis is known to be
frequently seen in patients with bronchiectasis. Among the mechanisms
causing this condition in patients with CF have been held responsible
are malnutrition, pancreatic failure, calcium malabsorption, Vitamin D
and K deficiency, long term use of systemic or inhaled steroids, delayed
puberty, chronic respiratory acidosis, cytokines increasing osteoclast
activation and decreased sex steroids; however, molecular mechanisms
playing role are yet unclear. In a study by Putman et al. mean BMD value
in vertebra and distal radius of young adults was demonstrated to be
always low in patients with CF and to be unchanged in a follow-up of 15
years in spite of the improvements in the respiratory functions of
patients with CF due to new developments in the follow-up of and vitamin
D support. Understanding the mechanisms in the development of
osteoporosis might allow new therapeutic approaches (19-22).

Bronchiectatic patients with CF and noncystic fibrosis have many common
features. Patients above 5 years of age and with bronchiectasis and who
had osteoporosis diagnosed by DEXA were included in the patient group in
this present study. The age of three of the patients included in the
study were 8.5 years or below, and the cause for this was considered to
be related with the required time for the development of osteoporosis.
Low BMD values in patients with cystic fibrosis and with normal
nutritional state, at earlier ages down to 6 years suggests that this
situation might be associated with bone metabolism (23).

RANKL expression is controlled by many factors such as glucocorticoids,
vitamin D and IL-1. Low RANKL levels in patients with immune deficiency
might be associated with the significantly low levels of vitamin D in
these patients compared to the control group.

OPG levels were lower in the patient group in this present study
compared to the control group, though not significant. Median OPG in
patients with PSD was found to be lower compared to the controls and
patients with immune deficiency, though not statistically significant.
No statistically significant differences were found in serum OPG levels
when the patients with PSD and control group were analyzed separately
and when patients with immune deficiency and PSD were analyzed
separately. OPG is known to be expressed in many tissues such as the
heart, kidneys and liver (12). We considered that osteoporosis seen in
patients with PSD might be due to the decreased expression of OPG in
lung tissue.

Similarly, no statistically significant difference was found in the
RANKL/OPG ratio between the patient and control groups. In this present
study, no significant correlation was found between serum RANKL and OPG
levels and DEXA z scores. Shead et al. in their study in adult patients
with CF, serum RANKL levels were analyzed in the period of clinical
stability of the patients, on the day prior to the start of antibiotics
during acute exacerbation and on day 14 of the completion of intravenous
antibiotic treatment. No significant difference was found in the RANKL
levels in the stable period of the patients compared to the controls;
however, RANKL levels on day 14 of the acute exacerbation was found to
be significantly high compared to the stable period in that study. Serum
OPG levels were found to be significantly low in patients with CF
compared to the controls in the stable period, and significantly high on
day 14 of the acute exacerbation (24). Ambroszkiewicz et al. in their
study in patients with CF, included patients with no lung findings, no
steroid treatment, and clinically stable patients as controls. Serum
RANKL values were found to be 2-folds in the patient group compared to
the controls, and OPG and OPG/RANKL levels were found to be lower
compared to controls in that study (25). The serum samples were
collected at the stable period of the patients and no comparison was
made with the acute exacerbation period and this might be the cause of
no significant difference in the RANKL, OPG and RANKL/OPG levels in this
present study.

Franchimont and Galluzzi et al. in their study serum OPG levels were
found significantly high in patients with Crohn's disease and Type 1
Diabetes Mellitus (26-27). Association of the changes in the
RANK/RANKL/OPG system with chronic inflammation might be the underlying
mechanism in osteoporosis in chronic diseases. We suggest that chronic
inflammation might be effective in the pathogenesis of the osteoporosis
developing in bronchiectasis patients.

Effects of inhaled and systemic corticosteroid use on bone health is a
subject of great interest. Corticosteroids cause decreased bone mineral
density by also effecting the RANK-RANKL-OPG system. A total of 5 g of
inhaled steroids is demonstrated to cause a loss of 1 SD in vertebral
BMD. General opinion on the systemic steroid use is that is causes low
BMD and morphometric fractures (28). In a study by Wasilewska et al. in
patients with nephrotic syndrome and using steroids, corticosteroids
were detected to dose-dependently elevate RANKL and RANKL/OPG ratio
(29). Corticosteroids and mostly inhaler corticosteroids were frequently
used in the patient group in this present study among patients with
bronchiectasis. Corticosteroid use was ignored when designing the
patient group. We consider that steroid use might have affected the
results of the study.

In vitro studies have been performed in patients with CF recently in
order to demonstrate the association between osteoporosis and RANKL/OPG
and CFTR gene mutation. These studies support that CFTR gene mutation
increases bone resorption due to chronic inflammation (25,30,31). If the
association of CFTR gene mutation and RANKL/OPG system becomes apparent
as a result of the future studies, it would be meaningful why no
association was found between the control group and the patient group
noncystic fibrosis in this present study.

Serum osteocalcin level which is a good predictor of bone production was
found to be low in the patient group compared to the control group,
however no statistical significance was found in this present study. No
statistical significance was found between the subgroups of immune
deficiency and PSD. Rossini et al. in their study in patients with CF
and vertebral fractures, found low osteocalcin levels in 36\% of the
patients (32). Ambroszkiewicz et al. in their study in patients with CF,
found low osteocalcin levels and high RANKL/OPG ratio in the patient
group compared to the control group and this was suggested to reflect
the increased bone destruction and decreased bone production (25). In
this present study, similarity of osteocalcin levels in patient and
control groups could be due to the clinical stability of the patients
and an achieved balance between bone production and destruction.

A powerful and negative correlation was found between the serum
osteocalcin and OPG levels in the patient group. An inverse correlation
was found between serum OPG level and BMD and osteocalcin level in a
study by Oh et al. in 80 Korean male patients between 42-70 years of age
(33). A powerful and positive correlation was found between OPG and
osteocalcin in patients with osteoporosis in a study
by\emph{Fahrleitner-Pammer}and found that low OPG levels were associated
with vertebral fractures (34).

Vitamin D levels were found to be low in both patient and control
groups, with no statistically significant difference between the groups
in this present study. Considering the previous studies, low levels of
vitamin D in the healthy control group might be attributed to the high
incidence of Vitamin D deficiency and insufficiency in Turkey. Vitamin D
level was found to be in the normal range (\textgreater{}30 ng/mL) in
only 12.3\% of the control group in a study was Dogru and Suleyman (35).
Vitamin D deficiency and insufficiency was detected in 48 \% of the
healthy control group composed of healthy individuals ina study by
Turkeli et al. (36). Vitamin D deficiency and insufficiency was found in
80.3 \% and 11.7 \%, respectively in healthy children included in the
study at the end of winter season in a study by Erol et al. performed in
Istanbul (37). Vitamin D levels were found to be significantly low in
the subgroup of patients with immune deficiency compared to the patients
with PSD and control group. When immune deficiency and control group was
compared, Ca and P levels were found to be significantly high in the
control group. We thought that this might be due to the prescription of
vitamin D and Ca preparations for patients in case when osteoporosis was
diagnosed subsequent to the required tests for osteoporosis due to the
clinical high incidence of osteoporosis and osteopenia in patients with
PSD.

Median serum Ca level was significantly different in the patient group
and the control group in this present study. Serum Ca level was found to
be significantly low in the subgroup with immune deficiency compared to
the PSD subgroup and control group. No significant difference was found
between the patient and control groups in terms of serum Ca levels in a
study by Ambroszkiewicz et al. in patients with CF (25). We suggest that
these results in this present study might be due to the low levels of
vitamin D in the control group and the subgroup of immune deficiency.

Median spot urine Ca/Cr ratio was statistically significantly different
between the patient and control groups in this present study. Spot urine
Ca/Cr ratio was found to be significantly high compared to the control
group. We found no positive results that might be helpful to associate
calciuria and OPG/RANKL; however, we thought that this might be
associated with other factors of osteoclastic activity and factors
causing increased bone resorption.

A powerful and positive correlation was found in QUS z scores and DEXA,
BMD z scores in the patient group. According to the literature data, QUS
is generally considered not to be a substitution of DEXA, but it could
be used as a screening method. Williams et al. found a powerful
correlation between QUS and DEXA in their study in obese patients with
CF; however, they concluded that QUS could not be used in place of DEXA,
especially in obese patients (38). Schepper et al. compared QUS, DEXA
and Perioheral Quantitative CT in adult patients with CF. QUS was deemed
not to replace DEXA in that study; but it was stated that it could be
used as a screening method in patients with a normal bone mass (39).
Similarly, Flohr et al. proved that QUS had no high specificity and
sensitivity to replace DEXA (40). Parallel to the literature findings, a
correlation was found in the bone US and DEXA values in the patient
group in this present study. This supports the applicability of QUS as a
screening test in patients with bronchiectasis since it is easily
applicable compared to DEXA in BMD measurements and includes no
radiation exposure.

A negative correlation was found between serum RANKL levels and ALP in
the patient group. A powerful and positive correlation was found between
NTX and bALP when the patient group was evaluated in itself and with the
control group. A positive and statistically significant correlation was
found between NTX and spot urine Ca/Cr in the patient group. A positive
and statistically significant correlation was found between the serum
osteocalcin and bALP, ALP, NTX, and Ca when the patient and control
groups were evaluated together. Positive and negative many correlations
between the bone production and destruction markers were found in this
present study in the patient group. This suggests a high bone turnover
in patients with bronchiectasis.

Our limitations were determination of serum OPG and RANKL levels are
challenging due to many reasons. The source of RANKL released in the
circulation is many and might be in many forms. RANKL is present in the
serum in free form and most of it as bound to OPG and the two molecules
have a circadian rhythm. Therefore, the measured values in the
circulation may not completely reflect the effects on the bone
microframe. Serum samples were preferred to be obtained in morning hours
in this present study; however, the study results might be affected from
the circadian rhythm.

In conclusion, we could not find the definite role of RANK/RANKL/OPG
system in the pathogenesis of osteoporosis developed in noncystic
fibrosis bronchiectasis patients in this present study. However, future
studies in larger series would be appropriate since the number of cases
is small in this study.

\textbf{Acknowledgements} : There are no conflict of interests in this
article.

\selectlanguage{english}
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