EVALUATION OF SERUM PENTRAXIN3 LEVEL IN IRAQI PATIENTS WITH DIABETIC NEPHROPATHY

Abstract

ABSTRACT : Diabetic nephropathy (DN) is a principle of microangiopathy in patients with T2DM and it is the main reason of kidney disease at the end stage in DM patients. nflammatory processes may be associated with pathophysiology in patients with diabetic nephropathy. The principal role of pentraxin 3 (PTX3), may be attributed to its ability to attenuate renal disorder in the case of T2DM. The objective of this work is to study the association of PTX3 with the
renal function in patients with diabetic nephropathy. Patients groups include 90 patients in addition to 30 healthy
subjects as control group, which were enrolled in this study. on the basis of the value of urine albumin-creatinine ratio
(ACR), diabetic patients were divided into three groups, ELISA kit was used to determine the level of serum PTX3.
Fasting blood glucose, blood urea, serum creatinine were measured by enzymatic method. Particle-enhanced Turbidimetric
inhibition immunoassay (PETINIA) was used to determine albumin in urine. Creatinine was determined using the
modified kinetic Jaffe technique. Ratio of albumin/creatinine (ACR) was calculated. The formula of ‘‘modification of
diet in renal disease’’ (MDRD) was applied to calculate of eGFR. The serum PTX3 levels in DN patients were found to
be significantly higher in patient groups than healthy group. A high significant increase in PTX3 level in patients group
with macroalbuminuria showed in comparison with the other studied groups. Also, results revealed a significant strong
positive correlated of PTX3 with each of ACR, Cr and blood urea while a negative correlated is recorded with eGFR.
PTX3 considers a good inflammatory marker for prediction in the case of diabetic nephropathy. It plays an important
role that attributed to the progression of diabetic nephropathy.

Full text

EVALUATION OF SERUM PENTRAXIN3 LEVEL IN IRAQI PATIENTS WITH
DIABETIC NEPHROPATHY
Sabreen Ali Mezil1, Kadhim K. Ghudhaib1 and Ali Abdulmajid Dyab Allawi2
1Department of Chemistry, College of Science for Women, University of Baghdad, Baghdad, Iraq.
2Department of Medicine, College of Medicine, University of Baghdad, Baghdad, Iraq.
(Accepted 24 September 2018)
ABSTRACT : Diabetic nephropathy (DN) is a principle of microangiopathy in patients with T2DM and it is the main
reason of kidney disease at the end stage in DM patients. Inflammatory processes may be associated with pathophysiology
in patients with diabetic nephropathy. The principal role of pentraxin 3 (PTX3), may be attributed to its ability to
attenuate renal disorder in the case of T2DM. The objective of this work is to study the association of PTX3 with the
renal function in patients with diabetic nephropathy. Patients groups include 90 patients in addition to 30 healthy
subjects as control group, which were enrolled in this study. on the basis of the value of urine albumin-creatinine ratio
(ACR), diabetic patients were divided into three groups, ELISA kit was used to determine the level of serum PTX3.
Fasting blood glucose, blood urea, serum creatinine were measured by enzymatic method. Particle-enhanced Turbidimetric
inhibition immunoassay (PETINIA) was used to determine albumin in urine. Creatinine was determined using the
modified kinetic Jaffe technique. Ratio of albumin/creatinine (ACR) was calculated. The formula of ‘‘modification of
diet in renal disease’’ (MDRD) was applied to calculate of eGFR. The serum PTX3 levels in DN patients were found to
be significantly higher in patient groups than healthy group. A high significant increase in PTX3 level in patients group
with macroalbuminuria showed in comparison with the other studied groups. Also, results revealed a significant strong
positive correlated of PTX3 with each of ACR, Cr and blood urea while a negative correlated is recorded with eGFR.
PTX3 considers a good inflammatory marker for prediction in the case of diabetic nephropathy. It plays an important
role that attributed to the progression of diabetic nephropathy.
Key words : T2DM, nephropathy, pentrxine3, microalbuminuria, macroalbuminuria.
Biochem. Cell. Arch. Vol. 18, No. 2, pp. 2473-2477, 2018 www.connectjournals.com/bca ISSN 0972-5075
INTRODUCTION
Diabetes with its characteristic hyperglycemia shows
long-term complications on several organs, therefore
regular monitoring of organ functions for early preventive
interventions of these complications is necessary (Karar
et al, 2015). Diabetic complications such as osteoporosis
in postmenopausal Iraqi women with T2DM, and Iraqi
patients with diabetic neuropathy were studied well in
our previous works (Kadhim K Ghudhaib et al, 2014,
2018; Hind S. Mohammed et al, 2018). Renal impairment
is one of such common complications of diabetes mellitus
(Gross et al, 2005). The major reason of kidney disease
progression in the case of diabetic nephropathy is
attributed to the angiopathy in glomeruli capillary and
the long period of diabetes. These factors consider a major
cause of morbidity and mortality in patients with Type 2
diabetes mellitus. This confirms the requirements of early
detection in patients with Type 2 diabetes which decrease
the risk of diabetic nephropathy (Roopakala et al, 2012).
The pathogenesis of DN was still not definite, but many
of researchers have pointed that its genesis was related to
glucose and lipid metabolism disorder, change of
hemodynamics, cytokines and inheritance and other
factors. Recently, more and more evidence suggests that
inflammation plays a role in the pathogenesis of DN
(Asmar et al, 2016). Microvascular damage in the case
of patients with T2DM considers as a first clinical feature
in characterization of disease, that can be identified on
the basis of proteinuria ‘albuminuria’. It was reported
that the losing in renal function is associated with
microalbuminuria and macroalbuminuria (Ninomiya et
al, 2009).
Pentraxin 3 (PTX3) is a secreted glycoprotein a lately
discovered inflammatory biomarker; belonging to the
pentraxin family (Liu et al, 2014). The production of
PTX3 is restricted in certain cells, which are including
epithelial cells, endothelial cells, fibroblasts, macrophages,
monocytes and which exist in vascular wall of heart, thus
PTX3 may be a proper predictor for atherosclerotic and
inflammatory changes in this type of tissues (Bottazzi et
al, 2010; Kaess et al, 2011). PTX3 is a member of family
that characterized by around 200 amino acid long called

2474 Sabreen Ali Mezil et al
‘pentraxin domain’ and it participates with other members
in eight amino acids called ‘pentraxin signature’ (Bonacina
et al, 2013). It leads to initiate inflammation, inhibit
angiogenesis, promote restenosis and plays a role in
formation of advanced atherosclerotic lesions (Shindo et
al, 2014). PTX3 attenuated kidney damage in mice with
DN by enhancing macrophage differentiation (Sun et al,
2015).
METHODS
Patients selection
90 patients with T2DM (45males and 45 females), in
age range (40-60) years, who attend to Baghdad teaching
hospital/Medical City Baghdad/Iraq, were enrolled in this
study. On the basis of albumin-creatinine ratio (ACR),
the patients were divided into three groups: 30 diabetic
patients with ACR < 30mg/g (normoalbuminuria group),
30 diabetic patients with ACR range of 30–300 mg/g
(microalbuminuria group) and the third patients group,
which consists of 30 diabetic patients with ACR>300 mg/
g (microalbuminuria group). Control group consist of 30
healthy subjects. The criteria of excluded cases include
patient with Type1DM, Pregnant women, chronic urinary
tract infection, malignant hypertension, congestive hard
failure, acute hyperglycemia, systemic lupus
erythematosis (SLE) and polycystic kidney disease.
Samples collection and methods
10 ml of venous blood samples were collected in
vacuum tubes with a clot activator. Blood samples were
centrifuged at 1500 rpm for 15 minutes. Serum was
separated and aliquots then stored at –20°C until analysis.
PTX3 was determined by the enzyme-linked
immunosorbent assay (ELISA) kits (Shanghai Yehua
Biological Technology Co. Ltd., China). Fasting blood
glucose, blood urea, serum creatinine were measured by
enzymatic method. First morning urine samples were
collected in a clean container free from detergent, urine
spots analyzed to estimated albumin and creatinine using
the particle-enhanced Turbidimetric inhibition
immunoassay (PETINIA), to determine the microalbumin
and uses a modified kinetic Jaffe technique to determine
urine creatinine, ratio of albumin/creatinine (ACR)
estimated from divide the value of urine microalbumin to
urine creatinine. The formula of ‘‘modification of diet in
renal disease’’ (MDRD) was applied to calculate of eGFR.
It is expressed in milliliters/ min. /1.73 m2: eGFR = 186
× S. Creatinine (mg/dl)-1.154 × (age)”0.203 × 0.742 (if female)
(Levey et al, 2000).
Statistical analysis
Data are expressed as means± SD. ANOVA used to
analyze the differences between more than two groups.
Linear regression analysis performed to assess the
relationship between different variables. SPSS program
version 20 used for statistical analysis, p value less than
0.05 considers a significant value.
RESULTS
Table 1 shows mean± SD values of age, BMI, FBG,
duration of diabetic (DM), ACR, GFR urea and creatinine.
The results revealed no-significance differences in age
between the studied groups (p>0.05), while significant
increase in body mass index (BMI), fasting blood glucose,
ACR, blood urea (BU), creatinine (Cr) in patient groups
(normoalbuminuria, microalbuminouria and
macroalbuminuria) than control group. Whereas, it was
noticed a significant decrease eGFR in patient groups than
healthy subjects. Furthermore, levels of blood urea (BU)
and creatinine (Cr) in patients with macro-albuminuria
are higher than other diabetic patient groups that include
normoalbuminuria and microalbuminuria.
Table 1 : Clinical characteristics of Patients and healthy subjects.
Parameters Control DM with normo- DM with micro- DM with macro- P value
albuminuria albuminuria albuminuria
No. 30 30 30 30 -
Age (year) 50.7 ± 3.4 51.2 ± 5.1 51.5 ± 5.9 53.4 ± 4.5 0.145
DM (year) -3.8 ± 1.5 7.7 ± 3.2b10.3 ± 4.4b,e <0.001
BMI 24.7± 1.9 29.2 ± 5.1a30.8 ± 4.2a29.9 ± 4.4a<0.001
FBS (mg/dl) 89.0 ± 6.5 201.4± 26.8a223.8 ± 36.1a259.3 ± 63.6a, b <0.001
S.Cr (mg/dl) 0.67± 0.10 0.78 ± 0.26 0.88 ± 0.27 3.81± 1.13a, b, c <0.001
B.U (mg/dl) 24.6 ± 5.4 26.2 ± 8.1 32.1 ± 9.3 127.9±39.9a, b, c <0.001
ACR (mg/g) 6.72± 5.86 12.21±7.28 137.04±65.1a, b 495.35±157.8a, b, c <0.001
eGFR 122.17±31.5 104.56±25.03d94.52±29.77a16.86±6.27a, b, c <0.001
FBS: fasting blood sugar, eGFR : estimated glomerular filtration rate (ml/ min./1.73 m2), ACR : albumincreatinine ratio, S.Cr : serum
creatinine, B.U : blood urea, BMI : body mass index, DM : duration of diabetes mellitus.
a: P<0.001 compared to control group, b: P<0.001 compared to normoalbuminuria group, c: P<0.001 compared to microalbuminuria
group, d: P=0.026 compared to control group, e = P=0.005 compared to microalbuminuria group.

Serum PTX3 concentrations
Serum PTX3 level is high significantly increase
(p<0.001) in diabetic patient groups in comparison with
the healthy subjects. Patients group with
macroalbuminuria (7.50±2.75 ng/ml) revealed high
significant increasing in serum PTX3 level (p<0.001)
compared with the other diabetic patient groups and
healthy subjects. Patients group with microalbuminuria
(3.43±1.27 ng/ml ng/ml) revealed significant increasing
in serum PTX3 level (p<0.001) compared with control
group (1.86±0.45 ng/ml). Furthermore, there is no-
significant difference in serum PTX3 level between patient
groups of normoalbuminuria (3.38±0.99 ng/ml) and
microalbuminuria (P>0.005), at the same time patients
group with normo albuminuria showed significant
increasing in serum PTX3 level (p=0.002) compared with
control group, as shown in Fig. 1.
Correlation Serum PTX3 with all variables in this
study
The correlation data of PTX3 with other variables in
this study are recorded in Table 2. The results revealed a
positive correlation of PTX3 with ACR in diabetic patients
Evaluation of serum pentraxin3 level in Iraqi patients with diabetic nephropathy 2475
groups (normoalbuminuria, microalbuminuria and
macroalbuminuria). In contrast, there is a negative
correlation of PTX3 with eGFR in patients with
macroalbuminuria, but PTX3 is positively correlated with
BMI in diabetic patients with micro and
macroalbuminuria groups.
DISCUSSION
In spite of consideration diabetic nephropathy as no
immune disease, it was reported to presence of
inflammatory and immunological mechanisms that
confirmed an essential role of their effects on development
and progression of this disease (Bussolati et al, 2003).
PTX3 expression is noticed in epithelial cells and renal
Table 2 : Correlation between PTX3 and various variables in DM patients.
DM with normoalbuminuria DM with micro albuminuria DM with macro albuminuria
Variables
rP value rP value rP value
Age 0.147 0.438 -0.014 0.944 0.208 0.270
BMI -0.006 0.974 0.744 <0.001* 0.374 0.042*
DM 0.289 0.121 0.162 0.393 -0.090 0.637
FBS -0.344 0.063 0.073 0.702 0.257 0.170
Cr.-0.465 0.010* 0.045 0.813 0.756 <0.001*
Urea -0.006 0.974 -0.172 0.365 0.0977 0.610
GFR 0.306 0.100 -0.055 0.771 -0.580 0.001*
ACR 0.555 0.001* 0.703 <0.001* 0.705 <0.001*
Linear regression analysis
r = correlation coefficient, *= significant difference
Fig. 1 : Levels of pentraxin 3 (PTX3) concentrations in all the
studied groups.
fibroblast of kidney tissues (Nauta et al, 2005; Wang et
al, 2016). Results obtained from the present study are in
agreement with results of other studies that found increase
in serum PTX3 with DN compared to control group
(Wang et al, 2016; Suliman et al, 2008). The results in
this study revealed that increase of serum PTX3 levels
associated with the presence of DN. This indicates that
serum PTX3 might be involved in the mechanism of the
development and progression of DN. The diabetic patients
with microalbuminuria in our study who had elevated
PTX3 levels showed a normal glomerular filtration rate.
These results are in accordance with those reported by El
eligi et al (2013), Stehouwer et al (2004), who found
significantly elevated in levels of pentraxin3 in patients
with microalbuminuria and with a normal renal function
when compared with healthy.
Mechanism of PTX3 role in diabetic nephropathy
pathogenesis is unclear, but several mechanisms that
published in previous studies referred to that increase of
permeability of glomerular basement membrane may
attributed to renal function disorders as a result of
endothelial dysfunction which consider an important
factor in development of DN (Ross et al, 1993). In

2476 Sabreen Ali Mezil et al
addition to that the PTX3 production occurred in
inflammation sites and regulated by stimulation of IL-1â
and TNF-á (Chen et al, 2012; Uzun et al, 2016).
The results in our study indicate a presence of negative
correlation in PTX3 level with renal function represented
by eGFR. The result is in agreement with Suliman et al
(2008); Doni et al (2012), Wang et al (2016). At the same
time, PTX3 is positively correlated with renal function
parameters, such as ACR, blood urea and Cr. This finding
is supported by previous investigation (Doni et al, 2012).
Accordingly, high level of PTX3 reflects kidney failure
and its rapid release due to its produced and stored in
vasculature (Deban et al, 2008; Jaillon et al, 2007).
Consequently, PTX3 might be implicated in renal
malfunction development and progression.
CONCLUSION
PTXE level is significantly increased in patient groups
than control group. Furthermore, it has a positive
correlated with ACR and negative correlated with eGFR.
Accordingly, it may play an important role that contributes
to the progression of diabetic nephropathy and its
association with renal function. Thus, it may be a better
inflammatory marker for prediction of this disease.
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