Archives of Medical Research 50 (2019) 91e97
ORIGINAL ARTICLE
Kruppel-Like Transcription Factor-4 Gene Expression and DNA Methylation
Status in Type 2 Diabetes and Diabetic Nephropathy Patients*
Zeynep Mine Coskun,a,1 Melike Ersoz,a,1 Mine Adas,b Veysel Sabri Hancer,c Serife Nur Boysan,d
Mustafa Sait Gonen,e and Aynur Acara
a
Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Demiroglu Bilim University, Istanbul, Turkey
Department of Endocrinology, Ministry of Health Okmeydani Research and Training Hospital, Health Sciences University, Istanbul, Turkey
c
Department Medical Genetics, Faculty of Medicine, Istinye University, Istanbul, Turkey
d
Department of Endocrinology, Faculty of Medicine, Demiroglu Bilim University, Istanbul, Turkey
e
Department of Endocrinology, Faculty of Cerrahpasa Medicine, Istanbul University, Istanbul, Turkey
b
Received for publication January 21, 2019; accepted May 24, 2019 (ARCMED_2019_68).
Background/Aim. Diabetic nephropathy (DN) is one of the most serious microvascular complications in diabetic patients. The kruppel-like transcription factor-4 (KLF-4) affects the
expression of genes involved in the pathogenesis of DN. The present study aims to identify
the KLF-4 expression and DNA methylation (DNAMe) status in patients with type-2 diabetes
(T2D) and DN and to reveal the contribution of the KLF-4 to the development of DN.
Material and Methods. The cohort study was performed with blood samples from 120
individuals; T2D group (n 5 40), DN group (n 5 40) and control group (n 5 40).
The expression level of the KLF-4 gene was analyzed using the real-time polymerase
chain reaction (qRT-PCR) and the methylation profile detected using the methylationspecific PCR (MS-PCR) technique.
Results. According to our findings, KLF-4 mRNA expression in the T2D group was 1.60
fold lower than in the control group ( p 5 0.001). In the DN group, the expression of
KLF-4 mRNA was 2.92-fold less than that of the T2D group ( p 5 0.001). There was
no significant alteration in the DNAMe status among the groups.
Conclusion. Our findings showed that regardless of the DNAMe status, KLF-4 gene
expression may play a role in the development of T2D and DN. This suggests that the
KLF-4 gene may be the target gene in understanding the mechanism of nephropathy,
which is the most important complication of diabetes, and planning nephropathyrelated treatments, but the data should be supported with more studies. Ó 2019
IMSS. Published by Elsevier Inc.
Key Words: Diabetic nephropathy, DNA methylation, Epigenetics, Kruppel-like transcription
factor-4, Type 2 diabetes.
Introduction
Type-2 diabetes (T2D) is the most common type of diabetes
mellitus (DM) and has been suggested to occur with multifactorial causes such as heredity, environment, and diet (1).
Conflict of interest: The authors declare there is no conflict of interest
Both authors contributed equally to this study
Address reprint requests to: Zeynep Mine Coskun, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Demiroglu Bilim University, 34394-Sisli, Istanbul, Turkey; Phone: (þ90) (212) 213 64 83,
FAX: (þ90) (212) 272 34 61; E-mail: zeynepminecoskun@gmail.com
1
The DM disease is primarily characterized by b cell
dysfunction and insulin resistance. Hyperglycemia, ketoacidosis and nonketotic hyperosmolar coma are serious metabolic disorders that occur in diabetic individuals (2).
Uncontrolled hyperglycemia induces macrovascular complications (stroke, peripheral arterial and coronary artery
diseases) and microvascular complications (diabetic nephropathy, retinopathy, and neuropathy) (3).
Diabetic nephropathy (DN) is serious microvascular
complications of T2D, which is defined as persistent microalbuminuria with or without a fall in glomerular filtration
0188-4409/$ - see front matter. Copyright Ó 2019 IMSS. Published by Elsevier Inc.
https://doi.org/10.1016/j.arcmed.2019.05.012
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Coskun et al./ Archives of Medical Research 50 (2019) 91e97
rate and induce end-stage renal disease (4). DN is associated with an elevated risk of diabetic complications progression and, increases the mortality rate in patients (5).
The Kruppel-like factors (KLFs) are a subclass of Cys2/
His2 zinc-finger DNA-binding proteins. Members of KLFs
have important regulatory functions during embryonic
development and play a role in various diseases. The KLFs,
transcription factors, are important regulators of glucose
and lipid metabolisms (6). KLF-4 is a transcription factor
expressed in a variety of tissues, including the epithelium
of intestine, skin, lung, and testis (7,8). Depending on the
target gene, KLF-4 can either activate or repress transcription (9). KLF-4 has a role in the process of nephron differentiation in embryonic kidneys (10).
DNA methylation (DNAMe) regulates gene expression
by upregulating, downregulating, or silencing of genes.
However, it is known that DNAMe commonly silences gene
expression and reduces transcription by affecting chromatin
structure (11,12). Dysfunction of DNAMe can lead a variety diseases including metabolic, cardiovascular and neurological disorders, and diabetes (13).
The current treatment strategies have provided benefit to
improving DN but unfortunately could not stop exactly its
progress. So, it is thought that the gene regulation mechanisms in the pathogenesis of DN are not fully understood.
Clinical observation and epidemiology data also show that
genetic predisposition is an important factor in the pathogenesis of DN. For this reason, we aimed to compare the
results of KLF-4 mRNA expression levels in the blood
among healthy, type-2 diabetic, and DN patients, to reveal
whether DNAMe is associated with KLF-4 mRNA
expression.
suffering from other causes of renal impairment or having
ESRD, cancer, new onset diabetes after organ transplantation, ischemic heart disease, cerebrovascular events, acute
illness at the time of the study were excluded from the
beginning of the study. The 120 patients were divided into
three groups. Control Group: Individuals were presented
fasting glucose levels !100 mg/dL and glycated hemoglobin (HbA1c) ! 5.7% (with normal glucose metabolism),
upon informed written consent. The control group without
risk factors for the development of Type-2 diabetes and
chronic kidney disease. They were considered as nondiabetics (14). Type-2 diabetes (T2D) Group: Patients that
were diagnosed by an endocrinologist and who monitored
their glycemic control by evaluation of HbA1c. The patients with the fasting glucose levels O100 mg/dL and glycated hemoglobin (HbA1c) O 5.7% were included in this
group. DN Group: Diabetic patients evaluated depending
on urine albumin/creatinine ratio (ACR) (mg/g creatinine).
ACR $30 mg/g was considered as patient with DN (15).
Diabetes and DN groups received therapy for diabetes,
but not control group (Figure 1).
Demographic and Clinical Characteristics in Study
Groups
The subjects answered a structured questionnaire about personal and family medical history, demographic characteristics (age, sex) and the use of medications. The clinical
examination consisted of blood pressure (mm of Hg),
weight (Kg), body mass index (BMI).
Laboratory Measurements and Lipid Profile of All groups
Materials and Methods
Ethics
The study protocol was approved by the Ethics in Human
Research Committee of the Istanbul Bilim University
(The number of ethical approval and date: 31e278,
26.05.2015), and was conducted according to the ethical
principles of the Helsinki Declaration. All volunteers were
informed about the aims and methods of the present study
and written informed consent was obtained from each
participates.
The blood samples were collected after a 12 h overnight
fast for the evaluation of fasting plasma glucose (mg/dL).
HbA1c, serum urea, creatinine, Na and K was measured
by Beckman Coulter AU2700 auto-analyzer (Beckman
Coulter Inc., USA). Moreover, albumin and protein were
determined in urine. Glomerular filtration rate (GFR) was
calculated as follows: GFR (mL/min/1.73 m2):175 x
Scr1.154x Age0.203 x 0.742 (If female). Serum lipid profile
(TC, TG, LDL, VLDL, and HDL) was measured photometrically by Beckman Coulter AU2700 auto-analyzer (Beckman Coulter Inc., USA).
Patients
RNA and DNA Extractions in Blood Samples
We evaluated 120 patients who hospitalized in the Department of Endocrinology, Ministry of Health Okmeydani
Research and Training Hospital and Medical Faculty of Istanbul Bilim University, between January 2016 and January
2017. The 120 patients aged from 25e75 years were classified according to American Diabetes Association (ADA)
criteria. Smokers, pregnant women, the patients with
Total RNA and DNA were isolated using RNA Extraction
Kit and DNA Isolation Kit (Hybrigen, R1051 and N1122
Turkey, respectively) from blood following the manufacturer’s instructions. The RNA and DNA purities were determined by the ratio of A260/A280 using the
spectrophotometer (NanoDrop Technologies). RNA and
DNA samples were stored at ‒80 C.
93
KLF-4 Expression in Diabetes and Diabetic Nephropathy
Figure 1. Study flow chart for case selection and recruitment. American Diabetes Association (ADA).
Gene Expression Analysis
RNA (0.5 mg) was converted into cDNA using reverse transcriptase according to the manufacturer’s instructions (Hibrigen,
Turkey). The cDNA was used as a template for quantitating
gene expression using SYBR Green real-time PCR kit (Hybrigen, Turkey) in CFX96 Touch Real-Time PCR Detection Systems (Bio-rad, USA) according to the manufacturer’s
instructions. GAPDH was used as normalization control and
the KLF-4 mRNA expression level was calculated using the formula 2DDCt values. The primers are shown in Table 1.
DNA Methylation Analyses
The methylation status of the KLF4 promoter CpG islands was
performed using methylation-specific PCR (MS-PCR). MSPCR was carried out on bisulfate-treated DNA. The isolated
DNAwas bisulfite-converted according to conventional protocol. Bisulfite-modified DNA was amplified by PCR using
0.2 mM of each primer (Table 1), 2 units of Hot Start Taq
DNA polymerase, and 0.2 mM of each dNTP per reaction.
Cycling programs were 95 C for 5 min, and then 40 cycles
of 95 C for 30 sec, 56 C for 30 sec, and 72 C for 30 sec, followed by a 5 min incubation at 72 C. The PCR products were
examined by gel electrophoresis in 1.5% agarose.
expressed as the mean standard deviation (SD).
ShapiroeWilk (SW) normality test was applied to determine the normality of the distribution. The normal data
analyzed for statistical significance using one-way analysis
of variance (ANOVA), followed by Tukey’s post-hoc test.
The Kruskal-Wallis and Mann-Whitney U nonparametric
test for nonparametric parameters were used to compare
the groups. p ! 0.05 was considered statistically
significant.
A post-hoc power analysis was achieved using G*Power
program (version 3.1.9.2 for windows, http://www.gpower.
hhu.de/en.html). It was performed using the following parameters: F test for ANOVA, sample size 5 120, alfa error 5 0.05 and medium effective size 5 0.3. The power
(1-b) of analysis was 0.835.
Table 1. Primer sequences used for the mRNA expression and DNA
methylation studies
Genes
Primer sequence (50 /30 )
KLF-4
F GAACCCACACAGGTGAGAAACC
R ATGCCTCTTCATGTGTAAG
F ACCCACTCCTCCACCTTTGAC
R TGTTGCTGTAGCCAAATTCGTT
F GGTTGATTATTTGAGGTTAGGTGTT
R CCCAAATAACAAAAATTACAAACA
F GTTGATTATTTGAGGTTAGGTGTTC
R CGAATAACGAAAATTACAAACGTA
GAPDH
Statistical Analysis
Un-methylated KLF-4
Statistical analysis was made using SPSS software (IBM
SPSS Statistics for Windows, Version 21.0.). All data were
Methylated KLF-4
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Coskun et al./ Archives of Medical Research 50 (2019) 91e97
Table 2. Demographic and clinical characteristics of the control and patient groups
Agea
Sex
Systolic blood pressurea
Diastolic blood pressurea
Weight (kg)a
BMIa
Control group
T2D group
DN group
p
45.65 12.0
20% male
80% female
116.55 13.07
77.70 11.24
70.79 14.67
25.65 4.74
56.92 9.69b
40% male
60% female
125.74 12.45
74.44 9.43
86.63 18.21c
31.32 5.45d
57.94 8.81b
50% male
50% female
133.52 9.43b
79.36 7.97
88.83 18.01b
33.65 8.32b
0.000
0.000
0.174
0.000
0.000
a
Means SD; Type-2 diabetes: T2D; diabetic nephropathy: DN.
p 5 0.000 vs. Control group; cp 5 0.003 vs. Control group; dp 5 0.006 vs. Control group.
b
Results
Patient Characteristics in Study Groups
Demographic and clinical characteristics of the groups are
seen in Table 2. Patient in T2D and DN groups were older
compared with control group ( p 5 0.000 for each), and
BMI (kg/m2) values in the T2D and DN groups were significantly higher than in control group ( p 5 0.006 and
p 5 0.000, respectively). Furthermore, control and patient
groups were similar in terms of diastolic blood pressure,
while systolic blood pressure was high in the DN group
compared to the control group ( p 5 0.000).
Blood and Urine Biochemistry
Biochemical measurement in blood and urine are presented
in Table 3. A significant increase in blood glucose and
HbA1c values were observed in T2D and DN groups as
compared to control group ( p 5 0.000 for each). In patients
with DN, serum urea and creatinine were significantly high
compared to control ( p 5 0.001 and p 5 0.004, respectively) and T2D ( p 5 0.01 and p 5 0.003, respectively)
groups. Patient and control groups were similar in terms
of Na, K, TC and LDL values. TG and VLDL levels significantly increased in T2D group when compared to control
group ( p 5 0.01 for each). Furthermore, TG and VLDL
levels were lower in the DN group than in the T2D group
( p 5 0.013). HDL levels in T2D and DN groups decreased
significantly compared to the control group ( p 5 0.000 for
each). In DN group, urine albumin-protein/creatinine ratio
was higher than the control and T2D groups ( p 5 0.000
for each). Similarly, urine albumin and protein levels in
DN group were higher than the control ( p 5 0.000 and
p 5 0.001, respectively) and T2D ( p 5 0.000 for each)
groups. Unlikely, GFR in DN group was lower than the
other groups ( p 5 0.001 vs. control group and p 5 0.009
vs. T2D group).
KLF-4 Gene Expression Analysis
KLF-4 gene expression in the circulation showed a 1.60
fold decrease in the T2D group as compared with the control group ( p 5 0.001). In the DN group, the decrease was
higher than T2D. KLF-4 gene expression reduced a 2.92
Table 3. Biochemical measurement in blood and urine
Control
Plasma glucose (mg/dL)a
HbA1ca
Serum ureaa
Serum creatininea
Naa
Ka
TC (mg/dL)a
TG (mg/dL)a
LDL (mg/dL)a
HDL (mg/dL)a
VLDL (mg/dL)a
Urine albumin-protein/creatinine ratioa
Urine albumina
Urine proteina
GFRa
a
88.02
5.46
31.32
0.83
141.12
4.53
200.21
122.46
120.38
57.71
24.49
36.23
10.22
9.95
98.48
10.40
0.31
21.40
0.53
2.04
0.33
46.86
61.24
43.19
17.01
12.24
14.68
8.81
3.22
3.46
T2D group
179.47
8.28
32.11
0.80
140.51
4.73
189.25
177.63
110.21
44.13
35.52
13.93
8.40
12.05
93.09
86.59b
2.02b
10.83
0.27
3.32
0.39
46.67
114.0g
35.18
13.69b
22.8g
3.91
6.85
1.64
3.38
DN group
165.67
8.29
50.62
1.29
148.54
4.75
185.68
163.08
118.83
42.68
32.61
971.30
59.11
78.70
74.93
62.72b
1.66b
32.71c,d
0.85e,f
46.27
0.50
48.51
66.97h
45.57
11.45b
13.39h
270.69b,i
30.05b,i
18.72c,i
6.22c,j
p
0.000
0.000
0.001
0.004
0.932
0.064
0.426
0.026
0.487
0.000
0.016
0.000
0.000
0.000
0.000
Means SD; Type-2 diabetes: T2D; diabetic nephropathy: DN.
p 5 0.000 vs. Control group; cp 5 0.001 vs. Control group; dp 5 0.01 vs. Diabetes group; ep 5 0.004 vs. Control group; fp 5 0.003 vs. Diabetes group;
g
p 5 0.01 vs. Control group; hp 5 0.013 vs. Control group; ip 5 0.000 vs. Diabetes group; jp 5 0.009 vs. Diabetes group.
b
KLF-4 Expression in Diabetes and Diabetic Nephropathy
Figure 2. The differences of kruppel-like transcription factor-4 (KLF-4)
mRNA expression level were shown in control, Type-2 diabetes (T2D)
and diabetic nephropathy (DN). Data are shown as the mean SD.
a
p 5 0.001 vs. control group, bp 5 0.001 vs. T2D group.
fold in the DN group when compared to T2D group
( p 5 0.001, Figure 2).
KLF-4 Methylation Status
The samples of 120 patients were used for methylation
analysis. In all samples, the amount of the unmethylated
PCR product was greater than the methylated ones as
shown in Figure 3. There were no significant changes in
methylation status of KLF-4 among control, T2D and DN
groups.
Discussion
More than 40% of patients with diabetes can develop DN
that is a serious microvascular complication and a major
cause of end-stage renal disease. Diabetes induces the
abnormal expression of genes involved in the pathogenesis
of DN via activating transcription programs in target cells
(16).
95
Individuals with fasting plasma glucose levels of 99 mg/
dL or less and HbA1c ! 5.7 are considered as a healthy
group and that individuals with plasma glucose levels of
126 mg/dL and higher, and HbA1c O 5.7 are considered
T2D group (17). According to Al-Rubean K, et al. (15),
the patients with DN were assessed by urine albumin/creatinine ratio $30 mg/g (ACR, mg/creatinine). In this study,
control, T2D and DN groups were formed based on these
values.
Proteinuria is a component of chronic kidney disease as
well as an important risk factor for cardiovascular diseases
(18). It is suggested that KLF-4 may provide a common
mechanism linking cardiovascular and renal diseases
because of reduction of KLF-4 expression in multiple tissues related to vascular and cardiac diseases (19,20). Thus,
the present study was planned to reveal the possible effects
of KLF-4 in the etiology of T2D and DN. The exposed to
high glucose significantly decreased KLF-4 mRNA levels
in human kidney proximal tubular cells. Likely in renal tissues of diabetic mice, KLF-4 mRNA levels were reduced.
Therefore, the researchers suggested that KLF-4 may be a
therapeutic target for DN (21). Similarly, it was reported
that KLF-4 expression, highly expressed in kidney glomerular podocytes, decreased in the diabetic animal models and
humans with proteinuria. The regulation of KLF-4 expression in impaired glomeruli through in vivo gene transfer
improved the nephrin expression and decreased proteinuria
(22). According to our findings, the level of KLF-4 mRNA
expression in the T2D group decreased compared to the
control group. The decreased level of KLF-4 mRNA
expression in the DN group was higher than in the control
and T2D groups. These findings suggest that KLF-4 may be
an effective gene in the pathogenesis of T2D and diabetic
kidney diseases.
Dysregulation in gene expression depending on DNA
promoter methylation status have been reported to be associated with many diseases (23). Dysregulated DNAMe is an
important factor in the reduction of gene expressions
involved in diabetes and metabolic syndrome, pancreatic
islet and skeletal insulin production, signaling, and energy
metabolism (24,25). Epigenetic changes may be altered
Figure 3. KLF-4 methylation status. First lane: 100 bp marker, other lanes: sample number with U (unmethylated) and M (Methylated), Type-2 diabetes:
T2D; diabetic nephropathy: DN.
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Coskun et al./ Archives of Medical Research 50 (2019) 91e97
by diet, lifestyle, and environmental factors. It has been
shown that hyperglycemia and free fatty acids can alter
the expression of genes associated with obesity-induced
diabetes by regulating DNAMe that is one of the epigenetic
mechanisms (26). Hyperglycemia induces DNA promoter
methylation that is often associated with decreased gene
expression and hyperglycemia altered the DNAMe status
and also caused abnormal DNAMe in the proximal tubules
of the diabetic kidney (26e28). Similarly, the exposure of
vascular endothelial and neuronal cells to hyperglycemia
caused to alteration in the DNAMe status of several genes
that have a role in the dysfunction of cells (29). In a study
on pancreatic islets of T2D patients, it was reported that
DNAMe in the insulin gene promoter is associated with
low levels of insulin and high levels of HbA1c (24).
There is the relationship between T2D and DNAMe, as
well as the relationship between the development of DN
and methylation status of different genes expressed in
different tissues. DNAMe profiles of microdissected tubules
of patients with DN showed differences in the methylation
status of various genes involved in fibrogenesis. The
expression of claudin-1 in podocytes and the expression
of sirtuin-1 in tubules decreased with DNAMe in DN
(30). The DNAMe status in kidney tubular epithelial cells
obtained from microdissection of chronic kidney disease
(CKD) compared to in the control group, significant
methylation alterations in 1.061 genes were observed in
DN patients (31). Brennan EP, et al. (32) identified specific
DNAMe patterns in human mesangial cells and proximal
tubular epithelial cells. In another study, various DNAMe
profiles were determined in saliva samples of patients with
end-stage renal failure (33).
It is known that DNAMe is tissue-specific. However,
Rakyan VK, et al. (34) emphasized that the DNAMe patterns slightly varied between different tissues. Teschendorff
AE, et al. (35) suggested that whole blood may be a reasonable option for determining whether genomic methylation
changes can be detected. Using DNAs isolated from peripheral blood cells of 192 diabetic patients with or without
renal disease, 19 CpG islands associated with DN risk were
identified in promoter regions of 14.495 genes (36). Pezzolesi MG, et al. (37) investigated about half a million
methylation sites in the blood cells of individuals with
and without CKD. Accordingly, between the patient and
control groups, in the DNAMe status of 23 genes were
found to be a significant difference, and six candidate genes
were reported to be related with renal disease. Genomic
DNA is hypomethylated in the liver of the diabetic rats
but not in the kidney, and also hypomethylation of DNA occurs in a tissue-specific manner (38). It is suggested that
DNA hypermethylation is effective in the pathogenesis of
type-2 diabetic rats, and this is specific to the genomic
DNA in the liver but not global DNAMe status and methylated CpG islands of the kidneys and heart (39). Xiao X,
et al. (2015) suggested that the hypermethylation of KLF-
4 promoter region contributes to the progression of renal
fibrosis (40). According to the other studies, KLF-4 expression was partly related with the hypermethylation of KLF-4
promoter region in renal cell carcinoma. The promoter hypermethylation of KLF-4 may cause to its expression suppression (41,42). In the present study, there were no
significant differences in methylation levels of KLF-4 gene
in both T2D and DN patients compared to the control
group. DNAMe is not an effective mechanism for the
reduction of KLF-4 mRNA expression in patients with
T2D and DN. It is suggested that unlike DNAMe, the other
epigenetic mechanisms including histone acetylation/
methylation, microRNAs and metabolic memory may be
effective in the changes of KLF-4 expression. In addition,
it may be more useful to identify tissue-specific methylation patterns associated with T2D and DN pathogenesis.
In conclusion, the data showed that the reduction of
KLF-4 expression level may be related with T2D and especially DN. However, it was found no evidence that
DNAMe-induced silencing is responsible for the reduction
of KLF-4 mRNA expression in genomic DNA samples
from T2D and DN patients. Due to the tissue-specific nature of the epigenetics, further researches should be performed on specific DNA methylation patterns for T2D
and DN in kidney tissue and different renal cells.
Acknowledgment
This study was supported by the Scientific Research Projects Coordination Unit of Istanbul Bilim University. Project no: 2015017.
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