CORRELATION BETWEEN p16, TGF-β AND NF-κB mRNA

EXPRESSION AND TUBULAR INJURY IN KIDNEY FIBROSIS

MODEL IN MICE

Iqbal Wahyu Dwiaji

16/393110/KU/18713
 CHAPTER I
INTRODUCTION
Background
• Kidney fibrosis is defined as a final common pathway of
chronic kidney disease (CKD). In Indonesia based on the
results of the Basic Health Research (Riskesdas) in 2018,
the prevalence of CKD was 0.38%
• The definitive treatment for CKD is still not obtained and so
is the definite disease progression, however many studies
in various model and organs seems to signify the
relationship and links between, inflammation, fibrosis and
possibly cell senescence. This has given a new wave of
kidney fibrosis treatment through anti inflammatory or anti
fibrotic therapy
• In spite of all this, the correlation between markers of cell
senescence, fibrosis, and also inflammation in kidney have
not been elucidated.
Problem Formulation
• How does the unilateral ureteral
obstruction (UUO) on day 3, 7 and 14
affect p16 mRNA expression?
• How does the unilateral ureteral
obstruction (UUO) on day 3, 7 and 14
affect TGF-β mRNA expression?
• How does the unilateral ureteral
obstruction (UUO) on day 3, 7 and 14
affect NF-κB mRNA expression?
• Is there any correlation between p16
expression and TGF-β as well as
between p16 expression and NF-κB in
mice with unilateral ureteral
obstruction (UUO) on day 3, 7 and 14
Research Objectives
• Elucidate the effect of unilateral ureteral
obstruction (UUO) on mRNA expression
p16 on day 3, 7 and 14.
• Elucidate the effect of ureteral
obstruction (UUO) on mRNA expression
TGF-β on day 3, 7 and 14.
• Elucidate the effect of unilateral ureteral
obstruction (UUO) effect on mRNA
expression NF-κB on day 3, 7 and 14.
• Elucidate the correlation between p16
and TGF-β as well as between p16
expression and NF-κB in mice with
unilateral ureteral obstruction (UUO) on
day 3, 7 and 14 in mice with unilateral
ureteral obstruction (UUO) on day 3, 7
and 14.
Study Originality
No Researcher Title Differences
1 Wolstein, et INK4a knockout mice exhibit increased fibrosis This experiment uses mice model with a
al., 2010 under normal conditions and in response to knockout gene, and only quantify p16.
unilateral ureteral obstruction
2 Chen, et al., Telbivudine attenuates UUO-induced renal The experiment used TGF-β, NF-κB as well
2018 fibrosis via TGF-β/Smad and NF-κB signaling other markers. Such as myofibroblast
markers and Il-1 and Il-6. Also the rats were
given the drug telbivudine.
3 Zhou, et al., Less Expression of Prohibitin Is Associated with The study used different timings on the
2012 Increased Paired Box 2 (PAX2) in Renal termination of animal. They used day 14
Interstitial Fibrosis Rats and 24. Also, this study measure the
expressions of PAX and PHB.
4 Tasanarong, Dual Inhibiting Senescence and Epithelial-to- This study used Smad2/3, S100A BMP-7 as a
et al., 2016 Mesenchymal Transition by Erythropoietin marker as well as p16 and TGF-β. Also this
Preserve Tubular Epithelial Cell Regeneration study used human erythropoietin as an
and Ameliorate Renal Fibrosis in Unilateral intervention.
Ureteral Obstruction
RESEARCH BENEFIT
• This study can add insight and knowledge into the effect of unilateral
ureteral obstruction (UUO) on the expression of mRNA p16, and TGF-β in cell
aging during renal fibrosis.

Student
• Increase insight regarding this particular field of kidney fibrosis and disease.
• In the future more studies can be done in this field by the institution with the
aim of increasing understanding of this diseases and possible finding more
cure.
Institution

• The results of this study are expected to broaden the medical knowledge
further about Chronic Kidney disease’s pathophysiology. So that we can
better diagnose and treat this disease as well as other medical problems
regarding the kidney.

Community
 CHAPTER II
LITERATURE REVIEW
Anatomy and Histology of The Kidney
• It is located above the waist with ribs 11 and 12
protect it partially. It lays behind the peritoneum
There are two distinct layer, the medulla and the
cortex
• The main function of the kidney, This process is
completed by three main processes, glomerular
filtration, tubular reabsorption, and tubular
secretion. This is done by the Nephron.
• Microscopically the kidney is made out of the
nephron, which is the functional structure of the
kidney. There are approximately one million
nephron units in one kidney
• Tubular nephron component starts from
Bowman's capsule and then proximal tubules
then Ansa Henle which functions to form
osmotic pressure gradients in urine to achieve
the proper concentration, and terminated by
distal tubules
Chronic Kidney Disease
• Chronic kidney disease (CKD) is defined when the value of Glomerular
Filtration Rate (GFR) is below the normal limit for more than 3 months
• CKD itself is a disease which causes a slow progression of kidney failure
development that can lasts for several years. In this disease, the kidneys
fail to maintain metabolic, fluid, and electrolyte balance that could
manifest into uremia or any other renal abnormalities
• According to the Indonesian Nephrologist Association (PERNEFRI) in 2012
are the two very common causes and these are hypertensive kidney
disease (35%) and diabetic nephropathy (26%).
Unilateral Ureter Obstruction (UUO)
• Unilateral Ureter Obstruction (UUO) is one of the
experimental methods with animal models to get a
picture of kidney fibrosis.
• UUO is done by ligation and cutting of the ureter
on one side, right or left. Ligation of the ureter
causes urine stasis, resulting in increased pressure
in the renal tubules.
• The UUO-obstructed kidney manifest in many
mechanical and physiological changes. These
changes include tubular dilation, activation of
Renin-Angiotensin System (RAS) system interstitial
expansion, loss of proximal tubular mass,
infiltration of leukocytes, tubular epithelial cell
death and presence of fibroblasts.
Kidney Fibrosis
• Kidney fibrosis is the final manifestation of all
progressive chronic kidney disease
• defined as the formation and accumulation of
excessive extracellular matrix (ECM),
• Fibrogenesis is a complex and dynamic
process but it can be divided into 4 processes.
Priming, activation, execution and
progression.
Cell Senescence
• Cell Senescence is defined when a cell has
stop dividing, or a permanent stage of cell
arrest.
• This mechanisms can be divided in two
main types. First one is called replicative
senescence, this is as a result of continuous
cell division. The other one is called stress
induced premature senescence (SIPS).
• These mechanisms invokes cell senescence
by activating p53/p21 and p16/pRb
pathways
• Senescent cells can a release a distinct
number of proteins termed the senescence-
associated secretory phenotype (SASP). Via
this set of proteins, these senescent cells
increases pro fibrotic and fibrotic effects
from other cells.
TGF- β, p16 and NF-κB
TGF- β
• Transforming growth factor
beta (TGF-β) is a cytokine
which has multiple function.
• TGF-β is a vital and pivotal
protein in creating and
accumulating ECM
• Angiotensin, oxidative stress
and chronic inflammation can
induced the production of
this cytokine
p16
• p16 is an important tumour
suppressing gene that inhibits
cyclin D–dependent protein
kinases it has a vital role in
the regulation of G1/S
transition
• p16 works by inhibiting cyclin
dependent kinase 4 (CDK4)
and cyclin dependent kinase 6
(CDK6) and stops them from
binding to cyclin D , resulting
the failure of the
phosphorylation of
retinoblastoma protein (pRb).
NF-κB
• NF-κB is a transcription factor
which serves as one of the
most vital mediator of
inflammation. It regulates
multiple aspects of innate and
adaptive immune functions.
• Angiotensin, oxidative stress
and LPS and pathogens can
induced the production of
this cytokine
Theoretical Framework Conceptual Framework
UUO
Notes:

Induced
Inhibit TUBULAR
RAS ACTIVATION
Being Studied OBSTRUCTION Dependent Variable

TUBULAR Independent TGF-β mRNA

DILATATION↑ Swiss
Variable expression
Background
Unilateral ureter NF-κB mRNA
TUBULAR EPITHELIAL Mice
INJURY obstruction expression
p16 mRNA
CYTOKINE
INFLAMMATION ↑
expression

OXIDATIVE
NF-κB TGF-b ↑
Controlled Variable
STRESS↑
Gender of rats
Initial weight of rats
CHRONIC
ECM ↑
INFLAMMATION

STRESS-INDUCED PREMATURE
SENESCENCE

p16

CDK6/4

RB

SENESCENCE
CELLULER FIBROSIS
 Hypothesis
1. Expression of p16 in mice with UUO is higher
compared with control group on day 3, 7
and 14.
2. Expression of TGF-β in mice with UUO is
higher compared with control group on day
3, 7 and 14.
3. Expression of NF-κB in mice with UUO is
higher compared with control group on day
3, 7 and 14.
4. There will be a positive correlation between
p16 and TGF-β as well as between NF-κB and
p16 in mice with unilateral ureteral
obstruction (UUO) on day 3, 7 and 14.
 CHAPTER III
METHOD OF RESEARCH
 Study Design and Setting
Study Design
• This study uses a quasi-experimental type of research with a post-test only controlled group
design research design. The study was conducted on research subjects in the form of
experimental animals that were manipulated and compared with the control group.

Study Setting
This study will be conducted on September 2018 – October 2019 at
• Anatomy Department of FK-KMK UGM for breeding and treatment of samples, termination of
samples, vascular remodeling analysis, electrophoresis,
• Integrated Research Laboratory of FKKMK UGM for quantification of RNA isolates, RT-PCR
documentation of electrophoresis results.
Study Subjects and Sample Size
• The subjects in this study were Swiss male strain mice 3-4 months old with 30-50 gram body
weight obtained from UGM LPPT.
• Subjects were divided into 4 groups according to the 3R principle (replacement, refinement,
reduction), namely:
Group 1 (SO), Group 2 (UUO3), Group 3 (UUO7), Group 4 (UUO14):
• We used Federer's formula to determine sample size
Federer's formula = (T-1) (N-1) ≥ 15
Abbreviation:
• T = Number of the group
• N = Number of the rats for each group
(4 – 1) (n – 1) ≥ 15
(4 – 1) (n – 1) ≥ 15
3 (n – 1) ≥ 15
n≥5+1
n≥6
The number of samples used for each group is 6 rats. So in total this study used 24 mice
Study Material and Measurement
Study Material
Food and drink for experimental
animals:
• Standard feed and drinking
water given ad libitum.
Ingredients for UUO and experimental
animal termination:
• Swiss strain male mice,
• Na-Phenobarbital 1:10 solution,•
• 70% alcohol,
• 0.9% sodium chloride (NaCl),
• Povidone iodine,
• Aquadest,
• 0.4 mm silk thread,
• Buffer 10% formalin,
• Later RNA.
Material for extracting RNA, making • GAPDH primer,
cDNA, PCR, and electrophoresis:
• Tris Borate EDTA (TBE),
• Genezol,
• Agarose gel and red gel.
• Diethylpyrocarbonate (DEPC),
• 70% alcohol, chloroform,
• Isopropanolol,
• Random primer,
RT buffer,
• Deoxyribonucleotide (dNTP),
• Reverent TraAce,
• taq master mix,
• p16 primer,
• TGF-β primer,
• NF-κB Primer
Study Tools
• Study Tools
• Gloves • RT-PCR machine
• Mask • 0,2 ml Eppendorf
• Ice pack • Micropipette
• Light Microscope • Pipette tip
• Optilab • Mice cages
• Set of minor surgical • Digital Scales
equipment • Mupid-2plus
electrophoresis
 Study Procedure

RT-PCR
UUO
DISTRIBUTION EXAMINATION
intervention SACRIFICE OF HARVESTING RNA CREATING
OF TREATMENT AND
and Sham THE RATS OF THE KIDNEY EXTRACTION cDNA
GROUP ELECTROPHOR
Operation
ESIS
 VARIABLE Operational Definition
• INDEPENDENT VARIABLE • Unilateral ureteral obstruction (UUO): This is
• Days of UUO modelled mice termination an animal model by ligating the right ureter
• DEPENDENT VARIABLE and renal pediculus proximal. The mice were
then terminated according to the groups with
• NF-κB mRNA expression
UUO3 terminated on day 3, UUO7 terminated
• p16 mRNA expression on day 7 and UUO 14 terminated on day 14.
• TGF-B mRNA expression • The p16, TGF-β, NF-κB, mRNA expressions:
• CONTROLLED VARIABLE These are the result of the analysis of band
• Strains densitometry from the electrophoresis of
• Gender Reverse-Transcriptase Polymerase Chain
• Age Reaction (RT-PCR) genes p16, TGF-β, NF-κB,
mRNA which are then compared with the
• Weight
GAPDH band as a house keeping gene and
calculated using lmageJ software
Statistic Method
• The data obtained analyzed statistically using the IBM® SPSS®
Statistics version 23 program.
• The results of the study are considered statistically significant if the
probability value (p) is <0.05. The following is the statistical test used
in this study:
• Normality test of data using Saphiro-Wilk.
• Numerical tests in group using One-Way ANOVA if the data
distribution is normal or Kruskal-Wallis if the data distribution is
not normal.
• Following that post-hoc LSD test are conducted. if the result is
significant between groups. Mann Witney will be used if
the result is not significant.
 CHAPTER IV
RESULT AND DISCUSSION
 Expression of NF-κB
• The mean of NF-κB /GAPDH on
NF-κB control group (SO) was (1.21±
0.5), the group was (1.86± 0.3),
GAPDH the UUO7 group was (2.20±
0.5), and the UUO14 group was
Control UUO3 UUO7 UUO14 (2.30± 0.9).
(SO)
NFKB/GAPDH • The data were distributed
3
*
normally.
*
2.5
• ANOVA value is significant
2
(p=0.01)
Arbitrary Unit

1.5
• Significant post hoc value on
1
between groups SO vs UUO7
0.5
(p=0.017) and also between SO
0
SO UUO3 UUO7 UUO14 vs UUO14 (p=0.010).
Experimental Group
 Discussion
Oxidative Stress and NF-κB
• Oxidative stress has been found in many types of renal injury, including in UUO-modelled mice. (Dendooven et al.,
2010). a positive correlation was observed between the levels of free radical oxidation markers in the obstructed kidney
tissue and in plasma (Klahr and Morrissey, 2002).
• The administration of antioxidant peptides to rats that suffered UUO was associated to a lower activation of NF-κB.
(Grande, Pérez-Barriocanal and López-Novoa, 2010). Further suggesting the link between oxidative stress and NF-κB
expression.
Angiotensin and NF-κB
• NF-κB is also activated by ANG II in the kidney (Klahr and Morrissey, 2002.) UUO induces RAS activation by lowering the
GFR due to increase pressure in afferent arterioles.
• NF-κB also induces angiotensinogen (Precursor of ANG II). Therefore, this provides an autocrine-reinforcing loop that
upregulates ANG II production.
• Studies have been shown that showed that administering ACE inhibitor, decreases NFκB expression in UUO modelled mice
(Nakatani et al., 2002).

ANG II NF-κB
Discussion
Angiotensin and NF-κB

• NF-κB is also activated by ANG II in the kidney (Klahr and Morrissey, 2002.) UUO induces RAS
activation by lowering the GFR due to increase pressure in afferent arterioles.
• NF-κB also induces angiotensinogen (Precursor of ANG II). Therefore, this provides an autocrine-
reinforcing loop that upregulates ANG II production.
• Studies have been shown that showed that administering ACE inhibitor, decreases NFκB expression
in UUO modelled mice (Nakatani et al., 2002). Further suggesting the link between RAS activation
and NFκB expression.

ANG II NF-κB
 Expression of TGF-β .
• The mean of p16/GAPDH expressions
TGF-β . on SO group was (0.43 ± 0.2), the
UUO3 group was (0.57± 0.1), the
GAPDH
UUO7 group was (0.60 ± 0.1), and the
Control UUO3 UUO7 UUO14 UUO14 group was (0.66 ± 0.3).
(SO)
TGFB/GAPDH
• The data were distributed normally.
4 * • ANOVA value is significant (p=0.01)
• Significant post hoc value on SO group
3.5

3
*
*
vs UUO3 (p=0.018), SO group vs UUO7
Arbitrary Unit

2.5

2 (p=0,03) , and SO group vs UUO14

1.5 (p=0,010).
1

0.5

0
SO UUO3 UUO7 UUO14
 Discussion
Angiotensin and TGF-β
• Previous studies had shown that Angiotensin played a significant role on the synthesis of cytokines and
growth factor. Particularly TGF-β (Klahr and Morrissey, 1998).
• This is particularly why ACE inhibitor and anti-oxidant have been proven to reduce kidney fibrosis in
mice (Nakatani et al., 2002)

• Cytokines and chemokines play a major role in the recruitment of inflammatory cells in the obstructed
kidney.
• A study showed that there is significant correlation between interstitial macrophage number and
cortical TGF-β1 expression levels that has been reported in the obstructed kidney. Showing the link
between inflammation and fibrosis.(Grande, Pérez-Barriocanal and López-Novoa, 2010).
Fibrosis and TGF-β
• It is documented well that TGF-β stimulates gene
expression of ECM components.
• Proliferation of ECM initially occurred as a way for
wound healing process but due to prolonged injury
pathological fibrosis could occur. This experiment
could signal the irreversible ECM disposition in UUO.
• TGF-β plays an important role in fibroblast
proliferation, inhibits the activities of proteases that
degrade the ECM, and increases the expression of
cell-surface integrins that interact with matrix
components. Thus increasing the deposition of ECM
(Nogueira et al., 2017)
• Moreover, the importance of TGF-β1 in renal fibrosis
is further supported by the findings that
overexpression of active TGF-β1 in rodent liver which
are capable of inducing the fibrotic response in
kidney(Meng et al., 2015).
 Expression of p16
• The mean of p16/GAPDH
P 16 expressions on SO group was
(0.43 ± 0.2), the UUO3 group
GAPDH was (0.57± 0.1), the UUO7
Control UUO3 UUO7 UUO14 group was (0.60 ± 0.1), and the
(SO) UUO14 group was (0.66 ± 0.3).
p16/GAPDH
0.8
*
• The data were distributed
0.7
*
* normally.
• ANOVA value is significant
0.6
Arbitrary Unit

0.5

0.4
(p=0.01)
0.3
• Significant post hoc value on SO
0.2
group vs UUO3 (p=0.018), SO
0.1

0
group vs UUO7 (p=0,03) , and
SO UUO3 UUO7
Experimental Group
UUO14
SO group vs UUO14 (p=0,010).
Expression of p16

Value TGF- Value of NF-

Β/GAPDH κB/GAPDH
Value p16/GAPDH Pearson .441 .465
Correlation
Sig. (2-tailed) .045 .039
N 21 20
 Discussion
P16 and Stress Induced Premature Cell Senescence
• P16 is a marker of cell senescence and the increase expression could
mean that the cell is undergoing senescence gradually. Since p16 is a
tumor suppressor gene that binds to CDK4 / CDK6 so that the cell cycle
will stop
• This could be due to reactive oxygen (ROS) species or the inflammation
process in the kidney. As all these aspect involves and causes senescence.
Also known as stress induced prematured senescencence. (De Magalhães
and Passos, 2018).
Fibrosis and p16 and Cell senescence
• through SASP (Senescence acquired secretory phenotype) p16 promote
the production of profibrotic cytokines from surrounding cells. (Valentijn
et al., 2017)
• These cytokines include interleukin 6, plasminogen activator inhibitor-1
(PAI-1) and TGF-β This could be the reason why there is a significant
correlation between p16 and tgfB
Limitations
Several limitations faced by this study are as follows:
• Lots of cytokines are involved in inflammation and fibrosis. Thus this
study is does not have a strong evidence to show the relationship
between inflammation and cytokines
• Many etiologies of CKD or injury cannot be demonstrated only by
UUO alone. Such as hypertension, diabetes, etc.
 CHAPTER IV
CONCLUSION AND SUGGESTION
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