Chlorogenic Acid Ameliorates Vascular Remodeling and Perivascular

Fibrosis in Kidney Fibrosis Model in Mice

Gabriella Bamba Ratih Lintin

, Nur Arfian

2 b

, Dwi Cahyani Ratna Sari

, Gina Andyka

Hutasoit

, Mohammad Salman

and Muhammad Mansyur Romi

Anatomy Department, Faculty of Medicine Universitas Tadulako, Palu

Anatomy Department, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta

Patology Anatomy Department, Faculty of Medicine Universitas Tadulako, Palu

Histology Department, Faculty of Medicine Universitas Tadulako, Palu

Keywords: Kidney Fibrosis, UUO, CGA, Vascular Remodeling, Perivascular Fibrosis

Abstract: Kidney Fibrosis is the common pathway final of Chronic Kidney Disease, which is characterized by vascular

remodeling and perivascular fibrosis. The Unilateral Ureteral Obstruction (UUO) model is used to cause

kidney fibrosis. Chlorogenic Acid (CGA) is an antioxidant as a renoprotective agent. However, fibrosis

perivascular and vascular remodeling has not analyzed yet. Study has objective to examine the effect of CGA

in vascular aspects, specifically vascular remodeling and perivascular fibrosis in kidney fibrosis. Material and

Methods: This research was a true experimental study. Unilateral Ureteral Obstruction (UUO) was performed

in swiss webster background mice (n=25, 2-3 months old, 20-30 g weight) to induce kidney fibrosis. The mice

were divided into five groups, SO (Sham Operation/control), U7 (UUO day-7), U14 (UUO day-14), UC7

(UUO+CGA day-7), and UC14 (UUO+CGA day-14). CGA 14 mg/kg body weight/day was induced

intraperitoneally. Vascular remodeling based on lumen area, mean wall thickness, and wall/lumen area ratio

(WLAR) and perivascular fibrosis of intrarenal arteries were quantified using Sirius Red staining. Study found

that UUO groups (U7 and U14) had significantly higher vascular remodeling, as shown by lower lumen area,

higher mean wall thickness and higher WLAR, and perivascular fibrosis, as shown by higher area compared

to SO group (p<0.05). On the other hand, CGA groups (UC7 and UC14) revealed lower vascular remodeling,

as shown by higher lumen area, lower WLAR, and perivascular fibrosis, as shown by lower area significantly

compared to UUO group (p<0.05). The mean wall thickness was lower, but the data was not significantly

different. Study conclude that CGA ameliorates kidney fibrosis through vascular remodeling and perivascular

fibrosis.

1 INTRODUCTION

Chronic Kidney Disease (CKD) is one of the world's

public health priorities because of its rapid increase in

the prevalence. Global Burden of Disease study in

2015, renal disease was the 12th leading cause of

death, with 1.1 million deaths worldwide. In

Indonesia, the high prevalence indicated by the

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enrollment of more than 15.000 new patients with

CKD in health insurance in 2011, 87% were End-

Stage Renal Disease (ESRD) patients (Perkumpulan

Nefrologi Indonesia, 2011). Decreased kidney

function and scar formation that occurs progressively

will direct CKD to the disease course's final stage,

ESRD (Mutsaers et al, 2015). Kidney fibrosis leads

to the ESRD as a final common pathway to CKD

(Fragiadaki & Mason, 2011). Kidney fibrosis is

Lintin, G., Arﬁan, N., Ratna Sari, D., Hutasoit, G., Salman, M. and Romi, M.

Chlorogenic Acid Ameliorates Vascular Remodeling and Perivascular Fibrosis in Kidney Fibrosis Model in Mice.

DOI: 10.5220/0010487000150020

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 15-20

ISBN: 978-989-758-499-2

characterized by interstitial fibrosis,

glomerulosclerosis, the formation and activation of

myofibroblasts cells (Duffield, 2014).

Unilateral Ureteral Obstruction (UUO) is the

most used experimental kidney fibrosis model,

especially in studies related to irreversible Acute

Kidney Injury (AKI) and CKD (Ucero et al, 2014).

The changing of hemodynamic in CKD may induce

vascular remodeling, which is influenced by

activation of the renin-angiotensin system (RAS),

endothelin-1 (ET-1), endothelial dysfunction,

oxidative stress and asymmetric dimethylarginine

(ADMA), and the anti-aging molecule Klotho (Briet

& Burns, 2012). Fibrosis also causes vascular

remodeling characterized by interstitial remodeling

and then vascular remodeling, resulting in reduced

blood supply to the kidneys, causing kidney failure

(Efstratiadis et al, 2009). Endothelin-1 (ET-1) and

Endothelial Nitric Oxide Synthase (eNOS), which

catalyzes the production of nitric oxide (NO) has a

significant role in vascular function as

vasoconstriction and vasodilatation agent (Schiffrin,

2012). It is becoming increasingly clear that an

imbalance between these two mediators is a

characteristic of endothelial dysfunction and is

essential in vascular remodelling

(Farris & Colvin,

2012).

Myofibroblasts are biomolecular markers and

terminally differentiated cells found in non-

pathological situations responsible for the synthesis

and accumulation of interstitial extracellular matrix

components during kidney fibrosis as the

pathogenesis of CKD (Farris & Colvin, 2012).

Perivascular fibroblasts and pericytes have

previously been identified as the major contributors

to the fibrosis then myofibroblast population in the

kidney, especially in vascular area

(Kramann &

Humphreys, 2014).

Chlorogenic acid is a phenolic acid with vicinal

hydroxyl groups on aromatic residues derived from

cinnamic acid esterification, including caffeic,

ferulic, and p-coumaric acids with quinic acid.

Much evidence has shown that chlorogenic acid has

many biological characteristics, including

antibacterial, antioxidant, and anticarcinogenic

activities, especially hypoglycemic, hypolipidemic,

and renoprotective effects

(Santana-Gálvez et al,

2017). Chlorogenic acid can improve kidney function

in 5/6 nephrectomy rats effectively due to its anti-

oxidation and inhibiting accumulation of

extracellular matrix

(Lou et al, 2016).

None of the existing studies have provided overall

effects from chlorogenic acid on fibrosis conditions

in several other organs, especially from the molecular

profibrotic and vasoactive substances. Therefore, this

study was conducted to examine CGA's effect on

vascular aspects, specifically vascular remodeling

and perivascular fibrosis in kidney fibrosis.

2 MATERIALS AND METHODS

This research was a true experimental study using a

post-test only controlled group design. Consists of

control and treatment groups. This study has obtained

permission from the Medical and Health Research

Ethics Committee Faculty of Medicine, Universitas

Tadulako, based on the Ethical Clearance certificate

number C.0942/UN28.1.30/KL/2018 on February 26,

2018.

2.1 Unilateral Ureteral Obstruction

Swiss Webster mice (n=25, 2-3 months old, 20-30 g

weight) were used for the experiments. Mice were

housed in the Department of Anatomy, Faculty of

Medicine, Universitas Tadulako in a cage with the

light-dark cycle of 12:12 hour, food and water ad

libitum. Unilateral Ureteral Obstruction (UUO) was

performed to induce kidney fibrosis. Mice were

anesthetized with Sodium Pentobarbital (0.1mL/10 g

weight) injected intraperitoneally. The right flank's

region was opened, and the right ureter was visualized

then double ligated, after that cut between the ligation

sides. Sham operation (SO) control group procedure

was used the same procedure except for ligating and

cutting the ureter, only for visualized.

2.2 Chlorogenic Acid Administration

Chlorogenic acid (Sigma-Aldrich C3878-1G) was

done with intraperitoneally injection with a dose 14

mg/kg body weight/day. Mice were divided into 5

groups. The distribution for each group: sham

operation (SO) group, was injected distilled water

intraperitoneally for 14 days as a control; mice with

UUO was injected distilled water intraperitoneally for

7 days, called group U7; mice with UUO was injected

with chlorogenic acid for 7 days called UC7 group;

mice with UUO was injected distilled water

intraperitoneally for 14 days called group U14; mice

with UUO was injected chlorogenic acid for 14 days

group UC14. Mice were euthanized on days 8 and 15.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

2.3 Kidney Harvesting

In this study, kidney harvesting mice were

anesthetized with Sodium Pentobarbital (0.1mL/10 g

weight) injected intraperitoneally after that abdomen

and thorax were opened. Perfusion was done with 0.9

% NaCl from the left ventricle. Right kidney tissues

were harvested, and the one-half side was fixated in

Normal Buffer. Formalin for 24 h, and used for the

paraffin-embedded tissue process.

2.4 Sirius Red Staining

Tissue slides in paraffin disk were cut in 4 mm

thickness. Paraffin sections were deparaffinized with

PBS washing. Tissue slides were given Sirius

Red working solution to the entire surface of tissue

for 1 hour. Afterward, the slides were soaked

sequentially in 100% ethanol and xylene 3 times each,

then mounted and incubated for 24 hours. Vascular

remodeling assessment based on lumen area, mean

wall thickness, wall area, and lumen area

ratio/WLAR, while perivascular fibrosis is based on

the difference between perivascular fibrosis and

intrarenal arteries blood vessel area. Ten to fifteen

intrarenal arteries with <50mm in diameter were

captured and used for quantification using ImageJ

software.

2.5 Statistical Analysis

Data were analyzed using the IBM SPSS

Statistics program. Data normality test was conducted

using the Shapiro-Wilk test. The homogeneity was

conducted using the Levene test, then the numerical

test using the One-Way ANOVA test for normal data

distribution and the Kruskal-Wallis test for abnormal

data distribution.

3 RESULTS

Vascular Remodelling and Perivascular Fibrosis Area

Unilateral Ureteral Obstruction (UUO) has known

can induce vascular remodeling based on the

quantification of Sirius Red staining (Figure 1A).

Chlorogenic acid effects to ameliorate vascular

remodeling that occurs in the condition of kidney

fibrosis. Quantitative analysis of the lumen area

showed lower significantly in the U7 and U14 groups

(p<0.05) compared to the SO group, then higher

significantly in the UC7 and UC14 groups (p<0.05)

compared to the U7 and U14 groups (Figure 1B).

Quantitative analysis of the mean wall thickness

showed higher significantly in the U7 and U14 groups

(p<0.05) compared to the SO group, then based on the

means data of UC7 and UC14 groups were lower

compared to the U7 and U14 groups (Figure 1C).

Quantitative analysis on WLAR showed higher

significantly in the U7 and U14 groups (p<0.05)

compared to the SO group, then based on the means

data of UC7 group was lower compared to the U7

group, whereas in the UC14 group (p<0.05) was

lower significantly compared to the U14 group

(Figure 1D).

Unilateral Ureteral Obstruction (UUO) has known

to induce perivascular fibrosis based on the

quantification of Sirius Red staining (Figure 1A).

Chlorogenic acid ameliorated perivascular fibrosis

that occurs in kidney fibrosis. Quantitative analysis in

the area of perivascular fibrosis showed higher

significantly in the U7 and U14 groups (p<0.05)

compared to the SO group, but based on the means

data of UC7 group was lower compared to the U7

group, whereas in the UC14 group (p<0.05) lower

significantly compared to the U14 group (Figure 1E).

Chlorogenic Acid Ameliorates Vascular Remodeling and Perivascular Fibrosis in Kidney Fibrosis Model in Mice

Figure 1. A. Histopatologic view of vessels with vascular remodelling and perivascular fibrosis, A: SO group, B: U7 group,

C: UC7 group, D: U14 group, E: UC14 group. B-E. Results of quantitative analysis of lumen area, mean wall thickness,

WLAR, and perivascular fibrosis. *=p<0.05 vs SO; #=p<0.05 vs U7; =p<0.05 vs U14.

4 DISCUSSION

Unilateral Ureteral Obstruction (UUO) is an

experimental kidney injury model that provides a

representative description of pathological conditions

in CKD because it can trigger kidney fibrosis

characterized by apoptosis, interstitial fibrosis,

glomerulosclerosis, and decrease of kidney mass

(Chevalier et al, 2009). There was an increase of ROS

in UUO due to decreased potential antioxidants,

which activated free radicals bioavailability so that

oxidative stress occurred

(Modaresi et al, 2015).

Oxidative stress, initiated due to an increase in ROS,

was a trigger factor for endothelial dysfunction,

marked by a decrease in NO level, vascular

remodeling, and cellular damage (Craige et al, 2015).

Vascular remodeling is a complex process involving

endothelial cells, smooth muscle cells, and fibroblast

cells. Vascular remodeling represents structural

changes like hypertrophy or hyperplasia of vascular

smooth muscle cells and extracellular matrix

components, which induce changing the artery's

mechanical function. Vascular remodeling can be

observed by assessing lumen area changes, mean wall

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

thickness, and the wall area/lumen area ratio

(Tanaka

& Laurindo, 2017).

In this study, the UUO group showed vascular

remodeling; meanwhile, in CGA group showed the

ameliorate of vascular remodeling (Figure 1). There

was an increase in ROS and an oxidation-reduction

reaction, by which c-Jun N-terminal Kinase (JNK)

was made and proliferation and hypertrophy leading

to vascular injury and vascular remodeling in UUO-

induced kidney fibrosis. Reactive Oxygen Species

(ROS) modulated intracellular Ca2+ level as a

primary factor of cellular activity (Görlach et al,

2015). Chlorogenic acid could inhibit the ROS-

modulated Ca2+ influx and restored the viability of

cells and endothelial cells. Other than that,

chlorogenic acid could suppress oxidative stress,

inflammation, apoptosis, and autophagy by

enhancing kidney regeneration (Domitrović et al,

2014). Chlorogenic acid was known to decrease JNK

pathway activation leading to inhibition of apoptosis,

contraction, migration, and inflammation and

reduction of oxidative damage induced by H2O2

(Yu

et al, 2016). We observed increasing vascular

remodeling with lower lumen area, higher mean wall

thickness, and WLAR in UUO might associate with

vasoconstrictor and vasodilator balance might play a

role in regulating vascular remodeling in UUO. Nitric

oxide (NO) plays an essential role in regulating vessel

tonus and remodeling

(Farris & Colvin, 2012).

Kidney vasculature also has a high sensitivity to NO.

NO released in the medulla induces local blood flow

and improves RBF in CKD model (Savard et al,

2012).

Besides affecting vascular remodeling,

chlorogenic acid also played roles in perivascular

fibrosis. We observed higher fibrosis perivascular

area in the UUO group. Meanwhile, CGA might

ameliorate fibrosis perivascular as shown by lower

fibrosis perivascular area in the CGA treated UUO

group (Figure 1). Perivascular fibrosis played active

roles in developing kidney fibrosis, which was

mediated by TGF-B induced myofibroblast

transformation (Kramann & Humphreys, 2014).

Increased ROS caused an imbalance between

oxidation-reduction and modulated the production of

TGF-B through Smad pathway (Liu & Desai, 2015).

Chlorogenic acid had anti-oxidative effects by

decreasing TGF-B gene expression and cytokines

responsible for fibrosis development through miR-21,

which regulated the Smad 7/TGF-B pathway. It

showed that chlorogenic acid was an antifibrosis

agents

(Yang et al, 2017). As a result, CGA treatment

ameliorated vascular remodeling; also reduced

perivascular fibrosis in kidney fibrosis.

5 CONCLUSION

In conclusion, that study highlighted the effect of

chlorogenic acid ameliorated vascular remodeling

based on wider lumen area, thinner mean wall

thickness and lower WLAR; ameliorated perivascular

fibrosis based on the lower area. For a further

research, it is necessary to measure vascular

remodeling using vessel myograph, which could

evaluate endothelial function due to vasoconstriction

and vasodilatation.

ACKNOWLEDGEMENT

The authors are grateful to Wiwit Ananda, Yuyun,

Shyntia, Maulida, and Mulyana in Anatomy

Department, Faculty of Medicine, Public Health, and

Nursing Universitas Gadjah Mada, which has helped

a lot in this research.

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