The Role of Chlorogenic Acid on Mice with Unilateral Ureteral

Obstruction Model: A Study toward Tubular Injury

Gina Andyka Hutasoit

, Nur Arfian

, Dwi Cahyani Ratna Sari

Gabriella Bamba Ratih Lintin

3 d

, Mohammad Salman

, Muhammad Mansyur Romi

Patology Anatomy Department, Faculty of Medicine Universitas Tadulako, Palu, Indonesia

Anatomy Department, Faculty of Medicine, Public Health, and Nursing

Universitas Gadjah Mada, Yogyakarta, Indonesia

Anatomy Department, Faculty of Medicine Universitas Tadulako, Palu, Indonesia

Histology Department, Faculty of Medicine Universitas Tadulako, Palu, Indonesia

Keywords: Unilateral Ureteral Obstruction (UUO), Tubular Injury, Chlorogenic Acid (CGA)

Abstract: Chronic kidney disease is a global public health problem with a poor prognosis of renal fibrosis. Injury to

tubular epithelial cells is the beginning of the occurrence of renal fibrosis. Unilateral Ureteral Obstruction

(UUO) is a representative method for obtaining models of renal fibrosis. Chlorogenic acid (CGA) is known

to have a renoprotective, anti-inflammatory and antioxidant effect. This study aims to examine the role of

CGA preventing tubular injury on mice with UUO model. This study was a quasi-experimental research with

post-test only with control group design. Subjects were 25 male adult Swiss Webster mice (2-3 months old,

20-30 g weight). Subjects were divided into five groups: SO (Sham Operation), 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. Tubular injury scores were examined with Periodic Acid Schiff staining. The data

obtained from this study were analyzed using SPSS. UUO group with CGA showed the tubular injury score

was lower than the UUO group without CGA with p-value < 0,005. The application of chlorogenic acid could

prevent tubular injury in mice model of UUO.

1 INTRODUCTION

Chronic renal disease was a global public health

problem with increased prevalence and incidence,

poor prognosis and high costs. According to the

Global Burden of Disease (2015), chronic renal

disease is the 18

leading cause of death in 2010 and

increased to 12

in 2015 (Neuen et al., 2017). The

global prevalence of chronic renal kidney is 13.4%

and tends to increased as the elderly population

increased, and the incidence of diabetes mellitus

(DM) and hypertension (Kemenkes, 2017). Central

Sulawesi Province was the region with the highest

prevalence of chronic kidney disease in Indonesia,



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which was 0.5%, followed by Aceh, Gorontalo and

North Sulawesi (Kemenkes, 2013).

Chronic kidney disease results from an injury

such as an obstruction. This injury causes renal

structure changes such as renal fibrosis, tubular

atrophy, apoptosis and inflammation (Ucero et al.,

2013). Fibrosis was an excessive deposition of

extracellular matrix proteins in tissues that caused

scar formation. The existence of structural changes

due to fibrosis in the kidneys results in renal

physiological function loss to become End-Stage

Renal Disease (ESRD) (Neuen et al., 2017).

Unilateral Ureteral Obstruction (UUO) was the

most representative experimental method for

obtaining renal fibrosis inflammation models (Ucero

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

The Role of Chlorogenic Acid on Mice with Unilateral Ureteral Obstruction Model: A Study toward Tubular Injury.

DOI: 10.5220/0010487700580062

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 58-62

ISBN: 978-989-758-499-2

et al., 2013). The UUO method was performed by

binding or unilateral ureteric ligation in experimental

animals. That could be subacute kidney injury

characterized by tubular cell injury, interstitial

inflammation, and increased intratubular pressure and

renal fibrosis inflammation (Ucero et al., 2013).

Tubular epithelial cells have multicellular structures

that bind together and acting as a barrier and

absorption/secretion. Injuries at tubular epithelial

cells were the beginning of renal fibrosis

(Guarino et

al., 2009). A tubular injury would make a decrease in

renal blood flow.

Chlorogenic acid was a compound polyphenol in

coffee which had contain a high antioxidant.

Chlorogenic acid effectively decreased the

metabolites of arachidonic acid, nitric oxide and the

production of proinflammatory cytokines compared

to ibuprofen

(Chauhan et al., 2012). This active

ingredient improved kidney dysfunction and kidney

injury induced by cisplatin that suppressed oxidative

stress, apoptosis, and autophagy by increasing kidney

regeneration (Domitrović et al., 2014). Research on

the role and utilization of chlorogenic acid to prevent

chronic renal disease was still not widely carried out.

The existing studies have not explained the various

pathways for preventing fibrosis in the incidence of

Chronic renal kidney, so this study needs to be done

to assess chlorogenic acid's role on the degree of renal

tubular injury used the UUO mice model so that it

could be considered as a new therapeutic agent.

Based on the above background, this study's

formulation problem was as follows: How is the

effect of chlorogenic acid on tubular injury scores at

the UUO mice model? General-purpose of this

research is to assess the role of administration

chlorogenic acid in preventing tubular injury in the

UUO mice model.

2 METHODS

This research was true experimental research used

post-test only controlled group design. Consists of

control and treatment groups. The subjects were 25

mature male Swiss Webster mice which were divided

into 5 groups, SO (Sham Operation/Control) group,

U7 (day-7 UUO + aquadest), U14 (day-14 UUO +

aquadest), UC7 (day-7 UUO + chlorogenic acid), and

UC14 (day-14 UUO + chlorogenic acid).

This study has obtained permission from the

Medical and Health Research Ethics Committee

Faculty of Medicine, Universitas Tadulako based on

the ethics feasibility certificate number

B.0941/UN28.1.30/KL/2018 on February 26, 2018.

2.1 Animal Model of Unilateral

Ureteral Obstruction (UUO)

Mice were anaesthetized using pentobarbital with a

dose of 0.1ml/10gBW/ times. After the anaesthesia

effect was achieved, the mice were placed on the

operating table with a pronation position. Mice hair

on the right-back was shaved then disinfected used

povidone-iodine, clamped with tweezers, then cut the

skin for ± 1.5 cm parallel to the body axis lumbar

region to the right lateral vertebrae region (region

flank). Cutting was done until the peritoneum visible.

Pinch the peritoneum with tweezers and then scissors

± 1 cm until the organs were visceral visible. Identify

the kidneys first, then used tweezers twisted over the

inferior pole and then pull the ureter up. Perform a

double ligation of the ureter in the inferior polus using

0.4 silk thread in the proximal area. The peritoneum

and skin were seen layer by layer using silk 0.4, then

gave povidone-iodine as an antiseptic.

2.2 Chlorogenic Acid Administration

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

injected in mice with a dose of 14 mg/kg BW/day

intraperitoneally. A total of 25 mice of swiss webster

strains weighing 20-30 grams, 2-3 months old were

used in this study. Mice were divided into 5 groups.

The distribution for each group as follows: sham

operation (SO) group, intraperitoneal distilled water

injection for 14 days as a control; mice with UUO

injected with distilled water intraperitoneally for 7

days called group U7; mice with UUO injected with

chlorogenic acid for 7 days called UC7 group; mice

with UUO injected with distilled water

intraperitoneally for 14 days called group U14; mice

with UUO injected with chlorogenic acid for 14 days

group UC14.

2.3 Histopathological Examination

with Staining of Periodic Acid

Schiff (PAS)

Mice kidney would make by paraffin blocks and cut

to 4μm thickness. The tissue slides were halarized and

drained with flow water, followed by oxidation with

periodic acid 0.5%for 5 minutes. The slides were

washed with flow water. The slides were soaked in

the reagent Schiff for 15 minutes and washed with

flow water. Counterstained with haematoxylin for 1

minute and then washed with flow water. The slide

was then dehydrated and mounted.

The Role of Chlorogenic Acid on Mice with Unilateral Ureteral Obstruction Model: A Study toward Tubular Injury

Figure 1: (A) Kruskal Wallis test p=0,000. *=p<0,05 vs SO, #=p<0,05 vs U7, ±=p<0,05 vs U14. (B) Microscopic findings of

the kidney with PAS staining. Description: White arrows show intraluminal cast in renal tubules that had atrophy. The black

arrow shows the brush border.

2.4 Statistical Analysis

Tubular injury scores classified from 0 to 4 (0 =

normal, 1 = tubular injury < 25% visual field, 2 =

tubular injury involving 25% -50% visual field, 3 =

tubular injury involving more than 51% -75% field

view, 4 = tubular injury involving > 75% visual field).

The assessment used a magnification of 400 times

and counted as many as 15 fields of view randomly

and did not overlap. This research variable has a ratio

scale.

Data were analyzed using the statistical

programme. Normality test used Shapiro-Wilk and

homogeneity test used the Levene Test. The

normality test for tubular injury scores was not

normally distributed, then used the Kruskal Wallis

test.

3 RESULTS

Histopathological examination of this study found

that there was good tubular appearance in the SO

group, with cuboidal epithelial cells in the proximal

tubule and distal, and the tubule's lumen still wide.

Besides that, the brush border was still intact, and no

protein was found cast in the lumen of the tubules.

While in the U7 and U14 groups tubular appearance

was found with various signs of injury such as the

presence of inflammatory areas, atrophic tubules, loss

of brush border in the proximal renal tubules, and

tubular dilatation with formation cast intraluminal.

Protein cast could be formed from podocyte damage,

resulting in protein escaping from glomerular

filtration, tissue hypoxia causes epithelial cell

apoptosis. Finally, together with the brush border, it

is released into the lumen. Compared to the UC7 and

UC14 groups that received chlorogenic acid, this

group clearly saw improvements in injury, namely the

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 )

brush border still attached to the epithelium with

intact epithelial cells and an inflammatory cell also

appear to be reduced. Tubular dilation in some

regions was still found with protein cast in the lumen,

but in small amounts when compared to the U7 and

U14 groups.

The tubular injury score results in the five sample

groups found that the U7 and U14 groups had a higher

tubular injury score compared to tubular injury scores

in the SO group. Simultaneously, tubular injury

scores in the UC7 and UC14 groups who received

chlorogenic acid were lower than those in the tubular

injury scores in the U7 and U14 groups who did not

get chlorogenic acid. This result shows that the UUO

model could cause a tubular injury that ends with

renal fibrosis. Kidney fibrosis occurs due to

obstruction, which causes an increased in intratubular

pressure and vasoconstriction in the kidneys, thus

ending with tissue ischemia. Also, in this study, it was

found that chlorogenic acid administration in the

UUO model can inhibit tubular injury, thereby

preventing renal fibrosis.

Statistical analysis results showed that there were

significant differences in tubular injury scores in the

U7 group (2,73±0,7) and U14 (3,77±0,18) compared

to the tubular injury scores in the SO (0) group. This

result shows that UUO was able to induce tubular

injury, which could end with renal fibrosis.

Comparison of tubular injury scores between the UC7

groups (1,33±0,15) and UC14 (1,32±0,29) showed a

significant difference compared to tubular injury

scores in the U7 and U14 groups. Tubular injury

scores indicated this result in the UC7 and UC14

groups given lower chlorogenic acid than tubular

injury scores in the U7 and U14 groups who were not

given chlorogenic acid.

4 DISCUSSION

In this study, it was found that chlorogenic acid had

roles in preventing tubular injury in laboratory UUO

mice to prevent renal fibrosis. This phenomenon

could be seen because the tubular injury score in

laboratory mice given chlorogenic acid was lower

than the tubular injury score in laboratory mice that

were not given chlorogenic acid.

According to the result, it was found that

unilateral ureter ligation in experimental animals

could cause kidney tubular injury, which resulted in

renal fibrosis. UUO method created a higher degree

of obstruction because ligation of one ureter caused a

total obstruction, increasing intratubular pressure and

causing secondary vasocontriction to the kidney,

leading to decreased GFR and tissue ischemia. This

condition caused further damage to kidney tubules so

that most tubular epithelial cells would undergo

apoptosis, resulting in atrophy (Ucero et al., 2013).

In this study, the tubular injury score was higher

in U7 and U14 groups which received UUO than in

SO group. An increased tubular injury score was

observed in exeperimental animals with longer UUO

(Tateishi et al., 2015). This result showed that UUO

was a representative model to create renal fibrosis

(Ucero et al., 2013). Other studies also explained

tubular injury caused by UUO by providing an

overview of tubular damage, tubular epithelial cells

apoptosis, fibrosis, and interstitial inflammation.

Increasing cell death could be seen in day-3 to day-14

after UUO, and specific histologic marker of chronic

kidney disease could be obtained after 1-2 weeks of

UUO. Cell death was started by an increased

hydrostatic tubular pressure accompanied by

increased free radical reactions and oxidative injuries

in proximal tubules (Xu et al., 2013). Moreover, the

tubular injury score was significantly higher in UC7

and UC14 groups which underwent UUO than in SO

group. Therefore, further study about the roles of

chlorogenic acid to tubular injury should be

conducted.

In this study, the tubular injury score was lower

in UUO groups, UC7 and UC14, which were given

chlorogenic acid than in UUO groups, U7 and U14,

which were not given chlorogenic acid. This result is

consistent with the previous study, which stated that

there were decreased tubular necrosis, intraluminal

cast, and tubular dilatation in laboratory mice given

chlorogenic acid (Domitrović et al., 2014). The

decrease in tubular injury score in groups given

chlorogenic acid was due to antioxidant, anti-

inflammatory, antibacterial, and anticarcinogenic

properties of chlorogenic acid (Naveed et al., 2018;

Tajik et al., 2017).

The limitation in this study is that it has not

investigated the role of chlorogenic acid in renal

fibrosis on epithelial cell markers and mesenchymal

cell markers that affect the occurrence of tubular

injury, therefore further research is needed in this

regard.

5 CONCLUSION

Tubular injury score in the UUO mice model given

chlorogenic acid was lower than the UUO mice

model without chlorogenic acid. Further research can

be sharpened by researching the role of chlorogenic

acid in the tubular injury and the analysis of the role

The Role of Chlorogenic Acid on Mice with Unilateral Ureteral Obstruction Model: A Study toward Tubular Injury

of chlorogenic acid in renal fibrosis by examining

other epithelial cell markers and other mesenchymal

cell markers.

ACKNOWLEDGEMENT

The authors are grateful to Wiwit Ananda, Cynthia,

Maulida, Yuyun and Mulyana in Anatomy

Departement Faculty of Medicine, Public Health, and

Nursing Universitas Gadjah Mada, which has helped

a lot in this research.

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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 )