Urine Protein, Creatinine, and Uacr Level in Pregnant Mus Musculus

Injected by Anti Qa2 as Endothelial Dysfunction Model to Induce

Preeclampsia

Meitria Syahadatina Noor,

Budi Santoso,

Triawanti,

Widjiati

Department of Public Health, Faculty of Medicine, Lambung Mangkurat University, Banjarmasin, Indonesia

Department of Obstetry and Ginaecology, Faculty of Medicine, Airlangga University, Surabaya, Indonesia

Department of Biochemistry, Faculty of Medicine, Lambung Mangkurat University, Banjarmasin, Indonesia

Department of Embriology, Faculty of Veterinary, Airlangga University, Surabaya, Indonesia

Keyword: Protein, Creatinine, UACR, Urine, Endothelial Dysfunction

Abstract: Preeclampsia is a condition which was identified by diastol blood pressure ≥ 90 mmHg in 20 weeks

of pregnant, and blood pressure in < 20 weeks of pregnant was still < 90 mmHg. Beside

hypertension, we also find proteinuria in preeclampsia after 20 weeks of pregnant. Preeclampsia

could cause low renal function that was indicated by creatinine and UACR level. Preeclampsia

happened from endothelial dysfunction. Endothelial dysfunction in Mus musculus could be made by

injecting anti QA2. This research was to analyze urine protein, creatinine, and UACR level in

endthelial dysfunction model in pregnant Mus musculus. The result was dose of anti QA2 could

cause the increasing of urine protein and UACR level, but not to urine creatinine level. The

conclusions were there was significant differences of urine and UACR protein level in pregnant Mus

musculus that was injected by anti QA an endothel dysfunction model that induced preeclampsia, but

not to urine creatinine level. The suggestions were preeclampsia must be detected, prevented and

treated as soos as possible, to prevent mechanism of endothel dysfuncyion in preeclampsia that could

cause low renal function.

1. INTRODUCTION

According to preeclampsia community guideline

(PRECOG), preeclampsia is a condition which is

identified by diastol blood pressure ≥ 90 mmHg in

20 weeks of pregnant, and blood pressure in < 20

weeks of pregnant was still < 90 mmHg. Beside

hypertension, we also find proteinuria in

preeclampsia after 20 weeks of pregnant.

Proteinuria is protein in urine ≥ 300 mg/litre or ≥ +1

in dipstick test, ratio of protein/creatinin ≥ 30

mg/mmol, or excretion of protein in urine ≥ 300 mg

in 24 hours (Milne et. al., 2005).

Signs of preeclampsia are: (Hladunewich dkk.,

2007)

a. Hypertension (blood pressure ≥ 140/90

mmHg). Hypertension in preeclampsia

happens because of disturbances of vasoactive

factors such as vasoconstrictor (endothelin,

tromboxan) that are higher than vasodilator

(nitric oxide/NO, prostacyclin).

b. Low GFR (glomerulus filtration rate): it

happens because the structure of glomerulus

changes after vasoconstriction. This condition

is showed by urine albumine and creatinine

ratio (UACR) level.

c. Proteinuria: it shows the difference between

preeclampsia and the other hypertension types.

Proteinuria happens because of the

disturbances in glomerulus filtration barier.

d. Coagulopathy and HELLP syndrome:

endothelial dysfunction in preeclampsia causes

light coagulopathy with high trombocyte

numbers, slow clothing time, and low

antrombine III. HELLP syndrome can happen

Noor, M., Santoso, B., Triawanti, . and Widjiati, .

Urine Protein, Creatinine, and Uacr Level in Pregnant Mus Musculus Injected by Anti Qa2 as Endothelial Dysfunction Model to Induce Preeclampsia.

DOI: 10.5220/0008790400390043

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 39-43

ISBN: 978-989-758-438-1

in 10% of severe preeclampsia. It increases

plasma concentration and trombocyte

activation.

Data of Indonesian Health Ministry in 2010-2013

showed that hypertension was the second cause of

maternal mortality in Indonesia after bleeding

(Kemenkes RI, 2014). High rate of preeclampsia

must be followed by effective preventive and

treatment that still need a lot of researches.

Some researches for treatment and prevention of

preeclampsia can not be done in human. So we need

animal model that will be similar to preeclampsia.

The researches about animal model of preeclampsia

were very variated and need to be confirmed with

cellular and clininal examanations.

One of researches about preeclampsia animal

model was endothelialial dysfunction model. It was

made by Sulistyowati et. al. (2010) as induction of

preeclampsia. That endothelial dysfunction model

was done by injecting anti QA2 (anti Human QA

Lymphocyte Antigen 2 Region). It blocked QA2

expression in placenta.

Placental QA2 expression was homolog to

human leucocyte antigen-G expression (HLA-G) in

human. Low HLA-G in trophoblast was a predictor

to endothelial dysfunction in preeclampsia. That

reasearch showed that endotel dysfunction model in

Mus musculus cauesd HSP70, VCAM-1 and matrix

metalloproteinase (MMP9) profiles that were similar

with women with preeclampsia (Sulistyowati et. al.,

2010). That research did not examine clinical

examanations that appeared from endothelial

dysfunction.

The goal of this research was making endothelial

dysfunction in pregnant Mus musculus that was

injected by anti QA2 and confirming urine

examanation (protein, creatinine, and UACR urine)

as one of preeclampsia clinical manifestations

because of endothelial dysfunction and low renal

function.

2. MATERIAL DAN METHOD

This research was true experimental with post test

only with control group design. This research used

female Mus musculus that was mated by male Mus

musculus. Female Mus musculus with positive

vaginal plug were used in the research. The vaginal

plug was the sign those female and male Mus

musculus were mated and the pregnant was called 0

day.

Mus musculus that were used must be 3 months,

healthy, bodyweight 15-25 grams, well moving, no

wound found in the body, and clear eye. This

research used 3 pregant Mus musculus/groups. The

duration of research was 2 weeks, consisted of

acclimatization, mating female and male Mus

musculus, intervention, and termination.

All of female Mus musculus were injected by

pregnant mare serum gonadothropine (PMSG) and

human chorionic gonadotropine (HCG) to equate

oestrus cycle. Female Mus musculus was injected

by 5 IU PMSG intra peritoneal, after 48 hours they

were injected again by HCG 5 IU intra peritoneal.

After that, female Mus musculus were mated by

male Mus musculus 1:1.

Tomorow morning after mating, female and male

Mus musculus were seperated. Female Mus

musculus were examined if they had positive vaginal

plug or not. Pregnant Mus musculus were who had

positive vaginal plug, and randomize into 7 groups

(3 pregnant Mus musculus/group).

The location was in Laboratory of Embriology,

Faculty of Veterinery, Airlangga University. This

research consisted of 7 groups: K0 (control, no

injection of anti QA2), K1 (anti QA2 10 ng), K2

(anti QA2 20 ng), K3 (anti QA2 30 ng), K4 (anti

QA2 40 ng), K5 (anti QA2 50 ng), and K6 (anti

QA2 60 ng).

K1 was injected by anti QA2 10 ng (0,1 ml) intra

peritoneal in the first day of pregnant, and examined

in the second day of pregnant. K2 was injected by

anti QA2 10 ng (0,1 ml) intraperitoneal in the first

and second day of pregnant, and examined in the

third day of pregnant. K3 was

injected by anti QA2

10 ng (0,1 ml) intraperitoneal in the first, second,

and third day of pregnant, and examined in the

fourth day of pregnant. K4 was injected by anti

QA2 10 ng (0,1 ml) intra peritoneal in the first,

second, third, and fourth day of pregnant, and

examined in the fifth of pregnant. K5 was injected

by anti QA2 10 ng (0,1 ml) intra peritoneal in the

first, second, third, fourth, and fifth day of pregnant,

and examined in the sixth day of pregnant. K6 was

injected by anti QA2 10 ng (0,1 ml) intra peritoneal

in the first, second, third, fourth, fifth, and sixth day

of pregnant, and examined in the seventh day of

pregnant.

Urine of Mus musculus was tooken in the

morning and was examined for protein, creatinin,

and UACR to analyze endothelial dysfunction and

low renal function.

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

3. RESULTS

3.1. Urine Protein Level

Table 1. Data of urine protein level in all groups of Mus

musculus with statistic results

GROUP MEAN SD p

VALUE

αVALUE

K0 0,633

0,236

K1 0,01

0,000 p=0,001

(Kruskal

Wallis

Test

)

0,05

K2 0,01

0,000

K3 1,333

0,392

K4 1,878

1,168

K5 0,835

0,147

K6 0,623

0,092

Notes:

p value < 0,05 means there is significant

differences. In each group, if there was

mean that had different letter code, it was

significant different. But if it had same

letter code, it was not significant

differences. (K0: without anti QA2, K1:

anti QA2 10 ng, K2: anti QA2 20 ng, K3:

anti QA2 30 ng, K4: anti QA2 40 ng, K5:

anti QA2 50 n

, K6: anti QA2 60 n

Urine protein level started to increase in K3 (anti

QA2 30 ng). Normality test with Shapiro Wilk α =

0,05 showed that not all of groups had normal

distribution. So, it was continued by Kruskall

Wallis test. The result of Kruskal Wallis test

showed there was significant differences among all

groups (Table 1). To know the different result

between 2 groups, the analyze was used U Mann

Whitney test α = 0,05.

3.2. Urine Creatinine Level

Table 2. Data of urine protein level in all groups of Mus

musculus with statistic results

GROUP MEAN SD p

VALUE

αVALUE

K0 0,054 0,012

K1 0,042 0,012 p=0,127

(Kruskal

Wallis

Test

)

0,05

K2 0,040 0,015

K3 0,040 0,018

K4 0,020 0,005

K5 0,070 0,012

K6 0,058 0,014

Notes: p value > 0,05 means there was no

significant differences (K0: without anti

QA2, K1: anti QA2 10 ng, K2: anti QA2

20 ng, K3: anti QA2 30 ng, K4: anti QA2

40 ng, K5: anti QA2 50 ng, K6: anti QA2

60 n

The highest level of urine creatinine was K5 (anti

QA2 50 ng). The results of normality test showed

that not all groups had normal distribution, so the

analyze was continued by Kruskall Wallis test. The

results were there was no significant differences

among all groups (Table 2).

3.3. Urine UACR Level

Table 3. Data of urine UACR level in all groups of Mus

musculus with statistic results

GROUP MEAN SD p

VALUE

αVALUE

K0 0,213

0,102

K1 0,287

0,148 p=0,003

(Kruskal

Wallis

Test

)

0,05

K2 39,433

1,616

K3 57,008

2,094

K4 50,100

2,428

K5 9,467

2,793

K6 18,850

1,034

Notes: p value <0,05 means there is significant

differences. In each group, if there was

mean that had different letter code, it was

significant different. But if it had same

letter code, it was not significant

differences. (K0: without anti QA2, K1:

anti QA2 10 ng, K2: anti QA2 20 ng, K3:

anti QA2 30 ng, K4: anti QA2 40 ng, K5:

anti QA2 50 ng, K6: anti QA2 60 ng).

Normality test with Shapiro Wilk α = 0,05 showed

normal distribution in all groups but not homogen.

So it was continued by Kruskall Wallis Test. The

results were in Table 3. It showed that p value <

0,05, there was significant differences among all

groups.

4. DISCUSSION

4.1 Urine Protein Level

Dose of anti QA2 that could increase of urine

protein started from 30 ng. Urine protein happened

as clinical manifestation of endothelial dysfunction

process. If it hapened in pregnant condition, it could

cause preeclampsia. Preeclampsia is complication in

pregnant that consists of hypertension and urine

protein. One of preeclampsia’s patogenesis is

endothelial dysfunction. Endothel is cell layer on

the vascular wall that leads to lumen. Endothel

functions were regulating vascular tonus,

fibrinolysis system, vascular growth, and preventing

trombosis (Dharma et. al., 2005).

Urine Protein, Creatinine, and Uacr Level in Pregnant Mus Musculus Injected by Anti Qa2 as Endothelial Dysfunction Model to Induce

Preeclampsia

Endothelial dysfunction happens because of

oxidative stress, inflammation, and

hypercholesterolemia. Oxidative stress and

inflammation are basic mechanisms of preeclampsia.

Endhotelial dysfunction causes disbalance of

vasoactive compounds that make hypertension.

Endothelial dysfunction also causes the increasing of

vascular permeability, so it affects excretion of

protein in urine (Dharma et. al., 2005).

Proteinuria is laboratory indicator that shows

early process of low renal function that still happens.

Urine protein level is an early indicator for

glomerulus disturbances (Fox et. al., 2013). In this

research, urine protein was found. This results

showed that injection of anti QA2 since dose of 30

ng could block placental QA2 expression. The

block of placenta QA2 expression made body gave

inflammation response that triggered endothelial

dysfunction. One of clinical manifestations of

endothelial dysfunction was detected by urine

protein in pregnant Mus musculus.

4.2. Urine Creatinine Level

The highest level of urine creatinine in this research

was in K5 (anti QA2 50 ng), but Kruskal Wallis test

showed there was no significant differences among

all groups. It meaned that dose of anti QA that was

given to Mus musculus could not increase significant

creatinine level. The asumption was dose of anti

QA that was given to Mus musculus had not made

low renal function yet in pregnant Mus musculus.

This result was almost the same with Lubis et. al.

(2017) that showed there was no significant

differences of creatinine level in preeclampsia and

normal pregnant. It was caused by process of

preeclampsia’s mechanism to reach low renal

function still needed some processes.

Preeclampsia women who suffered endothelial

dysfunction will decrease perfusion to many organs

include renal. If the perfusion still decreases, it will

damage renal especially in glomerulus as the

location of creatinine filtration. This damage could

increase creatinine level. If creatinine level had not

increased yet, so the endothelial dysfunction in Mus

musculus had not damaged glomerulus yet.

Creatinine is indicator of low renal function. It

was the result of creatine dan phosphocreatine

metabolisms. Creatinine is filtrated in renal

glomerulus and reabsoebed in renal tubuly. In

creatinine formation, there is no reuptake

mechanism in our body. So creatinine can be

excreted through renal (Alfonso et. al., 2016).

Dose of anti QA2 in this research could increase

urine protein but could not increase urine creatinine

yet, so the asumption was dose of anti QA that was

given had not damage renal glomerulus yet.

4.3. Urine UACR Level

UACR is included in chronic renal disease. This

ratio depends on albumine and creatinine level in

urine. Table 3 showed that there was significant

different of UACR among all groups. It was caused

by significant different of urine protein although

creatinine urine was not significant. This significant

value showed that the ratio of urine protein dan

creatinine in the early process of low renal function.

This results about UACR were simalr to Sogani

et. al. (2014). That research concluded that UACR

and serum uric acid levels as the prediction of

proteinuria in new onset hypertention and uric acid

in women with preeclampsia. UACR and serum uric

acid progressed from mild to severe condition.

UACR can asses renal diseases. UACR level

can also show the screening of microalbuminuria as

the predictor of cardiovascukar/renal diseases.

Microalbuminuria is defined as UACR > 2,5

mg/mmol in men and > 3,5 mg/mmol in women

(Fung et. al., 2017).

UACR level showed that endothelial dysfunction

that happened in pregnant Mus musculus could start

low renal function. So, we must be aware to

preeclampsia because the mechanism could cause

low renal function. It needed early and effective

prevention and treatment in preeclampsia.

5. CONCLUSIONS

The conclusions of this research were:

1. There was significant differences of urine

protein level in pregnant Mus musculus that

was injected by anti QA as endothelial

dysfunction model that induced preeclampsia.

2. There was no significant differences of urine

creatinine level in pregnant Mus musculus that

was injected by anti QA as endothelial

dysfunction model that induced preeclampsia.

3. There was significant differences of urine

UACR level in pregnant Mus musculus that

was injected by anti QA as endothelial

dysfunction model that induced preeclampsia.

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

6. SUGGESTIONS

Preeclampsia must be detected and prevented as

soos as possible and must be treated with effective

treatment as soon as possible, to prevent mechanism

of endothelial dysfuncyion in preeclampsia that can

cause low renal function.

ACKNOWLEDGEMENTS

We would like to say thank you very much to

Laboratory of Embriology, Faculty of Veterinary,

Airlangga University, Surabaya as the location of

this research.

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Urine Protein, Creatinine, and Uacr Level in Pregnant Mus Musculus Injected by Anti Qa2 as Endothelial Dysfunction Model to Induce

Preeclampsia