Improvement of Renal Proximal Tubules after Black Cumin (Nigella

Sativa) Extract Administration in Rat with CCl4-induced Chronic

Renal Damage

Fathiyah Safithri

1,*

, Desy Andari

and Fifa Yuniarmi

Department of Pharmacology, Faculty of Medicine, University of Muhammadiyah Malang Jalan Bendungan Sutami

No. 188A, Malang, East Java, Indonesia

Histology Department Faculty of Medicine, University of Muhammadiyah Malang, Jalan Bendungan Sutami No.

188A, Malang, East Java, Indonesia

Keywords: Black cumin extract, hyaline cast tubular, chronic renal damage, CCl

Abstract:

Chronic renal disease is a chronic pathological process in renal that accompanied by a progressive decline

in renal function and generally ends up as renal failure. Chronic inflammation and oxidative stress are key

factors in progression. Black cumin extract has antioxidant and anti-inflammatory effects that is thought to

repair renal tubular and inhibit progression of the damage. Objectives: to evaluate the improvement of

renal proximal tubules in rat with CCl4-induced chronic renal damage after black cumin extract

administration. Results: The result showed that the amount of hyaline cast in the proximal tubule

significantly decreased after black cumin extract adminstration. Conclusion: Black cumin extract

decreases the amount of hyaline cast in the proximal tubule. The antioxidant and anti-inflammatory

effects of Black cumin extract may modulate the improvement renal tubular.

1 INTRODUCTION

The improvement of renal function occurs in

patients with chronic renal disease who consume

black cumin extract (Nigella sativa) (Ansari,

Nasiruddin, Khan, & Haque, 2016). Chronic renal

disease is defined as abnormalities in the structure or

function of the renal that last more than three months

(Webster, Nagler, Morton, & Masson, 2017). The

prevalence of chronic kidney disease worldwide is

around 11-13% (Hill et al., 2016). Annually about

1.5% of patients with stage 3 and 4 chronic renal

disease will progress to grade 5 or end stage chronic

renal disease (renal failure) (Webster et al., 2017).

Chronic kidney disease is a multifactorial

disease. In developed countries, the most common

cause is diabetic nephropathy, while in developing

countries the most common cause is chronic

glomerulonephritis and intestinal nephritis. Some

risk factors for chronic kidney disease are

hypertension, diabetes mellitus, urinary tract

infections, urinary tract stones, age-related

congenital kidney disease, family history of chronic

kidney disease, obesity, cardiovascular disease, low

birth weight, autoimmune diseases such as systemic

lupus erythematosus (SLE) ) and drug poisoning or

other toxicity (Gunatilake, Seneff, & Orlando,

2019).

Injury mechanism due to toxicity to the kidneys

varies and all renal structures will be affected. The

tubulointerstitial compartment is the compartment

most commonly involved (Shirali & Perazella,

2014). Tubular epithelial cells are very sensitive to

anoxia and susceptible to toxins (Berger & Moeller,

2014). Injury due to toxic substances in renal will

activate macrophages to stimulate the release of

cytokines such as IL-1β, IFN- and TNF-α

(Akchurin & Kaskel, 2015). Continouse

inflammation of the renal progressively causes

severe kidney damage and eventually end up as

chronic renal failure. The proximal tubules damage

is characterized by narrowing of the tubules,

epithelial necrosis and the presence of hyaline cast.

Hyaline cast is a glycoprotein matrix derived from

renal tubular epithelial cells that shows an abnormal

condition in the renal tubules (Caleffi & Lippi,

2015).

Management of chronic renal failure includes

conservative therapy to prevent progressive

Saﬁthri, F., Andari, D. and Yuniarmi, F.

Improvement of Renal Proximal Tubules after Black Cumin (Nigella Sativa) Extract Administration in Rat with CCl4-induced Chronic Renal Damage.

DOI: 10.5220/0009120000210026

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 21-26

ISBN: 978-989-758-462-6

deterioration in renal function, symptomatic therapy

and renal replacement therapy. Pharmacological

therapy is also provided to control chronic

inflammation as a key factor in the progression of

chronic renal damage. Another approach for

repairing the tubular renal damage is reducing

proinflammatory mediators with natural materials,

such as black cumin seed (Yimer, Tuem, Karim, Ur-

Rehman, & Anwar, 2019).

Black cumin (Nigella sativa) is one of the

medicinal plants included in the Ranunculaceae

family, has long been known and used as a

medicinal plant. Black cumin contains more than

100 bioactives (multiple compounds) and not all

bioactive activities are known (Karaçil Ermumucu &

Şanlıer, 2017). The main components of black

cumin oil contained in many seeds that are thought

to act as anti-inflammatory are thymoquinone,

carvacrol, saponin, oleic acid and linoleic acid

(Parandin, Yousofvand, & Ghorbani, 2012). Invivo

research by Keyhanmanesh in 2010 showed that the

administration of thymoquinone in pigs with

pulmonary inflammation had an effect in the form of

improving the picture of the structure of pulmonary

histology through a decrease in IFN-γ

(Keyhanmanesh, Boskabady, Khamneh, & Doostar,

2010). Other studies have shown that carvacrol

given to mice that are inflamed on their fingers has

an improved effect through decreased IL-1β

production (Lima et al., 2013). Giving total saponins

from ginseng in pigs that have myocardial injury is

proven to reduce the IL-1β proinflammatory

mediator so that there is repair in damaged areas

(Aravinthan et al., 2015). Other studies also prove

that the administration of black cumin which has the

main content of linoleic acid and oleic acid can

reduce the production of IL-1β and TNF-α in mice

induced by dimethylbenzantresana so as to improve

the lung cells of damaged mice (Rahayu, Achmad,

& Ekowati, 2012).

Based on that phenomena, the researchers want

to prove whether the extract of black cumin seeds

which has anti-inflammatory and anti-oxidant

properties can reduce proximal tubular damage in

rats model of chronic kidney damage using CCl

This research was conducted to prove the curative

effect of black cumin on tubular damage after CCl4

exposure. Previous research on black cumin has

been more on the protective or preventive effects of

black cumin.

3 RESULTS AND DISCUSSION

Figure 1 show the hyaline cast tubular each group.

In A group /the normal groups (without induced

CCl4 and black cumin seed extract), the proximal

tubules appear normal, characterized by intact

cuboidal epithelium, a small number of necrotic

cells in the form of cells that have been lysis (lost)

and a little hyaline cast in the middle of the lumen.

In B group, the positive control group (only given

CCl4 1ml / kg BW / day without giving black

cumin), the epithelium of the proximal tubule cells

appears irregular and intact, the necrosis cells appear

more than normal cells and hyaline cast which is in

the middle of the lumen more than with normal

groups.

In C group (induced CCl4 + black cumin seed

extract) 1.2 g/kg BW/day), visible proximal tubular

cuboid epithelium began to be intact, necrotic cells

in the form of cariolysis and cell lysis (lost),

reduced, hyaline less cast than the positive control

group. In D group (induced CCl4 + black cumin

extract 2.4 g/kg BW/day), the proximal tubule

cuboid epithelium began to appear intact, more

normal cells than the positive control group and C

group, still obtained necrosis cells in the form of

cariolysis and cell lysis (lost) is reduced and hyaline

cast in the middle of the lumen.

In E group (induced CCl4 + black cumin

extract 4.8 g/kg BW/day), the proximal tubule

cuboid epithelium began to appear intact, more

normal cells than the positive control group, C and

D groups, still found necrotic cells in the form of

cariolysis and cell lysis (lost) is reduced and hyaline

cast in the middle of the lumen is approaching a

normal group.

Table 1: The mean of amount hyaline cast tubular in normal rat (A), rats induced CCl4 (B), and rat-induced CCl

and black

cumin extract administration (C, D, E).

The mean of amount hyaline cast tubular (mean±SD)

A. Normal 1.70±0.65

B. CCl

control 1 ml/kgBB) 6.33±1.14

C. (CCl

1 ml/kgBB + black cumin extract 1.2

g/kgBB) 4.97±1.10

HSIC 2019 - The Health Science International Conference

D. (CCl

1 ml/kgBB + black cumin extract 2.4

g/kgBB) 3.13±1.09

E. (CCl

1 ml/kgBB + black cumin extract 4.8

g/kgBB) 2.10±0.87



Figure 1. The hyaline cast tubular in normal rats (A), rats-induced CCl4 (B), and rats-induced CCl4 and black

cumin administration in dose 1,2; 2,4; 4,8 g/kgBB (C, D, and E) at 400x magnification. The yellow arrows

show positive hyaline cast tubular, marked as pink color on the tubular lumen.

The mean amount of hyaline cast tubular from all

groups are explaines in Table 1. CCl4 induction

significantly increases the mean amount of hyaline

cast tubular (p<0,05). Black cumin extract

administration in dose 1.2; 2.4; 4.8 g/kgBB)

significantly reduces the mean amount of hyaline

cast tubular (p<0,05).

In this study CCl

administration at a dose of 1

ml / kgBW 3 times a week for 8 weeks was proven

to cause kidney damage which was characterized by

increasing the average number of hyaline casts.

Previous studies of 1 ml / kg body weight of CCl

administered intraperitoneally three times a week for

8 weeks were also shown to cause damage to the rat

kidney, tubal dilatation, and appearance of foamy

epithelial cells in the tubular area. Kidney damage is

caused by toxic exposure to CCl

which is toxic

(Venkatanarayana, Sudhakara, Sivajyothi, & Indira,

2012). CCl

in the body will undergo a process of

biotransformation by the CYP2E1 enzyme forming

free radicals namely trichormormyl radical (CCl

These radicals will then react with oxygen (Khan,

Khan, & Sahreen, 2012).

The result of trichlormethyl radical reaction with

oxygen will cause trauma, lesions and inflammation

in the proximal tubules of mice so that it stimulates

pro-inflammatory mediators of T lymphocytes and

Natural Killer cells to produce INF-γ. IFN-γ will

then activate macrophages, these macrophages will

produce TNF-α and IL-1β, then an inflammatory

cascade will arise. This ongoing inflammation will

cause damage to the proximal tubule (El Boshy et

al., 2017)

In this situation, damage to the proximal tubule

will cause an increase in the permeability of the

glomerular membrane, thus allowing protein

(albumin) and substances dissolved in the plasma

bound to the protein to easily pass through it.

Increased permeability of the glomerular membrane

and disruption of the function of the proximal tubule

reabsorbtion cause an increase in protein in the urine

Improvement of Renal Proximal Tubules after Black Cumin (Nigella Sativa) Extract Administration in Rat with CCl4-induced Chronic

Renal Damage

causing hypoalbumin in the body (Dickson, Wagner,

Sandoval, & Molitoris, 2014).

In the histopathological picture of proximal

tubular damage one of which is characterized by an

increase in the average number of hyaline cast.

Hyaline casts are formed from several components

namely renal tubular epithelial cells, neutrophils,

eosinophils, which will stick to the surface or enter

the Tamm-Horsfall protein, where the Tamm-

horsfall protein is a glycoprotein matrix which is

sticky and only secreted by the proximal tubule. The

attachment of tubular renal components to the

epithelial cells, neutrophils, eosinophils on the

surface of the tamm-horsfall protein will cause the

formation of semi-solid lumps in the lumen and the

HE staining will appear pink, where in normal

circumstances no hyaline cast formation is found

because the process does not occur (Enhancement,

2012).

In this study the administration of black cumin

seed extract (Nigella sativa) at a dose of 1.2 g /

kgBB / day, 2.4 g / kgBB / day, 4.8 g / kgBB / day

can reduce the average number of hyaline cast. The

reduction in the mean of amount hyaline cast

tubular is thought to be caused by the active

compound content of black cumin seed extract

(Nigella sativa) which has anti-inflammatory and

antioxidant effect.The main components in black

cumin extract that have antiinflammatory effect are

thymoquinone, carvacrol, linoleic acid, oleic acid

and saponins which can inhibit pro-inflammatory

mediators such as TNF-α, IL-1β and IFN-γ (Hadi,

Kheirouri, Alizadeh, Khabbazi, & Hosseini, 2016) .

Inhibition of the mediator causes the formation of an

inflammatory cascade that can cause damage to the

kidney tubular epithelial cells and the emergence of

various macrophages that function as the main

components of hyaline cast formation.

Previous studies have shown that the

administration of thymoquinone to inflammatory

pigs' lungs by ovalbumin induction provides an

improved effect on the structure of pulmonary

histology by decreasing IFN-gamma

(Keyhanmanesh et al., 2010). Other studies have

also shown that carvacrol given to mice that have

inflammation in their fingers has an improved effect

through decreased IL-1β production (Lima et al.,

2013). Administration of the total saponin contained

in ginseng in pigs that have myocadial injury can

reduce proinflammatory mediators such as IL-1β so

as to repair damaged cells (Aravinthan et al., 2015).

Other studies also prove that the administration of

black cumin has the main content of linoleic acid

and oleic acid can reduce the production of IL-1β

and TNF-α in mice induced by dimethylbenz

antresana so as to improve the lung cells of damaged

mice (Rahayu et al., 2012). In another study it was

proven that the saponin content contained in Asian

ginseng extract could increase the number of new

blood vessels (angiogenesis) in the mandibular

socket of mice after tooth extraction via VEGF. An

increase in the number of new blood vessels can

increase the process of regeneration and repair of

renal tubular cells (Ahmad et al., 2013).

Black cumin or black cumin (Nigella sativa L)

contains several active compounds that have

antioxidant effect, the most important of which are

thymoquinone (30% -48%), thymohydroquinone,

dithymoquinone, p-cymene (7% -15%), carvacrol

(6% -12%) ), 4-terpineol (2% -7%), t-anethol (1% -

4) (Ahmad et al., 2013). Thymoquinone has a

protective effect on cells against damage caused by

oxidative stress (Abdelmeguid, Fakhoury, Kamal, &

Al Wafai, 2010). Thymoquinone works to inhibit

oxidative stress by increasing the activity of the

enzyme Super Oxide Demutase (SOD), the enzyme

glutathione and inhibits the lipid peroxidase reaction

(Leong, Rais Mustafa, & Jaarin, 2013) (Goyal et al.,

2017). Thymoquinone has been shown to

significantly reduce colonic MDA levels in mice by

induction of the Necrotizing enterocolitis (NEC)

model (Tayman et al., 2012). Likewise in a study

conducted by Fouda, 2008 proved that

Thymoquinone inhibits increased MDA levels in the

kidneys HgCl2-induced mice (Fouda, Daba, Dahab,

& Sharaf El-Din, 2008). Thymoquinone reduced

renal levels of MDA that were induced by

gentamicin (Samarghandian, Azimi-Nezhad,

Mehrad-Majd, & Mirhafez, 2015). In addition, black

cumin also contains flavonoids and fatty acids

including linoleic acid and oleic acid. Linoleic acid

and oleic acid have antioxidant effects that can

inhibit the lipid peroxidase reaction (El-Agbar, Naik,

& Shakya, 2018). While flavonoids function as

antioxidants by acting as electron donors which are

targets of free radicals [(Banjarnahor & Artanti,

2014).

The dosage of black cumin seed extract (Nigella

sativa.) Which gives a significant influence on the

improvement of proximal tubules in the kidney of

male white rats model of chronic kidney damage is a

dose of 2.4 gr/kgBB/day and 4.8 gr/kgBB/day.

There is a very strong and inversely related

relationship between administration of black cumin

extract (Nigella sativa) to the improvement of

proximal tubules in the kidney of male white rats, a

model of chronic kidney damage was shown by

Pearson correlation test = -0.829.

HSIC 2019 - The Health Science International Conference

4 CONCLUSIONS

Black cumin extract decreases the amount of hyaline

cast in the proximal tubule. The antioxidant and

anti-inflammatory effects of Black cumin extract

may modulate the improvement renal tubular.

ACKNOWLEDGEMENTS

The autors thanks Miftachurrahman, Slamet, and

Anto for their dedicated work in collecting data used

in this article as a part of the objective of our

research.

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