ORIGINAL�ARTICLE

ABSTRACT
Objective:	The	present	research	was	carried	out	to	observe	withdrawal	effects	of	energy	drinks,	whether	these	
histomorphological	changes	are	reversible	or	not.	
Study	Design:	Laboratory	based	experimental	study.	

st th
Place	and	Duration	of	Study:	The	research	was	carried	out	from	1 	July	to	30 	August	2019	at	national	institute	
of	health	Islamabad.	
Materials	and	Methods:	Total	thirty	adult	male	albino	rats	were	divided	into	3	groups	by	simple	random	
sampling,	with	ten	rats	in	each	group.	Group	I	was	control	group,	while	energy	drink	group	II	received	3.57ml/kg	
body	weight	red	bull	corresponding	to	one	can	of	energy	drink	(250ml)	in	humans	orally	for	eight	weeks.	Rats	in	
withdrawal	group	III	received	energy	drink	for	first	four	weeks	followed	by	normal	diet	and	water	for	last	four	
weeks.	After	eight	weeks,	rats	were	sacrificed	and	their	right	kidneys	were	removed.	Slides	were	prepared	using	
hematoxylin	eosin	and	Periodic	Acid	Schiff	Stain,	results	were	analyzed	by	SPSS.
Results:	The	results	showed	that	use	of	energy	drink	for	8	weeks	resulted	in	increase	in	weight	of	kidneys	along	
with	histological	alterations	in	renal	cortex	of	rat	kidneys.	Grade	4	(severe)	congestion,	hemorrhage,	loss	of	
brush	border	and	necrosis	was	observed	in	energy	drink	group	II.	Withdrawal	of	energy	drink	in	group	III	
resulted	in	weight	of	kidneys	near	to	control	group	along	with	significant	reduction	in	congestion,	hemorrhage,	
loss	of	brush	border	and	necrosis	grades	from	grade	4	to	grade	3	and	2	with	P≤0.05.
Conclusion:	Caffeinated	energy	drinks	are	having	damaging	effects	on	kidneys	of	albino	rats	and	these	
histological	changes	caused	by	caffeinated	energy	drinks	in	this	duration	of	study	and	in	low	doses	
corresponding	to	one	can	of	energy	drink	(250ml)	in	humans	are	reversible.

Key	Words:	Caffeinated, Energy Drink, Histological, Kidneys, Withdrawal.

available	 in	 Pakistan.	 First	 and	 most	 trendy	
caffeinated	energy	drink	to	be	introduced	is	red	bull.
Target	of	these	caffeinated	drinks	market	is	mainly	
youth.	In	Pakistan	majority	of	energy	drink	users	are	
youngsters	with	age	group	of	13-35	years.	EDs	claim	
to	provide	burst	of	energy	by	using	a	combination	of	
caffeine	 (chief	 active	 ingredient),	 guarana,	 yerba	
mate, 	 taurine, 	 glucose,	 fructose	 and	

3
glucuronolactone. 	Caffeinated	energy	drinks	(EDs)	
contain	 higher	 levels	 of	 caffeine	 along	 with	 other	
ingredients	that	are	not	commonly	found	in	sodas	
and	 juices,	 marketed	 as	 providing	 mental	 and	
physical	stimulation	especially	in	youth.	They	differ	
from	 soft	 or	 sport	 drinks	 due	 to	 their	 unique	

4
composition. 	
Caffeine	present	in	these	drinks	is	up	to	500	mg	per	
20	oz.	(600	mL)	serving	that	is	15	times	the	amount	of	
caffeine	present	in	a	12-ounce	(360	mL)	serving	of	
cola.	Beverages	that	contains	guarana	actual	levels	of	
caffeine	are	much	higher	than	levels	mentioned	on	

5
the	label. 	Youth	using	EDs	is	unaware	of	these	high	

Introduction
Energy	drinks	(EDs)	were	first	 introduced	in	UK	in	
1929	as	hospital	drink;	in	1980	they	were	promoted	
for	replenishing	lost	energy.	They	are	now	available	
in	>	140	countries	as	part	of	a	multi-	billion	dollar	

1
business. 	 In	 1960s	 they	 appeared	 in	 Asia	 and	

2
Europe. 	 There	 are	 diverse	 types	 of	 energy	 drinks	

Histological	Effects	of	Caffeinated	Energy	Drink	Consumption	and	Its	Withdrawal
on	Kidneys	of	Experimental	Rats

1 2 3 4 5 6
Syeda	Sara	Bano , Shabana	Ali ,	Rehana	Rana ,	Hussain	Ali ,	Ali	Ahmad ,	Tooba	Khurshid

Correspondence:
Dr. Syeda Sara Bano
Senior lecturer
Department of Anatomy
Shifa College of Medicine
Shifa Tameer-e-Millat University, Islamabad
E-mail:	banobokhari@hotmail.com

1
Department of Anatomy

Shifa College of Medicine
Shifa Tameer-e-Millat University, Islamabad
2,3,5,6 

Department of Anatomy
Islamic International Medical College
Riphah International University, Islamabad
4
Department of IT

NCA Rawalpindi.

Funding Source: NIL; Conflict of Interest: NIL
Received: November 19, 2019; Revised: June 05, 2020
Accepted: June 06, 2020

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levels	 of	 caffeine	 and	 its	 adverse	 effects	 that	 is	
alarming.
The	kidneys	are	the	organs	that	filter	waste	products	
from	 the	 blood.	 Kidneys	 are	 predominantly	
vulnerable	 to	 ischemic	 and	 toxic	 damage.	 Many	
studies	have	depicted	adverse	health	effects	of	EDs,	
one	of	them	is	hastened	progression	of	renal	micro	

6
vascular	 impairment	and	chronic	kidney	disease.	 	
Literature	illustrates	that	exposure	of	rats	to	energy	
drink	leads	to	kidney	damage	causing	renal	vascular	
congestion,	hemorrhage	of	interstitial	tissue,	focal	
atrophy	 and	 degeneration	 of	 lining	 epithelium	 of	

7
Proximal	 and	 Distal	 convoluted	 tubule. 	 There	 is	
limited	data	available	regarding	withdrawal	effects	
of	 these	 drinks,	 therefore	 present	 study	 was	
conducted	to	observe	nature	of	renal	damage	and	to	
observe	 changes	 after	 withdrawal	 of	 these	 drinks	

1
whether	these	changes	are	reversible	or	not. 		

Materials	and	Methods
This	 laboratory	 based	 experimental	 study	 was	

st th
conducted	from	1 	July	to	30 	August	2019	by	mutual	
collaboration	with	national	institute	of	health	(NIH)	
and	Islamic	international	medical	college	(anatomy	
department)	 after	 approval	 from	 ethical	 review	
committee	(ERC).
Thirty	 adult	 male	 healthy	 albinos	 Sprague	 Dawley	
rats	(n=10/group)	weighing	250±10	gm	were	used	in	
experiment.	They	were	housed	at	animal	house	of	
NIH	Chak	Shahzad	Islamabad	and	were	acclimatized	
to	laboratory	surroundings	with	free	food	and	water	
access	under	natural	dark	and	light	rhythms	prior	to	
commencement	of	study.	Female	rats	and	rats	with	
any	disease	or	pathology	were	excluded.	Total	thirty	
animals	were	divided	into	three	groups	each	group	
having	ten	rats	selected	by	simple	random	sampling	
and	treated	in	this	way:

The	slides	were	examined	under	light	microscope	in	
X10,	X40	Power.	Parameters	observed	were	weight	
of	kidneys,	congestion,	hemorrhage,	 loss	of	brush	
border,	 and	 necrosis.	 Using	 SPSS	 version	 21	 non	
parametric	data	was	analyzed	by	means	of	chi	square	
test.	 A	 p value	 of	 equal	 or	 less	 than	 0.05	 was	
considered	as	significant	value.

Results	
Renal	cortical	parenchyma	appeared	to	be	normal	in	
100%	rats	in	control	group	I,	while	25%	rats	showed	
minimal	congestion	 (grade	 1).	 In	 ED	 group	 severe	
congestion	(grade	4)	was	noted	in	62.5%	of	rats.	In	
withdrawal	 group	 62.5%	 lesions	 were	 moderate	
(grade	3)	showing	reversal	of	histological	alterations	
caused	by	EDs	(Fig:	1,	Table	I).

After	8	weeks	of	completion	of	experimental	study	
rats	were	dissected	and	kidneys	were	removed.	After	
fixation	and	embedding	transverse	sections	of	5	µm	
thickness	 were	 obtained.	 Staining	 was	 done	 with	
Hematoxylin	and	eosin	and	Periodic	Acid-Schiff	stain.	

Group	I

Group	II

Group	III

Fig	1:	Distribu�on	of	Renal	Cor�cal	Parenchymal	
Conges�on	Among	Different	Groups,	Group	I	K1R5	
Showing	No	Conges�on,	Group	II	K1R2	Showing	Severe	
Conges�on	And	Arrows	Indica�ng	Moderate	Conges�on	
n	Group	III	K1R7	(H&E,	X400).

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In	100%	rats	of	control	group	I	no	hemorrhage	was	
observed	in	renal	cortex	while	in	ED	group	severe	
hemorrhage	(grade	4)	was	noted	in	75%	of	rats.	In	
withdrawal	group	75%	lesions	were	mild	(grade	2)	
showing	significant	reduction	in	hemorrhage	grade	
when	compared	to	ED	group	(Fig:	2	and	Table	I).

Renal	cortex	appeared	to	be	normal	in	100%	rats	in	
control	group	I.	In	ED	group	severe	(grade	4)	loss	of	
brush	border	was	observed	in	PCT	of	87.5%	rats.	In	
withdrawal	group	mild	loss	of	brush	border	in	50%	of	
rats	 was	 seen	 showing	 significant	 reversal	 of	
histological	alteration	after	withdrawal	of	ED.	(Fig:	3,	
Table	II)

Group	I

Group	II

Group	III

Fig	2:	Distribu�on	of	Hemorrhage	In	Renal	Cor�cal	

Parenchyma	of	Different	Groups,	Group	I	K1R2	Showing

No	Hemorrhage,	Group	II	K1R8	Showing	Severe	

Hemorrhage	and	Arrows	Indica�ng	Mild	Hemorrhage	In

Group	III	K1R9	(H&E,	X400).

Table	I:	Distribu�on	of	Grades	of	Conges�on	and	

Hemorrhage	In	Control	and	Experimental	Groups	of	

Albino	Rats	By	Chi-Square	Test

Group	I

Group	II

Group	III
Fig	3:	Distribu�on	of	Loss	of	Brush	Border	In	PCT	Of	
Different	Groups,	Group	I	K1R5	Showing	Prominent	
Apical	Brush	Border,	Group	II	K1R9	Showing	Severe	Loss
of	Brush	Border	In	PCT	And	Arrows	Indica�ng	Mild	Loss
of	Brush	Border	In	Group	III	K1R1	(PAS,	X400).

In	 control	 group	 100%	 rats	 showed	 normal	 renal	
cortical	architecture.	In	ED	group	severe	(grade	4)	
necrosis	was	observed	in	100%	of	rats.	In	group	3	
moderate	(grade	3)	necrosis	was	seen	in	75.5%	rats	
showing	significant	reduction	in	grades	of	necrosis	
when	compared	to	energy	drink	group.	(Fig:	4	and	
Table	II)

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in	kidneys	of	male	rats.	In	this	study	we	observed	no	
congestion	with	normal	renal	cortical	parenchyma	in	
control	group.	In	ED	group	severe	congestion	in	renal	
cortical	 parenchyma	 was	 observed.	 In	 withdrawal	
group,	moderate	congestion	was	seen.	Impairment	
of	venous	outflow	due	to	inflammatory	mediators	
results	in	a	localized	increase	in	blood	to	different	
areas	of	kidney,	which	is	demonstrated	histologically	

8
as	congestion. 	Taiwo	et	al	documented	congestion	
after	 administration	 of	 energy	 drink	 in	 different	

9
doses	 to	 rabbits. 	 He	 documented	 that	 changes	
observed	 were	 reversible	 in	 28	 days	 duration	 of	

9
study. 	Similarly	results	has	been	supported	by	many	

10,11,12
other	studies.
In	our	study	severe	hemorrhage	was	observed	in	ED	
group.	 In	 rats	 of	 group	 III	 mild	 hemorrhages	 was	
seen.	Hemorrhage	observed	was	due	to	the	effect	of	
inflammation	leading	to	expansion	of	blood	vessels,	
as	 result	 vessels	 rupture	 and	 blood	 flows	 out.	 A	
previous	 study	 showed	 similar	 findings	 after	
administration	 of	 ED	 1ml/animal/day	 orally	 for	 4	

13
weeks. 	Comparable	findings	were	observed	in	liver	

14
of	 rats	 after	 administration	 of	 high	 dose	 of	 EDs. 	
Akande	also	proved	that	damaged	caused	by	ED	was	

15
reversible	in	28	days	duration.
The	 present	 study	 showed	 severe	 loss	 of	 brush	
border	of	PCT	in	ED	group,	while	in	withdrawal	group	
mild	loss	of	brush	border	was	observed	that	showed	
reversal	of	histological	changes	caused	by	red	bull	
after	withdrawal	of	ED.	This	might	be	explained	by	
vulnerability	 of	 cellular	 membranes	 to	 toxins,	
leading	to	decrease	cellular	production	of	ATP	and	
accumulation	of	reactive	oxygen	species	causing	cell	
damage	 and	 detachment	 of	 epithelial	 cell	 from	
basement	membrane.	Similar	results	were	shown	in	
albino	rabbits	by	Salih,	results	were	ascribed	to	high	

16
level	of	caffeine	in	EDs.
Most	common	cause	of	acute	kidney	injury	is	acute	
tubular	 necrosis	 that	 causes	 death	 of	 tubular	
epithelial	cells	leading	to	renal	failure.	In	group	III	
moderate	necrosis	was	seen	which	was	significantly	

17
reverted	 after	 withdrawal	 of	 ED. Coagulative	
pattern	 of	 necrosis	 was	 observed	 in	 various	
experimental	groups	which	is	more	common	in	toxic	

18
or	 ischemic	 injury. The	observed	necrosis	may	be	
due	to	carbon	dioxide	present	in	energy	drinks	that	
damages	 membranes	 of	 mitochondria,	 change	 in	

13
ATP	production,	hypoxia	and	cell	deah. In	another	

Group	I

Group	II

Group	III
Fig	4:	Distribu�on	of	Necrosis	In	Renal	Tubules	of	
Different	Groups,	Group	I	K1R3	Showing	No	Necrosis,
Group	II	K1R5	Showing	Severe	Tubular	Necrosis	and	
Arrows	Indica�ng	Moderate	Necrosis	In	Group	III	K1R7	
(H&E,	X400).

Discussion	
This	 study	 investigated	 the	 reversal	 of	 renal	
histomorphological	features	after	withdrawal	of	ED	

Table	II:	Distribu�on	of	Grades	of	Loss	of	Brush	Border
and	Necrosis	in	Control	and	Experimental	Groups	of	
Albino	Rats	By	Chi-Square	Test

p	value	≤	0.05

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study	 after	 administration	 of	 red	 bull,	 severe	
necrosis	 was	 observed	 in	 seminiferous	 tubules	 of	

19
male	rats.
In	this	study	we	observed	the	severity	of	renal	injury	
in	relation	to	time	duration	for	which	EDs	were	used.	
In	ED	group	significant	difference	was	observed	in	
weight	of	kidneys,	congestion,	hemorrhage,	loss	of	
brush	border	and	necrosis.	This	indicates	that	EDs	
leads	 to	 renal	 damage	 that	 is	 duration	 or	 time	
dependent,	since	more	damage	was	observed	in	ED	
group	 when	 compared	 to	 group	 III,	 it	 showed	
reversal	 of	 these	 histological	 features	 after	
withdrawal	of	ED.	Our	findings	are	consistent	with	
the	 results	 of	 study	 that	 proved	 that	 effects	 of	
caffeine	 on	 cell	 survival	 are	 highly	 time	 and	 dose	
dependent;	in	low	doses	it	increases	cell	survival	and	
at	 higher	 doses	 it	 increases	 super	 oxide	

20,21
production. 	 Also	 the	 study	 proved	 that	 chronic	

22
caffeine	intake	has	age	dependent	effects	on	brain.

Conclusion	
In	conclusion	caffeinated	energy	drinks	are	having	
damaging	 effects	 on	 kidneys	 of	 rats,	 besides	 that	
with	 low	 doses,	 corresponding	 to	 one	 can	 of	 ED	
(250ml	in	humans)	and	with	this	duration	of	study	
histomorphological	changes	caused	by	caffeinated	
EDs	are	reversible.	

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