Background: Renal ischemia-reperfusion injury (IRI) does not only affect kidneys, but also affects remote organs especially the liver. Stress may lead to further progression of renal and hepatic insults. This study was planned to explore the efficacy of olive oil (OO) on the assumed renal and hepatic changes of immobilization stress following renal IRI.

Methods: Seventy-seven adult male Wistar albino rats were divided into the following five groups; Sham-Operated Control group, Renal Ischemia-Reperfusion group, Stressed Renal Ischemia-Reperfusion group, OO-Supplemented Renal Ischemia-Reperfusion group) and OO-Supplemented Stressed Renal Ischemia-Reperfusion Group (OO+Stressed+IR). All animals were subjected to determination of renal and hepatic biochemical functions and histopathological examination.

Results: Renal IR significantly increased plasma creatinine and urea levels. This was associated with significant rise in serum levels of both ALT and AST, total bilirubin together with plasma level of TNF- $α$ $α$ , whereas the plasma level of albumin (ALB) was significantly reduced. In addition, histological disruptions of kidneys and livers were observed. Chronic immobilization stress aggravated the effects of renal IR. Also, renal and hepatic morphological changes were more worsened. Whilst, OO supplementation resulted in significant amelioration of renal and hepatic functions. Also, the kidney and liver morphologic lesions were attenuated.

Conclusion: Renal IR not only affected the renal functional and structural integrity but also the remote organ, the liver. Chronic immobilization stress rendered the kidney and liver more prone to injury. OO improved renal and hepatic dysfunction and morphological damage mediated by renal IRI, in control rats and in those exposed to chronic stress which can be exerted partially via its antioxidant, anti- inflammatory and nitric oxide (NO) reducing activities.

Keywords:

Abbreviations

ALT	:	Alanine transaminase
AST	:	Aspartate aminotransferase
AKI	:	Acute kidney injury
ALB	:	Albumin
eNOS	:	endothelial nitric oxide synthase
iNOS	:	inducible nitric oxide synthase
IRI	:	Ischemia-reperfusion injury
MDA	:	Malondialdehyde
NO	:	Nitric oxide
ROS	:	Reactive oxygen species
OO	:	Olive oil
TNF- $α$	:	Tumor necrosis factor alpha

Introduction

Renal ischemia-reperfusion injury (IRI) is considered as a common cause of acute kidney injury (AKI). AKI of ischemic nature was found to be the most common form of adults’ intrinsic kidney disease, also, it was found to have valuable effects on the individual’s morbidity and mortality.^1,2 The high mortality rate during AKI is mostly non-renal, being largely attributed to remote organ dysfunction. Numerous studies have shown the association between AKI and mild- to-moderate acute injury up to failure in organs remote from the kidney as the lung, liver and brain.^2,3

Stress is a well-known deleterious problem that can, not only, induce mental, physical and chemical responses in body⁴ but also can lead to oxidative injury at cellular level by affecting the (reduction–oxidation) REDOX state. Therefore, stress has been involved in the pathogenesis of several diseases.⁵

It was reported that restraint stress affected the integrity of the cells in many tissues, as liver heart, stomach and brain,^6,7 in addition to alleviating physical and psychiatric diseases.^8,9 Because the available literature dealing with the impact of chronic immobilization stress in models of renal IR is lacking, it was of interest to explore this effect which appeared novel.

Olive oil (OO), the principal fat of the Mediterranean diet, contributed to the superior health profile noticed in the Mediterranean populations.¹⁰ OO has been shown to have several health-promoting properties by several studies.^11,12 The beneficial effects of OO intake on health were mostly attributed to the high concentration of the healthy fat, the monounsaturated fatty acid (oleic acid), which represents the main component, and the phenolic compounds.^13,14

Recent studies have shed light on the impacts of virgin OO and/or its product (phenols) as a protective tool against oxidative and inflammatory stress-induced disorders, not only in human beings^15,16,17 but also experimentally, in rats^18,19,20 and in mice.^21,22,23 OO polyphenols have shown several biological activities, both in vivo and in vitro, as antioxidant, anti-inflammatory, anti-apoptotic, anti-aging, anti-carcinogenic, anti-neurodegenerative and anti- atherosclerotic agents.²⁴ Researchers, focused on the role of OO as a natural product and a source of polyphenols in ameliorating the hepatotoxicity^18,21 and nephrotoxicity in different animal models.^19,20,23

The management of renal IR injury remains a clinical challenge, despite the progress in the therapeutic approaches. To our knowledge, available information regarding the protective impact of OO consumption on hepatic and renal alterations of renal IR in rats exposed to chronic immobilization stress is lacking. We, therefore, tried to portray the efficacy of OO in such conditions in a rat model.

Material and Methods

This study was performed on 77 male adult albino rats, weighing 265–325g at the start of the study. Rats were bought from an animal farm in Helwan. Rats were housed in the Faculty of Medicine Ain Shams University Research Institute (MASRI), under standard boarding conditions; room temperature (22–25°C) and 12h light/dark cycle with free access to food and water. Rats were kept for one week prior to the experimental procedures for acclimatization. The meals were introduced regularly at 8am. Rats were fed a standard rat diet consisting of milk and green vegetables (7). Experimental work was done following the Guide for Care and Use of the Laboratory Animals and under the guidelines of the ICH, the Organization of Medical Sciences (IOMS) and United States Code of Federal Regulations and operates under the Federal-wide Assurance No. FWA 000017585. The protocol of this study took the approval of the Research Ethical Committee of the faculty of Medicine, Ain Shams University.

Experimental Protocol:

Animals were allocated randomly into the following five groups:

Group I: Sham-Operated Control Group (Sham Group) (17 rats):

Rats in this group remained undisturbed in their cages throughout the study period (4 weeks). Then, the rats underwent the same surgical steps as in the renal IR group without renal pedicles clamping.

Group II: Renal Ischemia-Reperfusion Group (IR group) (15 rats):

In this group, rats remained undisturbed in their cages throughout the study duration (4 weeks). Rats were then subjected to renal IR procedure (in the form of 45min of renal ischemia followed by 24h of reperfusion).

Group III: Stressed Renal Ischemia-Reperfusion Group (Stressed+IR group) (15 rats):

Rats in this group were subjected to immobilization stress by placing each rat separately in a tight animal restraining cage (Curtin Mathesson Scientific, regular size), in prone position at room temperature for 2h (10am:12am), 6 days/week for 4 weeks.²⁵ Then, the rats underwent the renal IR procedure.

Group IV: OO-Supplemented Renal Ischemia-Reperfusion Group (OO+IR group) (15 rats):

The supplemented OO was in the form of extra virgin OO (Safi) supplied by Elwatania for olive industries (Egypt). OO was supplemented in a dose of 12ml/kg/day, orally by gavage²⁶ at 9am, 6 days/week for 4 weeks. Then, rats underwent renal IR procedure.

Group V: OO-Supplemented Stressed Renal Ischemia-Reperfusion Group (OO+Stressed+IR group) (15 rats):

In this group, the rats received OO as in group IV. Then, 1h later to each OO dose supplementation, rats were separately subjected to immobilization stress as in group III. At the end of the study duration (4 weeks), the rats were subjected to the procedure of renal IR.

Renal Ischemia-Reperfusion Procedure:

The overnight fasted rats were anesthetized by intraperitoneal injection of xylazine (EPICO) (10mg/kg) together with ketamine hydrochloride (EPICO) (100mg/kg).²⁷ Renal IR was carried out following the method described by Gholampour et al. (2017).²⁸ The right and the left renal pedicles were clamped using non-traumatic vascular clamps for 45min.

Then, the animals were allowed to recover and were given free access to food and water.

Reperfusion was allowed for 24h.

Experimental Procedures:

On the day of sacrifice, the overnight fasted rats were anesthetized by intraperitoneal injection of pentobarbital sodium (El-Gomhoreya Co., Egypt) in a dose of 40mg/kg.²⁹ Then, a midline incision was done in the abdomen to expose the abdominal aorta for blood collection using polyethylene catheter.

After blood collection, both kidneys and liver were carefully dissected and weighted. The left lobe of the liver and the left kidney were both fixed in a solution containing 10% buffered formalin for subsequent histological examination. Then, the remaining lobes of the liver and right kidney were washed in cold saline, and then stored in parafilm at −80°C for later determination of tissue levels of malondialdehyde (MDA), nitrite and catalase activity.

(I)

Blood Biochemical Studies:

(A)

Evaluation of Renal Function:

		(1)	Plasma Creatinine Level was determined by colorimetric kinetic method by using creatinine kits supplied by bio-diagnostic, Egypt.³⁰
		(2)	Plasma Urea Level was determined using the enzymatic colorimetric Urease-Berthelot method by using kits supplied by bio-diagnostic, Egypt.³¹

(B)

Evaluation of Liver Function:

		(1)	Serum Activities of both Alanine Transferase (ALT) and Aspartate Transferase (AST) were measured colorimetrically by kits supplied by Bio-diagnostic, Egypt.³²
		(2)	Serum Alkaline Phosphatase (ALP) Activity was measured using colorimetric method by the use of kits supplied by bio-diagnostic, Egypt.³³
		(3)	Plasma Albumin (ALB) Level was assayed by colorimetric method using kits supplied by bio-diagnostic, Egypt.³⁴

(C)

Estimation of Plasma Level of Tumor Necrosis Factor-alpha (TNF- $α$ ):

In vitro quantitative determination of plasma TNF- $α$ level was performed using enzyme-linked immunoassay (ELISA) technique, (Infinite F50, Germany) by kits supplied by Wuhan EIAab $^{®}$ science Co., China.

(II)

Tissue Biochemistry Study:

Preparation of kidney and liver tissue homogenate:

The right kidney and the right lobe of liver were homogenized in 1ml phosphate buffered saline (Sigma-Aldrich)³⁵ per gram tissue, using the tissue homogenizer (Ultrasonic homogenizer, ILLnois, 60648, Cole-Parmer instrument Co., Chicago). Then, the sample was allowed to centrifuge for 20min at 10,000rpm. After that, the supernatant was removed, and stored at −80°C for later determination of MDA, catalase activity and nitrite level.

	(1)	Kidney and Liver Tissue Levels of MDA were determined colorimetrically by using the kits supplied by Bio-diagnostic, Egypt.^36,37
	(2)	Kidney and Liver Tissues Catalase Activity was estimated using the colorimetric method by kits supplied by Bio-diagnostic, Egypt.³⁸
	(3)	Kidney and Liver Tissue Levels of Nitrite were measured by the colorimetric method by the use of kits supplied by Bio-diagnostic, Egypt.³⁹

(III)

Histopathological Study (H&E examination):

The histopathological changes in the kidney were quantified by using the Endothelial, Glomerular, Tubular and Interstitial (EGTI) scoring system that was devised specifically for the animal research on the tissues of the kidney in context of injury. The scoring system was formed of the histological damage in the endothelial, glomerular, tubular and interstitial components.⁴⁰ (Table 1)

**Table 1. The EGTI histology scoring system.**
Tissue type	Damage	Score
Tubular	No damage	0
	Loss of Brush Border (BB) in less than 25% of tubular cells. Integrity of basal membrane	1
	Loss of BB in more than 25% of tubular cells, Thickened basal membrane	2
	(Plus) Inflammation, Cast formation, Necrosis up to 60% of tubular cells	3
	(Plus) Necrosis in more than 60% of tubular cells	4
Endothelial	No damage	0
	Endothelial swelling	1
	Endothelial disruption	2
	Endothelial loss	3
Glomerular	No damage	0
	Thickening of Bowman capsule	1
	Retraction of glomerular tuft	2
	Glomerular fibrosis	3
Tubulo/Interstitial	No damage	0
	Inflammation, hemorrhage in less than 25% of tissue	1
	(Plus) Necrosis in less than 25% of tissue	2
	Necrosis up to 60%	3
	Necrosis more than 60%	4

Regarding the liver histopathological scoring, liver specimens were measured, with a point counting method for hepatic injury severity by the use of the ordinal scale:

	Grade 0: Minimal or no evidence of injury.
	Grade 1: Mild injury consists of cytoplasmic vacuolation and focal nuclear pyknosis.
	Grade 2: Moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, loss of intercellular borders and mild to moderate neutrophil infiltration.
	Grade 3: Severe injury consists of disintegration of hepatic cords, hemorrhage and severe polymorphonuclear (PNL) leucocyte infiltration.

On average, 100 adjacent points on a 1mm² grid were graded for each specimen.⁴¹

Statistical Analysis⁴²:

Both the statistical data and the significance were determined by using the SPSS statistical package (SPSS Inc.), version 20.0. The statistical significance was performed using one-way ANOVA for the differences between the means of different groups. Then, the least significant difference test (LSD) was performed to determine the inter-groupal significance. A probability of $P < 0.05$ was considered statistically significant.

The relationships between variables were assessed by using Pearson’s correlation, a probability of $P < 0.05$ was considered statistically significant.

Results

Changes in kidney function tests (Table 2):

Table 2. Cumulative values of plasma levels of creatinine (mg/dl) and urea (mg/dl), serum levels of alanine transferase (ALT, U/ml), aspartate transferase (AST, U/ml) and alkaline phosphatase (ALP, IU/L) and plasma levels of albumin (Alb, g/dl) and total bilirubin (TB, mg/dl) in the different studied groups
Group	Creatinine	Urea	ALT	AST	ALP	Alb	TB
Sham-operated Control	0.629	42.14	42.33	111.08	266.1	4.094	0.096
	± 0.023	± 2.255	± 1.694	± 2.970	± 15.59	± 0.083	± 0.011
	(17)	(17)	(17)	(17)	(17)	(17)	(16)
Renal ischemia-reperfusion	1.579	114.09	107.1	144.9	262.1	3.428	0.157
	± 0.082	± 6.549	± 6.682	± 2.395	± 14.64	± 0.077	± 0.024
	(15)	(15)	(15)	(15)	(15)	(14)	(14)
P	<0.001	<0.001	<0.001	<0.001	NS	<0.001	<0.02
Stressed renal ischemia-reperfusion	1.889	142.6	143.7	165.9	282.1	3.414	0.195
	± 0.086	± 5.472	± 11.17	± 3.778	± 20.11	± 0.051	± 0.017
	(15)	(15)	(15)	(15)	(15)	(13)	(14)
P	<0.001	<0.001	<0.001	<0.001	NS	<0.001	<0.001
P₁	<0.001	<0.001	<0.01	<0.001	NS	NS	NS
Olive oil-supplemented renal ischemia-reperfusion	0.982	86.19	72.70	121.92	255.0	3.741	0.112
	± 0.047	± 2.879	± 2.820	± 4.508	± 19.90	± 0.084	± 0.010
	(15)	(15)	(15)	(15)	(15)	(15)	(13)
P	<0.001	<0.001	<0.01	<0.05	NS	<0.01	NS
P₁	<0.001	<0.001	<0.01	<0.001	NS	<0.01	NS
Olive oil-supplemented stressed renal ischemia-reperfusion	1.093	97.14	112.9	147.7	247.2	3.686	0.124
	± 0.040	± 5.708	± 8.901	± 2.756	± 13.25	± 0.063	± 0.015
	(15)	(15)	(15)	(15)	(15)	(13)	(14)
P	<0.001	<0.001	<0.001	<0.001	NS	<0.001	NS
P₁	<0.001	<0.02	NS	NS	NS	<0.05	NS
P₂	<0.001	<0.001	<0.01	<0.001	NS	<0.02	<0.01

Notes: Results are expressed as $mean \pm SEM$ .

In parenthesis is the number of observations.

P: Significance from sham-operated control group calculated by LSD at $P < 0.05$ .

P₁: Significance from renal ischemia-reperfusion group calculated by LSD at $P < 0.05$ .

P₂: Significance from stressed renal ischemia-reperfusion group calculated by LSD at $P < 0.05$ .

NS: Non-significant.

As compared to the Sham Group, the IR group exhibited a significant increase in both creatinine and urea plasma levels. Also, the stressed+IR group showed significant increase in the plasma creatinine and urea levels as compared to both IR and Sham Groups.

Upon OO supplementation, plasma levels of both creatinine and urea were significantly decreased in OO+IR group as compared to IR group. On the other hand, the plasma levels of creatinine and urea showed significant rise as compared to Sham Group.

OO+stressed+IR group revealed a significant reduction in both levels of creatinine and urea in plasma in comparison to both IR and stressed+IR groups. However, as compared to the Sham Group, the plasma levels of creatinine and urea exhibited significant increase.

Changes in liver function tests (Table 2):

Serum levels of ALT and AST were significantly elevated in the IR group as compared to Sham Group. Additionally, stressed+IR group exhibited a significant increase in both AST and ALT levels in comparison to both IR and Sham Groups. In OO+IR group, both ALT and AST levels were significantly reduced as compared to IR group, whereas they showed a significant elevation when compared to Sham Group. In OO+stressed+IR group, serum ALT and AST levels were significantly reduced compared to stressed+IR group, however, were significantly elevated as compared to Sham Group and increased insignificantly as compared to IR group.

In addition, serum ALP levels showed statistically insignificant changes in the different studied groups.

Plasma levels of ALB were significantly reduced in IR group as compared to the Sham Group. However, the plasma level of ALB showed statistically insignificant changes in stressed+IR group as compared to the IR group but was significantly decreased in stressed+IR group when compared to the Sham Group.

OO+IR group demonstrated a significant rise in plasma ALB level in comparison to IR group, but a significant decrease compared to the Sham Group. In OO+stressed+IR group, the plasma level of ALB showed a significant increase as compared to both IR and stressed+IR groups, whereas it was significantly reduced as compared to the Sham Group.

As regards plasma total bilirubin level, a significant increase was observed in IR group when compared to the Sham Group. Moreover, stressed+IR group exhibited elevation in total bilirubin plasma level that was statistically insignificant when compared to IR group but was statistically significant when compared to the Sham Group. Upon OO supplementation, the plasma level of total bilirubin showed no significant decrease in OO+IR group when compared to IR group, whereas significant reduction was observed in OO+stressed+IR group as compared to stressed+IR group. In both OO+IR group and OO+stressed+IR group, total bilirubin showed no significant changes compared to Sham Group.

Changes in plasma levels of TNF- $α$ :

Plasma levels of TNF- $α$ was significantly increased in IR group as compared to the Sham Group. Also, plasma TNF- $α$ was significantly increased in Stressed+IR group as compared to both IR and Sham Groups. In OO+IR group, plasma TNF- $α$ level was significantly reduced as compared to IR group, on the other hand, it showed insignificant changes, compared to the Sham Group. Additionally, in OO+stressed+IR group, plasma TNF- $α$ was significantly decreased when compared to stressed+IR group, whereas it was significantly increased as compared to the Sham Group (Table 3).

Table 3. Cumulative values of plasma level of tumor necrosis factor-alpha (TNF- $α$ , pg/ml), and kidney tissue and liver tissue malondialdehyde (MDA, nmol/g) level, catalase activity (U/g) and nitrite level ( $μ$ mol/g) in the different studied groups
		Kidney			Liver
Group	TNF- $α$	MDA	Catalase	Nitrite	MDA	Catalase	Nitrite
Sham-operated control	49.93	34.82	17.15	129.1	22.46	14.66	21.30
	± 1.245	± 4.541	± 1.060	± 5.771	± 1.842	± 1.190	± 1.175
	(15)	(16)	(16)	(16)	(17)	(17)	(17)
Renal ischemia-reperfusion	77.77	621.4	4.721	661.6	170.6	2.892	66.40
	± 3.156	± 22.23	± 0.524	± 18.92	± 12.26	± 0.281	± 5.429
	(15)	(14)	(14)	(14)	(15)	(15)	(15)
P	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.05
Stressed renal ischemia-reperfusion	155.4	787.9	7.055	902.9	413.5	1.289	325.9
	± 8.594	± 49.71	± 0.411	± 17.55	± 25.08	± 0.099	± 29.23
	(15)	(14)	(14)	(14)	(15)	(15)	(15)
P	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
P₁	<0.001	<0.001	<0.05	<0.001	<0.001	NS	<0.001
Olive oil-supplemented renal ischemia-reperfusion	54.13	203.8	7.053	311.1	78.23	7.407	53.39
	± 2.249	± 9.403	± 0.559	± 23.98	± 3.739	± 0.493	± 4.670
	(15)	(15)	(15)	(15)	(15)	(15)	(15)
P	NS	<0.001	<0.001	<0.001	<0.01	<0.001	NS
P₁	<0.01	<0.001	<0.05	<0.001	<0.001	<0.001	NS
Olive oil-supplemented stressed renal ischemia-reperfusion	74.31	367.2	11.37	383.7	214.8	5.553	162.0
	± 6.782	± 13.83	± 0.973	± 21.40	± 12.28	± 0.295	± 9.805
	(15)	(15)	(15)	(15)	(15)	(15)	(15)
P	<0.01	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
P₁	NS	<0.001	<0.001	<0.001	<0.05	<0.01	<0.001
P₂	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

Notes: Results are expressed as $mean \pm SEM$ .

In parenthesis is the number of observations.

P: Significance from sham-operated control group calculated by LSD at $P < 0.05$ .

P₁: Significance from renal ischemia-reperfusion group calculated by LSD at $P < 0.05$ .

P₂: Significance from stressed renal ischemia-reperfusion group calculated by LSD at $P < 0.05$ .

Changes in kidney tissue MDA level, catalase activity and nitrite level (Table 3):

Kidney tissue MDA showed significant elevation in the IR group as compared to the Sham Group. As compared to both IR group and the Sham Group, stressed+IR group, exhibited significant elevation in kidney tissue levels of MDA. In OO+IR group, kidney tissue levels of MDA were significantly reduced as compared to IR group. Also, in OO+stressed+IR group, kidney tissue MDA was significantly reduced when compared to both stressed+IR and IR groups. However, kidney tissue MDA was significantly elevated in both OO+IR and OO+stressed+IR groups in comparison to the Sham Group.

Regarding kidney tissue catalase activity, it was significantly decreased in IR group as compared to the Sham Group. In stressed+IR group, kidney tissue catalase activity showed significant rise compared to IR group but decreased when compared to Sham Group. In OO + IR group, kidney tissue catalase activity showed significant increase when compared to the IR group, however, was significantly decreased when compared to Sham Group. Also, in OO+stressed+IR group, kidney tissue catalase activity was significantly elevated as compared to both stressed+IR and IR groups, whereas it was significantly decreased compared to the Sham Group.

In comparison to the Sham Group, the IR group showed significant elevation in kidney tissue levels of nitrite, Also, stressed+IR group exhibited significant increase in the kidney tissue nitrite level, compared to IR and Sham Groups. In OO+IR group, the kidney tissue levels of nitrite were significantly decreased as compared to the IR group, however, it was elevated significantly, compared to the Sham Group. Also, in OO+stressed+IR group, kidney nitrite was significantly decreased when compared to both stressed+IR and IR groups, but exhibited significant elevation when compared to the Sham Group.

Changes in liver tissue MDA level, catalase activity and nitrite level liver tissue MDA showed a significant rise in the IR group when compared to the Sham Group.

Liver tissue MDA showed significant increase in stressed+IR group as compared to IR group and Sham Group. However, liver tissue MDA was significantly reduced in OO+IR group as compared to the IR group, whereas, it was significantly elevated when compared to the Sham Group. Likewise, in OO+stressed+IR group, liver tissue MDA was significantly decreased, compared to stressed+IR group, on the other hand, it was significantly increased when compared to both IR and Sham Groups (Table 3).

Compared to the Sham Group, liver tissue catalase activity exhibited significant reduction in the IR group. Liver tissue catalase activity showed a decrease, that was insignificant in IR group and significant in the Sham Group as compared to the stressed+IR group. In OO+IR group, liver tissue catalase activity was significantly increased when compared to the IR group, however, the catalase activity was decreased significantly when compared to Sham Group. Also, in OO+stressed +IR group, liver tissue catalase activity was significantly elevated as compared to the stressed+IR group and IR group, on the other hand, when compared to Sham Group, significant reduction was observed.

As regards liver tissue nitrite level, a significant increase was observed in its level in the IR group, when compared to the Sham Group. As compared to IR group and Sham Group, stressed+IR group revealed significant rise in liver tissue levels of nitrite. In OO+IR group, liver tissue nitrite level showed insignificant reduction as compared to the IR group, but insignificant elevation when compared to the Sham Group. In OO+stressed+IR group, liver tissue nitrite level was significantly reduced in comparison to stressed+IR group, but was significantly increased when compared to the Sham Group and the IR group.

Correlation studies: (Tables 4 and 5)

Table 4. Correlation studies between plasma creatinine, liver tissue levels of malondialdehyde (MDA), catalase and nitrite versus serum levels of alanine transferase (ALT), aspartate transferase (AST) and alkaline phosphatase (ALP) and plasma levels of albumin (ALB) and total bilirubin (TB) in the studied groups.
	ALT	AST	ALP	Alb	TB	Liver MDA	Liver Catalase	Liver Nitrite
Creatinine (n)	77	77	77	72	71	77	77	77
P	<0.001	<0.001	NS	<0.001	<0.001	<0.001	<0.001	<0.001
R	0.743	0.730	0.187	−0.558	0.525	0.767	−0.742	0.646
Liver MDA (n)	77	77	77	72	71
P	<0.001	<0.001	NS	<0.001	<0.001
R	0.720	0.772	0.102	−0.480	0.447
Liver catalase (n)	77	77	77	72	71
P	<0.001	<0.001	NS	<0.001	<0.01
R	−0.662	−0.697	−0.077	0.608	−0.394
Liver Nitrite (n)	77	77	77	72	71
P	<0.001	<0.001	NS	<0.01	<0.001
R	0.695	0.702	0.158	−0.363	0.416
TNF-alpha (n)	75	75	75	70	70	75		75
P	<0.001	<0.001	NS	<0.001	<0.01	<0.001		<0.001
R	0.598	0.716	0.225	−0.412	0.355	0.811		0.786

Note: Pearson correlation at $P < 0.05$ .

Table 5. correlation studies between plasma levels of creatinine, TNF-alpha and kidney tissue nitrite level versus kidney tissue nitrite versus kidney tissue levels of MDA, Catalase and nitrite in the studied groups.
	Kidney MDA	Kidney Catalase	Kidney Nitrite	Creatinine
Creatinine (n)	74	74	74
P	<0.001	<0.001	<0.001
R	0.856	−0.603	0.874
TNF-alpha (n)	72		72	75
P	<0.001		<0.001	<0.001
R	0.713		0.771	0.673
Kidney nitrite (n)	74
P	<0.001
R	0.920

Note: Pearson correlation at $P < 0.05$ .

(I) Correlations between plasma level of creatinine and other parameters (Tables 4 and 5)

Significant positive correlations were observed between plasma creatinine and each of the serum levels of ALT, AST and plasma level of total bilirubin, however, significant negative correlation was observed with ALB plasma level. Also, significant positive correlations existed between plasma-level creatinine and both liver tissue MDA and nitrite levels, however, significant negative correlation was observed with the liver tissue catalase activity (Table 4).

In addition, significant positive correlations were noticed between plasma creatinine level and kidney tissue levels of both MDA and nitrite, however, a significant negative correlation was observed with kidney tissue catalase activity (Table 5).

(II) Correlations between liver tissue parameters and liver function tests (Table 4)

Liver tissue MDA level showed significant positive correlations with each of serum ALT and AST levels and plasma total bilirubin level, but insignificant positive correlation with serum ALP level. However, liver tissue MDA level showed a significant negative correlation with plasma level of ALB.

Moreover, liver tissue catalase activity demonstrated significant negative correlations with each of serum levels of ALT and AST, and plasma level of total bilirubin, but insignificant negative correlation with serum level of ALP). However, a significant positive correlation was observed between liver tissue catalase activity and plasma ALB level.

Liver tissue nitrite level showed significant positive correlations with each of serum ALT and AST levels, and plasma total bilirubin level, but insignificant positive correlation with serum ALP level. However, liver tissue nitrite level showed a significant negative correlation with plasma ALB level.

(III) Correlations between plasma level of TNF- $α$ and other parameters (Tables 4 and 5)

Significant positive correlations were observed between plasma TNF- $α$ level and each of serum of ALT and AST levels, and plasma total bilirubin and creatinine levels, however, insignificant positive correlation was demonstrated with serum ALP level. However, TNF- $α$ showed significant negative correlation with the plasma level of ALB. Also, significant positive correlations were observed between plasma TNF- $α$ level and each of liver tissue nitrite and MDA levels (Table 4), and kidney tissue MDA and nitrite levels (Table 5).

(IV) Correlation between kidney tissue nitrite level and kidney tissue MDA level

Kidney tissue nitrite level showed a significant positive correlation with kidney tissue MDA level (Table 5).

Histopathological study:

Histopathological study of kidney:

Figure 1(a) shows that the glomerulus is intact (g) with thin-walled Bowman’s capsule (→) and absence of tuft retraction (★) (Glomerular score 0).

Figure 1(b) shows intact brush border of the tubular cells (t) with no basal membrane thickening (→), with no inflammation or cast formation (Tubular score 0). There is absence of visible interstitial (i) damage in the form of necrosis or inflammation (→) (Tubulo-Interstitial score 0).

Figure 1(c) shows uniform endothelium (e), with no endothelial cells swelling or disruption (→) (Endothelial score 0).

In Fig. 2(a), the glomerulus (g) shows the thickening of the Bowman’s capsule (★) together with tuft retraction of the glomerulus (→) (Glomerular score 2).

Figure 2(b) shows the tubular cells ( $t)$ with thickening of the basal membrane with loss of the brush border (★) in more than 25% of the tubular cells together with cast formation (c) (Tubular score 3), in addition to inflammation and hemorrhage (→) within the interstitial space, which is present in less than 25% of the tissues with no evidence of necrosis (Tubulo-Interstitial score 1).

Figure 2(c) shows endothelial cell (e) swelling (→) (Endothelial score 1).

Figure 3(a) shows the thickening of the Bowman’s capsule (★) of the glomerulus( $g)$ with glomerular tuft retraction (→) (Glomerular score 2).

In Fig. 3(b), the tubular cells (t) have thickening of the basal membrane together with loss of the brush border (★) in more than 25% of the tubular cells with cast formation ( $c)$ together with necrosis () in more than 60% of the cells (Tubular score 4). Additionally, hemorrhage (→) was seen within the interstitial (i) compartment together with necrosis () in up to 60% of the cells (Tubulo-Interstitial score 3). Figure 3(C) shows disruption (→) of the endothelial cell ( $e)$ (Endothelial score 2).

Figure 4(a) shows the glomerulus ( $g)$ thickening of the Bowman’s capsule (★) (Glomerular score 1).

In Fig. 4(b), the tubular cells (t) show that the basal membrane is intact with no cast formation or necrosis, whereas there is a loss of brush border (★) in less than 25% of tubular cells (Tubular score 1) with inflammation (→) in less than 25% of the interstitial tissue (i) with no hemorrhage or necrosis (Tubulo-Interstitial score 1).

Figure 4(c) shows that the endothelial cells (e) are intact (→) (Endothelial score 0).

In Fig. 5(a), the glomerulus (g) shows thickening of the Bowman’s capsule (★) with tuft retraction (→) (Glomerular score 2).

In Fig. 5(b), the tubular cells (t) show loss of brush border in more than 25% of tubular cells with thickening of the basal membrane (★) (Tubular score 2), together with inflammation (→) in less than 25% of the interstitial tissue ( $i)$ with no hemorrhage or necrosis (Tubulo-Interstitial score 1).

Figure 5(c) shows swelling (→) of the endothelial cells (e) (Endothelial score 1).

Histopathological study of liver:

Figure 6(a) shows the liver tissue sections of the sham-operated control group with a normal appearance of radiating cords of hepatocytes from the central vein (cv) with no evidence of injury as nuclear pyknosis, cytoplasmic vacuolation or neutrophil infiltration (Grade 0).

Figure 6(b) shows the liver tissue of renal ischemia-reperfusion group with moderate nuclear pyknosis (p), mild neutrophil infiltration (n) and cytoplasmic hypereosinophilia (e) (Grade 2).

Figure 6(c) shows the liver tissue of stressed renal ischemia-reperfusion group with severe PNL infiltration (n), moderate nuclear pyknosis (p) and hemorrhage (h) (Grade 3).

Figure 6(d) shows the liver tissue of OO-supplemented renal ischemia-reperfusion group showing mild nuclear pyknosis (p) and cytoplasmic vacuolation (v) (Grade 1).

Figure 6(e) shows the liver tissue of OO-supplemented stressed renal ischemia-reperfusion group showing cytoplasmic vacuolation (v) and mild nuclear pyknosis (p) (Grade 1).

Changes in kidney histopathological scores (Tables 6 and 8)

**Table 6. Kidney histopathological scores in the different studied groups**
	Glomerular				Tubular					Tubulo/Interstitial					Endothelial
	Score
Group	0	1	2	3	0	1	2	3	4	0	1	2	3	4	0	1	2	3
Sham- operated control (10)	10	–	–	–	10	–	–	–	–	10	–	–	–	–	10	–	–	–
Renal ischemia- reperfusion (10)	–	2	8	–	–	–	3	7	–	–	9	1	–	–	–	8	2	–
Stressed renal ischemia- reperfusion (10)	0	1	9	–	–	–	–	2	8	–	–	2	8	–	–	2	3	5
Olive oil-supplemented renal ischemia- reperfusion (10)	–	10	–	–	1	7	2	–	–	3	7	–	–	–	8	2	–	–
Olive oil-supplemented stressed renal ischemia-reperfusion (10)	–	4	6	–	–	2	4	4	–	–	7	3	–	–	–	9	1	–

Notes:Glomerular score: 0: No damage. 1: Thickening of Bowman’s capsule. 2: Retraction of glomerular tuft. 3: Glomerular fibrosis.

Tubular score: 0: No damage. 1: Loss of brush border (BB) in less than 25% of tubular cells. Integrity of basal membrane. 2: Loss of BB in more than 25% of tubular cells, thickened basal membrane. 3: (Plus) Inflammation, cast formation, necrosis up to 60% of tubular cells. 4: (Plus) Necrosis in more than 60% of tubular cells.

Tubulo/Interstitial score: 0: No damage. 1: Inflammation, hemorrhage in less than 25% of tissue. 2: (Plus) Necrosis in less than 25% of tissue. 3: Necrosis up to 60%. 4: Necrosis more than 60%.

Endothelial score: 0: No damage. 1: Endothelial swelling. 2: Endothelial disruption. 3: Endothelial loss.

Renal glomerular score showed a significant rise in both IR and stressed+IR groups when compared to the Sham Group, however, on comparing both stressed+IR group and IR group statistically insignificant changes were observed.

Upon OO supplementation, the glomerular score showed a significant reduction in OO+IR group when compared to the IR group, on the other hand, it was significantly increased when compared to the Sham Group.

Also, the glomerular score in OO+stressed+IR group showed a significant reduction as compared to the stressed+IR group, whereas, it was significantly elevated when compared to the Sham Group, and showed statistically insignificant changes when compared to the IR group.

The renal tubular score was significantly increased in both IR and stressed+IR groups as compared to the Sham Group. Also, it was significantly increased in stressed+IR group when compared to the IR group.

The renal tubular score was significantly decreased in OO+IR group when compared to the IR group, on the other hand, it was significantly elevated when compared to the Sham Group.

In addition, the tubular score showed significant reduction in OO+stressed+IR group as compared to both stressed+IR and IR groups, whereas it showed a significant rise compared to the Sham Group.

Concerning the renal tubulo/interstitial score, both IR and stressed+IR groups showed significant elevation in the renal tubulo/interstitial score when compared to the Sham Group, also, the score was significantly elevated in the stressed+IR group in comparison to the IR group.

Upon OO supplementation in OO+IR group, the renal tubulo/interstitial score demonstrated a significant reduction ( $P < 0.05$ ) as compared to the IR group, but exhibited a significant elevation ( $P < 0.001$ ) when compared to the Sham Group.

Additionally, the renal tubulo/interstitial score in the OO+stressed+IR group was significantly reduced when compared to the stressed+IR group, and was significantly increased when compared to the Sham Group, and exhibited insignificant changes as compared to the IR group.

As compared to the Sham Group, the renal endothelial score was significantly elevated in both the IR and the stressed+IR groups, also, as compared to the IR group, the renal endothelial score was significantly increased in the stressed+IR group.

The endothelial score was significantly decreased in the OO+IR group when compared to the IR group, however, in comparison to the Sham Group, statistically insignificant changes were observed.

Also, in the OO+stressed+IR group, the endothelial score exhibited a significant decline as compared to the stressed+IR group, however, it was elevated significantly when compared to the Sham Group. On comparing both IR and OO+stressed+IR groups, non-significant changes were observed.

Changes in liver histopathological score (Tables 7 and 8)

**Table 7. Liver histopathological scores in the different studied groups**
Group	Grade 0	Grade 1	Grade 2	Grade 3
Sham-operated control	10
Renal ischemia-reperfusion (10)		3	7
Stressed renal ischemia-reperfusion (10)		1	3	6
Olive oil-supplemented renal ischemia-reperfusion (10)		10
Olive oil-supplemented stressed renal ischemia-reperfusion (10)		7	3

Notes:Grade 0: Minimal or no evidence of injury.

Grade 1: Mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis.

Grade 2: Moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, loss of intercellular borders and mild to moderate neutrophil infiltration.

Grade 3: Severe injury with disintegration of hepatic cords, hemorrhage and severe polymorphonuclear leucocyte infiltration.

**Table 8. Cumulative values of kidney and liver histopathological scores in the different studied groups**
	Kidney Scores
Group	Glomerular	Tubular	Tubulo/Interstitial	Endothelial	Liver Scores
Sham-operated control	0.00	0.00	0.00	0.00	0.00
	± 0.00	± 0.00	± 0.00	± 0.00	± 0.00
	(10)	(10)	(10)	(10)	(10)
Renal ischemia-Reperfusion	1.80	2.70	1.10	1.20	1.70
	± 0.13	± 0.15	± 0.10	± 0.13	± 0.15
	(10)	(10)	(10)	(10)	(10)
P	<0.001	<0.001	<0.001	<0.001	<0.001
Stressed renal ischemia-Reperfusion	1.90	3.80	2.80	2.30	2.50
	± 0.10	± 0.13	± 0.13	± 0.26	± 0.22
	(10)	(10)	(10)	(10)	(10)
P	<0.001	<0.001	<0.001	<0.001	<0.001
P₁	NS	<0.001	<0.001	<0.001	<0.001
Olive oil-supplemented renal ischemia-reperfusion	1.00	1.10	0.70	0.20	1.00
	± 0.00	± 0.18	± 0.15	± 0.13	± 0.00
	(10)	(10)	(10)	(10)	(10)
P	<0.001	<0.001	<0.001	NS	<0.001
P₁	<0.001	<0.001	<0.05	<0.001	<0.01
Olive oil-supplemented stressed renal ischemia-reperfusion	1.60	2.20	1.30	1.10	1.30
	± 0.16	± 0.25	± 0.15	± 0.10	± 0.15
	(10)	(10)	(10)	(10)	(10)
P	<0.001	<0.001	<0.001	<0.001	<0.001
P₁	NS	<0.05	NS	NS	<0.05
P₂	<0.05	<0.001	<0.001	<0.001	<0.001

Notes: Results are expressed as $mean \pm SEM$ .

In parenthesis is the number of observations.

P: Significance from sham-operated control group calculated by LSD at $P < 0.05$ .

P₁: Significance from renal ischemia-reperfusion group calculated by LSD at $P < 0.05$ .

P₂: Significance from stressed renal ischemia-reperfusion group calculated by LSD at $P < 0.05$ .

NS: Non significant.

The liver grade showed a significant rise in the IR group, as compared to the Sham Group. Also, it was significantly elevated in the stressed+IR group when compared to the IR group and the Sham Group.

In OO+IR group, the liver grade showed a significant reduction when compared to the IR group, however, as compared to the Sham Group it showed a significant elevation.

Also, the liver grade in OO+stressed+IR group showed a significant reduction when compared to both IR and stressed+IR groups, and significant elevation when compared to the Sham Group.

Discussion

This study was planned to portray the effects of chronic immobilization stress on the changes in the kidney and liver tissues in a rat model of renal IR injury and explore the effect of OO supplementation.

Our results revealed that renal IR produced hepatic dysfunction and injury. The hepatic dysfunction induced by renal IR involved both impaired synthetic functions, as evidenced by low plasma ALB level, and impaired excretory function, as shown by the high total bilirubin level. Meanwhile, hepatocellular injury or damage was denoted by the high serum levels of liver functional indices including both ALT and AST, which are considered enzymatic markers of hepatic injury. Liver injury was, also, reflected by the hepatic histopathological data in the form of pyknotic nuclei (indicating the existence of hepatocytes necrosis), neutrophil infiltration and cytoplasmic hypereosinophilia (Fig. 6) as well as by the significantly increased hepatic histopathological score when compared to the Sham Group (Tables 7 and 8).

The hepatic results observed herein are in accordance with previous studies which showed that AKI triggered by IR leads to dysfunction and injury in mice,⁴³ rats⁴⁴ and pigs² livers.

Kidney dysfunction itself is one of the proposed mechanisms for liver affection. This explanation could be supported in this study by the presence of significant positive correlations between the plasma level of creatinine and each of serum ALT, AST, plasma total bilirubin and liver MDA and by significant negative correlations between the plasma level of creatinine and each of plasma ALB and liver catalase activity.

The renal IR group exhibited oxidant/antioxidant imbalance in liver tissue following the renal IR injury as evidenced by elevated levels of hepatic MDA together with declined hepatic catalase activity, an endogenous free radical scavenger with protective effect in the liver. These results are consistent with earlier reports.^45,46 Such imbalance could give plausible proof for the observed disturbances in hepatic functions.

This explanation is favored by the correlations that existed between liver function tests and liver MDA and catalase. Hepatic MDA showed significant positive correlations with each of serum levels of liver enzymes; ALT and AST, and the total bilirubin, as well as the presence of significant negative correlations between the liver enzymes and ALB. However, the reverse occurred with hepatic catalase. Catalase exhibited significant negative correlations with liver enzymes and total bilirubin but significant positive correlations with ALB.

This view is congruent with earlier studies showing that renal IR promotes oxidative stress, peroxidation of lipids together with the reduction of antioxidant capacity which results in hepatic structural and functional derangements⁴⁷ and hepatocyte death (necrosis and apoptosis).⁴³

Furthermore, the enhanced oxidative stress observed in the liver herein could have a role in the triggering and maintenance of hepatic inflammatory response. Neutrophil infiltration observed in the present hepatic histopathology study reflects the existence of an inflammatory state in the liver and hence, hepatocellular damage.

TNF- $α$ observed herein in the IR group could initiate the cellular inflammatory responses and resulted in induction of not only localized injury in the tissues but also, induced injury of remote organs, as it acts locally and at remote sites.⁴⁸ The significant positive correlation revealed in this study between liver tissue MDA and plasma TNF- $α$ supports the involvement of increased TNF- $α$ production and the activation of hepatic lipid peroxidation and subsequently hepatic tissue injury following renal IR.

In this work, renal IR group displayed significant elevation in liver tissue nitrite level as a part of the inflammatory process, which could be further supported by the presence of significant positive correlations between liver tissue nitrite and the plasma level of TNF- $α$ .

To our knowledge, studying the effect of stress on renal IR in our study is novel. The combination of chronic immobilization stress followed by renal IR in this study worsened the picture seen in renal IR group alone, as shown by the presence of the significantly increased plasma creatinine, urea and TNF- $α$ levels as well as the renal tissue levels of MDA and nitrite when compared to the IR group and the Sham Group. Catalase activity in the renal tissues in the stressed group showed significant elevation when compared to IR group but significantly declined when compared to the Sham Group.

Rats in the stressed group subjected to renal IR showed more deterioration in their kidney functions as compared to the non-stressed rats in the renal IR group as evidenced by the significantly increased creatinine and urea levels. Thus, the immobilization stress aggravated the ischemic renal impairment which was also proven by the greater degree of glomerular injury together with the acute tubular necrosis as the tubular and the tubulointerstitial necrosis exceeded more than 60% of the cells as well as endothelial cells disruption. Herein, the normal histological pattern of kidneys of the Sham Group was demonstrated in Fig. 1, while the histopathological deterioration of kidneys of the IR group as well as the stressed rats subjected to IR were exhibited in Figs. 2 and 3, respectively. The histological distortion was more profound in stressed rats (Fig. 3).

Oxidative stress denoted by high level of renal MDA could contribute to the renal dysfunction in rats exposed to chronic immobilization stress where urea and creatinine were elevated in their plasma. Consistently, stress was reported to cause oxidant/antioxidant imbalance.^49,50 Chronic immobilization was also found to increase reactive oxygen species (ROS) and to induce lipid peroxidation with subsequent tissue damage.⁵¹

Moreover, the catalase activity in renal tissue exhibited a rise in the renal IR group after exposure to stress. Upon exposure to stress, the observed high catalase could be an attempt to counteract the overproduction of oxidants but this function cannot be achieved as the renal MDA remained high upon exposure to immobilization stress and even surpassed the levels obtained in non-stressed rats in the renal IR group.

Also, the deleterious effects of chronic immobilization stress such as mitochondrial distortion, abnormal pathways of energy and enhanced apoptotic passages^52,53 could further explain the aggravated necrosis in the renal tubular and tubulointerstitial tissues induced by stress.

In this study, the elevation of TNF- $α$ in plasma of rats exposed to both chronic immobilization and renal IR reached double the value obtained in the unstressed rats in IR group, which could be attributed to the stress-induced activation of the sympathetic nervous system-adrenal medulla and the HPA axis, which in turn stimulates the secretion of catecholamines from adrenal medulla and glucocorticoids from the adrenal cortex, which have the ability to modulate both immune cells and cytokine production.^54,55

Moreover, exposure to chronic stress could produce a systemic inflammatory state that includes elevated concentrations of inflammatory mediators such as TNF- $α$ and IL-6, in the circulation and the liver as well,⁵⁶ which are potential sources of ROS.⁵⁷

In addition, rats exposed to chronic immobilization stress, in this study, demonstrated a significant rise in the renal tissue level of nitrite when compared to the unstressed rats in the renal IR group, despite the disruption of endothelial cells which was proved by renal histopathological examination together with the presence of significant elevation of endothelial injury score. Thus, from these results, endothelial dysfunction could be suspected and the high renal tissue level of nitrite could be of inducible origin, produced by iNOS and not eNOS. It has been reported that the oxidative stress could produce endothelial dysfunction through impairment of endothelium- dependent relaxation, induction of intracellular calcium overload and fragmentation of DNA.⁵⁸

The significant positive correlations of both parameters (TNF- $α$ and nitrite) with creatinine could reflect the possibility of the TNF- $α$ and nitric oxide (NO) involvement in the aggravation of renal function impairment associated to the chronic immobilization stress.

Exposure to chronic immobilization stress that is followed by renal IR, also, caused worsening of the injurious effect of renal IR on the liver, as a distant organ. It seems that immobilization enhanced the sensitivity of the liver rendering it more prone to injury and dysfunction on exposure to renal IR.

This was shown from the levels of liver enzymes (AST and ALT) that exhibited a greater rise in serum of rats exposed to the dual insult of stress and renal IR. This exaggerated hepatic functional impairment could be explained by stress-induced damage in liver tissue⁵⁹ and manifested by cellular degeneration including nuclear pyknosis (an indicator of necrotic hepatocytes), severe PNL infiltration (suggestive of increased inflammatory state) and hemorrhage together (Fig. 6) with significantly increased liver histopathological score compared to non-stressed renal IR.

The observed dramatic increase in liver tissue MDA reaching about 2.5-fold in stressed IR group compared to IR group together with decreased catalase to almost half its value in non-stressed group indicates severe oxidative stress in which excess generation of free radicals overwhelms the antioxidant capacity. This may lead to lipid peroxidation, protein oxidation and damage of DNA, resulting in cell death.^60,61

Furthermore, restraint stress was found to upregulate hepatic expression of TNF- $α$ , IL-1, IL-6 and interferon-gamma.⁶² The presence of significant positive correlations between the plasma level of TNF- $α$ and the liver tissue MDA herein could support this view.

The increased liver tissue MDA in the stressed IR rats could be attributed to the glucocorticoids which are generated during the stress response as a result of activation of HPA. Glucocorticoids accelerate the catabolic, inhibit the anabolic processes and cause elevation of the metabolic rate besides generating ROS. Interestingly, the liver is the primary peripheral target tissue for the action of glucocorticoids.⁶³

Meanwhile, the lowered activity of liver tissue catalase in the stressed IR group when compared to the Sham Group denotes suppressed antioxidant defense enzyme, and thus, its role in scavenging oxy-radicals and their products is impaired.

In addition, the liver tissue of stressed renal IR rats displayed severe leucocyte infiltration. The severe hepatic inflammation as indicated by severe PNL cells infiltration concomitant with the high hepatic MDA in the stressed renal IR group compared to the non-stressed IR group provides additional explanation for the amplified MDA hepatic content as PNL leucocytes were found to be a potential source of ROS and play a major role in the development of ROS- induced tissue injury.⁶⁴

Moreover, in this study, the exaggerated significant rise in hepatic nitrite content in the stressed IR group approaches about five-fold compared to the non-stressed ischemic group, simultaneous with the severe PNL infiltration indicated the production of large amounts of NO that can be toxic, prooxidant and pro-inflammatory and could be implicated in the progression of liver cell damage.

In rats subjected to immobilization stress together with renal IR injury, markers of inflammatory and/or oxidative stresses induced liver cell injury as evidenced by the increase in the levels of transaminases (AST and ALT). AST and ALT transfer aspartate and alanine, respectively, to ketoglutaric acid during the metabolism of amino acids, and they were released from the hepatocytes into the blood in cases of liver cell injury, leading to their elevation on blood tests.⁶⁵ ALT is primarily present in the liver and is thus more sensitive to the hepatocellular injury.⁶⁶

It was noted also in this work that further elevation of ALP or total bilirubin was not observed in the stressed IR group when compared to the unstressed renal IR group because the dysfunction was of the hepatocellular rather than the cholestatic pattern while ALP and total bilirubin usually elevate in cholestasis, and hepatobiliary injury.⁶⁶ Plasma level of ALB was also not significantly different in the stressed and non-stressed IR groups as it may need more time to exhibit significant change between both groups.

However, the ALP, total bilirubin and plasma ALB levels tended to be further deteriorated in stressed rats when compared to the renal IR group but did not reach significant levels.

OO supplementation resulted in attenuation of the deleterious effect provoked by renal IR alone or combined stress and renal IR on kidney and liver function and morphology. Amelioration of the renal dysfunction was evidenced by a significant decline in the plasma levels of both creatinine and urea, the indicators of renal function in the stressed renal IR group and renal IR group. These results were accompanied by histological findings (Figs. 4 and 5) and a significant decrease in renal histopathological score manifested by mitigation of tubular and tubulointerstitium necrosis and inflammation and reduction of glomerular and endothelial cell damages.

The beneficial effect of OO administration against liver dysfunction that occurred after renal IR as well as after combined stress and renal IR was reflected by the significant decrease in ALT and AST levels and the significant rise in plasma ALB. Also, the plasma level of total bilirubin was decreased to reach the control level. These findings indicate that both synthetic and excretory functions of the liver were improved by OO intake. Also, OO supplementation alleviated liver structural injury, as denoted by the lower hepatic pathological score in OO-supplemented groups when compared to their respective non-supplemented groups, as well as the significant amelioration in nuclear pyknosis, cytoplasmic degeneration and inflammatory cell infiltration with disappearance of hemorrhage.

OO supplementation to renal IR and stressed renal IR groups revealed suppression of MDA content and elevation of the renal and hepatic catalase activity, which reflects OO efficacy in reducing oxidative stress state caused by renal ischemic insult and by the combined effect of stress and renal ischemic insult locally in the kidney as well as in the remote organ, the liver.

This antioxidant effect of OO is achieved through free radical scavenging and reducing lipid peroxidation together with enhancing antioxidant capacity in both kidney and liver. Thus, OO helped in the restoration of oxidant and antioxidant balance, an effect which could provide an explanation for improvement of renal and liver functional and structural derangements that resulted from the ischemic renal injury alone or when combined with stress.

The aforementioned explanation agrees with the study of Jafaripour and his coworkers,⁶⁷ which attributed the lowered creatinine and urea levels in rats subjected to renal IR to the natural antioxidant effects of olive leaf extract that decreased IR-induced renal injuries.

The beneficial effects of OO could be attributed to the fact that OO possess polyphenolic compounds together with high concentration of monounsaturated fatty acids.¹³ The phenols present in OO act as potent antioxidant, free radicals scavengers and inhibitors of low density lipoprotein (LDL) oxidation.⁶⁸

Hydroxytyrosol was reported to be the principal antioxidant compound in OO and responsible for the beneficial properties of OO by the reduction of oxidative stress.⁶⁹ Extra virgin OO phenols, especially hydroxytyrosols, counteract ROS by acting through two main mechanisms: the first is by direct stabilization of radical molecules through electron removal and second is by activation of intracellular mechanisms promoting the elevation of antioxidant levels that are normally present in the cells.⁷⁰

OO, also, contains numerous antioxidants such as vitamin E, oleocanthal, carotenoids and oleuropein which inhibit LDL oxidation.⁷¹

In this study, plasma levels of TNF- $α$ was significantly reduced in both OO+IR and OO+stressed+IR groups, which reflects the efficiency of OO in limiting the inflammatory reactions induced by renal IR as well as by stress. Also, the anti-inflammatory effects of OO were apparent in the histopathology of kidney and liver in these groups (Figs. 5 and 6) which revealed a diminution of inflammation within renal tubulo-interstitium and the absence of PNL infiltration in the liver.

Such anti-inflammatory effect of OO could provide additional explanation for the beneficial role exerted by OO against the disruption of renal and hepatic structure and function induced by renal IR and, combined stress and renal IR. In this regard, the polyphenolic compounds and hydroxytyrosol contained in OO contributed to the beneficial effects of OO as antioxidant and anti-inflammatory agent.^24,72

Upon OO administration to the stressed renal IR group, nitrite content of both kidney and liver was significantly reduced compared to the untreated group. However, in OO supplemented renal IR group, nitrite content in kidney was significantly reduced but in the liver it was insignificantly reduced. The reduction in nitrite indicates a decreased generation of NO by OO intake which could be attributed to OO-mediated suppression of the oxidative state and inflammatory responses which are considered sources for the increased NO derived from iNOS in case of renal ischemic insult as well as in stress state.

The attenuation of NO, a free radical that may act as an inflammatory mediator by OO administration, could be implicated in the protective role of OO in the function and structure of both kidney and liver.

OO ameliorated renal function tests as plasma levels of both creatinine and urea were considerably reduced in the OO supplemented groups as compared to the renal IR group as well as the stressed renal IR group, though they did not approach the sham control levels. On the other side, the ameliorative effects of OO supplementation were less apparent regarding the hepatobiliary patterns of functions (ALP and total bilirubin) but were prominent regarding the hepatocellular patterns. The levels of AST and ALT showed a significant reduction in the OO supplemented renal IR group when compared to the renal IR group and in the OO supplemented stressed rats compared to their matching stressed IR group either as a direct potential or owing to the anti-inflammatory as well as the antioxidant effects of OO.

Conclusion

Renal IR, not only induced impairment of the renal functional and structural integrity, but also of the remote organ, the liver.

Chronic immobilization stress hypersensitized both the kidney and the liver rendering them susceptible to renal IR injury, and hence inflicting renal and hepatic functional and structural deterioration which amplified the detrimental effect of renal IR. Thus, the oxidative stress, the activated inflammatory response together with the enhancement of NO could participate in the aggravation of renal IR by the stress.

OO was efficient against the detrimental effects of renal IR injury, improving renal and hepatic dysfunction and morphological damage mediated by the renal ischemic insult in normal rats and in the face of chronic stress. Such ameliorating effect can be exerted via its antioxidant, anti- inflammatory and NO reducing activities.

Although, OO was proved as an effective nutritional tool, promoting reno-and hepato- protection against renal IR, in both normal and stressful situations, a full optimal state was not achieved. Therefore, further studies are strongly recommended concerning the use of higher doses or longer durations of OO administration as trials aiming to restore complete normalization.

Declarations

The ethical approval and the consent to participate:

The experimental protocols gained approval of the ethical approval committee, Ain Shams University, Cairo, Egypt. The ethical approval was organized and operated according to the guidelines of the ICH, the Anesthesiology and the Islamic Organization for Medical Science (IOMS), the United States Office for Human Research Protections and, the United States Code of Federal Regulations, operating under the Federal Wide Assurance No. FWA 000017585.
All the methods were performed in accordance with the relevant guidelines and regulations.
All the methods were reported in accordance with the ARRIVE guidelines.

Consent of Publication

Not applicable.

Conflict of Interest

The authors declare that the whole research was conducted without any commercial or financial relationships, which could be construed as a potential conflict of interest. On behalf of all authors, the corresponding author states that there is no conflict of interest.

Availability of Data and Materials

The data sets that are used and/or analyzed during this study are available from the corresponding author upon reasonable request.

Funding

Not applicable.

Author Contributions

MHE and MAA conceived and designed research and drafted and revised the paper. NSS and NNL performed the experiments and analyzed the data and shared in writing the paper. WB performed the histopathologic study and prepared the figures. All authors read and approved the final paper.

Acknowledgment

Not applicable.

ORCID

Noha Sobhy https://orcid.org/0000-0003-0864-345X

Mona A. Ahmed https://orcid.org/0000-0003-0864-345X

Noha N. Lasheen https://orcid.org/0000-0002-5418-2191

Walaa Baher https://orcid.org/0000-0003-2602-2083

Mohamed Hassan ElSayed https://orcid.org/0000-0002-8160-7976

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Received 20 September 2023

Accepted 7 January 2024

Published: 27 February 2024

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