INTRODUCTION

Ischemia-reperfusion (IRI) injury is a frequent clinical challenge induced by sudden temporary impairment of the blood flow to a particular organ. Acute kidney injury (AKI) induced by IRI is an unavoidable consequence in renal transplantation and multiple clinical settings. The pathophysiology of ischemic AKI is not fully understood. However, it is believed that, generation of vigorous oxidative stress and inflammatory mediators in response to hypoxia and re-oxygenation is the main core of tissue injury (^{Abdallah et al., 2018}).

Hypoxia and inflammation are two dependent processes inducing cell injury during ischemia. Hypoxia- inducible factor-1 (HIF-1α) is a vital transcription factor in cellular metabolic regulation in response to hypoxia (^{Choudhry & Harris, 2018}). During normoxic state, HIF- 1α undergoes hydroxylation, destabilization and degradation. Meanwhile, hypoxic conditions prevent HIF-1adestabilization, allowing its accumulation and nuclear translocation in order to induce the transcription of multiple protective genes (^{Shu et al., 2019}). Therefore HIF-1α participates in modulation of gene transcription to adapt cells for the hypoxic stressful conditions. However, HIF-1α is a double-edged sword. A body of evidence supports the presence of a direct relation between HIF-1α and the immune response activation (^{Cummins et al., 2016}). Inappropriate HIF-1α expression from renal tubulo- endothelial cells is associated with macrophage activation and nuclear factor-kB (NFkB) expression. Indicating a link between hypoxia and the induced renal tubulointerstitial inflammation (^{Li et al., 2019}).

Thymoquinone (TQ), 2-Isopropyl-5-methyl-1, 4- benzoquinone, is one of the promising medicinal plants with various therapeutic benefits including anti-oxidant and anti- inflammatory potential (^{Abuzinadah & Ahmad, 2020}; ^{Abdel-Daim et al., 2020}). The reno-protective effects of TQ have been demonstrated in various animal models of kidney diseases (^{Hammad & Lubbad, 2016}; ^{Elsherbiny et al., 2017}; ^{Al Fayi et al., 2020}). However, the regulatory role of thymoquinone on HIF-1ain renal IRI remains largely unknown.Our hypothesis was to study the mediating role of HIF-1α in the 3 hours and 24 hours post renal reperfusion rat model and to evaluate the renal protective efficacy of thymoquinone against IRI, and its modulatory role on HIF-1a.

MATERIAL AND METHOD

Animals and ethical consideration. Forty-eight adult male Wister albino rats (150-170g body weight) were enrolled in the current study. The study protocol was approved by the institutional animal care and use guidelines of Cairo University (IACUC, #:CU/III/F/22/20) according to the National Institutes of Health Guidelines. Rats were inbred and housed in normal light/dark cycles and maintained temperature at (25 ± 2 °C) with free access to water and standard rat chow.

Experimental design and study protocol. Following 2 days of acclimatization, the rats were allocated into 3 groups (n=16); 1) Sham operated group, 2) Renal ischemia reperfusion (IRI), and 3) rats exposed to renal IRI that were previously treated for 10 days with thymoquinone (TQ; Sigma-Aldrich, 274666) in a dose of 10 mg.kg^-1.day^-1 intravenous, dissolved in DMSO. Administration of thymoquinone started 10 days before the induction of ischemia (TQ-IRI) and injected immediately after reperfusion. Following the operation, each group was divided into two subgroups; 8 rats were euthanized after 3 hours and the remaining 8 rats at 24 hours (the protocol is demonstrated in Fig. 1).

Fig. 1 Timeline for the experimental protocol. 

Induction of bilateral renal IRI protocol. In brief, subcutaneous heparin 100U/Kg (Nile Co., Egypt) was injected to avoid renal vessel clotting during the ischemic phase. Rats then anesthetized by ketamine 50 mg/kg and xylazine hydrochloride 10mg/kg/i.p (Sigma, Egypt). Under aseptic precautions, a standard 3 cm midline laparotomy was performed. The intestines were retracted, and smooth clamps were placed carefully around both renal pedicles for 45 min during which the intestines were covered by a warm moist gauze to prevent dehydration. Afterward, the clamps were removed to allow reperfusion. The intestines were returned and the abdominal wall was sutured in layers. The sham group was subjected to the same operation procedures without renal pedicle occlusion.

Samples collection and preparation. At the indicated time either at 3rd or the 24^th hour following reperfusion and under pentobarbital (50 mg/kg i.p.) anesthesia, blood samples were drawn from the abdominal aorta. The collected blood samples were left to be clotted then centrifuged at 3000 rpm for 10 minutes for serum separation. The obtained serum samples were stored in -20 ºC till used for analysis.

RESULTS

Bilateral Renal IRI deteriorated renal functions and improved by thymoquinone. The measured blood urea nitrogen (BUN) and serum creatinine levels were increased markedly (P<0.001) following renal IRI at both 3 h and 24 h compared to the corresponding data of the sham group. The treatment with thymoquinone was accompanied by improved (P<0.001) renal functions at both 3 h and 24 h post-injury (Fig. 2).

Fig. 2 Renal functions determination through assessment of a) serum creatinine and b) blood urea nitrogen (BUN). IRI: ischemia and reperfusion injury, TQ: thymoqinone. *: refers to significance to Shame 3h, #: significant date compared to IRI-3h, $ significance to IRI-24h and @: refers to statistical significance to IRI-24h when p<0.05. 

Histological findings. EGTI scoring system of the renal cortex was assessed by light microscopic examination of the H&E sections at (x 200, x 400), demonstrated damage of renal cortex at 3h post-reperfusion (Figs. 3c,d). Loss of the brush margin of the tubular cells with focal necrosis (score 3) and glomerular tuft retraction (score 2) was detected. While tissue samples of IRI at 24h (Figs. 3i,j) showed cast formation and focal necrosis (score 3). Glomerular tuft retraction (score 2), inflammation and interstitial hemorrhage (score 1) compared to the normal cortical structure in the sham group (Figs. 3a,b,g,h). However, the signs of tissue injury were minimal by thymoquinone treatment at either duration. loss of tubular brush margin was less than 25 %, no cast formation or necrosis (Figs. 3e,f) and no damage/abnormality within the interstitial compartment (score 0) (Figs. 1 and 3k). Figure 3m demonstrates the total EGTI score.

Fig. 3 Light microscopic histological findings in the renal tissue sections stained with H&E (x 200 and x 400) magnifications. Images a & b as well as g & h demonstrate the normal cortical structure in the sham operated rats. Tissue injury manifestations are defined in the ischemia reperfusion injury groups (IRI) either at 3 h or 24h. Thymoquinone (TQ) treatment mitigated renal tissue injury and decreased the histological injury findings. The black arrow: pointed to glomerular retraction, red arrow head: intact brush margin, while the red arrow: loss of brush margin. The black circle demonstrates interstitial inflammatory infiltrates, blue ones for interstitial hemorrhage. The black arrow heads: determine cast formation and the asterisk: shows areas of cellular necrosis. The total EGTI score represented by the median and interquartile range. *: refers to significance to Shame 3 h, #: significant date compared to IRI-3h, $ significance to IRI-24 h and @: refers to statistical significance to IRI-24 h when p<0.05. 

Renal ischemia-inducedHIF-1a expression and mitigated by thymoquinone. Renal IRI markedly (P<0.001) elevated the protein level of HIF-1a that was recorded at 3 h following reperfusion and maintained elevated to the 24th hour compared to the sham group at both durations. Thymoquinone diminished the HIF-1a induction when measured either at 3 h or 24 h post-reperfusion (Fig. 4a).

Oxidative stress in the renal tissue of IRI group and suppression with thymoquinone. Disturbed renal tissue oxidation/reduction state was identified in the IRI group. The ROS content was increased following renal IRI, while the total antioxidant capacity was diminished compared to the sham 3 h and 24 h groups. Thymoquinone with its antioxidant potential fixed this disturbance, abrogated the elevated ROS levels, and restored the total antioxidant capacity (Figs. 4b,c).

Fig. 4 a) HIF-1a: the hypoxia inducible factor level in the renal tissue, b) renal tissue reactive oxygen species (ROS) determination and c) the total antioxidant capacity (TAC) content in the kidney tissues. IRI: ischemia and reperfusion injury, TQ: thymoqinone. *: refers to significance to Shame 3 h, #: significant date compared to IRI-3 h, $ significance to IRI-24 h and @: refers to statistical significance to IRI-24 h when p<0.05. 

Increased inflammatory markers by IRI and amelioration with thymoquinone. As expected, the inflammatory markers were upregulated in the renal tissue of the untreated rats. The levels of MCP-1, VCAM-1 and NFkBwere increased (P<0.001) following IRI at 3 h as well as at 24 h compared to the corresponding values of the sham group. Thymoquinone treatment for 10 days was effective in regulating pro-inflammatory markers and normalization of the VCAM-1 values were recorded at 3 h and at 24 h post-reperfusion (Fig. 5).

Fig. 5 Inflammatory markers determined in renal tissues. a) Nuclear factor (NFkB), b) monocyte chemo attractant protein (MCP-1) and c) vascular cell adhesion molecule (VCAM-1). IRI: ischemia and reperfusion injury, TQ: thymoqinone. *: refers to significance to Shame 3 h, #: significant date compared to IRI-3 h, $ significance to IRI-24 h and @: refers to statistical significance to IRI-24 h when p<0.05. 

Correlations. We determined a close correlation between HIF-1aand renal injury parameters; Creatinine (r=0.769, P<0.001), BUN (r=0.866, P<0.001), and the total EGTI score (r=0.893, P<0.001). In order to further support the link between HIF-1aand the oxidative stress, a strong positive correlation was detected between HIF- 1aandROS (r=0.881, P<0.001), whereas a negative association was determined to TAC (r= -0822, P<0.001). Furthermore, the renal HIF-1awas positively proportionate to and the NFkB level (r=0.872, P<0.001) (Fig. 6).

Fig. 6 Correlations between the hypoxia inducible factor (HIF-1) and a) serum creatinine, b) blood urea nitrogen (BUN), c) histological injury score, d) renal tissue reactive oxygen species (ROS) determination and e) the total antioxidant capacity (TAC) and f) Nuclear factor (NFkB) in the renal tissue. 

DISCUSSION

The principal findings in our study were that HIF- 1awas induced and accumulated in the renal tissues of the ischemia-reperfusion injury (IRI) group. HIF-1α was associated with the elevated serum creatinine and BUN biomarkers, as well as the total histological EGTI injury score. Thymoquinone treatment was valuable in diminishing the renal HIF-1α and the oxidation/inflammation biomarkers. Consequently, improvement of renal functions and the renal tissue histological picture at both 3 h and 24 h post-reperfusion.

Cellular stress during renal ischemia upregulates HIF- 1α. Although the precise pathophysiology of renal IRI is unclear, HIF-1α upregulation was associated with renal tubular damage (Li et al.). Several studies have emphasized the role of HIF-1α in mitochondrial dysfunction generating ROS, and induced tissue injury (^{Bellanti et al., 2016}; ^{Ma et al., 2017}). ROS generation mediates hypoxic renal tubular apoptosis and dysfunction. The results of ^{Yildiz et al. (2010}) study investigating renal ischemia for 45 min followed by reperfusion for 45 min caused significant renal function deterioration. They found an elevation of the total oxidant status (TOS), oxidative stress index (OSI), and myeloperoxidase (MPO) levels in the renal tissue and blood. These findings suggested that renal IRI enhances oxidative stress by modulating mitochondrial ROS generation and mitigating toxic cytokines production.

HIF-1α is important for myeloid cell development and differentiation, and further, itpotentiates cell-mediated inflammation (^{Cramer et al., 2003}). In the current study, we detected a strong positive correlation between renal tissue content of HIF-1α and NFkB. Interestingly, the transcription factors NFkB and HIF molecular activation is interdependent (Cummins et al.). NFkB plays a role in HIF-1α mRNA expression. During hypoxia, NFkB subunits bind to a specific site on the HIF-1α promoter, stimulating HIF-1α mRNA expression. In addition, mutation of this NFkB binding location on the HIF-1a promoter region abolished the hypoxic induction of HIF-1a (^{Belaiba et al., 2007}). Furthermore, NFkB-related neutrophil survival is dependent on the HIF-1α bioavailability (^{Walmsley et al., 2005}). Taken together, acomplex interplay between HIF-1α and NFkB is highly suggested in renal IRI pathogenesis. HIF-1α and NFkB potentiate inflammatory cascades that are responsible for this form of hypoxia-inducedrenal tubulo interstitial inflammation (Li et al.). NFkB mediated renal interstitial inflammation through stimulating MCP-1 (^{Donadelli et al., 2000}) and VCAM-1 expression (^{Pan et al., 2013}). These data support the elevated renal tissue inflammatory mediators VCAM-1 and MCP-1 reported in the current study.

Growing evidence has acknowledged the renoprotective effect of the multitargeted molecule thymoquinone, depending on its anti-oxidant and anti-inflammatory anti-apoptotic potential (Hammad & Lubbad; Elsherbiny et al.; Abuzinadah & Ahmad; Abdel-Daim et al.; Al Fayi et al.). Thymoquinone was effective in reducing glomerular degenerative changes and decreasing the tubular necrosis score in a rat model of renal IRI injury. Thymoquinone diminished cellular vacuolation, interstitial edema, hyperemia, hemorrhage and tubular necrosis (Yildiz et al.; ^{Mousavi, 2015}). However, the precise modulatory role of thymoquinone on HIF-1α activation in the setting of AKI has not been well addressed. These protective effects of TQ were previously explained, at least in part, by its antioxidant property and its ability to scavenge free radicals, protect cellular membranes against lipid peroxidation and increase the expression of tissue antioxidant genes. In line with our results, the treatment of rats with thymoquinone significantly ameliorated the renal oxidative stress indices. Thymoquinone inhibited malondialdehyde and enhanced glutathione and superoxide dismutase activities in renal tissues compared with the untreated IRI rats (^{Awad et al., 2011}). The improvement of mitochondrial function and enhancing ATP production (^{Nagi et al., 2011}) might be involved in its nephroprotective effects against oxidative tissue damage observed after acute renal IRI.

Furthermore, the anti-inflammatory potential of thymoquinone against renal injury has been characterized. Thymoquinone attenuated gene expression of a plethora of

pro-inflammatory cytokines. Thymoquinone inhibited the activity of myeloperoxidase and NFkB-p65 in the renal cortical tissues, in which histological analyses showed less inflammatory infiltrate in kidneys of treated rats (Yildiz et al.; Mousavi).

In summary, the current work provides novel insights into the molecular mechanism of thymoquinone against renal IRI. We demonstrated that thymoquinone inhibited HIF- 1a and can effectively protect against acute kidney. The protective effect of thymoquinone was assessed by improvement of blood urea, creatinine, histological EGTI score, and biomarkers of inflammation and oxidative stress.