Agathis robusta Bark Extract Protects from Renal Ischemia-Reperfusion Injury: Phytochemical, In Silico and In Vivo Studies

Background: Acute kidney injury (AKI) induced by renal ischemia-reperfusion injury (RIRI) is associated with a high incidence of mortality. Existing therapies are mainly supportive, with no available nephroprotective agent. The purpose of this study is to examine the potential protective effect of Agathis robusta Bark Extract (ARBE) in RIRI. Methods: The chemical composition of ARBE was examined by LC-ESI-MS/MS. Network pharmacology was utilized to identify the RIRI molecular targets that could be aimed at by the identified major components of ARBE. Experimentally validated protein–protein interactions (PPIs) and compound-target networks were constructed using the STRING database and Cytoscape software. Molecular docking studies were employed to assess the interaction of the most relevant ARBE compounds with the hub RIRI-related targets. Furthermore, ARBE was tested in a rat model of RIRI. Results: The phytochemical analysis identified 95 components in ARBE, 37 of which were majors. Network analysis identified 312 molecular targets of RIRI that were associated with ARBE major compounds. Of these 312, the top targets in the experimentally validated PPI network were HSP90, EGFR, and P53. The most relevant compounds based on their peak area and network degree value included narcissoside, isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside, among others. Docking studies of the most relevant compounds revealed significant interactions with the top RIRI-related targets. In the in vivo RIRI experiments, pretreatment of ARBE improved kidney function and structural changes. ARBE reduced the renal expression of p-NfkB and cleaved caspase-3 by downregulating HSP90 and P53 in rats exposed to RIRI. Conclusion: Taken together, this study revealed the chemical composition of ARBE, depicted the interrelationship of the bioactive ingredients of ARBE with the RIRI-related molecular targets, and validated a nephroprotective effect of ARBE in RIRI.


Introduction
Acute kidney injury (AKI) characterized by abrupt decline in renal function, is associated with several short and long-term complications and a high risk for mortality [1]. Renal ischemia-reperfusion injury (RIRI) is a common cause of AKI that occurs in clinical settings, e.g., kidney transplantation, heart surgery, and shock [2]. Management of this condition is mainly supportive, and survivors have markedly reduced health-related quality of life (HRQL) and continue to develop long-term adverse outcomes, including chronic kidney disease (CKD) [3]. Therefore, continuous search for novel nephroprotective agents, including natural products, in the setting of RIRI-induced AKI is essential.
Conifers constitute the main and most distinct set of living gymnosperms and include more than 600 species and 60-65 genera distributed in seven families, the Pinaceae, Cupressaceae, Podocarpaceae, Taxaceae, Cephalotaxaceae, Taxodiaceae, and Araucariaceae [4].

LC-ESI-MS/MS Profile
In the present work, the phytochemical profiling of A. robusta bark by LC-ESI-MS/ (negative and positive mode ESI) revealed the presence of 95 secondary metabolites, cluding mainly diterpenoid acids, biflavonoids, procyanidins, phenolic acids, and th derivatives, in addition to other classes, such as carboxylic acids, nucleobases, amino     To identify potential bioactive components, the 37 major compounds of ARBE were screened based on their pharmacokinetics and drug-likeness properties (Table S2). Indeed, most of these compounds showed higher bioavailability scores (OB > 0.55) and complied with Lipinski's rule of five, a rule of thumb to evaluate drug likeness. However, a few compounds did not meet the screening criteria perfectly, including the procyanidins, which are known to have multiple biological activities [63]. Therefore, we included all the major ARBE compounds in our study for a comprehensive analysis.

Molecular Targets of ARBE Major Compounds
To identify the molecular targets associated with the major constituents of ARBE, the Swiss Target Prediction database was utilized. This analysis resulted in 741 targets after the removal of duplicates (Table S3).

Molecular Targets of ARBE Associated with RIRI
To retrieve the molecular targets associated with RIRI, three disease-related databases, i.e., DisGeNeT, GeneCards, and OMIM, were accessed. The resulting targets were 1745 and were cut down to 1646 after removal of duplicates (Table S4). Among these targets, 312 (Table S5) overlapped with the 741 targets associated with ARBE major compounds ( Figure 3).

Pharmacokinetics of ARBE Major Compounds
To identify potential bioactive components, the 37 major compoun screened based on their pharmacokinetics and drug-likeness propertie deed, most of these compounds showed higher bioavailability scores (OB plied with Lipinski's rule of five, a rule of thumb to evaluate drug liken few compounds did not meet the screening criteria perfectly, including t which are known to have multiple biological activities [63]. Therefore, w major ARBE compounds in our study for a comprehensive analysis.

Molecular Targets of ARBE Major Compounds
To identify the molecular targets associated with the major constitue Swiss Target Prediction database was utilized. This analysis resulted in the removal of duplicates (Table S3).

Molecular Targets of ARBE Associated with RIRI
To retrieve the molecular targets associated with RIRI, three dise bases, i.e., DisGeNeT, GeneCards, and OMIM, were accessed. The resul 1745 and were cut down to 1646 after removal of duplicates (Table S4). A gets, 312 (Table S5) overlapped with the 741 targets associated with A pounds (Figure 3).

Protein-Protein Interaction (PPI) Network of the 312 Disease-Com
To reveal the potential molecular mechanisms of ARBE to protect work of the experimentally validated PPIs of the 312 disease-compounds was constructed in STRING database (Figure 4a, PPI). This network con connected by 598 edges. Then, we ranked the key essential targets in th on their degree value, i.e., the number of connecting edges. Indeed, a hig could pinpoint a more relevant target in the network. The top three HSP90AA1 (degree = 42), EGFR (degree = 39), and TP53 (degree = 33). T with their degree value are shortlisted in Table S6 and shown in Figure 4

Protein-Protein Interaction (PPI) Network of the 312 Disease-Compounds Targets
To reveal the potential molecular mechanisms of ARBE to protect from RIRI, a network of the experimentally validated PPIs of the 312 disease-compounds common targets was constructed in STRING database (Figure 4a, PPI). This network contained 312 nodes connected by 598 edges. Then, we ranked the key essential targets in the network based on their degree value, i.e., the number of connecting edges. Indeed, a higher degree value could pinpoint a more relevant target in the network. The top three targets included HSP90AA1 (degree = 42), EGFR (degree = 39), and TP53 (degree = 33). The top 20 targets with their degree value are shortlisted in Table S6 and shown in Figure 4b.

Top ARBE Compounds Associated with RIRI Targets
To determine the most significant compounds of ARBE associated with the 312 RIRI targets, we constructed a compound-target network in Cytoscape ( Figure 5). Then, we ranked the compounds based on degree value ( Table 2).

Top ARBE Compounds Associated with RIRI Targets
To determine the most significant compounds of ARBE associated with the 312 RIRI targets, we constructed a compound-target network in Cytoscape ( Figure 5). Then, we ranked the compounds based on degree value (Table 2).

Gene-Ontology (GO) and KEGG Pathway Enrichment Analysis of 312 Common Targets
To verify the relevant biological and functional characteristics of the 312 diseasecompound common targets, GO enrichment analysis was performed in biological processes (BP), molecular functions (MF), and cellular components (CC) based the number of targets that were enriched in those categories (Figure 6a-c). Top BP included signal transduction, inflammatory response and apoptotic process; Top MF included protein binding, enzyme binding, and protein kinase activity; Top CC included plasma membrane, cytoplasm, and nucleus. Detailed information of GO analyses is shown in Supplementary Tables S7-S9. Pharmaceuticals 2022, 15, x FOR PEER REVIEW 12 of 29 29 6-Hydroxycoumarin¬ † 44 29 3,4-Trihydroxy benzenepropanoic acid 44 29 Dehydroabietic acid 44 32 [(epi)catechin-(epi)gallocatechin] 43 32 Gallic acid 43 34 Pinusolidic acid 42 35 Hydroxypalmitic Acid 41 35 15-Hydroxypinusolidic acid 41 37 Carnosol  To recognize the possible pathways involved in the protective effects of ARBE in RIRI, KEGG pathway enrichment analysis of the 312 disease-compounds common targets was performed (p < 0.05). Top enriched pathways included lipid and atherosclerosis, HIF-1 signaling pathway, and PI3K-Akt signaling pathway (Figure 7). KEGG pathway results To recognize the possible pathways involved in the protective effects of ARBE in RIRI, KEGG pathway enrichment analysis of the 312 disease-compounds common targets was performed (p < 0.05). Top enriched pathways included lipid and atherosclerosis, HIF-1 signaling pathway, and PI3K-Akt signaling pathway (Figure 7). KEGG pathway results are presented in detail in Table S10.

Molecular Docking Study
To examine the interaction of ARBE compounds with key RIRI molecular t performed a molecular docking analysis. Ten compounds (Table 3), selected acc their peak area values and score based on degree value (Table 2), and the three t of RIRI (HSP90, EGFR, and p53) were included in the analysis.
Heat shock protein HSP 90-Alpha in complex with T5M (PDB code: 2XHX dermal growth factor receptor (EGFR) tyrosine kinase in complex with erlot code: 1M17) [65], and cellular tumor antigen P53 (PDB code: 3Q01) [66] were imp to provide insight on binding affinity of ARBE with the active pockets of the proteins.

Molecular Docking Study
To examine the interaction of ARBE compounds with key RIRI molecular targets, we performed a molecular docking analysis. Ten compounds (Table 3), selected according to their peak area values and score based on degree value (Table 2), and the three top targets of RIRI (HSP90, EGFR, and p53) were included in the analysis. Table 3. Docking details of the selected components of ARBE on HSP90A.

Component S Score Kcal/mol H-Bond Interactions
Pi-H Interactions Heat shock protein HSP 90-Alpha in complex with T5M (PDB code: 2XHX) [64], epidermal growth factor receptor (EGFR) tyrosine kinase in complex with erlotinib (PDB code: 1M17) [65], and cellular tumor antigen P53 (PDB code: 3Q01) [66] were implemented to provide insight on binding affinity of ARBE with the active pockets of the targeted proteins.

Docking with Epidermal Growth Factor Receptor (EGFR)
Docking studies reveal that 7-oxo-dehydroabietic acid, caffeic acid, narcissoside (Isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, syringetin-3-O-glucoside, 15hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4'-dimethyl ether, and ferulic acid on epidermal growth factor receptor (EGFR) tyrosine kinase (PDB code: 1M17) reached the binding site of the enzyme. In comparison to the co-crystallized ligand (AQ4: erlotinib), the docked components showed a good binding affinity, where the docking energy score ranged from −9.0112 to −5.1249 kcal/mol. The components confirmation within the active pocket stabilized by the H-bond interaction with the crucial amino acid residues. Table 4 shows the detailed amino acid residues involved in the interactions with the docked components. One of hydroxyl group of narcissoside (isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside acts as an anchor, forming an H-bond interaction with acidic ASP831. This is allowed the rest of the component structure to fill the active site properly, forming more H-bond interactions with MET742, GLN767, and MET769 in addition to a pi-H bind interaction with LEU694 ( Figure 9).

In Vivo Validation
To validate our in silico findings, we next tested whether ARBE is nephroprotective in an RIRI model in rats. RIRI in rats resulted in a decline in kidney function as indicated by increased serum creatinine and blood urea nitrogen, while pretreatment with ARBE improved kidney function (Figure 11a,b). Histopathological studies (Figure 11c) of the kidneys showed structural and pathological changes after RIRI, which were mitigated in the ARBE-pretreated animals. In Figure 11c, HE staining, sham kidneys demonstrated normal organized histological features of renal parenchyma with abundant records of apparent intact renal corpuscles (star), renal tubular segments with almost intact tubular epithelium (arrow), as well as intact vasculatures without abnormal morphological changes records; RIRI kidneys showed sever diffuse tubular epithelial loss and necrotic changes of different nephron segments (red arrow) alternated with abundant figures of degenerated pyknotic tubular epithelium with marked tubular dilatation. Significant

In Vivo Validation
To validate our in silico findings, we next tested whether ARBE is nephroprotective in an RIRI model in rats. RIRI in rats resulted in a decline in kidney function as indicated by increased serum creatinine and blood urea nitrogen, while pretreatment with ARBE improved kidney function (Figure 11a,b). Histopathological studies (Figure 11c) of the kidneys showed structural and pathological changes after RIRI, which were mitigated in the ARBE-pretreated animals. In Figure 11c, HE staining, sham kidneys demonstrated normal organized histological features of renal parenchyma with abundant records of apparent intact renal corpuscles (star), renal tubular segments with almost intact tubular epithelium (arrow), as well as intact vasculatures without abnormal morphological changes records; RIRI kidneys showed sever diffuse tubular epithelial loss and necrotic changes of different nephron segments (red arrow) alternated with abundant figures of degenerated pyknotic tubular epithelium with marked tubular dilatation. Significant congested glomerular tuft capillaries with significant dilatation of Bowman's spaces (star) with focal interstitial extravasation of blood. Marked records of intraluminal eosinophilic casts (yellow arrow) with moderate interstitial mononuclear inflammatory cells infiltrates (arrow head); kidneys of ARBE-pretreated rats showed significant protective efficacy on renal tubular epithelium with persistent moderate records of tubular epithelial degenerative changes (red arrow), alternated with relative higher records of apparent intact tubular segments (black arrow). Mild persistent records of tubular dilatations were shown as well as persistent dilatation of bowman's spaces (star). Moreover, there were minimal records of interstitial inflammatory cells infiltrate (arrow head) and intraluminal casts with intact vasculatures. In the immunohistochemical analysis, in comparison to the vehicle, ARBE pre-treatment in rats with RIRI significantly dampened renal inflammation and apoptosis as indicated by reducing p-NfKB and cleaved caspase-3 expression, respectively (Figure 11c,d). congested glomerular tuft capillaries with significant dilatation of Bowman's spaces (star) with focal interstitial extravasation of blood. Marked records of intraluminal eosinophilic casts (yellow arrow) with moderate interstitial mononuclear inflammatory cells infiltrates (arrow head); kidneys of ARBE-pretreated rats showed significant protective efficacy on renal tubular epithelium with persistent moderate records of tubular epithelial degenerative changes (red arrow), alternated with relative higher records of apparent intact tubular segments (black arrow). Mild persistent records of tubular dilatations were shown as well as persistent dilatation of bowman's spaces (star). Moreover, there were minimal records of interstitial inflammatory cells infiltrate (arrow head) and intraluminal casts with intact vasculatures. In the immunohistochemical analysis, in comparison to the vehicle, ARBE pre-treatment in rats with RIRI significantly dampened renal inflammation and apoptosis as indicated by reducing p-NfKB and cleaved caspase-3 expression, respectively ( Figure  11c,d). Next, we examined whether these nephroprotective activities of ARBE in RIRI are due to modulating the top RIRI molecular targets which were identified by network analysis and confirmed by docking. Therefore, we assessed the renal expression of two of these targets, i.e., HSP90 and P53, which have been shown to contribute to kidney dysfunction, inflammation, and cell death in previous studies [67,68]. Indeed, RIRI led to increased expression of both proteins in rat kidney, yet, pretreatment with ARBE attenuated this effect (Figure 11c,d).

Discussion
Based on phytochemical characterization followed by network pharmacology, docking, and preclinical validation, we here report a potential protective effect of ARBE against RIRI. This study revealed the most biologically significant components of ARBE and their possible molecular targets and mechanisms of actions in attenuation of RIRI-induced AKI.
The presence of such diverse chemical components in herbs makes it complex to assess the potential therapeutic action by the single component-single target paradigm [69]. Therefore, network pharmacology-based strategy could be a potential successful tool in this context. This approach utilizes a computational action plan to uncover the possible component-target-disease associations, and has been recognized as an efficient method in multiple previous studies [69][70][71][72]. Applying network pharmacology in this study led to the identification of 312 RIRI-related molecular targets that can be potentially aimed at by major components of ARBE. Of those targets, the most relevant hubs, based on experimentally validated PPIs, included molecular chaperones, i.e., HSPs, which regulate protein folding, intracellular transport, and repair or degradation [73]. Proteins involved in signaling pathways, cell survival, and cell division, e.g., EGFR and P53, were also included as significant targets [74,75].
Docking is a useful tool to predict the chemical reactivity of compounds towards molecular targets [76]. In this study, ten ARBE compounds with high peak area and degree value scores showed substantial binding with the top three RIRI targets in the experimentally validated PPI network, i.e., HSP90A, EGRF, and P53.
The molecular chaperone HSP90A is a homodimeric protein [77]. The HSP90A structure consists of three domains: N-terminal domain (N-domain), middle domain (M-domain), and C-terminal domain (C-domain). The N-domain, the catalytic domain, binds with ATP. M-domain associates both N-and C-domains, while the N-domain connects with its partner domain in the other subunit to form the dimer. The HSP90 chaperone cycle includes the turnover of ATP to ADP through the ATPase action in the N-terminal domain. The ATP binding site has been described by NMR and/or X-ray diffraction by the binding modes of a number of reported HSP90A inhibitors [78]. It was reported that resorcinol-bearing compounds are considered as lead compounds for discovering inhibitors or modulators of HSP90 [64,79]. The docked components have hydroxyl/phenolic groups exhibited interactions with the amino acids present in the adenine-binding site of HSP90A, proposing that ARBE may be effective in prevention or treatment of a diseases mediated by HSP90A.
The EGFR kinase domain (EGFR) has a characteristic bilobate-fold. The N-terminal domain is formed from mostly β-strands and one α-helix, whereas the larger C-terminal domain is formed from mostly α -helices. The two domains are separated by a cleft similar to those in which ATP, ATP analogues, and ATP inhibitors have been found to bind [65].
Docked components ARBE showed promising interactions through fitting into EGFR adenine pocket.
Cellular tumor antigen P53 (PDB code: 3Q01) was crystallized as a homodimer [66]. The resolved P53 protein structure has no co-crystallized ligand, so both Computed Atlas for Surface Topography of Proteins (CASTp) [80] and Site Finder module in MOE 2019.0102 were used to find potential 3D pockets for P53 protein. Molecular docking of the components within P53 active pocket revealed that they exhibited promising binding energies with potential activity on P53.
Computational analysis without experimental validation could be non-sufficient and meaningless. Thus, a preclinical model of RIRI was used in this study to examine the potential activity and mechanism of ARBE. This experiment revealed a nephroprotective effect of ARBE in RIRI by diminishing renal inflammation and apoptosis. These effects were due to downregulation of both HSP90 and P53 by ARBE. Indeed, previous studies showed that both HSP90 and P53 are upregulated after RIRI resulting in inflammation, oxidative stress, cell death, and structural changes. Both genetic and pharmacological inhibition of these proteins in RIRI are associated with better outcomes [67,68,81,82]. For instance, AT13387, an HSP90 inhibitor, ameliorated RIRI by abolishing Toll-like receptor 4 (TLR4)mediated NF-κB activation [67]. Furthermore, intravenous injection of synthetic siRNA to p53 after ischemic injury protected both proximal tubule cells and kidney function in rats [83]. In a clinical study (ClinicalTrials.gov identifier: NCT00802347), a single systemic administration of QPI-1002, a siRNA-based p53 inhibitor, reduced the incidence of delayed graft function in deceased donor allograft recipients by downregulating p53 following reperfusion [84]. For extraction, the dried powdered bark (280 g) was macerated with 70% ethanol (3 × 1 L). The extract was evaporated at reduced pressure to afford 80 g of viscous residue.

LC-ESI-MS/MS Analysis
The bark extract was reconditioned in (Water: Methanol: Acetonitrile, 50:25:25 v/v), and analyzed by LC-ESI-MS/MS using ExionLC (High flow LC, Sciex ®, Framingham, MA, USA), coupled with TripleTOF 5600+Time-of-Flight (IDA Acquisition, Sciex ® ) and Analyst TF 1.7.1 (LC-Triple TOF control, Sciex ® ). The injection concentration and volume were 2.5 µg/µL and 10 µL, respectively. The pre-column used consisted of In-Line filter disks (0.5 µm × 3.0 mm, Phenomenex ® , Torrance, CA, USA), while the used column was X select HSS T3 (2.5 µm, 2.1 × 150 mm, Waters ® , Milford, MA, USA) and the column temperature was set at 40 • C. The flow rate was 0.3 mL/min, and the elution was carried out using a buffer system of 1% methanol in 5 mM ammonium formate at pH 3 as solvent A for positive mode, at pH 8 as solvent B for negative mode and 100% of acetonitrile as solvent C. Gradient elution was carried out as follows: 90% solvent A or B and 10% of solvent C were injected for 20 min, then turned to 10% of solvent A or B to 90% of solvent C for the next 5 min, and finally, by the starting elution mixture was applied for the last 3 min. PeakView was employed for peaks extraction from the total ion chromatogram (TIC), on the basis of that the peaks should possess a signal-to-noise ratio greater than 5 (non-targeted analysis); in addition, the peak intensities of the sample-to-blank should be greater than 3. The interpretation of data was achieved using a ReifycsAbf (Analysis Base File) Converter for Wiff file conversion (Reifycs ® , Tokyo, Japan) and MS-DIAL 4.6 (RIKEN ® Tokyo, Japan). The compounds were tentatively identified according to their retention time, MS, and MS 2 fragmentation using PeakView TM software version 2.1, and the peak area values were calculated using the XIC Manager in this software. For each identified compound, extracted ion chromatograms (XICs) were automatically produced and compared to a user-defined threshold [85].
The molecular targets linked to the identified major compounds of AR were predicted using the SwissTargetPrediction (http://www.swisstargetprediction.ch/, accessed on 1 July 2022) database [87].

Construction of PPI and Compound-Target Networks
The common overlapping targets between ARBE major compounds and RIRI targets were identified in Microsoft Excel and represented as a Venn diagram.
Protein-protein interactions (PPI) network of the intersected targets between the AR compounds and RIRI-related targets was constructed using the STRING database Version 11.5 (https://string-db.org/, accessed on 2 July 2022) [92]. A confidence level of >0.4 in protein interactions was applied, and only experimentally validated PPI were included. A compound-target network was also constructed between the major compounds of AR and the intersected targets.
The Cytoscape 3.9.1 software program (NIGMS, USA) [93] was utilized to visualize the networks. The key essential targets and top compounds were ranked based on degree by applying the CytoHubba plugin contained in Cytoscape [94].

The GO Analysis and KEGG Pathway Enrichment
A database for Annotation, Visualization and Integrated Discovery (David database, https://david.ncifcrf.gov/tools.jsp, accessed on 3 July 2022) [95] was employed to perform GO analysis and KEGG pathway enrichment. A p-value < 0.05 was used as a cutoff and was corrected using a false discovery rate (FDR) error control technique.

Molecular Docking Study
Molecular docking studies of the following components: 7-oxo-dehydroabietic acid, caffeic acid, narcissoside (Isorhamnetin-3-O-rutinoside), isorhamnetin-3-O-glucoside, Syringetin-3-O-glucoside, 15-hydroxy-7-oxo-dehydroabietic acid, 6-O-p-coumaroyl ajugol, luteolin 7-rhamnoside, robustaflavone 7,4 -dimethyl ether, and ferulic acid were performed to evaluate their binding affinity with the targeted active sites of HSP90A, EGFR, and P53 proteins. Molecular Operating Environment MOE version 2019.0102 software (Chemical Computing Group, Montreal, CA) [96] was used for the docking studies. The exploited docking placement methodology is triangle matcher. Each ligand was allowed to be flexible, while the protein structure was kept rigid. The scores of the docking energy for the best-fitted poses of the components with the protein active pocket were recorded.
The score of docking energy (S; kcal/mol) and visual inspection of both two-dimensional and three-dimensional planes of the component-targeted protein interactions were exploited for the results analysis.

Animals, Ethical Statement, and Experimental Design
All experiments were approved by the Institutional Animal Care and Use Committee of Zagazig University (ZU-IACUC/3/F/111/2022). The procedures were carried out according to the National Institute of Health guidelines. A total of 24 Wistar rats (weighing 150-180 g) were used in this study. Prior to the experiment, all rats were allowed free access to standard diet and water and were subjected to a circadian rhythm with a 12 h day and 12 h night at an ambient temperature of 24~26 • C with 50~60% humidity for 1 week. The rats were randomly divided into three groups including a sham group (n = 8), a RIRI group (n = 8), and a RIRI group treated with ARBE (n = 8).

Induction of Renal Ischemia-Reperfusion (RIRI)
Renal ischemia-reperfusion injury (RIRI) was performed as previously described [98]. Briefly, ARBE (10 mL/kg, p.o., corresponding to 400 mg/kg) or vehicle was administered twice: 180 min and 15 min prior to ischemia. Dose was chosen based on a pilot study. Rats were anesthetized with thiopental sodium (Sigma Tec, Giza, Egypt) (25 mg/kg) administered i.p. before surgery. The left kidney was accessed through abdominal incision, and a non-traumatic vascular clamp was applied to the left renal artery for 60 min. Then, the clamp was removed to induce renal reperfusion. Finally, the abdominal cavity was closed. After the 24 h reperfusion period, the animals were decapitated for sample collection. The animals in the sham group underwent abdominal incision without clamping of the left renal arteries. Blood samples were collected via venipuncture and then were centrifuged, and the serum was stored at −80 • C for further analysis. Parts of the kidneys were fixed in 10% buffered formalin for histological and immunohistochemical studies. The rest of kidneys were snap frozen in liquid nitrogen and stored at −80 • C.

Kidney Function Assessment
Serum samples were assayed for blood urea nitrogen (BUN) and serum creatinine by using a Urea Nitrogen Kit (BioDiagnostic, Giza, Egypt) and the Creatinine Kit (BioDiagnostic, Giza, Egypt), respectively, according to the manufacturer's protocol.

Histopathology
Kidney tissue samples were fixed in 10% neutral buffered formalin for 72 h. Samples were processed in serial grades of ethanol, cleared in Xylene, then infiltrated and embedded into Paraplast tissue embedding media. Next, 5 µm thick serial sections were cut by rotatory microtome for demonstration of renal parenchyma in different samples and mounted on glass slides. Tissue sections were stained by Hematoxylin and Eosin as a standard staining method for blinded light microscopic examination by an experienced histologist.
Histological analysis was performed according to a previous study [99]. At least six non-overlapping fields were randomly selected and scanned from kidney tissue sections of each sample for the determination of relative area percentage of immunohistochemical expression levels of Cleaved Caspase 3, p-NFkB, HSP90, and P53 in immunohistochemically stained sections. All light microscopic examinations and data were obtained by using a Leica Application module for histological analysis, attached to a Full HD microscopic imaging system (Leica Microsystems GmbH, Wetzlar, Germany).

Statistical Analysis
GraphPad Prism version 9.4.1 (CA, USA) was used for the statistical analyses, and the results are presented as the means ± standard error of the mean (SEM). Comparisons between the groups were performed using the one-way ANOVA and Tukey's multiple comparison test; significance was accepted at p < 0.05.

Conclusions
Overall, our findings, using phytochemical, in silico network, and docking approaches, as well as further in vivo preclinical validation, show that ARBE could protect from RIRI by combating inflammation and apoptosis. Mechanistically, this might be due to the downregulation of HSP90 and P53. Yet, the effect of ARBE on other molecular targets cannot be excluded. Further drug discovery and preclinical and clinical studies are warranted on certain major components of ARBE, e.g., syringetin-3-O-glucoside and narcissoside, given their predicted interaction with multiple RIRI-related targets, in particular the hubs. These compounds may represent a base for the generation of new molecules that could be utilized as nephroprotective agents against RIRI.