Mitigation of hypobaric hypoxia induced renal inflammatory alterations by quercetin prophylaxis

Abstract

The objective of the present study is to decipher the role of NF-κB and its associated downstream genes involved in causing inflammation in kidneys of rats under acute hypobaric hypoxia exposure. The study also aims at finding out the efficacy of quercetin in comparison with dexamethasone in prevention of hypobaric hypoxia induced renal inflammation. Sprague Dawley (SD) rats (n = 6) were preconditioned with 50 mg/Kg BW of quercetin, 1 h prior to hypobaric hypoxia exposure (12 h). The results revealed a significant increase in reactive oxygen species (ROS) generation and malondialdehyde (MDA) (p < 0.001) levels along with down regulation of GPx and SOD levels in kidney tissue of hypobaric hypoxia exposed rats as compared to control. However, ROS production and MDA levels in kidney tissues were reduced significantly (p < 0.001) with enhanced antioxidant enzyme levels (GPx and SOD) in rats fed with quercetin as compared to hypobaric hypoxia exposed control (p < 0.001). Protein expression analysis through western blotting and EMSA performed in nuclear extracts of kidneys, exhibited a significant upregulation of NF-κB under hypobaric hypoxic stress as compared to control. Whereas pretreatment with quercetin aids in downregulation of NF-κB expression in kidneys of rats as compared to the hypobaric hypoxia exposed group. Further, quercetin prophylaxis significantly reduced the expression of pro-inflammatory cytokines (TNF-α, IL-2 and IL-6), increased the anti-inflammatory cytokines (IL-10, IL-4 and TGF-β) along with reduced expression of cell adhesion molecules (ICAM-1, VCAM-1, E-selectin and P-selectin) in kidneys of rats under hypobaric hypoxia as compared to hypobaric hypoxia control. However, pre-treatment with dexamethasone was not as effective as quercetin, in controlling the renal inflammation. Furthermore, improved hematological parameters such as WBC, RBC, and platelet count indicated that quercetin is a well-functioning effective molecule under hypobaric hypoxic exposure. The histopathological and TEM findings, manifested the structural changes in tubular arrangements in kidneys of rats, and these changes were found to be modified by quercetin prophylaxis under hypobaric hypoxic stress. Hence, the present study indicates that, quercetin can be considered as a potential phytochemical flavonoid moiety in preventing the acute renal inflammation under hypobaric hypoxia.

Graphical abstract

1. Introduction

The importance of molecular oxygen in the mammalian cell is unequivocal. At high altitudes, the prevailing low barometric pressure with low oxygen concentration (hypoxia) invites variety of cell signaling downstream genes to regulate redox imbalance.¹ Hypobaric hypoxia often leads to the development of pathological conditions, most of them involving hypobaric hypoxia induced-inflammatory processes. Inflammation is the key physiological response to hypoxic stress. Pathological complications such as Acute Kidney Injury (AKI), High Altitude Renal Syndrome (HARS) and Chronic Kidney Disorders (CKD) are some of the examples illustrating the adverse effects of hypobaric hypoxia on renal system.2, 3, 4

Cellular hypobaric hypoxia can trigger several inflammatory cytokines which causes not only tissue damage, but also initiate survival response. Although, hypobaric hypoxia-induced inflammation serves a protective role via initiation of immune response and tissue healing, but in excessive production, it results in numerous pathologies such as Acute Mountain Sickness (AMS), High altitude Pulmonary Edema (HAPE), High altitude Cerebral edema (HACE), kidney problems and cardiac disorders.^5,6 Acute and chronic hypobaric hypoxia exposure can produce maladaptive inflammation that can contribute to disease development.⁷ The key elements that are involved in regulating all the inflammatory responses are under the control of a transcription factor known as Nuclear Factor Kappa B (NF-κB). NF-κB is a heterodimer, consisting of p50 and p65 subunits and is often considered as master regulator of inflammation. Under normal conditions, NF-κB dimers in the cytoplasm are bound to their inhibitory components (IκB) which makes them inactive and therefore nonfunctional. However, upon appropriate stimulus (like hypoxia, heat, cold or any other stress), the IκB is phosphorylated and ubiquitinated via inhibitory kinase κ B (IKKB) complex. This degradation of IκB facilitates the translocation of NF-κB dimers into the nucleus of the cell and thus allow DNA binding.⁸ Involvement of NF-κB in pathogenesis of various inflammatory diseases has been proven in various animal and human studies.⁹ A sequence of signaling events, termed as canonical pathway of NF-κB activation, mediates the transcriptional induction of various pro-inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF- α), Interleukin-6 (IL-6) and Interleukin-1 (IL-1). These soluble components bind to their specific receptors and elicit crucial inflammatory processes that includes recruitment of monocytes and neutrophils to the site of injury leading to inflammation.^10,11 In general, inflammation is advantageous to the host however, uncontrolled inflammatory response can lead to excessive and prolonged tissue damage thus, results in acute or chronic inflammatory disorders. An intricate network of cytokines is generated in response to systemic endogenous or exogenous stimuli. It is the net effect of interactions between pro-inflammatory and anti-inflammatory cytokines that defines the nature of the physiological response against these stimuli. Anti-inflammatory cytokines are the modulatory elements that limit the potentially injurious effect of sustained or excessive inflammatory reactions. The principle anti-inflammatory molecules include Interleukin-4 (IL-4), Interleukin-10 (IL-10), and Transforming Growth Factor-beta (TGF-β). Under pathogenic conditions, these anti-inflammatory inhibitors either fail to provide sufficient control over pro-inflammatory mediators or over-compensate and inhibit the immune response thus, promote systemic infection.^12,13 Along with this imbalance between pro and anti-inflammatory cytokines under hypobaric hypoxic stress, a group of cellular adhesion molecules such as Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), E-selectin and P-selectin are extensively expressed under hypobaric hypoxia induced inflammation and contribute in causing the vascular leakage.¹⁴ Therefore, treatment with powerful anti-inflammatory agents to control inflammation may prove to be effective in diminishing the kidney injury and also aids in faster recovery.¹⁵

Quercetin, a polyphenol and a potent anti-inflammatory molecule may prove to be an appropriate remedy during hypobaric hypoxic exposure. It is a well-established phyto-molecule with numerous beneficial properties such as anti-inflammatory, anti-oxidant, anti-carcinogenic, anti-viral etc.¹⁶ Furthermore, dexamethasone is FDA approved corticosteroid medication used extensively for the treatment and prevention of high altitude illnesses. It acts as an anti-inflammatory and immunosuppressant agent and thus aids the acclimatization process.¹⁷ At present proper effective prophylactic treatment to prevent the hypobaric hypoxia induced renal inflammatory alternations is not available. Moreover, the most effective way to abolish these effects (hypobaric hypoxia induced problems), is to reduce the altitude exposure, and immediate descent to the lower altitudes. When it is not possible to reduce the high altitude exposure, due to some circumstances and ascend is crucial (as in the case of soldiers and in some business travelers, pilgrims or trekkers) it is better to treat the subjects with proper prophylaxis. It is always preferred to prevent the onset of renal inflammation under hypobaric hypoxia before the condition become worsen.

Hence, the present study was designed (i) to evaluate the prophylactic efficacy of quercetin in preventing the complications in kidneys of rats under hypobaric hypoxic stress in comparison with dexamethasone (ii) to assess the pro and anti-inflammatory cytokines i.e. TNF- α, IL-2, IL-6 and IL-10, IL-4, TGF-β respectively in kidneys of rats under hypobaric hypoxic stress (iii) we further intended to find out the protective efficacy of quercetin prophylaxis on cellular adhesion molecules i.e. ICAM-1, VCAM-1, E-selectin and P-selectin expression in kidneys of rats under low oxygen environment (iv) To analyze the alterations caused by hypobaric hypoxia on leukocytes, reticulocytes and platelets along with their renewal and restoration by quercetin and dexamethasone prophylaxis in kidneys of rats and (v) finally confirmation of these findings by histopathological and transmission electron microscopy analysis.

2. Material and methods

2.1. Chemicals and reagents

Quercetin and 2,7-Dichlorohydrofluroscein diacetate (DCFH-DA) were procured from Sigma Aldrich (St. Louis MO, USA), Dimethylsulphoxide (DMSO) from Sisco Research Laboratory (SRL, Maharashtra) along with Thiobarbituric acid (TBA) and Tricarboxylic acid (TCA). 5′5′-dithio-bis-(2- nitro-benzoic acid) (DTNB) from Sigma Aldrich. Dexamethasone was procured from Zydus Lifesciences Limited. All the other chemicals and reagents were of analytical grade.

2.2. Drug preparation

Drugs, quercetin (50 mg/kg BW) and dexamethasone (4 mg/kg BW) were freshly prepared by dissolving into a vehicle i.e., 0.5 % DMSO and sterile water, respectively. The drugs were administered orally to Sprague Dawley (SD) rats 1 h prior to the hypobaric hypoxia exposure.

2.3. Safety profile of quercetin

Quercetin is usually considered as a safe phyto-flavonoid moiety administered alone or in combination with other drugs.¹⁸ It is well known to inhibit pro-inflammatory cytokines and several enzymes thus aids in the reduction of inflammation in various physiological complications.¹⁹ Recently, critical importance of this flavonoid in combating deadly pandemic (SARS-CoV) has been established sturdily.²⁰ Numerous studies on safety profile of quercetin are performed and published till date. A study conducted on 200 male and female rats supplemented with different doses of quercetin (40–1900 mg/kg BW/day) demonstrated no alterations in physiological parameters of rats thus reflects optimum animal physiology.²¹ Previous literature reported the LD 50 value of quercetin to be 161 mg/kg BW after oral supplementation.²² The oral dose of quercetin is found to be the safest route of administration. However, if intravenous dose is administered, it is advised to monitor kidney functions regularly.²³ Hence, in this study oral administration of quercetin was considered as better route for all the experimental rats.

2.4. Experimental animals

Male Sprague Dawley rats of weight 180–200 g were obtained from central animal facility of DIPAS-DRDO, Delhi, India. Animals were kept in experimentally designed polypropylene cages of dimension 32in. × 24in. × 16in. provided with standard conditions (12 light/dark cycle, 25 ± 2 °C temperature & 55 ± 5 % relative humidity) and availability of animal chow and water ad libitum. All the animal studies and protocols are in accordance with standards provided in the guide for the Care and Use of Laboratory Animals (National Academy of Sciences, Washington, DC). Protocols involving animal studies were reviewed and sanctioned by the Institutional Animal Ethics Committee (IAEC), DIPAS, DRDO, Delhi, India, accredited to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (IAEC No.: DIPAS/IAEC/2017/19/EXT/22).

2.5. Experimental protocol

The experiments were performed with six groups (n = 6) of SD rats. (i) Group 1 include -normoxia control (N) that received only vehicle (ii) Group 2 – Hypoxia (H) received only vehicle and was exposed to hypobaric hypoxia for 12 h (iii) Group 3 – Normoxia + Quercetin (NQ) was supplemented with quercetin (50 mg/kg BW) without hypobaric hypoxia exposure (iv) Group 4 – Hypoxia + Quercetin (HQ) received quercetin (50 mg/kg BW) 1 h prior to hypobaric hypoxia exposure of 12 h (v) Group 5 – Normoxia + Dexamethasone (ND) was supplemented with dexamethasone (4 mg/kg BW) without hypobaric hypoxia exposure (vi) Group 6 – Hypoxia + Dexamethasone (HD) received dexamethasone (4 mg/kg BW) 1 h prior to hypobaric hypoxia exposure of 12 h. Efficacy of different doses of quercetin (25, 50, 100 and 200 mg/kg BW) was assessed in previous experiments of our lab, where 50 mg/kg BW of quercetin was found to be the most efficient dose in reducing the oxidative stress i.e. Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) and tissue injury marker i.e. Lactate Dehydrogenase (LDH) in kidneys of rats under hypobaric hypoxia. This dose significantly enhanced the antioxidant activity by increasing the levels of reduced glutathione (GSH) and improved the renal function parameters i.e. creatinine, Blood Urea Nitrogen (BUN) and uric acid in plasma of rats under hypoxic stress (12 h) among other tested doses.²⁴

The dose of dexamethasone (4 mg/kg BW) was selected on the basis of the previous studies.25, 26, 27 The earlier research conducted on the prophylactic efficacy of dexamethasone utilized 4 mg/kg BW of the drug every 6 h for a duration of 42 h, exposed at the height of 4570 m above sea level. Further, Montesinos et al. (2006) have reported that, intraperitoneal administration of 1.5 mg/Kg BW of dexamethasone 1 h prior to induction of inflammation in mice, significantly reduced the inflammation.²⁵ Another crucial study performed on human subjects revealed that oral intake of 0.5–8 mg/kg BW dexamethasone significantly decreased the post -operative edema. Based on these references and our laboratory experiments,²⁶ the dexamethasone dose (4 mg/kg BW) has been selected.

2.6. Hypobaric hypoxia exposure

The rats were exposed to hypobaric hypoxia in a simulated hypobaric chamber (Matrix, India) for 12 h at an altitude of 25,000 ft. Standardization of time duration was performed in previous experiments of our lab and among the tested hypobaric hypoxia durations (viz: 1 h, 3 h, 6 h, 12 h, 24 h and 48 h), 12 h was found to be the optimum time duration at which maximum renal damage had occurred.²⁴ Internal pressure was maintained at 280 mm Hg with fresh air flushing at the rate of 4 lit/h along with the relative humidity of 55 ± 5 % inside the hypobaric hypoxia chamber. Further, the partial pressure of oxygen (pO₂) in control rats was observed to be approximately 96 ± 2 mm Hg, whereas in hypobaric hypoxia exposed rats pO₂ was around 35 ± 2 mm Hg. This indicates that the animals were exposed to low barometric pressure at high altitude. Standard animal chow and water was made available ad libitum during hypobaric hypoxia exposure to rats. All animal experiments were performed with utmost care to minimize the sufferings on rats.

2.7. Method of sacrifice

After 12 h of hypobaric hypoxia exposure, rats were injected with anesthetic agents, Ketamine--Xylazine cocktail (80:20 mg/kg BW in ratio) intraperitoneal. Animals were sacrificed after proper sedation.²⁸

2.8. Preparation of sample

Animals were perfused with chilled 1X Phosphate-buffered saline (PBS) at the time of dissection. Kidney tissues were washed with 1X PBS and homogenized (10 %) in 0.154 M KCl containing DTT, PMSF and protease inhibitor cocktail (PIC) for performing biochemical estimations. Blood was collected by cardiac puncture from left ventricle of the heart. Plasma was separated and stored at −80 °C till further experimental analysis.

2.9. Biochemical assays

2.9.1. Measurement of oxidative stress

Estimation of reactive oxygen species (ROS) generation in kidney homogenate was carried out using DCFH-DA assay in kidneys of normoxic and hypobaric hypoxia exposed rats. DCFH-DA along with potassium dihydrogen buffer was added to tissue homogenates prior to incubation of 15 min at room temperature in dark. Fluorescence emitted by DCF formed as a result of oxidation of DCFH-DA in the presence of ROS was measured spectrophotometrically (Synergy H1, Biotek, Germany) at an excitation and emission wavelength of 485 nm and 530 nm, respectively.^29,30

2.9.2. Malondialdehyde (MDA) estimation

The assay involves condensation reaction of two molecules of TBA with one molecule of MDA which is formed as a byproduct of lipid peroxidation in kidney tissue homogenates of tested groups. The method consists of incubating samples at 80 °C with TBA, TCA and HCl for 1 h, the reaction mixture was further centrifuged at 3000 rpm for 10 min at 4 °C. The absorbance of the MDA-TBA adduct formed was measured spectrophotometrically at wavelength of 532 nm.^31,32

2.9.3. Antioxidants activity assessment

Antioxidants are the compounds that inhibit the production of free radicals or aids in their neutralization by impeding their oxidation or scavenging these unstable molecules. The antioxidant activity of Glutathione Peroxidase (GPx) and Superoxide dismutase (SOD) were carried out in accordance with the manufacturer's guidelines (EGPx-100, ESOD-100; BioAssay Systems, USA).

2.9.4. Pro-inflammatory cytokines

Pro-inflammatory cytokines are predominantly produced by activated macrophages and are involved in the up-regulation of inflammatory processes. Estimation of pro-inflammatory cytokines (TNF- α, IL-2 and IL-6) levels in kidney homogenate was done by Sandwich-Enzyme Linked Immunosorbent Assay kit (ELISA- Elabscience, USA) as per instructions provided by the manufacturer. All the samples and standards were tested in duplicates. The enzyme-substrate reaction was terminated by the addition of stop solution and optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The final concentrations of all the cytokines in kidney tissue homogenates were calculated by comparing OD of the samples to the standard curve.

2.9.5. Anti-inflammatory cytokines

The excessive production of pro-inflammatory cytokines is counter balanced by the release of series of anti-inflammatory molecules such as TGF- β, IL-4 and IL-10. The assessment of these cytokines was performed by Sandwich ELISA as per instructions provided by the manufacturer (Elabscience, USA). The final OD was measured spectrophotometrically (Synergy H1, Biotek, Germany) at a wavelength of 450 nm. The final concentrations of all the cytokines in kidney tissue homogenates were calculated by comparing OD of the samples to the standard curve.

2.10. Protein expression studies

Cytoplasmic and nuclear extracts were prepared using lysis (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl) and extraction buffers (20 mM HEPES, 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 25 % glycerol).³³ Protein quantification was estimated by Lowry's method (1951).³⁴ Western blot analysis was carried out for separation and identification of NF-κB, histone-3, TNF- α, ICAM-1, VCAM-1, E-selectin, P- selectin and β-actin (Santa Cruz Biotechnology Inc., USA in dilution 1:1000) protein expressions in nuclear and cytoplasmic extracts of kidneys of rats exposed to hypobaric hypoxia. The protein in kidney samples were separated using 10 % and 12 % SDS-PAGE on the basis of molecular weight. The separated proteins were then, electro-blotted to the nitrocellulose membranes and blocked with 5 % BSA dissolved in 1X PBST (pH 7.4) at RT for 1 h with gentle shaking. Membranes were then washed and incubated with respective primary antibodies over night at 4 °C. After 4–5 washings with PBST (Tween 0.1 %), the membranes were probed with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology Inc. USA in dilution 1: 20,000) at RT for 2 h. The blots were then thoroughly washed (4–5 times) with PBST. The membranes were developed using chemiluminescent peroxidase substrate (Luminata forte, Millipore U.S.A) and the bands were visualized in Chemidoc (UVP, Cambridge, U.K). The OD of the bands were quantified using lab works software (UVP-Bio Imaging systems, CA).

2.11. Electrophoretic mobility shift assay (EMSA) for DNA binding efficacy of NF-κB

Nuclear extracts were prepared using lysis buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl) and extraction buffer (20 mM HEPES, 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 25 % glycerol). The EMSA for NF-κB was carried out using a commercial kit (Thermo Scientific, USA). The samples were incubated with 10 mM Tris–HCl, pH 7.4, 50 mM NaCl, 50 mM KCl, 1 mM MgCl₂, 1 mM EDTA, 5 mM DTT and 10 ƞg of biotinylated double stranded oligonucleotide for EMSA of NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′); a mutant DNA sequence (5′-GCC TGG GAA AGT CCC CTC AAC T-3′) for 30 min at room temperature. Then, DNA-protein complexes were separated on native 6 % polyacrylamide DNA retardation gel and electro blotted onto positively charged nylon membranes. Biotinylated DNA/protein complexes were detected with peroxidase conjugated streptavidin and a chemiluminescent substrate kit (Thermo Scientific, USA).^26,35

2.12. Hematological analysis

Blood was collected from left ventricle by cardiac puncture followed by light anesthesia. Blood was collected in EDTA vacutainers (BD) and immediately analyzed through 5-part differential veterinary hematology analyzer (Spincell vet5 Compact, Spinreact, Spain).

2.13. Histopathological analysis

For histopathological slides preparation, all the experimental and control animals were perfused with 4 % paraformaldehyde after proper sedation. Kidneys were immediately removed and immersed in 4 % paraformaldehyde. The tissues were sectioned in to 5 μm thin sections and stained with hematoxylin & eosin (H & E staining). Finally, the images were captured using Olympus BX 50 microscope (Olympus, Japan).³⁶

2.14. Transmission electron microscopy (TEM)

To study the ultrastructure of quercetin or dexamethasone treated and non-treated renal tissues of rats, 1 cubic millimeter thin sections were cut and washed with 1x PBS buffer repeatedly. Further, the tissues were fixed in Karnovsky's fixative (0.5 % Glutaraldehyde +2.0 % Formaldehyde). Then the samples were washed in 0.1 M phosphate buffer (pH 7.0) and post fixed for 2 h in 1 % osmium tetroxide at 4 °C. The specimens were dehydrated with graded acetone and embedded in epoxy resin. Ultrathin sections were cut by ultramicrotome, stained with uranyl and lead acetate and examined under transmission electron microscope (Talos F200 G2, Thermo Fisher Scientific, USA) operated at 200 kV.³⁷

2.15. Statistical analysis

The results obtained were statistically analyzed using Graph Pad Prism software version 8, California, U.S.A. Comparisons between all the experimental groups of the study were made using one-way analysis of variance (ANOVA). Results were expressed as mean ± SD. Comparison between multiple groups were analyzed by One Way ANOVA followed by Bonferroni's multiple comparison and unpaired t-test was used to compare the data between the two groups. Differences were considered statistically significant for p < 0.05.³⁸

3. Results

3.1. Reactive oxygen species accumulation

ROS generated in all the experimental groups is depicted in Fig. 1 a. As a consequence of hypobaric hypoxic exposure, a significant increase in ROS production was observed in kidneys of hypobaric hypoxia exposed group in comparison with normoxia control group (2.7-fold ↑, p < 0.001). However, quercetin and dexamethasone pretreatment 1 h prior to hypobaric hypoxia exposure significantly restored the ROS production in kidneys of rats (1.8-fold ↓ and 1.5 fold ↓, respectively) as compared with hypobaric hypoxia exposed group. Further, quercetin and dexamethasone treated normoxia control animals exhibited no change in ROS generation in kidneys of rats. Both the drugs managed to attenuate the ROS generated in the kidneys of hypobaric hypoxia animals however, the reduction attained by quercetin was seen to be better than dexamethasone (1.8-fold ↓).

Fig. 1.

Effect of quercetin (50 mg/kg BW) and dexamethasone (4 mg/kg BW) supplementation on the production of (a) Reactive Oxygen Species (ROS) and (b) Malondialdehyde (MDA) in kidneys of rats exposed to hypobaric hypoxia at 7620 m for 12 h*p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD. Values are mean ± SD. N- Normoxia control, H- Hypobaric hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

3.2. Malondialdehyde adduct formation

As a result of hypobaric hypoxia induced excessive ROS production, lipid peroxidation via super-oxides interaction with membrane poly-unsaturated fatty acids (PUFA) leads to the over production of MDA. This can be evidently noted in Fig. 1 b where a significant elevation (p < 0.001) in MDA was observed in kidneys of hypobaric hypoxia exposed group when compared to normoxia control (2.5-fold ↑). However, a significant reduction in MDA levels in kidney tissues was seen in animal group pretreated with quercetin and dexamethasone as compared to hypobaric hypoxia exposed group (2-fold ↓ and 1.3-fold ↓, respectively). Drug pretreated normoxia groups (NQ and ND) exhibited no significant change in kidney MDA levels with respect to normoxia control. Prior administration of quercetin, reduced the kidney tissue MDA levels (by 1.5-fold ↓) more than the hypobaric hypoxia exposed dexamethasone treated group (p < 0.001).

3.3. Glutathione peroxidase estimation

Under hypobaric hypoxic conditions, antioxidant ability of body weakens which was apparently visible in results depicted in Fig. 2 a. GPx activity diminished significantly (p < 0.001) in kidney tissues of rats exposed to hypobaric hypoxia as compared to the normoxia control group (3.2-fold ↓). Although, quercetin and dexamethasone administered groups prior to hypobaric hypoxia exposure demonstrated a significant increment in kidney GPx levels (2.5-fold ↑ and 1.8-fold ↑, respectively) as compared with hypobaric hypoxia exposed group but the increase attained by quercetin was far more than dexamethasone (1.38-fold ↑). Drugs treated normoxia groups (NQ and ND) showed unmodified GPx levels in kidney tissues as compared to normoxia control.

Fig. 2.

Effect of quercetin (50 mg/kg BW) and dexamethasone (4 mg/kg BW) administration on the production of (a) Glutathione Peroxidase (GPx) *p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.05H vs HD, @p < 0.001 HQ vs HD and (b) Superoxide Dismutase (SOD) *p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD in kidneys of rats exposed to hypobaric hypoxia at 7620 m for 12 h. Values are mean ± SD. N- Normoxia control, H- Hypobaric Hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

3.4. Superoxide dismutase estimation

Similar to the results obtained in GPx activity, SOD activity experienced a significant upsurge (1.4-fold ↑ and 1.2-fold ↑) in kidneys of rats pretreated with quercetin and dexamethasone, respectively, as compared with hypobaric hypoxia exposed group that depicted a significant downfall (1.5-fold ↓) as compared with control group. However, no significant changes were observed in kidney SOD activity of drugs supplemented normoxic groups (NQ and ND) when compared with normoxia control group (Fig. 2 b). Further, quercetin treated hypobaric hypoxia exposed group (HQ) exhibited higher levels of kidney SOD activity than dexamethasone (1.1- fold ↑) treated hypobaric hypoxia exposed (HD) animals.

3.5. Pro-inflammatory cytokine activity

A significant elevation in the levels of TNF-α, IL-2 and IL-6 was observed in kidney tissue homogenates of hypobaric hypoxia exposed group with respect to normoxia control group (1.2-fold ↑, 1.5-fold ↑, 1.3-fold↑, respectively). The results obtained are described in Fig. 3 a, b and c, respectively. Quercetin administration under hypobaric hypoxic stress reduced these cytokines significantly (p < 0.001) in kidneys of rats in comparison with the hypobaric hypoxia exposed group (1.1-fold ↓, 1.25-fold ↓, 1.3-fold ↓, respectively). Animal group pretreated with dexamethasone observed a significant decrease in levels of TNF- α (1.1- fold ↓) in kidneys under normal conditions. The reduction achieved by dexamethasone under hypoxic stress in the levels of IL-2 in kidney tissues was far more than hypobaric hypoxia exposed group (4.4- fold ↓) (Fig. 3 b), but it was observed to be enhancing (1.2-fold ↑) the IL-6 levels in kidney tissue of rats (Fig. 3 c) as compared to hypobaric hypoxia exposed animals. Further, a significant reduction in dexamethasone treated normoxic group was observed in IL-2 kidney tissue levels in comparison with normoxia control (1.7- fold ↓). Kidney IL-2 levels in hypobaric hypoxia treated dexamethasone group decreased far beyond the IL-2 levels of normoxia control group (3.8- fold ↓) (Fig. 3 b.).

Fig. 3.

Production of (a) Tumor Necrosis Factor – α (TNF- α) (b) Interleukin −2 (IL-2) and (c) Interleukin −6 (IL-6) in kidneys of the rats supplemented with quercetin (50 mg/kg BW) and dexamethasone (4 mg/kg) prior to the hypobaric hypoxia exposure at 7620 m for 12 h. Values are mean ± SD. (a) *p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD, (b) *p < 0.05 N vs H, #p < 0.01H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD (c) *p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD. N- Normoxia control, H- Hypobaric hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

3.6. Anti-inflammatory cytokine determination

In order to balance the pro-inflammatory cytokines, body releases anti-inflammatory cytokines. The results of anti-inflammatory cytokines i.e., IL-10, IL-4 and TGF-β, of the present study are illustrated in Fig. 4 a, b and c, respectively. All the three cytokines were found to be reduced significantly (p < 0.001) in kidney tissues of hypobaric hypoxia exposed group when compared to normoxia control (1.1- fold ↓, 1.25- fold ↓, 1.5- fold ↓, respectively). Quercetin prophylaxis under hypobaric hypoxia showed a significant increase in IL-10 and TGF-β levels in kidneys of rats compared to hypobaric hypoxia control (p < 0.001). However, a slight but non-significant increase in IL-4 levels was noticed in kidneys of rats treated with quercetin under hypobaric hypoxia exposure compared to hypobaric hypoxia exposed control animals. Further, animal group pretreated with dexamethasone prior to hypobaric hypoxia exposure observed a significant decrease in kidney tissue IL-10 levels (0.5-fold ↓), a slight increase in IL-4 levels (1.04-fold ↑) and a significant decrease (0.6-fold ↓) (p < 0.001) in the levels of TGF-β compared to hypobaric hypoxia exposed animals. Further, a significant reduction in kidney tissue IL-4 and TGF-β levels were also observed in dexamethasone treated normoxia group when compared with normoxia control group (2.2-fold ↓ and 1.2-fold ↓, respectively) (Fig. 4b and c).

Fig. 4.

Anti-inflammatory Cytokines (a) Interleukin −10 (IL-10) (b) Interleukin −4 (IL-4) (c) Transforming Growth Factor – β (TGF- β) production in kidneys of rats supplemented with Quercetin (50 mg/kg BW) and Dexamethasone (4 mg/kg BW) prior to hypobaric hypoxia exposure at 7620 m for 12 h. Values are mean ± SD. (a) *p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD (b) *p < 0.05 N vs H, $p < 0.05H vs HD, @p < 0.001 HQ vs HD (c) *p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD. N- Normoxia control, H- Hypobaric hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

3.7. Protein expression

Hypobaric hypoxia induced inflammation was analyzed by the protein expression studies of NF-κB, TNF- α, ICAM-1, VCAM-1, E-selectin and P-selectin respectively (Fig. 5 a-h) in kidney tissues of rats. Consequentially, hypobaric hypoxia exposure enhanced the expression of NF-κB and its associated genes (TNF- α, ICAM-I, VCAM-I, E-selectin and P-selectin) significantly (p < 0.001) in kidneys of rats in comparison with the normoxia control. Pretreatment with quercetin prior to hypobaric hypoxia exposure facilitated the reduction of their expression in kidney tissues when compared with hypobaric hypoxia exposed group. Although, preconditioning with quercetin under normal conditions does not lead to any alteration in any of these proteins in comparison to normoxia control. Whereas normoxia group supplemented with dexamethasone (ND), showed increased levels of TNF- α, reduced levels of ICAM-1 and VCAM-1 in kidney homogenates of rats in comparison with the normoxia group, E-selectin and P-selectin protein expressions were same as that of normoxia. Quercetin aids in reducing P-selectin and E-selectin proteins expression in kidney tissues of rats exposed to hypobaric hypoxia in comparison to hypobaric hypoxia control group (Fig. 5f and g; n and o). However, dexamethasone administration was unable to reduce VCAM-1, E-selectin and P-selectin protein expressions in kidneys of hypobaric hypoxia exposed rats when compared to hypoxia control. Thus, dexamethasone appears to be less effective than quercetin as the reduction attained by quercetin was far better than dexamethasone. Histone −3 and β-actin (Fig. 5 b and h) blots determined in kidney tissue homogenates of rats were shown as housekeeping genes to represent nuclear and cytoplasmic protein expressions, respectively. The densitometry analysis of these proteins were depicted in Fig. 5 i-p.

Fig. 5.

Effect of quercetin (50 mg/kg BW) and dexamethasone (4 mg/kg BW) on protein expressions of (a) Nuclear Factor kappa- B (NFκB) (b) Histone 3 (c) Tumor Necrosis Factor-alpha (TNF- α) (d) Intercellular Adhesion Molecule 1(ICAM-1) (e) Vascular Cell Adhesion Molecule-1 (VCAM-1) (f) E -selectin (g) P -selectin (h) β-actin in kidneys of rats exposed to hypobaric hypoxia at 7620 m for 12 h. (i–p) Densitometry analysis of (i) NF-κB (*p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD) (j) Histone- 3 (k) TNF- α (*p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD) (l) ICAM-1 (*p < 0.001 N vs H, #p < 0.001H vs HQ, @p < 0.001 HQ vs HD) (m) VCAM-1 (*p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD) (n) E –selectin (*p < 0.001 N vs H, #p < 0.001H vs HQ, @p < 0.001 HQ vs HD) (g) P –selectin (*p < 0.001 N vs H, #p < 0.001H vs HQ, $p < 0.001H vs HD, @p < 0.001 HQ vs HD) (h) β - actin. N- Normoxia control, H- Hypobaric hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

3.8. EMSA analysis of NF-κB

The EMSA analysis showing the expression of protein bound DNA pertaining to NF-κB and its densitometry analysis in kidney tissues of rats is depicted in Fig. 6 a, b respectively. We observed a significant (p < 0.001) increase in NF-κB expression in the nuclear extract of kidney tissues in rats under hypobaric hypoxia exposure compared to normoxia control animals. Quercetin prophylaxis significantly reduced the NF-κB expression in nuclear extract of kidney homogenate as compared to hypobaric hypoxia exposed animals. However, dexamethasone treated animals under hypobaric hypoxia showed a significant increase in NF-κB expression in nuclear extracts of the kidneys compared to both hypobaric hypoxia and quercetin treated hypobaric hypoxia exposed animals. Expression of NF-κB remain unaltered in kidney homogenates of normoxia animals treated with both (NQ and ND) the drugs.

Fig. 6.

Effect of quercetin (50 mg/kg BW) and dexamethasone (4 mg/kg BW) on protein- DNA binding of Nuclear Factor kappa- B (NF-κB) in kidneys of rats exposed to hypobaric hypoxia at 7620 m for 12 h analyzed by Electron Mobility Shift Assay (EMSA) (a) Representative EMSA blot of NF-κB (b) Densitometry analysis of the blot. Values are mean ± SD. *p < 0.001 N vs H, #p < 0.001H vs HQ, @p < 0.001 HQ vs HD, $p < 0.001H vs HD. N- Normoxia control, H- Hypobaric hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

3.9. Changes in hematological parameters

Exposure to hypobaric hypoxia resulted in to a significant increase in White Blood Cells (WBC), lymphocytes, monocytes, neutrophils, Red Blood Cells (RBC), hemoglobin (Hb) and platelet as compared to normoxia control (p < 0.001) (Table 1). Quercetin and dexamethasone pretreatment prior to hypobaric hypoxia exposure maintained the levels of monocytes and Hb higher than the normoxia. Dexamethasone administration resulted into a significantly higher levels of lymphocytes, neutrophils, and platelets in hypobaric hypoxic conditions as compared with hypobaric hypoxia control group (p < 0.001). Quercetin supplementation also showed an increment in WBC, RBC and Hb under hypobaric hypoxic conditions in comparison to normoxia control. Further, dexamethasone supplementation under normal conditions decreased the WBC, Lymphocytes, Monocytes, Neutrophils and platelets significantly in blood samples of rats in comparison to normoxia group (Table 1).

Table 1.

Changes in hematological parameters of rats WBC (10^3/μL), Lymphocytes (%), Monocytes (%), Neutrophils (%), RBC (10^6/μL), Hemoglobin (Hb g/dL), Platelets (10^3/μL), supplemented with Quercetin (50 mg/kg BW) and Dexamethasone (4 mg/kg) prior to hypobaric hypoxia exposure at 7620 m for 12 h. Values are mean ± SD. ^ap < 0.001 N v/s H, ^bp < 0.01 N v/s H, ^cp < 0.05 N v/s H, ^dp < 0.01H v/s HQ, ^ep < 0.001H v/s HQ, ^fp < 0.001 N v/s ND, ^gp < 0.001H v/s HD, ^hp < 0.01H v/s HD, ⁱp < 0.001H v/s HD, ^jp < 0.01H v/s HD. WBC- White Blood corpuscles, RBD- Red Blood Corpuscles. N- Normoxia control, H- Hypobaric hypoxia, NQ- Normoxia + Quercetin, HQ- Hypobaric hypoxia + Quercetin, ND- Normoxia + Dexamethasone, HD- Hypobaric hypoxia + Dexamethasone.

S. No.	Parameters	N	H	NQ	HQ	ND	HD
1	WBC (10^3/μL)	9.85 ± 0.57	11.65 ± 0.39a	9.68 ± 0.66	9.97 ± 0.56d	8.87 ± 1.17	9.141 ± 0.93g
2	Lymphocytes (%)	71.2 ± 1.7	84.4 ± 3.31a	72.6 ± 2.4	70.92 ± 1.03e	66.9 ± 5.7	80.095 ± 4.7h
3	Monocytes (%)	4.05 ± 1.06	11.48 ± 0.8a	3.97 ± 0.72	6.05 ± 0.9e	2.26 ± 0.48	6.49 ± 1.06g
4	Neutrophils (%)	79.82 ± 2.7	88.27 ± 1.90b	79.6 ± 5.2	77.9 ± 7.05d	34.13 ± 2.04f	83.95 ± 1.93
5	RBC (10^6/μL)	6.58 ± 0.5	8.3 ± 0.53c	6.18 ± 0.8	7.82 ± 0.8	7.11 ± 0.88	6.54 ± 0.4i
6	Hb (g/dL)	13.94 ± 0.9	16.33 ± 0.9a	14.84 ± 0.4	18.23 ± 0.7d	14.17 ± 0.6	15.94 ± 0.9 h
7	Platelets (10^3/μL)	762.16 ± 31.1	937 ± 45.3a	764.33 ± 28.5	730.66 ± 38e	658.66 ± 49f	886.83 ± 22.7j

3.10. Histopathological analysis

Hypobaric hypoxia exposed renal sections of rats showed a degenerated and constricted glomerulus inside undefined Bowman's capsule, swollen renal tubules along with fluid accumulation when compared to normoxia rats which showed intact normal configuration of renal vasculature (Fig. 7 a, b (i) and b (ii)). Quercetin aids in protection of renal tissues under hypobaric hypoxia stress. The kidney tissue sections from the quercetin received group depicted an intact, well defined glomerulus with no fluid accumulation in renal tubules (Fig. 7 d). However, dexamethasone failed to prevent the damage occurred in kidneys caused by the hypobaric hypoxia and thus exhibited similar findings as that of hypobaric hypoxia exposed group (Fig. 7 f). Moreover, both the drugs (NQ and ND) did not show any significant histopathological alterations in kidneys under normoxic conditions (Fig. 7 c and e).

Fig. 7.

Hematoxylin and Eosin stained renal images depicting the degeneration of renal tubules, glomerulus and Bowman's capsule, swelling of renal tubules and fluid accumulation in kidneys of rats exposed to hypobaric hypoxia at 7620 m, 25 ± 2 °C for 12 h (Scale Bar: 50 μm, magnification: 20×) (a) Normoxia control group representing intact glomerulus and Bowman's capsule and tubules with no fluid retention (b) Renal images of hypobaric hypoxia exposed animals with degenerated Bowman's capsule (DG), ballooning of tubular cells with fluid accumulation (FA) evidencing the vascular leakage (c) Quercetin supplemented normoxia group depicting no significant changes with normal intact configuration, showing renal corpuscles and tubules (d) Kidney sections of Quercetin administered hypobaric hypoxia exposed group representing, no fluid accumulation (No vascular leakage) and degeneration in renal corpuscles and tubules (e) Dexamethasone administered normoxia rats illustrating no structural obliteration with no fluid retention (f) Images of dexamethasone supplemented hypobaric hypoxia exposed group showing the fluid accumulation and excessive degeneration of renal structure. NFA: No Fluid Accumulation, DG: Degenerated Glomerulus, FA: Fluid Accumulation.

3.11. Transmission Electron Microscopic analysis

Examination of ultrathin sections of renal tissues from rats exposed to hypobaric hypoxia revealed significant structural damage at the cellular level when compared to rats in the normoxia control group (Fig. 8 a. i, ii and b. i, ii, iii). These pathological changes were characterized by swollen and disrupted mitochondria and endoplasmic reticulum. Additionally, the podocyte foot processes exhibited fusion and irregular shapes, while the glomerular basement membrane appeared swollen and uneven in thickness. The endothelial fenestration lining the basement membrane showed structural disruption and irregularities. However, when rats were supplemented with quercetin, a notable improvement in protection in kidney against oxidative and inflammatory stress was observed. This supplementation (quercetin) restored the normal cell morphology, with regular podocyte foot processes and consistently constructed basement membranes. Furthermore, endothelial fenestrations at regular intervals were observed in the quercetin-supplemented hypobaric hypoxia exposed group (Fig. 8 d. i, ii).

Fig. 8.

Transmission Electron Microscopic (TEM) analysis (a-f, scale bar = 1 μm): (a) Normoxia (i and ii): Normoxia control group displayed normal kidney tissue morphology (b) Hypobaric hypoxia (i, ii and iii): Kidneys from hypobaric hypoxia exposed rats displayed swollen and irregular mitochondria, endoplasmic reticulum and basement membrane, effacement of podocytes and structurally abnormal endothelial fenestration (c) Normoxia + Quercetin (i and ii): Supplementation of quercetin under normal conditions displayed no noticeable changes in the structural arrangement of the kidney tissues (d) Hypobaric hypoxia + Quercetin (i and ii): Treatment with quercetin alleviated the glomerular obliterations caused by hypobaric hypoxia exposure to a level similar to that of the control group. (e) Normoxia + Dexamethasone (i and ii): Dexamethasone administered normoxic renal sections of rats exhibited similar cell morphology as that of control group (f) Hypobaric hypoxia + Dexamethasone (i and ii): Dexamethasone supplementation under hypobaric hypoxic stress eliminates the structural alterations caused by hypobaric hypoxia exposure. ER- Endoplasmic Reticulum, M-Mitochondria, N- Nucleus, P- Podocytes, Yellow arrow -Basement Membrane, White arrow head- Endothelial Fenestration.

In contrast, dexamethasone administered prior to hypobaric hypoxia exposed rats displayed normal mitochondria and endoplasmic reticulum. Nevertheless, these rats still exhibited a thickened and irregular basement membrane in their renal sections. Therefore, it was observed that dexamethasone did not provide complete protection against hypoxia-induced damage (Fig. 8 f. i, ii). Furthermore, when quercetin and dexamethasone were administered to the normoxia control groups, no adverse effects on glomerular morphology were observed, indicating the non-harmful nature of these drugs under normal conditions (Fig. 8 c. i, ii and 8. e. i, ii respectively).

4. Discussion

The study presented here illustrates the efficacy of quercetin and dexamethasone in amelioration of renal inflammatory damages caused by hypobaric hypoxia exposure. This study showed a significant increment in ROS and MDA production in kidneys of hypobaric hypoxia exposed groups as compared with normoxia control. Although, supplementation of quercetin and dexamethasone significantly reduced the ROS and MDA production in kidneys of rats, but dexamethasone was observed to be less effective than quercetin. Earlier studies have revealed similar findings and stated that dexamethasone significantly enhanced the ROS production and apoptosis via activation of endoplasmic reticulum stress in rats.³⁹ On the other hand, quercetin is well-known for its anti-oxidant properties along with its efficacy for enhancing endogenous anti-oxidant components.^40,41 Our experiments align with these findings, as quercetin supplementation significantly boosted the activity of renal GPx and SOD under hypoxic stress when compared to the hypoxia-exposed group without treatment. Studies in animals and cell lines have also shown that, quercetin can stimulate GSH synthesis.⁴² Moreover, the application of quercetin therapy in renal ischemia/reperfusion has also been observed to increase GSH levels and enhanced the antioxidant capacity, as reported in various experimental animal models.43, 44, 45^. In the present study, we observed that, dexamethasone also supported the production of antioxidant molecules (GPx and SOD) in kidneys of rats, but in comparison, it was not as effective as that of quercetin. Our previous studies reported that quercetin was able to reduce the hypobaric hypoxia induced increase in ROS and MDA levels and enhanced the antioxidant levels in brain²⁶ and lungs³⁸ of rats exposed to 24 h and 6 h under hypobaric hypoxia exposure, respectively. However, it is worth noting that, dexamethasone can have both antioxidant and pro-oxidant effects in different circumstances.^46,47

Exposure to hypobaric hypoxia can trigger an imbalanced inflammatory response, potentially leading to various pathological complications. It is reported that, Quercetin, inhibits the activation of NF-κB and its associated genes by blocking numerous pathways in response to inflammatory stimulants in different cell lines.48, 49, 50 Our earlier and recently published data reported that altered NF-κB under hypobaric hypoxia conditions can mediate an inflammatory cytokine response and also induce gene transcription of pro-inflammatory and cell adhesion molecules in lungs of rats.^38,51 Moreover, literature revealed that, quercetin is an inhibitor of oxidative stress as well as NF-κB activation in brain of rats exposed to hypobaric hypoxia for 24 h.²⁶ It is also reported that, quercetin reduces the expression of ICAM-1, IL-18, and IL-6 in ARPE-19 cells via inhibition of NF-κB phosphorylation process.^26,50 All these studies explained the mechanism by which quercetin blocks the phosphorylation of NF-κB thereby preventing the NF-κB entry into the nucleus.⁶

In our study, we observed a significant increase in pro-inflammatory cytokines such as TNF-α, IL-2, and IL-6 (Fig. 3) in kidneys of rats under hypobaric hypoxia exposure. Recent publications have linked inflammation occurring under hypoxic conditions to a wide range of human diseases. Additionally, many inflammatory diseases are commonly characterized by hypoxia, suggesting the involvement of hypoxia-dependent transcription factors.^52,53 Hence, the administration of anti-inflammatory molecules appears to be a promising approach to mitigate inflammation induced by hypoxia. We have observed that, pre-treatment with quercetin before exposure to hypobaric hypoxia significantly reduced the levels of pro-inflammatory cytokines. Previous studies (both in vitro and in vivo) have suggested that quercetin achieves these effects by inhibiting the production of TNF-α.^54,55 Furthermore, this plant flavonoid has demonstrated the ability to inhibit LPS-induced mRNA levels of TNF-α and IL-1α in glial cells, thereby reducing apoptotic neuronal cell death.⁵⁶ Another study focusing on quercetin's activity against H₂O₂-induced inflammation observed, a protective effects of this phyto-flavonoid against inflammation in human umbilical vein endothelial cells, attributing these effects to the downregulation of VCAM-1 and CD80 expression.^57,58 Our study is in accordance with these findings, as we observed a significant reduction in the upregulated protein expressions of ICAM-1, VCAM-1, P-selectin, and E-selectin in kidneys of rats following quercetin administration before hypobaric hypoxia exposure. The observed reduction in cell adhesion molecules could also be attributed to the decreased oxidative stress (ROS and MDA) in kidneys.

While dexamethasone is a known corticosteroid with anti-inflammatory properties, but it was less effective than quercetin in our study. It is well known that, dexamethasone works on the immune system and reduces the inflammation and therefore help in relieving the swelling, giddiness, redness and pain. But this drug has lot of side effects. Dexamethasone use, causes headache, blurred vision, dizziness or fainting, increased thirst or urination, tiredness, gastro intestinal problems, sleep disturbances.^26,59 Another fact is that, if this drug is discontinued at elevations before acclimatization, rebound problems can occur.⁶⁰ Even though pre-treatment with dexamethasone significantly reduced the levels of TNF-α in kidney homogenates, but failed to reduce IL-6 levels and also suppressed the kidney IL-2 levels more than normal values. Additionally, a significant reduction in all the aforementioned cytokines under normal conditions indicates an indiscriminate suppression of the immune response by dexamethasone. We also observed that although dexamethasone significantly reduced the expression of ICAM-1 that pointed towards its anti-inflammatory nature, but it could not reduce the expression of VCAM-1, P-selectin, and E-selectin in kidneys of rats under hypoxia. Similar findings were reported in a study carried out by Ionescu and colleagues (2011), where dexamethasone administration significantly reduced the plasma ICAM-I but not VCAM-I levels in laparoscopic cholecystectomy patients.⁶¹ The increased cell adhesion molecules are known to cause vascular leakage, perhaps this could be the reason due to which our histopathology studies have confirmed by showing the fluid accumulation in the renal tubules (Fig. 7 b i and ii; and 7. f) under hypoxia. However, in the present study, vascular leakage leading to fluid influx in to the renal tubules was not seen under quercetin prophylactic hypobaric hypoxia exposed rats indicating the protective role of quercetin in mitigating the inflammation in renal tubules. The observed anti-inflammatory effects exerted by quercetin was attributed to its inhibitory activity of NF-κB.

In order to maintain the immune response of the body, a well-balanced pro and anti –inflammatory cytokine milieu is required. The anti-inflammatory cytokine IL-10 in kidneys was drastically lowered in dexamethasone treated hypobaric hypoxia exposed rats as compared to hypobaric hypoxia control and quercetin treated hypobaric hypoxia exposed rats. Indeed, the main function of the IL-10 is to lessen the augmented pro-inflammatory cytokine levels in the body. Perhaps this could be the reason that dexamethasone treated hypobaric hypoxia exposed rats could not be able to reduce the IL-2 levels in kidneys of rats under dexamethasone treatment. Our study further observed a significant down regulation in the levels of IL-4 and TGF- β in kidneys of rats under hypobaric hypoxic conditions. Whereas, these anti-inflammatory cytokines were found to be significantly enhanced in kidneys of rats under quercetin supplementation (Fig. 4 b and c). TGF- β is known to be a pleiotropic cytokine; so, it can act, as both pro and anti-inflammatory molecule, but in our study we observed to be functioning as an anti-inflammatory molecule, as evidenced by reducing the inflammation in kidneys of rats. Literature revealed a decrease in the activation of TGF-β pathway via downregulation of TGF-β receptor type 2 (TGFBR2) under hypoxic conditions.⁶² It has been extensively studied as an anti-inflammatory molecule as TGF-β1-null mice displayed extreme inflammatory responses.⁶³ This is in consistent with the role of TGF-β in promoting the generation of immunosuppressive T-regulatory cells.^64,65

In the present study, we have observed an anti-inflammatory activity of IL-4 that could be due to its capability to inhibit pro-inflammatory cytokines such as TNF-α and IL-1 (Fig. 4 b) in kidneys of quercetin received hypobaric hypoxia exposed rats. Further the observed undue reduction in IL-10 and TGF-β levels in kidneys of rats supplemented with dexamethasone under hypobaric hypoxia indicates the enhanced inflammation and not able to prevent the vascular leakage due to enhanced cell addition molecules in the kidneys of rats as it was evidenced by histopathology studies and by the TEM imaging studies (Fig. 7 f and 8f, respectively). Another interesting observation noticed in the present study was that, the drastic reduction in TGF-β and IL-10 in dexamethasone treated rats under hypobaric hypoxia authenticated the uncontrolled inflammation in these animals as evidenced by drastic reduction in IL-2 followed by excessive production of IL-6 in the kidneys of rats (Fig. 3 b and c, respectively).

In the present study, the histopathological findings further revealed that, the reno-protective activity of quercetin over dexamethasone against hypobaric hypoxia induced damage. Further, we have noticed that treatment with quercetin aids in maintaining the normal vasculature of kidneys with intact glomerulus and renal tubules whereas hypobaric hypoxia exposed group treated with dexamethasone exhibited swelling in tubules along with unclear renal corpuscles. Simultaneously, TEM analysis of ultrathin sections of renal tissues further confirmed similar findings and suggested that, quercetin was able to maintain the cellular morphology with regular and even normal structural arrangements in kidneys. Whereas dexamethasone could not bring such satisfactory effects and therefore, not able to prevent the alterations caused by hypobaric hypoxia completely.

Further, quercetin succeeded in maintaining the hematological levels appropriately in comparison with dexamethasone. Increase in WBCs, RBCs and platelets count helps in improving and maintaining the oxygen demand and fluid electrolyte homeostasis under hypobaric hypoxic conditions.⁶⁶ Previous literature suggested, that quercetin enhances the RBC levels by increasing the production of erythropoietin.⁶⁷ Erythrocytes, being highly exposed to oxygen and given the high concentration of PUFA in their membranes, are extremely susceptible to oxidative stress. Quercetin can bind to RBCs and protect their membranes from oxidative damage and restore their endogenous glutathione content that was depleted by increased reactive oxygen species.^68,69 This study also observed an increased level of hemoglobin under quercetin treated hypobaric hypoxia exposed group. Administration of dexamethasone resulted in increased level of leukocytes in kidneys of rats under hypobaric hypoxic conditions, which indicates towards the premature release of these leukocytes from bone marrow and delayed apoptosis which might have led to organ dysfunction.^70,71 We also observed a lower cell count of WBC, lymphocytes, neutrophils and platelet count under dexamethasone treated normoxic group compared to normoxia control probably due to the excessively immunosuppressive nature of this drug.⁷¹ The prophylaxis with quercetin protected the rats by controlling the redox status in kidneys and there by protected the WBC, RBC and platelets from oxidative stress. Thus, the study observed that, quercetin as an effective and safe alternative to be supplemented prior to hypobaric hypoxia exposure.

In response to acute high altitude exposure, in order to cope up with the low O₂ availability, the body increases tissue oxygenation by enhancing the pulmonary ventilation leading to increased respiratory alkalosis.⁷² Since it is the kidney that compensate this alkalosis by excreting excess bicarbonate and retaining hydrogen ions to reduce respiratory alkalosis while maintaining the increased oxygenation.⁷³ It is obvious that, the magnitude of alterations in renal metabolism, oxidative stress, inflammation and physiological functions due to high-altitude exposure certainly impacts the health of the people working, travelling and living at high altitudes. Therefore, protecting the kidneys from inflammation is utmost important during acute high altitude exposure.

In this study, dexamethasone was used as a positive control to compare with the study drug i.e. quercetin. We have performed several biochemical assays comparing the effect quercetin and dexamethasone under hypobaric hypoxic stress. In our study dexamethasone significantly restored the oxidative imbalance and anti-oxidant activity in plasma of rats exposed to hypobaric hypoxia. It also facilitated the maintenance of some of the pro and anti-inflammatory cytokines in hypoxia exposed group. However, it also altered the levels of these cytokines under normal conditions that reflect towards its hyperactivity under normoxia control group. It either enhanced the levels of the cytokines to abnormal extent or reduced their levels to insufficiency in normoxia and hypoxia exposed groups. Studies suggest that dexamethasone promotes severe side effects such as hypertension, hyperglycemia, hydro-electric disorders and hypertension when administered through systemic route.⁷⁴ It has also been stated that long term usage of dexamethasone can downregulate the rate of skeletal muscle protein synthesis by increasing the rate of protein breakdown.⁷⁵ However, it was reported recently, that quercetin is able to protect the dexamethasone induced muscle atrophy and apoptosis by regulating the protein ratio of BAX and BCL-2 in C₂CL₂ cells.⁷⁶ Further, literature revealed that dexamethasone induces mitochondrial malfunction which leads to ATP deprivation in the cells.⁷⁷ Its involvement in causing oxidative stress by inducing ROS production which leads to DNA and protein damage has also been stated in previous studies.⁷⁸ Although, dexamethasone has its own remarkable set of anti-inflammatory benefits but at the same time shows numerous adverse effects as well. On the other hand, quercetin is a plant flavonoid which exerts its beneficiary effects (anti-oxidative property) by quenching the excessive ROS produced and directly inhibits the production of TNF- α, IL-18 and IL-1 as observed under hypobaric hypoxic stress.⁷⁹ Quercetin inhibits the production of inflammation producing enzyme i.e. cyclooxygenase and lipoxygenase.^80,81 Furthermore, Quercetin has a well demonstrated safety and tolerability profile in humans and thus comes under FDA approved GRAS (Generally Recognized as Safe) category.⁸² Our results provided substantial evidence to make use of quercetin for clinical applications. Several Clinical trials (CT) at different stages of CT-I, CT-II and CT-III for quercetin against several diseases (cancers, COPD, Inflammation and latest SARS-COV2) are still going on.83, 84, 85 Phase III trials conducted by Zwicker et al., 2019 on Thromboembolism of Vein in Colorectal, Non-small Cell Lung and Pancreatic Cancer (NC No.-NCT02195232) reported that, isoquercetin targeted the extracellular protein disulfide isomerase (PDI) and improved the markers of coagulation in advanced cancer patients. In near future, quercetin will come out to be an excellent phyto-molecule to treat several human diseases.⁸⁶

Our findings provided strong evidence to support the usage of Quercetin as a protective moiety to alleviate the oxidative stress induced renal inflammation under hypobaric hypoxia in comparison to Dexamethasone, which confers its effectiveness in preventing the renal inflammation under hypoxic stress. Therefore, it can be deduced from this study, that quercetin can be used as prophylactic measure to prevent the hypobaric hypoxia induced illness faced by soldiers, mountaineers, trekkers, pilgrims and high altitude sojourners as it facilitated the protection against inflammation in kidneys of rats at high altitude areas. The findings of this study are illustrated in Fig. 9.

Fig. 9.

Graphical demonstration of mitigation of hypobaric hypoxia induced renal inflammatory alterations by Quercetin prophylaxis: Acute (12 h) hypobaric hypoxia exposure activated the master regulatory gene of inflammation, Nuclear Factor kappa- B (NFκB). NFκB upregulation lead to the increment in pro-inflammatory cytokine levels i.e. Tumor Necrosis Factor-alpha (TNF α), Interleukin −2 (IL-2) and Interlukine-6 (IL-6) and downregulated the anti-inflammatory cytokines i.e. Transforming Growth Factor-beta (TGF-β), Interleukin-10 (IL-10) and Interleukin-4 (IL-4) in kidneys of rats. Excessive inflammation caused structural damage and disorientation of renal arrangement that further lead to vascular leakage (shown as fluid accumulation in (FA) histopathology studies) due to upregulated activation of cell adhesion molecules like Intercellular Adhesion Molecule 1(ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), P-selectin and E-selectin. Prophylaxis with quercetin, 1 h prior to hypobaric hypoxia exposure, not only aided in the reduction of pro-inflammatory cytokines but also appreciably facilitated the production of anti-inflammatory cytokines in the kidneys of rats. This protective effect of quercetin helped in maintaining the structural integrity of the kidneys under hypobaric hypoxic stress that prevented the renal damage caused by inflammation in rats at high altitude.

NF-κB - Nuclear factor kappa B, TNF-α- Tumor Necrosis Factor alpha, IL-2- Interleukin-2, IL-6- Interleukin-6, IL-10- Interleukin-10 IL-4- Interleukin-4, TGF- β-transforming growth factor beta, ↑- Upregulated, ↓- Downregulated, Ʇ- Attenuation.

5. Conclusion

The present study demonstrates that quercetin prophylaxis significantly aids in amelioration of oxidative stress and enhances the anti-oxidants in kidneys of rats under hypobaric hypoxic conditions. Quercetin prophylaxis reduced the inflammation by downregulating the NF-κB and its downstream pro-inflammatory cytokines, and facilitated an increase in the production of anti-inflammatory cytokines in kidneys of rats. The study also showed a comparative aspect between quercetin and dexamethasone and proved that quercetin is a better phyto-molecule in reducing the renal inflammation under hypobaric hypoxic stress without any side-effects against steroid therapy of dexamethasone under these circumstances. Therefore, it is inferred that quercetin mitigates the hypobaric hypoxia induced renal inflammatory alternations at high altitude areas.

Ethical approval

All the experiments involving animal studies were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), DIPAS, Delhi, India, accredited to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (IAEC No.: DIPAS/IAEC/2017/19/EXT/22).

Availability of data

All the relevant data is within the paper and its supporting information files.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.