Empagliflozin mitigates methotrexate-induced nephrotoxicity in male albino rats: insights on the crosstalk of AMPK/Nrf2 signaling pathway

Abstract

Background

The anti-diabetic drug, empagliflozin (EMPA), has many pleiotropic actions and is challenged recently to possess renoprotective properties. This renoprotective potential is proposed to be mediated via the activation of AMP-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. This research investigated the renoprotective potential and the mechanistic pathway of EMPA against methotrexate (MTX)-induced nephrotoxicity and evaluated the role of AMPK by utilizing an AMPK inhibitor, dorsomorphin (Dorso).

Methods

Thirty male Wistar rats, weighing 180–200 g, were divided equally into five groups. Group I represented the control group. Nephrotoxicity was induced in the remaining rats through the administration of a single intraperitoneal injection of MTX (20 mg/kg). Rats were then randomly assigned to: Group 2 (received MTX injection only); Group 3 (received MTX and EMPA 30 mg/kg/day); Group 4 (received MTX and Dorso 0.2 mg/kg/day), Group 5 (received MTX, Dorso, EMPA). After one week, blood samples were collected, the rats were euthanized, and renal tissues were harvested for biochemical and histomorphometric assessments.

Results

MTX produced a significant rise in serum creatinine and tissue MDA levels; an increase in BAX, p53, cytochrome-c expression; a reduction in Bcl2 level; and disruption of renal microarchitecture. In contrast, EMPA therapy in group 3, resulted in a significant improvement of all these parameters, correlated with significant increase in AMPK phosphorylation and Nrf2 expression. Importantly, the co-administration of Dorso, in group 5, prevented EMPA’s beneficial effects.

Conclusion

EMPA has a potential protective effect against MTX-induced toxicity through the activation of the AMPK/Nrf2 signaling pathway.

Background

Iatrogenic nephrotoxicity represents a notable cause of acute kidney injury. Methotrexate (MTX) is a pharmacological agent that functions as an anti-folate agent, principally by impeding the process of DNA synthesis. As an antimetabolite, it has been an effective chemotherapeutic drug for a variety of solid and hematological malignancies [1]. It also possesses anti-inflammatory, anti-proliferative, and immunosuppressive properties that can be used to treat rheumatoid arthritis, as well as other inflammatory and rheumatic diseases [2].

Although it has a broad spectrum of uses, MTX therapy is linked to several significant adverse events (AEs), even with low-dose therapy. These AEs, such as: bone marrow suppression, nephrotoxicity, and hepatotoxicity, may demand dose reduction or treatment discontinuation [2, 3].

Because MTX is mostly eliminated by the kidneys through the glomerular filtration process along with active transportation, renal toxicity is a predictable side effect. Actually, nephrotoxicity is the most concerning issues that limits its therapeutic utility [4]. Yet, the pathogenesis of MTX-induced nephrotoxicity remains unclear. Previous studies reported that MTX crystallization in the renal tubules is implicated in its nephrotoxicity. However, it is increasingly evident that oxidative damage, DNA suppression, inflammation, mitochondrial dysfunction, and apoptosis could have key roles in this nephrotoxicity [5,6,7].

Mitochondria have been identified as a target in MTX-induced tissue subcellular damage. MTX was found to: 1. possess an impact on mitochondrial redox regulation and energy production, 2. block the complexes I, II, and IV activities, as well as 3. cause mitochondrial enlargement and rupture [8]. Mitochondrial dysfunction and the resulting ATP depletion disrupt tight junctions, causing a subsequent disruption of renal functions [9]. In this context, finding a nephroprotective drug to mitigate renal toxicity is a great pressing need.

Sodium–glucose cotransporter 2 (SGLT-2) inhibitors represent a novel group of anti-diabetic treatments that inhibit sodium and glucose reabsorption from the proximal convoluted tubules, resulting in glycosuric and natriuretic effects [10]. Apart from their effect on the SGLT channel, new evidences revealed that SGLT-2 inhibitors have a variety of pleiotropic effects. SGLT-2 inhibitors were shown to be effective in improving inflammation [11], oxidative stress [12], apoptosis [13], ionic dyshomeostasis [14], and importantly, mitochondrial dysfunction [15, 16]. In view of these actions, SGLT-2 inhibitors have been explored in a variety of disorders such as heart failure, degenerative diseases, chronic kidney disease. Numerous studies have revealed that they have a protective effect on the heart and a potential to play a similar protective action on the kidneys [17,18,19].

The renoprotective activity of the SGLT-2 inhibitor, empagliflozin, has been evaluated in some studies with different pathologies, including diabetic nephropathy and renal ischemia caused by ureteric obstruction [20, 21]. Additionally, the EMPA REG OUTCOME Trial indicated that empagliflozin reduced the incidence of renal adverse events [22]. Accordingly, empagliflozin may be effective in preventing acute renal damage in special situations [23].

Several studies have found a link between SGLT-2 inhibitors and AMP-activated protein kinase (AMPK) activation, a kinase which is active during periods of low energy availability. To boost ATP production and minimize ATP consumption, it phosphorylates particular enzymes and growth control nodes [24, 25]. Beyond being a vital regulator of energy production in the cells, AMPK plays a crucial role in essential pathways like redox reduction and regulates specific elements of mitochondrial biology and homeostasis [25]. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor which regulates the gene expression of many protective proteins, including those involved in anti-oxidant, anti-inflammatory, and detoxification processes. The putative overlap in AMPK and Nrf2 signaling, either by direct phosphorylation or through the downstream events of the activated AMPK, is of great importance in lending the cells resilience against oxidative stress and boosting the redox mechanism [26].

Based on the aforementioned information and the expected molecular signaling pathway, this research was planned to assess the possible nephroprotective impact of empagliflozin against the MTX-induced kidney injury and to assess the possible mechanistic pathways including its effect on oxidative stress, apoptosis, mitochondrial dysfunction, and endothelial dysfunction among with studying its effect on Nrf2 expression. Also, we aimed to investigate the role of AMPK stimulation on all these parameters by using dorsomorphin (AMPK inhibitor).

Methods

Sample size calculation

As stated by Faul et al. [27], the sample size of this study was determined using the G*Power program (Version 3.1.9.2, by Franz Faul, Kiel, Germany). The primary outcome of the study was determined on the basis of a prior investigation carried out by Park et al. [28] to be the level of renal AMPK expression. Based on this proposal, a sample size of 30 rats, randomized into 5 groups (n = 6), attains a power of 90% to notice the effect size with an alpha level of 5%, when utilizing the one-way ANOVA test.

Animals and ethical statement

The study included 30 mature, healthy, 3 months aged, matched for age and weight (200–220) g, Wistar strain albino male rats. The rats were purchased and reared in the institutional animal house. The investigational protocol was reviewed and authorized by Cairo University’s Institutional Animal Care and Use Committee (CU-IACUC) (Approval number CU IIIF8619 on Jan 2020) in compliance with ARRIVE principles and the Guide for the Care and Use of Laboratory Animals procedure (NIH publication). The rats were housed in a standardized laboratory environment, with a temperature of 25 + 2 °C, a 12:12 light–dark cycle, and a relative humidity of 50 + 5%. They were kept in a plastic cage (33 cm × 40 cm × 17 cm). All the experiment measurements were taken during the light period (08:00–16:00). The investigational protocol was reviewed and authorized by the institutional animal care committee in compliance with ARRIVE principles and the Guide for the Care and Use of Laboratory Animals procedure (NIH publication). Water and chow pellets were accessible to the rats at all times without restriction.

Drugs

(1) Methotrexate ampoule 50mg (Haupt Pharma Gmbh Pharmaceutical Company, Germany) and (2) empagliflozin (Boehringer Ingelheim Pharmaceutical Company, Germany) were supplied as a tablet, dissolved in distilled water as a suspension in a concentration of 3%, and freshly prepared to be administrated in a dose of 30 mg/kg orally, (3) dorsomorphin (Dorso) powder (Sigma Pharmaceutical Company, Egypt) was dissolved in distilled water in a concentration of 0.02% and freshly prepared to be administrated in a dose of 0.2 mg/kg by intraperitoneal route.

Experimental design

Scheme 1 depicts the experimental schedule for this research. After one week of acclimatization, rats were assigned at random to one of five main groups of six rats each. Empagliflozin’s renoprotective ability was evaluated in the following manner:

Scheme 1

Clinicopathological characteristics

Group (1): Rats received a single intraperitoneal (I.P) injection of 0.1 ml saline and one ml distilled water orally daily throughout the experiment period and were designated as the control group; Group (2): Rats received a single (I.P) injection of MTX at a dose of 20 mg/kg (5) and one ml distilled water daily, along with an I.P injection of 0.1 ml saline throughout the experiment period. This group was nominated as MTX-nephrotoxic group; Group (3)/MTX/EMPA: Rats received a single I.P. injection of MTX at a dose of 20mg/kg, plus empagliflozin (EMPA) orally at a daily dose of 30 mg/kg [17] for one week, and an intraperitoneal injection of 0.1 ml saline daily starting from day one for one week; Group (4): Rats received a single I.P. injection of MTX at a dose of 20mg/kg, plus daily I.P. injection of Dorso in a dose of 0.2 mg/kg [29], and oral one ml distilled water daily starting from day one for one week; Group (5)/MTX-EMPA-Dorso: Rats received a single intraperitoneal injection of MTX at a dose of 20 mg/kg, plus a daily I.P injection of dorsomorphin at a dose of 0.2 mg/kg, and oral empagliflozin at a daily dose of 30 mg/kg starting from day one for 1 week. At the end of the experimental period (7 days), rats of every studied group were subjected to 24-h urine collection by placing them individually in metabolic cages (Orchid Scientific Ltd, India) with free access to food and water. The supernatant from urine samples was stored at − 20 °C after centrifugation. The rats were then euthanized using a 90 mg/kg ketamine–10 mg/kg xylazine cocktail (Scheme 2).

Scheme 2

Clinicopathological characteristics

Measurements

Renal function tests

Following euthanasia, a cardiac puncture was employed to collect blood samples. Following centrifugation of the blood, the serum was promptly transferred to 1.5-ml polypropylene tubes and stored at − 20 °C for the evaluation of renal function tests (serum urea, serum creatinine) via colorimetric assay, utilizing urea and creatinine detection kits (cat numbers URE118100 and CRE106120 for urea and creatinine, respectively) purchased from Bio-Diagnostic company, Egypt, and assessed according to the instructions provided by the manufacturer. The collected urine supernatants were used for measurement of creatinine clearance. Creatinine clearance was calculated using the formula:

$$\begin{aligned} {\text{Clearance }}\,{\text{creatinine }}\left( {{\text{ml}}/{\text{min}}} \right) \, & = {\text{ Creatinine }}\,{\text{in}}\,{\text{ urine }}\left( {\text{U}} \right) \, \left( {{\text{mg}}/{\text{dl}}} \right) \, \\ & \quad \times {\text{urine}}\,{\text{ volume }}\left( {\text{V}} \right) \, \,{\text{of}}\,{ 24}\,{\text{h}}\,{\text{ in }}\,{\text{ml}}/{\text{ Creatinine}}\,{\text{ in }}\,{\text{serum}}\, \, \left( {{\text{mg}}/{\text{dl}}} \right)\, \, \left( p \right) \, \times { 144}0 \\ \end{aligned}$$

1440 is the number of minutes in 24 h (60 min × 24 h = 1440 min).

Renal tissue homogenates

Kidney samples were homogenized; then, the supernatant was extracted and subsequently stored at − 80 °C.

Assessment of the oxidative stress markers and the mitochondrial protein cyclophilin d in the homogenates of renal tissue

To assess the extent and the impact of oxidative stress on renal tissue injury, and the potential renoprotective action of empagliflozin, malondialdehyde (MDA) and reduced glutathione (GSH) levels were measured using colorimetric assay [30, 31]. Specific colorimetric detection kits were used (Biodiagnostic Company, Egypt. Cat. No: GR 2510 for reduced glutathiones and Cat. No: MD 2528 for MDA) and assessed according to the instructions provided by the manufacturer. Additionally, the measurement of the tissue level of the mitochondrial protein-cyclophilin D (CypD) was assessed using ELISA kit from (My BioSource Co., San Diego, California, USA, Cat. No—MBS2097964 96 Tests).

Assessment of the expression level of renal BAX and Bcl2 genes by quantitative PCR (qPCR)

Quantitative PCR analysis was conducted to measure the levels of apoptotic and anti-apoptotic proteins, BAX (Bcl2 associated × protein) and Bcl2 (B cell lymphoma 2). The tissue homogenate was processed for total RNA extraction with Thermo Fisher Scientific Inc., Germany’s GeneJET Kit (#K0731), according to manufacturer instructions. Two-step method was used to synthesize complementary DNA (cDNA) using reverse transcriptase enzyme (#K4374966, Thermo Fisher Scientific, USA, for reverse transcriptase). An Applied Biosystem and software version 3.1 (StepOneTM, USA) was used for the real-time qPCR amplifier and descriptive analysis. The thermal cycling profile was 15 min at 45 °C for cDNA synthesis, followed by 5 min at 95 °C for reverse transcriptase inactivation and polymerase activation. Following that, 40 cycles of PCR amplification were performed. Each cycle included 15 s of DNA denaturation at 95 °C, 20 s of primer annealing at 55 °C, and 30 s of Taq polymerase extension at 72 °C. Table 1 shows the primer sequences for BAX and Bcl2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to standardize the data. The 2-ΔΔCT method was used to calculate the target gene’s relative expression based on the mean critical threshold (CT) of the housekeeping gene GAPDH [32].

$$\Delta {\text{ Ct }} = {\text{ Ct }}\,{\text{assessed }}\,{\text{gene }}{-}{\text{ Ct}}\,{\text{ reference}}\,{\text{ gene}}$$

$$\Delta \,\Delta \,{\text{ Ct }} = \, \Delta \, \,{\text{Ct }}\,{\text{sample }}{-}{\text{ Ct}}\,{\text{ internal }}\,{\text{control }}\,{\text{gene}}$$

$${\text{RQ }}\left( {{\text{relative}}\,{\text{ quantification}}\,{\text{ expression}}} \right) \, = { 2 }{-} \, \left( {\Delta \, \,\Delta \,{\text{ Ct}}} \right)$$

Table 1 Primers sequence for BAX, Bcl2, and the housekeeping gene GAPDH

Assessment of AMPK and p53 expression level by Western blot

Excised renal tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer, which consisted of 50 mM Tris HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS. A cocktail of phosphatase and protease inhibitors was added to protect protein integrity. Protein measurement was taken using the Bradford method [33]. 10 μg of protein from each sample was loaded and separated by SDS-PAGE and then transferred to a PVDF membrane. The membrane was then blocked with 5% bovine serum albumin (BSA). Subsequently, the membrane was incubated at 4 °C overnight on a roller shaker with the primary antibodies for the P-Thr172-AMPK antibody (Abcam, USA, catalog number #ab32047) and the p53 antibody (Abcam, catalog number #ab26). The levels of AMPK and p53 in all investigated samples were quantified using densitometric analysis of the immunoblot, with beta-actin (a housekeeping protein) employed as a control sample for protein normalization. Bio-Rad’s ChemiDoc MP imaging system (version 3) was used for this analysis.

In vitro experiment on isolated aortic ring to assess endothelial dysfunction

The thoracic aortic artery was cleansed of connective tissue and sliced into 2–3-mm-long rings. The rings were suspended in 10 ml organ baths (Manufacturer: Panlab, Spain Model Number: LE11200) at a temperature of 37 °C. The organ baths contained a Krebs bicarbonate solution with the following composition: NaCl (119 mM), KCl (4.7 mM), KH₂PO₄ (1.18 mM), MgSO₄ (1.18 mM), CaCl₂ (1.25 mM), NaHCO₃ (25 mM), and d-glucose (11 mM). The solution was oxygenated with 95% O₂ and 5% CO₂ and had a pH of 7.4. Ring was attached to isometric force recording transducers (Manufacturer: Panlab, Spain Model number: TR1201) from the upper sides, while the lower sides were fixed to stationary hooks. A data acquisition system (Manufacturer: ADinstruments, Australia Model number: Po Werlab 4/30 with Lab Chart Pro (product#ML866/p)) was used to record contractions [34]. A dose concentration curve of phenylephrine-induced contraction was performed on isolated aortic rings from normal rats to determine the submaximal contraction produced by phenylephrine. After reaching a plateau of contraction, cumulative concentrations of acetylcholine (Ach) were applied. The concentration that resulted in a 50% reduction of the phenylephrine-induced contraction was extrapolated. This concentration of Ach was then applied to pre-contracted aortic rings with the extrapolated submaximal concentration of phenylephrine from rats in groups 2–5. The percentage of relaxation produced by Ach from the maximum contraction was measured.

Histopathological examination of the kidney

Specimens of each kidney were cut off and preserved in neutral buffered formalin for 24 h. They were then embedded in paraffin after being washed, dried using ethyl alcohol serial dilutions, and cleared in xylene to evaluate changes in histopathology. Subsequently, transverse sections of 4–5 μm thickness from paraffin blocks were placed on glass slides and stained using hematoxylin and eosin (H&E) [35].

Immunohistochemical studies

Immunohistochemical studies were conducted to detect the expression of Nrf2 and cytochrome-c oxidase (cyt-c) on paraffin sections of kidneys from different study groups according to method adopted by Abd El-Rahman and Fayed [36]. The tissue slices were incubated for 60 min at 37 °C with monoclonal antibodies against Nrf2 and cyt-c (Abcam, Cambridge, MA, USA, ab31163 and ab204790) at a dilution of 1:200 and 1:100, respectively. Following a PBS wash, the sections were incubated for 60 min at 37 C with a secondary antibody (Dako, Carpinteria, CA, USA). Incubation was then performed using biotinylated horseradish peroxidase H and Avidin DH complex from the Vectastain ABC peroxidase kit (Vector Laboratories Inc., Burlingame, CA, USA). The immunological reactivity of each marker was measured as the optical density of positively stained (brown) patches in randomly selected high-power (X400) microscopic fields using ImageJ software from the National Institutes of Health, version 1.46a, Bethesda, MD. All pathological evaluations were carried out by an investigator who was blinded of the identity of the samples to avoid bias.

Statistical analysis

The Statistical Package for the Social Sciences (SPSS) version 28 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. The data are presented as the mean (M) ± standard deviation (SD) for the normally distributed quantitative variables or median and range for non-normally distributed quantitative variables. The Shapiro–Wilk test was employed to ascertain the normal distribution of the data. To determine significant differences between the means of the measured parameters in the study groups, analysis of variance (ANOVA) with multiple comparisons post hoc test was used in normally distributed quantitative variables, while the nonparametric Kruskal–Wallis test was employed for non-normally distributed quantitative independent variables, followed by Dunn’s multiple comparison test. A statistically significant p value of less than 0.05 was evaluated.

Results

Assessment of renal function tests

As depicted in Table 2, after induction of nephrotoxicity with a single I.P injection of MTX, MTX-induced nephrotoxicity was revealed in group 2, which exhibited a significant deterioration of kidney functions represented by an elevation in the mean value of both serum creatinine and serum urea in comparison with their corresponding values in group1. Moreover, a marked decline in the calculated creatinine clearance was recorded among the MTX nephrotoxic group 2 in comparison with its corresponding values in group 1 (p < 0.0001). Empagliflozin-treated group 3 showed a significant decline in both serum creatinine and serum urea as compared to group 2 (p < 0.0001). Furthermore, a significant elevation in creatinine clearance was reported in the empagliflozin-treated group 3 in comparison with group 2 (p < 0.0001). On the other hand, in group 5, which received MTX + EMPA + Dorso, there was a significant elevation in the mean values of both serum urea and creatinine (p < 0.0001). This was accompanied by a significant decline in the median value of calculated creatinine clearance in comparison with group 3 (p < 0.0001), Table 2.

Table 2 The mean serum creatinine (mg/dl), mean serum urea (mg/dl), and the median values of creatinine clearance(ml/min) in different studied groups of male albino rats

Assessment of oxidative stress markers (MDA and GSH) and (cyclophilin D) in renal tissue

MTX significantly raised renal tissue levels of MDA in the MTX-nephrotoxic group 2, with a remarkable decline in the mean renal tissue level of GSH (p < 0.0001, in comparison with their corresponding values in the control group 1). However, administration of empagliflozin to the MTX-EMPA group 3 showed a significant decline in the mean tissue MDA level (p < 0.0001) with an elevation in the mean renal tissue level of glutathione (p < 0.0001) in comparison with group 2, while dorsomorphin administration with empagliflozin in group 5 showed a significant elevation in the mean tissue level of MDA (p < 0.001), along with a significant decline in the mean tissue GSH level (p < 0.001) compared to the MTX-EMPA group 3 (Fig. 1A, B).

Fig. 1

A The mean renal tissue MDA level (mmol/gm tissue), B the mean tissue glutathione (mmol/gm tissue), C the mean cyclophilin D level in renal tissue (ng/gm tissue). Values represent means ± SD (n = 6). A significant difference is reported when p is less than 0.05. Data analyzed by one-way ANOVA followed by post hoc Tukey’s test. Groups sharing same letter are insignificantly different. MTX methotrexate, EMPA empagliflozin, Dorso dorsomorphin, G group

Regarding tissue levels of the mitochondrial protein CypD, there was a significant rise in the mean value of CypD among group 2 (p < 0.0001) relative to the mean value in group 1. Conversely, in group 3, empagliflozin therapy showed a significant decrease in CypD level in comparison with MTX-nephrotoxic group 2 (p < 0.0001) and reached a level approximate to that of group 1. Additionally, in group 5, the administration of dorsomorphin with empagliflozin showed a significant increase in CypD levels (p < 0.05) compared to the MTX-EMPA group 3, as shown in Fig. 1C.

Assessment of renal tissue expression of apoptosis markers, BAX and Bcl₂ by quantitative PCR analysis

This study revealed a considerable upregulation of the gene of Bax protein (p < 0.0001) in the MTX-nephrotoxic group 2, in comparison with group 1, with a concomitant decline in Bcl2 Mrna gene expression level (p < 0.0001), resulting in a significant increase in the Bax/Bcl2 ratio. Empagliflozin treatment in MTX-EMPA group 3 significantly suppressed the MTX-induced elevation in Bax mRNA gene expression and reduction in Bcl2 mRNA gene expression (p < 0.0001). Furthermore, dorsomorphin administration with empagliflozin in MTX-EMPA-Dorso group 5 showed a significant elevation of Bax mRNA gene expression (p < 0.05), while it showed a significant reduction of Bcl2 mRNA gene expression (p < 0.0001) in comparison with the empagliflozin-treated group 3 (Fig. 2C).

Fig. 2

A The mean tissue phosphorylated p-Thr172 AMPK expression levels (relative expression to ß-actin) in different studied groups of male albino rats, B the mean values of p53 relative expression in renal tissue (relative expression to ß-actin) in different studied groups of male albino rats, C the mean values of BAX and Bcl-2 relative expressions (relative to housekeeping gene GAPDH) in renal tissue in different studied groups of male albino rats, and D scanned quantitative Western blot gel of phosphorylated AMPK, P53, and the housekeeping gene ß-actin. Values represent means ± SD (n = 6). A significant difference is reported when P is less than 0.05. Groups sharing the same letter are insignificantly different. Data analyzed by one-way ANOVA followed by post hoc Tukey’s test. MTX methotrexate, EMPA empagliflozin, Dorso dorsomorphin, G group

Assessment of renal tissue expression of p53 protein and AMPK by Western blot analysis

MTX-nephrotoxic group 2 exhibited significant elevation in the mean value of p53 expression (p < 0.001) with a significant decline in AMPK phosphorylation (p < 0.001) as in comparison with normal control group 1, while MTX-EMPA group-3 showed a significant decrease in the mean value of p53 expression with a significant increase in phosphorylated AMPK level in comparison with MTX-nephrotoxic group 2 (p < 0.0001). On the other hand, MTX-EMPA-Dorso group 5 showed a significant change compared to group 3 as regard both AMPK and P53 expression, Figure 2A, B, and D.

Histopathological results

Assessment of empagliflozin on histopathological morphometric changes by H&E

Renal histopathological changes are displayed in Fig. 3a–e. The control group showed normal tubular and glomerular renal architecture (Fig. 3a), while MTX produced a considerable perturbation of renal architecture (Fig. 3b1, b2). Pathological features in the MTX-nephrotoxic group 2 were mainly in the form of tubular necrosis, vacuolar degeneration, nuclear pyknosis, and dispersed apoptosis. In this group of rats, the renal glomeruli showed an increase in the thickness of the Bowman’s capsule with inflammatory cells. There were also casts in the Bowman’s space and a small amount of fibrinous exudate with intertubular vascular congestion. Additionally, the glomerular tufts displayed focal thickening of the glomerular basement membrane together with vacuolation of their endothelial linings (Fig. 3b1, b2).

Fig. 3

Photomicrographs of H&E-stained sections of kidney tissues showing a control rat showing normal histological structure of the renal tubules (RT) as well as the renal glomeruli. (RG), b1 and b2 MTX-nephrotoxic rat showing thickening of the kidney capsule (KC), vacuolar degeneration (arrow) and necrosis (dotted arrow) of tubular epithelium, congestion of inter-tubular vessels (short arrow), b2 proliferation of partial epithelium of Bowman’s capsule (thin arrow), focal thickening of the glomerular basement membrane (thick arrow), and granular cast formation in the tubular lumens (short arrow). c1, c2 MTX + EMPA-treated rat showing c1 mild degenerative changes (arrow) of renal tubular epithelium and few eosinophilic material in few Bowmans’ spaces (dotted arrow), c2 mild granular (arrow) and vacuolar (thin arrow) degeneration, scattered necrosis (dotted arrow) of renal tubular epithelium. d1, d2 MTX-Dorso-administrated rat showing d1 segmental thickening of the glomerular basement membrane (arrow) and perivascular edema (dotted arrow), d2 moderate vacuolar degeneration of the renal tubular epithelial linings with scattered necrosis (arrow), multifocal thickening of the glomerular basement membrane (short arrow). e1 and e2 renal section in MTX + Dorso + EMPA administrated rat showing e1 moderate degree of tubular epithelial degeneration and necrosis (arrow) and attempts of hyaline cast formation (short arrow) in the lumen of some tubules, e2 moderate tubular epithelial vacuolar degeneration (dotted arrow), focal thickening of glomerular basement membrane (short arrow). MTX methotrexate, EMPA empagliflozin, Dorso dorsomorphin, G group

Pathological changes induced by MTX markedly improved to near normal in the MTX-EMPA group 3, showing almost complete restoration of the glomerular and tubular structure except mild degenerative changes of the renal tubules that were still observed in few sections. These changes observed were in the form of minor granular and vacuolar degeneration, scattered necrosis of tubular lining epithelium, a limited number of desquamated cells, and few luminal casts (Fig. 3C1, C2). Notably, the administration of dorsomorphin with empagliflozin to group 5 remarkably averted the improved histopathological features attained by empagliflozin in group 3. The examination of renal tissues of this group revealed moderate tubular epithelial degeneration, necrosis, desquamation, and early hyaline cast formation in the lumen of some tubules with the focal thickening of the glomerular basement membrane as depicted in Fig. 3E1, E2.

Immunohistochemical assessment of the expression of cytochrome-c and Nrf2

The release of cyt-c was significantly increased among the MTX-nephrotoxic group 2 (p < 0.0001) in comparison with group 1. However, empagliflozin-treated group 3 showed a significant regression of cyt-c release induced by MTX (p < 0.0001). Moreover, dorsomorphin administration in group 5 significantly increased cyt-c expression compared to the MTX-EMPA group 3 as displayed in Fig. 4.

Fig. 4

Photomicrographs of immunohistochemical staining of kidney tissue for Nrf-2 expression in various experimental groups, showing mild increased expression in MTX-nephrotoxic rats, significant increased expression in MTX + EMPA treated rats, as well as mild and moderated increased expression in MTX + Dorso and MTX + Dorso + EMPA rats as presented by the quantitative estimation of expression of Nrf-2. Values represent means ± SD (n = 6). A significant difference is reported when p is less than 0.05 and determined by one-way ANOVA followed by post hoc Tukey’s test. Groups sharing the same letter are insignificantly different. MTX methotrexate, EMPA empagliflozin, Dorso dorsomorphin, G group

Regarding the expression of Nrf2, it was significantly higher in rats of group 2 subjected to MTX nephrotoxicity relative to the control rats in group 1 (p < 0.0001), while empagliflozin-treated group 3 showed a significant increase in the antioxidant defense of Nrf2 expression compared to the MTX-nephrotoxic group 2 (p < 0.0001). Interestingly, MTX-EMPA-Dorso group 5 showed a significant suppression to the increased Nrf2 expression achieved by empagliflozin treatment in group 3 (p < 0.0001) as displayed in Fig. 5.

Fig. 5

Photomicrographs of immunohistochemical staining of kidney tissue for cytochrome-C expression in various experimental groups showing marked expression MTX-nephrotoxic G2, MTX + Dorso G4, and MTX + Dorso + EMPA compared to decreased its expression in MTX + EMPA-treated rats as presented by the quantitative analysis of optical density of cytochrome-c expression. Values represent means ± SD (n = 6). A significant difference is reported when P is less than 0.05 and determined by one-way ANOVA followed by post hoc Tukey’s test. Groups sharing the same letter are insignificantly different. MTX methotrexate, EMPA empagliflozin, Dorso dorsomorphin, Cyt-c cytochrome-c oxidase, G group

Assessment of empagliflozin on the percentage of aortic relaxation in response to cumulative dose of acetylcholine

Phenylephrine concentration that produced the submaximal contraction of the isolated aortic ring (40 µg/10 ml bath) was applied to isolated aortic rings of rats of groups 2–5; then Ach, at a concentration of 60 µg/10 ml bath, was applied. This concentration of Ach was chosen as that caused a 50% relaxation of maximum aortic ring contraction by phenylephrine in the normal control group. The percentage of relaxation of the contracted aortic ring caused by this fixed concentration of Ach was estimated in the different studied groups.

MTX-injected rats in group 2 showed failure of relaxation of aortic rings in response to Ach with 0% relaxation of the maximum aortic rings’ contraction induced by phenylephrine, while empagliflozin therapy in group 3 showed a significant rise in the percentage of relaxation of their aortic rings in response to Ach, with a median relaxation value of 34% compared to the median of MTX-nephrotoxic group 2 (p < 0.05). Dorsomorphin co-administration with empagliflozin in group 5 significantly decreased the percentage of relaxation of the aortic ring by Ach relative to group 3, with a median value of 3.5% (p < 0.05), regressing its previously attained beneficial effect on the endothelial dysfunction as depicted in Fig. 6.

Fig. 6

The percentage of aortic relaxation produced by Ach (60 mg/10 ml bath) on phenylephrine-induced contractions of the aortic ring in different studied groups of male albino rats. Values represent median with min and max, data analyzed by Kruskal–Wallis test. A significant difference is reported when p is less than 0.05. Groups sharing same letter are insignificantly different. MTX methotrexate, EMPA empagliflozin, Dorso dorsomorphin, G group

Discussion

MTX-induced nephrotoxicity is a serious adverse drug event that limits the use of this drug, despite playing a crucial function in the therapeutic regimens of many auto-immune and cancer diseases. The pathophysiology of MTX-induced nephrotoxicity seems to be multifactorial and is not yet fully elucidated. However, emerging data suggest involvement of oxidative stress, mitochondrial dysfunction, endothelial dysfunction, and apoptosis in the development of nephrotoxicity [5, 6].

Thus, addressing these pathways marks an exciting new era area for research to develop an effective protective medication against MTX-induced nephrotoxicity.

Emerging data showed that empagliflozin has many pleiotropic effects including reno and cardioprotective effects in many cardiovascular and renal disorders [37, 38].

According to certain studies, empagliflozin can activate AMPK, an essential regulator of cell energy hemostasis [39, 40]. AMPK serves as a crucial mediator of many pathways, including redox generation, fatty acid oxidation, inflammatory production, cell apoptosis, mitochondrial function, and autophagy [25, 41]. It also regulates endothelial function through boosting endothelial nitric oxide synthase (eNOS) [42, 43].

Recent studies have reported a relationship between AMPK and Nrf2, a critical transcriptional factor in sustaining mitochondria’s structural and functional integrity through enhancing the expression of several anti-oxidant and anti-apoptotic proteins [44, 45]. Nrf2 and AMPK are functionally linked and have cascade-like effects. The studies have referred to an AMPK-driven enhanced Nrf2 signaling axis via direct phosphorylation of Nrf2 or downstream events of the activated AMPK [26, 46, 47].

Consequently, empagliflozin was predicted to demonstrate a renoprotective potential against methotrexate-induced nephrotoxicity. We hypothesized that the mechanism by which empagliflozin exerts its beneficial effect is mediated by AMPK activation either directly or through activation of the Nrf2 signaling pathway.

In the current study, the oxidative stress pathway was assessed by measuring MDA and glutathione levels in kidney tissue. Expression of p53, BAX, and Bcl2 was used to assess the apoptosis pathway in different groups. Both cyt-c expression and CypD levels in renal tissues were used as indicators for mitochondrial dysfunction. Furthermore, an in vitro experiment was done to assess the endothelial function by investigating the relaxing ability of acetylcholine on a pre-contracted isolated aortic ring induced by phenylephrine. The role of AMPK activation/blockage in regulating levels of oxidative stress markers and CypD, gene expression of Bax/Bcl2, and expression of p53 and cyt-c was assessed, along with its impact on the Nrf2 expression level, through the use of the AMPK inhibitor dorsomorphin.

In the present study, MTX injection to rats of group 2 resulted in a significant renal injury with a significant increase in serum creatinine and urea, with reduced estimated creatinine clearance. This was confirmed by the histopathological perturbation observed in this group, which is in accordance with other studies [3, 48, 49].

Additionally, MTX injection to rats of group-2 caused a significant greater tissue MDA levels and lower tissue GSH levels, implicating the role of oxidative stress in the MTX nephrotoxic effect. This is in concordance with many studies [3, 49]. Oxidative stress can then induce cell injury via different mechanisms including lipid peroxidation, protein oxidation, antioxidant enzyme inactivation, and DNA damage, resulting in cell damage [50].

Enhanced cyt-c release and increased CypD level were also reported in this group. The release of cyt-c from the mitochondria indicates a significant mitochondrial damage. In such cases, mitochondrial dysfunction may also contribute to MTX-induced nephrotoxicity. A significant increase in CypD protein level increased the susceptibility of isolated mitochondria to the permeability transition triggered by Ca²⁺ and oxidative stress [51].

Furthermore, increased BAX with decreased Bcl2 gene expression and increased p53 expression was observed in the MTX-nephrotoxic group 2 rats. These data support the concept that apoptosis is involved in the pathogenesis of MTX toxic actions. This is in agreement with other studies [49, 52]. In response to stress stimuli, p53 protein translocates to the outer mitochondrial membrane causing Bax to be released mediating mitochondrial dysfunction and ending with apoptosis and cell death. In addition, p53 also controls necrotic cell death through interacting with CypD and dynamin-related protein 1 (Drp1) [53].

In the current study, MTX injection to nephrotoxic group 2 rats caused failure of pre-contracted aortic rings to relax in response to acetylcholine, indicating development of endothelial dysfunction with inability of endothelial cells to release nitric oxide. Consistent with this finding, other studies also reported the ability of MTX to promote vascular endothelial dysfunction either via increasing oxidative stress or directly injuring the endothelium. In their studies, they have found that MTX can also cause oxidative stress by inducing endothelial dysfunction in the form of endothelium shedding, increased oxidative stress, lower serum nitrite levels, and increased aortic collagen deposition was all associated with methotrexate exposure [54, 55]. This implicates the interplay between oxidative stress and endothelial dysfunction in the development of MTX-induced nephrotoxicity.

Conversely, in this research, administration of empagliflozin to rats of group-3 resulted in a significant improvement in the renal function tests in the MTX injected rats together with an improvement in the distorted renal architecture induced by MTX as shown by histopathological examination.

Empagliflozin administration to this group also produced a significant improvement in oxidative stress markers (MDA and GSH), apoptotic markers (p53, Bax/Bcl2), with significant decrease CypD, and cyt-c in comparison with the MTX-nephrotoxic group 2, implicating significant effects of empagliflozin against oxidative stress, mitochondrial dysfunction, and apoptosis induced by MTX. These are in accordance with many previous studies [56,57,58,59,60]. Furthermore, empagliflozin-treated group 3 showed a significant improvement in MTX-induced endothelial dysfunction, as evidenced by the significant increase in the percentage relaxation of the pre-contracted aortic rings. This finding was similar to what was reported in another study [61]. In their study, they reported that empagliflozin could improve endothelial function and prevent aortic stiffness through inhibition of membrane-anchored matrix metalloproteinase activation, resulting in a positive effect on periarterial fibrosis.

This group also showed enhanced Nrf2 expression levels. This is in accordance with another study conducted by Osman et al., which demonstrated the beneficial renoprotective effect of empagliflozin therapy against oxidative stress, apoptotic pathway, and inflammation with an increase in renal Nrf2/PPARγ (peroxisome-proliferator activator receptor γ)/HO1expression (heme oxygenase 1) [59].

The enhanced Nrf2 expression caused by empagliflozin together with the improvement reported in oxidative markers, apoptotic and anti-apoptotic proteins, mitochondrial protein CypD, and cyt c in this group (group-3), implicating that all these beneficial effects could be linked with the increased Nrf2 signaling pathway.

Another study conducted by Radwan et al. [62] using curcumin against MTX-induced nephrotoxicity, reported that it had antioxidant, anti-inflammatory, and anti-apoptotic activities through controlling the expression of renal Nrf2/HO-1.

However, in the current study we did not assess other Nrf2-related parameters like antioxidant response element (ARE), PPARγ, and HO-1 expression.

In the current study, empagliflozin administration to group 3 rats was associated with an increase in the expression of phosphorylated AMPK. The elevated phosphorylated AMPK expression reported in this group was most importantly found to be linked to all the beneficial effects reported including enhanced Nrf2 expression and the improvement reported against oxidative stress, apoptosis, mitochondrial dysfunction, and vascular reactivity.

On the other hand, the marked reported improvement produced by empagliflozin in group 3 rats was suppressed with the concomitant treatment by the AMPK inhibitor dorsomorphin in group 5 rats, with significant deterioration of renal function tests, oxidative markers, p53, Bax/Bcl2, CypD, cyt-c, percentage of aortic ring relaxation induced by Ach, and Nrf2 expression levels, suggesting that the renoprotective actions of empagliflozin are entirely dependent on AMPK-mediated actions that are also regulating the Nrf2 signaling pathway. This AMPK-dependent Nrf2 activation was recently reported in a study done by Lu et al. [39] on a diabetic nephropathy rat model.

Although Ala et al. [60], in contrast to our findings, ascertained that empagliflozin did not increase phosphorylated AMPK expression in rats with ischemic reperfusion I/R injury, the inconsistency between results could be ascribed to variations in the experimental model, the degree of the renal insult, the timeline of the experimental design, and the duration of exposure to the nephrotoxicity-induced agent. However, further well-designed clinical trials are required to validate the future potential of empagliflozin prescription to protect vulnerable patients from MTX-induced nephropathy.

Conclusion

In conclusion, our findings elucidated that empagliflozin represents a new promising therapeutic modality against the nephrotoxicity induced by MTX. Anti-oxidant, anti-apoptotic, improving mitochondrial dysfunction, and improving vascular function are all possible mechanistic pathways in its effect. It is suggested that all these effects could be attributed to the increased Nrf-2 expression induced by empagliflozin. Empagliflozin’s renoprotective effect and all these protective properties are entirely dependent on AMPK-mediated actions which also control the Nrf2 signaling pathway.

Limitations of the study

In this study, we did not assess the inflammatory pathway as a documented pathway in the pathogenesis of MTX-induced nephrotoxicity. Another limitation was that the molecular pathways and post-translational genes involved in the signaling pathway between AMPK and Nrf2 were not assessed. Also, histopathological examination of ultrastructural pathological changes in kidney tissues, like mitochondrial changes, cannot be assessed by a light microscope alone and needs the use of an electron microscope. It would be better to estimate protein expression of all proteins using Western blot assay rather than qPCR which estimates mRNA expression and immunohistochemistry which is less accurate. Also, assessment of endothelial dysfunction needs to be verified by more accurate measures through estimation of eNOS level, hemodynamic studies, or performing in vitro study on renal artery rather than aorta. Furthermore, the total AMPK level was not measured in addition to the phosphorylated form. So, we recommend further experimental and clinical studies on empagliflozin be conducted to verify these molecular mechanisms.