Kidney protective effect of sitagliptin in 5-fluorouracil-challenged rats

The possible protective effect of sitagliptin (SIT) against nephrotoxicity induced by a single dose 5-fluorouracil (5-FU) (150 mg/kg, i

Cellular responses to oxidative, inflammatory, and apoptotic insults are greatly controlled by the fine balance between the anti-inflammatory nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and the pro-inflammatory nuclear factor-κB (NF-κB) pathway (Wardyn et al. 2015;Liu et al. 2017).Previous investigations showed that 5-FU-induced nephrotoxicity caused by a marked disruption in the balance between the Nrf2 and NF-κB pathways, which intensify oxidative stress, inflammation responses, and cell apoptosis (Arab et al. 2018;Ahmad Ansari et al. 2023).
Sitagliptin (SIT), the dipeptidyl peptidase-4 inhibitor, is commonly used for treatment of patients with type 2 diabetes mellitus.SIT also showed significant antioxidant, anti-inflammatory, and antiapoptotic activities.Previous investigations revealed that SIT significantly mitigated nephrotoxicity caused by gentamicin (Abuelezz et al. 2016), cisplatin (Abdelrahman 2017), adenine (Abdelrahman et al. 2019), and methotrexate (Afkhami Fard et al. 2023), and alleviated streptozotcin-induced diabetic nephropathy (Ashraf et al. 2023) in rats.However, to the best of our knowledge, the protective effect of SIT against renal toxicity induced by 5FU was not yet investigated.Therefore, this study was performed to detect the potential protective effect of SIT against 5-FU-induced nephrotoxicity in rats, and to reveal the mechanisms underlying this effect.

Medications
SIT and 5-FU powders were purchased from Sigma-Aldrich, USA.SIT was prepared in carboxymethylcellulose (CMC), 0.5% solution, and 5-FU was dissolved in physiological saline.The doses of SIT and 5-FU used in the current work were selected from previous studies (Rashid et al. 2014;Abdelrahman et al. 2019).

Laboratory animals and study protocol
Male Sprague-Dawley rats (weight 200 ± 10 g, each) were purchased from National Research Center, Giza, Egypt.Rats were housed in standard facilities, and supplied with ordinary chew and tap water ad libitum.They were left to acclimatize for 1 week before experiments.The rats were randomly assigned into 4 equal groups (n = 8, each), as follows: -First group (control) received daily CMC (vehicle of SIT), p.o., for 7 days, and a single i.p. injection of physiological saline (vehicle of 5-FU) on 6 th day.-Group 2 received daily CMC, p.o., for 7 days, and a single dose of 5-FU (150 mg/kg, i.p.) on 6 th day.-Group 3 received SIT (5 mg/kg/day, p.o.) for 7 days, and a single dose of 5-FU (150 mg/kg, i.p.) on 6 th day.-Group 4 received SIT (10 mg/kg/day, p.o.) for 7 days, and a single dose of 5-FU (150 mg/kg, i.p.) on 6 th day.

Sampling and biochemical studies
On the 8 th day, rats were euthanized by thiopental (70 mg/ kg.i.p.), and blood samples were collected via cardiac punctures.They were centrifuged at 5000 rpm for 5 min, after being clotted.Serum creatinine was measured by a colorimetric kit (Biodiagnostic, Egypt), and serum neutrophil gelatinase-associated lipocalin (NGAL) was determined by an ELISA kit (R&D Systems, USA).The kidneys were dissected out, and their dried weight was measured.The right kidneys were homogenized in cold 0.05 M, KH 2 PO 4 buffer (pH 7.4), and centrifuged at 5000 rpm for 10 min.Colorimetric kits were used to determine MDA and TAC (Biodiagnostic, Egypt), and cleaved caspase-3 (R&D Systems, USA) in the resulting supernatant.In addition, ELISA kits were used to measure kidney TNF-α and IL-1β (R&S Systems, USA), NF-κB p65 (Novus Biologicals, USA), Bax, Bcl-2, and Nrf2 (LS Bio-Sciences, USA).

Statistics
Data analysis was performed using one-way ANOVA followed by Tukey test for post hoc comparisons using GraphPad Prism Software Program (version 6.01).Results shown as mean ± S.E.M., and significance was considered at p < 0.05 level.

Histopathology findings
5-FU caused marked distortion of the normal kidney histology in the form of renal tubular dilatation, necrosis and desquamation of epithelial cells, inflammatory cell infiltration, interstitial edema, and multiple areas of coagulative necrosis (Fig. 4).On the other hand, SIT (5 and 10 mg/kg) mitigated the kidney tissue injury, preserved the renal architecture, and significantly reduced renal tubular injury score in 5-FU-challenged rats (Fig. 4).

Discussion
The current study, in agreement with the previous ones, showed that 5-FU-inuced renal toxicity is due to oxidative stress, exhaustion of endogenous antioxidant defences, and increased production of MDA, the end product of peroxidation of lipid biomembranes (Gelen et al. 2021;Ahmad Ansari et al. 2023).Similarly, activation of inflammatory cascades and increased generation of inflammatory cytokines were proved to participate in kidney injury induced by 5FU (Arab et al. 2018;Ahmad Ansari et al. 2023).Prior investigations, in accordance with the present one, also revealed that inflammatory cytokines, particularly TNF-α, up-regulated the NFκB pathway leading to further tissue injury.Phosphorylation of the cytoplasmic inhibitor of nuclear factor-κB (IκB) results in release of the active NF-κB p65 unit, which translocate to the nucleus and activate gene transcription of inflammatory cytokines, and augment the inflammatory responses (Arab et al. 2018;Wan et al. 2021).The current study showed that SIT provided significant antioxidant and anti-inflammatory effects, as evidenced by reduction of MDA, preserved TAC, and suppression of TNF-α and IL-1β production in rat kidneys.This can be related in part to the ability of SIT to suppress NADPH oxidase, the main source of ROS generation during oxidative stress (Jo et al. 2018).
The intrinsic mitochondrial-dependent cell apoptotic pathway is controlled by the balance between Bcl-2 (the main anti-apoptotic protein), and Bax (the main pro-apoptotic protein).
Increased Bax/Bcl-2 ratio causes disruption of mitochondrial membrane permeability, and release of apoptogenic factors into the cytosol, which eventually activate caspase-3-dependent execution phase apoptosis (Parson and Green 2010).Similar to the current study, previous investigations revealed that activation of mitochondrial-dependent apoptotic pathway with subsequent events were implicated in 5-FU-induced renal toxicity (Liu et al. 2018;Ahmad Ansari et al. 2023).Consistent with the current results, it was also reported that SIT provided significant antiapoptotic effect by inhibiting the intrinsic apoptotic pathway, with subsequent reduction of Bax/Bcl-2 ratio and caspase-3 activity (Famurewa et al. 2023).
The fine balance between the anti-inflammatory Nrf2 pathway and the pro-inflammatory NF-κB pathway is the main factor regulating the homeostasis of cellular responses to oxidative stress, inflammation, and apoptosis.Nrf2 up-regulates cellular defenses against oxidative and inflammatory, and apoptotic insults (Wardyn et al. 2015).Contrarily, NF-κB is responsible for activation of inflammatory cascades (Liu et al. 2017).The previous studies, in agreement with the present one, demonstrated that 5FU administration caused a significant increase of NF-κB p65 and a significant decrease of Nrf2 in rat kidneys (Arab et al. 2018;Ahmad Ansari et al. 2023).The current study also showed that SIT significantly restored the balance between

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the two pathways, as evidenced by the significant increment of Nrf2, and the significant decrement of NF-κB p65 in kidneys of rats challenged with 5-FU.Similarly, prior investigations showed that SIT provided antioxidant, anti-inflammatory, and antiapoptotic effects through up-regulation of Nrf2 pathway and down-regulation of NF-κB pathway (Abo-Haded et al. 2017;Famurewa et al. 2023).

Conclusions
SIT significantly mitigated renal injury and dysfunction caused by 5-FU in rats through its antioxidant.
anti-inflammatory, and antiapoptotic effects.SIT modulated the balance between Nrf2 and NF-κB pathways in a favorable way to counteract oxidative stress, inflammatory reactions, and apoptotic responses implicated in the pathogenesis of 5-FU-induced nephrotoxicity.