Therapeutic Potential of Ertugliflozin in Renal Ischemia/Reperfusion Injury by Modulating Nrf2 and Necroptosis Pathways: A Preclinical Study in Rats

Introduction: Ischemia-reperfusion injury (IRI) is a significant clinical challenge that often leads to acute kidney injury (AKI), adversely affecting renal function and patient outcomes. Recent advancements in pharmacotherapy have highlighted the potential of sodium-glucose cotransporter-2 (SGLT2) inhibitors in providing renal protection. Objectives: This study aimed to investigate the protective effects of bexagliflozin on renal function in a rat model subjected to IRI. Materials and Methods: In an experimental study, 28 male rats, weighing between 200-300 g, were utilized in this experimental study and divided into four distinct groups. The sham group underwent identical anesthesia and surgical procedures without the induction of ischemia. The IRI group experienced 30 minutes of bilateral renal ischemia followed by 24 hours of reperfusion. The dimethyl sulfoxide (DMSO) group, serving as a vehicle for bexagliflozin, received an oral dose two hours before the ischemia induction and subsequently underwent the same reperfusion protocol. In the bexagliflozin pretreated group, rats were administered bexagliflozin at a dosage of 3 mg/kg orally two hours before ischemia induction, followed by 30 minutes of bilateral renal ischemia and 24 hours of reperfusion. After the reperfusion period, all rats were subjected to a laparotomy to collect blood and kidney samples, including urea, creatinine, interleukin-6 (IL-6), Akt, glutathione (GSH), caspase, light chain 3-B (LC3-B), kidney injury molecules-1 (KIM-1), and histopathological renal tubular injury. Results: The study findings indicated that both the IRI and IRI+DMSO groups experienced significant renal impairment compared to the sham group, as evidenced by elevated levels of serum urea, creatinine, caspase, Akt, LC3-B, KIM-1, and IL-6, alongside decreased GSH levels. In contrast, the IRI + bexagliflozin treatment group demonstrated notable protective effects against renal injury, reflected in lower levels of these parameters and reduced renal tubular injury scores compared to the IRI and IRI+DMSO groups. Furthermore, bexagliflozin was associated with a smaller increase in GSH levels relative to the other groups, underscoring its potential therapeutic role in alleviating renal damage linked to IRI. Conclusion: Bexagliflozin demonstrated promising protective effects against renal injury, as evidenced by lower levels of injury markers and reduced renal tubular damage. These findings suggest that bexagliflozin may serve as a viable therapeutic option for mitigating renal damage associated with IRI, warranting further investigation into its clinical applications.

Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1

The transcription factor NF-E2-related factor 2 (Nrf2) plays a critical role in the mammalian response to chemical and oxidative stress through induction of phase II detoxification enzymes and oxidative stress response proteins. We reported that Nrf2 expression was activated by deltamethrin (DM), a prototype of the widely used Parathyroid pesticides, in PC12 cells. However, no study has examined Nrf2 nuclear translocation and free radical production, two hallmarks of oxidative stress, in the mammalian brain in vivo. To this end, we examined translocation of Nrf2 and production of free radicals in rat brain exposed to DM. Indeed, DM initiated nuclear translocation of Nrf2 in a dose-dependent manner. Furthermore, Nrf2 translocation was accompanied by the expression of heme oxygenase-1 gene, an Nrf2-regulated gene linked to free radical production. Deltamethrin exposure promoted free radical formation in rat brain and reactive oxygen species generation in PC12 cells. Translocation of Nrf2 may be a response to DM-dependent induction of free radicals and DM may act as a mammalian neurotoxin by initiating oxidative stress.

The antioxidant-activated transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the induction of cytoprotective genes against chemical toxicity and oxidative injuries. The role of phosphorylation in Nrf2 activation has been suggested but remains elusive. We report that phenolic antioxidant/pro-oxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. In vitro treatment with lambda phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. In vivo (32)Pi-phosphorylation resulted in (32)Pi-labeling of the Nrf2 protein in the presence of tBHQ that can be dephosphorylated by lambda phosphotase, indicating that the slower migrating form is a phosphorylated Nrf2 protein and the faster form an unphosphorylated Nrf2. Unphosphorylated Nrf2 predominated in the cytoplasm, whereas the phosphorylated form preferentially localized in the nucleus. Nuclear Nrf2 can be dephosphorylated by lambda phosphotase in vitro and be converted to the faster migrating form, implicating phosphorylation of Nrf2 in the cytoplasmic-nuclear translocation of the protein. Deletional analyses from both the carboxyl- and amino-ends revealed the transcription activation (TA) domains Neh4 (Nrf2-ECH homology 4) and Neh5 (Nrf2-ECH homology 5) as a major region necessary for the phosphorylation. The TA domains are characterized by the presence of multiple phosphorylation sites of casein kinase 2 (CK2). Moreover, CK2 phosphorylated the TA domains in vitro. Treatment with CK2 inhibitor 2-dimethylamino-4,5,6,7,-tetrabromo-1H-benzimidazole (DMAT) blocked the induction of endogenous target genes of Nrf2 in cells and inhibited the TA activities of both the full length and the TA domains of Nrf2 to a large extent. Finally, phosphorylation of the TA domains correlated with the nuclear translocation of Nrf2 that was inhibited by DMAT in a concentration-dependent manner. The findings demonstrated that phosphorylation of Nrf2 at the TA domains by CK2 is an integral component of Nrf2 activation necessary for the nuclear localization and transcription activation function of Nrf2 in neuroblastoma cells.

Acteoside inhibits high glucose-induced oxidative stress injury in RPE cells and the outer retina through the Keap1/Nrf2/ARE pathway

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient of red pepper, is reported to have antimutagenic and anticarcinogenic properties. However, the mechanisms underlying its chemoprotective effects remain largely unresolved. In the present study, we found that capsaicin induced expression of heme oxygenase-1 (HO-1) in HepG2 cells. Capsaicin treatment resulted in a transient increase in the phosphorylation of Akt and subsequently nuclear translocation of NF-E2-related factor 2 (Nrf2), enhancing its binding to antioxidant response element (ARE). HepG2 cells treated with capsaicin exhibited increased production of reactive oxygen species (ROS). Prior exposure of cells to N-acetyl-L -cysteine blocked not only the ROS production but also the nuclear translocation of Nrf2 and its ARE binding, as well as HO-1 induction by capsaicin. Immunoblot analysis showed that whereas the level of HO-1 protein was elevated, that of NAD(P)H:quinone oxidoreductase (NQO1) was decreased after the treatment with capsaicin or the inhibitor of NQO1, dicumarol. We hypothesize that quinone metabolites or other reactive forms of capsaicin may bind covalently to NQO1 and thereby inhibit its activity, leading to production of ROS. This, in turn, would trigger the activation of Akt via phosphorylation, increase the nuclear translocation and ARE binding of Nrf2, and upregulate the expression of HO-1.

Nimesulide is a widely prescribed nitroaromatic sulfoanilide-type nonsteroidal anti-inflammatory drug that, despite its favorable safety profile, has been associated with rare cases of idiosyncratic drug-induced liver injury (DILI). Because reactive metabolites have been implicated in DILI, we aimed at investigating whether hepatic bioactivation of nimesulide produces a protein-reactive intermediate in hepatocytes. Also, we explored whether nimesulide can activate the transcription factor Nrf2 that would protect from drug-induced hepatocyte injury. We found that [(14)C]-nimesulide covalently bound to human liver microsomes (<50 pmol/mg under standard conditions) or immortalized human hepatocytes in a sulfaphenazole-sensitive, rifampicin-inducible manner; yet the overall extent of binding was modest. Although exposure of hepatocytes to nimesulide was not associated with increased net levels of superoxide anion, nimesulide (100 microM, 24 h) caused nuclear translocation of Nrf2 in a sulfaphenazole-sensitive manner, indicating a role of electrophilic metabolites. However, knockdown of Nrf2 with siRNA did not make the cells more sensitive to nimesulide-induced cell injury. Similarly, exposure of wild-type C57BL/6x129 Sv mice to nimesulide (100 mg/kg/day, po, for 5 days) was associated with nuclear translocation of immunoreactive Nrf2 in a small number of hepatocytes and induced >2-fold the expression levels of the Nrf2-target gene Nqo1 in wild-type but not Nrf2-null mice. Nimesulide administered to Nrf2(-/-) knockout mice did not cause increases in serum ALT activity or any apparent histopathological signs of liver injury. In conclusion, these data indicate that nimesulide is bioactivated by CYP2C to a protein-reactive electrophilic intermediate that activates the Nrf2 pathway even at nontoxic exposure levels.

Hypoxia/reoxygenation induces synthesis of reactive oxygen species (ROS) which can attack macromolecules and cause brain injury. The transcription factor, nuclear factor (erythroid-derived 2)-like 2, (Nrf2), ia potent activator of genes with an antioxidant responsive element and Nrf2 can counteract oxidative injury by increasing expression of several antioxidative genes in response to ROS stress. Here, we show that activation of the δ-opioid receptor (DOR) increasedNrf2 protein expression and translocation, thereby leading to cytoprotection. We used HEK293t cells exposed to 0.5% O2 for 16 h and then reoxygenated for 4 h as a model of hypoxia-reperfusion (H/R) injury. Real time PCR, Western blotting, siRNA and immunohistochemical techniques were used to follow Nrf2 expression and activity. Cell viability and damage (as LDH leakage) were also measured. H/R injury triggered Nrf2 translocation into the nucleus and up-regulated expression of several downstream genes, relevant to antioxidation, such as quinone oxidoreductase (NQO1). Incubation with the DOR agonist UFP-512 enhanced Nrf2 protein expression and translocation and up-regulated its downstream genes in normoxia and further increased Nrf2 expression and translocation after H/R, protecting the cells against loss of viability and damage. The effect of UFP-512 on Nrf2 nuclear translocation was blocked by the DOR antagonist, naltrindole. Also, DOR-mediated cytoprotection was strongly inhibited after transfection of HEK293t cells with Nrf2 siRNA. The DOR agonist UFP-512 was cytoprotective against H/R injury and this effect was partly dependent on DOR-mediated increase in Nrf2 function.

Nordihydroguaiaretic acid induces Nrf2 nuclear translocation in vivo and attenuates renal damage and apoptosis in the ischemia and reperfusion model

Renal ischemia/reperfusion injury is a critical clinical problem caused by kidney and heart surgery and can lead to acute kidney injury (AKI). Bevacizumab is a humanized monoclonal antibody that binds to circulating soluble isoforms of VEGF-A, thereby inhibiting the activation of VEGF molecularpathways and eliciting antiangiogenic effects. This study assessed the nephroprotective potential of bevacizumab in a rat model of renal ischemia/reperfusion injury (I/R). Twenty-four Sprague-Dawley rats were allocated into four groups: Sham, I/R, I/R + normal saline, and I/R + bevacizumab. The sham group was subjected to laparotomy without I/R induction. The I/R, I/R + normal saline, and I/R + bevacizumab groups were subjected to 30min of bilateral renal ischemia, followed by 24h of reperfusion. The rats in the I/R + normal saline and I/R + bevacizumab groups were administered normal saline (vehicle for bevacizumab) and 0.1mg/kg bevacizumab via intraperitoneal injection 60min before ischemia, respectively. Renal damage markers (creatinine and KIM-1), inflammatory and oxidative markers (TNF-α, IL-1β, NF-κB, F8-isoprostane and SOD), and an apoptotic marker (caspase-3) were measured via ELISA. Nrf2 and MLKL were assessed by IHC, and RIPK1 and HO-1 were assessed by RT‒qPCR, in addition to histological examination and molecular docking. Compared with the sham group, the I/R and I/R + normal saline groups presented significant increases in creatinine, KIM-1, NF-κB, TNF-α, IL-1β, F8-isoprostane, caspase-3, RIPK1, and MLKL and a reduction in SOD. Compared with those in the sham group, the histological findings in the I/R and I/R + normal saline groups revealed notable structural damage. Conversely, bevacizumab significantly reduced renal damage, inflammatory marker levels, cellular death, and histopathological findings. In bevacizumab-treated rats, the nuclear translocation of Nrf2 and HO-1 increased. Moreover, molecular docking analysis revealed that bevacizumab interacted with Keap1. Bevacizumab has nephroprotective effects against renal IRI by diminishing inflammation, necroptosis, apoptosis, and necrosis through the activation of the Nrf2/HO-1 pathway and the inhibition of the RIPK1/MLKL pathway.

Delayed Remote Ischemic Preconditioning Produces an Additive Cardioprotection to Sevoflurane Postconditioning Through an Enhanced Heme Oxygenase 1 Level Partly Via Nuclear Factor Erythroid 2-Related Factor 2 Nuclear Translocation

Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: Roles of endothelial nitric oxide synthase and endothelial progenitor cells

The aim: To determine whether Tilianin (TIL) may have Nephroprotective effects on bilateral renal IRI in rats by analyzing kidney function biomarkers U and Cr, inflammatory cytokines like TNF α and IL-1β, antioxidant marker total anti-oxidant Capacity (TAC), anti-apoptotic markers caspase-3, and histopathological scores. Materials and methods: 20 rats divided into even 4 groups as: Sham group: Rats underwent median laparotomies without having their ischemia induced. Control group: Rats had bilateral renal ischemia for 30 minutes, followed by 2 hours of reperfusion. Vehicle group: 30 minutes prior to the onset of ischemia, rats were given a pretreatment of corn oil and DMSO. Tilianin treated group: Rats administered Tilianin 5 mg/kg for 30 min prior to ischemia induction, then IRI. Results: The study found that the serum levels of TNF, IL-1, caspase-3, urea and creatinine, as well as TNF and creatinine in the Tilianin group were significantly lower than those of the control and vehicle groups. On the other hand, it revealed that TAC levels are remarkably higher in the Tilianin group than they are in the control and vehicle groups. Conclusions: This study concluded that Tilianin have a Nephroprotective effect via multiple impacts as anti-inflammatory, anti-apoptotic, and anti-oxidant agents.

Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury