Introduction
Nephrotoxicity could be induced by various substances in a variety of patterns and mechanisms. Tubular necrosis, interstitial nephritis, crystal nephropathy, angiopathy, changes in intraglomerular hemodynamics, rhabdomyolysis, and fibrosis are the most common signs of drug-induced nephrotoxicity (DIN), which can lead to renal failure (Ghane Shahrbaf and Assadi
2015; Gao et al.
2021). DIN reduces the efficacy of drugs and complicates the treatment of serious cancers (Kintzel
2001), renal transplantation (de Mattos et al.
2000) and several metabolic disorders (Raza and Naureen
2020).
Among the drugs that have a nephrotoxic effect is amikacin (AMK) (Azırak
2023). AMK is a semisynthetic, broad-spectrum aminoglycoside antibiotic derived from kanamycin A. Despite its strong antibacterial activity, low cost, rapid onset of action, synergy with beta-lactam antibiotics, and low resistance, nephrotoxicity and ototoxicity limit AMK clinical applicability (Wargo and Edwards
2014). Multiple pathophysiological effects, including inflammation, inhibition of specific transporters, induction of oxidative stress and apoptosis, vascular alterations, contribute to AMK-induced renal toxicity (Prajapati and Singha
2010; Hlail et al.
2020).
Gamma-aminobutyric acid (GABA) is a non-protein amino acid that is primarily found in the central nervous system especially the brain, where it functions as a significant inhibitory neurotransmitter (Olsen and Betz
2006). Notable GABA biological activities include anti-diabetes, anti-hypertension, anti-cancer, antioxidant, antimicrobial, anti-allergenic and anti-inflammatory properties (Han et al.
2007; Prud’homme et al.
2013; Ngo and Vo
2019). In addition, GABA was reported to protect the intestine, liver and kidney from toxin-induced injury (Rashmi et al.
2018). GABA was demonstrated to be capable of trapping reactive intermediates during lipid peroxidation (Deng et al.
2010).
Nanoparticles (NPs) are remarkable drug delivery systems which are utilized due to their stability, unique properties (e.g., biodegradability, biocompatibility, and nontoxicity) in comparison to bulkier equivalents (Ravishankar and Jamuna
2011; Divya and Jisha
2018). Chitosan nanoparticles (CSNPs) possess the properties of CS and NPs, including nontoxicity, biodegradability, antimicrobial properties, interface effects, small size, and quantum size effects (Ingle et al.
2008). CSNPs can also deliver medicines or macromolecules to targeted sites via controlled release and modifying their pharmacokinetics (López-León et al.
2005; Shi et al.
2011,
2012; Perera and Rajapakse
2013). This study aimed to synthesize and characterize AMK–GABA–CSNPs, then evaluate their intraperitoneal (i.p.) injection in Sprague–Dawley rats for effective administration with minimum side effects. We compared the renoprotective effects of unloaded AMK–GABA combination with that of AMK–GABA–CSNPs.
Discussion
AMK-induced nephrotoxicity is mainly caused by oxidative injury due to renal cortical lipoperoxidation and peroxide generation (Kaynar et al.
2007). As the oxidative injury is one of the primary causal factors relating to AMK-induced renal injury. The current work attempts to diminish the oxidative injury via co-administration of GABA, a powerful antioxidant, with AMK loaded on CSNPs. CSNPs can resolve the majority of the initial problems with drug delivery, including fewer side effects, poor bioavailability, accurate distribution and slow release to the site of action resulting in excellent pharmacological efficiency and a therapeutic outcome (Mikušová and Mikuš
2021).
There have been several reports of AMK nanoparticles with various structures. AMK was encapsulated using gold nanostars (GNS) (Aguilera-Correa et al.
2022). Poly (lactic acid-co-polyethylene glycol) (PLA-PEG), low-molecular-weight poly (lactic acid) (PLA), and with poly (vinyl alcohol) (PVA) were also applied in AMK NPs (Glinka et al.
2021). Reddy and Damodharan (
2022) also optimized AMK CSNPs with a double efficacy as pure AMK. Rahmati et al. (
2022) confirmed the synthesis of AMK-loaded niosome nanoparticles with enhanced antibacterial activity. To the best of our knowledge, the present study is the first to successfully prepare AMK–GABA–CSNPs by the ionic gelation method. We applied this procedure as it is the most frequently used technique to develop CSNPs. In addition to being easy to utilize, this technique does not alter the drug’s chemical structure (Hoang et al.
2022). Shilpa et al. (
2012) suggested the formation of GABA–CSNPs by bond formation between the amino group of CS and the carboxyl group of GABA. The particle size was 77.5 ± 16.5 nm. So that the AMK–GABA–CSNPs synthesized here were within the range of 1 to 100 nm, which defines the nanoparticle scale in accordance with the Standard Terminology Relating to Nanotechnology E 2456-06 (ASTM
2006). This range is smaller than the size range recorded in a previous study by Reddy and Damodharan (
2022), whose particles presented 168–297 nm when formulated as AMK CSNPs. This finding suggests that nanoparticle suspension preparation standards should be taken into consideration. The ZP was + 38.94 ± 2.65 mV. The positively charged amine group of the main component, chitosan, is the cause of the NPs’ positive character. The formed AMK–GABA–CSNPs’ positive zeta potential reflects the higher stability of the nanoparticles used in the formulation. Reddy and Damodharan (
2022) synthesized AMK CSNPs with ZP range from +34.65 mV to +43.24 mV. Ghaffari et al. (
2010) also recorded positive ZP ranging from +4 mV to +16 mV after preparation of AMK-loaded solid lipid nanoparticles. In addition, the FTIR results of AMK–GABA–CSNPs showed that GABA and AMK were effectively loaded into the CSNPs. The resulting spectra for CS, AMK and GABA were in agreement with that obtained in earlier reports (Suresh et al.
2008; Zareie et al.
2019; Abdel-Hakeem et al.
2022).
An efficient antioxidant possesses reducing power due to its capacity to transfer electrons. The synthesized AMK–GABA–CSNPs had significantly higher antioxidant activity compared to CSNPs and ascorbic acid as control. The reducing power of the AMK–GABA–CSNPs can be due to the large number of hydrogen ions produced from GABA molecules. Falah et al. (
2021) also suggested a significant antioxidant efficiency of GABA via its ability to neutralize DPPH radicals. According to Liu et al. (
2016), enzymatic hydrolysis and decarboxylation of GABA cause the amino acids to split apart and release tyrosine amino acids in the terminal C peptide, which become capable of donating electrons and stopping the free-radical chain reaction.
When applying biomaterials, effective blood coagulation must be taken into consideration. As hemorrhage and thrombosis may result from an imbalance between pro- and anti-coagulation activities. This study used PT and PTT assays to determine how AMK–GABA–CSNPs affected blood hemostasis. Extrinsic and intrinsic blood coagulation pathways are represented by PT and PTT, respectively. In the present study, the formulated AMK–GABA–CSNPs showed no significant effectiveness in accelerating blood coagulation time (PT and PTT), indicating no interruption of the thromboembolism in the vascular system. Earlier studies suggested the interaction of the positively charged amino group along the CS molecule with the negatively charged erythrocytes and platelets to change the microstructure of hemoglobin and make blood more viscous (and prolong PT and PTT) (de Lima et al.
2015; Wang et al.
2019,
2021). However, the formation of a bond between the amino group of CS and the carboxyl group of GABA may explain the obtained results of normal PT and PTT for AMK–GABA–CSNPs. In consonance with our findings, Tyurenkov et al. (
2014) reported in vivo improvement of the hemostasis system with GABA derivatives administration via changes in calcium levels and calmodulin activity in platelets. In addition, Jaccob et al. (
2019) suggested that AMK had no apparent impact on the PTT clotting time and that the only way it could extend PT was at high concentrations, as it could prevent the production and activation of fibrinogen and inhibit the endogenous clotting factor in order to prevent platelets from aggregating.
Hemolytic activity was carried out to assess the safety of the produced AMK–GABA–CSNPs. Considering the results obtained, it can be stated that even at high concentrations AMK–GABA–CSNPs have no hemolytic effect and are appropriate for systemic administrations with promising in vitro anti-inflammatory activity. In agreement with the present results, Abdel-Hakeem et al. (
2022) described erythrocytes as osmometers that lyse in response to changes in the blood’s osmotic and physical parameters and confirmed that gentamicin–ascorbic acid CSNPs did not exhibit hemolytic activity.
The MTT assay measures mitochondrial activity, which is directly related to cell viability Dead cells cannot change the MTT tetrazolium salt to colored formazan crystals, whereas metabolically active cells can. The results of the MTT experiments supported AMK–GABA–CSNPs' good cell compatibility, as there is no evidence of cytotoxicity and well tolerated by cells up to concentration of 240.7 µg/ml.
In the present investigation, we established an AMK-induced acute nephrotoxicity model using Sprague–Dawley rats and verified that AMK injection at 20 and 30 mg/kg aggravated symptoms of renal injury after 3 weeks. Elevated levels of BUN, CRE and UA indicated renal dysfunction (Gounden et al.
2020). The dose-dependent elevations of BUN, CRE and UA due to AMK injection were consistent with previous reports (Batoo et al.
2018; Madbouly et al.
2021; Azırak
2023). Whereas unloaded AMK–GABA combination had recoded a protective effect as it suppressed serum levels of BUN, CRE and UA. In agreement with our findings, Lee et al. (
2022) suggested GABA protective effect against cisplatin-induced nephrotoxicity via inhibition of tubular dilation and hemorrhage. Interestingly, AMK–GABA–CSNPs injections (20 or 30 mg/kg) recorded amelioration of kidney functions with higher significance compared to unloaded AMK–GABA combinations. Shilpa et al. (
2012) attributed this to chitosan's positive charge, high cell binding affinity, and the prolonged GABA effect of GABA when loaded on CS due to lower exposure to the internal body environment.
It has been proposed that AMK-induced nephrotoxicity was triggered due to the production of highly destructive free radicals like superoxide anions, H
2O
2, and hydroxyl radicals (Batoo et al.
2018). In harmony with this theory, our findings demonstrated AMK injection markedly increased lipid peroxidation (LPO), indicated by higher MDA, and inhibited GSH and SOD’s antioxidant activity in renal homogenates, making the kidneys more susceptible to oxygen radical stress.
The intraperitoneal injection of 50 mg/kg GABA with AMK (20 or 30 mg/kg) potentiated the antioxidant activity of SOD and GSH and suppressed MDA. This demonstrates that GABA can defend against AMK-induced nephrotoxicity. In agreement with our results, Ali et al. (
2015) reported the ameliorative action of oral GABA on SOD and GSH and protection against cisplatin-induced nephrotoxicity through increasing its urinary excretion, with no effect on the drug's therapeutic efficacy by boosting its urine excretion. Furthermore, Sasaki et al. (
2006) reported the protective action of GABA against oxidative stress during chronic renal failure caused by nephrectomy. In comparison to the unloaded AMK–GABA combination, AMK–GABA–CSNPs demonstrated a significantly enhanced renal antioxidant system as it almost normalized MDA and SOD levels with a significant increase of GSH activity. These findings suggest that the loading on CSNPs benefits the antioxidant properties of GABA. This may be attributed to the improved solubility and bioavailability of GABA after loading. In addition, CSNPs’s synergistic effect contributes high antioxidant activity and free radical scavenging capacity, which can reduce lipid peroxidation (MDA levels) and boost the antioxidant defense system, protecting renal tissue from free radical damage induced by AMK (Samadarsi and Dutta
2020; Halawa et al.
2022).
As the pathogenesis of acute renal injury is believed to be seriously influenced by inflammation, the present study monitored the inflammatory immune response. Because of direct contact with reactive oxygen species (ROS), renal vascular endothelial cells and tubular epithelium were involved in the early initiation and extension of inflammatory responses in the injured kidney. This is done via the release of various local inflammatory cytokines such as IL-1β, IL-4, IL-6, IL-17, IL-18, TNF-α and IFN-γ. The injured kidney’s proximal tubules, lymphocytes, neutrophils, and macrophages develop IL-18, a proinflammatory cytokine. Caspase-1 activates IL-18 which induces the synthesis of many cytokines and chemokines that trigger activation of T helper cells and lymphocyte proliferation (Akcay et al.
2009). The accumulation of ROS in renal tissue activates nuclear factor kappa B (NF-kB), triggering the inflammatory signaling cascade and express TNF-α. TNF-α enhances the production of other inflammatory cytokines, especially IFN-γ, IL-1β and IL-6 which are powerful proinflammatory cytokines that play a role in inflammatory renal damage (Kumar et al.
2015; Tripathi and Alshahrani
2021; Farid et al.
2023). IL-17 is a proinflammatory cytokine that is primarily produced by T helper 17 (Th17) a subset of CD4 + T cell. A broad spectrum of factors, including cytokines like IL-6 and IL-1β, influence the differentiation of CD4 + cells to Th17 cells (Wang et al.
2023). IL-17 is elevated in blood and renal lysate in case of nephrotoxicity (Collett et al.
2022). The actual role of IL-4 during renal injury is complex and contrasting to be described. As some reports have found IL-4 to play a significant role in enhancing the recovery of tubular damage in renal disease by inducing M2 macrophage phenotypic polarization (Zhang et al.
2017). On the other hand, other reports suggest that the IL-4 receptor α chain/STAT6 pathway promotes fibrosis and renal disease progression (Liang et al.
2017). The present work recorded a significant increase in renal levels of IL-1β, IL-4, IL-6, IL-17, IL-18, TNF-α and IFN-γ that may potentiate AMK-induced nephrotoxicity. These cytokines were reported to elevate due to drug-induced nephrotoxicity (Saeed et al
2022; Chen et al.
2022; Dari et al.
2023). In this investigation, we discovered that the unloaded AMK–GABA combination dropped the renal levels of IL-1β, IL-4, IL-6, IL-17, IL-18, TNF-α and IFN-γ in correlation with AMK dose. The capacity of unloaded AMK–GABA combination to reduce inflammation and alleviate oxidative stress may be connected. GABA exerts its potent anti-inflammatory effects by inhibiting numerous signaling pathways that contribute to the generation of proinflammatory cytokines. As previously reported, one of these effects is that GABA is a paracrine and autocrine signaling molecule that activates GABA receptors on immune cells. GABA modulates immune function via attaching to GABA
A receptors, which modify proliferation and suppress the release of numerous cytokines (about 47 cytokines) (Bhandage et al.
2018). Besides, GABA
A receptor engagement is a powerful inhibitor of NF-kB activation by the kidneys' intracellular ROS generation after AMK therapy. As a result, the observed decrease in TNF-α, IL-1, and IL-6 levels following GABA therapy can be explained (Zhang et al.
2022). In agreement with our results, early studies revealed that GABA alter both innate and adaptive immunity as it lower Th17, CD4 + T cells and CD8 + T cells, promoted CD4 + and CD8 + regulatory T cell (Treg) responses and shift natural killers (NKs), dendritic cells (DCs), and macrophages toward anti-inflammatory phenotypes (Tian et al.
2014, 2019; Bhandage et al.
2020; Zhang et al.
2021).
Since previous investigations proved that CSNPs have various anti-inflammatory properties (Kim et al.
2004; Friedman et al.
2013; Jhundoo et al.
2020), we exactly studied whether the AMK-induced inflammatory cytokines could be regulated in the presence of GABA–CSNPs. Interestingly, we found that AMK–GABA–CSNPs minimized the renal inflammatory cytokines levels in a manner that was more significant than that of unloaded AMK–GABA combinations. The augmented anti-inflammatory activity detected for AMK–GABA–CSNPs may be attributed to the combining of the anti-inflammatory properties of GABA with CSNPs' intrinsic anti-inflammatory action in the same delivery platform. Similar to our study results, Liu et al. (
2020) and Mohyuddin et al. (
2021) revealed down-regulation of plasma levels of TNF-α, IL-1β, IL-6 and IL-10 in mice receiving the oral administration of CS. Yang et al. (
2016) stated that CS suppresses the manifestation of inflammatory genes by inhibiting TLR4/NF-kB signaling pathways. Nomier et al. (
2022) outlined that CSNPs provided considerable protection and improved CCl4-induced nephrotoxicity by lowering TNF-α and IL-1β.
According to the histopathology studies, AMK injection (20 or 30 mg/kg) was related to changes in the renal histological construction, particularly infiltration of inflammatory cell, degradation of tubular epithelial lining, and tubular necrosis. These findings corroborate recent findings (Madbouly et al.
2021; Saeed et al
2022) that AMK causes significant degenerative alterations in the kidney due to the accumulation of inflammatory mediators and ROS in the renal cortex. Unloaded AMK–GABA combinations decreased the histopathological alterations by lowering inflammation, necrosis, and fibrosis. These findings are comparable with that of Lee et al. (
2022), who found that GABA significantly improved histological symptoms of nephrotoxicity such as tubular dilatation, renal hypertrophy, hemorrhage, and collagen deposition in cisplatin-induced nephrotoxicity. The anti-fibrogenic properties of GABA may be attributed to its antioxidant properties and the lowering of proinflammatory cytokines. Sasaki et al. (
2007) revealed that GABA inhibits transforming growth factor-beta (TGF-β1) and fibronectin expression in renal tubules by binding to GABA
A and GABA
B receptors, which in turn reduce renal fibrosis. The administration of AMK–GABA–CSNPs revealed synergistic anti-inflammatory and anti-fibrogenic effects. In agreement with our findings, Qiao et al. (
2014) illustrated the ability of CS-based nanocomplex to enhance specific renal targeting and could be used to design drug delivery systems that reduce renal fibrosis. Wu et al. (
2022) suggested that CS exert excellent inhibitory effects on fibronectin, collagen deposition, and TGF-β1/Smad signal pathway.