Nephroprotective Effects of Caffeine, Vanillin, and Their Combination against Experimental AlCl3-Induced Renal Toxicity in Adult Male Wistar Rats

Aluminum (Al) is known to be a nephrotoxic metal that can cause renal toxicity in both humans and animals. The use of functional foods has been reported to have significance in managing the toxic effects associated with such metals. This study aimed to assess the potential protective effects of caffeine, vanillin, and their combination in mitigating AlCl3-induced renal toxicity in adult male Wistar rats. A total of thirty (30) adult male Wistar rats weighing between 150 and 200 g were randomly divided into five groups, each consisting of six rats (n = 6). Group 1 served as the control, while the remaining treatment groups received a daily oral dose of 100 mg/kg AlCl3 for a duration of 21 days. In addition, groups 3–5 were coadministered 50 mg/kg body weight (bw) of caffeine, vanillin, and a combination (50/50 mg/kg bw) of both substances, respectively. In the results, AlCl3-treated showed a significant (p < 0.05) increase in serum biomarkers such as ALT, ALP, urea, and creatinine, and a significant (p < 0.05) decrease in serum total proteins (TPs). The renal tissue's antioxidant system, including SOD, CAT, GPx, and GSH, exhibited a significant (p < 0.05) reduction, accompanied by an elevated MDA level. However, the administration of caffeine, vanillin, and their combination resulted in a significant (p < 0.05) decrease in serum ALT, ALP, urea, and creatinine, and a significant (p < 0.05) increase in serum TP. Furthermore, following the treatment, there was a significant (p < 0.05) increase in renal SOD, CAT, GPx, and GSH levels, along with a reduction in the MDA level. In addition, the treatment for 21 days caused a significant (p < 0.05) reversal to the altered histomorphological architecture. These findings suggest that caffeine, vanillin, and their combination could potentially be an effective regimen in managing AlCl3-induced renal toxicity.


Introduction
Aluminum (Al) is a well-known environmental toxicant and a reactive waste product generated by industrial activities [1].It is widely distributed and commonly found in various food products [2].Te potential risks associated with exposure to Al have raised signifcant concerns, highlighting the need for attention [3].Epidemiological research has demonstrated that metal toxicants, including Al, play a crucial role in the development of chronic kidney toxicity [4].Te organ kidney is critical in maintaining homeostasis, detoxifcation, and excretion of both drugs and toxic metabolites; it appears to be a major target organ for toxicants [5].Te kidneys have the primary responsibility of eliminating the majority of Al compounds that enter the body through contaminated food, water, and environmental factors [6].Consequently, the kidneys are the primary target for Al bioaccumulation.At elevated concentrations, Al accumulates in renal tissue and leads to nephrotoxicity [7].
Furthermore, numerous in vitro and in vivo experimental investigations have demonstrated that Al negatively infuences cellular components, leading to the production of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radical [8].In addition, Al disrupts mitochondrial function, resulting in the excessive generation of highly reactive free radicals/electrophiles [9].Tese ROS can further damage cellular constituents such as lipids, proteins, and DNA, and interfere with the redox regulatory system [10].However, recent studies suggest that neutralizing ROS is a crucial strategy in preventing or impeding the progression of pathological processes associated with Al exposure [11,12].Te roles of antioxidants or other compounds that can scavenge or inhibit the formation of ROS have been reported in the reduction or neutralization of ROS [13].By reducing the levels of ROS, this strategy restores the balance of oxidative stress and prevent further damage to cellular components or cellular dysfunction [14].Recent reports have highlighted the signifcance of functional foods in managing pathological conditions associated with ROS by providing additional bioactive compounds and functional properties [15,16].Functional foods are known to contain natural antioxidants such as vitamins (e.g., vitamin C and E), minerals (e.g., selenium and zinc), phytochemicals (e.g., polyphenols and carotenoids), and other bioactive compounds [17].Tese antioxidants directly interact with ROS, neutralizing them by donating electrons or hydrogen atoms and preventing their detrimental efects on cellular components [18].
Cafeine, a bioactive molecule found in cofee [19], has antioxidant and anti-infammatory activities [20,21].It has been demonstrated to decrease lipid peroxidation, reduce ROS growth, and improve mitochondrial function in a variety of biological processes [22].Similarly, vanillin (4-hydroxy-3-methoxybenzaldehyde), a primary aromatic component of vanilla [23], is found in a wide range of processed foods, medications, and perfumes [24].According to research, vanillin has antioxidant, anti-infammatory, anticarcinogenic, and mutagenic efects [25,26].However, there are little data on the possible therapeutic benefts of these drugs on aluminum-induced kidney damage.As a result, the focus of this study was to look into the nephroprotective potential of cafeine, vanillin, and their combination against aluminum chloride (AlCl 3 )-induced kidney damage in adult Wistar male rats.

Chemicals and Reagents
Used.Chemicals and reagents such as AlCl 3 and thiobarbituric acid (TBA) were procured from Sigma-Aldrich, Inc., (Saint Louis, MO, USA) while vanillin and cafeine were sourced from British Drug Houses (BDH) Chemical Ltd., Poole, England.Other biochemical assay kits such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), total proteins (TPs), urea, and creatinine assay kits were procured from Randox Laboratory Ltd., Crumlin (Antrim, Northern Ireland, UK).

Sample Preparation.
Te stock solutions (concentrations 50 mg/kg each) of cafeine and vanillin were prepared in distilled water and kept in refrigerator (at −8 °C) throughout the treatment period following a modifed protocol of Akomolafe et al. [21].

Animal Treatment.
A total of thirty adult male Wistar rats (n � 30) weighing between 150 and 200 g were obtained from the animal house at Afe Babalola University in Ado-Ekiti, Ekiti State, Nigeria.Te animals were allowed to acclimatize under humane conditions for a period of 7 days at room temperature before the commencement of the study, which spanned 21 days (3 weeks).Troughout the duration of the study, experimental animals were housed at room temperature with a 12-hour light/dark cycle and provided with unrestricted access to pelletized animal feed (ABUAD LIVESTOCK FEED, produced by the Afe Babalola University Farm in Ekiti State, Nigeria; consisting of ingredients such as corn starch, soybean oil, dextrin starch (maltodextrin), casein, vitamins, and choline bitartrate) and water ad libitum.Te animal treatment procedures conducted in this study adhered strictly to the Principles of Laboratory Animal Care [27].

Ethical Clearance.
Te use of experimental animals (rats) involved in this study was carried out with a strict compliance to the ethical guidelines for the best practice issued by the Ethical Clearance Committee (ECC) of Afe Babalola University with the applied ethical code: ABUAD/ ACA/126.

Al Exposure Protocol.
Exposure to Al for the induction of nephrotoxicity was carried out according to a modifed method described by Kumar et al. [28].Experimental rats received a daily oral dose of 100 mg/kg AlCl 3 bw (dissolved in water) and 100 mg/kg cafeine, vanillin, and their combination for a period of twenty-one days.

Biochemical Assays.
Determination of serum biomarkers such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity assays were carried out using the method described by Reitman and Frankel [29], while serum total proteins (TPs), urea, and creatinine were determined according to the user manuals (Randox reagent kits).Enzymatic antioxidant parameters of the kidney tissues such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and nonenzymatic antioxidant parameter such as reduced glutathione (GSH) and malondialdehyde (MDA) contents produced were determined using the method described by Oloyede et al. [30].

Histological Study.
Histological alterations in the renal tissue samples were analyzed according to the method used by Oloyede et al. [30].Samples were isolated from the animals after sacrifce and preserved in formalin (10%) solution.Tereafter, tissues were embedded in parafn.A microtome was used to collect micrometerthick parafn sections, and tissues were then stained with hematoxylin and eosin (H&E).Examination of possible changes was carried out under a light compound microscope.

Statistical Analyses.
Te collected data were subjected to statistical analysis using the one-way analysis of variance (ANOVA) test in the SPSS software (Evaluation Version 16.0, SPSS Inc., Chicago, IL, USA).Post hoc comparisons were conducted using the Duncan multiple range test when necessary.Graphical representations of the data were generated using the GraphPad Prism 9.0 program (GraphPad Software, San Diego, CA, USA).Statistical signifcance was determined at a p value of less than 0.05, and the results were presented as the mean ± standard error of the mean (SEM) from 6 trial determinations (n � 6).

General Observation.
At the end of the experimental window, animals in AlCl 3 -induced nephrotoxicity group showed slight weakness, reduction in weight and feed consumption as well as furs loss when compared to the normal rats (control), vanillin-, and cafeine-treated groups.It should also be noted that no mortality was recorded in this study based on the diferent treatments among either the AlCl 3 -induced nephrotoxicity group or other groups.

Discussion
Nephrotoxicity is a condition in which the kidneys detoxifcation and excretion functions are impaired as a result of toxicants or drug-induced damage [31].Al is a wellknown toxicant that can accumulate in various tissues of mammals, including the kidneys, liver, heart, blood, bones, and brain [32].Among these tissues, the kidneys are particularly vulnerable to Al exposure, as they play a crucial role in preventing its bioaccumulation through excretion mechanisms [33].Te objective of this study was to evaluate the nephroprotective efects of cafeine, vanillin, and their combination in preventing and/or reducing kidney damage induced by AlCl 3 in adult male rats.Tis was achieved by assessing various biochemical parameters related to renal function in an experimental model of induced renal toxicity.
According to a study reported by Ghlissi et al. [34], parameters such as ALT and AST are considered indicators of nephrotoxicity.In addition, Abadi et al. [35] found that elevated serum levels of transaminases (ALT and AST) are often released from the cell cytosol due to cellular necrosis, indicating cytotoxicity.When an organ experiences cellular degeneration or damage, the plasma levels of these markers tend to increase [36].Te observed increase in serum activities of AST and ALT, as seen in Figures 1(a) and 1(b), can be attributed to the cytotoxic efect of Al on the kidney membrane permeability [37], which could lead to the leakage of these enzymes from the cytosol into bloodstream, along with disruptions or dysfunction in their activation [38].Conversely, a slight decrease in total protein levels observed in AlCl 3 -induced rats may be due to an increase in intracellular Al concentration, which has been shown to reduce protein synthesis [39], aligning with the fndings of Nawel et al. [40].However, the administration of cafeine, vanillin, and their combination resulted in decreased activities of ALT and AST, as well as an increase in plasma protein concentration (Figure 1).Tis suggests a nephroprotective efect of cafeine and vanillin against AlCl 3induced nephrotoxicity, possibly through the preservation of renal membrane permeability or their ability to reduce intracellular Al concentration.Tis efect is further supported by the signifcant improvement observed in the renal histological architecture, as indicated in Figure 4.
Serum creatinine and urea concentrations are widely utilized as indicators of injury in studies involving nephrotoxicity or renal dysfunction [41].Creatinine is a metabolic byproduct generated in muscles from high-energy storage compounds such as creatine phosphate [42].It is eliminated from the bloodstream via glomerular fltration in the kidneys and subsequently excreted in urine with minimal reabsorption by the renal tubules [43].Conversely, urea is a nitrogenous waste product that undergoes fltration by the Biochemistry Research International glomerulus, reabsorption by the renal tubules, and excretion in urine [44].Te plasma concentration of urea is dependent on the rate of excretion by the kidneys [45].Te elevation of creatinine and urea levels observed in the AlCl 3 -induced nephrotoxicity (Figure 2) may indicate severe renal injury associated with Al exposure, leading to a reduction in glomerular fltration rate and impaired clearance of serum creatinine, as well as abnormal retention of urea [46].However, treatment with cafeine, vanillin, and their combination resulted in a signifcant reduction in plasma urea and creatinine levels, indicating potential renoprotective efects against Al-induced kidney toxicity.Tis protective efect could be attributed to their ability to inhibit arginase induction, which is involved in urea production, or to their ability to improve glomerular fltration rate, thereby facilitating proper clearance of creatinine and urea [47].Intracellular antioxidant systems play a critical role in protecting against nephrotoxicity induced by heavy metals [48].Under normal physiological conditions, the balance between the generation and elimination of reactive oxygen species (ROS) maintains cellular function, while disturbances in redox homeostasis can lead to oxidative stress [49].[46].Tis fnding corroborates the report of Liu et al. [54].However, administration of cafeine, vanillin, and their combination caused an increase in the levels of these proteins with a concomitant decrease of MDA possibly by mitigating Al-induced ROS proliferation, thereby promoting protection in the kidney against nephrotoxic efect of Al [55].Furthermore, a normal histoarchitecture of the kidney plays a key role in its excretory and homeostatic functions [56].In our study, a variety of structural alterations were noticed in the renal histology of the AlCl 3 -induced groups, an efect that probably have refected Al-induced oxidative assault and derangement of both the proximal and distal convoluted tubules (Figure 4) [57,58].In this study, treatment with cafeine and vanillin signifcantly improved and restored renal histological status such as normal glomerulus, moderate degeneration of the proximal, and distal convoluted tubules with a decrease in urinary space.However, the group treated with the combination of vanillin and cafeine revealed an increase in urinary space and mild degeneration of the glomerular cells.Tis efect probably indicated that the combination had no signifcant efect improving the urinary space in nephrotoxicity.

. Conclusion
Tis study evaluated nephroprotective efects of cafeine, vanillin, and their combination in AlCl 3 -induced nephrotoxicity.Our fnding revealed that all these compounds demonstrated signifcant improvement in biochemical parameters and restoration of histoarchitectural status, which are crucial for normal excretory and homeostatic functions of the kidneys.Terefore, cafeine, vanillin, and their combination could be efective nephroprotective regimens in the management of AlCl 3 -related renal toxicity.
Experimental animals were euthanized by mild exposure to diethyl ether, after overnight fasting, following the withdrawal of feed and water.Blood samples were immediately collected by direct heart puncture into plain sample bottles.Blood samples were subsequently centrifuged at 1800 ×g at 27 °C for 10 min to obtain the sera used for the various biochemical analyses.2.7.2.Preparation of TissueHomogenate.Kidney tissues were subsequently dissected and rinsed in 0.1 M tris bufer (pH 7.4), blotted with flter paper, and placed on ice.Each sample was weighed and subsequently homogenized in 0.1 M tris bufer (1 : 5 w/v).Te homogenate was centrifuged at 3000 rpm for 10 min with the pellet discarded and the supernatant kept in refrigerator for various biochemical assays.
3.1.1.Efects of Cafeine, Vanillin, and Teir Combination on Serum Biomarkers and Total Proteins during AlCl 3 -InducedRenal Toxicity in Adult Male Rats.Figures1(a)-1(c) illustrates the efects of cafeine, vanillin, and their combination on serum-specifc biomarkers and TP levels during AlCl 3induced renal toxicity in adult male Wistar rats.Te results demonstrate a signifcant (p < 0.05) increase in AST and ALT activities, along with a signifcant (p < 0.05) reduction in TP levels of the AlCl 3 -induced nephrotoxicity compared to the normal control.However, treatment with 50 mg/kg cafeine, vanillin, and their combination resulted in a signifcant (p < 0.05) reduction in the activities of serum AST and ALT, accompanied by a signifcant (p < 0.05) increase in TP levels, compared to both the normal control and the AlCl 3 -induced nephrotoxicity group.
increase in MDA levels of the AlCl 3 -induced nephrotoxicity compared to normal.However, administration of 50 mg/kg cafeine, vanillin, and their combination caused a signifcant (p < 0.05) increase in the activities of SOD, CAT, and GPx activities as well as GSH levels with a signifcant (p < 0.05) increase in MDA level when compared to AlCl 3 -induced nephrotoxicity.Biochemistry Research International3.1.4.Efects of Cafeine, Vanillin, and Teir Combination onHistological Architecture of AlCl 3 -Induced Renal Toxicity in Adult Male Rats.Figures 4(a)-4(e) shows the efects of cafeine, vanillin, and their combination on histological status of AlCl 3 -induced renal toxicity in adult male rats.As indicated in Figure1(a), AlCl 3 -induced nephrotoxicity showed enlargement of the urinary space, DCT, and PCT compared to normal control with a normal architecture of the glomerulus showing moderate loss of PCTand DCTcells.However, group administered with 50 mg/kg bw vanillin revealed a slightly enlarged urinary space, loss of cuboidal cells in some DCT and PCT with 50 mg/kg bw cafeine-