Chloroquine, an aminoquinoline, which was formerly the first-line drug for the treatment of malaria for many years until it was discouraged due to drug resistance strains of malaria parasite has been re-purposed for the treatment of many infectious, immunologic and inflammatory disorders because of its inherent property. This study evaluates the possible therapeutic potential of chloroquine, its combination with steroid in the treatment of acrylamide-induced nephropathy. Five groups of male Wistar rats were used for the study: Group 1 rats were used as the untreated control; group 2 rats were intoxicated with oral 2 mg/kg/day of acrylamide (ACR) for 14 days. Following the induction, Groups 3, 4 and 5 rats were treated with chloroquine (25 mg/kg/day), chloroquine + prednisolone (25 mg/kg/day + 5 mg/kg/day) prednisolone (5 mg/kg/day) for 28 days respectively. Urine volume, protein estimation and highly sensitive biomarkers of renal injury (cystatin C, KIM-1, urea, and creatinine) were assessed. In addition, inflammatory and complement factors were also evaluated. Results showed that chloroquine, and its combination with prednisolone attenuated the biochemical parameters of kidney injury as well as reversing inflammation and complement activation. Furthermore, the anti-apoptotic potential of this drug was demonstrated by immunohistochemistry analysis, which revealed BAX down-regulation, caspase 3, and 9 expressions. Also, ACR-induced histological changes were significantly reversed by this drug. Thus, highlighting the promising therapeutic potential of chloroquine in mitigating ACR-induced nephropathy, which was probably mediated via anti-inflammatory and anti-apoptotic mechanisms.

• Chloroquine; Nephropathy; Acrylamide; Prednisolone; Apoptosis; Inflammation; Complement factors

Idris AO, Olufunke O (2024) Chloroquine Attenuates Acrylamide-Induced Nephropathy in Male Wistar Rats. J Clin Nephrol Res 11(2): 1121.

Acrylamide (ACR) is a monomeric substance derived from carbohydrate-rich foods fried at very high temperature for prolonged period of time. ACR is a potential carcinogenic substance which is formed during heating of food products containing carbohydrates and asparagine [1]. Prior to 2002, the predominant mode of exposure to ACR was thought to be only occupational, but research released by the Swedish National Food Agency reported the presence of acrylamide in many food items that are commonly consumed [2,3]. French fries, potatoes chip, pop corns, processed cereals, and also baked foods have been documented to have a high level of ACR [4]. These varieties of food are favourites in developed Western nations. However, there are increasing number of developing nations in Africa and Asia that have adopted Western style of fast food diet. In addition to ACR content in food, other forms of exposure to ACR include the following: its use in the production of polyacrylamide which serves as a flocculants in water treatment, use in paper and plastic industries and as an additive in cosmetic industry. ACR is used in the production of polyacrylamide gels for electrophoresis, and also used in the petroleum industry [2].

A survey released by the European Food Safety Authority (EFSA) in 2015. Entailed the estimation of the proportion of ACR in many food products. ACR contents in fried potatoes was reported to be very high with value up to 1.0 mg/kg while the highest quantities of ACR was detected in coffee which contained 4.5 mg/kg. It was concluded that ACR-induced toxicity required urgent attention [5].

ACR may enter drinking water if polyacrylamide is used in the treatment process. It can be found in soils, but is rarely found in air except for cigarette smoke. Animal studies demonstrated the effect of ACR on the reproductive potentials to include reduced fertility among male rats. The International Agency for Research on Cancer (IARC), National Toxicology Program (NTP), and the Department of Health and Human Services declared ACR as both a carcinogen, and a mutagen. The maximum daily limit of ACR exposure in drinking water has been put at 1.5mg/L. With the legal limit of 0.3 mg/m3 for in air averaged over an 8-hour work day as the threshold for exposure [6]. A review of the literature has described the various toxicities of exposure to ACR to include neurotoxicity, carcinogenicity, mutagenicity and reproductive toxicity. In addition, the renal toxicity of ACR has been documented [7-9]. Idris et al., described the development of renal disease in the litters of rats whose mothers were exposed to acrylamide. The exposure to ACR through various sources by humans poses a significant risk to health globally [10].

The mechanism of ACR formation in starchy foods is by the Mailard reaction which has been described as a reaction which involves reducing sugars and the amino acid, asparagine [2,11]. ACR demonstrates negative influence on scavengers of free radicals, by potentiating the metabolism of glycidamide by the CYP450 pathway through inhibition of glutathione-S- transferase enzyme and undergoes oxidative biotransformation by cytochrome P4502E1 (CYP2E1) to a more potent and highly reactive molecule that initiates cellular toxicity [12]. The pathway results in an epoxide derivative, glycidamide, which is more reactive towards DNA, proteins, and with hemoglobin. Glycidamide can be hydrolyzed to glyceramide, it can also be detoxified via enzymatic, glutathione-S-transferase or non- enzymatic pathways to form acrylamide- and glycidamide- glutathione conjugates, with the resultant formation of two mercapturic acid products excreted in the urine [13].

A detailed mechanism of acrylamide-induced neurotoxicity has been elucidated, and showed to include the induction of apoptosis through its effect in collapsing the mitochondrial and release of pro-apoptotic proteins (caspase 9 and 3), BAX and down-regulation of anti-apoptotic agent, BCL-2 [14]. Although the potential molecular mechanism reported for ACR-related nephrotoxicity is multifaceted; oxidative stress, inflammation and glomerulonephritis have been implicated [9]. Studies involving detailed evaluation of ACR-induced nephropathy is essential to understanding the possible treatment options for this ACR toxicity.

Drug re-purposing of currently used drugs for new therapeutic indication is a trend that is gaining ground in the field of medicine. Chloroquine and hydroxychloroquine, are aminoquinolines, traditionally used for the treatment of malaria has been re-purposed to treat many infectious, immunologic and inflammatory disorders including anti-phospholipid syndrome, lupus nephritis, amoebic liver abscess, HIV, Q-fever and Whipples’ disease. This is due to their inherent anti-inflammatory, immune- modulatory, and anti-infective properties. Both drugs have strong anti-proliferative effect on T-cells, chloroquine also has role in innate immunity, and they are able to block the interactions of toll-like receptors (TLR), to reduce innate immune response [15]. The evaluation of synergistic effect of chloroquine with prednisolone, an anti-inflammatory corticosteroid, is a research area that may be beneficial in treatment of kidney disease [16].

This study, thus, examined the possible ameliorative potential of chloroquine CQ and combination of chloroquine with prednisolone CQ + PRED on ACR-induced nephropathy in male Wistar rats.

Experimental Animals and Housing

The experiment was conducted on 50 young adults male Wistar rats (10-12 weeks old and weighing between 160-180 grams) that were purchased from the Animal House of Osun State University, Osogbo, Osun State, Nigeria. The rats were maintained under standard environmental conditions (23-25?, 12h/12h light/dark cycle) and were sheltered in standard polypropylene cages that were kept in the Animal House of the Faculty of Basic Medical Sciences, University of Ilorin, Ilorin, where the research was carried out. They were fed with clean water and standard rat chows. Acclimatization was observed for a period of two weeks before the start of the experiment.Ethical Clearance was obtained from the research ethics committee of the Faculty of Basic Clinical Sciences, University of Ilorin. (UERC/ASN/2021/2290).

Experimental Design

• The experimental design was divided into two phases: Induction phase and Experimental phase.

Induction Phase: This phase involved the induction of chronic glomerulonephritis (CGN) and tubulo-interstitial nephritis (TIN). Ten rats were used, 2 mg/kg of ACR was administered to each of the rats daily by oral gavage for a period of 14 days, followed by sham handling until day 28 when they were sacrificed for histopathological studies. Histopathologic studies done on the kidneys confirmed the presence of glomerulonephritis and tubulo-interstitial nephritis. This was done in order to confirm that acrylamide can induce nephropathy.

Experimental Phase: The remaining 40 rats were divided into 5 groups of 8 rats in each group. Each group of rats was induced for ACR-induced CGN and TIN as earlier described except the control group which was not induced. Quantification of 24-hour urinary output was done weekly and 24-hour urinary protein was collected. The body weights of the rats were measured before and at the conclusion of the experiment. Following induction, a group was placed on observation, which represent the induced group, while the other groups were treated with varying daily doses of drugs for 28 days (Table 1).

Table 1: Animal Groups and their Treatments

Groups	8 rats in each group they received daily dose of drugs for 28 days
Group 1	Control
Group 2	Induced (2mg/kg/day for 14days)
Group 3	Chloroquine (25mg/kg/day) (CQ)
Group 4	Chloroquine+Prednisolone(25mg/kg +5mg/kg/day) (CQ+PRED)
Group 5	Prednisolone 5mg/kg/day (PRED)

The dose for chloroquine was selected according to the method used by Kopanska [17]. Dose of prednisolone was selected according to Kumar [18].

On day 28, all experimental animals were sacrificed under inhaled light halothane anesthesia, about 4-5 ml of whole blood from each rat was collected by cardiac puncture into lithium heparinized tubes, and centrifuged at 4000g for 15 min at −4 °C, using a cold centrifuge (Centurium Scientific, Model 7886). The plasma was collected into separate plain tubes for the assessment of electrolytes and markers of renal function.

Measurement of Body Weight

Body weights of the rats were measured with the aid of a  digital weighing balance (Hanson, China) to assess the weight gain or loss in each group.

Kidney Function Assays

A. Determination of Total Protein in the Urine.

The total protein in the urine of the rats was determined according to laboratory protocol as described by Lowry et al [19].

B. Estimation of Specific Kidney Biochemical Parameters

Plasma Urea, Creatinine, Cystatin C, and KIM-1 were determined using specific rat ELISA kits purchased from Elabscience (Crumlin, Co. Antrim, USA), (Catalog No: E-EL-R3056), (Catalog No: E-EL-R3123), (Catalog No: E-EL-R2764) and (Catalog No: E-EL-R3019) respectively. The assays were conducted by following the manufacturers specifications.

Assays for Assessment of Inflammatory Markers

A.   Assays of Plasma C Reactive Protein.

Plasma CRP was determined using specific rat ELISA kit purchased from Elabscience (Catalog No: E-EL-R0506) (USA) following manufacturers’ instruction. Wells were prepared according to instructional manual, biotinylated Detection Ab working solution was added to each well. Conjugate working solution was added followed by incubation. The optical density (OD value) of each well was determined at once with a micro- plate reader set to 450 nm.

B.      Assays of Plasma Interleukin 1 beta –1(IL-1β)

Plasma IL-1β was determined using specific rat ELISA kit purchased from Elabscience (Catalog No: E-EL-R0012) (USA) following the manufacturers’ instruction. Wells were prepared according to instructional manual, biotinylated Detection Ab working solution was added to each well. Conjugate working solution was added followed by incubation. The optical density (OD value) of each well was determined at once with a micro- plate reader set to 450 nm.

C. Assays of Plasma tumor necrosis factor-alpha (TNF-α)

Plasma TNF-α was determined using specific rat ELISA kit purchased from Elabscience (Catalog No: E-EL-R2856) (USA) following manufacturers’ instruction. Wells were prepared according to instructional manual, biotinylated Detection Ab working solution was added to each well. Conjugate working solution was added followed by incubation. The optical density (OD value) of each well was determined at once with a micro- plate reader set to 450 nm.

Estimation Immunology Markers

A.      Assays of Plasma complement 3

Plasma C3 was determined using specific rat ELISA kit purchased from Elabscience (Catalog No: E-EL-R0687) (USA) following the manufacturers’ instruction. Wells were prepared according to instructional manual, biotinylated Detection Ab working solution was added to each well. Conjugate working solution was added followed by incubation. The optical density (OD value) of each well was determined at once with a micro- plate reader set to 450 nm.

B.      Assays of Plasma complement 4

Plasma C4 was determined using specific rat ELISA kit purchased from Elabscience (Catalog No: E-EL-R0256) (USA) following manufacturers’ instruction. Wells were prepared according to instructional manual, biotinylated Detection Ab working solution was added to each well. Conjugate working solution was added followed by incubation. The optical density (OD value) of each well was determined at once with a micro- plate reader set to 450 nm.

Immunohistochemical   studies   of   kidney   tissues for caspase-9, caspase-3, BCL-2 and BAX levels and expressions

The right kidney was harvested for this purpose. Assessment of Caspase-3, Caspase-9, BCL-2 and BAX Levels and Expressions. This was done following the Manufacturer’s procedures contained in the product leaflets accompanying the test kits. Small section of the right kidney was trimmed off and was fixed in 10% formalin neutral buffer for histological processing and paraffin embedding. 4–5-μm thin sections of the tissue were micro-sectioned, floated, and mounted on charged glass slides. The slides were labeled, placed in oven at 50–60° C for 20–30 min to melt excess paraffin. Slides were further deparaffinized and prepared for heat-induced antigen retrieval in citrate buffer solution (10 mM citric acid, pH 6.0). The staining was performed using Thermo Scientific Pierce™ 36000 Peroxidase IHC Detection Kits with slight modification of the procedure. Endogenous peroxidase activity was quenched by incubating kidney tissues for 30 min in Peroxidase Suppressor and washed three times in wash buffer, following which blocking buffer was added to the slides and incubated for 30 min. This was followed by addition of primary antibodies: Caspase-3, or Caspase-9 or, BCL-2 or BAX monoclonal antibodies at a dilution of 1: 100, and left overnight in a humidified chamber at 4° C. Slides were washed two times for 3 min with wash buffer and incubated with biotinylated secondary antibody for 30 min. The slides were washed thrice for 3 min each with wash buffer, and incubated with avidin/ streptavidin–horseradish peroxidase conjugate for another 30 min, and washed three times for 3 min each with wash buffer. The tissues were incubated with metal-enhanced DAB (3,3′ diaminobenzidine) substrate working solution for 5 min. The slides were rinsed with distilled water and treated with Mayer’s hematoxylin stain for 1–2 min at room temperature. and the slides washed several times with distilled water. and were with cover slips and dibutylphthalate polystyrene xylene (DPX) mountant for histology.

Scoring-Based Stain Intensity

Photomicrographs were taken with AmScope MU900 9MP USB2.0 Microscope Digital Camera attached to Fisher Science  Education™ 160-Series University/Laboratory Compound Microscope (Model: S2387, Fisher Scientific, California, United States). The images were quantified for staining intensity using Fiji (open source image processing package based on Image J) software [20].

Histopathological Studies

The left kidney of the rats was fixed in 10% buffered formalin, dehydrated in graded alcohol, cleared by xylene and embedded in paraffin wax. The tissues were then cut into 3–4m thick sections on a microtome. Cut sections were placed on slides and stained with haematoxylin and eosin. The slides were examined under a light microscope (Olympus CH; Olympus, Tokyo, Japan) and photomicrographs were taken with a Leica DM 750 camera at ×400 magnifications. All sections were evaluated for the degree of tubular and glomerular injury and necrosis.

Statistical Analysis

All values were expressed as means ± S.E.M. The statistical analysis was performed using 1-way ANOVA followed by Neumann–Keul’s post hoc test for comparison between groups. Differences were considered significant when p < 0.05. The data were analyzed using the statistical package program Stat Graph Pad version 9.0 (Graph Pad Software Inc., California, USA).

Effect of chloroquine and chloroquine plus prednisolone on body weight

Figure 1A, revealed the percentage weight loss observed weight in all group of animals both induced and treated except the control group.

Figure 1: Effect of Chloroquine and prednisolone on average body weight expressed as percentage change in weight of rats, and on average protein excretion during the 4 week of experimental phase. Each value is expressed as mean+SEM. *P<0.05 vs induced. Control, Induced (2 mg/ kg/day of ACR), CQ- chloroquine (25 mg/kg/day), CQ+PRED – Chloroquine +Prednisolone (25 mg/kg/day + 5 mg/kg/day)

The chloroquine + prednisolone achieved a slight increase in body weight. There was a statistical difference (p<0.05) in the weight changes of the chloroquine, chloroquine + prednisolone treated groups compared to the induced. In contrast, there was no significance difference (p<0.05) between the prednisolone-treated group compared to the induced.

Effect of Chloroquine on proteinuria in acrylamide- induced renal disease

Figure 1B, in the control group, animal’s urine was devoid of protein throughout the course of the experiment. Treated rats induced with ACR, demonstrated a progressive rise in the level of proteinuria throughout the experimental phase, which rose up to as high as above 1000 mg/dl of albuminuria [Figure 2].

Figure 2: Effect of Chloroquine on the plasma urea (mmol/l) and on the plasma creatinine (µmol/l) in Acrylamide-induced renal disease. Each bar is expressed as mean and SEM (N=8), Significant difference denoted by * p < 0.05 vs. control, αp < 0.05 vs. induced, β not significant p< 0.05 (CQ VS CQ+PRED) δ significant p < 0.05 (CQ VS CQ+PRED) by One-way ANOVA followed by Turkey’s post hoc test. Control, Induced (2mg of ACR), CQ- Chloroquine (25mg/kg), CQ+PRED – Chloroquine +Prednisolone (25mg/kg +5mg/kg)

The administration of chloroquine, chloroquine + prednisolone in this experiment was effective in reversing the level of proteinuria significantly (p<0.05) compared to the induced. However, the prednisolone-treated group failed to demonstrate significance difference (p<0.05) compared to the induced [Table 2].

Table 2: Effect of Chloroquine and prednisolone on average urine volume (ml/24hr) collected over the 4-week period of experiment on Acrylamide-induced renal disease. Each value is expressed as mean+SEM. *p<0.05 vs induced. Control, Induced (2 mg/kg/day of ACR), CQ- Chloroquine (25 mg/kg/day), CQ+PRED – Chloroquine +Prednisolone (25 mg/kg/day +5 mg/kg/day

WEEKS	CONTROL mls/24hr	INDUCED mls/24hr	CQ mls/24hr	CQ+PRED mls/24hr	PRED mls/24hr
1	3	1.8	1.8	2.0	1.6
2	2.8	1.6	2.0	2.0	1.8
3	2.8	1.4	2.2	2.2	1.9
4	3.2	1.0	2.2	2.4	1.7
MEAN	2.95	1.45	2.05*	2.15*	1.75

Estimation of urine volume in control, induced and treated groups of animals

There was a decline in the 24-hour urinary volume of animals induced with acrylamide. Chloroquine and chloroquine + prednisolone significantly p<0.05 increase the volume compared to the induced group [Table 2]. However, prednisolone-treated group failed in this regard, although there was a slight rise in the urine volume.

Effect of chloroquine on the serum urea in acrylamide- induced nephropathy

Chloroquine,  chloroquine  plus  prednisolone  reduce significantly (p<0.05) serum urea concentration when compared with the ACR-induced animals [Figure 2A]. This result demonstrated the positive effect of these drugs in ameliorating the level of urea. However, treatment of rats with prednisolone was not effective to cause significant reduction (P<0.05) in plasma urea.

Effect of drug treatment on the serum creatinine in ACR-induced nephropathy

Chloroquine, chloroquine plus prednisolone were able to reduce significantly (p<0.05) [Figure 2B] the serum creatinine when compared with the induced. Interestingly, there was statistical significance (p<0.05) between the chloroquine and chloroquine plus   prednisolone   treated   groups,   therefore, the potency of chloroquine plus prednisolone treated group over those administered with chloroquine only group was demonstrated [Figure 2B]. Moreover, this demonstrated the positive effect of the drugs in ameliorating the level of creatinine. Conversely, prednisolone action was not effective to cause significant (p<0.05) reduction in plasma creatinine [Figure 2B].

Effect of drug treatments on the serum Cystatin C

Chloroquine, chloroquine plus prednisolone significantly (p<0.05) reduced the serum cystatin C levels [Figure-3A] when compared with the acrylamide-induced group of rats. Cystatin C, a marker of glomerular filtration rate (GFR) was significantly reduced (p<0.05) in the chloroquine-only treated group. However, prednisolone treatment failed to significantly (p<0.05) lowers the blood levels of cystatin C.

Figure 3: Effect of Chloroquine on the plasma Cystatin and the plasma KIM 1 (ng/ml) in Acrylamide-induced renal disease. Each bar is expressed as mean and SEM (N=8), Significant difference denoted by * p < 0.05 vs. control, αp < 0.05 vs. induced, β not significant p<0.05 (CQ VS CQ+PRED) by One-way ANOVA followed by Turkey’s post hoc test. Control, Induced (2mg of ACR), CQ- Chloroquine (25mg/kg), CQ+PRED – Chloroquine +Prednisolone (25mg/kg +5mg/kg)

Effect of drug treatment on the serum KIM-1 in acrylamide-induced nephropathy

Serum KIM-1 level was significantly attenuated (p<0.05) [Figure 3B], in the chloroquine and Chloroquine plus Prednisolone treated groups of rats when compared to the induced and control groups. Prednisolone-treated group did not achieve significant (P<0.05) reduction of the expression of KIM-1 in the plasma caused ACR induction.

Effect of drug treatments on the level of plasma complement factors C3 (ng/ml)

Chloroquine, Chloroquine plus Prednisolone, and prednisolone caused significant elevation in the complement factor, C3 (p<0.05) compared to the induced and control groups [Figure 4A]. No statistical significance (P<0.05) observed between choloroquine and chloroquine + prednisolone treated group [Figure 4A].

Figure 4: Effect of Chloroquine on the plasma complement factors C3 and C4 (ng/ml) in Acrylamide-induced renal disease. Each bar is expressed as mean and SEM (N=8), Significant difference denoted by * p < 0.05 vs. control, α p < 0.05 vs. induced, β not significant p> 0.05 (CQ VS CQ+PRED) by One-way ANOVA followed by Turkey’s post hoc test. Control, Induced (2mg of ACR), CQ- Chloroquine (25mg/kg), CQ+PRED – Chloroquine +Prednisolone (25mg/kg +5mg/kg)

Estimation of serum plasma complement factors C4 (ng/ml) levels in acrylamide-intoxicated rats.

Figure 4B showed that chloroquine, chloroquine plus prednisolone, and prednisolone only treatments caused significant elevation in the complement factor, C4 (p<0.05) compared to the induced and control groups. Although there was no statistical significance (p<0.05) in chloroquine treated groups compared to chloroquine plus prednisolone treated group.

Estimation of plasma CRP levels in ACR-induced nephropathy

Plasma levels of CRP significantly reduced (p<0.05) in the animals treated with chloroquine, chloroquine plus prednisolone, and prednisolone groups when compared to the induced [Figure 5A].

Figure 5: Effect of Chloroquine on the plasma TNF-1α, CRP, IL-1β (ng/ml) in Acrylamide-induced renal disease. Each bar is expressed as mean and SEM (N=8), Significant difference denoted by *p < 0.05 vs. control, αp < 0.05 vs. induced, β not significant p<0.05 (CQ VS CQ+PRED) by One-way ANOVA followed by Turkey’s post hoc test. Control, Induced (2mg of ACR), CQ- Chloroquine (25mg/kg), CQ+PRED – Chloroquine +Prednisolone (25mg/kg +5mg/kg)

Estimation of plasma TNF-alpha levels in ACR-induced nephropathy

Plasma levels of TNF-α significantly fell (p<0.05) in the animals treated with chloroquine, chloroquine plus prednisolone, and prednisolone groups when compared to the induced [Figure 5B].

Estimation of plasma IL-1β levels in the acrylamide- induced nephropathic rats

Plasma levels of IL-1β was significantly reduced (p<0.05) following treatment of ACR-induced animals with chloroquine, or chloroquine plus prednisolone, or prednisolone only and also in comparison to the induced animals. [Figure-5C].

Effects of Chloroquine, Chloroquine+Prednisolone, and Prednisolone only treatments, on the expression of renal tissue caspases-3 in ACR-treated rats

Caspase-3 expressions in ACR-induced rats [Figure 6b], following treatment of animals with chloroquine, chloroquine+ prednisolone or prednisolone only are as depicted [Figure 6c-6e],   

Figure 6: Representative photomicrographs of immunohistochemical expression of caspase-3 in kidney tissue of rats (magnification ×400). (A) Control, (B) Induced (Acrylamide 2mg/kg/day), (C) Chloroquine 25mg/kg/day, (D) Chloroquine + prednisolone 25 mg/kg/day+5mg/kg/day, (E) Prednisolone 5mg/kg/day, (F) intensity score (I.S) of caspase-3 expression, mean ± SEM (n =8), and significant difference denoted by *p < 0.05 vs. induced yp < 0.05 CQ vs CQ + Pred, ? p> 0.05 vs control by one-way ANOVA followed by Turkey’s post hoc test

respectively.   Kidney   sections   from    ACR-induced rats showed enhanced expression of caspase 3 [Figure 6b] compared to the control group [Figure 6a]. Quantification of the immunohistochemical intensity demonstrated profound (p<0.05) decrease in the intensity score suggesting reduced caspase-3 expression in CQ, CQ+PRED- treated groups compared to the induced group [Figure 6f]. However, there was no significant difference (p<0.05) between the CQ-treated group and the control, indicating the potency of the drug in suppressing Caspase-3. However, there was no significant difference (p<0.05) between the prednisolone treated group compared to the induced.

Caspase-9 expressions in ACR-induced kidneys is depicted in [Figure 7b], and kidney tissues treated with chloroquine, chloroquine + prednisolone and prednisolone are as depicted in [Figures 7c-7e] respectively.

Figure 7: Representative photomicrographs of immunohistochemical expression of caspase-9 in kidney tissue of rats (magnification ×400). (A) Control, (B) Induced (Acrylamide 2mg/kg/day), (C) Chloroquine 25mg/kg/day, (D) Chloroquine + prednisolone 25 mg/kg/day+5mg/kg/day, (E) Prednisolone 5mg/kg/day, (F) intensity score (I.S) of caspase-9 expression, mean ± SEM (n =8), and significant difference denoted by *p < 0.05 vs. induced βp < 0.05 CQ vs CQ + Pred, by one-way ANOVA followed by Turkey’s post hoc test

ACR-induction resulted in enhanced   expression   of caspase 9 [Figure 7b] compared to the control group [Figure 7a]. Quantification of the immunohistochemical intensity demonstrated profound (p<0.05) decrease in the intensity score suggesting reduced Caspase-9 expression in CQ treated groups compared to the induced group [Figure 7f]. However, there were no significant difference (p<0.05) between the chloroquine+prednisolone, prednisolone- treated group compared to the induced.

The photomicrographs of immunohistochemical staining for BCL-2 expressions in kidney tissues are as shown in Figure-8. While control rats did not show significant BCL-2 expression (p<0.05) compared to the induced, there was no significant difference (p<0.05) between treated groups versus the ACR- induced rats [Figure 8].

Figure 8: Representative photomicrographs of immunohistochemical expression of BCL-2 in kidney tissue of rats (magnification ×400). (A) Control, (B) Induced (Acrylamide 2mg/kg/day), (C) Chloroquine 25mg/kg/day, (D) Chloroquine + prednisolone 25 mg/kg/day+5mg/kg/day, (E) Prednisolone 5mg/kg/day, (F) intensity score (I.S) of BCL-2 expression, mean ± SEM (n =8), and no significant difference p<0.05 by one-way ANOVA followed by Turkey’s post hoc test

Effects of Chloroquine, chloroquine+prednisolone, or prednisolone treatment, on renal tissue BAX expressions in the ACR-induced RATS

The photomicrographs of immunohistochemical staining for BAX expressions in kidney tissues are as shown in [Figure 9], while the normal rats did not show significant BAX expression, there was significant decrease (p<0.05) between the Chloroquine- treated group vs induced [Figure 9c], chloroquine +prednisolone vs induced [Figure 9d], and prednisolone vs induced [Figure 9e].

Figure 9: Representative photomicrographs of immune-histochemical expression of BAX in kidney tissue of rats (magnification ×400). (A) Control, (B) Induced (Acrylamide 2mg/kg/day), (C) Chloroquine 25mg/kg/day, (D) Chloroquine + prednisolone 25 mg/kg/day+5mg/kg/day, (E) Prednisolone 5mg/kg/day, (F) intensity score (I.S) of BAX expression, mean ± SEM (n =8), and significant difference denoted by *p < 0.05 vs. induced αp < 0.05 vs control, by one-way ANOVA followed by Turkey’s post hoc test

Effect of  drug  treatments  on the kidneys: histopathologic evaluation of representative sections.

No observable lesion was seen in the histological sections of the control group, as the glomeruli, Bowman’s Capsule, tubules and   interstitium   all   appeared   normal   [Figure 10 control].

Figure 10: Photomicrograph of a representative histological sections of the kidneys of experimental rats stained with (Haematoxylin and Eosin stain and x400 magnification). The control group section revealed no remarkable histological lesion in the glomeruli, Bowman’s capsule, tubules and interstitial all appeared normal. Kidneys from induced group of rats showed infiltration of chronic inflammatory cells in the glomerulus and along the tubules (black arrow and blue arrow) indicating CGN and TIN respectively. The chloroquine (CQ) treated group revealed random glomerular atrophy with distension of Bowman’s capsule (green arrow), tubules appeared normal. CQ + prednisolone treated group, showed fewer tubular epithelial damage (blue arrow). Yellow arrow proximal convoluted tubules, green arrows Bowman’s capsule.

ACR induction resulted in severe infiltration with chronic inflammatory cells in the mesangium of the glomerulus, depicting a state of chronic glomerulonephritis. In addition, there was tubular epithelial necrosis and degeneration with associated moderate interstitial nephritis. [Figure-10induced]. The chloroquine-treated group showed random glomerular atrophy and distension of Bowman’s space. [Figure 10 chloroquine]. There was resolution of chronic inflammatory cells and fewer signs of tubular necrosis. The chloroquine+prednisolone-treated group revealed random glomerular atrophy, tubular epithelial necrosis [Figure 10 CQ+PRED]. Prednisolone-treated group [Figure 10 pred], showed random glomerular atrophy with severe tubular epithelial necrosis.

In this study, the development of ACR-induced CGN and TIN was confirmed during an induction phase of the study. Each of ten rats were administered, 2 mg/kg of ACR daily by oral gavage for a period of 14 days, followed by sham handling until day 28 when they were sacrificed for histopathological studies to ascertain ACR-induced CGN and TIN. Abrogation of ACR-induced CGN and TIN in treated rats by the administration of chloroquine, and a combination of chloroquine + prednisolone was demonstrated in this present study using both qualitative and quantitative methods.

The role of both inflammation and immunology in the pathogenesis of ACR-induced renal injury have been evaluated. Complement activation is an established pathogenetic pathway in the development of immune-complex glomerular disease as well as other renal diseases, such as tubule-interstitial fibrosis, haemolytic uraemic syndrome and transplant injury [21]. Important mediators include plasma C3 and C4 where low levels of these complements are detected in those with immune mediated renal diseases [21].

Rats administered with ACR showed reduction in weight as compared with the control, this is could be attributable to the role of chronic inflammation caused by ACR and its catabolic tendency. This result is in consonance with a previous report by Swamy et al [22] who observed a similar reduction in body weight in animals treated with ACR. Treatment with drugs did not result in weight increase during the 28 days of treatment except, in the chloroquine+prednisolone group, which achieved slight weight gain, and chloroquine-treated group had a significant (p<0.05) weight gain compared to the rats that were induced with ACR and received no other drug treatment.

Wistar rat’s urinary protein contains less than 30g/dl, it is unusual for protein excretion to exceed 150g/dl in urine except in pathological disease state (nephropathy) [23]. Nephropathy occurred following induction with ACR evidenced by the appearance of proteinuria in excess of 30 g/dl following the intake of ACR, indicating that, ACR is capable of causing disruption of glomerular membrane to allow proteins to leak into the urinary tubules. Results from this study showed the attenuating potential of chloroquine and chloroquine+prednisolone in reducing the urinary protein excretion in which the ACR-induced group of rats treated with chloroquine, and those treated with chloroquine + prednisolone had a drastic fall in proteinuria to less than 500mg/ dl. This result, showed the anti-proteinuric effect of chloroquine, hence, a possible candidate for reversing glomerulopathy. In addition, animals induced with ACR had a significant drop in their 24-hour urinary output as compared to the control, and those treated with chloroquine and chloroquine+prednisolone. This is possibly attributed to the effect of ACR in reducing the GFR. This was supported by EFSA and Friedman [7,9], which demonstrated the nephrotoxic potential of ACR.

This study further demonstrated chloroquine as a good alternative drug in the treatment of renal disease characterised by its potential at reversing or blocking the biochemical, inflammatory, and immune markers of reno-toxicity. Quantitative measurement of renal function relies strongly on GFR which can be indirectly measured by substances that are filtered but not secreted or reabsorbed by the kidneys [21]. Traditional renal biochemical parameters; urea and creatinine which are common markers of assessing renal dysfunction have some limitations, as the body mass index, disease state and age alters the values of these parameters [24]. It was observed that there was significant attenuation (p < 0.05) in the levels of urea and creatinine in the treated groups except the prednisolone-only-treated group of rats. This further supports the reno-protective property of chloroquine and its effect in renal disease.

Furthermore, we employed newer, highly sensitive, and specific markers of renal damage; Cystatin C, KIM-1, whose levels are independent of muscle mass, type of diet, and a critical immune factor in detecting tubular and glomerular damage [25].

KIM-1 is a type 1 membrane protein expressed by proximal tubular epithelial cells but not the glomeruli in acute tubular necrosis. It is a sensitive biomarker in acute kidney injury whose level has been demonstrated to rise in chronic kidney diseases [25]. Results from this study demonstrated that KIM-1 was significantly elevated in the ACR-induced group of rats, and provides further support that ACR causes renal damage, more particularly tubular damage. Interestingly, chloroquine and chloroquine plus prednisolone treatment of ACR-induced rats suppressed damage caused by ACR, as shown by the significant low levels of KIM-1. Also, the histopathologic sections of the kidneys of chloroquine-treated rats revealed fewer inflammatory cells deposition in the glomeruli and tubular cells.

Cystatin C, is a more reliable marker of GFR than creatinine [24]. The role of cystatin C in the evaluation of renal diseases cannot be over-emphasized, being a low molecular weight protein freely filtered by the glomerulus and metabolized by proximal convoluted tubules, so that it does not return to the blood. It is a sensitive and specific marker of glomerular filtration rate. It is a more accurate evaluator of renal function than the traditional biochemical parameters, urea and creatinine, as its plasma level is not influenced by diet and body mass index [24]. Therefore, the significant elevation of cystatin C level recorded in induced animals compared to the control is another indication of renal damage. In contrast, the significant reduction in the plasma level of cystatin C observed in chloroquine, and chloroquine+prednisolone treated rats demonstrates a potent evidence of the abrogative potential of these drugs at reversing the kidney damage. However, prednisolone possessed limited ability, as demonstrated in the treated group, to reverse ACR- induced renal damage.

Activation of the classical complement pathway and the immune system contribution has been demonstrated in the pathogenesis of glomerular diseases, as results obtained from evaluation of the complement system was supportive of this fact, which revealed significantly low levels of C3 and C4 of in ACR-induced group of rats when compared to the control. The significant elevation in the complement factors in the treated groups; chloroquine, chloroquine+prednisolone and prednisolone, also point to the therapeutic potentials of these drugs in restoring the immune functions of rats with nephropathy.

Other mechanistic pathways of induction of nephropathy were explored, as previous research demonstrated the effect of ACR in causing oxidative stress, and inflammation through elaboration of inflammatory markers such as CRP, TNF-α, and IL-1β, [26]. IL-1β is an important marker in cardiovascular and renal disease. It binds to IL-1 receptor resilting in immune activation and fever [27]. IL-1β is a key mediator of inflammation in the kidneys and it has been shown that use of its antagonist has resulted in fall in the level of this inflammatory marker [27]. The significant reduction in levels of IL-1β demonstrated by chloroquine treatment suggests that this inflammatory marker elaborated by ACR was suppressed by chloroquine alongside prednisolone. Similar results were observed in other key pro- inflammatory markers, TNF-α, and CRP. Inflammatory markers are drivers of both acute and chronic inflammatory cells in renal damage [28]. Kidneys of ACR-induced rats revealed many pro-inflammatory cells deposited along the glomerulus and tubules, further supporting the mechanism of induction of renal damage, caused by this ACR. Conversely, the suppression of these inflammatory cells in the chloroquine, and chloroquine+ prednisolone treated rats further buttressed the mechanism of these anti-inflammatory drugs and their effectiveness in the treatment of inflammation related kidney diseases.

The role of apoptosis in the pathogenesis of acrylamide- induced neurotoxicity has also been previously reported [14,29], showed that mitochondrial malfunctioning is one of the mechanisms responsible for induction of apoptosis by acrylamide in human cells. This process, which involves the activation of caspase 9 and up regulation of Bax/BCL-2 ratio is responsible for the ACR-induced apoptotic neuronal changes. Another signalling pathway involved in this apoptotic process is the activation of mitogen-activated protein kinase (MAPK), which includes extracellular regulated protein kinases (ERK), c jun N-terminal kinase (JNK) and p 38 protein genes [30]. Inactivation of ERK and JNK and P38 are essential for inducing apoptosis by acrylamide [30]. In current study, we explored the mitochondrial malfunction pathway mechanism of induction of apoptosis by ACR in damaged rat kidneys. Interestingly, ACR caused upregulation of caspase 3, 9 and Bax in the induced animals. Our results showed that ACR

-induced renal injury occurs through apoptosis, although there was no alteration of BCL-2 proteins. The ability of chloroquine and chloroquine+prednisolone to suppress this apoptotic process by causing significant inhibition of these proteins further confirmed the therapeutic potentials of these drugs in reversing ACR-induced apoptosis and thus, renal damage.

In addition, the chloroquine, chloroquine + prednisolone- treatment demonstrated effectiveness at reversing varying histopathological alterations caused by ACR on the kidney architecture. Induction of rats with ACR resulted in severe proliferative chronic inflammatory cells, interstitial nephritis, as well as tubular necrosis. These changes were significantly reversed in groups of rats treated with these drugs. The effect was much pronounced in the chloroquine-treated rats, as the kidney sections have minimal observable lesions

The current study demonstrated that multi-faceted mechanisms are at play in the action of chloroquine in reversing renal damage induced by ACR, namely: anti-apoptotic, anti- inflammatory, and immune-mediated pathways. One would have expected a synergistic effect of chloroquine+ prednisolone to produce dramatic result in the treatment of the renal damage. However, the results obtained from the possibility of an adjuvant potential in this study was not satisfactory, as this group failed to show consistent significant differences between the chloroquine- treated and chloroquine+prednisolone-treated groups. Combination with prednisolone failed to demonstrate additional benefits, in spite of the fact that prednisolone treatment demonstrated anti-inflammatory property at inhibiting inflammatory markers, CRP, TNF-α AND IL-1β. Further research should elucidate the reasons for the failure of the expected adjunctive potential of the combination therapy.

In conclusion, findings of this study highlight the promising therapeutic potential of chloroquine as a repurposed drug in the management of ACR-induced nephropathy which were possibly mediated via anti-inflammatory, anti-apoptotic and immune- mediated mechanisms.

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