Effect of Perfluorooctanoic Acid on Kidney Function in Diabetic and Non-Diabetic Male Guinea Pigs

The main sources of exposure to PFOA are water, soil, and animal-origin food (meat, fish, and dairy products). The aim of this study to evaluate the renal function following oral gavage of sub-lethal dose of PFOA in diabetic and non-diabetic guinea pigs. The experiment run for 4 weeks, total of 40 male guinea pigs, ( Cavia porcellus ), were randomly selected and grouped into four equal groups. The first group (G1) served as the negative control; 2nd group (G2) alloxan induced diabetic, 3rd group (G3) non-diabetic was exposed to PFOA at 100 mg/kg BW orally/daily and 4th Group (G4) was diabetic guinea pig exposed to PFOA at 100 mg/kg BW orally/daily. Serum creatinine and histopathological alterations in the kidney tissue were evaluated. Serum creatinine concentrations were significantly increased ( P <0.05) in G3 and G4 exposed to PFOA. High serum creatinine levels were suggesting impairment in kidney function. Impaired kidney function was confirmed through histopathological changes such as glomerular atrophy, severe necrosis

increase in concern about the potential harmful effects of PFOA and its salts on both the environment and living organisms (9,10).Researchers revealed that PFOA accumulate mainly in the kidney and causes nephrotoxicity with impairment kidney functions (6).Moreover, diabetes (DM) is one of the health conditions which can be caused by Perfluorooctanoic acid (PFOA) (11).
The toxicokinetic of diverse PFAS members are influenced by their chemical composition, chain length and branching (12).PFOA is easily absorbed orally, as well as inhalation and cutaneous routes (13).Animal studies have found that both long-and short-chain PFAS are rapidly absorbed when given orally to the animal model.In toxicokinetic studies, substances are evaluated based on the time that required for 50% of the chemical to fall of its initial concentration half-life (t ½).For example, the rate of absorption in the digestive tract of rats has been calculated to be < 2 h.Notably, female rats absorbed PFOA at a rate faster than in male rats about 1.1 h vs. 10 h.The mechanisms underlying the differences in toxicokinetics variables between males and females is unknown, but it is supposed to include hormonal variations influencing the absorption of these chemical materials (14) PFOA is easily absorbed following oral exposure and is mostly preset in liver, kidney and blood of rats.It can easily cross the bloodplacenta barrier and pass to the fetus, where it is mainly present in the liver (15).PFAS bind to the serum albumin and proteins in plasma such as gamma globulin, transferrin, alpha-globulin, beta-lipoproteins and alpha-2macroglobulin in both rat and human plasma, but the affinity for albumin to binding in rats is more than in human (16,17).The absorbed PFAS distributed extensively from plasma into the soft tissues.The variance in PFAS plasma half-lives between species is most likely due to changes in the density, nature and the activity of renal tubular organic anion transporters (OATs) among species, as well as the differences in affinity of PFAS (17,18).PFOA is not broken down within the body (not metabolized) (19,20).PFAS are mostly excreted in urine, with minor levels expelled in the feces and in breast milk.The elimination half-life of PFAS compounds has shown to be quicker in females than in males rats (14) The association between serum PFOA and diabetes prevalence was documented (21).Based on the biodegradability and bioaccumulation properties of PFOA in animals and human tissues, concerns are increasing about the harmful effects of PFOA on kidney function.The aim of this study is to investigate PFOA effects on the kidney of diabetics and non-diabetics guinea pigs.

Ethical Approval
Ethical approval was granted through the local committee of the animal care and use at the College of Veterinary Medicine in the University of Baghdad (Number 828 at 9/4/2023) before starting this study.

Animals
The experimental animal model selected for this study was Cavia porcellus guinea pigs.Forty male guinea pigs about 12-14 week of age with range weight 500-550 g, were obtained from the animal house of College of Veterinary Medicine, University of Baghdad.A standard diet of commercial pellets, vegetables, vitamin C and tap water were available.The animals apparently healthy and kept under hygienic environment at 22±4 °C in air-conditioned room, a relative humidity of 50±10%, and the light system was 12/12 h.light/dark cycle (22,23).The air of the place was continuously changed by the ventilation vacuum (24).The place was cleaned every three days to avoid any stress and all animals were observed daily for clinically apparent signs of disease or injury.

Induction of Diabetes in Guinea Pigs
Following overnight fasting, guinea pigs were injected intraperitoneally (IP) with 200 mg/kg of alloxan monohydrate in 24 h.intervals for three days, respectively in order to induce diabetic and maintain the increasing blood sugar levels in the experimental diabetic guinea pigs.Blood glucose test was performed before and after the injection as described in (26,27) with modification.Following restraint of the animal, a blood drop from the ear is placed on the strips, and the sugar is measured with a glucometer.Diabetes mellitus was induced in twenty guinea pigs represented in Group 2 and Group 4, respectively.Guinea pigs with fasting blood glucose concentrations over 200 mg/dL were considered as diabetic (28) and involved in this study.

Experimental Design
The experimental animals (40 male guinea pigs) were divided randomly and equally into 4 groups.Group 1 (G1) was represented as a negative control treated with DW. 1 mL orally for 4 weeks.Group 2 (G2) induced diabetic male guinea pigs using alloxan monohydrate served as a positive control.Alloxan induce hyperglycemia which can be sustained for less than month.The sub-lethal toxicity of PFOA dose was calculated below the literature reported LD50 200 mg/kg in guinea pigs (25).Non-diabetic animals in Group 3 (G3) received 100 mg/kg BW of PFOA.Group 4 (G4) diabetic male guinea pigs received 100 mg/kg BW of PFOA orally by using stomach gavage.The duration of the experiment was 4 weeks.

Samples and Laboratory Tests
The first blood collection from the heart was performed immediately before the 1 st dosing and then animals in each group had blood collecting every week after exposure at 1, 2, 3 and 4 weeks.After manual restraint of guinea pigs, blood samples (2.5 mL) were collected from each animal by cardiac puncture.Blood was deposited into tubes without anticoagulant then, blood samples were centrifuged at 3000 rpm for 15 min (29).The serum samples were kept in polyethylene Eppendorf tubes at -20 °C (30) which were used for assessing creatinine concentrations.

Histopathological Examination
After the completion of dosing for 4 weeks, guinea pigs were euthanized by using an overdose of ketamine (95 mg/mL) with xylazine (5 mg/mL, 1.5 mL/kg BW) (31) and then necropsies.Samples for histopathology collected from the kidney and kept in 10% formalin (32).The samples were processed for paraffin embedding.Then, sectioning was performed using microtomy.Finally, the sections were stained by hematoxylin and eosin (H&E).The slides were examined as described by (33).All the micro-pathological changes in treatment groups were assessed and compared with the control group.

Statistical Analysis
The data of the experiment were analyzed using the GraphPad Prism Statistical (version 8.0.2).Two-way ANOVA and Tukey's multiple comparison test were performed to evaluate significant differences among means of the groups.The results were expressed as mean ± stander errors and P≤0.05 was considered statistically significant (34).

Clinical Signs
Guinea pigs that exposed to PFOA represented by G3 and G4 showed signs of emaciation, hypoactivity and unhealthy hair appearance.Alloxan induces diabetes in G2 shows polyuria, polydipsia and excessive water drinking.

Histopathological Findings of Kidney
To study the histopathological alterations of PFOA in normal and diabetic male guinea pigs, tissue sections from the kidney were examined microscopically to determine any changes following PFOA toxicity.
The first group (control negative group) shows no noticeable lesions (Figure 2).The second diabetic (control positive group) shows infiltration of inflammatory cells and hemorrhage (Figure 3 A) with dilation of Bowman's space and sloughing of renal tubules (Figure 3 B).Guinea pig's kidney that treated orally with 100 mg/kg BW of PFOA (G3 and G4) shows necrotic changes with sloughing in the tubular epithelium lining, glomerular atrophy, vacuolar degeneration, infiltration of inflammatory cells (Figure 4 A, B, Figure 5 A-C).In addition, formation of hyaline casts in the renal tubular lumen (Figure 4 B).

DISCUSSION
This study investigates the renal histopathological changes associated with creatinine levels following oral gavage of sub-lethal dose of PFOA in diabetic and nondiabetic guinea pigs.Our results revealed that PFOA lead to rose in serum creatinine levels as show in Figure 1.These results are in accordance with (35,36) which demonstrated that PFOA causes renal impairment and significant elevation of creatinine concentrations.Moreover, a study showing elevation concentrations of serum creatinine at dose 0.05 mg/kg of PFOA in rats (37).Furthermore, the presence of PFOA in the water at concentrations of (30 mg/L) for a period of 30 days in Nile tilapia (Oreochromis niloticus) caused a considerable rise in creatinine levels (38).PFOA caused alterations in renal microvascular endothelial-cell permeability by increasing production of reactive oxygen species (ROS) (39).When the oxidation processes of reactive oxidative species are stronger than the antioxidant response within the cell, the oxidative stress will occur (40).
In addition, diabetic rats treated with perfluorooctanoic acid showed extensive kidney damage with marked fibrosis and inflammation of glomerular mesangial cell due to disturbance metabolisms of amino acids and purines (41).Epigenetic alterations which promote expression of fibroblast activation markers were reported in kidney tissues of mice exposed to PFOA (42) The non-significant differences between non-diabetic group (G3) and diabetic group (G4) that both toxicated with PFOA indicated that PFOA is responsible for increasing creatinine concentrations with or without development of diabetes.
Several reports indicated the pathological effects of alloxan in renal tissues.Alloxan treated mice that caused kidney damage and degeneration to proximal and distal convoluted tubules and degeneration in glomerulus with increased capsular space (43).These results are in accordance with the current study in diabetic guinea pigs following the injection by alloxan monohydrate 200 mg/kg BW.Earlier it was reported that PFOA caused necrotic alteration in epithelial lining of renal tubules with accumulation of eosinophilic substance in tubular lumen in albino rats at dose 20-40 mg/kg BW by intragastric intubation daily for month (44).Exposure to PFOA changed numerous pathways that related to kidney diseases, including induction of oxidative stress, peroxisome proliferator activated receptor pathway, inflammatory pathway, autophagy pathway and DNA methylation (45).Moreover, the main nephrotoxicity mechanism in fish treated with 0.2 mg/kg PFOA was mediated by the oxidative damage and production of ROS (46).In addition, to increased production of free radicals, disturbance in serum lipid and glucose concentrations following PFOA exposure in guinea pigs was reported (47).The histopathological changes in the kidney such as necrosis, diffuse vacuolar degeneration and glomerular atrophy following PFOA administration (100 mg/kg BW, daily for 4 weeks) were related strongly with serum creatinine elevation (Figure 1).In fish, PFOA exposure influence on both the excretory and hemopoietic parts of the kidney with dilated glomerular capillary bed and loss of typical folding pattern (48).Furthermore, different doses of PFOA (0.05, 0.1, 0.5, and 1 mg L −1 ) for 7, 14, and 21 days lead to swelling of mitochondria, hydropic endoplasmic reticulum and vacuolization in zebrafish kidney (49).PFOA also has been identified as a kidney carcinogenic that may have significant effects on public health for many individuals exposed to this widespread and extremely persistent chemical worldwide (50).It is believed that PFOA induces kidney carcinogenesis by activation signaling pathway of Peroxisome proliferator-activated receptors (PPARα) (51,52).Another hypothesis supposed that PFOA induced epithelial-mesenchymal transition in the kidney by increasing the expression of genes correlated with epithelial-mesenchymal transition with promotion of cell migration (53).
In vitro studies are recommended to well-known the impact of long-term PFOA exposure on the kidney including the toxicological information and mechanism of toxicity under stressed conditions (54,55) In conclusion, after 4 weeks, positive associations of PFOA with serum creatinine, have been observed in nondiabetic and diabetic guinea pigs exposed to PFOA.Furthermore, PFOA lead to disturbances in the kidney performance and nephrotoxicity in diabetic and nondiabetic animals.

Figure 1 .
Figure 1.Serum creatinine concentrations in the control, alloxan induced diabetic, treatment animals with PFOA at 100 mg/kg BW and diabetic guinea pigs exposed to PFOA at 100 mg/kg BW during the experimental period of 4 weeks, n=10, * P < 0.05