L- Arginine Supplementation Protections Sodium Fluoride-Induced Nephrotoxicity And Hypertension By Suppressing Mineralocorticoid Receptor and Angiotensin Converting Enzyme Activity

Sodium uoride (NaF) is one of the neglected environmental toxicants that has continued to silently cause toxicity to both humans and animals. NaF is universally present in water, soil and the atmosphere. The persistent and alarming rate of increase in cardiovascular and renal diseases and disorders caused by chemicals such as sodium uoride (NaF) in mammalian tissues have led to the use of various drugs for the treatment of these diseases. This study aims at evaluating the renoprotective and antihypertensive effects of L- Arginine on NaF-induced nephrotoxicity. Thirty male Wistar rats (150 – 180 g) were used in this study. The rats were randomly divided into ve groups of six rats each as Control, NaF (300 ppm), NaF + L- Arginine (100 mg/kg), NaF + L- Arginine (200 mg/kg), and NaF + Lisinopril (10 mg/kg), respectively; orally for eight days. Histopathological examination and immunohistochemistry of renal angiotensin converting enzyme (ACE) and mineralocorticoid receptor (MCR) were performed. Markers of renal damage, oxidative stress, antioxidant defence system, and blood pressure parameters were determined. L- Arginine signicantly (p <0.05) ameliorated the hypertensive effects of NaF. The systolic, diastolic and mean arterial blood pressure of the treated groups were signicantly (p< 0.05) reduced compared with the hypertensive group. This nding was concurrent with signicantly increased serum bioavailability of nitric oxide in the hypertensive treated groups. Also, there was signicant reduction in the level of blood urea nitrogen (BUN) and creatinine in the serum of the hypertensive rats treated with L- arginine. There was signicant (p<0.05) reduction in markers of oxidative stress such as hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA) and protein carbonyl (PCO) and concurrent increase in the levels of antioxidant enzymes in the kidney of hypertensive rats treated with L arginine. The results of this study suggest that L- Arginine normalized high blood pressure, reduced oxidative stress, reduced the expression of renal ACE and MCR, and improved nitric oxide production. Thus, L- Arginine holds promise as a potential therapy against hypertension and renal damage.


Introduction
Sodium uoride (NaF) is one of the neglected environmental toxicants that has continued to silently cause toxicity to both humans and animals . NaF is universally present in water, The present study elucidated the molecular mechanism of action of anti-hypertensive action of L-Arginine in a toxicant-induced hypertensive and nephrotoxic rat model.

Experimental Animals and Design
Thirty male Wistar rats (150 -180 g) were used in this study, the rats were randomly divided into ve groups of six rats each as Control, NaF (300 ppm), NaF + L-Arginine (100 mg/kg), NaF + L-Arginine (200 mg/kg), and NaF + Lisinopril (10 mg/kg), respectively; orally for eight days. The concentration of NaF (Oyagabemi et al. 2021) and the dosages of L-Arginine (Saad 2021) and Lisinopil (Oyagabemi et al., 2021) were chosen based on the previous literature. The animals were also fed with rat cubes ad libitum and water was supplied liberally. The rats were kept in wire mesh cages under controlled light cycle (12 h light/12 h dark) and fed with commercial rat chow ad libitum and liberally supplied with water. The blood of the rats was taken on the 8 th day and rats were sacri ced on the 9 th day.

Ethical approval
The study was conducted following guidelines approved by the Animal Care and Use Research Ethics Committee (ACUREC) of the University of Ibadan (Approval number: UIACUREC/ 19/124).

Blood pressure measurement
The systolic (SBP), diastolic (DBP), and mean arterial (MAP) blood pressures were determined noninvasively in conscious animals by tail plethysmography using an automated blood pressure monitor (CODA S1, Kent Scientific Corporation, Connecticut, USA). The blood pressure parameters were obtained by an indirect method of blood pressure measurement as recently reported from our laboratory (Oyagbemi et al. 2019).

Serum preparation
The serum was obtained from whole blood collected into anticoagulant free sample bottles following a post-collection waiting period of 60 mins. Thereafter, the serum was kept at a 4 o C temperature.
Determination of serum markers of renal damage Serum creatinine and blood urea nitrogen (BUN) were determined following the manufacturer's instructions in the purchased Randox® kits (Randox® Laboratories Ardmore, United Kingdom).

Preparation of renal post mitochondrial fractions
The kidney and testes were quickly excised, rinsed, weighed and homogenized with homogenizing buffer (0.1M phosphate buffer, pH 7.4) using a Te on homogenizer. The homogenate was centrifuged at 10,000 g for 10 minutes at -4 O C.

Biochemical assays
Estimation of renal oxidative stress Hydrogen peroxide generation was determined according to the method of Wolff (1994). The reaction mixture was subsequently incubated at room temperature for 30 minutes. The mixtures were read at absorbance of 560 nm and H 2 O 2 generated was extrapolated from H 2 O 2 standard curve. The Malondialdehyde (MDA) content as an index of lipid peroxidation was quanti ed in the PMFs of cardiac and renal tissues according to the method of Varshney and Kale (1990). The absorbance was measured against a blank of distilled water at 532 nm. Lipid peroxidation was calculated with a molar extinction coe cient of 1.56 × 10 5 /M/cm. Protein carbonyl (PCO) contents in the renal and cardiac tissues were measured using the method of Reznick and Packer (1994). The absorbance of the sample was measured at 370 nm. The carbonyl content was calculated based on the molar extinction coe cient of DNPH (2.2 104 cm1 M1) and expressed as nmoles/mg protein while vitamin C contents were measured as earlier described (Jacques-Silva et al. 2001).

Renal antioxidant status
The Superoxide dismutase (SOD) assay was carried out by the method of Misra and Fridovich (1972), with slight modi cation (Oyagbemi et al. 2015). The increase in absorbance at 480 nm was monitored every 30 s for 150 s. The one unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline to adrenochrome. Reduced glutathione (GSH) was estimated by the method of Jollow et al. (1974). Glutathione peroxidase (GPx) activity was also measured according to Beutler et al. (1963). Glutathione S-transferase (GST) was estimated by the method of Habig et al. (1974) using 1-chloro-2, 4-dinitrobenzene as substrate. The protein and nonprotein thiol contents were determined as described by Ellman (1959).

Estimation of serum nitric oxide concentration and total protein
The serum nitric oxide concentrations were measured spectrophotometrically at 548 nm as previously described (Olaleye et al. 2007). Protein concentration was determined by the Biuret method of Gornal et al. (1949), using bovine serum albumin (BSA) as standard.

Histopathology
Small pieces of kidney were xed in 10% formalin, embedded in para n wax, and sections of 5-6 mm in thickness were made and thereafter stained with hematoxylin and eosin (H&E) for histopathological examination according to the methods as previously described (Drury et al. 1976). Thereafter, the sections were examined with light microscopy. Immunohistochemistry Immunohistochemistry was done as described by Oyagbemi et al. (2019). Antibodies against renal angiotensin converting enzyme (ACE) and mineralocorticoid receptor (MCR) were probed in the heart using 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System with DAB solution (Catalog number: E-IR-R217 from Elabscience Biotechnology®, China). The kidney samples were xed with 10% paraformaldehyde, embedded in para n and sectioned at a thickness of 5 μm. The slides were subsequently dewaxed in xylene (100%) solution for 2 minutes and afterward, hydration was carried out in different concentrations of ethanol (100%, 90%, and 80%) for 2 minutes each. The hydrated slides were rinsed and put in a PBS buffer tank for 5 mins. The antigen retrieval was performed with citrate buffer solution containing 2.1 g of citric acid monohydrate and 14.75 g of trisodium citrate dehydrate adjusted to pH 6.0 in microwave oven. Endogenous peroxide (H 2 O 2 block) was carried out following manufacturer's instructions as directed on the kit (E-IR-217C). Drops of H 2 O 2 were added to cover the sections and incubated in humidifying chamber at room temperature for 10 min. The slides were rinsed afterwards and put back in the PBS tank for 5 min. Goat serum (E-1R-R217A) was added onto the slides to prevent nonspeci c binding and incubated in humidifying chamber at room temperature for 30 mins. After 30 mins of incubation, the tissues were probed with primary antibodies viz-a-viz Angiotensin Converting Enzyme1 polyclonal antibody (E-AB-16159: 1:500 Dilution) and anti-mineralocorticoid receptor polyclonal antibody (E-AB-70261: 1:500 Dilution), and incubated for 2 hours at room temperature.
Following incubation, the slides were rinsed with PBS and secondary antibody labelled (E-1R-R217B) was added, and the slides were incubated in humidifying chamber at room temperature for 20 min. Thereafter, the slides were rinsed and immersed in PBS tank for 5 min. Finally, a few drops of the substrate diaminobenzidine (DAB) was added at room temperature for 10 s; 50 µL of DAB concentrate (E-1R-R217D) + 1 mL DAB solution (E-1R-R217E) in the dark. The reaction was terminated with deionized water and slides were immersed in hematoxylin (Sigma-Aldrich, USA) for 3 s before rinsing with PBS. The slides were placed in 80%, 90%, and 100% of ethanol, and then xylene (100%) for 2 minutes each. Slides were removed, allowed to dry and a DPX mountant was applied. Sections were observed with a light microscope (Leica LAS-EZ®) using Leica software application suite version 3.4 equipped with a digital camera.

Statistical Analysis
Data obtained were analyzed with One-way ANOVA with Dunnett's post-test at a 95% con dence limit. All values are expressed as mean ±S.D. The test of signi cance between two groups was estimated by Student's t test.

Sodium uoride intoxication on kidney weight and kidney relative weight
The results from gure 1 showed a signi cant (P<0.05) reduction in relative body weight of rats intoxicated with NaF and those co-administered with either L-Arginine or Lisinopril. Similarly, there was a signi cant (P<0.05) reduction in relative kidney weight of rats administered only NaF. However, L-Arginine supplementation and Lisinopril co-administration showed signi cant restorative effect on the relative kidney weight to near normal values (Figure 1).

Hemodynamic parameters
The blood pressure parameters measured in the present study indicated signi cant (P<0.05) increases in the values of systolic blood pressure (SBP), diastolic blood pressure (SBP) and mean arterial pressure (MAP) of rats intoxicated with NaF ( Figure 2). On the other hand, there was dose-dependent reduction in the values of SBP, DBP and MAP of rats intoxicated with NaF and treated with L-Arginine and Lisinopril respectively ( Figure 2). Lisinopril co-administration gave a better reduction of blood pressure parameters as recorded in gure 2.

Renal antioxidant defence system
From table 1, L-Arginine supplementation was found to signi cantly improve the activities of renal GPx, GST, SOD, PSH and vitamin C content, respectively. Our results showed that NaF intoxication signi cantly (P<0.05) reduced activities of renal GPx, GST, SOD, and vitamin C content in comparison to the control (Table 1). Furthermore, there were signi cant increases in the activity of renal SOD, PSH and NPSH content, but not statistically signi cant when compared to the control (Table 1).
In table 2, we recorded signi cant reductions in the activities of GPx, GST, SOD, and GSH, NPSH and vitamin C content in cardiac tissues of NaF intoxicated rats. However, L-ARG co-administration at 100 mg/kg and 200 mg/kg enhanced the activities of cardiac SOD and GPx together with the content of GSH, PSH and NPSH, respectively. It was interesting to observe that there was no appreciable improvement in the cardiac content of vitamin C except in the rats administered Lisinopril (Table 2). It is worth noting that treatment with Lisinopril gave better improvement in both cardiac and renal antioxidant defence systems (Tables 1 and 2).

Markers of renal damage and oxidative stress
We also observed that intoxication with NaF caused a signi cant (P<0.05) increase in the values of serum blood urea nitrogen (BUN) and creatinine when compared to the control and rats co-administered with L-Arginine (100 mg/kg and 200 mg/kg) as indicated in gure 3. The nephron-protective effect of L-Arginine was demonstrated as indicated with signi cant reduction in the serum levels of BUN and creatinine in comparison to the NaF intoxicated group (Figure 3).
In gure 4, renal malondialdehyde (MDA) which is the product of lipid peroxidation, in NaF intoxicated rats, increased signi cantly as compared to the control group. There was signi cant reduction in the MDA content of L-Arginine and Lisinopril co-administered rats when compared to the NaF alone treated rats ( Figure 4). Our data also revealed an exaggerated content of protein carbonyl (PCO) in NaF only administered rats in comparison to the control (Figure 4). The free radical scavenging action of L-Arginine was demonstrated by signi cant reduction in the content of renal PCO when compared to NaF only rats (Figure 4). Also in gure 4, the administration of NaF caused signi cant reduction in nitric oxide (NO) bioavailability relative to the control. Again, L-Arginine supplementation caused signi cant improvement in NO bioavailability similar to that of Lisinopril (Figure 4).

Histopathology and immunohistochemistry
The histopathology of the kidney revealed mild tubular necrosis in rats intoxicated with NaF, while rats coadministered with L-Arginine and Lisinopril showed no visible lesion ( Figure 5). The renal immunohistochemistry of mineralocorticoid receptor (MCR) revealed higher expression of MCR in NaF intoxicated rats relative to the control ( Figure 6). However, lower expression of MCR was observed in L-Arginine and Lisinopril treated rats when compared to the NaF alone rats (Figure 6). It is important to note that lower expression of MCR was recorded in rats that received 100 mg/kg of L-Arginine relative to those that received 200 mg/kg of L-Arginine and Lisinopril ( Figure 6).
In another experiment, our study revealed higher expression of angiotensin converting enzyme (ACE) in renal tissues of rats intoxicated with NaF when compared to the control (Figure 7). Interestingly, cotreatment with either L-Arginine or Lisinopril reduced the expression of ACE relative to the NaF intoxicated rats (Figure 7).

Discussion
This study showed that L -arginine ameliorates NaF-induced hypertension in male Wistar rats. This can be corroborated by statistically signi cant reduction in systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure across the treated groups when compared with the hypertensive untreated rats. Our ndings also con rmed earlier reports on toxicity of NaF on cardiovascular system It was evident from our study that L-Arginine signi cantly increased NO bioavailability and reversed high blood pressure precipitated by NaF intoxication.
We observed from our study that NaF intoxication caused signi cant increase in blood urea nitrogen (BUN) and creatinine levels. The increase in BUN and creatinine has been associated with various degrees of renal injuries (Nasiruddin et al. 2020;Ni et al. 2021). The observed nephrotoxicity by NaF might be due to free radical generation and increase protein catabolism with concomitant systemic oxidative damage. This nding might also suggest extensive glomerular damage and tubular epithelial cell damage that are positively correlated with exaggerated levels of BUN and creatinine. Surprisingly, treatment with L-Arginine signi cantly attenuated these deleterious effects by the reduction in BUN and creatinine levels across treated groups in comparison to the Na intoxicated group. This therefore indicates the nephropretective effect of L-Arginine against nephrotoxicity induced by NaF intoxication.
Our study therefore is in support of nephropretective effect of L-Arginine against nephrotoxicity and hepatorenal damage ( The ability of L-Arginine to mitigate oxidative stress in hypertensive rats was also demonstrated in the present study. Renal markers of oxidative stress including hydrogen peroxide (H 2 O 2 ) generated, malondialdehyde and protein carbonyl contents increased signi cantly in NaF-induced hypertensive rats compared with the control. The exaggerated production of H 2 O 2 as classic example of reactive oxygen species (ROS) has been reported during oxidative stress with ultimate damage to proteins, nucleic acids, against NaF-induced renal protein carbonylation might be associated with the antioxidant activity of L-Arginine which prevents protein oxidation. Protein carbonylation, one of the most harmful irreversible oxidative protein modi cations has been considered as a major hallmark of oxidative stress-related disorders including aging and several age-related disorders (Fedorova et al. 2014). From this study, we can propose that L-Arginine could be found applicable in the management of aging and several agerelated disorders against protein oxidation and crosslinking.
Glutathione in its reduced form is an important intracellular antioxidant that protects against a variety of oxidant species (Masella et al. 2005). The protective mechanisms of glutathione against oxidative stress can be through detoxi cation of enzymes such as glutathione peroxidase against oxidative stress and scavenging hydroxyl radicals and singlet oxygen directly (Masella et al. 2005). Glutathione peroxidase (GPx) is a selenium-containing enzyme that catalyzes detoxi cation of lipid hydroperoxide and hydrogen peroxides to water and oxygen (O 2 ). The reduction in the activity of GPx would lead to a concurrent increase in hydrogen peroxide with subsequent tissue damage (Espinoza et al. 2000;Farhat et al. 2018).
Superoxide dismutase (SOD) on the other hand catalyzes the dismutation of the supeoxide anion radical to hydrogen peroxide (Pizzino et al. 2017).
Our data also showed signi cant decrease in the activity of enzymatic and non-enzymatic antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), reduced glutathione (GSH) and vitamin C in NaF intoxicated hypertensive group, con rming the involvement of oxidative stress in the pathogenesis of hypertension. Treatment of the hypertensive rats with L-Arginine at 100 mg/kg and 200 mg/kg brought about signi cant improvement in the of antioxidant defence system. However, the increase in reduced glutathione level in the renal tissues of the hypertensive rats treated with L-Arginine was not signi cant except in the group treated with 10 mg/kg Lisinopril. The reduction in the levels of markers of oxidative stress and concurrent increase in antioxidant enzymes may suggest an ability of L-Arginine to scavenge free radicals and mitigate oxidative stress associated with NaF toxicity.
The signi cant decrease in the activity of SOD and GPx in the hypertensive group may subsequently lead to an increase in superoxide anion radical and H 2 O 2 levels, thereby potentiating oxidative stress as a major factor in the progression of hypertension. The accumulation of the superoxide anion radical was also sequel to the observed decrease in the activity of SOD. Hence, increasing levels of the superoxide anion radical might enhance the uncoupling of endothelial nitric oxide synthase (eNOS) with a resultant reduction in NO bioavailability. Furthermore, superoxide anion radical is also capable of reacting with NO to form peroxynitrite, which is a cytotoxic signaling molecule (Wu et  Our study revealed overactivation of MCR by NaF intoxication as recorded with higher expression of renal MCR. The observed higher expression of MCR could be positively correlated with exaggerated high blood pressure obtained in rats administered only NaF. From our data, L-Arginine or Lisinopril co-administration with NaF caused reduction in the expression of MCR. This might be indicative of renoprotective and antihypertensive action of L-Arginine and Lisinopril, respectively. The amino acid L-Arginine could be found applicable for the management of toxicant-induced nephrotoxicity.
Recently, science has taken the advantage of selectively inhibiting angiotensin converting enzyme (ACE) as a therapeutic target for preventing CKD and better management of hypertension (Puspita et   . In this study, we also investigated renal immunolocalization of ACE following NaF intoxication. The immunohistochemistry revealed higher expression of renal ACE in rats administered with NaF relative to the control and rats co-administered with either L-Arginine or Lisinopril. The increased in the expression of ACE was similar to that of MCR as stated above; meaning that NaF nephrotoxicity might be through over activation of MCR and ACE signaling pathway. The over activation of these pathways could actually be responsible for the nephrotoxicity and hypertension. The ability of L-Arginine to block the activities of MCR and ACE could be maximized as novel therapeutic agent in the management and treatment of kidney damage and associated hypertension.

Conclusion
The results of this study showed that L-Arginine normalized high blood pressure, reduced oxidative stress, improved renal antioxidant defence system, offered protection against renal damage, and nephrotoxicity and improved nitric oxide bioavailability thereby serving as a precursor to nitric oxide production. Thus, L-Arginine could serve as a potential alternative therapy against toxicant-induced oxidative stress, nephrotoxicity, and hypertension via increase in the supply of endogenous nitric oxide.