Evaluation of the nephroprotective and antioxidant efficacy of spirulina extract fortified with moringa leaves and finger millet in arsenic-treated wistar rats

Introduction

Industrialization has severely polluted water bodies by releasing untreated chemical waste. Major pollutants include heavy metals, pesticides, petroleum products, and polychlorinated biphenyls. Since water is a strong solvent, these toxic substances easily dissolve and contaminate aquatic systems. Among these, heavy metal and metalloid contamination in drinking water is especially concerning. Toxic elements such as arsenic (from natural sources, smelting, and power plants), mercury (from chlor-alkali plants, lamps, and hospital waste), cadmium (from zinc smelting, batteries, e-waste, and paint sludge), and lead (from batteries, paints, smelting, and coal-based power plants) are widely present and alarming.

DNA repair processes by production of reactive oxygen species (ROS). It also influences epigenetic changes and activates specific signal transduction pathways, resulting in abnormal gene expression (Ratnaike, 2003) [4] . Spirulina is a microscopic and filamentous cyanobacterium that belongs to family Oscillatoriaceae and has a long history of use as food and food supplement. Multiple studies have demonstrated that Spirulina possesses a wide range of health-promoting properties, including anti-inflammatory, anticancer, antibacterial, antioxidant, and immunemodulatory effects. Additionally, early research has reported promising results regarding its hypolipidemic, antidiabetic, anti-obesity, and antianemic potential (Kulshreshtha et al., 2008) [5] . The leaves of Moringa oleifera, rich in polyphenols, flavanols, phenolic acids, kaempferol, quercetin, and essential vitamins and minerals, exhibit antioxidant, free radical-chelating, and anti-inflammatory properties, which help reduce hepatic and renal damage and support the normalization of related biomarkers (Razis et al., 2014) [6] . Finger millet possesses strong antioxidant activity due to its rich phenolic and flavonoid content, along with enzymatic and non-enzymatic antioxidants such as catalase and vitamins E and C. Phenolic acids, flavonoids, and tannins in the seed coat act as radical scavengers and metal chelators, while its anti-inflammatory effects mediated through inhibition of enzymes like A5-lipoxygenase and xanthine oxidase contribute to reduced hepatic enzyme and renal biomarkers levels (Kumar et al., 2021) [7] . Spirulina, Moringa leaves, and finger millet are recognized for their exceptional phytonutrient, vitamin, and mineral composition, offering high-energy nutrition with a low glycemic index. These super foods possess strong antioxidant, anti-inflammatory, and immune-enhancing properties that contribute to overall health improvement. Fortification of microalgal Spirulina extract with Moringa leaves and finger millet is expected to enhance its therapeutic potential by developing a synergistic, nutrientdense formulation with improved functional and protective properties. Hence, the present study was designed to assess the nephro-protective effects of Spirulina extract fortified with finger millet and Moringa leaves against arsenicinduced nephro-toxicity in Wistar rats.

Materials and Methods

The research was carried out on adult healthy male Wistar albino rats. Adult male Wistar rats, each weighing between 150 to 200 grams, were obtained from the Chromed Biosciences Private limited Labs Plot No. C-38, KIADB, Industrial area, Hirehalli, Mydala, Tumkur, Karnataka-572168 (Reg. No. 2171/PO/RcBiBt/S/22/CCSEA). All the rats were acclimatized to standard laboratory conditions at Small Animal House facility, Hassan for seven days prior to initiation of the experiment and maintained at 25±2 ℃ housing temperature and relative humidity of 50 to 70 per cent and to laboratory conditions of 12-hour light/dark cycle throughout the study period and provided with regular standard pellet diet along with free access to deionized drinking water ad libitum throughout the course of the experiment. All the protocols were adhered as per the guidelines of the Committee for Control and Supervision of Experiments on Animals for care and use of laboratory animals and were approved by the Institutional Animal Ethics Committee at the Veterinary College in Hassan (HVC/IAEC/11/2025).

Preparation of test items and mode of administration:

Sodium arsenite was obtained from Jai Maruthi Scientific Bangalore, manufactured by NICE Chemicals Private Limited, Kochi. It was administered orally at a dose of 10 mg/kg body weight, in normal saline, daily for 60 days. MSFM in powder form was received from Department of Studies in Food Technology, Davangere University, and Karnataka. It was used at the dose rates of 100, 200, and 400 mg/kg body weight. Telmisartan procured from Intas Pharmaceutical Limited, Ahmedabad, India was administered orally daily at a dose of 10 mg/kg body weight.

• Experimental protocol: This study consisted of six groups (Group I to VI), comprising of six rats in each group. Group I (normal control) rats received normal saline orally daily for 60 days. All the rats in Group II (disease control) to VI rats received sodium arsenite@10mg/kg body weight orally daily for 60 days. In addition to sodium arsenite, Group III (reference control) rats received Telmisartan@10mg/kg body weight orally for 60 days; while Groups IV, V and VI rats received MSFM@100mg/kg, @200mg/kg and @400mg/kg body weight orally daily for 60 days, respectively. • Parameters assessed: Body weight (g) were assessed every week. All animals were examined twice daily for any signs of clinical abnormalities or symptoms. At the end of the study, all rats were ethically euthanized by administering an intramuscular overdose of Ketamine and Xylazine. Blood samples were drawn from the retro-orbital plexus and collected into EDTA vials for hematological analysis and serum vials for biochemical evaluation.

• Estimation of antioxidant enzymes:

The representative tissue samples from kidney were dissected and washed with Normal saline to remove any tissue debris and blood clots. The collected kidney samples were homogenized in a solution of ice-cold 0.1M Phosphate buffered saline (pH 7.4) at 4ºC and centrifuged for 10 minutes at 15,000 rpm. The supernatants were stored at -80ºC for analysis of superoxide dismutase, catalase, and thiobarbituric acid reactive substances.

• Histopathological analysis: Kidney tissue from rats of all the groups was subjected to histopathological studies. The tissue was fixed using 10 per cent Neutral Buffered Formalin solution and sections were prepared using paraffin blocks and stained with hematoxylin and eosin stain. Similarly, the standard procedure of Masson's trichome staining was employed to stain required histopathological sections (Suvarna et al., 2018) [8] . • Histopathological scoring for Kidney: The Kidney samples were examined in random microscopic areas semi-quantitatively under high power fields and the number of changes were assessed by the counting twenty non overlapped fields for the same slide of each animal. The extent of damage and the severity of lesions in the kidney were assessed semi-quantitatively (Al Forkan et al., 2016) [9] with slight modification as follows;

Results

• General observation: Group I rats remained healthy and active throughout the experiment. Group II rats exhibited clinical signs such as reduced feed intake, reduced body weight, ruffled hairs, dehydration, restlessness and were difficult to handle. The rats of Group III to VI manifested similar clinical signs as that of disease control rats, but with reduced intensity and frequency.

• Body weight: The mean body weights in grams with standard error of mean at different time intervals of 0 day and 1 st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th and 8 th week of the experiment have been presented in Table 1. Group I rats remained healthy and active throughout the period of experiment and demonstrated steady and progressive enhancement in their body weight over the course of experiment. Body weight of Group II rats were decreased significantly from 3 rd week to 8 th week in comparison to Group I rats. Group III rats showed a significant and consistent increase in body weight from the 4 th to 8 th week compared to Group II. MSFM-treated rats (Groups IV-VI) exhibited a consistent and significant (p<0.05) increase in body weight throughout the study compared to Group II. Also, Group VI showed improvement comparable to Group I normal control rats throughout the experiment period. increase in serum creatinine of Group II rats in comparison to Group I rats. Among the MSFM treated groups, there were no significant difference in mean creatinine values. A significant (p<0.05) reduction in creatinine levels was observed in Groups VI when compared to Group II. However, the reductions observed in Groups III to V were not statistically significant in comparison to Group II rats. • Blood Urea Nitrogen (BUN): There was a significant (p<0.05) increase in BUN of Group II rats in comparison to Group I rats. The mean values of BUN in treatment groups (III, V and VI) showed significant improvement in comparison to Group II rats. While, Group IV rats showed no improvement in comparison to Group II rats. However, Group VI rats showed decrease in the BUN values which was statistically significant in comparison to Group II rats and were comparable to that of Group I rats. comparison to Group II rats. Furthermore, Group VI exhibited a significant (p<0.05) increase over Group III to V rats and was comparable to that of Group I rats. • Superoxide dismutase (SOD): There was a significant (p<0.05) decrease in superoxide dismutase enzyme activity of Group II rats in comparison to Group I rats. The mean values of Group III to VI were significantly (p<0.05) increased in comparison to Group II rats and were comparable to that of Group I rats.

Histopathology

The kidneys of the disease control group (Group II) showed varied histopathological changes characterized by tubular degeneration ranging from moderate to severe degree. Kidney showed mild to moderate degree of congestion and haemorrhage involving both cortex and medulla. The proximal and distal convoluted tubules showed degenerative changes characterised by severe cellular swelling to vacuolation with reduced tubular lumen size in few tubules. Occasionally, few tubules showed eosinophilic debris in their lumen. The glomerular architecture was slightly altered with reduced urinary space. The degenerative changes of tubules were accompanied by loss of cilia and infiltration of inflammatory cells in moderate degree in renal interstitial area consisting mainly of mononuclear cells viz lymphocytes and macrophages (Fig. 1 to 6). The blood vessels in the renal parenchyma showed thickening of tunica media (Fig. 7 and8).

The kidneys of the reference control group rats (Group III) and treatment Groups (IV to VI) showed improved tubular architecture with decreased swelling of renal tubules and reduced vacuolations of renal tubular epithelial cells when compared to Group II rats. The degree of congestion and haemorrhages were mild to minimal when compared to Group II rats (Fig. 9 to 12).

Histopathological score of kidney

The mean HP severity score of all sectioned kidney samples was assessed for lesions like tubular cell swelling to vacuolar degeneration, cortical haemorrhage and congestion, medullary haemorrhage and congestion, mononuclear cell infiltration, eosinophilic debris in the lumen of tubules and thickening of blood vessels. The mean HP severity score values of kidney with standard error of mean are presented in Table 6. The mean HP score of Group III to VI were significantly (p <0.05) lower than mean HP score of Group II disease control rats.

Discussion

In the current investigation, exposure to sodium arsenite caused diminished feed intake, reduced body weight gain, rough hair coat, and mild hair loss. These symptoms were comparable to those previously reported in rats subjected to arsenic toxicity (Goyal, 2012) [11] . Our observation of decreased body weight following sodium arsenite exposure was consistent with the findings of earlier researchers (Dhar et [11,12,13,14] . The gradual decline in body weight observed in the sodium arsenite-treated group may be attributed to the progressive development of anorexia resulting from toxemia (Biswas et al., 2000) [15] . Kaltreider et al. (2001) [16] found that even low levels of arsenic can interfere with the glucocorticoid hormonal system. This system plays a crucial role in regulating glucose levels and managing carbohydrate, fat, and protein metabolism. Disruption of glucocorticoid function may therefore lead to metabolic disturbances, contributing to either weight loss or gain.

In the present study, sodium arsenite exposure led to significant alterations in hematological parameters, including reductions in TEC, Hb, PCV, TLC, and MCHC, along with elevations in MCV and MCH, consistent with previous reports (Kannan et [17,9,18] . The decrease in PCV, Hb, RBC, and MCHC observed in arsenic-exposed animals can be attributed, in part, to arsenic's interference with heme biosynthesis. Arsenic is known to inhibit aminolevulinic acid dehydratase, a critical enzyme in the conversion of δ-aminolevulinic acid to porphobilinogen, thereby disrupting porphyrin and heme formation. This impairment reduces hemoglobin availability and affects erythroid cell maturation, ultimately suppressing erythropoiesis and contributing to anemia (Ola-Davies and Akinrinde, 2016) [19] . Furthermore, arsenic induces oxidative stress in erythrocytes through cellular redox cycling, generating reactive oxygen species that damage the erythrocyte membrane, promote methemoglobin formation, and trigger hemolysis and cell death (Sato et al., 1998) [20] .

Additional mechanisms, such as interference with folic acid absorption, which is essential for DNA synthesis and red blood cell production and its role as a capillary toxin that increases erythrocyte fragility, further exacerbate the hematological deficiencies observed in arsenic toxicity (Biswas et al., 1998) [21] . Collectively, these multifaceted effects of arsenic contribute to the marked anemia and altered hematological profile noted in exposed animals. The marked decline in TLC levels in this study may be due to the direct cytotoxic effects of arsenic on the bone marrow, which suppress hematopoietic activity and consequently lead to erythrocytopenia and leukopenia (Sajan et al., 2010) [22] . The increase in MCV and MCH observed after arsenic exposure typically indicates a macrocytic response linked to anemia. The decline in MCHC levels in the arsenic-treated group may result from multiple factors, including disrupted heme biosynthesis, oxidative stress mediated haemoglobin degradation, and the development of macrocytic anemia, collectively contributing to a reduced haemoglobin concentration per erythrocyte (Ola-Davies and Akinrinde, 2016) [19] . A significant elevation in the mean serum levels of creatinine and BUN was recorded in the present study, which are consistent with the earlier observations ( [23,24,25] . In the present study, the observed elevation in plasma urea and creatinine levels following arsenic exposure reflects impaired renal function, most likely resulting from glomerular damage and reduced filtration efficiency. Arsenic undergoes biotransformation to monomethylarsinic and dimethylarsinic acids, which are primarily excreted through glomerular filtration and partially reabsorbed by proximal tubular cells. The accumulation of these metabolites within renal tubular cells can induce pronounced oxidative stress, leading to tubular cell injury and subsequent nephrotoxicity (Sinha et [26,27,28,29,30] .

In the present study, a significant elevation in kidney TBARS levels accompanied by a marked reduction in CAT and SOD enzyme activities was observed, indicating increased lipid peroxidation and oxidative stress in rats. These findings are consistent with earlier reports (El-Demerdash et al., 2009: Zhang et al., 2014) [13,31] . The pronounced rise in MDA levels in the arsenic-exposed group reflects enhanced lipid peroxidation resulting from excessive generation of reactive oxygen and nitrogen species. Arsenic promotes ROS production by disrupting mitochondrial electron transport, activating NADPH oxidase, promoting oxidative metabolism of its intermediates, and releasing redox-active iron from ferritin. The observed decline in enzymatic antioxidants such as SOD and CAT may be attributed to direct inhibition of their activity or depletion of essential cofactors by arsenic and its metabolites (Hu et al., 2020) [32] . The kidneys of the disease control group showed varied histopathological changes characterized by tubular degeneration ranging from mild to severe. The renal tubules showed degenerative changes characterised by cellular swelling to vacuolation, eosinophilic debris in their lumen.

The glomerular architecture was slightly altered with reduced urinary space. The degenerative changes of tubules were accompanied by loss of cilia and infiltration of inflammatory cells in moderate degree consisting mainly of mononuclear cells viz lymphocytes and macrophages. The blood vessels in the renal parenchyma showed thickening of tunica media. Comparable histopathological alterations have been documented by Liu et al. (2000) [33] , Fahmy et al.

(2008) [34] and Gora (2014) [35] . Improvement in treatment groups (Group IV to VI) may be attributed to the antioxidant, anti-inflammatory antiapoptotic properties of MSFM. Spirulina is rich in a diverse array of antioxidants, including β-carotene, tocopherol, chlorophyll, and phycobiliproteins such as phycocyanin, cryptoxanthin, and allophycocyanin, all of which collectively enhance its free radical-scavenging capacity and mitigate oxidative stress (El-Naggar et al., 2018) [36] . Among these, phycocyanin a predominant biliprotein containing the tetrapyrrole chromophore phycocyanobilin plays a pivotal role in Spirulina's antioxidant efficacy. It exhibits strong free radical-quenching activity along with significant metalbinding and chelating abilities, facilitating the detoxification and removal of heavy metals from the body. Furthermore, C-phycocyanin modulates inflammatory responses by inhibiting the phosphorylation of p38 MAPK, thereby down regulating cytokine synthesis and attenuating inflammation (Aladaileh et al., 2020) [37] . Abd Elhalim et al. (2017) [38] demonstrated that Spirulina prevents apoptosis by up regulating Bcl-2 mRNA expression. This effect is mediated through increased intracellular Bcl-2 protein levels, which protect against oxidative stress by inhibiting mitochondrial release of cytochrome-C. As a result, Bcl-2 overexpression enhances the cellular capacity to regulate ROS and supports the activity of endogenous antioxidant enzymes, thereby preventing ROS-induced Bcl-2 depletion and reducing cell death.

According to Mundkar et al. (2022) [39] , the antiinflammatory properties of Moringa are primarily attributed to its glucosinolates. During hydrolysis, these compounds yield isothiocyanates, which interact with Keap-1 and facilitate the nuclear translocation of Nrf-2. Inside the nucleus, Nrf-2 activates the antioxidant response element, triggering a phase II antioxidant defense that suppresses NF-κB activation and reduces the release of pro-inflammatory cytokines such as interleukins and TNF-α. In addition, Moringa modulates apoptosis by increasing Bcl-2 expression while decreasing Bax, cytochrome-c, and caspase-3 levels, thereby protecting cells from oxidative stress-induced damage. Finger millet is a rich source of flavonoids (quercetin, catechin, gallocatechin, epicatechin, epigallocatechin,

and phenolic acids (gentisic, vanillic, gallic, syringic, salicylic, protocatechuic, p-hydroxybenzoic, chlorogenic, caffeic, sinapic, p-coumaric, trans-cinnamic, and ferulic acids). These bioactive compounds exert strong antioxidant and anti-inflammatory effects (Udeh et al., 2017) [40] . The present study demonstrates that microalgal Spirulina extract fortified with finger millet and Moringa leaves powder provides significant, dose-dependent protection against sodium arsenite-induced nephrotoxicity, with the higher doses (200 and 400 mg/kg body weight) exhibiting greater protective effects. The observed improvements in biochemical markers, antioxidant status, and histopathological outcomes underscore its potential as a therapeutic adjunct in arsenic toxicity. Nevertheless, additional mechanistic investigations and clinical evaluations are warranted to fully establish its efficacy and translational relevance.

Acknowledgement

Acknowledgments None Financial support & sponsorship None

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