Proper functioning of the kidney is essential for homeostasis and elimination of toxic metabolites through urine [39, 40]. In current scenario, renal disorder is a global public health concern and a multi-factorial disease entity with both unmitigated excessive generation of oxidants, reduced antioxidant capacity and diminished clearance of pro-oxidant radicals [13, 41]. Climate change coupled with intensive agriculture practices involving non-judicious applications of pesticides such as DM as well as increased volume of elemental pollutants such as F− in soil and ground water cumulatively impose heavy impact on terrestrial as well as aquatic life forms [42, 43]. Kidney cells face very high F− ions concentrations making them susceptible to F− toxicity [44, 45] and exposure to DM causes pronounced ill effects on kidney . Simultaneous exposure of F− and DM potentiated the redox imbalance induced renal damage when compared to exposure to either chemical alone [47, 48]. Urea is the end product of protein metabolism formed in liver whereas CR is formed from creatine after breakdown of muscle protein but both are excreted via kidneys and their elevated levels indicate diminished renal functioning. Uric acid on the other hand is the final breakdown metabolite of purine processing and its high serum levels point towards renal malfunction. Disturbances in electrolyte homeostasis including disruption in concentration of Ca and P and increased levels of BUN, CRE and UA are considered sensitive indicators of renal malfunctioning. In the current study all the above biochemical renal markers were elevated in all toxicant administered rats which are concurrent with the previous findings [5, 46, 49]. Alterations in biochemical parameters were also recorded upon fluoride and arsenic exposure in earlier studies [5, 50]. Analogous findings were registered during a study by Mahjoubi-Samet et al.  in which DM induced nephrotoxicity in adult rats and their suckling pups. Further, in the present study, levels of CR, BUN and UA in co-exposed groups were significantly higher from that seen in individual toxicant exposed rats indicating greater abnormalities in renal physiology in rats co-exposed to F− and DM. Interestingly, our results also highlight that continuous co-exposure can lead to significant renal impairment. However, it was found that with ZO extract supplementation, the toxicant administration led changes in levels of renal biomarkers were significantly reversed. Nitric oxide is a vasodilator which relaxes muscles of blood vessel walls leading to increase in blood circulation. Its levels alter in response to oxidative stress and inflammation in body. Clinically, it has been observed that lower levels of NO predispose to various cardiovascular diseases like arteriosclerosis and hypotension [51, 52].
Significant reduction in NO levels was observed after administration of F− and DM alone. Moreover, combination toxicant group recorded further depletion in NO levels. Similar findings were documented by Miranda et al.. Also, ZO addition ameliorated toxicant brought about significant reduction in NO levels in contrast quercetin was less effective in restoring NO levels in the combination toxicant group. Exposure to F− stimulates ROS production and the ensuing oxidative stress hampers antioxidant enzyme activity, energy metabolism and ion transport across plasma membranes . Acute fatal F− toxicity diminishes calcium ionic concentration in blood which affects contractability of cardiac cells causing cardiac arrest , whereas prolonged exposure to F− leads to electrolyte imbalance with significant reduction of Ca and magnesium levels in plasma . In the present study, after administration of DM or F−, the plasma levels of both ions (Ca and P) were decreased and the depreciation was more significant after the co-exposure of DM and F. However, diazinon administration in rats led to a fall in calcium levels without effecting phosphorus concentrations . Derangements in blood calcium and phosphorus levels were also registered by Sharma et al.  in rats after F− intoxication. Rat ameloblasts witnessed increased calcium influx raising intracellular calcium levels upon exposure to high F− concentrations  which may lead to imbalance in Ca and P ratio. Another study revealed F− induced influx of calcium inside cells causing drop in calcium levels in plasma . Further, kidney being primarily responsible for F− excretion, damaging effects due to F− induced alteration in renal antioxidant enzymes as appreciated in the present work might have also interfered with absorption and renal clearance of phosphate. Since latter’s altered concentration affects Ca levels, it may have also contributed towards steep fall in plasmatic calcium concentration. Taken together, all these could have resulted in a sharp drop in Ca levels in blood in our work.
In the present study, administration of F− and DM alone and in combination induced reduction in concentrations of both enzymatic and non-enzymatic antioxidants but increased lipid and protein peroxidation. However, these changes were significantly dampened after ginger administration. GSH performs various functions in living organisms. Apart from acting as a carrier of active thiols group such as cysteine residues, it also acts as a co-factor for GST and GPx [59, 60]. A decrease in the TAS spurs decrease in parameters such as TTH, AE and AChE. Also, there is strong evidence that compounds having oxidant activity can not only modulate AChE activity but also repress its gene expression . Exposure to herbicidal agent clomazone has been shown to decreased AChE and antioxidant levels but at the same time increased lipid peroxidation in erythrocytes . Moreover, upsurge in AOPP and MDA concentrations portend oxidative damage to cellular proteins and lipids respectively. Additionally, in the event of oxidative insult, activity of AE, which grants protection to lipids against their peroxidation, may be severely curbed. Large scale fluctuations in lipid and protein peroxidation along with depletion of oxidant scavengers may unfold into frank tissue damage. Similar to the depletion in levels of TAS, TTH, AE, AChE, CAT, SOD, GPx, GR and rise in MDA and AOPP our study. Concurrent findings were also reported by Khan et al. , who studied toxic effects of deltamethrin and F− in rats. Reduction in CAT, GSH and NO levels in rat were also were reported by Miranda et al., after F− toxicity. Likewise, various studies reported decrease in CAT, SOD and GSH in response to toxicant pesticides and metals administrations in response to oxidative damage [14, 50, 63].
A wide variety of alterations such as degeneration, necrosis, and haemorrhage in renal parenchyma were noticed histologically in our study which is most likely the result of toxicant induced oxidative damage. Histopathological changes were most severe in the dual toxicant administered group receiving maximum doses of both the extraneous compounds. Similar changes have been observed by [46, 64, 65]. Similarly, Sharma et al.  also found that chronic exposure to F induced significant changes in oxidative stress parameters and histomorphology of hepatic, renal and cardiac tissues in Wistar rats. Various experimental studies have indicated that simultaneous exposure to phytochemicals (flavonoids) and toxicants reduces toxicant induced renal damage [39, 60, 66]. Correspondingly, ginger administration bolstered antioxidant profile in our experimental rats. In the present work, ginger extract supplementation reversed deviations in plasma biomarkers of renal injury, renal TAS, AE, AChE, GPx, GR, AOPP and MDA levels alongside renal histoarchitecture in all toxicant groups of our study thereby authenticating the presence of antioxidant, anti-apoptotic and anti-inflammatory properties in ginger as suggested in earlier in-vitro and in-vivo studies . Nephroprotection was also imparted by essential oils of ZO and turmeric rhizomes against cadmium-induced toxicity in rats in a prior report . Furthermore, commendable antioxidant and chelating properties of ZO against cadmium nephrotoxicity were also propounded in a study done by Gabr et al.. Maghsoudi et al., also promulgated ginger as a therapeutic remedy for renal dysfunctions. Correspondingly, essential oils from ginger were found to mitigate renal damage induced by administration of cadmium and acetaminophen [68, 71]. Similarly, studies revealed that supplementation of ginger extract effectively countered toxic changes such as sloughing of tubular epithelium, dilatation of renal tubules and interstitial fibrosis in renal parenchyma induced by fructose consumption [68, 71]. 6-gingerol an active component of ginger efficiently alleviated kidney dysfunctions, oxidative stress and histopathological changes induced by mercuric chloride in male rats . In a nutshell, use of ZO in the present investigation conferred noticeable protection against the oxidative damage unleashed by dual toxicant exposure which was better than that seen following quercetin treatment. Taken together, the results of this study highlight the potential perils of simultaneous intake of toxic dose of DM and F− on kidney functioning in animals and humans. It is particularly concerning for people and animals in places where DM is being used in agricultural practices without much caution and at the same time naturally F− contaminated ground water is utilized for irrigation and drinking. Besides, use of ginger must be encouraged among populations in such areas to counter harmful effect of instantaneous exposure to F− and DM.