*3.1. Oxidative Stress*

*3.1. Oxidative Stress*  Oxidative stress is caused by an imbalance between the excessive free radical generation and insufficient antioxidant defense [29,30]. It is frequently observed in CKD [31– 33], and has become a diagnostic factor [34]. A number of studies documented that *M. oleifera* has antioxidative properties to protect and/or alleviate cellular damage (Table 1 and Figure 2). *M. oleifera* extracts and compounds, particularly quercetin, kaempferol, isothiocyanates, rutin, myricetin, ascorbic acid, and β-carotene, showed antioxidant potentials either via direct scavenging of free radicals [35]. Oxidative stress is caused by an imbalance between the excessive free radical generation and insufficient antioxidant defense [29,30]. It is frequently observed in CKD [31–33], and has become a diagnostic factor [34]. A number of studies documented that *M. oleifera* has antioxidative properties to protect and/or alleviate cellular damage (Table 1 and Figure 2). *M. oleifera* extracts and compounds, particularly quercetin, kaempferol, isothiocyanates, rutin, myricetin, ascorbic acid, and β-carotene, showed antioxidant potentials either via direct scavenging of free radicals [35].





### **Table 1.** *Cont.*

MDA, Malondialdehyde; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; STZ, streptozotocin (C8H15N3O7); GSH: glutathione; CAT, catalase; SOD, superoxide dismutase; GPx, Glutathione peroxidase; IL-1β, Interleukin 1 beta; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; AOPP, advanced oxidation protein products; PC, protein carbonyls; NO, nitricoxide; H2O2, hydrogen peroxide; 8-OHdG, 8-hydroxy-2-deoxyguanosine; MPO, myeloperoxidase; CRP, C-reactive protein; MTX, methotrexate; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; TAC, total antioxidant capacity; LPP, lipid perioxidation products; TPCC, total protein carbonyl content; ALAD, delta-amino levulinic acid dehydratase; BUN, Blood urea nitrogen; KIM-1, transmembrane tubular protein; Bcl-2, B-cell lymphoma 2; TGF-β, transforming growth factor beta; CHOL, Cholesterol; ORAC, oxygen radical absorbance capacity; and APAP, acetaminophen. ↑, increased; ↓, decreased.

> Methanol extract of *M. oleifera* reduced the oxidative stress in STZ induced male rats by lowering the production of MDA, ROS, LDL, and CHOL, which increase the risk of CKD [36,54]. Methanol extract also lowered the generation of MDA, AOPP, NO, H2O2, GPx, and GST, all of which induce oxidative stress in ischemia-induced Wistar rats [29]. Another study showed that metabolic extract reduced the levels of BUN and creatinine, and total protein is increased in CKD patients [42]. Ethanolic extract of *M. oleifera* inhibits oxidative stress and atherosclerosis in CKD by lowering LDL [20]. 8-OHdG causes oxidative stress to DNA and promotes cancer [56], ameliorated by the ethanolic extract of *M. oleifera* [56]. Ethanol extracts decrease the plasma creatinine level by enhancing the process of creatinine clearance [30]. Plasma sodium and potassium levels were raised after treating nickelinduced Wistar rats with ethanolic extract of *M. oleifera* [34]. Ethanolic extract detoxified plasma by reducing the bilirubin levels (indirect/direct), urea levels, etc., in ML-induced male Sprague Dawley rats [48]. HO-1 and Nrf2 expression were stimulated by leaf extract of *M. oleifera* at dosages of 300 and 400 mg/kg body weight, respectively [25,41]. Leaf extracts up-regulated the level of total thiol TiO2NPs induced male albino rats, which play an important role in antioxidant protection [41]. Leaf extract of *M. oleifera* also downregulated

the oxidative stress generating mediators in sodium fluoride (NaF)-induced *Oreochromis niloticus*, gentamicin-induced rabbit, and APAP-treated mice [23,42,57].

*M. oleifera* alcoholic extract reduced oxidative stress by lowering the lipid peroxidation, and ROS in iodide injected rabbits [51]. Furthermore, fermented leaf extract of *M. oleifera* boosts the antioxidant activity in bacteria-induced mice [53]. *M. oleifera* extract reduced the manifestation of MDA, indicating that the free radicle overproduction was reduced in both Tilmicosin and Hg induced rats. Abarikwu et al. showed that SOD level was increased after treatment with *M. oleifera* in tilmicosin induced rats [40]. Hydroalcoholic root extract raised blood sugar, antioxidant enzyme activities, and G-6-phase activities, which protect the kidney from nephropathy in Beryllium-induced rats [45]. Seed powder reduced free radical species, TPCC, metal content, and increased ALAD activity in lead-treated rats [57]. In arsenic-treated rats, seed powder of *M. oleifera* considerably increased antioxidant function including GSH, CAT, and ALAD [46].

### *3.2. Inflammation*

The kidney is responsible for maintaining whole-body homeostasis. Kidney disease is characterized by inflammation as a major pathology [58–60]. Acute or chronic disease such as ischemia, toxins, or inflammation affects kidney tubules, causing kidney fibrosis that is associated with reduction of GFR in kidneys [61]. Kidney injury is linked to the production of cytokines levels, which prolongs the acute phase of kidney disease [62]. Moreover, chronic inflammation is regarded as a comorbid condition in CKD [63]. Many plants have an anti-inflammatory action through active substances such as hesperidin, diosmin, withaferin, fucoidan, thymoquinone, etc. [64–67]. Here, the anti-inflammatory effects of *M. oleifera* has been discussed. *M. oleifera* has been reported to exhibit strong inflammatory activity (Table 1 and Figure 3). Methanolic extract of *M. oleifera* reduced inflammation in STZ induced male Wister rats by down-regulating the tumor necrosis factor (TNF-α), IL-6, and MCP-1, an important chemokine [36,54]. Tang et al. investigated the effects of ethanolic extract of *M. oleifera* in metformin-induced mice and observed that the *M. oleifera* declines the production of inflammatory markers and the expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) by reducing the phosphorylation of mitogen-activated protein kinase (MAPK) pathway [20]. Ethanolic extract of *M. oleifera* down-regulates the inflammatory cytokines in CoCl2-induced rats, including NO, which is involved in the pathogenesis of inflammation [37]. Leaf extract of *M. oleifera* inhibits inflammatory cytokines production and regulates the inflammation by inhibiting NF-kB [25]. It was also observed that inflammation in Tilmicosin (Til) induced rats was reduced by *M. oleifera* extracts [39]. *M. oleifera* leaf extract protects against interstitial kidney inflammation with fibrosis by down-regulating KIM-1 in TiO2NPs induced male albino rats [41]. *M. oleifera* extract increases the secretion of cortisol, adrenaline, Treg cells, NK, and leptin, promoting anti-inflammatory cytokines and regulating the immune system [47]. *M. oleifera* treatment reduced the expression of KIM-1, TIMP-1, and TNF-α in ML-induced male Sprague Dawley rats [48]. TNF-α, an inflammatory cytokine that stimulates IL-1; IL-6, downregulated by *M. oleifera* in Seabream (*Sparus aurata*); and activated TGF-β, elicits anti-inflammatory effects [49]. *M. oleifera* also reduced the inflammatory cytokines in APAP-treated mice, where APAP induces AKI [50]. Fermented extract of leaves also reduces the Nrf2 in *Salmonella*-induced mice [53].

Moringa seed's phytochemicals can reduce the production of nitric oxide (NO) and the gene expression of LPS-inducible iNOS and interleukins 1β and 6 (IL-1β and IL-6) compared to curcumin [68]. Flavonoids have been shown to be effective inhibitors of nitric oxide synthase type 2 (NOS-2) actions, and it also inhibits protein tyrosine kinase action that is involved in the NOS-2 expression at the molecular level [69–71]. Flower extract can cause the activation of pro-inflammatory proteins such as toll-like receptors. In the flowers, quercetin and kaempferol can inhibit the signal transducer and activator of transcription 1 (STAT-1) and the NF-*κ*B pathways [72,73]. *M. oleifera* flowers contain 80% hydroethanolic, a potent agent of anti-inflammation in the NF-*κ*B signaling pathway [74]. Scientists discovered that phenolic glycosides suppress inducible iNOS expression and NO production

in mouse macrophage cells, as well as COX-2 and iNOS proteins [75,76]. *Moringa* extracts eventually down-regulate the inflammatory mediators because its seeds and flowers contain many bioactive compounds. Each of these compounds has its individual effects.

### *3.3. Fibrosis*

Kidney fibrosis is defined as a radical harmful connective tissue deposition on the kidney parenchyma, which leads to renal dysfunction. Epithelial to mesenchymal transition (EMT) is the main mechanism of kidney fibrosis, and the TGFβ-1-SMAD pathway and hypoxia are known as the main modulator of EMT [32,77]. TGF-β-induced expression of fibronectin, type I collagen, and PAI-1 rat kidney fibroblast cells is reduced by *M. oleifera* extract [55]. Furthermore, moringa root extract selectively inhibited TGF-β-induced phosphorylation of SMAD4 and ERK expression. These results suggest that moringa root extract may reduce renal fibrosis by a mechanism related to its antifibrotic activity in rat kidney fibroblast cells. Oral administration of *M. oleifera* seed extract reduced CCl4-induced liver fibrosis in rats [78].

### *3.4. Other Pathologies Those Are Associated with Kidney Diseases*

Autophagy has a critical role in kidney physiology and homeostasis [79], and, thus, its regulation is an important determinant of kidney diseases [61]. AKI or CKD causes mitochondrial damage, but damaged mitochondria begin to accumulate in response to these types of stimuli. Autophagy protects the kidney through the removal of ROS-producing mitochondria [80–82]. Apoptosis is a type of programmed cell death in which cells are killed by a controlled system. It is an energy-dependent complex process [83]. It contributes to develop AKI, even organ failure [84]. Ischemia/reperfusion (I/R) induces apoptosis or necrosis in the kidney and loss of tubular cells, leading to decreased GFR [85,86]. Renal tubular cells express cell surface 'death receptors' of TNF-α which is responsible for inducing apoptosis [87]. Also, ROS production in kidney disease is responsible for promoting apoptosis [86].

TNF-α inducer of apoptosis, also increased the expression of apoptosis-related molecules which was down-regulated by ethanol extract of *M. oleifera* in CoCl2-treated rats [37,88]. Leaf extract at a dose of 300 mg/kg body weight reduced the expression of caspase-9, the precursor of caspase-3, leading to apoptosis [25,89]. Bcl-2 inhibited apoptosis by blocking cytochrome c release and preventing caspase activation [90] while it was up-regulated by ethanol extract of *M. oleifera* in ML-induced rats. *M. oleifera* also reduced the expression of TIMP-1, which is involved in renal fibrosis and apoptosis [48].

### **4. Prospects for** *M. oleifera* **in Drug Development**

Researchers are targeting the development of drugs from natural sources instead of the synthetic drug because natural sources have fewer side effects than synthetic sources. Nigerian scientists proved that *M. oleifera* is a beneficial herb and causes no harm to the body and kidneys [91]. Another study reported that higher doses of *M. oleifera* created toxicity in rats, but a moderate level dose of *M. oleifera* is safe [92]. *M. oleifera* has been shown to alleviate diabetic nephropathy in alloxan-induced rats [93]. Acetaminophen causes hepato-renal toxicity, which can be cured by *M. oleifera* treatment at the dosage of 500 mg/kg [94]. *M. oleifera* reduced necrosis, dilatation of renal tubules in Cd-induced rats, where Saleh et al. suggested that *M. oleifera* could be used as an herbal drug [95]. *M. oleifera* leaf extracts reduced oxidative stress, kidney, and liver damage [96]. A randomized placebo-controlled study suggested that *M. oleifera* leaf capsules can be used to control blood sugar level and blood pressure level [97]. Moreover, aqueous extracts of *M. oleifera* can reduce metal (As (III), Cd, Ni and Pb) toxicity and showed the protective effects in *Saccharomyces cerevisiae* [98].

The rich phytochemical profile and advances in biotechnological techniques have made this tree indispensable for opening a new era in medical science. An in vitro propagation technique provides new insights into developing more effective, eco-friendly, and biodegradable products using mass multiplication and production techniques. Though efficiency in in vitro propagation techniques for *M. oleifera* has been established, there are still gaps in

the production of metabolites and those specific metabolites in the human body. The use of biotechnological approaches will help in the commercialization of important plant products. There is no doubt that biotechnological protocols will allow great research to make *M. oleifera* one of the essential solutions for various health issues including kidney diseases.

### **5. Conclusions**

Kidney function declines with age, and aging-related kidney complications proportionately increase. Their side effects limit the effectiveness of existing drugs for treating kidney diseases and, therefore, natural compounds with fewer side effects are being evaluated. The literature discussed in this review suggests that *M. oleifera* alleviates several pathological factors associated with kidney diseases, including inflammation and oxidative stress. However, a mechanism associated with protective potential of *M. oleifera* against kidney diseases has been provided in this study (Figure 4). *Plants* **2021**, *10*, 2818 12 of 17

**Figure 4.** Protective mechanisms of *M. oleifera* against kidney injury. *M. oleifera* increased the production of catalase (CAT); superoxide dismutase (SOD); glutathione peroxidase (GPx); glutathione (GSH); total antioxidant capacity (TAC); delta-amino levulinic acid dehydratase (ALAD); and G-6- Pase, which facilitated oxidative stress reduction by activating glutathione (GSH), a non-protein thiol that suppresses free radicals. GSH suppresses the oxidative stress situation. *M. oleifera* also suppressed oxidative stressors caused by ROS, H2O2, GSSG, and LPO by inhibiting MDA, LPP, TPCC, BUN, Creatinine, and NO. Bcl-2 was similarly produced by stress stimuli and was linked to the suppression of necrosis, induced by *M. oleifera*. *M. oleifera* inhibited the expression of Caspase-9, a protein involved in the formation of caspases. Following NF-kB, stress stimuli also increased CRP expression. NF-kB then moved from the cytosol to the nucleus, bound to DNA, and activated inflammation-related proteins. *M. oleifera* inhibited the mechanism by which inflammation factors **Figure 4.** Protective mechanisms of *M. oleifera* against kidney injury. *M. oleifera* increased the production of catalase (CAT); superoxide dismutase (SOD); glutathione peroxidase (GPx); glutathione (GSH); total antioxidant capacity (TAC); delta-amino levulinic acid dehydratase (ALAD); and G-6-Pase, which facilitated oxidative stress reduction by activating glutathione (GSH), a non-protein thiol that suppresses free radicals. GSH suppresses the oxidative stress situation. *M. oleifera* also suppressed oxidative stressors caused by ROS, H2O<sup>2</sup> , GSSG, and LPO by inhibiting MDA, LPP, TPCC, BUN, Creatinine, and NO. Bcl-2 was similarly produced by stress stimuli and was linked to the suppression of necrosis, induced by *M. oleifera*. *M. oleifera* inhibited the expression of Caspase-9, a protein involved in the formation of caspases. Following NF-kB, stress stimuli also increased CRP expression. NF-kB then moved from the cytosol to the nucleus, bound to DNA, and activated inflammation-related proteins. *M. oleifera* inhibited the mechanism by which inflammation factors were produced, hence, reducing inflammation. *M. oleifera* has been linked to a reduction in the progression of kidney disease.

gression of kidney disease.

were produced, hence, reducing inflammation. *M. oleifera* has been linked to a reduction in the pro-

This study discusses the insights of *M. oleifera* against kidney diseases including AKI and CKD, which have not been reported previously. In addition, further studies are needed to confirm the effects of the bioactive phytochemicals (vitamins, alkaloids, polyphenols, isothiocyonates, glucosinolates, tannins, and saponins) of *M. oleifera* against kidney diseases. We anticipate that the points raised in this review will provide a future research direction for understanding how pharmacological interventions based on natural products could modulate kidney disease. In contrast, it would shed light on how *M. oleifera*based drugs would potentially be a kidney protective agent in treating aging-associated kidney abnormalities. Considering the harmful effects of synthetic resources and their non-renewable nature, the use of natural resources as a source of medicine has received a lot of attention in recent years. *M. oleifera* based medicine would be an excellent protective agent against several risk factors associated with kidney diseases.

**Author Contributions:** Conceptualization, M.J.U., A.M. and M.A.R.; data curation, T.A. and M.A.I.A.; funding acquisition, M.J.U.; methodology, T.A., M.A.I.A. and A.F.; initial draft, T.A. and M.A.R.; supervision, M.J.U.; writing—review and editing, T.A., M.A.R., M.A.H. and M.J.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work acknowledges National Research Foundation (No. 2020R1I1A1A01072879 and 2015H1D3A1062189), and Brain Pool program funded by the Ministry of Science, and ICT through the National Research Foundation (No. 2020H1D3A2A02110924), Korea.

**Conflicts of Interest:** There are no conflicts of interest regarding this work.

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