Background: Acute kidney injury (AKI) is a common and potentially fatal complication encountered during a variety of kidney surgeries. Renal ischemia/reperfusion (I/R) injury is the predominant mechanism of AKI in this setting. Hence, controlling I/R injury is a key research imperative as it is directly related to the prognosis of patients. Summary: In the last decade, studies in vitro and in animal models have demonstrated that flavonoids can significantly alleviate I/R-induced AKI through a variety of pathways, including anti-oxidative stress, anti-inflammation, anti-cell death, inhibition of endoplasmic reticulum stress, and alleviation of mitochondrial dysfunction. Based on the extensive role of flavonoids in ischemia-reperfusion injury, the lack of drugs entering the clinic so far is a question worthy of consideration. Key Messages: This review summarizes the available evidence pertaining to the protective effect of flavonoids against renal I/R injury and discusses their potential clinical application in renal I/R injury.

© 2022 S. Karger AG, Basel

Introduction

Acute kidney injury (AKI), a syndrome characterized by rapid loss of renal excretory function, is diagnosed based on reduction in urine output and accumulation of nitrogen metabolism end products (urea BUN and creatinine) or both [1]. The reported incidence of AKI in patients undergoing kidney surgeries is 2.03% [2]. In cases of sepsis, the prevalence of AKI at intensive care unit admission was reported to be as high as 42% [3]. AKI is categorized into three types, i.e., prerenal (caused by inadequate renal perfusion), intrarenal/endogenous (caused by direct intrinsic renal injury), and postrenal (caused by urinary tract obstruction) [4]. Renal ischemia/reperfusion (I/R) injury belongs to the intrarenal type of AKI [4, 5].

Renal I/R injury is caused by sudden temporary impairment of blood supply to the kidney followed by restoration of blood flow and re-oxygenation, which causes a series of pathological changes, including glomerular damage and extensive tubular damage, manifested by tubular epithelial cell detachment, swelling, vacuolization, nuclear sequestration, tubular dilatation, brush border defects, tubular formation, interstitial edema, medullary congestion, and necrosis [6-8]. During I/R, the damaged tissue produces excessive reactive oxygen species (ROS), causing oxidative stress, and the blood flow in the period of reperfusion generates oxygen free radicals which cause apoptosis and cell death through lipid peroxidation and oxidative damage to proteins and DNA, ultimately resulting in kidney tissue damage [9]. In addition, endothelial damage, leukocyte infiltration, and production of inflammatory mediators play an important role in I/R injury [10]. Renal I/R injury is a common complication of renal surgery, including partial nephrectomy, renal transplantation, and renal artery angioplasty. However, there are no specific therapeutic agents for control of renal I/R injury in clinical settings. Therefore, development of effective and specific drugs for renal I/R injury is a key research imperative.

The term flavonoids refers to a class of compounds with a 2-phenyl chromogenic ketone as the basic parent nucleus, and its concept refers broadly to a class of compounds with two benzene rings (A and B rings) interlinked by three carbon atoms. Based on the degree of oxidation of the three-carbon chain between the A and B rings, the presence or absence of a substituent at the 3-position, and other characteristics, flavonoids are classified into seven subclasses, including flavones (e.g., luteolin, apigenin), flavonols (e.g., quercetin, rutin), isoflavones (e.g., genistein), flavanones (e.g., naringin, hesperidin), flavanols (e.g., catechins and procyanidins), anthocyanins (e.g., cyanidin), and chalcones (e.g., hydroxysafflor yellow A [HYSA]). Flavonoids are widely distributed in angiosperms and are important active ingredients in traditional Chinese medicine, with multiple pharmacological effects, including antioxidant [11], anti-inflammatory [12], antibacterial [13], antiviral [14], anticancer [15], antidiabetic [16], and immune function modulation [17]. There is growing evidence that flavonoids have significant protective effects against I/R-induced AKI. These effects are mediated through a variety of pathways and targets, including anti-oxidative stress, anti-inflammation, anti-cell death, inhibition of endoplasmic reticulum (ER) stress, and alleviation of mitochondrial dysfunction (shown in Fig. 1). This review summarizes the available evidence of the protective effects of flavonoids against I/R-induced AKI, focusing on the potential mechanisms to provide a reference for further clinical studies.

Fig. 1.

Protective effects of flavonoids in renal ischemia-reperfusion injury. The red circles represent different bioflavonoids in the relevant regulatory pathway. (1) Luteolin, (2) apigenin, (3) baicalin, (4) baicalein, (5) chrysin, (6) quercetin, (7) rutin, (8) fisetin, (9) hesperidin, (10) naringin, (11) genistein, (12) EGCG, (13) proanthocyanidins, (14) anthocyanidins, (15) HYSA.

Protective Role of Flavonoids against Renal I/R InjuryAnti-Oxidative Stress Injury

The special structural characteristics of the kidney make it vulnerable to hypoxic injury. During I/R injury, renal tissue produces large amounts of ROS, which exceed the body’s scavenging ability. ROS cause oxidative cellular damage by reacting with various cellular structural components [18], which leads to cellular damage through lipid peroxidation in mitochondria, lysosomes, and plasma membranes, thus impairing the structural and functional integrity of the membrane [19]. Malondialdehyde (MDA) and thiobarbituric acid-reactive substances are identified as markers of oxidative stress [19]. Endogenous free radical metabolizing enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase, and glutathione (GSH) are crucial for scavenging ROS. Flavonoids were found to play an antioxidant role by boosting the activity of these enzymes. Apigenin (4 mg/kg) was shown to increase SOD and glutathione peroxidase activity and reduce MDA in a rat model of renal I/R injury through activation of the JAK2/STAT3 signaling pathway [20]. In another study, injection of baicalin (30 mg/kg) via tail vein half an hour before reperfusion in SD rats was found to reduce the levels of advanced oxidation protein products and MDA [21]. Quercetin showed protective effects in the SD rat renal I/R model through attenuation of oxidative stress by improving the levels of SOD, CAT, and GPx [22]. Several other studies have shown that pretreatment with flavonoids can rescue I/R injury. In a renal I/R model in Wistar albino rats, pretreatment (1 h before ischemia) with rutin (1 g/kg) significantly attenuated renal insufficiency, inhibited MDA production, and restored the depleted GSH levels and manganese superoxide dismutase activity [23]. Pretreatment with 100 mg/kg of naringin daily for 7 days before renal ischemia in SD rats significantly enhanced SOD, CAT, and GPx activities [24]. Pretreatment with naringenin (400 mg/kg) significantly reduced the elevated thiobarbituric acid-reactive substance levels due to renal I/R and recovered the depleted antioxidant enzymes in the kidney [8]. Genistein [25], epigallocatechin gallate (EGCG) [26], proanthocyanidins [27, 28], and anthocyanins [29] have also been demonstrated to attenuate renal I/R injury in a similar way.

In addition, flavonoids can reduce oxidative stress by modulating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Nrf2 is a redox-sensitive transcription factor that regulates the expression of several cytoprotective and antioxidant genes [30]. In the SD rat renal I/R model, luteolin (50 mg/kg) reduced ROS accumulation by modulating the Nrf2 signaling pathway [31]. In an in vitro model of renal I/R injury established by H2O2 stimulation of HK-2 cells, pretreatment with baicalein (100 μmol/L) was found to promote the expression level of the antioxidant transcription factor Nrf2 [32]. Naringin was shown to reduce I/R-induced oxidative stress by enhancing the expression level of Nrf2 in renal tissues and preventing ECG changes and myocardial damage following renal IR injury [33]. Furthermore, proanthocyanidin pretreatment was found to enhance the expression of anti-oxidative stress proteins through the Nrf2/HO-1 signaling pathway and to significantly reduce oxidative stress damage in the kidney tissue of ICR mice during the period of renal I/R injury [34].

Notably, nitric oxide (NO) is considered to be an essential mediator of the pathological and physiological processes of renal I/R injury [35]. Its synthesis is modulated by nitric oxide synthase (NOS), which has three isozymes: endothelial inducible NOS (iNOS), neuronal NOS, and endothelial NOS (eNOS). Studies have demonstrated that renal I/R activates iNOS [36] and that NO production is harmful to the kidney during renal ischemia [37]. Flavonoids have been shown to inhibit peroxynitrite production by suppressing iNOS activity and NO production. In the Wistar rat renal I/R injury model, rutin (1 g/kg) pretreatment significantly attenuated the level of elevated nitrite/nitrate and cGMP in plasma, as well as the formation of iNOS and 3-nitrotyrosine in renal tissue [38]. In an SD rat renal I/R model, intravenous administration of hesperidin (10 or 20 mg/kg) for a continuous period of 14 days after surgery resulted in a significant reduction in iNOS expression in the kidney tissue of the hesperidin-treated group [39]. Administration of proanthocyanidins (100 mg/kg) for a continuous period of 7 days prior to I/R significantly reduced I/R-induced tissue nitrite/nitrate levels, suggesting that the proanthocyanidins may protect against renal I/R injury by reducing nitrite stress [40]. In contrast to the nephrotoxic effects of iNOS, eNOS-induced NO plays an organ-protective role through processes such as blood flow retention, vasodilation, scavenging of ROS, and inhibition of platelet aggregation and leukocyte adhesion [41, 42]. Both in vitro and in vivo research indicates that oligomeric proanthocyanidin-rich grape extract increases eNOS and thus exerts a protective effect against ischemic AKI [43].

Anti-Inflammatory Injury

Inflammatory response is an essential pathophysiological process during I/R-induced renal injury. Intricate interactions between endothelial cells, epithelial cells, cytokines, and inflammatory mediators result in sustained injury during acute tubular necrosis [44]. Therefore, inhibition of the inflammatory response is an important strategy for the prevention of renal I/R injury. Natural flavonoids have demonstrated great potential in the treatment of I/R-induced AKI due to their anti-inflammatory effects. Tumor necrosis factor-α (TNF-α) is the upstream of the inflammatory cascade and initiates the upregulation of chemokines and cytokines. IL-6 and IL-1β are the downstream of the inflammatory cascade and can damage renal cells directly [45]. Pretreatment of Swiss albino mice with luteolin (100 mg/kg) for 7 days before renal ischemia was found to significantly reduce the expression levels of TNF-α, IL-6, and IL-1β in renal tissues [46]. HMGB1 is a pro-inflammatory cytokine that functions primarily via the RAGE and TLR receptors [47]. Luteolin (40 mg/kg) pretreatment reduced the levels of NF-κB activation and HMGB1 expression in the SD rat renal I/R model [7]. Both in vivo and in vitro studies have demonstrated the protective effect of apigenin against I/R-induced inflammatory damage in renal cells by upregulating miR-140-5p and downregulating CXCL12, and thus inhibiting activation of the NF-κB pathway [48]. Chrysin (100 mg/kg) can exert anti-inflammatory effects on renal I/R injury through inhibition of the IκBα/NF-κB signaling pathway [49]. In the F344 rat renal I/R model, intraperitoneal injection of quercetin (1, 30 mg) 2 h before surgery significantly decreased the high expression of chemokines MCP-1, RANTES and macrophage-associated cytokine AIF, thereby reducing renal inflammatory infiltration [50]. In the SD rat renal I/R model, EGCG reduced renal I/R injury through inhibition of NF-κB, induction of renal p38 mitogen-activated protein kinase activation, and suppression of inflammation [51]. In addition, EGCG also protected against renal I/R injury by inhibiting macrophage infiltration and upregulating HO-1 [52].

Toll-like receptor 4 (TLR4) signaling is considered to be another critical pathogenic mechanism of renal I/R injury [53]. Activation of TLR4 facilitates the generation of chemokines and pro-inflammatory cytokines, leukocyte migration and infiltration, and renal tubular epithelial cell apoptosis [54]. Intraperitoneal injection of baicalin (10 mg/kg, 100 mg/kg) 30 min before ischemia in a Wistar rat renal I/R model resulted in significant downregulation of MyD88, TLR2/4, and NF-κB signaling, as well as attenuated the ensuing pro-inflammatory response [55]. In a renal I/R model in SD rats, intravenous administration of hesperidin (10 or 20 mg/kg) for a continuous period of 14 days after surgery resulted in a dramatic decrease in the expression of NF-kB, p65, p-IkBa, TLR4 protein, and iNOS in renal tissues, suggesting that the protective effect of hesperidin against I/R-induced renal injury may be mediated via the TLR4/NF-κB/iNOS pathway [39]. Genistein (15 mg/kg) was also shown to reduce renal I/R-induced inflammatory response by decreasing the levels of TNF-α and TLR4 expression [25]. In the C57Bl/6J mouse renal I/R model, anthocyanidin pretreatment downregulated IL-6, IL-1 β, MCP-1, TNF-α, and TLR4 expression during renal I/R injury [29]. Furthermore, cellular and animal studies have also demonstrated a similar effect of HYSA [56].

Anti-Cell Death

Among cell death events, some modes of programmed cell death, involving cell necrosis, apoptosis, and autophagy, are the pathophysiological consequences of renal I/R injury [57]. Targeting the cell death program appears to be an attractive therapeutic strategy which retains cellular function directly. Most flavonoids exert antiapoptotic effects in renal I/R injury. Reduced apoptosis is correlated with the inactivation of the caspase cascade. As the downstream effector of this cascade, caspase-3 directly mediates apoptosis after activation by a variety of upstream signals [58] and is considered a key indicator of apoptosis during I/R injury [59, 60]. In the Swiss albino mouse kidney I/R model, luteolin pretreatment significantly reduced the expression of Bax (the pro-apoptotic protein) and increased the expression of Bcl-2 (the antiapoptotic protein), while decreasing the level of caspase-3 expression, thereby reducing apoptosis in kidney tissue [46]. PI3K/Akt signaling pathway regulates renal repair after I/R injury, and apigenin (20 mg/kg) pretreatment has been shown to protect against renal I/R injury and prevent tubular apoptosis in vitro and in vivo by activating PI3K/Akt-mediated mitochondria-dependent apoptotic signaling pathway [61]. In addition, inhibition of renal apoptosis by suppressing the activation of the Fas/FasL pathway may be another protective mechanism of apigenin against renal I/R injury [62]. Furthermore, genistein (15 mg/kg) was shown to exert its antiapoptotic and proliferative effects via the SIRT1/p53 axis, and thus ameliorates I/R-induced renal injury [6]. Among the flavonoids, baicalin [32, 55], chrysin [49], quercetin [63], fisetin [64], hesperidin [39], naringin [65], EGCG [51], proanthocyanidins [34], anthocyanins [29], and HYSA [56] were also demonstrated to have protective effects against renal I/R injury by inhibiting apoptosis.

Autophagy has been demonstrated to protect against renal I/R injury [66]. In a mouse model of I/R-induced kidney injury, quercetin (5 and 10 mg/kg) was shown to activate the AMPK-regulated autophagic signaling pathway, as evidenced by upregulation of AMPK phosphorylation, downregulation of mTOR phosphorylation, and elevation of LC3-II protein levels. This effect was also confirmed in a chemical cell hypoxia model of LLC-PK1 cells [67]. Notably, ferroptosis, a newly discovered form of cell death that is peroxidation driven, iron dependent, and non-apoptotic, has been suggested as a promising target for treating renal I/R injury [68]. In a recent study, quercetin was shown to ameliorate I/R-induced AKI by inhibiting ferroptosis and subsequent inflammation [69].

Others

Ischemic hypoxia impairs mitochondrial oxidative phosphorylation, resulting in impairment of ATP synthesis and cellular energy-dependent processes, ultimately causing cell death [70]. In post-ischemic cells, mitochondria are subsequently exposed to high levels of oxygen free radicals and Ca2+. Such elements may lead to progressive deterioration of mitochondria during the reperfusion phase [71]. Therefore, reversing mitochondrial dysfunction is very important in the prevention and treatment of renal I/R injury. Fisetin was shown to successfully reverse mitochondrial dysfunction and reduce mitochondrial mass due to renal I/R injury in a Wistar rat renal I/R model. Intraperitoneal administration of fisetin (20 mg/kg) 15 min before surgery attenuated I/R-induced mitochondrial dysfunction and restored the reduction in mitochondrial copy number while reversing Pgc1α gene expression, reducing mitochondrial fission, increasing mitochondrial fusion and mitochondrial autophagy to protect against I/R-related decline of mitochondrial mass in renal tissue [64].

ER stress is another mechanism investigated in the context of I/R injury. ER is an intracellular organelle that plays an essential role in protein homeostasis, including in the synthesis, folding, modification, and degradation of proteins [72]. Exposure of cells to harmful stimuli leads to accumulation of unfolded or misfolded proteins in the ER, triggering ER stress [73]. In vivo and in vitro studies have demonstrated the crucial role of ER stress in I/R-induced renal injury [74]. Therefore, inhibition of ER stress is another potential therapeutic strategy for renal I/R injury. Luteolin (40 mg/kg) treatment was found to significantly reduce the levels of GRP78, CHOP, ATF-4, and XBP-1s expression in a SD rat renal I/R model. This suggested that the protective effect of luteolin against I/R-induced renal injury may be mediated via suppression of ER stress [7]. Similarly, the protective effect of baicalin against renal I/R injury may also be mediated via inhibition of ER stress [32].

Flavonoids ameliorate not only renal I/R-induced AKI but also remote complications of renal I/R injury in other organs. In the SD rat renal I/R model, quercetin (10 mg/kg) pretreatment attenuated I/R-induced liver dysfunction manifested by improvements in levels of alanine aminotransferase and aspartate aminotransferase [75]. Another study demonstrated the protective effect of quercetin against renal I/R-induced cardiac injury, and this protective effect was potentially achieved through inhibition of the RhoA/ROCK pathway and modulation of hydrogen sulfide production [76]. In addition, naringin (100 mg/kg) pretreatment was shown to improve solitary bundle nucleus (NTS) electrical activity and stress reflex sensitivity (SBR) in rats with renal I/R injury [77].

Comparison of Flavonoids with Other Compounds against Renal I/R Injury

Other than the flavonoids, other phytochemicals such as polysaccharides have also been shown to have protective effects against renal I/R injury. Hericium erinaceus polysaccharide was pre-administered to mice by gavage at a dose of 300 mg/kg for 15 days prior to renal ischemia, showing that Hericium erinaceus polysaccharide pretreatment significantly reduced I/R-induced renal oxidative damage and increased antioxidant activity [78]. In the C57B6J mice renal I/R model, it was shown that low molecular weight fucoidan may inhibit the MAPK signalingpathway to suppress the apoptotic pathway, thereby ameliorating renal I/R injury [79].Furthermore, the protective effect of Ganoderma lucidum polysaccharide peptide against renal I/R injury may be attributed to the inhibition of NADPH oxidase-dependent production of ROS and the increase of free radical-scavenging capacity for the balance of the oxidation/antioxidant system, improving mitochondrial dysfunction and ER stress-dependent apoptosis [80]. It is thus clear that the mechanisms by which phytochemical polysaccharides improve renal I/R injury are similar to those of flavonoids. However, there are some difficulties in comparing their efficacy. First, the renal I/R model used in different studies was not identical; second, the mode, timing, and dose of administration likewise vary from study to study. Therefore, more scientific studies are needed to further compare the efficacy of flavonoids with other compounds against renal I/R injury.

Conclusion

Ischemia-reperfusion-induced AKI is a key concern in clinical kidney surgeries. Studies have demonstrated the great potential of flavonoids in the prevention of renal I/R injury. In this review, we summarize the evidence from recent preclinical studies exploring the mechanism of the protective effect of flavonoids against renal I/R injury (shown in Table 1). The underlying mechanisms include anti-oxidative stress, anti-inflammation, anti-apoptosis, alleviation of mitochondrial dysfunction, and inhibition of ER stress. The specific mechanisms involve a variety of signaling pathways, such as the Nrf2 pathway, TLR2/4 pathway, NF-κB pathway, p38 MAPK pathway, JAK2/STAT3 pathway, PI3K/Akt pathway, Bcl-2 pathway, and Fas/FasL pathway.

Table 1.

Mechanisms of the protective effect of flavonoids against renal ischemia-reperfusion injury

In most studies, flavonoids were administered prior to ischemia, with a few studies administering both before ischemia and after reperfusion. Combined with the clinical and pathophysiological manifestations of renal I/R injury in these two stages, the ischemic stage showed decreased renal function and sublethal damage to the tubular epithelium and endothelium, while the reperfusion stage showed extensive tubular damage accompanied by an intense inflammatory cascade [81]. The pretreatment with flavonoids significantly improved the renal functional and histological damage caused by renal I/R injury, suggesting that flavonoids play an important role in both the ischemic and reperfusion stages.

However, there are no reports of clinical application of flavonoids in this context. This is because most of the studies on the above compounds have been conducted in animals or cells. There is a paucity of clinical studies, which are essential for clinical translation. Studies have indicated poor bioavailability of flavonoids, and the clinical dosage, dosage formulation, administration method, and other key factors are also unstudied. Moreover, the toxicity profile of flavonoids is not well characterized. Lastly, the structure-function relationship of flavonoids is undefined, which may constrain further structural modification in order to improve their efficacy. Nonetheless, the available evidence suggests a great potential for clinical application of flavonoids.

Conflicts of Interest Statement

The authors declare that they have no conflicts of interest.

Funding Sources

This work was supported by The Science and Technology Planning Project from The Youth Project of Science and Technology Project from the Department of Education of Jiangxi Province (GJJ190819); Department of Science and Technology of Ganzhou City (GZ2019ZSF051); The Doctoral Research Start-up Fund from First Affiliated Hospital of Gannan Medical University (QD073).

Author Contributions

Tianpeng Xie, Junrong Zou, and Peng Peng designed the thesis and outline for the review. Bin Zhong conducted the literature search. Peng Peng drafted the manuscript. Tianpeng Xie, Guoxi Zhang, and Xiaofeng Zou reviewed the manuscript and polished the grammar. All authors contributed to the manuscript revision and approved the submitted version.

Data Availability Statement

No data were used to support this study.

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