A systematic literature search was performed in PubMed, Web of Science, Embase, and Science Direct from their inception date to February 2021. Keywords included “ischemia/reperfusion injury,” “ischemia-reperfusion injury,” “I/R injury,” “melatonin,” “N-acetyl-5-methoxytryptamine,” and “MT,” “MLT.” All object types were limited to animals, and there are no restrictions on language.

Two investigators (DRL and YM) screened the titles and abstracts of studies identified for this review independently. Download the full text of the screened articles to determine whether they meet the predetermined inclusion and exclusion criteria. Agreement of the study selection was determined using the quadratic-weighted kappa value (Kw). The result was accepted if the Kw value was >0.75. Disagreements were resolved through discussion with the third reviewers (YP).

Data was extracted from the included studies independently by two reviewers (DRL and LTY), agreement of the data was determined using the quadratic-weighted kappa value (Kw). The result was accepted if the Kw value was >0.75. Disagreements were resolved through discussion with the third reviewers (YP). The following relevant information of each study was extracted in Table 1: (1) study's characteristics (i.e., first author's name, publication year, country), (2) animals (i.e., number of included animals, strain, gender, weight/week age), (3) I/R injury (i.e., kidney excised or not, duration of ischemia, left or right, unilateral or bilateral), (4) interventions (i.e., group, dosage and time, administration route, timing, and duration), and (5) data about BUN, SCr, MDA, MPO, SOD, and GSH. The mean results, standard deviation (SD), as well as the sample size of animals in each group were collected.

If any data were only displayed by graphs, the GetData Graph Digitizer 2.24 was adopted to estimate the results. Besides, in order to avoid human error, two reviewers (DRL and LTY) independently extracted relevant data from the papers. If the error was within the acceptable range (error ≤ 1% average data), the average data of two were used. Otherwise, the third investigator (MY) would extract the data again, and take the average of two data which were more close.

The methodological quality was assessed by using the CAMARADES 10-item checklist: (1) peer-reviewed journal; (2) temperature control; (3) animals were randomly allocated; (4) blind established model; (5) blinded outcome assessment; (6) anesthetics used without marked intrinsic neuroprotective properties; (7) animal model (diabetic, advanced age or hypertensive); (8) calculation of sample size; (9) statement of compliance with animal welfare regulations; (10) possible conflicts of interest (Macleod et al., 2004).

Two reviewers (DRL and LTY) assessed the risk of bias. Bias was marked as high or low risk, as well as “unclear” indicated that the risk of bias was unclear. The symbol “+” was used to marked low risk, and it was also recorded as the point of quality score. Agreement over the quality assessment was determined using the Kw. The Kw value >0.75 was accepted. Disagreements were resolved through discussion with the third reviewers (YP).

If at least three studies reported the results of the same outcome, the data were summarized, thus we evaluated six outcomes separately (BUN, SCr, MDA, MPO, SOD, and GSH). First, we conducted a meta-analysis for studies comparing melatonin group to control group. The Review Manager 5.3 software was adopted to analyzed data. The results in this report were described as weighted mean difference (WMD) of measurements with same unit or standardized mean difference (SMD) using different units. We analyzed the pooled data to evaluate the therapeutic action of melatonin. The heterogeneity of included studies was evaluated by I². The fixed effect models were adopted, if the heterogeneity was not obvious (i.e., p > 0.1; I² ≤ 50%); when p ≤ 0.1; I² > 50%, the source of heterogeneity was tried to detect by sensitivity analysis. Otherwise, random effect models were used (Higgins and Green, 2011).

Subgroup analysis was conducted between different ischemia duration (≤ 45 min, or > 45 min), times of administration (single or multiple), unilateral or bilateral I/R injury, dosage (< 10, 10, or > 10 mg/kg), administration time (before ischemia, or after reperfusion), and risk of bias (< 4, or ≥ 4). If we include at least 10 studies in a meta-analysis related to primary outcomes, funnel plots were used to test the potential risk of publication bias (Higgins and Green, 2011).

The process of inclusion and exclusion is shown in Figure 1. Two hundred seventeen publications were identified after search. After removing duplicates and performing title and abstract screening, 35 papers were selected for full-text screening, and 31 met the inclusion criteria (Sener et al., 2002; Kunduzova et al., 2003; Sahna et al., 2003; Rodríguez-Reynoso et al., 2004; Aktoz et al., 2007; Kurcer et al., 2007a,b; Fadillioglu et al., 2008; Ersoz et al., 2009; Sinanoglu et al., 2012; Ahmadiasl et al., 2013, 2014a,b; Sezgin et al., 2013; Cetin et al., 2014; Sehajpal et al., 2014; Hadj Ayed Tka et al., 2015; Oguz et al., 2015; Yilmaz et al., 2015; Yip et al., 2015; Banaei et al., 2016a,b; Chang et al., 2016; Souza et al., 2018; Chen et al., 2019a,b; Shi et al., 2019; M El Agaty and Ibrahim Ahmed, 2020; Wang et al., 2020; Yang et al., 2020; Zahran et al., 2020). Of the four publications excluded at the full-text level, two did not conduct renal I/R injury to AKI (Li et al., 2009; Zhu et al., 2017), and the other two used melatonin combined with other treatments (mesenchymal stem cell-derived exosomes, or nitric oxide synthase inhibitor) (Deniz et al., 2006; Alzahrani, 2019). The Kw value of the 2 reviewers was 0.915, after discussion with the third review author, the differences were resolved by consensus.

The majority of these studies were conducted in Turkey (n = 12), while the remaining in China (n = 6), Iran (n = 5), Egypt (n = 2), Brazil (n = 1), USA (n = 1), India (n = 1), France (n = 1), Mexico (n = 1), and Tunisia (n = 1) (Table 2). A unilateral nephrectomy was performed before contralateral renal I/R injury in 12 studies (Sener et al., 2002; Sahna et al., 2003; Rodríguez-Reynoso et al., 2004; Kurcer et al., 2007a; Sinanoglu et al., 2012; Ahmadiasl et al., 2013, 2014a,b; Sezgin et al., 2013; Oguz et al., 2015; Banaei et al., 2016a,b). Most of the studies conducted unilateral renal I/R injury, except 10 studies performed bilateral renal I/R injury (Ersoz et al., 2009; Sehajpal et al., 2014; Hadj Ayed Tka et al., 2015; Yip et al., 2015; Chen et al., 2019a,b; Shi et al., 2019; M El Agaty and Ibrahim Ahmed, 2020; Wang et al., 2020; Zahran et al., 2020). The duration of ischemia ranged from 25 to 75 min, then the reperfusion from 1 to 72 h. The sample size of a single group ranged between 5 and 12. The melatonin administration was < 10 mg/kg in eight studies (Kunduzova et al., 2003; Sahna et al., 2003; Kurcer et al., 2007b; Fadillioglu et al., 2008; Cetin et al., 2014; Sehajpal et al., 2014; Yilmaz et al., 2015; Wang et al., 2020), 10 mg/kg in 16 studies (Sener et al., 2002; Rodríguez-Reynoso et al., 2004; Aktoz et al., 2007; Kurcer et al., 2007a; Ersoz et al., 2009; Sinanoglu et al., 2012; Ahmadiasl et al., 2013, 2014a,b; Sezgin et al., 2013; Sehajpal et al., 2014; Oguz et al., 2015; Banaei et al., 2016a,b; Souza et al., 2018; Shi et al., 2019), and > 10 mg/kg in eight studies (Hadj Ayed Tka et al., 2015; Yip et al., 2015; Chang et al., 2016; Chen et al., 2019a,b; M El Agaty and Ibrahim Ahmed, 2020; Yang et al., 2020; Zahran et al., 2020). Most studies took melatonin intraperitoneal injection, except one with subcutaneous injection (Sener et al., 2002), one with intravenous injection (Kunduzova et al., 2003), and another one with oral administration (M El Agaty and Ibrahim Ahmed, 2020). Single-dose administration was conducted in 18 studies (Sener et al., 2002; Kunduzova et al., 2003; Sahna et al., 2003; Rodríguez-Reynoso et al., 2004; Kurcer et al., 2007a; Ersoz et al., 2009; Ahmadiasl et al., 2013, 2014a,b; Cetin et al., 2014; Sehajpal et al., 2014; Hadj Ayed Tka et al., 2015; Oguz et al., 2015; Banaei et al., 2016a,b; Souza et al., 2018; Wang et al., 2020; Zahran et al., 2020), while the others used multiple-dose administration (Deniz et al., 2006; Aktoz et al., 2007; Kurcer et al., 2007b; Fadillioglu et al., 2008; Sinanoglu et al., 2012; Sezgin et al., 2013; Yilmaz et al., 2015; Yip et al., 2015; Chang et al., 2016; Chen et al., 2019a,b; Shi et al., 2019; Yang et al., 2020). Two independent reviewers assessed the data, with a Kw of 0.817. Consensus was reached on 100% of the occasions when the reviewers initially disagreed with the third reviewer.

The Kw value of the 2 reviewers was 0.903, consensus was reached on 100% of the occasions, and the risk of bias was in Table 2. The overall quality of research methodology is good, as 21 (80.65%) out of these 31 studies were labeled as high quality. All of them were peer reviewed publication, and applied appropriate animal model, and anesthetic without significant intrinsic vascular protection activity, but none of them reported sample size calculation, and only 10 studies did not report control of temperature (Sener et al., 2002; Kunduzova et al., 2003; Sinanoglu et al., 2012; Sehajpal et al., 2014; Yilmaz et al., 2015; Chen et al., 2019b; M El Agaty and Ibrahim Ahmed, 2020; Wang et al., 2020; Yang et al., 2020; Zahran et al., 2020). Randomization was conducted in 14 studies (Aktoz et al., 2007; Kurcer et al., 2007b; Fadillioglu et al., 2008; Ersoz et al., 2009; Sinanoglu et al., 2012; Sezgin et al., 2013; Yilmaz et al., 2015; Yip et al., 2015; Souza et al., 2018; Chen et al., 2019a,b; Shi et al., 2019; M El Agaty and Ibrahim Ahmed, 2020; Yang et al., 2020), then no study reported blinded induction of model, and blinded assessment of the outcome was applied in 15 studies (Sahna et al., 2003; Ersoz et al., 2009; Sinanoglu et al., 2012; Ahmadiasl et al., 2013, 2014a,b; Cetin et al., 2014; Yilmaz et al., 2015; Yip et al., 2015; Banaei et al., 2016a; Chang et al., 2016; Souza et al., 2018; Shi et al., 2019; Yang et al., 2020). Most of the studies reported compliance with animal welfare regulations (Sahna et al., 2003; Wang et al., 2020), while 10 studies reported potential conflicts of interest and study funding (Sener et al., 2002; Rodríguez-Reynoso et al., 2004; Aktoz et al., 2007; Ersoz et al., 2009; Ahmadiasl et al., 2013; Sehajpal et al., 2014; Yilmaz et al., 2015; Yip et al., 2015; Banaei et al., 2016a,b; Wang et al., 2020).

The BUN was reported in 21 studies, then animals treated with melatonin showed a greater reduction in BUN level compared with controls (21 studies, n = 324; WMD = −30.00; 95% CI = −42.09 to −17.91; p < 0.00001; Figure 2), with random-effects model. Subgroup analysis suggested that multiple administration of melatonin could not reduce the BUN level in renal I/R injury model compared with controls (11 studies, n = 160; WMD = −26.51; 95% CI = −36.86 to 16.15; p < 0.00001; Table 3), while single administration was the opposite (10 studies, n = 164; WMD = −28.92; 95% CI = −61.27 to 3.43; p = 0.08; Table 3). The other subgroup analysis found that the reduction of BUN level did not differ between different ischemia duration (≤ 45 min, or > 45 min), unilateral or bilateral I/R injury, dosage (< 10, 10, or > 10 mg/kg), administration time (before ischemia, or after reperfusion), or risk of bias (< 4, or ≥ 4). The subgroup analysis also concluded that enhancing the dosage of melatonin did not improve the protective effect of BUN compared to dosage of 10 mg/kg (10 studies, n = 148; WMD = −38.07; 95% CI = −70.19 to −5.94; p = 0.01; Table 3), and > 10 mg/kg (6 studies, n = 92; WMD = −15.25; 95% CI = −22.76 to −7.74; p < 0.0001; Table 3).

The pooled results from 20 studies showed a significant reduction in SCr level with melatonin treatment (20 studies, n = 288; WMD = −0.91; 95% CI = −1.17 to −0.66; p < 0.00001; Figure 3), with random-effects model. Subgroup analysis suggested that the reduction of SCr level did not differ between different ischemia duration (≤ 45 min, or > 45 min), times of administration (single or multiple), unilateral or bilateral I/R injury, dosage (< 10, 10, or > 10 mg/kg), administration time (before ischemia, or after reperfusion), and risk of bias (< 4, or ≥ 4). The subgroup analysis also concluded that enhancing the dosage of melatonin did not improve the protective effect of SCr compared to dosage of 10 mg/kg (7 studies, n = 122; WMD = −1.18; 95% CI = −1.85 to −0.51; p = 0.0006; Table 4), and > 10 mg/kg (6 studies, n = 94; WMD = −0.57; 95% CI = −0.80 to −0.34; p < 0.00001; Table 4).

Antioxidative effects were measured in most of the studies, except 9 studies (Kurcer et al., 2007b; Sinanoglu et al., 2012; Oguz et al., 2015; Yilmaz et al., 2015; Banaei et al., 2016b; Chang et al., 2016; Chen et al., 2019a,b; Wang et al., 2020). The MDA level (20 studies, n = 332; SMD = −2.76; 95% CI = −3.52 to −2.01; p < 0.00001; Figure 4) and the MPO level (6 studies, n = 88; SMD = −3.78; 95% CI = −6.60 to −0.95; p = 0.009; Figure 5) were significantly reduced after melatonin administration, while the SOD level (10 studies, n = 152; SMD = 1.79; 95% CI, 0.54 to 3.05; P = 0.005; Figure 6), and GSH level (6 studies, n = 98; SMD = 2.58; 95% CI, 1.33–3.82; P < 0.0001; Figure 7) were increased compared with control.

The funnel plots of BUN and SCr were asymmetrical, hinting at a high risk of publication bias (Figures 8A,B).

This is the first preclinical meta-analysis of melatonin in the treatment of renal I/R injury. Thirty-one studies compared melatonin to placebo controls were enrolled. The overall quality of all the included studies was good, as there were 21 (80.65%) out of 31 studies were considered high quality.

Our findings indicated that melatonin can significantly improve renal function (BUN and SCr) and reduces oxidative stress (decreasing MDA and MPO, increasing SOD and GSH) of renal post-I/R injury in animals studies. The subgroup analysis also concluded that 10 mg/kg might be the appropriate dosage of melatonin for renal post-I/R injury, while enhancing the dosage of melatonin did not improve the therapeutic action of melatonin for renal I/R injury, and multiple administration might be more effective. Otherwise, the marked benefits of melatonin were not affected by either the duration of ischemia, duration of reperfusion, unilateral or bilateral I/R injury, timing of administration, or methodological quality.

At present, there are various mechanisms to explain the origin of tissue damage. An important initiating step in renal I/R injury is the uncontrolled ROS production during reperfusion (Kim et al., 2010). From here, some injury cascades are activated, including loss of endothelial function and programmed leukocyte death (such as apoptosis and autophagy). The activation of the innate and acquired immune system can be achieved through the risk or injury related molecular patterns associated with toll like receptor (TLR) binding and the activation of complement system, resulting in the occurrence of fibrosis associated with chronic transplantation dysfunction (Ben Mosbah et al., 2010). While in this situation, melatonin can function as an antioxidant stress molecule could attenuate apoptosis, autophagy, inflammation, and fibrillation in renal I/R injury (Table 5, Figure 9).

Oxidative stress and other states may cause ER stress. Under the condition of ER stress, ER chaperone, glucose-regulated protein (GRP) 78, leaving from ER membrane, and combining the free misfolding proteins, results in the activation of protein kinase-(PKR-) like ER kinase (PERK), inositol-requiring enzyme 1, and activating transcription factor 6, so as to reconstruct ER homeostasis (Ben Mosbah et al., 2010). Hadj Ayed Tka et al. revealed that melatonin treatment could inhibit ER stress in an experimental model of renal I/R injury (Hadj Ayed Tka et al., 2015). Melatonin inhibits ER stress by suppressing cell apoptosis. Melatonin can play a protective role by inhibiting phosphorylated c-Jun NH2 terminal kinase (JNK), as well as C/EBP homologous protein (CHOP). The CHOP and JNK activation, as downstream events of ER stress, can cause mitochondrial mediated apoptosis by promoting Bax aggregation in mitochondria (Yang et al., 2014; Yan et al., 2018).

The TLRs act critical roles in the pathogen immune response, and TLR4 is an important regulator related to the inflammatory reaction in I/R injury (Zhang et al., 2016). The myeloid differentiation primary response 88 (MyD88) pathways is the TLR4-induced downstream pathways, then the former pathway promotes the expression of inflammation-associated genes by activating the transcription factors, nuclear factor (NF)-κB. It regulates the expression of a variety of inflammatory factors, such as interleukin (IL)-1β, and IL-6 (Su et al., 2019). Chang et al. and Yang et al. showed that melatonin could down-regulate the pathway of TLR4-MyD88-NF-κB and reduced the deliver of IL-6, IL-1β, as well as tumor necrosis factor (TNF)-α, eventually leading to the reduction of inflammation in the injured area (Chang et al., 2016; Yang et al., 2020). Otherwise, the activation of NF-κB is also enhanced according to growth arrest-specific 6 (Gas6)/Axl. In the early stage of I/R injury, Gas6/Axl inhibits its downstream mediator nuclear factor-erythroid-2-related factor 2 (Nrf2) and NF-κB to attenuate inflammation and oxidative stress after I/R in multiple organs (Llacuna et al., 2010; Ruiz et al., 2013). Oxidative stress and inflammation are closely related, as they jointly construct a vicious circle, that is, oxidative stress causes inflammation, which leading to the recruitment and activation of immunocytes. On the other hand, inflammation promotes oxidative stress by activating leukocytes and resident cells to produce ROS and reactive nitrogen species (He et al., 2017). Chen DQ et al., indicated that melatonin could upregulate Gas6/Axl to down-regulate NF-κB/Nrf2 signaling to reduce oxidative stress, as well as inflammation in the renal I/R injury early stage (Chen et al., 2019b).

Autophagy plays a cardinal contribution in renal I/R injury. Injurious autophagy may lead to the release of excessive ROS and pro apoptotic factors, which ultimately cause cell death (Kurusu and Kuchitsu, 2017; Liu et al., 2019). Yang et al. suggested that a major pathway for melatonin-induced autophagy in I/R injury involves activating the TLR4-MyD88 signaling pathway (Yang et al., 2020). Mitogen-activated protein kinases (MAPK) pathways is actived by the MyD88 pathway (Liu et al., 2018). Mammalian target of rapamycin complex 1 (mTORC1), a convergence point for many upstream pathways, including MAPKs, regulates autophagy. Yang et al. revealed that melatonin-induced autophagy among renal I/R mice was dependent on downregulation of mTORC1 by its upstream activator, the TLR4/MyD88 (Yang et al., 2020).

Otherwise, it is known that phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway perform a core effect to regulate cell survival, as well as cell growth, and proliferation (Hassan et al., 2013). Another important finding of Chang YC was that PI3K/AKT/mTORC1 signaling pathway was significantly upregulated in the I/R kidney, while melatonin can significantly inhibit this signal pathway (Chang et al., 2016). It was claimed that the activation of PI3K/AKT/mTOR signaling under the I/R condition is the internal response of tissue or organs to I/R (i.e., the more serious, the stonger response), and melatonin could reverse this situation (Chang et al., 2016).

Fibrillation plays a leading role in renal I/R injury to accelerate kidney damage from AKI to CKD condition. The Wnt/β-catenin, as well as transforming growth factor-β (TGF-β)/Smad pathways perform important effects in the fibrosis of CKD (Yang et al., 2010; Meng et al., 2015). Under damage conditions, Wnt/β-catenin signaling is activated (Chen et al., 2017; Wang et al., 2017), and its sustained activation can promote renal fibrosis to induce the transition from AKI to CKD (Xiao et al., 2016). Fibrotic, as well as profibrotic genes, including twist, snail1, fibroblast-specific protein 1, and plasminogen activator inhibitor-1, are the downstream molecules related to Wnt/β-catenin signaling. TGF-β1 phosphorylate Smad2 and Smad3 to activate receptors through the combination with its adaptor, Smad anchor with its receptors TGF-β receptor I and II (Wang et al., 2017). The phosphorylated Smad2 and Smad3 pass through Wnt/β-catenin signaling to regulate the expression the profibrotic factor. Smad4 is also critical to TGF-β1-mediated fibrogenesis (Morishita et al., 2014), while Smad7, as the negative regulation factor of the TGF-β/Smad pathway, could inhibit phosphorylate Smad2 and Smad3 (Meng et al., 2012). Chen et al. demonstrated that melatonin performed extensive inhibitory effects on the TGF-β/Smad and Wnt/β-catenin pathways to prevent the AKI from progressing to CKD (Chen et al., 2019b).

Otherwise, in AKI to CKD condition, Gas6/Axl mentioned earlier serves as a profibrotic route and accelerates fibrogenesis (Bárcena et al., 2015). Then, melatonin could downregulate Gas6/Axl signaling pathway to prevent the fibrosis of the kidney (Chen et al., 2019b).

Combining the advantages of systematic review and traditional review, the study pooled the previous data to show that melatonin is a promising drug for renal I/R injury, and summarized the mechanism of melatonin. A deeper understanding of the mechanism of melatonin to treat renal I/R injury would not only help to explore the pathological mechanisms during renal I/R injury, but also contribute to the investigation of new effective drugs for further development and related translational research.

Inevitably, the article also has some limitations. First, since we cannot obtain the data of individual animals, the study can only be meta-analyzed at the overall level of each study (based on mean and standard deviation). The renal I/R injury models used of each study are different, different ischemia times for instance. The simple hybrid method is not the most appropriate method, but we can't analyze all the inconsistencies hierarchically. Then, the funnel chart is asymmetric, so the potential publication deviation cannot be ignored, which may affect the accuracy of the results.