Tanshinone IIA Prevents Acetaminophen-Induced Nephrotoxicity by Stimulating the Eux of its Toxic Intermediate Metabolite From Kidney

It’s known that acetaminophen (APAP) overdose often leads to hepatotoxicity and nephrotoxicity. Previous studies have shown that Tanshinone IIA (Tan IIA) could protect against APAP-induced hepatotoxicity via activating the Nrf2 pathway. In the present study, we further investigated the preventative effect of Tan IIA to APAP-induced nephrotoxicity. Oral supplementation of Tan IIA (30 mg/kg/day) prior to APAP intoxication (200 mg/kg) dramatically reduced APAP-induced nephrotoxicity as evidenced by histopathological evaluation and serum CRE levels. Tan IIA pretreatment promoted the eux of the toxic intermediate metabolite N-acetyl-p-benzoquinone imine (NAPQI) from mouse kidney, thus reducing its injury to tissue. After Tan IIA pretreatment, signicant induction of Nrf2, Mrp2, and Mrp4 mRNA and protein expression was detected in Nrf2 +/+ mice kidneys, however, much lower increase of Mrp2 and Mrp4 mRNA and protein expression was observed in Nrf2 −/− mice kidneys. Consistent with our observations in vivo, Tan IIA increased the mRNA and protein expression of Nrf2-MRP2/4 pathway and promoted the nuclear Nrf2 accumulation compared with APAP treatment alone in HK-2 cells. Collectively, the ndings in this study demonstrated that Tan IIA facilitates the clearance of toxic intermediate metabolite NAPQI from the kidney by up-regulating the Nrf2-Mrp2/4 pathway, thereby, performing a preventive effect against APAP-induced nephrotoxicity.


Introduction
Acetaminophen (APAP), a worldwide used acetanilide antipyretic and analgesic drug, is commonly used for cold or in uenza-induced fever and headache. APAP is safe at therapeutic dosages, however, it's known to cause hepatic and renal damage in humans when administered in overdoses (Chen et  but studies have shown that NAC has no effect on APAP-induced nephrotoxicity, and may even aggravate renal damage (Stern et al., 2005a;Stern et al., 2005b). Therefore, the development of the more effective and safer drugs against APAP toxicity is urgent in clinics.
In recent years, natural plant medicine has become a future hope for cure of a variety of primary disease or drug-induced disease (Liang et  In many theories about APAP toxicity, there is a hypothesis that the mechanism of APAP-induced nephrotoxicity is that when APAP is ingested in a toxic amount, excessive amounts of APAP and its toxic intermediate called N-acetyl-p-benzoquinone imine (NAPQI) are excreted by the kidney and contribute to kidney damage (Saleem and Iftikhar, 2019 Inc. (Suzhou, China). Nrf2 +/+ and Nrf2 −/− mice were generated by mating Nrf2 +/− male and female mice. All experimental mice were maintained in laboratory animal facility with free access to food and water under 12 h light/dark cycles. All animal procedures were carried out in accordance with the Guide for the The mice were sacri ced 3 h after APAP administration. The blood and kidney samples were collected from sacri ced animals. Serum samples were isolated from the blood after centrifugation at 3000×g for 10 min. Kidney samples were either xed in neutral formalin solution or snap frozen at -70°C.
Experimental design for pharmacokinetic study Page 5/22 144 Nrf2 +/+ male mice were assigned to 2 groups (each group consisted of 72 mice) in pharmacokinetic study: Group 1: Nrf2 +/+ mice were orally administrated with 1 week of 0.5% CMC-Na. At 15 min after the last treatment, mice were gavaged with a single dose of APAP (200 mg/kg).
Group 2: Nrf2 +/+ mice were orally administrated with 1 week of Tan IIA (30 mg/kg/d). At 15 min after the last treatment, mice were gavaged with a single dose of APAP (200 mg/kg).
Blood samples were collected from heart into heparinized tubes and kidney samples were collected before the dose of APAP and at the following intervals after the dose of APAP: 5, 10, 20, 30, 45 min, 1, 1.5, 2, 3, 6, and 12 h. About 0.5 mL of plasma at each of point was harvested after centrifuging the blood samples at 4450 ×g for 5 min. Kidney homogenate was made with 7 weight units of cold normal saline and 1 weight unit of tissue. All samples were stored at -70°C until analysis.

Assessment of pharmacokinetic parameters
The validated HPLC-MS/MS method was applied to determine plasma and kidney concentrations of APAP and its metabolites in mice used in the pharmacokinetic study. Pharmacokinetic parameters were calculated via non-compartmental analysis using Phoenix WinNonlin (version 6.3, Pharsight Corp, Mountain View, CA, USA).

Serum biochemistry assay
The level of serum creatinine (CRE) were determined using commercially available test kits and an automated biochemical analyzer in State Key Laboratory of Biotherapy, Sichuan University.

Histological examination
Kidney tissues were xed in neutral formalin solution for at least 48 h, then embedded in para n and sectioned at 4 µm. Tissue sections were stained with hematoxylin & eosin (HE) and histopathologic changes of tissues were viewed by a light microscope.
Cell culture and treatment HK-2 cells were cultured in a 1:1 mixture of Dulbecco's modi ed Eagle and Ham F12 (DMEM/F12; Gibco, USA) medium supplemented with 10% of fetal bovine serum (FBS), 100 µg/mL of penicillin/streptomycin mixture at 37°C in a 5% of CO 2 atmosphere. HK-2 cells were seeded in 6-well plates at an initial density of  Table. Cells were harvested for further analysis after transfection for 24, 48, or 72 h.

RNA isolation and RT-qPCR analysis
Total RNA was isolated from the mouse kidney and HK-2 cells using TRNzol reagent (TIANGEN, Beijing, China) according to the manufacturer's instructions. The cDNA was produced by using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan) and was ampli ed by a Hieff™ qPCR SYBR Green Master Mix (YEASEN, Shanghai, China). Thermal cycling was performed on a LineGene 9620 Fluorescence Quantitative Detection system (Bioer, Hangzhou, China). The sequences of primers are listed in Table. GAPDH/Gapdh was used as an internal control. The changes of mRNA quantity for the genes relative to the control were calculated using the 2 −△△CT method.

Protein lysate preparation and WB analysis
Total proteins were extracted from mouse kidney and HK-2 cells using RIPA lysis buffer (Wanlei, Shenyang, China). Nuclear and cytoplasmic proteins were extracted from HK-2 cells using the nuclear and cytoplasmic protein extraction kit (Wanlei, Shenyang, China). Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (Wanlei, Shenyang, China). The procedures of WB analysis were conducted as previous (Wang et al., 2017). The membranes after protein visualizing with electrochemiluminescence (ECL) (Thermo Scienti c, USA) were scanned using SageCapture software with MiniChemi imaging system (Sagecreation, Beijing, China). The bands density of was determined using ImageJ software (National Institutes of Health, USA).

Statistical analysis
Data were expressed as the mean ± standard error of mean (SEM) and analyzed using one-way ANOVA followed by a least signi cant difference (LSD). The Kruskal-Wallis test was used when variances were unequal. P values less than 0.05 were considered to be signi cant. SPSS v22.0 software (SPSS, USA) was used for the statistical analysis.

Results
Effect of Tan IIA on histological changes and serum enzyme levels in APAP treated mice vehicle control group, (2) APAP (200 mg/kg) group, (3) Tan IIA (10 mg/kg/d) + APAP (200 mg/kg), (4) Tan IIA (30 mg/kg/d) + APAP (200 mg/kg) group. Light microscope observation of mice kidneys in control group showed normal morphology of renal parenchyma. Histological lesions including dilatation of Bowman's space with glomerular atrophy, casts, and brush border of lumen surface fell off were clearly observed in APAP treated group. In Tan IIA pretreated group, despite the presence of mild incomplete brush border in the proximal tubules, casts was minimal and glomeruli maintained a much better morphology compared with the APAP treated group (Fig. 1A). Signi cant increase in the plasma CRE levels was noted in APAP treated group in comparison with the control, and pretreatment of high dose of Tan IIA to mice treated with APAP prevented the APAP-induced increase in CRE levels (Fig. 1B). These results indicate that Tan IIA could preserve the renal function after APAP overdose.
Effect of Tan Fig. 2A and B). We then assayed for Nrf2-targeted genes Mrp2 and Mrp4. We found that mRNA expression of Mrp2 and Mrp4 followed the same pattern as Nrf2 with increased levels following Tan IIA pretreatment ( Fig. 2A). Similar results were observed in the protein levels (Fig. 2B). These results indicate that the preventive effects of Tan IIA on APAP-induced nephrotoxicity may result from its ability to induce the Nrf2-Mrp2/4 pathway.
Effect of Tan IIA on pharmacokinetic pro les of APAP and its metabolites in mice Based on the induction of Mrp2 and Mrp4 in mice kidneys by Tan IIA, we speculated that the pharmacokinetic pro les of APAP and its metabolites may have changed. Therefore, we performed a pharmacokinetic study to investigate the effects of Tan IIA on the pharmacokinetic pro les of APAP and its metabolites in mice. As reported in our previous study, after pretreatment of Tan IIA, t 1/2 and V z of APAP in mice were signi cantly lower than those in control group, indicating that Tan IIA enhanced the elimination and affected the distribution of APAP in vivo (Zhang et al., 2020). As illustrated in Fig. 3A, the exposure of APAP had no difference between the Tan IIA pretreated group and the control group, and pretreatment of Tan IIA increased the exposure of APAP-gluc and APAP-sul in mouse kidney compared with the control group ( Fig. 3C and D). However, in Tan IIA pretreated group, the exposure of toxic intermediate NAPQI in mouse kidney was lower than those in the control group (Fig. 3B), indicating that Tan IIA reduced the accumulated toxicity of NAPQI in the kidney.
Effect of Tan IIA on APAP-induced nephrotoxicity in Nrf2 de cient mice In order to verify that Nrf2 plays a key role in reducing APAP-induced nephrotoxicity, Nrf2 −/− and Nrf2 +/+ mice were applied in the therapeutic study. Two genotypes of mice were respectively divided into three groups, each containing 6 animals. Dose selection and treatment duration were based on the published study(Zhang et al., 2020). We found that Nrf2 −/− mice were highly sensitive to APAP-induced nephrotoxicity as a result of the worse histopathological signs (more widespread of dilatation of Bowman's space with glomerular atrophy, casts, and brush border of lumen surface fell off) were shown in Nrf2 −/− mice kidneys (Fig. 4A). Compared to the vehicle-treated mice, serum CRE concentration increase in Nrf2 −/− mice was higher than that in Nrf2 +/+ mice after treated with APAP alone. Tan IIA reduced the increase of serum CRE level in mice of both two genotype groups, however, serum CRE level was only marginally decreased in Nrf2 −/− mice while it returned to normal in Nrf2 +/+ mice (Fig. 4B).
Effect of Tan IIA on the expression of the Nrf2-MRPs pathway in HK-2 cells Previous results indicated that Tan IIA could induce the expression of the Nrf2-Mrps pathway in mice, we next used human normal kidney cells (HK-2) to investigate the effect of Tan IIA on the expression of the Nrf2-MRPs pathway. We rst performed an RNA interference experiment for Nrf2. As shown in Fig. 5A-F, reduction of Nrf2 mRNA and protein led to a signi cant decrease in MRP2 and MRP4 expression at both mRNA and protein levels, especially when transfection with Nrf2 target siRNA for 48h in HK-2 cells. Compared with control and APAP treated alone group, signi cant increases in mRNA and protein expression of Nrf2 and MRP2/4 were observed in HK-2 cells receiving combined treatments of diverse concentrations of Tan IIA or positive control CUR (Fig. 5G-L). Moreover, the maximum Tan IIA concentration treated group exerted the highest upregulation. In contrast, though treated with Tan IIA, the mRNA and protein expression of MRP2 and MRP4 was not increased while Nrf2 was knockdown by Nrf2 target siRNA in HK-2 cells (Fig. 5M-N). These results indicate that Nrf2 is a positive regulator for MRP2 and MRP4 expression in HK-2 cells, and con rm the role of Tan IIA in the expression of the Nrf2-MRPs pathway.
Effect of Tan IIA on Nrf2 nuclear translocation in HK-2 cells Nrf2 is a nuclear transcription factor. Phosphorylation of Nrf2 serine 40 (p-Nrf2) enable it to escape or release from Keleh-like ECH-associated protein 1 (Keap1), known as an inhibitor of Nrf2. Translocating to the nucleus is a sign of Nrf2 activation, and Nrf2 in nucleus could perform an antioxidant response element (ARE)-driven regulation on target genes (Niture et al., 2014). To examine the activation of Nrf2 by Tan IIA, the nuclear and cytoplasmic content of p-Nrf2 and nuclear translocation of Nrf2 were examined in HK-2 cells.
Nuclear and cytoplasmic extracts were prepared from HK-2 cells treated with different compounds and were subjected to western blot analysis using anti-Nrf2 antibody. Compared with control group, APAP group was associated with a decrease in the nuclear content of p-Nrf2 after treated with APAP for 24, 48, or 72 h. In contrast, combined treatments with Tan IIA along with APAP were characterized by an increased nuclear content of the p-Nrf2 protein (Fig. 6A-C). As illustrated in Fig. 6D, the results of cell immuno uorescence analysis also showed much more accumulation of Nrf2 in the nucleus of HK-2 cells treated with APAP along with Tan IIA for 48 h than that treated with vehicle or with APAP alone. These results indicate that Tan IIA can stimulate the activation and nuclear translocation of Nrf2, a prerequisite step for the induction of ARE-driven detoxi cation and antioxidant genes, which confers protection against injury from toxic substances.

Discussion
Evidence developed over the past decades of years have suggested that APAP overdose can cause liver injury and even death due to liver failure. As a result, there have been a lot of researches on APAP-induced hepatotoxicity and the therapeutic strategies of it. However, other evidences have declared that renal insu ciency occurs in approximately 2-10% of patients with APAP overdose (Stollings et al., 2016). As kidney injury cases caused by APAP are becoming more common clinically, and some acute renal failure cases even perform no signs of liver injury (Ammenti et al., 1999) In the present study, the doses of APAP and Tan IIA were insistent with the previous research which exploring the protective effect of Tan IIA on APAP-induced hepatotoxicity (Zhang et al., 2020). Severe histological lesions and markedly increased serum level of CRE indicated that the dose of 200 mg/kg of APAP could cause nephrotoxicity in mice, and Tan IIA performed a preventative effect to APAP-induced nephrotoxicity by an observation of the much lower pathologic alterations and the near control level of CRE in Tan IIA pretreatment group. Excessive NAPQI in liver or kidney due to APAP overdoses can cause intracellular glutathione (GSH) depletion, which is an early event of hepatotoxicity and nephrotoxicity (Fisher and Curry, 2019). As we have proved that Tan IIA reduced the accumulation of NAPQI in mice liver, the present study further evidenced that the content of NAPQI in mice kidney was also decreased by Tan IIA, thus preventing nephrotoxicity.
Nrf2 cytoprotective adaptive response has evolved to be a powerful protective strategy for organisms against exposure to environmental toxins and toxicants, such as tobacco smoke, air pollution, dietary carcinogens, and pharmaceuticals (Osburn and Kensler, 2008). Nrf2 up-regulates the expression of the numerous cytoprotective genes as well as other antioxidant genes that attenuate cellular damage from xenobiotic toxicity. The high sensitivity of Nrf2-de cient mice to APAP-induced hepatotoxicity was con rmed by the results of a greater severity in liver injury than the wild-type mice and the death in the homozygous knockout mice only (Enomoto et al., 2001). Similarly, our previous and current ndings also demonstrated that, compared with Nrf2 +/+ mice, Nrf2 −/− mice expressed more severe hepatotoxicity and nephrotoxicity after administration of a parallel dose of APAP. Due to the contribution to cellular defense system, Nrf2 has become an important target to prevent a variety of cellular damage. Exposure of mice to oltipraz protected the kidney against injury from cisplatin by Nrf2-dependent induction of miR-125b levels (Joo et al., 2013). Tan IIA performed a protective effect against liver injury caused by APAP through increasing Nrf2 and its target detoxi cation enzyme genes (Wang et al., 2016). Similar ndings were reported in our previous study indicating that Nrf2-MRPs pathway play a vital role in preventing APAPinduced hepatotoxicity (Zhang et al., 2020). Furthermore, we have performed the luciferase reporter assays and chromatin immunoprecipitation assays to investigate how Nrf2 acts on MRP2 and MRP4, and the results of two assays indicated that Nrf2 could increase MRP2 and MRP4 promoter activity by binding to the certain AREs in MRP2 and MRP4 promoter, thus forming the Nrf2-MRPs signaling pathway (Zhang et al., 2020). The ndings of RNA interference experiment and mRNA/protein expression research on Nrf2-MRPs in this study were in agreement with the previous claim. Moreover, the upregulation of Mrp2 and Mrp4 by pretreatment of Tan IIA was observed in mice kidney, which is a prerequisite to promote the excretion of NAPQI from the kidneys.

Conclusions
The results in this study demonstrate that Tan IIA has the ability to protect against APAP-induced nephrotoxicity. This protective effect is positively correlated with the induction and nuclear accumulation of the transcription factor Nrf2, which increases the expression of Mrps, thus lightening the chemical burden of the kidney by decreasing the distribution of APAP and its metabolites in kidney. These ndings make Tan IIA a good candidate for future use in preventing or treating APAP-induced nephrotoxicity. Additionally, previous and current evidences have suggested that Tan IIA can ameliorate the APAPinduced hepatotoxicity and nephrotoxicity by enhancing the excretion of NAPQI from the tissues through the upregulation of the Nrf2-Mrps pathway. Besides, the alternations of pharmacokinetic pro les of APAP and its metabolites also implied that Tan IIA may affect the expression of speci c metabolic enzymes. Since studies have shown that APAP is mainly metabolized by cytochrome P-450 (CYP-450) enzyme system into toxic intermediate NAPQI (Laine et al., 2009), it's meaningful to further investigate the effect of Tan IIA on CYPs in the process of protecting against APAP-induced liver or kidney injury.