Baicalein acts as a nephroprotectant that ameliorates colistin-induced nephrotoxicity by activating the antioxidant defence mechanism of the kidneys and down-regulating the inflammatory response

Abstract

Background

Nephrotoxicity is the major adverse effect patients experience during colistin therapy. The development of effective nephroprotective agents that can be co-administered during polymyxin therapy remains a priority area in antimicrobial chemotherapy.

Objectives

To investigate the nephroprotective effect of baicalein, a component of the root of Scutellaria baicalensis, against colistin-induced nephrotoxicity using a mouse model.

Methods

C57BL/6 mice were randomly divided into the following groups: control, baicalein 100 mg/kg/day (administered orally), colistin (18 mg/kg/day administered intraperitoneally) and colistin (18 mg/kg/day) plus baicalein (25, 50 and 100 mg/kg/day). After 7 day treatments, histopathological damage, the markers of renal functions, oxidative stress and inflammation were examined. The expressions of Nrf2, HO-1 and NF-κB mRNAs were also further examined using quantitative RT–PCR examination.

Results

Baicalein co-administration markedly attenuated colistin-induced oxidative and nitrative stress, apoptosis, the infiltration of inflammatory cells, and caused decreases in IL-1β and TNF-α levels (all P < 0.05 or 0.01) in the kidney tissues. Baicalein co-administration up-regulated expression of Nrf2 and HO-1 mRNAs and down-regulated the expression of NF-κB mRNA, compared with those in the colistin alone group.

Conclusions

To the best of our knowledge, this is the first study demonstrating the protective effect of baicalein on colistin-induced nephrotoxicity and apoptosis by activating the antioxidant defence mechanism in kidneys and down-regulating the inflammatory response. Our study highlights that oral baicalein could potentially ameliorate nephrotoxicity in patients undergoing polymyxin therapy.

Introduction

Polymyxin (polymyxin B and colistin) lipopeptide antibiotics are used as last-line therapy against problematic Gram-negative pathogens that are resistant to almost all currently available antibiotics.¹ Available population pharmacokinetic and pharmacodynamic data indicate that the current recommended dosage regimens of polymyxins achieve suboptimal plasma concentrations and that higher doses are needed to achieve effective killing and prevent resistance.² Nephrotoxicity is the major dose-limiting factor that limits effective colistin therapy and occurs in ∼60% of patients.^3,4 Therefore, the development of effective nephroprotective agents that can be co-administered during polymyxin therapy remains a priority area in antimicrobial chemotherapy.

Our immunohistochemical and correlative microscopy studies revealed that polymyxins could significantly accumulate in the kidneys.^5,6 Understanding the pathways involved in kidney tubular cell death during polymyxin-induced nephrotoxicity is crucial for the development of nephroprotective agents. Our group has pioneered this area in reporting a series of studies that showed that polymyxin-induced apoptosis is mediated through the death receptor (up-regulation of Fas, FasL and Fas-associated death domain), mitochondrial (down-regulation of Bcl-2 and up-regulation of cytochrome C and Bax) and endoplasmic reticulum (up-regulation of Grp78/Bip, ATF6, GADD153/CHOP and caspase-12) pathways in cultured renal tubular cells and animals.^7–9 In cultured renal tubular cells, polymyxin B treatment induces a concentration-dependent loss of mitochondrial membrane potential, morphology changes and the generation of reactive oxygen species (ROS).⁷ Elevated expression levels of the cyclin-dependent kinase 2 and phosphorylated JNK and autophagy were also observed in kidney tissues of the colistin-treated mice.^8,10 Overall, these fundamental mechanistic studies have greatly facilitated the development of key polymyxin-nephroprotectant discovery platforms.

Baicalein is a component of the root of Scutellaria baicalensis, which possesses many beneficial pharmacological activities including anti-inflammatory, antioxidative, antimacrobiotic and immuno-regulatory; it has a time-honoured place in Asian traditional medicine for the treatment of diseases of the liver, kidney and cardiovascular system.^11–13 Baicalein supplementation has been reported to attenuate cisplatin and myocardial ischaemia-induced nephrotoxicity via the inhibition of oxidative stress and inflammatory responses.^11,14 Recently, Choi et al.¹⁵ reported that baicalein suppresses hydrogen peroxide-induced DNA damage and apoptosis in HT22 murine hippocampal neuronal cells by activating the nuclear factor-erythroid 2-related factor 2 (Nrf2) gene, a master transcription factor regulating the cellular oxidant stress response.^15,16 Coincidently, we reported that pharmacologically induced activation of Nrf2 and its downstream gene haem oxygenase-1 (HO-1) could protect against polymyxin-induced nephrotoxicity in rodent models.^16,17 In addition, baicalein treatment of mice has been shown markedly to attenuate renal fibrosis via anti-inflammatory mechanisms involving down-regulation of the NF-κB and mitogen-activated protein kinase pathways.¹⁸ The present study describes the nephroprotective activity of baicalein against colistin-induced nephrotoxicity in a mouse model and examines the molecular mechanisms (Figure 1a).

Figure 1.

Baicalein attenuates colistin-induced nephrotoxicity in mice. (a) Pathways of polymyxin-induced nephrotoxicity in mouse kidney tissues and the putative nephroprotective mechanism of baicalein. (b) and (c) Levels of serum BUN and CRE, respectively, in mice treated with colistin and/or baicalein. Results are presented as the mean ± SD (n = 10 in each group). **P < 0.01 compared with the untreated control. ^#P < 0.05 and ^##P < 0.01 compared with the colistin treatment group. Bai, baicalein.

Materials and methods

Chemicals

Colistin (sulphate) was purchased from Zhejiang Shenghua Biology Co., Ltd (20 400 U/mg, Zhengjiang, China). Baicalein (purity ≥98%) was obtained from Aladdin Reagent Co., Ltd (Shanghai, China). All other chemicals were analytical grade.

Animals

C57BL/6 mice (female, 6–8 weeks, 18–22 g) were purchased from Vital River Animal Technology Co., Ltd (Beijing, China). Mice were housed in a room maintained at a temperature of 23 ± 2°C and relative humidity of 50 ± 10% with a 12 h light/dark cycle. Mice were acclimatized for 1 week prior to use and had free access to food and water during the experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee at the China Agricultural University.

Experimental design

Mice were randomly divided into six groups (n = 10 in each group), as follows: control, baicalein 100 mg/kg/day (baicalein 100 group); colistin 18 mg/kg/day (colistin group); colistin 18 mg/kg/day plus baicalein 25 mg/kg/day (colistin + baicalein 25 group); colistin 18 mg/kg/day plus baicalein 50 mg/kg/day (colistin + baicalein 50 group); and colistin 18 mg/kg/day plus baicalein 100 mg/kg/day (colistin + baicalein 100 group). The colistin (sulphate) dose was administered intraperitoneally twice daily.¹⁰ Baicalein was suspended in 0.5% carboxyl methyl cellulose sodium and given orally 2 h prior to colistin administration. In the vehicle control group, mice were orally administrated with the equal volume of 0.5% carboxyl methyl cellulose sodium and twice intraperitoneal injections of saline. In the baicalein alone group, mice were orally administrated with baicalein at 100 mg/kg/day. All groups were administered their respective treatments for 7 consecutive days. After 12 h following the last dose, mice were euthanized by intraperitoneal sodium pentobarbital (80 mg/kg) (Sigma–Aldrich, St Louis, MO, USA), blood samples were collected and the serum was separated by centrifugation (3000 g for 15 min) and stored at −80°C until assayed. Kidney tissue samples were sectioned and flash frozen for subsequent biochemical, histopathological, ELISA and quantitative RT–PCR (qRT–PCR) studies.

Serum blood urea nitrogen (BUN) and creatinine (CRE) assays

The levels of serum BUN and CRE were examined by an automatic analyser (Hitachi 7080, Hitachi High-Technologies Corporation) using the standard diagnostic kits (Shanghai Kehua Bio-engineering Co., Ltd, Shanghai, China).

Measurement of markers of oxidative stress in kidney tissues

To examine the biomarkers of oxidative stress, 10% (w/v) kidney tissue homogenate was prepared as previously described in detail.⁸ The supernatants were collected by centrifugation (3000 g, 15 min, 4°C) and assayed for the levels of malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH) and the activities of superoxide dismutase (SOD), catalase (CAT) and inducible NO synthase (iNOS) using commercial kits (Nanjing Jiancheng Institute of Biological Engineering) according to manufacturer’s instructions. Protein concentration in the supernatant was measured using the bicinchoninic acid protein assay kit (Beyotime, Haimen, China).

Histopathological examination

Kidney tissue samples were randomly selected from four mice and fixed in 10% neutral buffered formalin for at least 48 h. The samples were de-waxed in xylene and rehydrated in a series of graded alcohols and then embedded in paraffin. These samples were then cut into 5 μm thick sections and stained with haematoxylin–eosin for histopathological analysis as we have previously described in detail.¹⁹ A semi-quantitative evaluation of kidney injury was carried out and a semi-quantitative score (SQS) was used according to our previous study.¹⁹ Scores of 0, +1, +2, +3, +4 and +5 corresponded to no change, mild change, mild to moderate change, moderate change, moderate to severe change and severe change, respectively.

Measurement of caspase-9 and -3 activities and TNF-α and IL-1β levels

The collected kidney tissue samples were lysed using lysis buffer (1:10 w/v) for 15 min on ice, then centrifuged at 15 000 g for 10 min at 4°C. The isolated supernatants were used to determine the activities of caspase-3 and caspase-9 (Beyotime, Haimen, China) and the levels of TNF-α and IL-1β (R&D Systems, Minneapolis, MN, USA) using commercial ELISA kits according to the manufacturers’ instructions, respectively.

qRT–PCR examination

Total RNA from kidney tissue samples was extracted using the TRIzol^® reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. The cDNA was synthesized from 2 μg of total RNA using the Prime Script RT-PCR kit (Takara, Dalian, China). The quality of RNA was verified by evaluating the OD at 260/280 nm. The PCR primers used are detailed in Table 1. The qRT–PCR was performed using an AB7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). Target gene expression was normalized to GAPDH.

Table 1.

Primer sequences used for quantitative real-time PCR

Gene	Direction	Primer sequence (5′ to 3′)
Nrf2	forward	5′-CAC ATT CCC AAA CAA GAT GC-3′
	reverse	5′-TCT TTT TCC AGC GAG GAG AT-3′
HO-1	forward	5′-CGT GCT CGA ATG AAC ACT CT-3′
	reverse	5′-GGA AGC TGA GAG TGA GGA CC-3′
NF-κB	forward	5′-CAC TGT CTG CCT CTC TCG TCT-3′
	reverse	5′-AAG GAT GTC TCC ACA CCA CTG-3′
GAPDH	forward	5′-ACA GTC CAT GCC ATC ACT GCC-3′
	reverse	5′-GCC TGC TTC ACC ACC TTC TTG-3′

Statistical analyses

All results are presented as mean ± SD. The statistical analyses were performed using SPSS V16.0 (SPSS Inc., Chicago, IL, USA) and the differences between groups were compared with one-way ANOVA followed by Dunnett’s multiple comparison procedure. P < 0.05 was regarded as statistical significance.

Results

Baicalein ameliorates colistin-induced nephrotoxicity

In the colistin alone treatment group, serum BUN and CRE significantly increased to 14.7 mmol/L and 83.6 μmol/L (both P < 0.01) (Figure 1b and c), respectively, compared with the vehicle control group. In the colistin + baicalein 50 group, serum BUN and CRE decreased to 11.1 mmol/L and 72.3 μmol/L (both P < 0.05), respectively; in the colistin + baicalein 100 group, serum BUN and CRE decreased to 8.2 mmol/L and 53.8 μmol/L (both P < 0.01), respectively. Notably, treatment with baicalein (100 mg/kg/day for 7 days) did not affect the serum BUN and CRE levels, compared with the vehicle control group (Figure 1).

Kidney histopathological examination showed that baicalein supplementation markedly attenuates colistin-induced tissue damage (Figure 2). The kidneys of mice treated with colistin alone displayed extensive damage seen as marked tubular degeneration, necrosis, tubular dilation, cast formation and infiltration of inflammatory cells (Figure 2c); SQS 3.0 ± 0.81 (P < 0.01) (Figure 2g). Baicalein supplementation attenuated the colistin-induced kidney damage in a dose-dependent manner, particularly in the colistin + baicalein 50 and colistin + baicalein 100 groups, seen as a marked attenuation of the infiltration of inflammatory cells and tubular dilatation and necrosis in the renal cortex (Figure 2e and f); SQS 1.8 ± 0.50 (P < 0.05) and 1.3 ± 0.50 (P < 0.01), respectively (Figure 2g). There were no marked histopathological changes in the baicalein alone group (Figure 2b), which showed comparable histology to the vehicle control group (Figure 2a).

Figure 2.

Representative histopathological changes in kidneys of mice treated with colistin and/or baicalein. (a) Control group: no damage. (b) Baicalein group: no damage. (c) Colistin (18 mg/kg/day) group: extensive damage. (d) Colistin (accumulated dose 126 mg/kg) plus baicalein (accumulated dose 175 mg/kg): intermediate damage. (e) Colistin plus baicalein (accumulated dose 350 mg/kg): mild damage. (f) Colistin plus baicalein (accumulated dose 700 mg/kg): minor damage. (g) SQS values are presented as the mean ± SD (n = 4). **P < 0.01 compared with the untreated control. ^#P < 0.05 and ^##P < 0.01 compared with the colistin treatment group. Filled arrows indicate marked tubular degeneration, necrosis and tubular dilation, arrowheads indicate cast formation and the open arrow indicates infiltration of inflammatory cells. Haematoxylin–eosin staining. Magnification: ×20. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Baicalein ameliorates colistin-induced oxidative stress in kidney tissue

Table 2 shows that colistin treatment significantly induced oxidative stress in the kidney tissues of mice, seen as a marked increase in the levels of MDA, iNOS and NO to 1.79 mmol/mg of protein, 1.31 U/mg of protein and 9.56 μmol/g of protein (all P < 0.01), respectively; significantly decreased activities of SOD and CAT and the level of GSH to 64.4 U/mg of protein, 78.5 U/mg of protein and 48.4 mmol/mg of protein (all P < 0.01), respectively. Baicalein supplementation, particularly at 50 and 100 mg/kg/day, markedly attenuated all of these colistin-induced biomarkers of oxidative stress (Table 2). The biomarkers did not appreciably change between the baicalein and vehicle control groups (Table 1).

Table 2.

Impact of baicalein supplementation on the levels of oxidative and nitrative stress markers in the kidney tissues of mice treated with colistin

Biomarker	Treatment group
	control	baicalein 100	colistin	baicalein 25 + colistin	baicalein 50 + colistin	baicalein 100 + colistin
MDA (mmol/mg of protein)	1.32 ± 0.32	1.27 ± 0.21	1.79 ± 0.31**	1.68 ± 0.34	1.62 ± 0.37#	1.54 ± 0.45##
SOD (U/mg of protein)	86.5 ± 12.1	93.4 ± 8.92	64.4 ± 7.41**	67.6 ± 12.3	73.8 ± 9.45#	77.6 ± 12.5##
CAT (U/mg of protein)	110.5 ± 13.4	114.7 ± 12.6	78.5 ± 9.70**	83.5 ± 14.2	95.7 ± 15.1#	100.5 ± 10.5##
GSH (mmol/mg of protein)	67.1 ± 7.42	72.1 ± 10.6	48.4 ± 7.64**	52.3 ± 8.11	56.4 ± 12.1#	59.1 ± 5.28#
iNOS (U/mg of protein)	0.62 ± 0.27	0.58 ± 0.17	1.31 ± 0.31**	1.26 ± 0.43	0.94 ± 0.29#	0.78 ± 0.24##
NO (μmol/g of protein)	6.32 ± 0.89	5.67 ± 1.21	9.56 ± 1.08**	9.12 ± 1.21	7.63 ± 0.95##	7.10 ± 0.82##

Results are presented as the mean ± SD (n = 10 in each group).

^**P < 0.01 compared with the untreated control.

^#P < 0.05 and ^##P < 0.01 compared with the colistin treatment group.

Baicalein attenuates colistin-induced activation of caspase-9 and -3 in kidney tissue

Colistin treatment significantly increased the activities of caspase-9 and -3 to 2.44- and 3.23-fold (both P < 0.01), respectively, compared with the vehicle control group (Figure 3a and b). Baicalein supplementation markedly attenuated colistin-induced activation of caspase-9 (Figure 3a) and -3 (Figure 3b) in a dose-dependent manner. In the colistin + baicalein 50 and colistin + baicalein 100 groups, caspase-9 decreased to 1.91- and 1.62-fold and caspase-3 decreased to 2.36- and 1.97-fold (all P < 0.01), respectively, compared with the colistin group. There was no marked change in the activities of caspase-9 and -3 in the baicalein alone treatment group (Figure 3a and b).

Figure 3.

Baicalein attenuates colistin-induced activation of caspase-9 (a), caspase-3 (b) and the inflammatory mediators IL-1β (c) and TNF-α (d) in the kidney tissue of mice treated with colistin. ELISA results are presented as the mean ± SD (n = 10 in each group). **P < 0.01 compared with the untreated control. ^#P < 0.05 and ^##P < 0.01 compared with the colistin treatment group.

Baicalein attenuates colistin-induced increase in IL-1β and TNF-α levels in kidney tissue

Colistin treatment significantly increased the levels of IL-1β and TNF-α, which were markedly attenuated by baicalein co-administration in a dose-dependent manner (Figure 3c and d). The significant attenuating effect was most pronounced in the colistin + baicalein 100 group, where the IL-1β levels decreased from 49.3 to 30.2 pg/mg of protein (P < 0.01) (Figure 3c) and the TNF-α levels decreased from 37.2 to 18.8 pg/mg of protein (P < 0.01) (Figure 3d). Baicalein treatment alone showed a mild decrease in the basal levels of IL-1β and TNF-α compared with that in the vehicle control group (Figure 3c and d).

Baicalein up-regulates the expression of Nrf2 and HO-1 mRNA and down-regulates the expression of NF-κB mRNA in kidney tissue

Interestingly, treatment of the mice with baicalein or colistin alone and in combination significantly increased the expression of Nrf2 and HO-1 mRNAs (Figure S1, available as Supplementary data at JAC Online). The effect was more pronounced with the colistin/baicalein combinations. In contrast, the expression of NF-κB mRNA significantly decreased in the colistin + baicalein 50 and colistin + baicalein 100 groups, compared with the colistin alone group. Unlike the results with the Nrf2 and OH-1 mRNAs, the expression of NF-κB mRNA did not markedly change in the baicalein only treatment group.

Discussion

MDR Gram-negative bacteria have become a crisis in hospitals in the USA and worldwide due to their proclivity to spread rapidly and the diminishing therapeutics available to treat infections effectively due to these pathogens. Polymyxin B and colistin are among the few remaining antibiotics that are effective against these deadly ‘superbugs’. Unfortunately, dose-limiting nephrotoxicity remains the Achilles’ heel for effective polymyxin therapy. Accordingly, the development of strategies to ameliorate this unwanted side-effect and thereby improve polymyxin therapy is of the utmost importance. Baicalein (5,6,7-trihydroxyflavone) is a polyphenolic flavonoid component of the Baikal Skullcap root that possesses a multitude of pharmacological activities.^{11,13,15,20–22} In the present study, we provide demonstrable proof that oral baicalein supplementation markedly reduces colistin-induced nephrotoxicity in a mouse model. In mice intravenously administered a nephrotoxic dose of colistin (18 mg/kg/day, 7 days; accumulated dose 126 mg/kg), co-administration of oral baicalein (25–100 mg/kg/day) produced a dose-dependent decrease in the serum levels of the kidney function markers, BUN and CRE, and ameliorated the histopathological damage caused by colistin (Figures 1 and 2).

We have previously shown that oxidative stress plays a critical role in polymyxin-induced nephrotoxicity in vitro in cultured proximal tubular cells and in vivo using our mouse nephrotoxicity model.^5,8,23,24 In line with these findings, here we show that decreased SOD and CAT activities and lower levels of GSH were detected in the kidney tissues of the colistin-treated mice (Table 2). Moreover, the lipid peroxidation marker MDA and nitrative stress-related NO and iNOS activities were significantly increased in the kidney tissues of the colistin-treated mice. Supplementation with baicalein strongly diminished these adverse oxidative/nitrative changes, with the nephroprotective effect being most pronounced at the 100 mg/kg/day baicalein dose (Table 2). Nrf2 is a transcription factor that centrally regulates oxidative stress response genes.²⁵ The activation of Nrf2 effectively acts to attenuate cellular oxidative damage by activating genes that encode Phase II detoxifying enzymes and antioxidant enzymes, such as CAT, SOD, HO-1 and glutathione peroxidase.²⁵ In the present study, we show that baicalein treatment activates Nrf2 expression and its downstream gene HO-1 (Figure S1), as well as increasing SOD and CAT activities (Table 2). These data indicated that the Nrf2/HO-1 pathway, in part, contributes to the nephroprotective effect of baicalein against colistin. Baicalein supplementation was shown to alleviate doxorubicin-induced cardiotoxicity in mice via suppression of oxidative stress, the inflammatory response and apoptosis.¹⁸ The cardioprotective mechanism of baicalein appeared to involve the restoration of doxorubicin-induced decreases in myocardial antioxidants [GSH, NAD(P)H:quinone oxidoreductase 1, SOD and CAT] and increase in iNOS by up-regulating the Nrf2/HO-1 and down-regulating the NF-κB pathway.¹⁸ In addition, Nrf2 activation can lead to inhibition of the oxidative stress driven by the NF-κB nuclear translocation-mediated inflammatory response via HO-1 end-products (i.e. bilirubin).²⁶ On the contrary, the NF-κB p65 subunit can also repress the Nrf2-antioxidant response element pathway at the transcriptional level by depriving CREB binding protein from Nrf2 and facilitating recruitment of histone deacetylase 3 to MafK.²⁷ Our results (Figure S1) indicate that the activation of the Nrf2/HO-1 pathway may partly contribute to the ability of baicalein to inhibit the NF-κB-mediated inflammatory response.

The antioxidant and anti-nitrative properties of baicalein were also shown to be fundamental for its ability to alleviate cisplatin-induced nephrotoxicity in mice.¹¹ The strong antioxidant properties of baicalein are partly due to the three 5,6,7 position OH-groups in its structure (Figure 1), which allow the compound to scavenge ROS via sacrificial oxidation of these groups.^12,28 Furthermore, baicalein’s ability to chelate redox-active metal ions such as Fe²⁺/Fe³⁺ also contributes to its antioxidant effect.^14,28 Overall, the direct radical scavenging activity of baicalein and its ability to increase the resistance of the kidneys by activating their intrinsic antioxidant defence mechanisms, are major factors that are responsible for the observed dose-dependent reduction of colistin-induced nephrotoxicity.

Our previous studies identified that the mitochondrial, death receptor and endoplasmic reticulum pathways are involved in polymyxin-induced nephrotoxicity in mice and in cultured rat and human kidney proximal tubular cells.^7–9 Caspase-3 is a key apoptotic mediator, which can be activated by both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways.²⁹ Caspase-9 is an important mediator in the mitochondrial apoptosis pathway.²⁹ Here we show that baicalein supplementation significantly attenuated colistin-induced activation of caspase-3 and -9 in kidney tissue (Figure 3a and b). Indeed, several other studies have reported that the multitude of pharmacological activities of baicalein are inextricably linked to its role in modulating mitochondrial function and dynamics.^{11,12,14,20,22}

Inflammation appears to play an important role in the progression of polymyxin-induced nephrotoxicity.²⁹ Accumulation of ROS can activate NF-κB, a central transcriptional mediator of the pro-inflammatory cytokine response.³⁰ In the present study, the colistin-induced inflammatory response in the kidney tissues of mice was characterized by the infiltration of inflammatory cells, up-regulated NF-κB expression and the release of the pro-inflammatory cytokines IL-1β and TNF-α (Figure 3c and d). Baicalein supplementation markedly attenuated NF-κB expression and decreased IL-1β and TNF-α to control levels (Figure 3c and d). Coincidently, the hepatoprotective effects of baicalein against d-galactosamine/LPS-induced acute liver failure were reported to involve its ability to inhibit the aforementioned inflammatory pathways.³¹ In rodents, baicalein is reported to have poor bioavailability;^32,33 however, a recent Phase I trial in 72 healthy subjects showed that oral baicalein (100–2800 mg daily) displays favourable pharmacokinetic profiles, is safe and well tolerated.³⁴ Notably, various formulations (e.g. baicalein-loaded nanostructured lipid carriers) have been developed to improve the stability and bioavailability.^35,36 Alternatively, to avoid potential bioavailability issues, baicalein could be co-administered intravenously together with the polymyxin dose.

To the best of our knowledge, we provide the first demonstrable proof that baicalein ameliorates colistin-induced nephrotoxicity in mice by inhibiting oxidative/nitrative stress, apoptosis and inflammation in kidney tissue. Baicalein supplementation may represent a promising approach for the prevention of nephrotoxicity in patients receiving polymyxin therapy.