Prostaglandin F2α analogue, bimatoprost ameliorates colistin-induced nephrotoxicity

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Biomedicine & Pharmacotherapy 168 (2023) 115446

Available online 31 October 2023

Prostaglandin F2 α analogue, bimatoprost ameliorates colistin-induced nephrotoxicity

Lina Joo

^a^,^b^,¹

, Hye Yun Jeong

^c^,¹

, Dong Hyuck Bae

^a^,^b

, Joo Hyun Jee

^a^,^b

, Woo Hee Choi

^a^,^b^,^d

, Hye-Youn Kim

^e

, Sejoong Kim

^f^,^g

, Dong-Ho Yang

^c

, Heon Yung Gee

^e

, SeongGyeong Jeon

^a^,^b

, Yun-Gil Roh

^h

, Jongman Yoo

^a^,^b^,^d^,^*

aDepartment of Microbiology, CHA University School of Medicine, Seongnam, the Republic of Korea

bCHA Organoid Research Center, CHA University, Seongnam, the Republic of Korea

cDivision of Nephrology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam, the Republic of Korea

dR&D Institute, ORGANOIDSCIENCES LTD., Seongnam, the Republic of Korea

eDepartment of Pharmacology, Yonsei University College of Medicine, Seoul 03722, the Republic of Korea

fDepartment of Internal Medicine, Seoul National University Bundang Hospital, Seongnam-si, Gyeonnggi-do 13620, the Republic of Korea

gDepartment of Internal Medicine, Seoul National University College of Medicine Seoul, 03080, the Republic of Korea

hProgram in Health Policy, Chung-Buk National University, Republic of Korea

A R T I C L E I N F O Keywords:

Bimatoprost Colistin Nephrotoxicity Drug repurposing Oxidative stress Apoptosis

A B S T R A C T

Colistin (polymyxin E) is an antibiotic that is effective against multidrug-resistant gram-negative bacteria.

However, the high incidence of nephrotoxicity caused by colistin limits its clinical use. To identify compounds that might ameliorate colistin-induced nephrotoxicity, we obtained 1707 compounds from the Korea Chemical Bank and used a high-content screening (HCS) imaging-based assay. In this way, we found that bimatoprost (one of prostaglandin F2α analogue) ameliorated colistin-induced nephrotoxicity. To further assess the effects of bimatoprost on colistin-induced nephrotoxicity, we used in vitro and in vivo models. In cultured human proximal tubular cells (HK-2), colistin induced dose-dependent cytotoxicity. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, indicative of apoptosis, was higher in colistin-treated cells, but this effect of colistin was ameliorated by cotreatment with bimatoprost. The generation of reactive oxygen species, assessed using 2,7-dichlorodihydrofluorescein diacetate, was less marked in cells treated with both colistin and bimatoprost than in those treated with colistin alone. Female C57BL/6 mice (n =10 per group) that were intraperitoneally injected with colistin (10 mg/kg/12 hr) for 14 days showed high blood urea nitrogen and serum creatinine concentrations that were reduced by the coadministration of bimatoprost (0.5 mg/kg/12 hr). In addition, kidney injury molecule-1 (KIM1) and Neutrophil gelatinase-associated lipocalin (NGAL) expression also reduced by bimatoprost administration. Further investigation in tubuloid and kidney organoids also showed that bimatoprost attenuated the nephrotoxicity by colistin, showing dose-dependent reducing effect of KIM1 expression. In this study, we have identified bimatoprost, prostaglandin F2α analogue as a drug that ameliorates colistin-induced nephrotoxicity.

1. Introduction

Multidrug-resistant (MDR) gram-negative bacteria are now widely distributed, such that they have become a significant public healthcare

concern, especially for critically ill patients in the intensive care unit [1, 2]. Infections with MDR gram-negative bacteria most frequently occur following prolonged hospitalization, and are associated with a higher cost and a higher mortality rate [3].

Abbreviations: MDR, Multidrug-resistant; PGE2, prostaglandin F2 α; HCS, high-content screening; HK-2, human proximal tubular cells; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling WST-1, water-soluble tetrazolium salt; DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; LTL, Lotus tetragonolobus lectin; KIM1, kidney injury molecule-1; NGAL, Neutrophil gelatinase-associated lipocalin; ROS, reactive oxygen species; PH, phase contrast; E-cad, E-cadherin.

* Corresponding author at: Department of Microbiology, CHA University School of Medicine, 59 Yatap-ro, Bundang-gu, Seongnam-si, 13496, the Republic of Korea.

E-mail address: [email protected] (J. Yoo).

1 These authors contributed equally to the work as co-first authors.

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

https://doi.org/10.1016/j.biopha.2023.115446

Received 1 May 2023; Received in revised form 21 August 2023; Accepted 4 September 2023

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Colistin, a polymyxin antibiotic, is the principal option for the treatment of MDR gram-negative bacterial infections[4] and was initially used therapeutically in the 1950s. However, the intravenous use of colistin has gradually been abandoned in most countries since the 1980s because of the high risk of nephrotoxicity [5]. As no novel antibiotics are expected to become available in the near future for the treatment of infections caused by MDR gram-negative bacteria, its use has been undergoing a revival [6]. There have been various attempts to identify compounds that might protect against colistin-induced nephrotoxicity. As the oxidative stress, apoptosis, and inflammatory pathways are known to provide the principal mechanism of colistin-induced nephrotoxicity, most previous studies have focused on identifying compounds that could protect against the activation of these pathways.

Although some compounds, including vitamin C, vitamin E, and astaxanthin, have been shown to ameliorate this oxidative stress, their utility could not be confirmed using conventional markers of renal function, such as serum creatinine concentration [7–9].

Drug repurposing, also known as drug repositioning, is used for speeding up finding novel drug, as it is the method identifying novel application for investigational or approved drugs. It is not only cheaper but also safer drug discovery and development strategy compared to conventional procedures [10]. For this strategy, this present study used a high-content screening (HCS) imaging-based assay [11–14] of fully automated microscopes and image analysis software [11]. We analyzed FDA-approved 1707 compounds from the Korea Chemical Bank, and identified bimatoprost as a candidate substance for the amelioration of colistin-induced nephrotoxicity.

Bimatoprost is a synthetic analogue of prostaglandin F2α used for treatment of glaucoma in ophthalmology [15]. As previous studies have showed the antioxidative properties and apoptosis suppression of prostaglandin F2α analogue [16,17], we investigated whether bimatoprost would attenuate the nephrotoxic effects of colistin reducing activation of the oxidative stress and apoptotic pathways in cellular and animal models. Additionally, we also used Prostaglandin F2α analogue, Pros- taglandin F2a tris salt to explore whether these effects are similar to other Prostaglandin F2a analogue. The protective effectiveness of bimatoprost on colistin-induced kidney injury were further confirmed in renal organoids and tubuloids.

2. Methods

2.1. Chemicals and reagents

A compound library of 1707 compounds was obtained from the Korea Chemical BanK (Daejeon, Korea). Bimatoprost (S1407) was purchased from Selleck Chemicals for the treatment of cells and from Bio- synth Carbosynth (FB18599) for animal experiments. Colistin sulfate (CAS 1264–72–8, 19,000 IU/mg) and Prostaglandin F2α tris salt (P0424) was purchased from Sigma-Aldrich.

2.2. Cell culture

The human proximal tubule epithelial cell line HK-2 was purchased from the Korean Cell Line Bank of the Seoul National University College of Medicine. Cells were cultured in RPMI 1640 medium containing 10 % fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Welgene, Gyeongsan-si, Korea). The cultures were maintained at 37 ^◦C and in a 5 % CO2 atmosphere.

2.3. HCS imaging-based assay

To screen for an anticytotoxic effect, 4000 cells were seeded per well into clear, flat-bottomed 96-well plates (SPL) and cultured in 100 μl cell culture medium containing 10 % FBS, 100 U/ml penicillin, and 100 μg/

ml streptomycin. After 24 hr of incubation, 5 μM of each chemical library compound was added to each well for 24 h, and then colistin was

added for a further 48 h. Subsequently, the cells were stained with Hoechst 33342 (H1399) or propidium iodide (PI, P21493) to identify nuclear defects and cell death by apoptosis, and the effects of each compound were quantified. As a secondary screen, cells were simulta- neously treated with each of the compounds and colistin in 100 μl of cell culture medium containing 10 % FBS, 100 U/ml penicillin, and 100 μg/

ml streptomycin. After 48 hr of incubation, cells were stained with Hoechst and PI. HCS was then used to evaluate the Hoechst-stained area.

2.4. High-content imaging

Imaging was carried out using a Cytation5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) in 5 % CO₂and at 37 ^◦C. Hoechst (1 μg/ml final concentration) was imaged using an excitation wavelength of ~377 nm and an emission wavelength of ~447 nm. PI (1 μg/

ml final concentration) was imaged using an excitation wavelength of

~531 nm and an emission wavelength of ~647 nm. Image analysis was performed using Gen5 software (BioTek).

2.5. Cell viability (WST-1) assay

Cell viability was determined using a EZ-cytox Cell Viability Assay Kit (EZ-1000, Daeil Biotech Co. Ltd., Suwon, Korea) using the water- soluble tetrazolium salt (WST-1) principle. HK-2 cells were treated with colistin (400 μg/ml) and bimatoprost (20 μM) for 48 hr, and then 20 μl WST-1 reagent were added to the medium, cells were incubated at 37 ^◦C and in a 5 % CO2 atmosphere for 2 hr, and the absorbance was measured at 450 nm.

2.6. TUNEL assay

Apoptosis was assessed by TdT-mediated dUTP nick-end labeling (TUNEL) using the DeadEnd™ Fluorometric TUNEL System, according to the manufacturer’s protocol (Promega, Madison, WI, USA). TUNEL- positive cells were identified using a Nikon Eclipse Ti2 (Nikon, Tokyo, Japan).

2.7. Assessment of ROS generation

The intracellular ROS concentration was measured using the cell- permeable fluorogenic probe 2

′

,7

′

-dichlorodihydrofluorescein diacetate (DCF-DA), according to the manufacturer’s protocol (ROS0300; OZ Biosciences, San Diego, CA, USA). Cells were incubated 4 ×10³per well in 96-well plate. After 48 hr, colistin (400 μg/ml) and bimatoprost at a final concentration of 10 μM or vehicle (1 % DMSO in PBS) at 37 ^◦C for 24 hr. Subsequently, DCF-DA solution was added to the medium, and the cells were incubated at 37 ^◦C for a further 30 min. The cells were washed with PBS. Then, 100 μl/well PBS was added and fluorescence measured with a fluorescence microplate reader (SpectraMax id5, USA) using an excitation wavelength of ~485 nm and an emission wavelength of

~535 nm. The method for Prostaglandin F2a tris salt is described in Supplementary Methods.

2.8. Antimicrobial susceptibility testing

Gram-negative bacteria, E. coli DH5-α 155065, were obtained from Biozoa Biological Supply (Seoul, Korea). Bacterial cultures were revived and maintained in LB Broth (Miller) and incubated at 37 ℃ in a CO2 incubator for 24 h. Following this, a secondary culture was carried out for an additional 6 h. The bacteria were then seeded onto 100pi dishes at a concentration of 5 ×10^5 CFU/ml. A detailed methods on determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) is provided in the Supplementary Material.

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Biomedicine & Pharmacotherapy 168 (2023) 115446

2.9. Mice

C57BL/6 N mice (female, 6–8 weeks, 18–20 g) were purchased from Raon Bio. They had free to access food and water, and were housed under specific pathogen-free conditions. The experimental protocol was approved by the CHA University Institutional Animal Care and Use committee (IACUC#210072).

2.10. Establishment of a model of colistin-induced nephrotoxicity and bimatoprost treatment

To determine the effects of bimatoprost on colistin-induced nephrotoxicity, Mice were acclimated to their environment for 3 days and then randomly allocated to four groups (n =10 per group) that were administered intraperitoneal injections as follows: (1) Control group (saline +1 % DMSO), (2) Colistin group (colistin 10 mg/kg/12 hr), (3) Bimatoprost group (bimatoprost 0.5 mg/kg/12 hr), and (4) Bimatoprost +Colistin group (colistin 10 mg/kg/12 hr +bimatoprost 0.5 mg/kg/12 hr).The mice were injected with each compound for 14 consecutive

days, after which blood samples were collected for the measurement of blood urea nitrogen (BUN) and serum creatinine and stored at − 80 ^◦C.

The mice were then sacrificed by cervical dislocation, and both kidneys were harvested and fixed in 4 % paraformaldehyde (PFA) for subsequent histological analysis.

2.11. Measurement of BUN and serum Cr

The BUN and serum Cr concentrations were measured using an Abbott i-STAT handheld portable clinical analyzer (Abbott Point-of- Care, East Windsor, NJ, USA) and i-STAT CHEM 8 + cartridges (Abbott Laboratories).

2.12. Immunofluorescence

The kidneys were fixed in 4 % PFA and embedded in paraffin.

Paraffin Section (5 μm thick) were deparaffinized in xylene and rehy- drated in a graded ethanol series. For immunofluorescence analysis, permeabilization was performed using 0.2 % Triton-X-100 in PBS, and 5 Fig. 1. Image-based assay using HCS (High-content-screening) for 1st Hit drug screening methods and 2^ndHit drug discovery. A) Schema of the High-content- screening (HCS) image based assay. B) Representative images of top 14 compounds second screening. Merged images with Hoechst 33342 (blue) staining and cell death using propidium iodide (PI) staining (scale bar= 200µm). C) Example of Gene5 nuclei masking algorithms (scale bar= 100µm). D) The chemical structure of bimatoprost. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparison test. *, p<0.05, **, p<0.01,

***, P<0.001.

L. Joo et al.

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% bovine serum albumin (BSA) in DPBS (Welgene) was used to reduce nonspecific binding. After incubation with primary antibodies at 4 ^◦C overnight, the sections were incubated with secondary antibodies at room temperature for 2 hr. Finally, 1 μg/ml Hoechst 33342 (Millipore Sigma, Burlington, MA, USA) was used for nuclear staining. The antibodies used were anti-KIM-1 (1:100; AF1817; R&D systems), LTL (1:200; FL-1321–2; Vector Laboratories), Alexa Fluor® 488 dye (1:400, Invitrogen), and Alexa Fluor® 594 dye (1:400, Invitrogen). Images were acquired using a Nikon Eclipse Ti2 (Nikon, Tokyo, Japan).

2.13. Transmission electron microscopy

The cortex sections of the left kidneys for electron microscopy were isolated to observe ultrastructure of mice kidneys. The detailed methods for tissue preparation and microscopy equipment are in the supple- mental appendix.

2.14. Culture of human kidney tubuloid

The Institutional Review Board of the authors’ institute approved this study (IRB number: B-2003–601–302). Human urine sample was centrifugated for 20 min at 600xg, the pellet was washed with basal medium (ADMEM/F12 supplemented with 1 % (vol/vol) penicillin/

streptomycin, HEPES and GlutaMAX). After a second centrifugation step, the pellet was embedded in growth factor-reduced Matrigel (Corning, #356231) or Basement Membrane Extract (BME, R&D Sys- tems, #3533), and polymerized for 1 hr in a 37 ℃ incubator. Tubuloid medium contained basal media supplemented with N-acetylcysteine (1 mM, Sigma), 1.5 % (vol/vol) B27, EGF (50 ng/ml), FGF-10 (100 ng/ml), Rho-kinase inhibitor Y-27632 (10 μM), A8301 (5 μM), primocine (0.1 mg/ml) supplemented with 10 % Rspo1-conditioned medium. For

nephroprotective assay, tubuloids were culture in basal medium sup- plement with bimatoprost or curcumin.

2.15. Differentiation for human kidney organoid

The human iPS(IMR90)-4 (Wicell, #WISCi004-B) were maintained in mTeSR1 (STEMCELL Technologies, # 85850) in 6-well tissue culture plates coated with 0.5 % (vol/vol) iMatrix511 silk (Matrixome,

#MX892021) in a 37 ℃ incubator containing 95 % air and 5 % CO₂. hiPSCs were passaged using ReLeSR (STEMCELL Technologies, #05872) at 1:10 split ratio every 7 days according to the manufacturer’s protocol.

For induction to kidney nephron progenitor organoid, IMR90 (80–90 % confluence) were briefly washed on with DPBS and dissociated into single cells with accutase (STEMCELL Technologies, #07920). The serum-free hiPSC differentiation medium consisted of DMEM⋅F12 supplemented with 2 % (vol/vol) B27, 2 % (vol/vol) GlutaMAX, 1 % (vol/

vol) ITS (insulin-transferrin-selenium), 2 % (vol/vol) non-essential amino acids, 1 % (vol/vol) penicillin/streptomycin, 90 µM 2-mercaptoe- tahnol. The dissociated cells were reaggregated at a density of 2 ×10⁴ cells onto 96 V-bottom well plate with the ROCK inhibitor Y27632 (10 µM), activitn A (1 ng/ml), FGFb (20 ng/ml) and BMP4 (1 ng/ml). After 24 hs (on day 1), cell aggregates were transfer onto 96 well U bottom low-attachment plate and the medium was changed to 10 µM CHIR99021. After 48 hrs, the medium containing Y27632 and CHIR99021 was switched, half of the culture medium volume was refreshed with new medium on day 5. On day 7, the medium was replaced with medium containing Y27632 (10 µM), retinoic acid (1 nM), activin A (10 ng/ml), BMP4 (3 ng/ml), and CHIR99021 (3 µM). On day 10, the medium was replaced with medium containing Y27632 (10 µM), CHIR99021 (1 µM), and FGF9 (5 ng/ml). On day 13, for liquid-air interface culture, induced nephron progenitor cell spheroids were Table 1

1st Screening Hit compound list.

Number General Name Max Phase Disease area Target

1 Dibucaine hydrochloride approved Neurology/psychiatry CALM1, SCN10A,SCN5A

2 Bimatoprost approved Ophthalmology AKR1C3, PTGER1, PTGER3, PTGFR

3 c-Kit-IN-1 Phase 3 Cancer c-Kit inhibitor; c-Met/HGFR inhibitor

4 ATB 346 Phase 2 Nervous system COX inhibitor

5 GSK2981278 Phase 2 Immune system ROR agonist

6 Fexinidazole Phase 3 Immune system Others

7 TD-139 Phase 2 Respiratory system/Metabolism galec�n

8 Ibiglustat Phase 3 Others Transferase inhibitor

9 Ethacrynic acid approved Cardiology, Gastroenterology,

Nephrology, Rheumatology ATP1A1, SLC12A1

10 Methylprednisolone approved

Endocrinology, Rheumatology, Dermatology, Infec�ous disease, Allergy, Hematology, Neurology/

Psychiatry, Gastroenterology

NR3C1

11 Pibeserod hydrochloride Phase 2 Metabolism 5-HT Receptor antagonist

12 Darifenacin hydrobromide approved Neurological Disease Muscarinic M3 Receptor Antagonists 13 Tacrine hydrochloride approved;withdrawn Neurology/ psychiatry ACHE, BCHE

14 (+/-)-AMG 487 Phase 2 Inﬂamma�on/immumology Chemokine CXCR3 Receptor Antagonists The top 14 compounds include information of General Name, Max phase, Disease area, and Target.

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transferred on to the upper chamber of 3.0 µm pore size of Transwell (Corning) supplied with KR medium supplemented DMEM-F12 supplemented with 2 % (vol/vol) GlutaMAX, 2 % (vol/vol) non-essential amino acids, 1 % (vol/vol) penicillin/streptomycin, 5 % (vol/vol) knockout serum replacement, 50 µM 2-mercaptoethanol. In vitro nephroprotective property of bimatoprost or curcumin against colistin were treated in 3D kidney organoids cultured in the KR medium after day 24 of differentiation.

2.16. Immunostaining of kidney organoids

Kidney organoids were processed for paraffin sectioning using standard protocols. Kidney organoid were fixed by submersion in 4 % formaldehyde at 4 ^◦C overnight. Organoids embedded in paraffin were sectioned at 5 µm thicknesses. Kidney tubuloids culture in chamber slide were fixed by 4 % PFA at RT for 40 min. Deparaffinized organoid section or tubuloids were incubated in blocking buffer containing 1 % BSA for 1 hr at RT. The sections were incubated with primary and then fluorophore-tagged secondary antibodies were 1:100 and 1:1000 diluted with 0.1 % BSA, respectively. Confocal images of kidney organoids were obtained with a Carl Zeiss LSM780 instrument and ZEN software was used for image processing. Fluorescence microscopy image of kidney tubuloids were obtained with a leica microscopy and LAS X software was used. LTL (Vector Labs, #FL-1321), alexa fluor 488 phalloidin (Invi- trogen, #A12379), anti-AQP2 (Santa Cruz, Sc-9882), anti-E-cadherin (BD Biosciences, #610181), anti-KIM-1 (R&D systems, #AF1750) and anti-nephrin (Progen, #GP-N2) antibodies were purchased from

commercial sources.

2.17. Statistical analysis

Data are expressed as the mean ±SD or ±SEM and were obtained from experiments performed in triplicate. Groups were compared using Student’s t-test or one-way analysis of variance (ANOVA) and Tukey’s or Bonferroni’s test post hoc using Prism software version 5.0 (GraphPad, San Diego, CA, USA). P <0.05 was considered to represent statistical significance.

3. Results

3.1. HCS imaging-based screening shows that bimatoprost ameliorates colistin-induced nephrotoxicity

To identify compounds with a protective effect against colistin- induced nephrotoxicity, we screened a library of 1707 compounds obtained from the Korea Chemical Bank. The effects on nuclear morphology were examined using Hoechst 33258 staining, and cell death was identified using PI staining (Fig. 1A). The top 14 compounds identified using the primary HCS (Table 1) were subjected to a second screening, during which each of these compounds were added alongside colistin. The viability of the cells treated with each of the top 14 compounds was calculated, and the results are shown in Fig. 1B. This showed that bimatoprost was the most effective compound at reducing colistin- induced apoptosis (Fig. 1B). On the basis of the HCS imaging-based Fig. 2. Bimatoprost ameliorates colistin induced nephrotoxicity in human proximal tubular (HK-2) cells. A) Statistical analysis of cell viability evaluated with PI in bimatoprost only treatment. B) Statistical analysis of cell viability rate in bimatoprost pre-treatment and colistin. C) Statistical analysis of cell viability rate in Bimatoprost and Colistin co-treatment of. D) Representative images of co-treatment of bimatoprost and colistin. Merged images with Hoechst 33342 (blue) staining and cell death using propidium iodide (PI) staining (scale bar=200µm) All experiments were duplicated for three times. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparison test. *, p<0.05, **, p<0.01, ***, P<0.001.

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assay (Fig. 1C), we hypothesized that bimatoprost might represent an effective means of ameliorating colistin-induced nephrotoxicity (Fig. 1D).

3.2. Bimatoprost ameliorates colistin-induced nephrotoxicity in human proximal tubular (HK-2) cells

To evaluate the toxicity of bimatoprost, the cell viability of HK-2 cells was determined using WST-1 assay following bimatoprost treatment. Cells were treated with 1μM, 5μM, 10μM, 50μM, or 100μM bimatoprost, and control cells were treated with 1 % DMSO. “In our experiments, exposure to a high concentration of Bimatoprost resulted in a significant reduction in cell viability when compared to the control group. (Fig. 2A).

To investigate the protective effect of bimatoprost on colistin- induced nephrotoxicity, the effects of bimatoprost on cell viability were examined in colistin-treated HK-2 cells. After 24 hr incubation with 1μM, 5μM, 10μM, 50μM, or 100μM bimatoprost, HK-2 cells were treated with 400μg/ml colistin, and it was shown that 10μM bimatoprost significantly reduced cell death vs. the other groups.

(Fig. 2B).

In addition, we investigated the effects of simultaneous treatment of the cells with bimatoprost and colistin (1μM, 5μM, 10μM, 50μM, or 100μM bimatoprost with 400μg/ml colistin). This showed that 10μM bimatoprost significantly increased cell viability and reduced apoptosis by more than two-fold (Fig. 2C, D).

Thus, bimatoprost protects against colistin-induced kidney damage by reducing cell death by apoptosis.

3.3. Bimatoprost protects against colistin-induced loss of cell viability, apoptosis, and oxidative stress in human proximal tubular cells, showing similar effects to other Prostaglandin F2α analogue

The exposure of HK-2 cells to colistin reduced their viability, according to a WST-1 assay. The simultaneous treatment of the cells with bimatoprost prevented the effect of colistin, inhibiting the reduction in cell viability (Fig. 3A). The administration of colistin markedly increased the apoptosis of the cells, assessed using a TUNEL assay, but bimatoprost significantly inhibited the colistin-induced apoptosis (Fig. 3B, C).

We next measured ROS accumulation in colistin-treated cells using DCF-DA. The fluorescence signal was higher in the colistin-treated group than in the control group, but this was significantly reduced by co- treatment with bimatoprost (Fig. 3D).

We also investigated whether other Prostaglandin F2α analogue also shows the same results as bimatoprost. We used Prostaglandin F2a tris salt and identified the similar protective effect of this Prostaglandin F2α analogue on colistin-induced nephrotoxicity. (Supplementary Fig. 1 A- C). In addition, We demonstrated that treatment of Prostaglandin F2α tris salt also effectively inhibited the apoptosis induced by colistin (Supplementary Fig. 1 D,E) and reduced ROS formation. (Supplemen- tary Fig. 1F).

To investigate the primary target of bimatoprost, we additionally used inhibitor of Prostaglandin receptor exploring the effect on bimatoprost-treatment effect. When PTGFR was inhibited using AL- 8810 (5uM), bimatoprost failed to safeguard against colistin-triggered nephrotoxicity, indicating PTGFR as its principal target for renal Fig. 3.Effects of Bimatoprost on colistin-induced renal oxidative stress and apoptosis in human proximal tubular (HK-2) cells. A) Cell viability using WST-1 assay B) and C) Transferase dUTP nick-end labeling (TUNEL) staining. Merged images with Hoechst 33342 (blue) staining and TUNEL (green) staining (scale bar=250µm) D) Determination of ROS generation via detection of DCF-DA by fluorescence microtiter plate reader (excitation: ~485 nm/ emission: ~535 nm). All experiments were duplicated for three times. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparison test. *, p<0.05, **, p<0.01,

***, P<0.001.

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protection. This was further echoed in ROS level assays where trends showed a significant link between PTGFR and bimatoprost’s action mechanism. Despite treatment with indomethacin and Flufenamic Acid, bimatoprost retained its renoprotective capabilities against colistin, hinting that PTGER1 and AKR1C3 may not be its primary targets.

(Supplementary Fig. 2A,B).

These results suggest that bimatoprost protects cells against colistin- induced apoptosis and oxidative stress, which are the effects demonstrated by Prostaglandin F2α analogues.

3.4. Bimatoprost does not affect the antibacterial effect of colistin Antimicrobial disk susceptibility testing was performed to determine whether bimatoprost influences the antibacterial effect of colistin against E. coli. The effects of four paper disks containing colistin 10 mg, colistin 10 mg+bimatoprost 10 mg, bimatoprost 10 mg, and saline +DMSO 1 % (control) were compared (Fig. 4A). At the end of the incubation period, the diameters of the areas of bacterial growth inhibition were measured for each disk. Co treatment with colistin and bimatoprost was associated with similar activity against E. coli to colistin alone. In contrast, neither the negative control nor bimatoprost were effective against E. coli (Fig. 4B). Additionally, through the MIC/MBC test, we confirmed that bimatoprost does not influence the antibacterial effect (See Supplementary Table 1).

These results suggest that bimatoprost could be used concomitantly with colistin because it would reduce the host cell damage induced by colistin but not its antibacterial effect.

3.5. Bimatoprost ameliorates colistin-induced nephrotoxicity in mice C57BL/6 N mice were randomly allocated to four groups, which were administered compounds as follows: saline +1 % DMSO, colistin 10 mg/kg/12 hr, bimatoprost 0.1 mg/kg/12 hr, or colistin 10 mg/kg/

12 hr +bimatoprost 0.1 mg/kg/12 hr. After colistin treatment, the kidneys appeared whiter in color than those of control mice, suggesting that colistin induces renal damage in mice. This morphological appearance was ameliorated in mice that were also administered bimatoprost (Fig. 5A). During the colistin treatment period, the mice had lost weight after the sixth day, whereas control, bimatoprost, and bimatoprost/colistin-treated mice did not show a significant weight change (Fig. 5B). Although it was not statistically significant, the levels of BUN and serum Cr increased by colistin treatment (10 mg/kg/12 hr), but the addition of 0.1 mg/kg/12 hr bimatoprost ameliorated these effects (Fig. 5 C, D).

Immunofluorescence was used to evaluate the expression of Kim-1 (Fig. 6A, C) and NGAL (Fig. 6B, D), which are markers of proximal tubular damage. The expression of both Kim-1 and NGAL was significantly increased by colistin treatment, and there was little or no immunelabeling in the vehicle and bimatoprost-treated mice. Immu- nolabeling for both markers in the proximal tubule was significantly reduced by bimatoprost co-treatment (Fig. 6A–D).

We observed electron microscopy to investigate change of mitochondrial ultrastructure in mice kidneys. Compared with the kidneys from control, the numbers of autophagosome and multi lamellar body increased in the kidneys treated with colistin. However, the observation using TEM also revealed the reduced formation of autophagosomes and multi-layered structures in the kidney treated with colistin and bimatoprost (Supplementary Fig. 3).

These results show that bimatoprost ameliorates colistin-induced nephrotoxicity in animal model.

3.6. Bimatoprost ameliorates colistin-induced nephrotoxicity in tubuloid and kidney organoids

To investigate colistin-induced renal damage in tubuloids which were established from the urine of a healthy donor, we added 1μM, 3μM, 10μM, 30μM, 100μM, 300μM and 1000μM of colistin to determine EC50. The expression of KIM1 increased as colistin concentration increased, and EC50 was 230.1±14.62μM (Supplementary Fig. 4A) When 230μM colistin and 3μM, 10μM and 30μM bimatoprost, or curcumin were co-treated, KIM1 expression was significantly reduced compared to colistin only and the protective effect of bimatoprost or curcumin was dose-dependent. (Fig. 7A). We compared the protective effect of bimatoprost to that of curcumin, which is already known to be effective on colistin-induced nephrotoxicity in rat model [18]. by quantifying the KIM1 intensity. The IC50 values of bimatoprost (4.006±17.37μM) and curcumin (5.908±12.15μM) were not statistically different (Fig. 7A, Supplementary Fig. 4B). However, 30μM of curcumin could not be analyzed because it destroyed the cuboidal epithelium structure of tubuloids, which was not observed with 30μM bimatoprost.

We further investigated the protective effect of bimatoprost on colistin-induced nephrotoxicity in kidney organoids. The KIM1 expression increased depending on concentration of colistin with EC50 of 156.4±16.2μM (Supplementary Fig. 5A) The 1000μM of colistin were not included in the calculation of EC50 value due to degradation of kidney organoid structures. The nephrotoxicity induced by colistin was attenuated by co-treatment with bimatoprost, and the protective effect of bimatoprost treatment was concentration-dependent (Supplementary Fig. 5B). KIM1 expression was significantly reduced by 30μM curcumin, but not by 10μM curcumin, whereas bimatoprost exhibited the protective effect starting from 10μM. IC50 of bimatoprost (5.543±6.293) was statistically significantly lower than curcumin (17.06±3.691) (Supplementary Fig. 5B). Additionally, we conducted the Fig. 4. The effect of bimatoprost on the antibacterial susceptibility test. A)

Discs containing the drugs were placed on Mueller–-Hinton agar plates that had been inoculated with E. coli (DH5-α 155065) The contents of the numbered discs are as follows: 1, Bimatoprost (10 mg); 2, Control (10 mg); 3, Colistin (10 mg); 4. Colistin (10 mg) and Bimatoprost (10 mg) B) The bar graph showing the diameter of inhibition (Scale bar=10 mm) All experiments were duplicated for three times.

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characterization of kidney organoids and kidney tubuloids. Through immunofluorescence, we were able to confirm the significant expression of characteristic markers specific to both the kidney and tubuloids (Supplementary Fig. 6A,B).

These findings suggest that bimatoprost could alleviate the nephrotoxicity induced by colistin also in kidney tubuloids and organoids.

4. Discussion

In the present study, we have shown that bimatoprost ameliorates colistin-induced nephrotoxicity through effects on oxidative stress and apoptosis using in vitro and in vivo models.

Although the exact mechanism of the nephrotoxicity of colistin is unclear, the ‘detergent theory’ provides one potential explanation [4,19, 20]. Because colistin contains cations and has high affinity for lipids, it readily binds to phospholipids in cell membranes, increasing their permeability. Tubular damage occurs when this results in renal cell distension and lysis, which is the result of an electrostatic interaction leading to membrane instability and greater permeability. Findings of previous studies are consistent with this theory because this phenome- non was found not be specific to kidney cells but also extended to red blood cells and liver cells [21,22].

The effects of colistin have also been attributed to its reabsorption in proximal tubule cells. Colistin is reabsorbed both by an endocytic pro- cess and by transporters located in the proximal tubule, and this reabsorption exposes renal tubular cells to a high colistin concentration.

Recent studies have focused on the intracellular accumulation of colistin, and have suggested that colistin-induced nephrotoxicity may be mediated through oxidative stress and apoptotic cell death [23,24]. Dia et al. showed that colistin treatment increases the levels of a marker of oxidative stress in a mouse model. They also examined the role of the mitochondrial pathway, the key pathway of apoptotic cell death in colistin-induced nephrotoxicity, and confirmed its involvement in

colistin-induced apoptosis by demonstrating the upregulation of cyto- chrome C and Bax and the downregulation of bcl-2 expression [25].

There have been a number of attempts to identify compounds that protect against colistin-induced nephrotoxicity, and various antioxidants and anti-inflammatory drugs have been evaluated, including N- acetylcysteine, ascorbic acid, vitamin E, and melatonin. Recently, Aslan et al. showed that ascorbic acid and dexpanthenol, an antioxidant that is similar to ascorbic acid and has been shown to reduce amikacin nephrotoxicity in animal models, ameliorate the nephrotoxic effect of colistin. However, most previous studies of the potential protective effects of antioxidants have been performed using in vitro or animal models, and the few human studies that have been conducted did not show sufficient efficacy. Although one observational cohort study of patients with severe sepsis or septic shock showed that the co administration of ascorbic acid with colistin had a nephroprotective effect, the study sample was very small, and the possibility of nephrotoxic effect of agents other than colistin was not fully excluded [26].

In the present study, we used an HCS imaging-based assay to identify substances that ameliorated colistin-induced nephrotoxicity. HCS per- mits the rapid and systemic characterization of compounds, which is not feasible using other biochemical assays or conventional cell-based ap- proaches [12]. This can be used to screen large chemical libraries and yields quantitative information. In addition, such an imaging-based cellular high-throughput method can provide pharmacokinetic information regarding solubility in a cellular context, and can enable compounds to be identified at the single-cell level without significant fluorescent artifacts or cytotoxicity [27]. Using this screening method, we identified bimatoprost as a compound that ameliorates the nephrotoxic effect of colistin.

Bimatoprost is a synthetic prostamide analog that is related to prostaglandin F2a and is topically injected for the treatment of glaucoma. Its beneficial effect on intraocular pressure is achieved by increasing the outflow of aqueous humor through pressure-sensitive and Fig. 5.Effect of Bimatoprost on kidney morphology, body weight and kidney function on colistin induced nephrotoxicity. A) Photomicrograph showing the effect of Bimatoprost on the gross morphology of the colistin-treated mice kidney. B) Graph of body weight change of mice. (C, D) Changes of the renal function represented by BUN and serum creatinine (Cr). The results are represented as means plus standard deviations (SD) (error bars) (n=10). Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparison test. *, p<0.05.

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pressure-insensitive pathways [28]. In previous in vitro studies of the toxic effects of commercial prostaglandin analogs, it was shown that they caused a significant reduction in inflammatory cytokine production [29,30]. In addition, these analogs had significant protective effects that were mediated principally through a reduction in ROS production [16].

Recently, there have been suggestions that prostaglandin analogs might also have a protective effect on neurons that is independent of their effect on intraocular pressure [31,32,33]. Yamagishi et al. demonstrated that bimatoprost significantly improves retinal ganglion cell survival in a hypoxic environment by reducing apoptotic cell death, suggesting that this drug modifies apoptotic cell signaling [17]. In the present study, we have also shown that apoptosis, indicated by the number of TUNEL-positive cells, was lower in the colistin plus bimatoprost group than in the colistin only group. ROS generation also decreased when treated with combination of colistin and bimatoprost. These effects of bimatoprost to ameliorate the nephrotoxicity of colistin were also identified in animal model.

In this study, we also utilized kidney tubuloid and organoid models to investigate the effect of candidate drug, in addition to in vitro primary cell culture and in vivo animal model. Organoids are miniature organs derived from pluripotent stem cells or adult tissue resident stem cells [34], and are bridging the gap between conventional in vitro cell culture

and in vivo animal model [35]. As the organoids mimic the same intrinsic pattern of organ itself, they are increasingly adopted powerful system in a wide range of studies for investigating not only for development but also for adult repair and homeostasis [36]. The organoid-based models derived from patients have higher sensitivity and stability restoring the real environment of human species compared with traditional drug screening models. As the previous studies which investigated the protective drug for colistin-induced nephrotoxicity have limitations that they could not apply the candidate drugs on clinical studies based on human subjects, the results of this study using human kidney organoid models might be the bridging the gap between pre-clinical and clinical trial of drug investigation.

5. Conclusion

In conclusion, we have shown that the nephrotoxic effects of colistin, which involve the induction of oxidative stress and apoptosis, are ameliorated by bimatoprost. Therefore, the administration of bimatoprost alongside colistin might ameliorate the renal damage induced by the latter. Further studies are warranted regarding drug reformulation to prepare bimatoprost for safe and effective systemic use.

Fig. 6.Immunofluorescence microscopy image of Colistin induced nephrotoxicity, with or without Bimatoprost treatment. A) Immunostaining of Kim-1. Merged images of LTL (Lotus tetragonolobus lectin) for proximal tubules (green), Kim-1 (red), and Hoecsht 33342 (blue). (Scale bar=250µm) B) Immunostaining of NGAL.

Merged images of LTL (Lotus tetragonolobus lectin) for proximal tubules (green), NGAL (red), and Hoecsht 33342 (blue). (Scale bar=250µm). C) Quantification of fluorescence intensity of KIM-1. D) Quantification of fluorescence intensity of NGAL. All experiments were duplicated for three times. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparison test. *, p<0.05, ***, P<0.001.

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Funding

This work was supported by the Technology Innovation Program (or Industrial Strategic Technology Development Program-3D-TissueChip Based Drug Discovery Platform Program) (20009773, Commercializa- tion of 3D Multifunction Tissue Mimetics Based Drug Evaluation Plat- form) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea); and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2018R1D1A1B07045947).

※ MSIT: Ministry of Science and ICT and National Research Foundation (NRF) of Korea grant funded by the Korean government (MSIT) (No.

NRF-2018R1D1A1A02047589).

CRediT authorship contribution statement

Jongman Yoo and Hye Yun Jeong devised the conceptual ideas.

Jongman Yoo, Lina Joo and Hye Yun Jeong contributed to research design. Dong Hyuck Bae, Joo Hyun Jee, Seong Gyeong Jeon and Woo Hee Choi performed in vivo and in vitro studies. Hye-Youn Kim and Heon Yung Gee performed tubuloid and organoid studies. Lina Joo, Hye Yun Jeong, Dong Hyuck Bae, Yang Dong-Ho and Sejoong Kim analyzed data. Jongman Yoo, Heon Yung Gee, Yang Dong-Ho, Sejoong Kim and Yun-Gil Roh contributed to discussion. Lina Joo and Hye Yun Jeong wrote the article. All authors approved the final version of the article.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

All data are included in the manuscript and/or supporting materials.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2023.115446.

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