Potential Efficacy of Proteasome Inhibitor, Delanzomib, for the Treatment of Renal Fibrosis

Abstract

Renal fibrosis is scarring and tissue hardening caused by the excess deposition of extracellular matrix proteins in response to chronic inflammation. Renal fibrosis is the primary cause of a progressive loss of renal function, and is an important therapeutic target because it ultimately leads to end-stage renal failure, which can be treated only by either dialysis or kidney transplantation. There is no effective treatment that specifically targets renal fibrosis. Myofibroblasts are known to evade apoptosis by activating molecular mechanisms in response to pro-survival biomechanical and growth factor signals from the fibrotic microenvironment. In this study, we screened and selected compounds that selectively cause cell death in myofibroblasts in vitro and studied their possible potency against renal fibrosis in a mouse model. Several proteasome inhibitors induced selective cell death in myofibroblasts differentiated from the human fibroblast cell line (MRC5). The in vivo antifibrotic effect of Delanzomib (Dz), one of the proteasome inhibitors most sensitive to myofibroblasts in vitro, was investigated in a Unilateral Ureteric Obstruction (UUO) mouse model. Treatment with Dz decreased the expression levels of the actin-alpha-2 (ACTA2) and collagen-type-1-alpha-1 (COL1A1) genes in the kidney, which are common fibrosis markers. These results suggest that Dz might be a compound that suppresses renal fibrosis by inducing selective cell death of myofibroblasts, although further investigation is required.

INTRODUCTION

Chronic kidney disease (CKD) is recognized as a leading public health problem worldwide. CKD is identified as kidney damage or a glomerular filtration rate (GFR) lower than 60 mL/min/1.73 m² for three months or more.¹⁾ The prevalence of CKD continues to increase, and the current estimated global prevalence is 13.4% (11.7–15.1%).²⁾ There is no effective treatment for CKD, and there are only treatments that can slow the progression of the disease. Therefore, as the disease progresses, the kidneys no longer function, a condition that is known as end-stage kidney disease (ESKD); either dialysis or a kidney transplant is needed for survival. It is estimated that between 4.902 and 7.083 million people with ESKD need renal replacement therapy,²⁾ and the cost of treatment for CKD is enormous worldwide. Progressive CKD is associated with several complications that interact with higher prevalence and intensity as kidney function reaches lower levels.^3–5) Thus, developing a treatment for CKD is an urgent issue.

The progression of CKD is characterized by fibrosis, which is independent of the underlying disease and involves the deposition of extracellular matrix proteins in interstitial spaces.^6–8) Myofibroblasts are differentiated from fibroblasts around an injury site when the tissue is injured⁹⁾ and are characterized by the expression of α-smooth muscle actin (α-SMA).¹⁰⁾ Myofibroblasts typically disappear via apoptosis after wound closure.¹¹⁾ In fibrotic tissues, on the other hand, myofibroblasts are resistant to apoptosis in response to stimuli from the fibrotic microenvironment¹²⁾ and continue to produce extracellular matrix proteins.¹³⁾

Myofibroblasts are critical players in fibrosis, which makes them a primary therapeutic target.¹⁴⁾ The transforming growth factor beta (TGF-β) signaling pathway is one of the critical signaling pathways in myofibroblast differentiation and activation.^15,16) Neutralizing antibodies and small molecule inhibitors have been developed to target TGF-β signaling, but those studies have failed to demonstrate therapeutic efficacy on fibrotic kidney disease.¹⁷⁾ Alternative strategies have been proposed to selectively remove myofibroblasts while causing less damage to other normal cells. These studies have shown that the induction of apoptosis in myofibroblasts ameliorates fibrosis.^18,19) Thus, selective inhibition of myofibroblast survival could be an effective strategy to ameliorate fibrosis.

In the present study, we screened for compounds that could selectively inhibit myofibroblast survival compared with normal fibroblasts. We found that some proteasome inhibitors are highly sensitive to myofibroblasts. Furthermore, we used Delanzomib to study the possible potency of in vivo renal fibrosis, because it is one of the proteasome inhibitors most sensitive to myofibroblasts in vitro. The results suggest that Delanzomib may be a compound that might be used to suppress renal fibrosis by inhibiting myofibroblast survival, although further investigation is required.

MATERIALS AND METHODS

Compounds

A compound library consisting of 806 entries with identified molecular targets was selected. Some (36) of the 806 compounds inhibited TGF-β1-induced myofibroblast survival by more than 50% at a concentration of 10 µM, and these are summarized in Table 1.

Table 1. IC₅₀ Values of Each Compound against Fibroblasts and the Myofibroblasts Derived from MRC5

Compound	IC50 (µM)
	Fibroblast	Myofibroblast
Delanzomib	13.1	0.2
ABT-737	60.8	3.5
Ixazomib	1.8	0.2
Bortezomib	0.5	0.1
DCC-2036	8.3	3.8
BI 2536	7.3	3.5
CC-223	9.7	4.9
KU-0063794	4.4	2.5
A-1331852	3.0	1.7
AS1517499	6.1	3.7
Triclosan	6.3	4.4
GZD824	6.1	4.4
NMS-873	5.5	4.0
CB-5083	1.7	1.3
Niclosamide	5.7	4.6
ONX-0914	1.2	1.0
Fingolimod	3.5	3.0
MK-886	10.5	9.9
Mitoxantrone hydrochloride	8.4	8.1
Ivacaftor	5.5	5.5
Navitoclax	3.5	3.5
PF-3758309	8.5	9.4
TAK-901	4.6	5.4
JTC-801	3.9	4.7
Afuresertib	3.1	3.8
Tegaserod	3.6	4.6
Cinacalcet	3.8	5.0
SB 271046	6.9	9.4
WYE-125132	4.9	6.7
REF-000598	5.4	7.3
UNC2025	1.9	2.7
CHIR-124	6.8	9.8
ALK-IN-1	3.4	5.2
PF-03814735	5.5	8.3
Adomeglivant	1.7	2.7
BMS-754807	1.8	4.7

IC₅₀ values for both myofibroblasts and fibroblasts for the 36 compounds that inhibited cell viability of MRC5-derived myofibroblasts by more than 50% at a concentration of 10 µM are shown.

Cell Culture

MRC5 (normal human lung fibroblasts) was purchased from the Japanese Collection of Research Bioresources (JCRB) (Osaka, Japan), and cultured in Minimum Essential Media (MEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. NRK49F (normal rat renal fibroblasts) was also obtained from JCRB, but was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% FBS, 1% penicillin/streptomycin, and 1% MEM Non-Essential Amino Acids Solution at 37 °C in a humidified 5% CO₂ atmosphere.

Western Blot

Total protein was extracted from the cells using 200 µL of M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, MA, U.S.A.). The amounts of extracted proteins were quantitatively determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) after centrifugation at 24250 (× g) for 20 min at 4 °C. The extracted proteins were denatured at 100 °C for 5 min in 4 × sodium dodecyl sulfate (SDS) Sample Buffer (Merck, Darmstadt, Germany), separated by electrophoresis on 10% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, CA, U.S.A.), and electrotransferred onto 0.45-µm polyvinylidene difluoride membranes (Merck). Then, the membranes were blocked overnight at 4 °C in TBST (1 × Tris-buffered saline (TBS) containing 0.05% Triton X-100 and 5% bovine serum albumin (BSA)). The blocked membrane was incubated with primary antibodies that were either rabbit monoclonal anti-β−actin antibody (4970S, Cell Signaling Technology, MA, U.S.A.) at a dilution of 1 : 1000 or mouse monoclonal anti αSMA antibody (ab7817, Abcam, Cambridge, U.K.) at a dilution of 1 : 300 at room temperature (r.t.) for another 1 h. After being washed in TBST, the membranes were incubated with either anti-mouse immunoglobulin G (IgG), HRP-linked antibody (Cell Signaling Technology) at a dilution of 1 : 1000 or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology) at a dilution of 1 : 3000 for 30 min at r.t. The membranes were then washed again with TBST. Finally, the protein bands were visualized using a SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). The optical density of the protein bands was read using a LAS-4000 Imaging System (GE Healthcare, IL, U.S.A.). Quantification of signal density was analyzed using ImageQuant TL Software (GE Healthcare).

Immunofluorescence Staining for αSMA in the Cells

MRC5 was plated at a density of 3  ×  10³ cells per each well in 96-well plates. After 24 h of incubation without FBS, the cells were treated for 72 h with TGF-β1 (1, 3 or 10 ng/mL) or a vehicle. Then, the cells were washed with phosphate buffered saline (PBS) and fixed with 4% PFA in PBS for 15 min at r.t. The cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at r.t. Subsequently, the cells were washed and incubated for 1 h at r.t. in PBS containing 5% normal goat serum (Abcam). The cells were washed and incubated overnight at 4 °C with rabbit polyclonal anti-αSMA antibody (ab5694, Abcam), and diluted 1∶200 in PBS containing 1% normal goat serum. After washing with PBS, the cells were incubated for one hour at r.t. with goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (ab150077, Abcam), diluted 1∶1000, and Hoechst 33342 (10 mg/mL) (Thermo Fisher Scientific), diluted 1∶2000 in PBS containing 1% normal goat serum. Individual cells were acquired using the CellVoyager CV6000 (Yokogawa, Tokyo, Japan).

Cytotoxicity Assay

The viability of MRC5 and NRK49F was determined using either the “Cell” ATP Assay reagent (Toyo Bnet, Tokyo, Japan) or CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, WY, U.S.A.) according to the instructions recommended by the supplier. Either MRC5 cells or NRK49F cells were plated at a density of 5  ×  10³ cells per each well in 96-well plates, incubated for 24 h without FBS, and then exposed to TGF-β1 (1 ng/mL) for 72 h, as described above. Dimethyl sulfoxide (DMSO) or Staurosporine (STS) was added to each compound, and the cells were further incubated at 37 °C for 48 h. After adding the same volume of ATP assay reagent or CellTiter-Glo^® Reagent as the cell medium (Promega), the plates were shaken. The amount of ATP extracted from the cells was quantified via the firefly luminescence method. The luminescence intensity was measured using an ARVO X Light (PerkinElmer, Inc., MA, U.S.A.). STS was used as a positive control in the cytotoxicity assay to determine the maximum cytotoxicity. The cytotoxicity rate of the test compound was calculated using the following formula.

  

• X: the cytotoxicity rate of the test compound (%)
• S: the luminescence intensity of the positive control (STS) sample
• T: the luminescence intensity of the test sample
• D: the luminescence intensity of the negative control (DMSO) sample

Animal Model of Renal Interstitial Fibrosis

C57BL/6J male mice (aged 9 weeks; Charles River Laboratories, Kanagawa, Japan) were used in this study. This animal study was performed in accordance with the Guidelines for the Animal Care and Use of Otsuka Pharmaceutical Co., Ltd. The study protocol was approved by the Institutional Animal Care and Use Committee of the Otsuka Pharmaceutical Co., Ltd. (Permit No. 21-0036). All experiments were conducted with respect to the welfare of the animals to minimize their suffering, as follows: Animals were treated carefully by experts who had undergone education and training on how to maintain a healthy environment and reduce distress. The Unilateral Ureteral Obstruction (UUO) model was employed to induce renal fibrosis with the left ureter ligated.²⁰⁾ Briefly, surgery was performed under a combination anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol). The left ureter was visualized following a flank incision and either ligated using a 5.0 silk suture at two points along its length (UUO-operated animals) or similarly manipulated without ligation (sham-operated animals). Delanzomib (Dz) (MedChemExpress, NJ, U.S.A.) was administered orally at 3 or 10 mg/kg once daily for one week beginning the day after surgery. The kidneys were collected, rinsed with isotonic saline, and cut in half. One was stored at −80 °C for the measurement of hydroxyproline. The other was immersed in RNA later (Thermo Fisher Scientific) overnight at 4 °C and used for gene expression analysis as described below.

Gene Expression Analysis

For the cells, total RNA was extracted using an RNeasy Mini kit (Qiagen, Venlo, Netherlands). Reverse transcription was performed using a QuantiTect Reverse Transcription Kit (Qiagen). For the tissues, total RNA was extracted from the kidney homogenate using a Maxwell^® RSC simplyRNA Tissue Kit (Promega) according to the instructions. Reverse transcription was performed using a PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa Bio, Shiga, Japan).

Real-time PCR was performed for gene expression analysis using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) for 40 cycles, which consisted of denaturation for 3 s at 95 °C and annealing with extension for 30 s at 60 °C. At the end of the 40 cycles, samples were heated to 95 °C to verify that a single PCR product was obtained during amplification. The threshold cycle (Ct) was calculated using the instrument’s software (7500 Fast System SDS Software). Analysis of the relative mRNA expression was performed using the ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin beta (ACTB) (Thermo Fisher Scientific) served as a reference for normalization. The Primers for gene expression were obtained from TaqMan Gene Expression Assays (Thermo Fisher Scientific).

Measurement of Hydroxyproline

The frozen renal tissue was added to 0.5 mL of PBS (−) and homogenized (2000 rpm for 30 s × 4) at r.t. using a multi-bead shocker with zirconia beads (Yasui Kikai, Osaka, Japan). An aliquot of the homogenate (200 µL) was incubated at 120 °C for approximately 20 h with an equal volume of 12 N HCl. The samples were centrifuged (22480 × g, r.t., 10 min). The supernatant (5 µL) was applied to 96-well plates. Stepwise diluted hydroxyproline standard cis-4-Hydroxy-L-proline solutions (5 µL) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were also applied to the 96-well plates in the same manner. Then, 100 µL of chloramine T reagent (62 mM chloramine T, 10% N-propanol in citric/acetic acid buffer) was added to each well and reacted for 20 min at r.t. Then, 100 µL of Ehrlich’s reagent (2.5 g p-dimethylaminobenzaldehyde in 9.3 mL N-propanol, 3.9 mL 70% perchloric acid) was added to each well and further incubated at 60 °C for about 30 min. The absorbance was measured at 560 nm using a SpectraMax^® iD5 (Molecular Devices, CA, U.S.A.). A calibration curve was prepared from the absorbance of the standard solutions, and the concentration of hydroxyproline in the samples was calculated. A portion of the same kidney homogenate was used to determine the protein concentration using the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific).

Statistical Analysis

Data are presented as the mean ± standard deviation (S.D.). The differences between two groups and among more than two groups were analyzed using an unpaired t-test and Dunnett’s test. Synergistic effects were evaluated with the p-value of the interaction effect analyzed by two-way ANOVA. The differences were considered significant at 5% in the two-tailed test. SAS software was used for all statistical analyses.

RESULTS

Induction of Myofibroblasts by TGF-β1 in MRC5 Fibroblasts

Myofibroblasts are characterized by the expression of αSMA encoded by ACTA2 and produce extracellular matrix components such as type 1 collagen (COL1A1).^21–23) Without TGF-β1, MRC5 (human lung fibroblast) showed almost no expression of αSMA. Treatment with TGF-β1 (1, 3, or 10 ng/mL) increased the expression level of αSMA in MRC5 (Supplementary Fig. 1). Even at lower concentrations (1 ng/mL), TGF-β1 markedly induced the expression of αSMA. In the absence of TGF-β1, Supplementary Fig. 1B shows that MRC5 manifested the typical morphology of cultured fibroblasts with a dendritic shape. By contrast, MRC5 treated with TGF-β1 showed more flattened shapes with larger cell sizes and expressed α-SMA stress fibers that crossed into the cytoplasm. In addition, treatment with TGF-β1 (1 ng/mL) significantly increased gene expression of the myofibroblast marker ACTA2 and the fibrosis marker COL1A1.²³⁾ These findings indicate that treatment with TGF-β1 (1 ng/mL, 72 h) induces a myofibroblast differentiation of MRC5 in vitro. Thus, in subsequent studies, MRC5 treated with 1 ng/mL of TGF-β1 for 72 h was used as myofibroblasts.

Higher Sensitivity of TGF-β1-Induced Myofibroblasts to Proteasome Inhibitors Compared with Fibroblasts

To search for the compounds that inhibit the survival of TGF-β1-induced myofibroblasts, cell viability was assayed following incubation in the presence of each of the 806 in-house compounds. As shown in Table 1, a small portion (36) of the 806 compounds inhibited the induced survival of myofibroblasts by more than 50% at a concentration of 10 µM. In each compound, the IC₅₀ value for both the induced myofibroblasts and fibroblasts were also summarized (Table 1, Supplementary Fig. 2). Among these compounds, 20 compounds had higher IC₅₀ values in the myofibroblasts compared with that in the fibroblasts. This indicates that 20 compounds exerted selective survival inhibitory effects on myofibroblasts. Accordingly, their selectivity was compared using the ratio of IC₅₀ values in the myofibroblasts to those in the fibroblasts as an index. Seven compounds exhibited a higher ratio (> 2). Three of the seven compounds, Dz, Ixazomib (Iz), and Bortezomib (Bz), were proteasome inhibitors. The effects that these selected proteasome inhibitors—along with that of a proteasome inhibitor, marizomib (Mz) (not included in the library)—exert on the viability of TGF-β1-induced myofibroblasts were further studied (Fig. 1). The IC₅₀-based selectivities of Dz, Mz, Iz, and Bz in the myofibroblasts from MRC5 were 146, 17, 18, and 10-fold higher than in the fibroblasts, respectively. On the other hand, the selectivities of Dz, Mz, Iz, and Bz in the myofibroblasts were low in NRK49F and 6, 2, 9, and 3-fold higher than in the fibroblasts, respectively. Dz was subjected to the next in vivo experiment since it showed more than one order of magnitude higher selectivity for MRC5-derived myofibroblasts than for other compounds.

Fig. 1. Sensitivity of TGF-β1-Induced Myofibroblasts (MF) to Proteasome Impairments

Fibroblasts (FB) (MRC5 and NRK49F) were incubated with different concentrations of Delanzomib (Dz), Marizomib (Mz), Ixazomib (Iz), and Bortezomib (Bz) for 48 h. Myofibroblasts (MF), derived from MRC5 and NRK49F, as shown in Fig. 1, were also incubated with different concentrations of Dz, Mz, Iz, and Bz for 48 h. After the incubation, the cell viability of FB or MF was determined. Data represent the means ± S.D. (N = 3).

Inhibition of Gene Markers Related to Renal Fibrosis by Dz in UUO Kidneys

The effect of Dz, which showed selective and strong cytotoxicity for myofibroblasts derived from both MRC5 and NRK49F (Fig. 1), was examined using the UUO mouse model.²⁰⁾ The UUO model was validated by evaluating an increase in the expression levels of myofibroblast marker gene ACTA2, fibrosis marker gene COL1A1, and hydroxyproline level. In the mouse UUO model, the daily oral administration of Dz (10 mg/kg) for one week tended to suppress increases in the gene expression levels of a myofibroblast marker ACTA2, and significantly suppressed increases in the fibrosis marker COL1A1 (Fig. 2A). However, the daily oral treatment did not suppress increases in the level of hydroxyproline, a marker of fibrosis progression, in the range of Dz dosages we used in this study (Fig. 2B). The results of gene expression analysis might suggest that Dz treatment decreases the number of myofibroblasts and thereby inhibits fibrosis if the tissue concentration of Dz in the kidney might be increased with kidney-selective delivery methods.

Fig. 2. Effects of Treatments with Delanzomib on Gene Expression Levels of ACTA2 and COL1A1 in UUO-Induced Renal Fibrosis

A: Relative gene expression levels of ACTA2 and COL1A1 in UUO kidneys treated with either 3 or 10 mg/kg of Dz once daily for one week. Target gene expression was normalized to GAPDH. Data represent the mean of fold-gene expressions relative to a sham group (means ± S.D. (N = 4–12)). B: Amount of hydroxyproline in UUO kidneys treated with either 3 or 10 mg/kg of Dz once daily for one week. Data represent the means ± S.D. (N = 4–12). Data were analyzed via Two-Way-ANOVA, followed by a Dunnett’s test. ** p < 0.01 versus sham group; ⁺⁺ p < 0.01 versus ipsilateral kidney in the mice UUO model.

DISCUSSION

This study demonstrated that Dz induces the selective in vitro cell death of TGFβ1-induced myofibroblasts, which are critical for fibrosis promotion (Fig. 1). In addition, treatment with Dz tended to suppress the elevation of ACTA2 gene expression levels, a myofibroblast marker, and significantly suppressed the elevation of COL1A1 gene expression levels, a fibrosis marker, in the mouse UUO model (Fig. 2). These results suggest that Dz is a potential candidate for treating renal fibrosis. Proteasome inhibitors including Dz are known to induce apoptosis in cancer cells. Dz has been reported to induce apoptosis of hepatocellular carcinoma cells through the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eIF2α/ATF4/CHOP signaling of endoplasmic reticulum stress.²⁴⁾ Zhu et al. reported that proteasome inhibitor MG132 also induces apoptosis in TGFβ1-induced myofibroblasts.²⁵⁾ These factors imply that proteasome inhibitors such as Dz might be promising drug candidates for treating renal fibrosis by causing cell death in myofibroblasts.

The mouse UUO model generates progressive renal fibrosis.²⁶⁾ In this study, we applied this model to investigate the potential efficacy of Dz, which was selected by in vitro screening, for the treatment of progressive fibrosis in vivo. As shown in Fig. 2, although Dz suppressed the increased UUO-induced expression of fibrosis-related genes, it did not suppress the levels of hydroxyproline, a marker protein that confirms sufficient symptomatic improvement. Another group has reported that the effects of Bz, another of the proteasome inhibitors, on the UUO model were weaker than those on an aristolochic acid nephropathy (AAN) model.²⁷⁾ This led to a concern that the UUO-induced kidney pathological changes might be too severe. Other than the UUO-model, other models are available: the AAN model, the ischemia-reperfusion injury (IRI) model,²⁸⁾ and the Adriamycin model.²⁹⁾ Investigations to evaluate the in vivo efficacy of Dz in other pathological models with various levels of fibrosis are planned.

Based on the results shown in Table 1, three compounds (Dz, Iz, and Bz) were selected, because they showed higher selectivity to MRC5-derived myofibroblasts in vitro. Dz is known to reversibly inhibit standard proteasomes (β1, β2, β5).³⁰⁾ Iz and Bz reversibly inhibit mainly the β5 proteasome subunit.^31–33) By contrast, Mz irreversibly inhibits standard proteasomes (β1, β2, β5).³³⁾ Three compounds (Dz, Iz, and Bz) have a boronic acid structure. In contrast, Mz has no boronic acid structure. Although Mz was not in the compound list, we added Mz to see whether there are any differences depending on the structure. As shown in Fig. 1, sensitivity to the TGF-β1-induced myofibroblasts from either human lung fibroblast cells (MRC5) or rat kidney fibroblast cells (NRK49F) was investigated. Both Dz and Iz showed a higher level of selectivity for TGF-β1-induced myofibroblasts from NRK49F. All four compounds showed higher selectivity to the TGF-β1-induced myofibroblasts from MRC5. In this study, only Dz showed higher sensitivities to both TGF-β1-induced myofibroblasts. Therefore, in this study, Dz was selected for further in vivo study.

In addition to proteasome inhibitors, throughout our in vitro screening study ABT-737 was the other top-hit compound (Table 1). ABT-263 is a BH3 mimetic similar to ABT-737, which selectively induces apoptosis in myofibroblasts and suppresses dermal fibrosis.¹⁹⁾ This could confirm that the in vitro assay we constructed in this study is a suitable method for screening compounds that selectively induce cell death in myofibroblasts, which is a significant cause of fibrosis.

Some antifibrotic agents have been used or studied in clinical settings,³⁴⁾ but most have shown a low level of bioavailability and are not specific to kidneys, which results in a need to increase the dosage during treatment in order to achieve a positive therapeutic effect. Multiple dosing, however, strongly limits their availability due to severe side effects.^35,36) The results of the current study also suggest that Dz would inhibit the increase in the level of hydroxyproline, which is a marker of fibrosis progression, if its tissue concentration in the kidney could be increased with kidney-selective delivery methods. Many drug delivery systems have been introduced to achieve the selective delivery of compounds to kidneys.^37–39) For example, nanoparticles (NPs) coated with low molecular-weight chitosan could be accumulated selectively in kidneys.⁴⁰⁾ Also, glutathione (GSH)-modified gold nanoparticles are known to explicitly accumulate in the kidneys, particularly in the renal proximal tubules.⁴¹⁾ Despite their specificity to kidneys, these systems target renal tubular epithelial cells rather than myofibroblasts, which directly contribute to the fibrosis process. In combination with myofibroblast-targeted delivery systems,^35,42) a proteasome inhibitor such as Dz might be a useful drug for renal fibrosis via inducing the cell death of myofibroblasts, which are critical for fibrosis promotion. Accordingly, we reached the conclusion that Dz, a proteasome inhibitor, might be a potential candidate for the treatment of kidney fibrosis.

Acknowledgments

The authors would like to thank Dr. Ikuo Murakami, Haruka Takata, and Yoshino Kawaguchi for their helpful advice. The authors are grateful to James L. McDonald for his helpful advice in developing the English manuscript.

Conflict of Interest

A.S-A., K.H., G.N. and Y.Y. are employees of the Otsuka Pharmaceutical Co., Ltd. The authors report no other conflicts of interest in this work.

Supplementary Materials

This article contains supplementary materials.

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