Male C57BL/6 mice were purchased from the Central Laboratory for Experimental Animals, Inc. (Osaka, Japan) and housed in a temperature (22±2°C) and humidity (40±20%)-controlled room under a 12 h light-dark cycle. The mice were given free access to tap water and standard laboratory chow (MF; Oriental Yeast Co., Ltd., Tokyo, Japan). All animals were cared for in accordance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals 8th Edition (National Institutes of Health, Bethesda, MD, USA), and the present study was approved by the ethics committee of the Kawasaki Medical School (Kurashiki, Japan; approval no. 13-051).

Male mice (age, 8 weeks; weight, 23–28 g) were randomly divided into two groups, vehicle-treated (n=8) and colchicine-treated (n=14). Colchicine (0.5 mg/kg/day; cat. no. C9754; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was diluted into saline and administered by ALZET^® osmotic pumps (model no. 1004; Durect Corporation, Cupertino, CA, USA) over 3 weeks, which were implanted under the skin of the dorsal region. The UUO procedure was conducted 7 days following the pump insertion, by ligation of the left ureter as previously described (18). The right, non-UUO-operated kidneys were used as control samples. A total of 14 days following UUO, the body weight was recorded, the mice were sacrificed, and their kidneys were carefully extracted and weighed. Blood samples were obtained at the time of death for measurement of serum creatinine and serum alanine aminotransferase. The levels of alanine aminotransferase and creatinine were measured by a commercial laboratory at SRL, Inc. (Tokyo, Japan) using an automatic inspection device (DRI-CHEM, FDC-700; Fujifilm Corporation, Tokyo, Japan).

Kidney tissues were fixed in 4% paraformaldehyde overnight at room temperature. Then, tissues were dehydrated in graded alcohols and embedded in paraffin using standard techniques. Kidney sections (4 µm thick) were prepared from paraffin-embedded tissues. The sections were de-waxed, re-hydrated by standard techniques and stained with Masson's trichrome stain. Images were then captured using a BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). For immunohistochemistry, deparaffinized serial sections were rehydrated in PBS and subjected to antigen retrieval by microwave heating (500 W, 15 min). Sections were washed with PBS and incubated in 0.3% hydrogen peroxide (in 1X PBS) to block endogenous peroxidase activity, prior to further incubation with the primary antibodies for 1 h at 37°C in a humidified chamber. Primary antibodies against vimentin (dilution, 1:500; rabbit polyclonal; cat. no. sc-7557R; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and α-smooth muscle actin (α-SMA; dilution, 1:100; rabbit polyclonal; cat. no. ab5694; Abcam, Cambridge, UK) were used and detected using the Histofine Simple Stain MAX-PO kit (Nichirei Corporation, Tokyo, Japan) and 3,3′-diaminobenzidine (Sigma-Aldrich; Merck KGaA). The severity of tubulointerstitial injury was evaluated by examining 10 optical fields in randomly selected tissue samples. Images of scarred areas (stained blue with Masson's trichrome) and areas with positive staining for vimentin and α-SMA (brown) were quantified using a color image analyzer (Win ROOF version 5.6; Mitani Co., Fukui-city, Fukui, Japan). Glomeruli, tubules and blood vessels of the cortex were excluded. Results were presented as a % of the relative volume of the scanned interstitium.

Total RNA extraction and RT-qPCR were performed as described previously (20). Briefly, total RNA was isolated from kidneys using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), followed by digestion with DNase (Sigma-Aldrich; Merck KGaA). cDNA was synthesized from total RNA (1 µg) using Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific, Inc.) with oligo (dT)_12–18 as a primer (Thermo Fisher Scientific, Inc.). Reverse transcription was performed for 50 min at 37°C according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). Primers and probes for TaqMan analysis were designed using sequence information from GenBank (National Institutes of Health) (21) and the Primer3 online software (http://frodo.wi.mit.edu/primer3/; accessed July 1, 2015). Primer and probe sequences are listed in Table I. TaKaRa Premix Ex Taq (Takara Bio, Inc., Otsu, Japan), with a final reaction volume of 20 ml, was used for the TaqMan probe-based RT-PCR reaction, which was performed on a Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following cycling conditions: 2 min at 50°C, 10 min at 95°C, 55 cycles of 15 sec at 95°C and 1 min at 60°C. The level of mRNA expression in each sample was quantified using the absolute quantification standard curve method (22). Plasmid cDNA of each gene was used to prepare absolute standards. The concentration was measured using the A₂₆₀, which was converted to the number of copies using the molecular weight of the DNA. Each mRNA expression levels were normalized to those of the housekeeping 18S ribosomal RNA gene.

NRK-49F cells, a fibroblastic clone of normal rat kidney cells, were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (FBS), 1% non-essential amino acids and 1% penicillin/streptomycin (all Sigma-Aldrich; Merck KGaA). Confluent cells at passages 4–10 were used in subsequent experiments.

Effects of colchicine on cell viability were evaluated using the Premix (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) Cell Proliferation Assay system (cat. no. MK400; Takara Bio, Inc.). LDH levels (an indicator of cell injury) were measured using the LDH Cytotoxicity Detection kit (cat. no. MK401; Takara Bio, Inc.). These assays were conducted according to the manufacturer's protocols. Cells (1×10³ cells) were seeded in 96-well plates and were co-incubated with 100 nM angiotensin II (cat. no. A9525; Sigma-Aldrich; Merck KGaA) and 0–10 nM colchicine (cat. no. C9754; Sigma-Aldrich; Merck KGaA) for 24 h, prior to performing the WST-1 or LDH assays.

RhoA activity was assessed using the RhoA G-LISA Activation Assay Biochem kit (cat. no. BK121; Cytoskeleton, Inc., Denver, CO, USA) according to the manufacturer's protocol. Cells were incubated with 0 to 10 nM colchicine at 37°C for 30 min, followed by treatment with 100 nM angiotensin II. The cells were collected for analysis 12 min post-stimulation. Cells were lysed with lysis buffer containing Tris pH 7.5, MgCl₂, NaCl, nonionic detergent and SDS. Cell lysates were then incubated in a Rho-GTP affinity plate with binding buffer for 30 min at 4°C prior to further incubation with the anti-RhoA primary antibody for 45 min at room temperature. Following plate washing with kit wash buffer, the appropriate secondary antibody was added and incubated at room temperature for 45 min. Following incubation, the plate was washed with kit wash buffer again then the mixed horseradish peroxidase detection reagent was added into each well. The luminescence signals were detected using a microplate luminescence reader (23). All antibodies and reagents used for the RhoA activity assay were provided by the Cytoskeleton, Inc. kit.

Cells were grown to confluence in 10 cm plates, and a sterile 200 µl pipette tip was used to scratch the cell layer. The cells were washed twice with PBS, after which medium supplemented with 100 nM angiotensin II, 0.5% FBS and 0–10 nM colchicine was added. An inverted microscope (IX81N; Olympus Corporation, Tokyo, Japan) was used to assess the cells at 0 and 20 h. Cell migration rates (%) were calculated using the following formula: [(0 h scratch area-20 h scratch area)/0 h scratch area]x100.

Actin fibers were stained with Acti-stain 555 phalloidin (cat. no. PHDH1-A; Cytoskeleton, Inc.). Fibroblasts were cultured as aforementioned prior to the wound-healing assay, and actin staining was performed according to the manufacturer's protocol. Briefly, the culture medium was removed, and the cells were gently washed with PBS prior to fixation with paraformaldehyde (3.7%) for 10 min. The cells were washed again with PBS, permeabilized with 0.5% Triton X-100 for 5 min, washed, and stained with phalloidin for 30 min in the dark at room temperature. Nuclei were stained with Hoechst 33342 (cat. no. H3570; Thermo Fisher Scientific, Inc.), and images of the cells were captured using a BZ-9000 fluorescence microscope (Keyence Corporation).

In vitro experiments were performed in duplicate and independently repeated three times. Data are expressed as the mean ± standard error of the mean. Statistical significance was evaluated using a two-tailed unpaired Student's t-test for comparisons between two groups, or one-way analysis of variance with Tukey-Kramer post hoc tests for comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

No significant renal or liver dysfunction was observed in the UUO-operated animals, as indicated by measurements of serum creatinine and alanine aminotransferase, respectively (Table II). In the UUO kidneys, fibrosis was observed mainly in the interstitium surrounding atrophic or dilated tubules (Fig. 1A). Masson's trichrome staining demonstrated that colchicine treatment significantly suppressed renal interstitial fibrosis compared with the vehicle treatment (Fig. 1A). The number of interstitial cells expressing vimentin, a mesenchymal marker, was significantly increased in the UUO kidneys compared with the control kidneys; however, this effect was attenuated by colchicine treatment (Fig. 1B). Expression of the myofibroblast marker α-SMA was also significantly increased in the UUO kidneys compared with the control kidneys, and this UUO-mediated increase was also attenuated by colchicine treatment (Fig. 1C).

The effects of colchicine treatment on the mRNA expression levels of profibrotic genes fibronectin and α1 type I collagen were examined in the UUO and control kidneys. The results demonstrated that the expression levels of fibronectin and α1 type I collagen were higher in the UUO kidneys compared with the control kidneys (Fig. 2). However, expression of these genes in the UUO kidneys was significantly attenuated by colchicine treatment compared with the vehicle treatment (Fig. 2). In addition, the mRNA expression levels of the epithelial adhesion molecule E-cadherin were decreased in the UUO kidneys compared with the control kidneys; however, its expression was not affected by colchicine treatment (Fig. 2).

The effects of colchicine on normal fibroblast activities were further examined in vitro, by measuring the cell viability and RhoA activity of NRK-49F normal rat kidney fibroblast cells. Angiotensin II, colchicine, or co-stimulation with both, had no significant effects on the viability (Fig. 3A) or death (Fig. 3B) of NRK-49F cells. RhoA activity in NRK-49F cells was significantly increased by angiotensin II stimulation (Fig. 3C); however, colchicine treatment significantly suppressed this angiotensin-II-mediated RhoA activity in a dose-dependent manner (Fig. 3C).

Cells were stimulated with angiotensin II in the presence or absence of colchicine and migration was examined in vitro 20 h after wounding. Colchicine treatment inhibited the NRK-49F cell migration in a concentration-dependent manner (Fig. 4A and B). When cells were stained with fluorescent phalloidin for visualization of actin fibers, formation of filopodia was upregulated in the NRK-49F cells treated with angiotensin II, whereas colchicine treatment suppressed this effect (Fig. 4C).