Animal experiments
The FVB mice (5–6 weeks old; Orient Bio, Gyeonggi-Do, Korea) were housed under specific pathogen-free conditions in a temperature-controlled environment with a 12-hour light/dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Yonsei University College of Medicine, Seoul, Korea (IACUC approval number: 2021 − 0218). Animal research throughout the current study was completed and reported in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. For the initial unilateral IRI and second-stage contralateral nephrectomy surgery, the animals were anesthetized by inhalation of 1–3% isoflurane, and their body temperature was regulated during surgery using a heated thermoplate (Jeung Do Bio & Plant Co, Korea). On day 0, unilateral IRI was induced by clamping the left kidney pedicle with an atraumatic vascular clamp (Fine Science Tools, Foster City, CA, USA) for 42 min, while the right kidney was kept intact. After 2 weeks, the right kidneys were removed under the same anesthesia and surgical protocols. The mice that underwent uIRIx were randomly assigned to either the control or rosuvastatin group. They were orally administered the vehicle (0.5% methylcellulose) or rosuvastatin (10 mg/kg) daily for four weeks. The kidneys were collected at the end of the experiments, fixed in a 4% paraformaldehyde solution, and embedded in paraffin, as previously described.43 Blood samples were collected through cardiac puncture, and serum creatinine concentrations were determined using a Fuji Dri-Chem 4000i system (Fujifilm, Japan). The urinary albumin concentration was measured using an ELISA kit (catalog no. ab-108792, Abcam, Cambridge, MA, USA), while the urinary creatinine concentration was measured using a creatinine colorimetric kit (catalog no. 500701, Cayman Chemical, Ann Arbor, MI, USA). The urine albumin-to-creatinine ratio was calculated as the urinary albumin concentration divided by the urinary creatinine concentration. Blood pressure was measured using a computerized non-invasive tail cuff system (BP-2000 Blood Pressure Analysis System; Visitech Systems, NC, USA). To minimize induced stress, the animals were accustomed to the instrument for four consecutive days prior to definitive measurements. This consisted of a minimum of 10 repeated measurements per mouse.
Kidney tissue pathological analysis
To analyze kidney fibrosis, kidney sections were stained with Masson’s trichrome and Sirius red. The areas positive for Masson’s trichrome and Sirius red were evaluated relative to the unit area using MetaMorph image analysis software (Molecular Devices, CA, USA). Immunohistochemistry for α-SMA (R&D Systems, Minneapolis, MN, USA) and F4/80 (Abcam, Cambridge, MA, USA) was performed to identify α-SMA-positive myofibroblasts and F4/80-positive macrophages. The quantification of these cells was based on the number of cells per high power field. Microscopic assessment was performed in a blinded manner, with 20 randomly selected fields from each slide section examined at 400 × magnification, as previously described.44
MDCK cell culture, RNA knockdown of HOXA13, and rosuvastatin treatment
The MDCK cells were obtained from the Korean Cell Line Bank (Seoul, South Korea) and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 100 U/ml penicillin G, and 100 mg/ml streptomycin. The cells were incubated at 37°C in a humidified environment with 5% CO2. To achieve HOXA13 knockdown, RNA interference (RNAi) was utilized as detailed previously.25 Specifically, sub-confluent MDCK cells were transfected with either Stealth RNAi Negative Control Med GC (Invitrogen, Carlsbad, CA, USA) or predesigned small interfering RNA targeting canine HOXA13 using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). The small interfering RNA sequences targeting HOXA13 were: sense 5′-UUUGUUUGUAGCAUAUUCCCGUUCG-3′ and antisense 5′-CGAACGGGAAUAUGCUACAAACAAA-3′. Post-transfection, cells were cultured in serum-free medium for 24 hours, then treated with recombinant human TGF-β1 (5 ng/ml, R&D Systems, Minneapolis, MN, USA), with or without rosuvastatin (10 µM), in 1% FBS-containing medium for an additional 48 hours.
Immunofluorescent staining of MDCK cells
The treated MDCK cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and immediately fixed with 4% formaldehyde diluted in PBS for 15 min at room temperature. This was followed by permeabilization with 0.2% Triton X-100 for 10 min. The cells were subsequently blocked with 1% bovine serum albumin for 30 min at room temperature. After blocking, the cells were incubated with corresponding antibodies overnight at 4°C, and rhodamine-conjugated antibodies against rabbit IgG were used to detect primary antibodies. The immunostained cells were visualized using an LSM-780 META confocal microscope (Carl Zeiss, Oberkochen, Germany) at 488 nm for GFP and 647 nm for rhodamine. Fluorescence intensity was assessed by examining at least five fields per section at 400 × magnification using MetaMorph digital image analysis.
Quantitative RT-PCR analysis for USAG-1, BMP-7, HOXA13, TGF-β1 and collagen 1
Total RNA was isolated from kidneys or MDCK cells with TRIzol reagent (Invitrogen, Life Technologies, CA, USA) following the manufacturer's instructions, and then 2µg total RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Bedford, MA, USA). The cDNA was then amplified in the ABI 7500 sequence detection system (Applied Biosystems, Bedford, MA, USA) using Power SYBR® Green PCR Master Mix (Applied Biosystems, Bedford, MA, USA) with the following cycling conditions: 40 cycles of 95°C for 5 sec, 58°C for 10 sec, and 72°C for 20 sec. The sequences of primers were as follows: canine TGFβ-1 (forward: 5′- TGGACACGCAGTACAGCAA-3′, reverse: 5′- TAGTACACGATGGGCAGTGG-3′), canine collagen 1 (forward: 5′-GTGTGTACAGAACGGCCTCA-3′, reverse: 5′-TCGCAAATCACGTCATCG-3′), canine USAG-1 (forward: 5′-GGAGGCAGGCATTTCAGTAA-3′, reverse: 5′-TGTACTTGGTGGAACGCAGT-3′), canine BMP-7 (forward: 5′-CTGGTGTTCGACATCACAGC-3′, reverse: 5′-CATGAAGGGCTGCTTGTTCT-3′), canine HOXA13 (forward: 5′-GGGAGAAAGAAGCGCGTC-3′, reverse: 5′-CGTCGTGGCTGATATCCG-3′), and canine glyceraldehyde-3-phosphate dehydrogenase (GADPH) (forward: 5′-TTCCACGGCACAGTCAAG-3′, reverse: 5′-ACTCAGCACCAGCATCAC-3′). Target gene expression was normalized to that of GADPH and quantitative analyses were conducted using the ΔΔcycle threshold method and StepOne Software version 2.2.2.
Western blot analysis for fibrosis markers, USAG-1/BMP-7, and Smad signaling molecules
Total cell lysates were prepared by lysis of mouse kidney tissues or MDCK cells with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA) and the protein contents were measured using the Bradford assay (Bio-Rad, 500–0006). Equivalent amounts of each protein extract were heat denatured in 5 × sample buffer (2% sodium dodecyl sulfate, 62.5mM Tris, pH 6.8, 0.01% bromophenol blue, 1.43mM mercaptoethanol, and 0.1% glycerol), separated on 12% polyacrylamide gels, and electrophoretically transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking, membranes were treated with the appropriate antibodies, including E-cadherin (1:1000; BD, Franklin Lakes, NJ, USA), fibronectin (1:5000; Santa Cruz, Dallas, TX, USA), α-SMA (1:10000; R&D, Minneapolis, MN, USA), collagen 1 (1:1000; Santa Cruz, Dallas, TX, USA), vimentin (1:5000, Abcam, Cambridge, MA, USA), BMP-7 (1:1000, Abcam, Cambridge, MA, USA), USAG-1 (1:1000, Thermo Fisher Scientific, Waltham, MA, USA), Smad3, pSmad3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), Smad1/5/9, pSmad1/5/9 (1:1000, Cell Signaling Technology, Danvers, MA, USA), α-tubulin (1:10000, Abcam, Cambridge, MA, USA), and actin (1:10000, Santa Cruz, Dallas, TX, USA). Immunostaining was performed using chemiluminescent reagents (SuperSignal West Pico Luminol/Enhancer solution; Thermo Fisher Scientific, Waltham, MA, USA) and Agfa medical X-ray film, and the band density was measured using NIH Image J software.
Statistical analysis
Data are expressed as mean ± standard deviation. Statistical differences among the groups were determined using the Mann–Whitney U test. Statistical analyses were performed using SPSS for Windows version 25.0 (SPSS Inc., Chicago, IL, USA). Results of comparisons with P-values < 0.05 were considered statistically significant.