Reagents.
General reagents were purchased from Wako Pure Chemicals (Osaka, Japan) or Nakalai Tesque (Kyoto, Japan).
Animals.
Seven-week-old male Wistar rats (Clea Japan: Tokyo, Japan), at least n=6/per group were used. Rats were treated with three mixed anaesthetics: domitor (0.15 mg), dolmicum (2 mg), butorphanol (2.5 mg) in saline 1.45 mL) at 0.25 mL per 100 g body weight. Sham group underwent sham operation and 1K group underwent right nephrectomy only. In the 1K-IR group, two weeks after right nephrectomy (1K), for simulating renal ischemia-reperfusion injury, the left renal arteriovenous system was occluded using a clamp for 45 min and then reperfused. The FK-23 and LFK were provided by Nichinichi Pharmaceutical Co Ltd (Mie, Japan). The FK-23 (3 %), LFK (0.3% or 3%) were then fed after nephrectomy ad libitum with mixed pellets of in CE-2 (Clea Japan: Tokyo, Japan), respectively. They were dissected under intraperitoneal administration of mixed anesthetic agents [medetomidine (0.15 mg/0.15 ml/kg), midazolam (2 mg/0.4 ml/kg), butorphanol (2.5 mg/0.5 ml/kg), and saline (1.45 ml/kg)] at 3 or 8 weeks after CKD treatment (12 and 17 weeks old at autopsy). Blood was collected from the inferior vena cava (IVC), and tissues were collected after perfusion with saline via the IVC. The samples (plasma, urine, tissues, and feces) were immediately stored at –80 °C until further analyses. Partly, kidneys, heart and liver were fixed in 10% neutral buffered formalin solution (Wako Pure Chemicals: Osaka, Japan). Animal experiment protocols were approved by the Use of Laboratory Animals Committee of Osaka Metropolitan University (Approval number: 18053). The study of animal experiments was carried out in accordance with the ARRIVE guidelines and national animal experiment guidelines.
Biochemical analysis.
Plasma BUN and creatinine were measured subcontracted to SRL Inc. (Tokyo, Japan). Plasma uremic substances were analyzed by LC-MS system (ExionLC-AD/X500R, SCIEX). Standard reagents were purchased as described below.
Phenylsulfate (PS) and p-cresylsulfate (PCS) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Indoxylsulfate (IDS) and indoxylsulfate 13C6 were purchased from Sigma-Aldrich. PCS-d6 and creatinine (CRE) were purchased from Toronto Research Chemicals. CRE was purchased from Nacalai Tesque (Kyoto, Japan). The samples brought in were diluted 10-fold with isotope-labeled internal standard aqueous solution. PS, PCS, IDS, and CRE were analyzed using an LC-MS system (ExionLC-AD/X500R, SCIEX). The LC column was an InertSustain AQ-C18 HP column (2.1 mm×150 mm, 3 μm; GL Science). A gradient condition consisting of 2 mM ammonium bicarbonate in ultrapure water (A) and methanol (B) was adopted. The gradient, expressed as changes in mobile phase B, was as follows: 0 min, start at 5% B, 0 – 15 min, a linear increase from 5% to 50% B; 15 – 16 min, a linear increase from 50% to 100% B; 16 – 21 min, hold at 100% B; 21–26 min, equilibration at 5% B. The flow rate of the mobile phase was 0.2 mL/min. The high-resolution multi-reaction monitoring (MRMHR) mode was used for the quantitation. The m/z values were shown in Supplemental Table S2. The working parameters for the ESI source were the following. The ion spray voltage was –4500 V (negative-ion). The source temperature was maintained at 300 °C. The settings for the nebulizer and heater gases were 40 psi and 60 psi, respectively.
Histochemical analysis.
The tissues collected were fixed with formaldehyde. They were then embedded in paraffin, cut into thin sections (4 µm), and mounted on silane-coated glass slides. After deparaffinization for immunohistochemistry, the tissue was immersed in Target Retrieval Solution 10X Concentrate (pH 6: S1699) (Dako: Tokyo, Japan) diluted 10-fold with dH₂O and autoclaved at 121 °C for 5 min for antigen activation. After cooling, the samples were rinsed and then stained using VECTASTAINE UNIVERSAL ABC-AP kit (VECTOR, Burlingame, CA). Then the slides were incubated overnight at 4 °C using primary antibody (a-SMA or HNE) diluted in Can Get Signal immunostain solution A (Toyobo, Osaka, Japan). After overnight incubation, the samples were incubated for 30 min with biotin-labeled universal antibody for color development, alkaline phosphatase substrate kit (Vector Laboratories, Inc. Newark, CA) was used and nuclear staining was performed with hematoxylin. Azan staining or Masson trichrome staining was performed at the laboratory of Osaka Metropolitan University.
Preparation of the cytosol stock solution.
Kidney homogenates (20%) were prepared using RIPA Buffer (pH 7.4) with protease inhibitor cocktail tablets (1 tablet/10 mL, Roche: Basel, Switzerland) on ice for 20 s at a speed of 30 krpm using a homogenizer (Microtech Nition: Chiba, Japan). The sample was then centrifuged (Kubota Shoji: Tokyo, Japan) at 4 °C and 14000 rpm for 30 min. The supernatant was collected and used as the cytosol stock solution. Protein concentrations were measured using the BCA protein assay kit (Thermo Fisher Scientific: Waltham, MA).
Western blot.
Cytosol solution was prepared by adding ultrapure water and 6× SDS Buffer (0.35 M Tris-HCl, 10% SDS, 15% 2-mercaptoethanol, 30% glycerol, 0.06% bromophenol blue), and the sample was heated at 95 °C for 5 min.
SDS-PAGE was performed as previously described26. Primary antibodies (Supplemental Table. S3) were diluted in Can get signal solution 1 and then their solutions were applied at 4 °C overnight. After incubation with secondary antibody in Can get signal solution 2 at room temperature for 1 h and extensive washing, blots were placed in Amersham ECL™ Prime Western Blotting Detection Reagent (Cytiva; Tokyo, Japan), allowed to react for 2 min and then imaged with LuminoGraph1 WSE (ATTO, Tokyo, Japan). Blots were measured using CS Analyzer (Ver. 3.0, ATTO) to determine the emission intensity of each band. Band intensities of the same samples were normalized by b-actin.
RNA isolation from the kidney and quantitative RT‑PCR.
Total RNA was isolated from kidney samples using NucleoSpin® RNA (Macherey-Nagel. Düren, Germany) according to the manufacturer’s instructions. cDNA was synthesized by reverse transcriptase PCR (RT-PCR) using a ReverTra Ace qPCR RT kit (Toyobo: Osaka, Japan). Quantitative PCR (qPCR) was performed using gene-specific primers or TaqMan Gene Expression Assays (Thermo Fisher SCIENTIFIC: Waltham, MA, USA) (Supplemental Table S4) and the THUNDERBIRD Probe qPCR Mix (Supplemental Table S5, Toyobo: Osaka, Japan).
PCR was performed using 7500 Fast (Applied Biosystems, Foster City, CA) with the following cycling conditions: denaturation at 95 °C for 15 s and 60 °C for 60 s for 40 cycles. All qPCR targets were quantified based on standard curves ran on the sample plate. The mRNA levels of specific genes were normalized to those of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase of the same sample.
Metagenome analysis of rectal feces samples.
Metagenomic analysis of the bacterial flora in feces was performed using 16S rRNA sequencing with SheepMedical (Tokyo, Japan).
Statistical Analysis.
Statistical analyses were performed using Statcel 4 (OMS Publications) or Excel statistics with multiple comparison tests. Statistical significance was determined using the Tukey–Kramer method, and differences were considered significant at p<0.05.