Male C57BL/6J mice were obtained from SLC Japan Inc., Shizuoka, Japan. The mice were housed with food and water available ad libitum in light- and temperature-controlled environments. The mice were randomly divided into four groups at 10 weeks of age: sham-operated mice (sham, n = 6), induced-CKD mice (CKD, n = 6), induced-LVH mice (LVH, n = 6), and induced-CKD and induced-LVH mice (CKD/LVH, n = 6). At 10 weeks, TAC was performed for mice in the LVH and CKD/LVH groups. Briefly, the mice were anesthetized with isoflurane, and medetomidine, midazolam, and butorphanol were intraperitoneally administered. They were intubated with 22 G vascular catheters and then artificially ventilated (Small Animal Ventilator MK-V100, Muromachi Kikai). The thorax was opened, the aorta was exteriorized and a ligature was placed around the aorta as previously described . The needle used for ligation was 27 G. At 11 weeks, a left 2/3 nephrectomy was performed, and one week later, a right nephrectomy was also performed for mice in the CKD and CKD/LVH groups. For mice in the sham group, sham operations were performed at 10, 11, and 12 weeks of age. Echocardiographic parameters were measured and 24-hour urine samples were collected from each mouse using a metabolic cage before the mice were sacrificed. At 16 weeks, these mice were sacrificed under anesthesia. Blood samples for serum measurements were collected from the right ventricle, and the hearts and bones were removed for RNA extraction and histomorphological analysis.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Experiment Facility Ethics Committee, Graduate School of Medicine, Kobe University (Permit Number: P160707-R2). All surgery was performed under anesthesia with 1.5 % isoflurane with intraperitoneal medetomidine, midazolam, and butorphanol premedication (0.15 mg/kg medetomidine, 2 mg/kg midazolam, and 2.5 mg/kg butorphanol. Efforts were made to minimize suffering by following the ARRIVE guidelines for reporting experiments involving animals [10, 11]. A randomized protocol was used in the experiments with a total of 24 mice.
Blood And Urine Measurement
Collected blood was centrifuged for 15 min at 3,000 rpm and stored at -80℃ until analysis. Serum creatinine (Cr) levels were measured using a Fuji Dri-chem 3500 (FUJIFILM Japan, Tokyo, Japan). Serum phosphate, intact parathyroid hormone (iPTH), intact fibroblast growth factor 23 (iFGF23), and aldosterone levels were measured using ELISA (phosphate: FUJIFILM Japan, Tokyo, Japan; iPTH: Cloud-Clone Corp., Houston, Texas, USA; iFGF23: Kainos Laboratories, Inc., Tokyo, Japan; Aldosterone: Abcam, Cambridge, UK). Urinary excretion of albumin (uAlb), urinary Cr levels, urinary angiotensinogen (uAGT) levels, and 8-hydroxydeoxyguanosine (u8-OHdG) were also determined using ELISA (uAlb: FUJIFILM Wako Shibayagi Corporation, Gunma, Japan; u-Cr: FUJIFILM Wako Pure Chemicals Corporation, Osaka, Japan; uAGT: MyBioSource, San Diego, CA, USA; u8-OHdG: Japan Institute for Control of Aging, Shizuoka, Japan). Serum 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D) levels were measured using 25D125I radioimmunoassay kit (DIAsource ImmunoAssays S.A., Nivelles, Belgium) and a TFB 1,25D radioimmunoassay kit (Immunodiagnostic Systems Ltd., Boldon, UK), respectively.
Blood Pressure Measurements
Systolic blood pressure (BP) was measured using tail-cuff plethysmography (Model MK-2000; Muromachi Kikai Co. Ltd., Japan). The mice rested for 15min to reduce stress-induced BP elevation. The mean value was determined using multiple readings (at least 10). The BP evaluation was performed at the end of the study period.
The mice were mildly anesthetized with 1.0 % isoflurane. Echocardiography was performed using a commercially available echocardiographic system (F37; Hitachi Aloka Medical, Ltd, Tokyo, Japan). The wall thickness and ejection fraction (EF) were measured using a two-dimensional short-axis view of the left ventricle (LV) at the papillary muscle level. These studies were performed at 16 weeks.
Histological And Immunohistochemical Analyses
The hearts were removed, weighed, and fixed in 10 % formaldehyde. The paraffin block was made by immersing the samples in ethanol, xylene, and paraffin in the embedding device at room temperature. The paraffin blocks were cut into 2-µm sections and stained with hematoxylin-eosin for routine histology and Sirius red for morphometric studies.
FGF23, angiotensin II, angiotensin converting enzyme (ACE), and angiotensin converting enzyme 2 (ACE2) expression in the heart were assessed with anti-FGF23 monoclonal antibodies (R&D Systems, Minneapolis, MN, USA), anti-angiotensin II monoclonal antibodies (Novus Biologicals, LLC, Littleton, Colorado, USA), anti-ACE monoclonal antibodies (Abcam, Cambridge, UK), and anti-ACE2 monoclonal antibodies (R&D Systems, Minneapolis, MN, USA). In brief, heart slices were preincubated with blocking agents and then incubated with the primary antibodies mentioned above for 60 min at room temperature (ACE, FGF23) and overnight at 4 ℃ (ACE2, angiotensin II). Biotinylated Anti-rat and Anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) and an avidin: biotinylated enzyme complex (VECTASTAIN Elite ABC Reagent; Vector Laboratories, Inc., Burlingame, CA, USA) for FGF23 and ACE2, and a universal immunoperoxidase polymer (Histofine Simple Stain mouse MAX PO, anti-rat and anti-rabbit; Nichirei, Tokyo, Japan) for ACE and angiotensin II were used for immunostaining.
The percentage of positive areas of Sirius red staining, relative immune-positive area, and cardiomyocyte width and area in the LV sections were calculated as mean values in 20 randomly selected microscopic fields using ImageJ (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA). As for assessing the immune-positive area, the relative value was defined as that in each group or that in the sham group. All evaluations were performed in a blinded manner.
RNA Extraction And Real-time Polymerase Chain Reaction
As previously reported [12, 13], total RNA was extracted from the mouse heart and bone samples with an ISOGEN kit (Wako Pure Chemicals Industries, Ltd, Osaka, Japan) according to the manufacturer’s instructions. ReverTra Ace™ qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan) was used to create cDNA using an oligo-dT primer as recommended. The synthesized cDNA was stored at -80℃ until analysis with quantitative PCR. mRNA expression was examined with real-time PCR using Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with the SYBR Green Assay with Thunderbird SYBR qPCR Mix (TOYOBO Co., Ltd., Osaka, Japan) following the manufacturer’s protocol. The analysis was performed with the relative quantification method of the Applied Biosystems 7500 Real-Time PCR Software. The relative amount the mRNA samples was normalized to GAPDH mRNA. For PCR analysis, we used the following primers: α-myosin heavy chain (α-MHC), (5′-ATGTTAAGGCCAAGGTCGTG-3′, 5′-CACCTGGTCCTCCTTTATGG-3′), β-myosin heavy chain (β-MHC), (5′-AGCATTCTCCTGCTGTTTCC-3′, 5′-GAGCCTTGGATTCTCAAACG-3′), atrial natriuresis peptide (ANP), (5′-AGGCAGTCGATTCTGCTTGA-3′, 5′-CGTGATAGATGAAGGCAGGAAG-3′), brain natriuresis peptide (BNP), (5′-TAGCCAGTCTCCAGAGCAATTC-3′, 5′-TTGGTCCTTCAAGAGCTGTCTC-3′), collagen type I α2 (Col1a2), (5′-GCAGGTTCACCTACTCTGTCCT-3′, 5′-CTTGCCCCATTCATTTGTCT-3′), collagen type III α1 (Col3a1), (5′-TCCCCTGGAATCTGTGAATC-3′, 5′-TGAGTCGAATTGGGGAGAAT-3′), connective tissue growth factor (CTGF), (5′-CACAGAGTGGAGCGCCTGTTC-3′, 5′-GATGCACTTTTTGCCCTTCTTAA-3′), FGF23, (5′-ACAAGGACACCTAAACCGAACAC-3′, 5′-AGCTACTGACTGGTCCTATCACA-3′), AGT, (5′-TCTCTTTACCCCTGCCCTCT-3′, 5′-GAAACCTCTCATCGTTCCTTG-3′), AT1R, (5`-CCATTGTCCACCCGATGAAG-3′, 5′-TGCAGGTGACTTTGGCCAC-3′), ACE, (5′-CCCTAGAGAAAATCGCCTTCTTG-3′, 5′-CGAAGATACCACCAGTCGAAGTT-3′), ACE2, (5`-TGGGCAAACTCTATGCTG-3′, 5′-TTCATTGGCTCCGTTTCTTA-3′), Renin, (5′-CCTCTACCTTGCTTGTGGGATT-3′, 5′-CTGGCTGAGGAAACCTTTGACT-3′), GAPDH, (3′-GCAAAGTGGAGATTGTTGCCA-5′, 3′-AATTTGCCGTGAGTGGAGTCA-5′).
Collected data were analyzed using the computer software application IBM SPSS statistics version 25.0 (IBM Corp., Armonk, NY, USA) for all statistical analyses. Values are presented as means ± SEM. The Mann–Whitney U test was used to analyze the significance of the differences between the two groups. The Kruskal-Wallis test was used to assess the differences between the four groups. Pearson’s and Spearman’s correlation coefficients were used to analyze relationships between the variables. A P value of < 0.05 was considered statistically significant.