Mannitol

To examine the protective effects of Tre, cell viability in the DCD model was assessed using CCK-8 (Dojindo) and Propidium Iodide (PI)/Hoechst 33342 (Dojindo) staining. Cell viability was measured using the CCK-8 assay after reperfusion and calculated as previously described. For PI/Hoechst 33342 (Dojindo) staining, after 1 h reperfusion, cells were washed with PBS (Gibco) once and incubated with 100 μL PBS (Gibco) containing 0.67 μg/mL PI (Dojindo) and Hoechst 33342 (Dojindo) dye in each well for 30 min according to the manufacturer’s instructions. PI (Dojindo) was used for dead cell staining, which is shown in red, and Hoechst (Dojindo) was used for all cell staining, which is shown in blue. Images were obtained using the APX100 Digital Imaging System (EVIDENT CORPORATION, Tokyo, Japan), and cell numbers were counted using ImageJ software Version 1.54 (U. S. National Institutes of Health, Bethesda, MD, USA). The percentage of live cells was calculated as the live cell number divided by the total cell number. To exclude the effect of osmolarity, we also used mannitol (50 mM, FUJIFILM Wako Pure Chemical Corporation) and sucrose (50 mM, FUJIFILM Wako Pure Chemical Corporation) as negative controls during the preconditioning and ischemia phases. Subsequently, we tested the cell viability after reperfusion by CCK-8 (Dojindo).

Liver tissues were weighed to approximately 100 mg and added to 10 volumes (w/v) of 20 mM HEPES buffer (pH 7.2) (Dojindo Morecular Technologies, Inc., Kumamoto, Japan) with 1 mM EGTA (Kanto Chemical., Co., Ltd., Inc., Osaka, Japan), 210 mM mannitol (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), and 70 mM sucrose (Fujifilm Wako Pure Chemical Corp., Osaka, Japan). Suspensions were then homogenized, and the resulting homogenates were centrifuged at 1500× g at 4 °C for 5 min. Supernatants were further centrifuged at 14,000× g at room temperature for 15 min to concentrate the samples using an Amicon concentrator (Merck Millipore Ltd., Tokyo, Japan) with a molecular weight cut-off of 10,000. The samples obtained were stored at −80 °C until used. Prior to the SOD activity assay, filtrates were thawed under running water.
SOD activity was assessed using a previously reported method [31 ]. Activity was measured using a commercial ELISA kit (Superoxide Dismutase Assay Kit, Cayman chemical, Ann Arbor, MI, USA, inter-assay coefficient of variation 3.7%, dynamic range 0.005–0.05 units/mL SOD). This assay was performed according to the manufacturer’s instructions. One unit of SOD was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.

Intracellular metabolites were measured by CE-TOFMS (Agilent Technologies, Palo Alto, CA, USA) as previously described^{24 (link),50 (link),51 (link)}. In brief, cells were washed twice with 5% (w/v) mannitol (FUJIFILM Wako Pure Chemical Industries) and dissolved in 600 µL of methanol containing internal standards (25 μM each of methionine sulfone, ethane sulfonic acid, and d-Camphor-10-sulfonic acid). The homogenate was mixed with 200 μL of Milli-Q water and 400 μL of chloroform. After centrifugation, the separated methanol–water layer was ultra-filtrated through a Millipore 5-kDa cutoff filter (Millipore, Bedford, MA, USA) to remove proteins. The filtrate was lyophilized, dissolved in 25 μL of Milli-Q water containing internal standards (200 μM each of 1,3,5-benzenetricarboxylic acid and 3-aminopyrrolidine), and analyzed using CE-TOFMS. The raw data were processed with MasterHands^{52 (link)}. Metabolite identities were assigned by matching their m/z values and migration times with those of standard compounds.

Normal rat kidney cells (NRK-52E), a tubular epithelial cell line derived from rat kidney, were directly purchased from the Japanese Collection of Research Bioresources (JCRB No. IFO50480, Osaka, Japan). Both mannitol and urea for adjusting the osmolarity in the medium were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Y-27632, ROCK (Rho-associated protein kinase) inhibitor, was also obtained from FUJIFILM Wako Pure Chemical. Antibodies for α-SMA, vinculin, E-cadherin, and GAPDH were purchased from DAKO (Glostrup, Denmark), Invitrogen (Carlsbad, CA), abcam (Cambridge, MA), and Cell Signaling Technology (Danvers, MA), respectively.

NIL was purchased from Tokyo Chemical Industry Co., Ltd. (Saitama, Japan). BAC was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan), and 0.4% Benoxil, 0.5% phenylephrine and 0.5% tropicamide were provided by Santen Pharmaceutical Co., Ltd. (Osaka, Japan). HPβCD and MC were kindly donated by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan), respectively. SUPER FIX™ rapid fixative solution was purchased from Kurabo Industries, Ltd. (Osaka, Japan), and the ELISA Insulin Kit was obtained from Morinaga Institute of Biological Science, Inc. (Kanagawa, Japan). Mannitol and streptozotocin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available. Wistar rats aged 7 weeks were provided by Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). STZ rats were prepared by injecting Wistar rats intraperitoneally with 100 mg/kg streptozotocin twice on two consecutive days, and then housing them for two weeks after the last injection before use in this study. All experiments were performed in accordance with the ARVO resolution on the use of animals in research, and were approved by the Kindai University Faculty of Pharmacy Committee Guidelines for the Care of Laboratory Animals (project identification code KAPS-25-003, 1 April 2013).

We isolated HPMCs from human omentum as described previously.¹⁸ (link) Harvesting of the omentum was permitted by the Medical Ethics Committee of Hiroshima Graduate School of Biomedical Science (E-2161). Written Informed consent was obtained from each patient. HPMCs were maintained in M199 medium (Lonza, Basel, Switzerland) containing 10% fetal bovine serum and penicillin-streptomycin. The cells were seeded into 6-well plates. At subconfluence, HPMCs were incubated for 24 hours in M199 medium and then treated with 4.25% high glucose or identical concentrations of Mannitol for up to 48 hours. Mannitol was purchased from Fujifilm wako chemicals (Osaka, Japan) and glucose solution was purchased from Sigma-Aldrich (St. Louis, MO, USA).

MH, mannitol, MC, and the Magnesium B test kit were provided by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Carteolol and Benoxil (0.4%) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and Santen Pharmaceutical Co., Ltd. (Osaka, Japan), respectively. BAC was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Sterilized Tear Production Measuring Strips and fluorescein were provided by Showa Yakuhin Kako Co., Ltd. (Tokyo, Japan) and Alcon Japan (Tokyo, Japan), respectively. Cell Count Reagent SF was purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals used were of the highest purity commercially available.