Supplementary Materials and Methods Gene Expression Analysis To compare the relative amount of of selected gene expression between sham and mice given renal ischemia-reperfusion injury (IRI), ncounter Analysis System was used (Nanostring, Seattle, USA). In brief, mice were sacrificed 4 hrs post reperfusion, and total RNA was isolated from kidney tissue and splenic B cells sorted out using FACSAria III (BD). The experiment using ncounter GX Mouse Immunology V1 kit (Nanostring) was performed by PhileKorea Technology, Inc. (Seoul, Korea), following the manufacturers instructions. The full list of analyzed genes can be found in http://www.nanostring.com/support/literature?cat=gene_lists. Among the genes with significantly changed expressions, cytokine, chemokine and their receptor genes were selected in Supplementary Tables 1-2. Magnetic Resonance Imaging MRI experiments were performed with a 9.4 T/160 mm Agilent MRI scanner (Agilent Inc., Palo Alto, CA) using a millipede RF volume coil (inner diameter, 3.5 cm). Mice were anesthetized through a mask via spontaneous inhalation of 1.5% 2.0% isoflurane and placed in the handling system in the prone position. Automatic first-order shimming was performed to minimize B0 in homogeneities prior to MRI scanning. Coronal T2-weighted fast spin-echo images were obtained (repetition time [TR] = 4500 ms; kzero = 4; echo spacing = 12 ms; 16 segments; echo train length = 8; effective echo time [TE] = 48 ms; 4 averages; matrix = 128 128; field of view = 25 30 mm; slice thickness = 1.2 mm). DTI was performed using a spin-echo sequence with a Jones6 software (TR/TE = 2500/31.3 ms; slice thickness = 1.2 mm; 4 averages; matrix = 96 128; gradient amplitude = 16.4 G/cm; duration = 4 ms; separation 1
= 10 ms; target b value = 300 s/mm; 6 directions). ADC and FA maps were processed using VNMRJ software (Agilent Technologies). Regions of interest (ROIs) were carefully drawn over the renal cortex, corticomedulla, and medulla in a T2-weighted image. ADC and FA values were measured and analyzed in each ROI. 2
Supplementary Tables Table S1. Fold changes of cytokines/chemokines and their receptors gene expression of renal IRI kidneys compared with that of healthy kidneys (n=2) Up-regulated Gene Name Common name Ratio Down-regulated Gene Name 3 Common name Ratio CX3C chemokine Il6 Interleukin 6 146.53 Cx3cr1-5.32 receptor 1 tumor necrosis factor chemokine (C-X-C Cxcl1 85.74 Tnfsf10 (ligand) superfamily, -5.16 motif) ligand 1 member 10 Interleukin 1 receptor, Il1r2 29.48 Il15 Interleukin 15-2.17 type II (IL1R2) stromal cell-derived Ccl2 Ccl7 Il1rn Ccl3 Ccl4 Tnfrsf1b Tnfsf18 Ccl9 chemokine (C-C motif) 20.47 Cxcl12 ligand 2 Chemokine (C-C motif) 7.41 ligand 7 interleukin-1 receptor 6.44 antagonist Chemokine (C-C motif) 6.31 ligand 3 Chemokine (C-C motif) 5.65 ligand 4 Tumor necrosis factor receptor superfamily 4.64 member 1B Tumor necrosis factor ligand superfamily 4.54 member 18 Chemokine (C-C motif) 3.86 ligand 9 Il1b Interleukin-1 beta 3.44 Il1r1 Il4ra Cxcl10 Il6ra Cxcr4 Interleukin 1 receptor, 3.33 type I interleukin 4 receptor, 3.28 C-X-C motif 2.65 chemokine 10 interleukin 6 receptor, 2.64 C-X-C chemokine 2.22 receptor type 4 factor 1 (SDF-1) also -2.13 known as C-X-C motif chemokine 12
Table S2. Fold changes of cytokines/chemokines and their receptors gene expression of splenic B cells from renal IRI mice compared with that of healthy (n=2) Gene Name Up-regulated Common name Ratio 4 Gene Name Down-regulated Common name Il7r interleukin-7 receptor 241.12 Il18 Interleukin-18 (interferongamma inducing factor) Tumor necrosis factor receptor superfamily Il18r1 interleukin-18 receptor member 13C 60.09 Tnfrsf13c 1 (B-cell-activating factor receptor or BAFF receptor) Il2rb Tumor necrosis factor Interleukin-2 receptor 42.03 Tnfrsf13b receptor superfamily subunit beta member 13B Ccl5 Cxcr6 Tnfsf8 Il1rl2 Il6st Ccr9 Il18rap Il6ra Tnfsf10 Il2ra Tnfrsf4 Ifngr1 Cxcr3 Tnfaip3 Il27ra Chemokine (C-C motif) ligand 5 C-X-C chemokine receptor type 6 tumor necrosis factor (ligand) superfamily, member 8 Interleukin-1 receptorlike 2 interleukin 6 signal transducer C-C chemokine receptor type 9 Interleukin 18 receptor accessory protein interleukin 6 receptor, tumor necrosis factor (ligand) superfamily, member 10 interleukin 2 receptor, tumor necrosis factor (ligand) superfamily, member 4 Interferon gamma receptor 1 (IFNGR1) C-X-C chemokine receptor type 3 tumor necrosis factor, -induced protein 3 Interleukin 27 receptor, 35.45 Cxcr5 34.99 Il10ra 19.03 Il12a 11.75 11.44 11.26 8.02 4.33 4.18 3.33 2.85 2.44 2.25 2.16 1.95 C-X-C chemokine receptor type 5 Interleukin 10 receptor, subunit Interleukin-12 subunit Ratio -5.47-4.49-2.99-2.92-2.07-2.05
Supplementary Figure Legends Figure S1. The results of a serum creatine assay. (A) Mice were administered per os with designated inhibitors and, 24 hours later, sera were collected for a creatine assay. The error bars represent the SEM. n=2 5 mice. H, healthy; V, vehicle control; LY, LY294002; AS, AS252424; IC, IC87114. The numbers indicate the doses in milligrams/kilogram. (B) Mice were administered per os with the designated inhibitors 30 min prior to anesthesia and, 4 hrs post-ischemia, sera were collected for a creatine assay. The error bars represent the SEM. n=4 6 mice/group. H, healthy; S, sham; (-), negative control given renal IRI without any reagents; V, vehicle control. (C) Mice were administered per os with designated inhibitors 30 min prior to anesthesia and, 48 hours post-ischemia, sera were collected for a creatine assay. The error bars represent the SEM. n=5 7 mice. (-), negative control given renal IRI without any reagents; AS, 5 mg/kg AS252424. Figure S2. Less ATN in the AS252424-treated kidneys following renal IRI. Mice were administered per os with designated inhibitors 30 min prior to anesthesia, and the kidneys were harvested for PAS staining 24 hours post-ischemia. The magnification of the far left panels is 40, whereas that of the other images is 200. Arrows indicate ATN. H, healthy; S, sham; (-), negative control given renal IRI without any reagents; V, vehicle control, LY, 10 mg/kg LY294002; AS, 5 mg/kg AS252424. Figure S3. The number of kidney-infiltrated Gr-1 + cells were reduced by AS252424. Four hours post-ischemia, single-cell suspensions of the kidneys were prepared for flow cytometry. The numbers of Gr-1 + cells were calculated. The results are from three independent experiments. n=3 8 mice/group. The error bars represent the SEM. 5
Figure S4. MRI results could predict recovery earlier than serum creatine or histology. (A) MRI images before and after (0, 3, 8, 24 and 48 hr) renal IRI of an untreated kidney. The T2-weighted images were obtained as anatomical images and ADC and FA maps were processed. ROIs at three regions (red line, cortex; yellow line, cortico-medulla; blue line, medulla) were drawn to measure ADC and FA values. (B) The time-course change in ADC values. (C) The time-course change in FA values. N=6 7 mice/group. The error bars represent SEM. *P<0.05. 6
A Figure S1 B 7
Figure S2. 8
Figure S3. 60000 50000 40000 30000 20000 10000 0 S V LY AS IC 9
Figure S4. 10