Volume 94, Issue 3, Pages 524-535 (September 2018) A sodium-glucose cotransporter 2 inhibitor attenuates renal capillary injury and fibrosis by a vascular endothelial growth factor–dependent pathway after renal injury in mice  Yifan Zhang, Daisuke Nakano, Yu Guan, Hirofumi Hitomi, Akiyoshi Uemura, Tsutomu Masaki, Hideki Kobara, Takeshi Sugaya, Akira Nishiyama  Kidney International  Volume 94, Issue 3, Pages 524-535 (September 2018) DOI: 10.1016/j.kint.2018.05.002 Copyright © 2018 International Society of Nephrology Terms and Conditions

Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 1 Time course of changes in blood urea nitrogen (BUN), creatinine clearance, and histological damage score in the kidney after ischemia-reperfusion. The (a) blood urea nitrogen levels, (b) creatinine clearance, and (c) histological damage kidney scores were measured on days 1, 3, and 7 in luseogliflozin (luseo)- or vehicle-treated groups of mice that underwent a sham-operation or ischemia-reperfusion (n = 5–8). n.o., not observed; n.s., not significant. *P < 0.05 versus sham-operated group, #P < 0.05 versus vehicle-treated group. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 2 Assessment of renal fibrosis development following luseogliflozin treatment at 1 week after renal ischemia-reperfusion injury. (a) Schematic of the experiment schedule. The left kidney was subjected to unilateral renal ischemia for 30 minutes. Luseogliflozin was administered 6 hours after reperfusion and then daily until day 7. Kidney samples were collected at day 7. Representative images of Sirius red–stained sections from the ischemic kidney of vehicle-treated mice (b, cortex; c, outer medulla) or luseogliflozin (luseo)-treated mice (d, cortex; e, outer medulla) at 1 week after reperfusion. Representative images of Sirius red–stained sections from the contralateral kidneys of (f) vehicle- or (g) luseo-treated mice. (h) The relative difference in Sirius-red–positive area in the kidney at 1 week after reperfusion (n = 7). (i) The transforming growth factor-β (TGF-β) levels in the kidneys of the vehicle- or luseo-treated groups at 1 week after reperfusion (n = 8–9). Bar = 100 μm. *P < 0.05 versus contralateral control, #P < 0.05 versus vehicle-treated group. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 3 Assessment of renal fibrosis development following luseogliflozin treatment at 4 weeks after renal ischemia-reperfusion injury. (a) Schematic of the experiment schedule. The left kidney was subjected to unilateral renal ischemia for 30 minutes. Luseogliflozin was administered 6 hours after reperfusion and then daily until day 7. Kidney samples were collected at 4 weeks after reperfusion. Neither luseogliflozin nor vehicle was administered during the period of 1 to 4 weeks after reperfusion. Representative images of (b,d) Sirius-red–stained or (c,e) Azan-stained renal outer medulla from (b,c) vehicle-treated or (d,e) luseogliflozin (luseo)-treated mice at 4 weeks after reperfusion. The relative differences in (f) Sirius red or (g) Azan staining-positive area (n = 5–6). Bar = 100 μm. *P < 0.05 versus contralateral control, #P < 0.05 versus vehicle-treated group. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 4 Assessment of renal function following luseogliflozin treatment at 4 weeks after renal ischemia-reperfusion injury. (a) Schematic of the experiment schedule. The left kidney was subjected to unilateral renal ischemia for 30 minutes. Luseogliflozin was administered 6 hours after reperfusion and then daily until day 7. Neither luseogliflozin nor vehicle was administered during the period of 1 to 4 weeks after reperfusion. At 4 weeks after reperfusion, the mice underwent nephrectomy of the contralateral right kidney. Urine samples were collected from 6 to 30 hours after uninephrectomy. (b) Plasma creatinine level (left), 24-hour urine volume (center), and creatinine clearance (right) after uninephrectomy at 4 weeks after unilateral ischemia-reperfusion. *P < 0.05 versus vehicle-treated group. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 5 Effect of luseogliflozin treatment on endothelial rarefaction after renal ischemia-reperfusion injury. Vehicle- or luseogliflozin (luseo)-treated mice were analyzed at day 7 after ischemia-reperfusion injury. (a) A representative image (left) and the corresponding congestion-hemorrhage scores (right) of the renal outer medulla. Yellow arrows indicate congestion and hemorrhage (n = 6). Bar = 50 μm. (b) Representative images and (c) corresponding quantification of the area positive for anti-CD31 antibody in the contralateral and ischemic kidneys (n = 8). Bar = 300 μm. Bottom images show enlargements of the boxed areas in the top images. (d) Representative images of the pimonidazole staining in the renal cortex (n = 4). (e) Quantification of the renal vascular endothelial growth factor-A (VEGF-A) mRNA expression in the contralateral and ischemic kidneys (n = 9–10). Bar = 100 μm. *P < 0.05 versus contralateral control. #P < 0.05 versus vehicle-treated group. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 6 Effect of treatment with sunitinib. Vehicle- or luseogliflozin (luseo)-treated mice were co-treated with sunitinib, a vascular endothelial growth factor (VEGF) receptor inhibitor, during days 3 to 7 after ischemia-reperfusion (n = 5–12). The (a) congestion-hemorrhage scores, (b) Sirius red–positive areas, (c) renal transforming growth factor-β (TGF-β) mRNA expression levels, (d) NG2-positive areas, and (e) CD31-positive areas of these mice. Open symbols: contralateral kidney; closed symbols: ischemia-reperfusion kidney. n.s., not significant. *P < 0.05 versus vehicle control. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 7 Glucose uptake in the kidney. Representative images (left) and quantification (right) of the uptake of a fluorescent glucose analogue, NBDG, in mouse proximal tubules at day 3 after ischemia-reperfusion (IR) (n = 7). Bar = 50 μm. *P < 0.05 versus vehicle, #P < 0.05 versus sham, †P < 0.05 versus interaction. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 8 Glucose transporter 2 (GLUT2) expression in the membranous fraction of the kidney. Western blots show the GLUT2 protein expression in the ischemia-reperfusion kidneys (at day 3). Representative (a) Western blots and (b) quantitative data are shown (n = 4). Note: there are multiple bands for GLUT2 due to glycosylation. The bands that are indicated by an arrow, which are detectable mainly in the cell membrane and are potentially glycosylated, were analyzed, and the resulting data are shown in Figure 8b. #P < 0.05 versus sham. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 9 Effect of glucose on vascular endothelial growth factor-A (VEGF-A) mRNA expression. (a) VEGF-A mRNA expression in mProx24 cells cultured with media containing different concentrations of glucose (n = 9–12). Note: 17.5 mmol/l is the glucose concentration in the standard medium for mProx24 cells. *P < 0.05 versus 0 mmol/l glucose, #P < 0.05 versus 5 mmol/l glucose. (b) Effects of cytochalasin B (GLUT2i) treatment on VEGF-A mRNA in luseogliflozin (luseo)-treated mProx24 cells (n = 6). *P < 0.05 versus vehicle, †P < 0.05 versus luseo. (c) Effects of GLUT2 siRNA on VEGF-A mRNA in luseo-treated mProx24 cells (n = 6). *P < 0.05 versus vehicle, †P < 0.05 versus luseo. (d) Effects of luseo on VEGF-A mRNA in mProx24 cells under CoCl2-induced hypoxia (for 20 hours) and CoCl2-free reoxygenation (for 6 hours) in media with various glucose concentrations (n = 10–18). #P < 0.05 versus vehicle + normoxia in 5 mmol/l glucose, †P < 0.05 versus vehicle control in hypoxia-reoxygenation. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 10 Working hypothesis for the sodium-glucose cotransporter 2 (SGLT2) inhibitor-induced depletion of glucose uptake in proximal tubules. Under normal (uninjured) conditions, glucose is taken up through SGLT2 and transferred to the interstitial space via glucose transporter 2 (GLUT2). Although SGLT2 inhibition may transiently reduce the intracellular glucose level under normal conditions, GLUT2, an equilibration transporter, transfers glucose from the interstitial side to the intracellular side, so the intracellular glucose level will return to a level similar to that in the interstitial space. In contrast, the GLUT2 level is reduced in injured proximal tubular cells and is insufficient to equilibrate the intracellular glucose level following treatment by SGLT2 inhibitors. Thus, under these conditions, treatment with SGLT2 inhibitors induces the reduction of glucose uptake and stimulates the expression of vascular endothelial growth factor-A (VEGF-A). The VEGF-A may act to maintain neoangiogenesis and prevent endothelial rarefaction. Kidney International 2018 94, 524-535DOI: (10.1016/j.kint.2018.05.002) Copyright © 2018 International Society of Nephrology Terms and Conditions