Dapagliflozin, SGLT2 Inhibitor, Attenuates Renal Ischemia-Reperfusion Injury

Abstract

Dapagliflozin, a new type of drug used to treat diabetes mellitus (DM), is a sodium/glucose cotransporter 2 (SGLT2) inhibitor. Although some studies showed that SGLT2 inhibition attenuated reactive oxygen generation in diabetic kidney the role of SGLT2 inhibition is unknown. We evaluated whether SLT2 inhibition has renoprotective effects in ischemia-reperfusion (IR) models. We evaluated whether dapagliflozin reduces renal damage in IR mice model. In addition, hypoxic HK2 cells were treated with or without SGLT2 inhibitor to investigate cell survival, the apoptosis signal pathway, and the induction of hypoxia-inducible factor 1 (HIF1) and associated proteins. Dapagliflozin improved renal function. Dapagliflozin reduced renal expression of Bax, renal tubule injury and TUNEL-positive cells and increased renal expression of HIF1 in IR-injured mice. HIF1 inhibition by albendazole negated the renoprotective effects of dapagliflozin treatment in IR-injured mice. In vitro, dapagliflozin increased the expression of HIF1, AMP-activated protein kinase (AMPK), and ERK and increased cell survival of hypoxic HK2 cells in a dose-dependent manner. In conclusion, dapagliflozin attenuates renal IR injury. HIF1 induction by dapagliflozin may play a role in renoprotection against renal IR injury.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Effects of dapagliflozin on renal function (S(n = 5), sham; S + Dapa (n = 5), sham + dapagliflozin; IR(n = 7), vehicle-treated renal IR mice; IR + Dapa (n = 7), dapagliflozin-treated IR mice).

Dapagliflozin pretreatment reduced BUN and serum creatinine in IR-injured mice. *P < 0.05 vs. vehicle-treated sham, #P < 0.05 vs. vehicle-treated IR. Bar represents mean ± s.d.(Standard deviation).

Fig 2. Representative kidney section stained for hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS).

(S(n = 5); S + Dapa (n = 5), IR(n = 7), IR + Dapa (n = 7)). (A) H&E stain, yellow arrows indicate cell debri and tubular necrosis. Yellow arrowheads indicate inflammatory cells. Original magnification, 200×. (B) Semi-quantitative analysis of tubule interstitial injury in wild-type and dapagliflozin- and/or albendazole-treated mice 24 h after renal IR injury (C) PAS stain. Yellow arrows indicate necrotized tubules or cast formation. Yellow arrowheads indicate loss of brush border or dilated tubules. Black arrow indicates brush border. Original magnification, 200×. (D) Semi-quantitative analysis of tubular injury in wild-type and dapagliflozin- and/or albendazole-treated mice 24 h after renal IR injury. *P < 0.05 vs. vehicle-treated sham, #P < 0.05 vs. vehicle-treated IR. Bar represents mean ± s.d.

Fig 3. Effects of dapagliflozin on HK2 cell survival and apoptosis.

(A) Dapagliflozin significantly increased the cell survival in hypoxic HK2 cell in a dose-dependent manner. (B) Representative western blot (B-a). Dapagliflozin significantly decreased the Bax expression and Bax/Bcl2 ratio in hypoxic HK2 cells (B-b and d). However, Bcl2 expression was unaffected by dapagliflozin in hypoxic HK2 cells (B-c). Dapagliflozin significantly decreased the PARP expression in hypoxic HK2 cells (B-e) *P < 0.05 vs. normoxic HK2 cells, #P < 0.05 vs. control hypoxic HK2 cells. Bar represents mean ± s.d. All cellular experiment repeated three times for cell viability test and western blot.

Fig 4. Effects of dapagliflozin on apoptosis in IR-injured kidneys (S(n = 5); S + Dapa (n = 5), IR(n = 7), IR + Dapa (n = 7)).

(A) Western blot analysis shows dapagliflozin decreased Bax expression and increased Bcl2 expression in IR-injured kidneys. Dapalgliflozin decreased PARP expression in IR-injured kidneys. (B) Representative kidney section. Dapagliflozin decreased the TUNEL-positive cells in an IR-injured kidney. *P < 0.05 vs. sham kidney, #P < 0.05 vs. IR kidney. Bar represents mean ± s.d.

Fig 5. Effects of dapagliflozin on HIF1 in HK2 cells.

(A) Representative Western blot: dapagliflozin increased HIF1 expression and phosphorylation of AMPK and ERK in hypoxic HK2 cells. *P < 0.05 vs., dapagliflozin nontreated normoxic HK2 cells, #P < 0.05 vs. normoxic HK2 cells. **P < 0.05 vs. control hypoxic HK2 cells. (B) Representative Western blot: hypoxia increased HIF1 expression and decreased SGTL2 expression. Dapagliflozin pretreatment decrease SGLT2 expression in hypoxic HK2 cells. *P < 0.05 vs., dapagliflozin nontreated normoxic HK2 cells, #P < 0.05 vs. normoxic HK2 cells. **P < 0.05 vs. control hypoxic HK2 cells. (C) Dapaglilfozin pretreatment increase HIF1 expression in normoxic and hypoxic HK2 cells compared to control and hypoxic control HK2 cells respectively. Albendazole decreased HIF1 expression in dapagliflozin-treated hypoxic HK2 cells. *P < 0.05 vs., control HK2 cells, #P < 0.05 vs.control hypoxic HK2 cells. **P < 0.05 vs. albendazole nontreated—dapagliflozin treated hypoxic HK2 cells. (D) Albendazole decreased cell survival in dapagliflozin-treated hypoxic HK2 cells. *P < 0.05 vs., control hypoxic HK2 cells #P < 0.05 vs.dapagliflozin treated hypoxic HK2 cells. (E) Dapagliflozin decreased glucose uptake in hypoxic HK2 cells. *P < 0.05 vs., hypoxic HK2 cells. Bar represents mean ± s.d. All cellular experiment repeated three times for western blot, cell viability test, and glucose uptake test.

Fig 6. Effects of dapagliflozin on HIF1 in IR-injured kidneys (S (n = 5), sham; S+Dapa (n = 5), sham+dapagliflozin; IR (n = 7); vehicle-treated renal IR mice; IR+Dapa (n = 7), dapagliflozin-treated IR mice; IR+Dapa+ABZ (n = 7), albendazole- and dapagliflozin-treated IR mice).

(A) Representative Western blot: dapagliflozin increased HIF1 expression in IR-injured kidneys. Albendazole decreased HIF1 expression in dapagliflozin-treated IR-injured kidney. (B) Representative Immunohistochemistry of HIF1. Dapagliflozin increase the HIF1 stained area in tubular area of IR kidney. Original magnification, 200× (C) The effects of dapagliflozin on renal function. Albendazole treatment elevated BUN and serum creatinine in dapagliflozin-treated IR mice. (D–F) Representative kidney section: albendazole treatment increase tubulointerstitial injury and TUNEL-positive cells in IR-injured kidneys. *P < 0.05 vs. vehicle-treated sham, **P < 0.05 vs. vehicle-treated IR, #P < 0.05 vs. dapagliflozin-treated IR. Bar represents mean ± s.d.