Previous studies have demonstrated intrarenal hypoxia in patients with diabetes. Hypoxia-inducible factor (HIF)-1 plays an important role in hypoxia-induced tubulointerstitial fibrosis. Recent clinical trials have confirmed the renoprotective action of SGLT2 inhibitors in diabetic nephropathy. We explored the effects of an SGLT2 inhibitor, luseogliflozin on HIF-1α expression in human renal proximal tubular epithelial cells (HRPTECs). Luseogliflozin significantly inhibited hypoxia-induced HIF-1α protein expression in HRPTECs. In addition, luseogliflozin inhibited hypoxia-induced the expression of the HIF-1α target genes PAI-1, VEGF, GLUT1, HK2 and PKM. Although luseogliflozin increased phosphorylated-AMP-activated protein kinase α (p-AMPKα) levels, the AMPK activator AICAR did not changed hypoxia-induced HIF-1α expression. Luseogliflozin suppressed the oxygen consumption rate in HRPTECs, and subsequently decreased hypoxia-sensitive dye, pimonidazole staining under hypoxia, suggesting that luseogliflozin promoted the degradation of HIF-1α protein by redistribution of intracellular oxygen. To confirm the inhibitory effect of luseogliflozin on hypoxia-induced HIF-1α protein in vivo, we treated male diabetic db/db mice with luseogliflozin for 8 to 16 weeks. Luseogliflozin attenuated cortical tubular HIF-1α expression, tubular injury and interstitial fibronectin in db/db mice. Together, luseogliflozin inhibits hypoxia-induced HIF-1α accumulation by suppressing mitochondrial oxygen consumption. The SGLT2 inhibitors may protect diabetic kidneys by therapeutically targeting HIF-1α protein.
BackgroundRecent large-scale clinical studies demonstrate that sodium-glucose cotransporter 2 (SGLT2) inhibitors protect the diabetic kidney. However, clinical and animal studies have not shown the changes of the total glomeruli in the whole kidney treated with SGLT2 inhibitors.MethodsWe performed computed tomography (CT) imaging on mice using synchrotron radiation to investigate the impact of luseogliflozin, a SGLT2 inhibitor, on the number and volume of glomeruli in the whole kidney.FindingsWe did not observe a significant difference in the total glomerular number (Nglom) among mice. Luseogliflozin redistributed the number of glomeruli in different regions, accompanied by the normalization of diabetes-augmented renal volume (Vkidney). Diabetic db/db mice had a larger glomerular volume in the mid-cortex than did control db/m mice, and luseogliflozin increased the glomerular volume in all renal cortical zones of the whole kidney in db/db mice. According to the multivariate regression analysis, hemoglobin A1c level was the most relevant determinant of Vkidney, not Nglom or mean glomerular volume (Vglom), indicating that hyperglycemia induced renal (tubular) hypertrophy, but not glomerular enlargement. Luseogliflozin increased hypoxia in the juxtamedullary region, sustained upregulated renal renin expression and plasma renin activity, and failed to decrease albuminuria by downregulating megalin in db/db mice.InterpretationBased on our findings, SGLT2 inhibitors may alter glomerular distribution and size in addition to their glucose-lowering effects, presumably by affecting oxygen metabolism and humoral factors.FundFunding for this research was provided by The , the Japan Diabetes Foundation, and .
Maternal overnutrition affects offspring susceptibility to nonalcoholic steatohepatitis (NASH). Male offspring from high-fat diet (HFD)-fed dams developed a severe form of NASH, leading to highly vascular tumor formation. The cancer/testis antigen HORMA domain containing protein 1 (HORMAD1), one of 146 upregulated differentially expressed genes in fetal livers from HFD-fed dams, was overexpressed with hypoxia-inducible factor 1 alpha (HIF-1alpha) in hepatoblasts and in NASH-based hepatocellular carcinoma (HCC) in offspring from HFD-fed dams at 15 weeks old. Hypoxia substantially increased Hormad1 expression in primary mouse hepatocytes. Despite the presence of three putative hypoxia response elements within the mouse Hormad1 gene, the Hif-1alpha siRNA only slightly decreased hypoxia-induced Hormad1 mRNA expression. In contrast, N-acetylcysteine, but not rotenone, inhibited hypoxia-induced Hormad1 expression, indicating its dependency on nonmitochondrial reactive oxygen species production. Synchrotron-based phase-contrast micro-CT of the fetuses from HFD-fed dams showed significant enlargement of the liver accompanied by a consistent size of the umbilical vein, which may cause hypoxia in the fetal liver. Based on these findings, a maternal HFD induces fetal origins of NASH/HCC via hypoxia, and HORMAD1 is a potential therapeutic target for NASH/HCC.
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The renoprotective mechanism of SGLT2 inhibitors has not been fully elucidated. Tubular hypoxia is major driving force for proximal tubulopathy in diabetic kidney. In addition, diabetes causes dysregulation of AMP-activated protein kinase (AMPK) in kidney cortex. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on hypoxia inducible factor-1α (HIF-1α) expression and AMPK phosphorylation in cultured human renal proximal tubular epithelial cells (HRPTECs) and on tubulointerstitial pathological changes in diabetic mice. Luseogliflozin inhibited hypoxia (1%O2, 24h)-induced HIF-1α protein expression in HRPTECs in a dose-dependent manner (1-100µM). In addition, luseogliflozin increased AMPKα protein phosphorylation (Th172) in normoxic and hypoxic condition. Intriguingly, luseogliflozin suppressed oxygen consumption rate (OCR) measured by oxygen quenching phosphorescent probe by 69% in HRPTECs from normoxic condition. Hypoxia decreased OCR by 52% and luseogliflozin further decreased OCR by 33% from hypoxic condition. Moreover, luseogliflozin rescued hypoxic state in HRPTECs even under hypoxic conditions assessed by a hypoxia-sensitive dye, pimonidazole. These data indicate luseogliflozin inhibits HIF-1α expression through suppression of mitochondrial OCR, which leads to restore intracellular hypoxia. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin ameliorated tubular injury compared to non-treated db/db mice. Furthermore, immunostaining of HIF-1α and fibronectin in cortical tubules revealed that luseogliflozin decreased HIF-1α and fibronectin expression in db/db mice. In conclusion, luseogliflozin decreases mitochondrial OCR and ameliorates tubular fibrosis at least partly by suppressing HIF-1α expression and activating AMPK in renal proximal tubules of diabetic mice. These results may provide a novel renoprotective target of SGLT2 inhibitor. Disclosure R. Bessho: None. Y. Takiyama: None. T. Takiyama: None. T. Ota: None.
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