Abstract:Objective To investigate the protective effects and potential mechanisms of Henagliflozin on renal injury in mice with obesity-related glomerulopathy (ORG).Methods Male C57BL/6 mice were fed a high-fat diet to establish the ORG model and were then randomly allocated into an ORG group (n = 6) and an ORG + Henagliflozin group (n = 6). Mice fed a standard chow served as normal controls (NC group, n = 6). Body weight, blood glucose, blood ketone levels, and the urinary microalbumin-to-creatinine ratio (UACR) were monitored; insulin tolerance tests (ITT) and glucose tolerance tests (GTT) were performed, and the corresponding areas under the curve (AUCs) were calculated. The following were assessed: renal histopathology and ultrastructural changes, as well as serum levels of β-hydroxybutyrate (BHB), creatinine (UCr), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Additionally, renal levels of BHB, total antioxidant capacity (TAOC), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), α-smooth muscle actin (α-SMA), interleukin-6 (IL-6), interleukin-1β (IL-1β), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were evaluated.Results Body weight and Lee's index were significantly higher in the ORG group and ORG + Henagliflozin group than in the NC group (P < 0.05), while these two indicators were markedly lower in the ORG + Henagliflozin group relative to the ORG group (P < 0.05). Fasting blood glucose was elevated in the ORG group compared with the NC group (P < 0.05), and Henagliflozin intervention significantly reduced fasting blood glucose in the ORG + Henagliflozin group versus the ORG group (P < 0.05). Intraperitoneal insulin tolerance test was conducted at week 23 to detect blood glucose at 0, 30, 60 and 120 min. The results revealed statistically significant differences: (1) blood glucose levels varied significantly across different time points (P < 0.05); (2) blood glucose differed remarkably among the three groups (P < 0.05), with the ORG group presenting the highest glucose concentration; (3) the dynamic changing trends of blood glucose showed significant intergroup differences (P < 0.05). The area under the ITT curve was increased in the ORG group relative to the NC group (P < 0.05). ITT-AUC was significantly decreased in the ORG + Henagliflozin group compared with the ORG group (P < 0.05), and no statistical difference was detected between the ORG + Henagliflozin group and the NC group (P > 0.05). Intraperitoneal glucose tolerance test was performed at week 24 to measure blood glucose at 0, 30, 60 and 120 min. Statistical differences were observed as follows: (1) blood glucose values differed significantly at distinct time points (P < 0.05); (2) intergroup blood glucose levels had obvious discrepancies (P < 0.05), and the ORG group had the highest blood glucose; (3) significant differences existed in the glucose variation trends among groups (P < 0.05). GTT-AUC was significantly higher in the ORG group than in the NC group (P < 0.05). Henagliflozin treatment lowered GTT-AUC in the ORG + Henagliflozin group versus the ORG group (P < 0.05), and there was no significant difference between the ORG + Henagliflozin group and the NC group (P > 0.05). Serum total cholesterol, triglyceride and low-density lipoprotein cholesterol levels were elevated, whereas high-density lipoprotein cholesterol was reduced in the ORG group compared with the NC group (all P < 0.05). After Henagliflozin intervention, TC, TG and LDL-C were decreased, and HDL-C was increased in the ORG + Henagliflozin group relative to the ORG group (all P < 0.05). Urine albumin/creatinine ratio, serum creatinine and blood urea nitrogen were all higher in the ORG group than in the NC group (P < 0.05); all three renal function markers were significantly lower in the ORG + Henagliflozin group than in the NC group (P < 0.05). Fasting blood ketone level, as well as serum and renal β-hydroxybutyrate concentrations, were significantly higher in the ORG + Henagliflozin group than in the ORG group (P < 0.05). Oil Red O staining showed massive red lipid droplet deposition in renal tubular epithelial cells of ORG mice, which was markedly alleviated after Henagliflozin administration. Hematoxylin-eosin (HE) staining demonstrated irregular glomerular morphology, dilated capillary tufts, thickened mesangial matrix and edematous and deformed renal tubules in ORG mice; all the above renal structural injuries were obviously ameliorated by Henagliflozin treatment. Renal superoxide dismutase (SOD), total antioxidant capacity (TAOC) and glutathione peroxidase (GSH-Px) activities were significantly lower, while malondialdehyde (MDA) content was higher in the ORG group relative to the NC group (all P < 0.05). Henagliflozin intervention upregulated SOD, TAOC and GSH-Px activities and downregulated MDA levels in renal tissues of ORG mice (all P < 0.05). The relative protein expression levels of renal interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly increased in the ORG group compared with the NC group (P < 0.05), and were markedly suppressed in the ORG + Henagliflozin group versus the ORG group. Immunohistochemical staining revealed abundant expression of the renal fibrotic marker α-smooth muscle actin (α-SMA) in ORG mice, and Henagliflozin intervention decreased α-SMA protein expression. Total and nuclear protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in renal tissues were significantly downregulated in the ORG group compared with the NC group (P < 0.05), and were remarkably elevated in the ORG + Henagliflozin group relative to the ORG group (P < 0.05).Conclusion Henagliflozin alleviates glucose and lipid metabolism disorders and ameliorates renal injury in ORG mice, potentially through activation of the Nrf2/HO-1 signaling pathway via ketogenesis.
