Additive protection by LDR and FGF21 treatment against diabetic nephropathy in type 2 diabetes model

Abstract

The onset of diabetic nephropathy (DN) is associated with both systemic and renal changes. Fibroblast growth factor (FGF)-21 prevents diabetic complications mainly by improving systemic metabolism. In addition, low-dose radiation (LDR) protects mice from DN directly by preventing renal oxidative stress and inflammation. In the present study, we tried to define whether the combination of FGF21 and LDR could further prevent DN by blocking its systemic and renal pathogeneses. To this end, type 2 diabetes was induced by feeding a high-fat diet for 12 wk followed by a single dose injection of streptozotocin. Diabetic mice were exposed to 50 mGy LDR every other day for 4 wk with and without 1.5 mg/kg FGF21 daily for 8 wk. The changes in systemic parameters, including blood glucose levels, lipid profiles, and insulin resistance, as well as renal pathology, were examined. Diabetic mice exhibited renal dysfunction and pathological abnormalities, all of which were prevented significantly by LDR and/or FGF21; the best effects were observed in the group that received the combination treatment. Our studies revealed that the additive renal protection conferred by the combined treatment against diabetes-induced renal fibrosis, inflammation, and oxidative damage was associated with the systemic improvement of hyperglycemia, hyperlipidemia, and insulin resistance. These results suggest that the combination treatment with LDR and FGF21 prevented DN more efficiently than did either treatment alone. The mechanism behind these protective effects could be attributed to the suppression of both systemic and renal pathways.

Keywords: fibroblast growth factor-21; inflammation; insulin resistance; low-dose radiation; oxidative stress.

Fig. 1.

Establishing a type 2 diabetic mouse (DM) model using the HFD/STZ (high-fat diet/streptozotocin) model. C57BL/6J mice were fed a HFD (40% of calories from fat) for 12 wk to induce obesity. Body weight (A), glucose tolerance (B), and insulin sensitivity (C) were examined. Three days after injection of STZ, blood glucose was measured (D). Mice were regarded as diabetic once hyperglycemia was observed (>12 mmol/l). Data are presented as means ± SE; n = 9 per group. GTT, glucose tolerance test; ITT, insulin tolerance test. *P < 0.05 vs. HFD group; #P < 0.05 vs. DM group.

Fig. 2.

Effects of combination treatment strategy on hyperglycemia and insulin resistance. Blood glucose levels (A) and insulin tolerance (B) were examined in each group. LDR, low-dose radiation; FGF, fibroblast growth factor. Data are presented as means ± SE; n = 9 per group. *P < 0.05 vs. HFD group; #P < 0.05 vs. DM group; &P < 0.05 vs. LDR group; $P < 0.05 vs. FGF21 group.

Fig. 3.

Effects of combination treatment strategy on diabetes-induced histopathological changes in the kidneys of mice. Representative images of hematoxylin and eosin (A, H&E) staining, periodic acid-Schiff (B, PAS) staining, and Masson's Trichrome staining (C, Masson) to detect renal pathological changes, glomerulosclerosis, and collagen deposition, respectively. Magnification ×400. Glomerulosclerosis and collagen accumulation were examined in PAS- or Masson's-stained slices, respectively, and quantified using Image-Pro Plus 6.0 software (D and E). Renal connective tissue growth factor (CTGF) expression was measured using Western blotting (F). Renal hypertrophy was examined by assessing the change in the kidney weight-to-tibia length ratio (G). Data are presented as means ± SE; n = 9 per group. *P < 0.05 vs. HFD group; #P < 0.05 vs. DM group; &P < 0.05 vs. LDR group.

Fig. 4.

Effects of combination treatment strategy on diabetes-induced oxidative stress in kidneys. Expression of oxidative damage markers 3-nitrotyrosine (3-NT; A) and renal malondialdehyde (MDA; B) were measured using Western blotting and ELISA, respectively. Data are presented as means ± SE; n = 9 per group. *P < 0.05 vs. HFD group; #P < 0.05 vs. DM group; &P < 0.05 vs. LDR group; $P < 0.05 vs. FGF21 group.

Fig. 5.

Effects of combination treatment strategy on renal inflammation in type 2 diabetic mice. Renal tissues were collected from the different groups at the indicated times, and intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor-α (TNF-α), and plasminogen activator inhibitor 1 (PAI-1) expression was measured using Western blotting (A–C). Data are presented as means ± SE; n = 9 per group. *P < 0.05 vs. HFD group; #P < 0.05 vs. DM group; &P < 0.05 vs. LDR group; $P < 0.05 vs. FGF21 group.

Fig. 6.

Effects of combination treatment strategy on expression of renal nuclear factor E2-related factor 2 (Nrf-2) and its downstream targets in type 2 diabetic mice. Renal tissues were collected from the different groups at indicated times, and superoxide dismutase-1 (SOD-1), NAD(P)H:quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1), and Nrf-2 expression was measured using Western blotting (A–D). Data are presented as means ± SE; n = 9 per group. *P < 0.05 vs. HFD group; #P < 0.05 vs. DM group; &P < 0.05 vs. LDR group; $P < 0.05 vs. FGF21 group.