Ferroptosis Enhanced Diabetic Renal Tubular Injury via HIF-1α/HO-1 Pathway in db/db Mice

Abstract

Background: Ferroptosis is a recently identified iron-dependent form of cell death as a result of increased reactive oxygen species (ROS) and lipid peroxidation. In this study, we investigated whether ferroptosis aggravated diabetic nephropathy (DN) and damaged renal tubules through hypoxia-inducible factor (HIF)-1α/heme oxygenase (HO)-1 pathway in db/db mice.

Methods: Db/db mice were administered with or without ferroptosis inhibitor Ferrostatin-1 treatment, and were compared with db/m mice.

Results: Db/db mice showed higher urinary albumin-to-creatinine ratio (UACR) than db/m mice, and Ferrostatin-1 reduced UACR in db/db mice. Db/db mice presented higher kidney injury molecular-1 and neutrophil gelatinase-associated lipocalin in kidneys and urine compared to db/m mice, with renal tubular basement membranes folding and faulting. However, these changes were ameliorated in db/db mice after Ferrostatin-1 treatment. Fibrosis area and collagen I were promoted in db/db mouse kidneys as compared to db/m mouse kidneys, which was alleviated by Ferrostatin-1 in db/db mouse kidneys. HIF-1α and HO-1 were increased in db/db mouse kidneys compared with db/m mouse kidneys, and Ferrostatin-1 decreased HIF-1α and HO-1 in db/db mouse kidneys. Iron content was elevated in db/db mouse renal tubules compared with db/m mouse renal tubules, and was relieved in renal tubules of db/db mice after Ferrostatin-1 treatment. Ferritin was increased in db/db mouse kidneys compared with db/m mouse kidneys, but Ferrostatin-1 reduced ferritin in kidneys of db/db mice. Diabetes accelerated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived ROS formation in mouse kidneys, but Ferrostatin-1 prevented ROS formation derived by NADPH oxidases in db/db mouse kidneys. The increased malondialdehyde (MDA) and the decreased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GSH-Px) were detected in db/db mouse kidneys compared to db/m mouse kidneys, whereas Ferrostatin-1 suppressed MDA and elevated SOD, CAT, and GSH-Px in db/db mouse kidneys. Glutathione peroxidase 4 was lower in db/db mouse kidneys than db/m mouse kidneys, and was exacerbated by Ferrostatin-1 in kidneys of db/db mice.

Conclusions: Our study indicated that ferroptosis might enhance DN and damage renal tubules in diabetic models through HIF-1α/HO-1 pathway.

Keywords: diabetic nephropathy; ferroptosis; heme oxygenase-1; hypoxia-inducible factor-1α; renal tubular injury.

Figure 1

Physical and biochemical characteristics in db/m, db/db, and db/db+Fer1 groups. (A). Body weight. (B). Kidney weight. (C). Blood glucose. (D). Insulin. (E). Serum creatinine (SCR). (F). Urinary albumin-to-creatinine ratio (UACR). Male mice, n = 9/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 2

Renal tubular injury in db/m, db/db, and db/db+Fer1 groups. (A, B). Representative photographs and quantification of kidney injury molecular-1 (KIM-1) (A) and neutrophil gelatinase-associated lipocalin (NGAL) (B) in mouse kidneys measured by western blot. (C, D). Quantification of urinary KIM-1 (C) and NGAL (D) levels measured by ELISA. (E) Representative photographs of mouse kidneys by hexamine silver staining staining. Male mice, n = 6-9/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 3

Renal fibrosis in db/m, db/db, and db/db+Fer1 groups. (A–C). Representative photographs and quantification of renal fibrosis by Masson’s staining (blue) (A) and Sirius red staining (red) (B) (six sections per mouse were analyzed). (D). Representative photographs and quantification of collagen I in mouse kidneys measured by western blot. Male mice, n = 6/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 4

The expression of hypoxia-inducible factor (HIF) -1α and heme oxygenase (HO) -1 in mouse renal tissues. (A, B). Representative photographs and quantification of HIF-1α (A) and HO -1 (B) in mouse kidneys measured by western blot. Male mice, n = 6/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 5

Iron content in db/m, db/db, and db/db+Fer1 groups. (A, B). Representative photographs and quantification of iron content (blue) in kidneys by Lillie staining (six sections per mouse were analyzed). (C). Representative photographs and quantification of ferritin heavy chain in mouse kidneys measured by western blot. (D–F). Quantification of serum iron ion (D), ferritin (E), and transferrin (F) measured by ELISA. Male mice, n = 6–9/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 6

ROS formation in mouse renal tissues. (A, B). Representative photographs and quantification of ROS formation (red) in mouse kidneys by dihydroethidium staining (six sections per mouse were analyzed). (C). Representative photographs and quantification of gp91 phox in mouse kidneys measured by western blot. Male mice, n = 6/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 7

Lipid peroxidation and GPX4 in mouse renal tissues. (A–D). Quantification of malondialdehyde (MDA) (A), superoxide dismutase (SOD) (B), catalase (CAT) (C) and glutathione peroxidases (GSH-Px) (D) in mouse kidneys. (E). Representative photographs and quantification of glutathione peroxidase 4 (GPX4) in mouse kidneys measured by western blot. Male mice, n = 6–9/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.

Figure 8

Renal apoptosis in db/m, db/db, and db/db+Fer1 groups. (A, B). Representative photographs and quantification of renal apoptosis by TUNEL assay (six sections per mouse were analyzed). (C). Representative photographs and quantification of cleaved caspase-3 in mouse kidneys measured by western blot. Male mice, n = 6/group. *P < 0.05, vs db/m group; ^# P < 0.05, vs db/db group. Db/m, db/m mice; db/db, db/db mice without Ferrostatin-1 treatment; db/db+Fer1, db/db mice with Ferrostatin-1 treatment. Data are means ± S.E.M.