SRS 16-86 promotes diabetic nephropathy recovery by regulating ferroptosis

Abstract

Diabetic nephropathy (DN) is a common complication of diabetes mellitus (DM), and cell death plays an important role. Ferroptosis is a recently discovered type of iron-dependent cell death and one that is different from other kinds of cell death including apoptosis and necrosis. However, ferroptosis has not been described in the context of DN. This study explored the role of ferroptosis in DN pathophysiology and aimed to confirm the efficacy of the ferroptosis inhibitor SRS 16-86 on DN. Streptozotocin injection was used to establish the DM and DN animal models. To investigate the presence or occurrence of ferroptosis in DN, we assessed the concentrations of iron, reactive oxygen species and specific markers associated with ferroptosis in a rat model of DN. Additionally, we performed haematoxylin-eosin staining, blood biochemistry, urine biochemistry and kidney function analysis to evaluate the efficacy of the ferroptosis inhibitor SRS 16-86 in ameliorating DN. We found that SRS 16-86 could improve the recovery of renal function after DN by upregulating glutathione peroxidase 4, glutathione and system x_c ^-light chain and by downregulating the lipid peroxidation markers and 4-hydroxynonenal. SRS 16-86 treatment could improve renal organization after DN. The inflammatory cytokines interleukin 1β and tumour necrosis factor α and intercellular adhesion molecule 1 were significantly decreased following SRS 16-86 treatment after DN. The results indicate that there is a strong connection between ferroptosis and the pathological mechanism of DN. The efficacy of the ferroptosis inhibitor SRS 16-86 in DN repair supports its use as a new therapeutic treatment for DN.

Keywords: diabetic nephropathy; ferroptosis; ferroptosis inhibitor.

FIGURE 1

Establishment of the DM and DN animal models. (a) A schematic representation of the animal model. (b, c) Blood glucose (b) and body weight (c) after STZ treatment. Data are shown as means ± SD; n = 6/group.

FIGURE 2

Urinary biochemical indices were used to evaluate renal function. Twenty‐four hour urine volume (a) and 24‐h UTP (b) after STZ treatment. (n = 6/group; ns: P > 0.05; ****P < 0.0001.)

FIGURE 3

Reduced iron overload and ROS by ferroptosis inhibition. The content of iron overload (a) and expression of ROS (b) in all groups. (n = 6/group; ****P < 0.0001.)

FIGURE 4

Expression changes of xCT–GSH–GPX4–4HNE pathway. (a) Representative western blot images of xCT, GPX4 and 4‐HNE. (b–d) Quantifications of western blot band of xCT (b), GPX4 (c) and 4‐HNE (d). (e) Quantification of GSH assessment. (f) Expression of MDA. (n = 6/group; **P < 0.01; ***P < 0.001; ****P < 0.0001.)

FIGURE 5

Reduction of DN inflammation by ferroptosis inhibitor. (a) Representative western blot images of TNF‐α, IL‐1β and ICAM‐1. (b–d) Quantification of western blot band of TNF‐α (b), IL‐1β (c) and ICAM‐1 (d). (n = 6/group; *P < 0.05; ***P < 0.001; ****P < 0.0001.)

FIGURE 6

(a) The renal organizational structure in control group, DN group and DN‐SRS group. (b–d) The score of glomerular and tubular damage, as well as fibrosis in control group, DN group and DN‐SRS group. (n = 6/group; ***P < 0.001; ****P < 0.0001.)

FIGURE 7

Improved renal function recovery after DN via the inhibition of ferroptosis. Urine biochemical indicators including 24‐h UTP (a) and ACR (b) in the DN‐SRS group were markedly lower than those in the DN group (n = 6/group; **P < 0.01; ****P < 0.0001.)

FIGURE 8

Improved renal function recovery after DN via the inhibition of ferroptosis. The blood biochemical indicators including UA (a), urea (b) and creatinine (CRE) (c) in the DN‐SRS group were markedly lower than that in the DN group. (n = 6/group; *P < 0.05; **P < 0.01; ****P < 0.0001.)