Mitochondria-Targeted Peptide SS31 Attenuates Renal Tubulointerstitial Injury via Inhibiting Mitochondrial Fission in Diabetic Mice

Abstract

Objective: Renal tubular injury is an early characteristic of diabetic nephropathy (DN) that is related to mitochondrial dysfunction. In this study, we explore the effects and mechanisms of mitochondria-targeted peptide SS31 on renal tubulointerstitial injury in DN.

Method: 40 C57BL/6 mice were randomly divided into control group, STZ group, STZ+SS31 group, and STZ+normal saline group. SS31 was intraperitoneally injected to the mice every other day for 24 weeks. Renal lesions and the expression of Drp1, Mfn1, Bcl-2, Bax, Caspase1, IL-1β, and FN were detected. In in vitro studies, HK-2 cells were incubated with different concentrations of D-glucose (5, 30 mM) or combined with SS31 and Drp1 inhibitor Midivi1. Mitochondrial ROS, membrane potential, and morphology have been detected to evaluate the mitochondrial function.

Results: Compared with diabetic mice, the levels of serum creatinine and microalbuminuria were significantly decreased in the SS31 group. Renal tubulointerstitial fibrosis, oxidative stress, and apoptosis were observed in diabetic mice, while the pathological changes were reduced in the SS31-treatment group. SS31 could decrease the expression of Drp1, Bax, Caspase1, IL-1β, and FN in the renal tissue of diabetic mice, while increasing the expression of Mfn1. Additionally, mitochondria exhibit focal enlargement and crista swelling in renal tubular cells of diabetic mice, while SS31 treatment could partially block these changes. An in vitro study showed that pretreatment with SS31 or Drp1 inhibitor Mdivi1 could restore the level of mitochondrial ROS, the membrane potential levels, and the expressions of Drp1, Bax, Caspase1, IL-1β, and FN in HK-2 cells under high-glucose conditions.

Conclusion: SS31 protected renal tubulointerstitial injury in diabetic mice through a decrease in mitochondrial fragmentation via suppressing the expression of Drp1 and increasing the expression of Mfn1.

Figure 1

Effects of SS31 on biochemical index in diabetic mice. (a–c) Body weight, blood glucose, and proteinuria levels in mice from 0 to 24 weeks. (d) Renal malondialdehyde (MDA) concentrations of various groups. (e) Renal superoxide dismutase (SOD) concentrations of various groups. (f) Renal glutathione peroxidase (GSH-PX) concentrations of various groups. Data are presented as mean ± SD, ∗P < 0.01 vs. STZ groups, ^#P < 0.01 vs. control groups. n = 10.

Figure 2

Effects of SS31 on renal tubulointerstitial damage in diabetic mice. (a) Renal tissue stained with HE, PAS, Masson trichrome, and immunohistochemical analysis of fibronectin (FN) (magnification ×400). (b) Tubulointerstitial damage scores, ∗P < 0.01 vs. STZ groups, ^#P < 0.01 vs. control groups, n = 3. (c) Glomerular damage scores, ∗P < 0.01 vs. STZ groups, and ^#P < 0.01 vs. control groups, n = 3. (d) Western blot analysis of FN protein. (e) Each bar graph represents the ratios of FN to β-actin, ∗P < 0.01 vs. STZ groups, ^#P < 0.01 vs. control groups, n = 3.

Figure 3

Effects of SS31 on apoptosis in renal tissue of diabetic mice. (a) TUNEL-IHC staining (upper panels) and immunohistochemical analysis of Bcl-2 (middle panels) and Bax (lower panels) in mouse renal tissue in various groups (magnification ×400). (b) Bar graphs represent quantification of tissues stained with TUNEL, ∗P < 0.01, vs. STZ groups, ^#P < 0.01, vs. control groups, n = 3. (c) Western blot analysis of Bcl-2 (upper panel) and Bax (middle panel) protein expression. (d and e) Each bar graph represents the densitometric analyses of Bcl-2 to β-actin (d) and Bax to β-actin (e). ∗P < 0.01 vs. STZ groups, ^#P < 0.01 vs. control groups, n = 3.

Figure 4

Renal IL-1β, Caspase1, Mfn1, and Drp1 expression in diabetic mice following SS31 treatment. (a) Renal immunohistochemical staining with anti-IL-1β antibody (upper panel), anti-Caspase1 antibody (middle panel), anti-Drp1 antibody (middle panel), and anti-Mfn1 (lower panel) (magnification ×400). (b) Western blot analysis of Mfn1 (upper panel), Drp1 (middle panel), Caspase1 (middle panel), and IL-1β (bottom panel) protein expression. (c–f) Densitometric analyses of the Western blotting results: IL-1β to β-actin (c), Caspase1 to β-actin (d), Drp1 to β-actin (e), and Mfn1 to β-actin (f). Values are mean ± SD, ∗P < 0.01, vs. STZ groups, ^#P < 0.01 vs. control groups, n = 3.

Figure 5

Effects of SS31 on mitochondrial morphology in the kidney of diabetic mice and mitochondrial ROS and mitochondrial membrane potential in HK-2 cells exposed to HG after SS31 administration. (a) EM analysis showed that the diabetic mouse renal tissues displayed obvious mitochondrial morphological changes; these changes were reversed by SS31 treatment (magnification ×5,000). (b) Representations of mitochondrial ROS levels (upper panel) and mitochondrial membrane potential (MMP, bottom panel) in HK-2 cells exposed to HG treatment with SS31 or Mdivi1 pretreatment (magnification ×400). (c) Relative percentages of fragmented mitochondria in the four groups. ∗P < 0.01 vs. STZ groups, ^#P < 0.01 vs. control groups, n = 3. (d, e) Quantification of mitochondrial ROS production as measured with MitoSox Red staining (d) and MMP as measured with TMRE staining (e). ∗P < 0.01 vs. HG groups, ^#P < 0.01 vs. LG groups, n = 3.

Figure 6

Effects of SS31 on Drp1, Mfn1, Caspase1, and IL-1β protein expression in HK-2 cells exposed to HG. (a) IF analysis of Drp1 expression and mitochondrial morphology in HK-2 cells exposed to HG conditions and pretreated with SS31 or Mdivi1. (b) Western blot analysis of Mfn1 (upper panel), Drp1, Caspase1 (middle panel), and IL-1β (bottom panel) protein expression in HK-2 cells exposed to HG conditions and pretreated with SS31 or Mdivi1. (c–e) Densitometric analyses of the Western blotting results: Drp1 to β-actin (c), Mfn1 to β-actin (d), Caspase1 to β-actin (e), and IL-1β to β-actin (f). The data are presented as mean ± SD, ∗P < 0.01 vs. HG groups, ^#P < 0.01 vs. LG groups, n = 3.