Diabetic nephropathy (DN) is a severe complication of diabetic patients [
Recently, a novel antioxidative peptide targeted on mitochondria named MTP131 or SS31 was designed [
Human proximal tubular epithelial cells (HK-2 cells) were cryopreserved at the Institute of Kidney Disease, Central South University. SS31 was synthesised and provided by Chinapeptide Co. Ltd. (Shanghai, China). Streptozocin (STZ) was obtained from Sigma-Aldrich (USA). The selective Drp1 inhibitor Mdivi1 (ab144589) was obtained from Abcam (UK). Anti-fibronectin (FN) antibody (sc-52331), anti-Bcl-2 antibody (sc-56015), anti-IL-1
A total of 40 eight-week-old C57BL/6 mice (about 20 g body weight) were purchased from Slyke Jingda Biotechnology Company (Hunan, China), then they were divided into 4 groups. The control group was injected with sodium citrate buffer only (
Renal tissue sections were cut for hematoxylin-eosin (H&E), periodic acid-Schiff (PAS), and Masson's staining as described previously; glomerular and tubular injury was analyzed using a semiquantitative scoring system as previously described [
Blood glucose was tested using a blood glucose monitor (Roche Accu-Chek, Germany) every two weeks. Mice were placed in individual metabolic cages for a 24-hour urine collection. A mouse urine albumin ELISA kit (Bethyl Laboratories, USA) was used to measure urine albumin concentrations. Serum creatinine, triglyceride, and cholesterol levels were measured by an automated biochemical analyzer (Hitachi 7600, Japan).
Mouse renal tissue sections (3
HK-2 cells were maintained in media containing 5-30 mM D-glucose and other interventions: HK-2 cells maintained in 5 mM D-glucose (LG), HK-2 cells maintained in 30 mM D-glucose (HG), HK-2 cells treated with HG plus SS31 (100 nM), HK-2 cells treated with HG plus Mdivi1 (50
Briefly, after the fractionated proteins were transferred onto a nitrocellulose membrane, they were incubated with various primary antibodies anti-FN (1:1,000), anti-Bcl-2 (1:1,000), anti-Bax (1:1,000), anti-IL-1
After HK-2 cells were treated with various agents, the cells were first immersed in MitoTracker Red (1:1000) solution, then the cells were fixed and permeabilized. After that, the cells were incubated with primary antibody (Drp1, 1:100 dilution). The cells were then incubated with FITC-conjugated secondary antibody and DAPI, and finally a confocal laser scanning microscope was used to capture images (Zeiss LSM 780).
A LSM 780 META laser scanning microscope (Zeiss LSM780) was used to complete the confocal microscopy examination. The LSM 510 software (Zeiss) was used for image analysis [
We used transmission electron microscopy (EM) to observe mitochondrial morphology. Briefly, renal cortices were minced into 1 mm3 pieces, then renal cortices were fixed with 2.5% glutaraldehyde; lastly, thin sections were prepared for EM to delineate the mitochondrial morphology in tubules.
HK-2 cells were incubated with MitoSOX and examined by confocal microscopy to assess mitochondrial ROS levels. The cells were stained with TMRE and examined by confocal microscopy to evaluate the perturbations of MMP (ΔΨm). Mean fluorescence intensity was calculated based on 10 randomly selected fields.
MDA content as well as SOD and GSH-PX activities were determined by chemiluminescence methods as previously described [
We used SPSS 16.0 software to analyze the experimental results. The results were expressed as
At the end of 24 weeks, 3 mice in the STZ group died, 3 mice in the STZ+SS31 group died, and 2 mice in the STZ+NS group died. Administration of SS31 for 24 weeks had no effect on body weight and blood glucose levels (Table
Effect of SS31 on blood glucose, renal function, total cholesterol, and triglyceride and weight of STZ-induced diabetic mice.
Group | BW (g) | KW (mg) | KW/BW (mg/g) | BS (mmol/l) | Scr ( |
BUN (mmol/l) | Proteinuria ( |
Urine volume (ml) | Proteinuria | |
---|---|---|---|---|---|---|---|---|---|---|
Control | 10 | |||||||||
STZ | 10& | |||||||||
STZ+saline | 10& | |||||||||
STZ+SS-31 | 10& |
Note: BS: blood glucose; BW: body weight; KW: kidney weight of the right kidney; Scr: serum creatinine; BUN: blood urea nitrogen; TC: total cholesterol; TG: triglyceride. &3 mice in the STZ group died, 2 mice in the STZ+saline group died, and 3 mice in the STZ+SS31 group died during 24 weeks.
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
It showed that SS31 treatment significantly alleviated mesangial matrix proliferation compared with untreated diabetic mice as indicated by HE, PAS staining, and glomerular damage scores (Figures
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,
As shown in Figure
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,
IHC staining showed that renal IL-1
Renal IL-1
As shown in Figure
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.
Immunofluorescence studies indicated that HG increased Drp1 expression; additionally, MitoTracker staining showed increased mitochondrial fragmentation in HK-2 cells exposed to HG conditions (Figure
Effects of SS31 on Drp1, Mfn1, Caspase1, and IL-1
The present study indicates that SS31 ameliorates renal tubulointerstitial injury in diabetic mice, which might be due to an antioxidant action, as well as decreasing mitochondrial fragmentation then restoration of mitochondria morphology via suppressing the expression of Drp1 and increasing the expression of Mfn1 in renal tubular epithelial cells.
The mitochondria target peptides included SS01, SS02, SS20, and SS31; the structural motif of these peptides was an alternation of aromatic residues and basic amino acids. It has been shown that SS31 could concentrate more than 1000-fold in the mitochondria [
Conventional wisdom suggested that glomerular injury was the major source of DN; however, recent studies indicated that tubulointerstitial lesions also closely correlated with the progression of DN, and the tubulointerstitial injury has been described as diabetic tubulopathy [
The kidney is an organ needing continuous energy consumption due to the excretion and reabsorption process that existed in the renal tubule; there was a large amount of mitochondria both in the tubular and glomerular cells, particularly in the proximal tubular cells [
Mitochondria are a class of highly shape-changed organelles which constantly undergo fusion and fission. In physiological conditions, they were elongated and filamentous, but the shape changed to fragment under stress including various kidney diseases [
Because of a variety of reasons, there still existed several drawbacks in this study; first, we just examined the effects of SS31 on oxidative stress and apoptosis, while other effects such as anti-inflammatory effect had not been evaluated. Second, as we discussed above, mitochondrial fusion and fission were regulated by several factors (e.g., Drp1, Mfn1, OPA1, Mfn2, and Fis1), but in this study, we just examined the inhibiting effect of SS31 on Drp1 and the increasing effect of SS31 on Mfn1. Third, in the vitro experiment, we found that SS31 could inhibit the expression of Drp1 in HK-2 cells under HG condition, and the inhibiting effect was similar with Mdivi1; however, the results were suggestive and not cause-and-effect. In the future study, we will further investigate the more detailed molecular mechanism about SS31 regulating mitochondrial dynamics.
In conclusion, our data showed that SS31 could protect renal tubulointerstitial injury and reduce ROS and apoptosis in diabetic mice, which might be due to the decrease in mitochondrial fragmentation via suppressing the expression of Drp1 and increasing the expression of Mfn1.
The data used to support the findings of this study are available from the first author and corresponding author upon reasonable request.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Shi-kun Yang, Ai-mei Li, and Ya-chun Han contributed equally to this work.
This study was supported by grants from the National Natural Science Foundation of China (81500558, 8197033544, 81870498, and 81600566), Natural Science Foundation of Hunan Province (2018JJ3785), the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University (JY201521), and Zhejiang Natural Science Foundation (LQ16H050001).