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. 2024 Dec;46(2):2381597.
doi: 10.1080/0886022X.2024.2381597. Epub 2024 Jul 22.

Mesenchymal stem cell-derived exosomes ameliorate diabetic kidney disease through NOD2 signaling pathway

Yinghui Wang  1   2 , Donglin Lu  1   2 , Shasha Lv  1   2 , Xiangchun Liu  1   2 , Gang Liu  1   2   3
Affiliations

Mesenchymal stem cell-derived exosomes ameliorate diabetic kidney disease through NOD2 signaling pathway

Yinghui Wang et al. Ren Fail. 2024 Dec.

Abstract

Background and aims: Diabetic kidney disease (DKD) is one of the most common complications of diabetes. It is reported that mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) may have great clinical application potential for the treatment of DKD, but the underlying mechanism has not been illustrated. To clarify the effect of MSC-Exo on NOD2 signaling pathway in podocytes under high glucose (HG) and DKD, we conduct this study.

Methods: We co-cultured podocytes and MSCs-Exo under 30 mM HG and injected MSCs-Exo into DKD mice, then we detected the NOD2 signaling pathway by western blot, qRT-PCT, immunofluorescence, transmission electron microscopy and immunohistochemistry both in vitro and in vivo.

Results: In vitro, HG lead to the apoptosis, increased the ROS level and activated the NOD2 signaling pathway in podocytes, while MSCs-Exo protected podocytes from injury reduced the expression of inflammatory factors including TNF-α, IL-6, IL-1β, and IL-18 and alleviated the inflammatory response, inhibited the activation of NOD2 signaling pathway and the expression of it's downstream protein p-P65, p-RIP2, prevented apoptosis, increased cell viability in podocytes caused by HG. In vivo, MSCs-Exo alleviated renal injury in DKD mice, protected renal function, decreased urinary albumin excretion and inhibited the activation of NOD2 signaling pathway as well as the inflammation in renal tissue.

Conclusion: MSCs-Exo protected the podocytes and DKD mice from inflammation by mediating NOD2 pathway, MSCs-Exo may provide a new target for the treatment of DKD.

Keywords: Diabetic kidney disease; NOD2; exosomes; mesenchymal stem cells.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Identification and localization of HUC-MSCs derived exosomes. (a) Representative Western blot gel documents showing the protein expression of TSG101, CD63, CD81 in MSCs-Exo. (b) Morphology of MSCs-Exo under transmission electron microscopy (bar = 100μm). (c) Nanoparticle tracking method showing the diameters and concentrations of MSCs-Exo. (d,e) Representative immunofluorescent staining and summarized data showing the uptake of PKH67-labeled MSCs-Exo by podocytes at different time points (12, 24 and 48 h) (n = 3, bar = 10μm).
Figure 2.
Figure 2.
Protective effects of MSCs-exo on podocytes. (a,b) The viability of podocytes under different doses of HG and AGEs for 48 h (n = 3). (c,d) Respective viability of podocytes treated with increasing doses of MSCs-Exo under 30 mM HG and 200 μg/ml AGEs for 48 h (n = 3). (e,f) Summarized data showing podocyte apoptosis under HG (30 mM) with or without MSCs-Exo (30μg/ml) for 48 h determined by flow cytometric analysis (n = 3). (f,h) Summarized data showing podocyte apoptosis under 200 μg/ml AGEs with or without MSCs-Exo (30μg/ml) for 48 h determined by flow cytometric analysis (n = 3). (i) Representative images of ROS changes in podocytes under HG (30 mM) with or without MSCs-Exo (30μg/ml) for 48 h visualized by DHE (red) staining and nuclei (blue) staining (bar = 20μm). (j) Representative confocal microscopic images of F-actin using phalloidine (red) staining, nephrin (green) staining and nuclei (blue) staining in podocytes under HG (30 mM) with or without MSCs-Exo (30μg/ml) for 48 h (n = 3, bar = 20μm). Exo: HUC-MSCs derived exosomes; NG: normal glucose; HG: high glucose; AGEs: advanced glycation end products; Data are represented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.
Figure 3.
Figure 3.
The expression of NOD2 in podocytes and DKD mice. (a) Relative mRNA level of NOD2 in podocytes under different concentrations of HG for 48 h (n = 3). (b, c) Representative western blot gel documents and summarized data showing the protein levels of NOD2 in podocytes under different concentrations of HG for 48 h (n = 3). (d) Relative mRNA levels of NOD2 in the kidney from HFD/STZ-induced DKD mice and db/db mice (n = 3). (e, f) Representative Western blot gel documents and summarized data showing the protein levels of NOD2 in the kidney from HFD/STZ-induced DKD mice and db/db mice (n = 3). NG: normal glucose; HG: high glucose; data are represented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.
Figure 4.
Figure 4.
MSCs-Exo specifically inhibit NOD2 signaling activation in podocytes under HG. (a) Levels of proinflammatory factors (TNF-α, IL-6, IL-1β, and IL-18) expression in supernatants from podocytes with different treatments for 48 h (MSCs-Exo: 30μg/ml; HG: 30mM; n = 3). (b, c) Representative immunofluorescent staining and summarized data showing the effect of MSCs-Exo (30 μg/ml) on the expression of NOD2 in podocytes with different treatments for 48 h (MSCs-Exo: 30μg/ml; HG: 30mM for 48 h; n = 3; bar = 10μm). (e) Relative mRNA level of NOD2 and podocyte marker-nephrin in podocytes with different treatments for 48 h (MSCs-Exo: 30μg/ml; HG: 30mM for 48 h; n = 3). (d, f) Representative Western blot gel documents and summarized data showing the protein levels of nephrin, NOD2, p-P65 and p-RIP2 in podocytes with different treatments for 48 h (MSCs-Exo: 30μg/ml; HG: 30mM for 48 h; n = 3). NG: normal glucose; HG: high glucose; Exo: HUC-MSCs derived exosomes. Data are represented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.
Figure 5.
Figure 5.
MSCs-Exo ameliorate renal injury in DKD mice. (a) Representative immunofluorescent staining showing the localization of PKH67-labeled Exo (n = 3, bar = 20μm). (b) Representative images of Masson staining showing the renal fibrosis in kidneys (n = 4, bar = 20μm). (c) Representative images of PAS staining showing the typical glomerular structure changes in kidneys (n = 4, bar = 20μm). (d) Physical and biochemical parameters of four groups of mice (n = 4). Exo: HUC-MSCs derived exosomes; Ins: insulin; Con: db/m mice; DKD: db/db mice; DKD + Exo: db/db mice with Exo; DKD + Ins: db/db mice with insulin. Data are represented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 6.
Figure 6.
Protective effects of MSCs-exo in DKD mice. (a, c) Representative immunofluorescent staining and summarized data showing the expression of podocyte marker: nephrin in the kidney from different groups of mice (n = 4, bar = 20μm). (b, d) Representative immunofluorescent staining and summarized data showing the WT-1+ podocytes in the kidney from different groups of mice (n = 4, bar = 20μm). (e) Representative photomicrographs showing morphological changes in podocytes foot processes in different groups of mice by transmission electron microscopy (TEM) analyses (n = 4, bar = 1μm, 10000×). (f) Quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice by TEM analyses (n = 4). Exo: HUC-MSCs derived exosomes; Ins: insulin; Con: db/m mice; DKD: db/db mice; DKD + Exo: db/db mice with Exo; DKD + Ins: db/db mice with insulin. Data are represented as mean ± SD, *p < 0.05l **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant.
Figure 7.
Figure 7.
MSCs-Exo specifically inhibit NOD2 signaling activation in DKD mice. (a) Levels of proinflammatory factors (TNF-α, IL-6, IL-1β, and IL-18) expressionin in different groups of mice (n = 4). (b) Representative immunohistochemical images of NOD2 in four groups of kidneys (n = 4, bar = 20μm). (c) Relative mRNA level of NOD2 in four groups of kidneys (n = 3). (d) (e) Representative western blot gel documents and summarized data showing the protein levels of NOD2, p-P65 and p-RIP2 in four groups of kidneys (n = 3). Exo: HUC-MSCs derived exosomes; Ins: insulin; Con: db/m mice; DKD: db/db mice; DKD + Exo: db/db mice with Exo; DKD + Ins: db/db mice with insulin. Data are represented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 8.
Figure 8.
Graphical abstract. MSCs-Exo protected the podocytes from inflammation by mediating NOD2 pathway. P: phosphorylation.
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This work was supported by the national natural science foundation of China [82370720]; the national key R&D program of China [2022YFC2703804]; fostering foundation of the second hospital of Shandong University [2022YP46].

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