Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

Figure 1 High-dose glucose-induced nephrin endocytosis promotes podocyte injury and endocytosis inhibitors block this effect. A and B: Western blot analysis of cytoplasmic nephrin, membrane nephrin, and total nephrin in podocytes after different treatments and densitometric quantification of these results (n = 3); C: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue: Nuclei; red: Cytoskeleton); D: Adhesion of podocytes after different treatments (n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 400; scale bar: 20 μm) and quantification of these results (n = 3). ^aP < 0.05; ^bP < 0.001. HG: High-dose glucose.

Figure 2 Rab5 promotes podocyte injury by increasing nephrin endocytosis, Rab5 silencing blocks this effect, and Rab5 upregulation promotes this effect. A and B: Western blot analysis of Rab5 in podocytes after different treatments and densitometric quantification of these results (n = 3); C and D: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) in podocytes after different treatments and densitometric quantification of the C-nephrin/T-nephrin ratio (n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 1000; scale bar: 50 μm) and quantification of these results (n = 3); G: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue nuclei; red: Cytoskeleton); H: Adhesion of podocytes after different treatments (n = 3). ^aP < 0.05; ^bP < 0.001. HG: High-dose glucose; NC: Negative control.

Figure 3 Rab5-mediated nephrin endocytosis by podocytes depends on Rab5 activation. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes after different treatments and quantification of these results (n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes after different treatments (magnification: × 630; Scale bar: 20 μm); E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes after different treatments and quantification of these results (n = 3); G and H: Western blot analysis of cytoplasmic nephrin and total nephrin in podocytes after different treatments and quantification of the C-nephrin/T-nephrin ratio (n = 3). ^aP < 0.05; ^bP < 0.001. HG: High-dose glucose.

Figure 4 IL-6 secretion by HK-2 cells contributes to the HG-induced injury of podocytes. A and B: Western blot analysis of E-cadherin, vimentin, and α-SMA in HK-2 cells after different treatments and quantification of these results (n = 3); C: ELISA measurements of IL-6 after different treatments (n = 3); D: Method used to examine the effect of HK-2 conditioned medium on podocytes; E: Representative confocal microscopy images of podocytes after culture in different conditioned media (magnification: × 1000; scale bar: 50 μm; blue: Nuclei: Red: Cytoskeleton); F: Quantitative analysis of podocyte adhesion after culture in different conditioned media (n = 3); G and H: Representative electron microscopy images of podocyte spreading after culture in different conditioned media (magnification: × 400; scale bar: 20 μm) and quantification of these results (n = 3). ^aP < 0.05; ^bP < 0.001. HG: High-dose glucose; NAb: Neutralizing Ab.

Figure 5 IL-6/Rab5 signaling mediates nephrin endocytosis in podocytes by promoting crosstalk between HK-2 cells and podocytes. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes cultured in different conditioned media and quantification of these results (n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes grown in different conditioned media (magnification: × 630; scale bar: 20 μm); E: Co-immunoprecipitation of nephrin and active Rab5 in podocytes grown in different conditioned media and quantification of these results (n = 3; F: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) after growth in different conditioned media and quantification of the C-nephrin/T-nephrin ratio (n = 3). ^aP < 0.05; ^bP < 0.001. HG: High-dose glucose; NAb: Neutralizing Ab.

Figure 6 Nicotinamide mononucleotide alleviates podocyte injury in diabetic mice. A and B: Blood glucose and HbA1c levels in the different groups; C: Systolic and diastolic blood pressure in the different groups; D and E: Representative PAS staining (magnification × 400; scale bar: 50 μm) and quantification of the glomerular surface area in different groups (n = 10 glomeruli/group); F: Representative electron microscopy images of podocyte foot process effacement in the different groups (magnification × 10,000; scale bar: 100 μm); G: Urinary albumin excretion (albumin/creatinine ratio) in the different groups (n = 5); H and I: Co-localization images and quantification of the epithelial-to-mesenchymal transition, indicated by altered E-cadherin and α-SMA expression, in the different groups (magnification × 630; scale bar: 20 μm); J and K: Immunohistochemical staining and quantification of glomerular expression of Rab5-GTP in the different groups (magnification × 400; scale bar: 20 μm); L and M: Immunofluorescence co-localization and quantification of nephrin and active Rab5 (Rab5-GTP) in the different groups (magnification × 630; scale bar: 20 μm). Data are expressed as mean ± SEM. ^aP < 0.05; ^bP < 0.001. DN: Diabetic nephropathy; NMN: Nicotinamide mononucleotide; AOC: Area of coverage.

Figure 7 Nicotinamide mononucleotide protection depends on crosstalk between proximal tubular epithelial cell and podocytes. A and B: Western blot analysis of epithelial-to-mesenchymal transition markers in HK-2 cells that received different treatments and quantification of these results (n = 3); C: IL-6 secretion by HK-2 cells that received different treatments (n = 3); D: Co-immunoprecipitation of active Rab5 in podocytes cultured in different conditioned media and quantification of these results; E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes cultured in different conditioned media and quantification of the results (n = 3); G and H: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) from podocytes cultured in different conditioned media and quantification of C-nephrin/T-nephrin ratio (n = 3); I: Representative confocal microscopy images of podocytes cultured in different conditioned media (magnification: × 1000; scale bar: 50 μm; red: Cytoskeleton; blue: Nuclei); J: Quantification of podocyte adhesion after culture in different conditioned media (n = 3); K: Representative electron microscopy images of podocyte spreading after culture in different conditioned media (magnification: × 400; scale bar: 20 μm); L: Quantitative analysis of podocyte spreading after culture in different conditioned media (n = 3). ^aP < 0.05; ^bP < 0.001. HG: High-dose glucose; NMN: Nicotinamide mononucleotide.

Figure 8 Possible mechanism of crosstalk between proximal tubular epithelial cells and podocytes and effect of nicotinamide mononucleotide during diabetic nephropathy. High-dose glucose induces the epithelial-mesenchymal transition and secretion of IL-6 by proximal tubular epithelial cells (PTECs). Podocytes sense this IL-6, and this leads to the binding of internalized nephrin with active Rab5, followed by disruptions of the cytoskeleton, podocyte adhesion, and podocyte spreading. nicotinamide mononucleotide blocks signaling from the PTECs, decreases the binding of nephrin with active Rab5, and ameliorates cellular damage. EMT: Epithelial-mesenchymal transition; NMN: Nicotinamide mononucleotide.