Acute hyperglycemia induces podocyte apoptosis by monocyte TNF-α release, a process attenuated by vitamin D and GLP-1 receptor agonists

Figures

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  Fig. 1.. Acute hyperglycemia does not injure podocytes directly but induces a monocyte/macrophage secretome that drives podocyte damage/apoptosis. Human immortalized podocytes were exposed to: (A, B) euglycemic (5 mM D-glucose, green dots) or hyperglycemic (HG: 16.7 mM D-glucose, blue dots) conditions for 24 h; (C, D) conditioned media from human normal monocytes exposed to 5 or 16.7 mM glucose for 6 h; (E, G) conditioned media from THP1 cells (macrophage phenotype) exposed to 5 or 16.7 mM glucose for 24 h. (A, C, D) Podocyte injury (Normalized podocalyxin:β-actin protein expression in euglycemia (green dots), and hyperglycemia-induced fold reduction in podocalyxin:β-actin ratio (blue dots) from at least 3 independent experiments; (B, D, F) Podocyte apoptosis (Normalized basal caspase 3/7 activity in euglycemia (green dots) and hyperglycemia-induced fold-increases in caspase 3/7 activity (blue dots). Each dot represent the average of quadruplicate measurements; the number of dots indicate the number of individuals or independent experiments. *p < 0.05.

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  Fig. 2.. Acute hyperglycemia-induced monocyte TNF-α secretion causes podocyte apoptosis in a vitamin D-dependent manner. (A) Normalized TNF-α secretion from peripheral blood monocytes from 11 normal human subjects exposed ex vivo for 6 h to euglycemic (5 mM D-glucose, green dots) conditions, and individual fold increases in TNF-α secretion in response to hyperglycemia (HG, 16.7 mM D-glucose, blue dots); (B) Normalized TNF-α secretion in 24 h by THP-1 macrophages cultured under euglycemic conditions (green dots), and fold increases in TNF-α secretion in response to hyperglycemia (blue dots) (from n = 4 independent experiments); (C) Podocyte apoptotic rate (normalized caspase 3/7 activity) after 24 h exposure to conditioned media from peripheral blood monocytes from 6 normal individuals, each exposed to hyperglycemic conditions (dark blue dots) + /− TNF-α neutralizing antibody (light blue dots) for 6 h; (D) Podocyte apoptotic rate (normalized caspase 3/7 activity; n = 5) after 24 h exposure to conditioned media from THP-1 macrophages exposed to hyperglycemic conditions (dark blue dots) + /− TNF-α neutralizing antibody (light blue dots) for 24 h; (E) Fold differences in monocyte TNF-α…

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  Fig. 3.. Inhibition of glycolysis, ER and oxidative stress attenuates acute hyperglycemia-induced monocyte TNF-α secretion. TNF-α secretion (N = 5) by 50,000 human monocytes cultured under hyperglycemic conditions (HG: 16.7 mM D-glucose, dark blue bar) with and without the glycolysis inhibitor 2-deoxyglucose (2DG red bar), the ER stress inhibitor 4-phenylbutyric acid (PBA, light blue bar), or the oxidative stress inhibitor N-acetylcysteine (NAC, yellow bar) for 6 h. Bars and error bars represent Mean + SEM, *p < 0.05.

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  Fig. 4.. ER and oxidative stress activation in human monocytes mediate acute hyperglycemia-induced iRhom2/Adam17 expression. (A, B) ADAM17 and its partner for TNF-α release iRhom2 mRNA expression in human monocytes cultured either under euglycemic (5 mM D-glucose, green bar) or hyperglycemic (HG: 16.7 mM D-glucose, dark blue bar) conditions for 6 h (n = 8); ADAM17 or iRhom2 mRNA expression in human monocytes cultured under (C,D) ER inhibitors (PBA n = 6) or (D, E) oxidative stress inhibitor (NAC; n = 6). Bars and error bars represent mean + SEM. *p < 0.05.

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  Fig. 5.. GLP-1 receptor activation ameliorates podocyte apoptosis by attenuating monocyte/macrophage TNF-α secretion through a reduction in iRhom2 and ADAM17 expression. (A-C) TNF-α secretion (n = 5), iRHOM2 (n = 6) and ADAM17 (n = 6) expression in human monocytes cultured under hyperglycemic (HG: 16.7 mM D-glucose) conditions for 2 h (dark blue dots or bars) + /− 10 nM GLP-1 RA (light blue dots or bars); (D) TNF-α secretion from THP-1 macrophages exposed to hyperglycemic (HG, 16.7 mM D-glucose) conditions (dark blue dots) or HG + GLP-1 RA (light blue dots) for 24 h (n = 4); (E) caspase 3/7 activity in podocytes exposed for 24 h to conditioned media from THP-1 macrophages cultured under hyperglycemic (HG, 16.7 mM D-glucose, dark blue dots) conditions or HG + GLP-1 RA (light blue dots) for 24 h (n = 4). Each dot represents the average of duplicate measurements per individual or experimental condition, and the number of dots indicates the total number of individuals or independent experiments. Bars and error bars indicate Mean ± SEM of 3 independent experiments per experimental condition. *p < 0.05..