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Interleukin-20 targets podocytes and is upregulated in experimental murine diabetic nephropathy

View Article: PubMed Central - PubMed

ABSTRACT

Interleukin (IL)-20, a proinflammatory cytokine of the IL-10 family, is involved in acute and chronic renal failure. The aim of this study was to elucidate the role of IL-20 during diabetic nephropathy development. We found that IL-20 and its receptor IL-20R1 were upregulated in the kidneys of mice and rats with STZ-induced diabetes. In vitro, IL-20 induced MMP-9, MCP-1, TGF-β1 and VEGF expression in podocytes. IL-20 was upregulated by hydrogen peroxide, high-dose glucose and TGF-β1. In addition, IL-20 induced apoptosis in podocytes by activating caspase-8. In STZ-induced early diabetic nephropathy, IL-20R1-deficient mice had lower blood glucose and serum BUN levels and a smaller glomerular area than did wild-type controls. Anti-IL-20 monoclonal antibody (7E) treatment reduced blood glucose and the glomerular area and improved renal functions in mice in the early stage of STZ-induced diabetic nephropathy. ELISA showed that the serum IL-20 level was higher in patients with diabetes mellitus than in healthy controls. The findings of this study suggest that IL-20 induces cell apoptosis of podocytes and plays a role in the pathogenesis of early diabetic nephropathy.

No MeSH data available.


Functions of IL-20 in mouse podocytes. (a–c) Mouse podocytes were treated with hydrogen peroxide (0.5 mM), glucose (25 mM) and TGF-β1 (20 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR using primers specific for IL-20 to amplify the transcripts. GAPDH served as an input control. *P<0.05, **P<0.01 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (d) Mouse podocytes were treated with IL-20 (100, 200 or 400 ng ml−1) and glucose (25 mM) for 72 h. The cells were fixed with ethanol, stained with propidium iodide (PI) and analyzed by flow cytometry. The percentages of dead cells (M1 region) are shown. *P<0.05 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (e) Mouse podocytes were treated with glucose (25 mM) and IL-20 (200 ng ml−1) for 24 h. The cells were stained with the TUNEL agent. The data are representative of three independent experiments. Magnification: × 200. Scale bars, 30 μm. (f) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with IL-20 (400 ng ml−1) for 24 h. Total cell lysates were analyzed by western blotting with specific antibodies against caspase-8 and β-actin. The data are representative of three independent experiments. (g) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with mIL-20 (200 ng ml−1) for the indicated time periods. Total cell lysates were analyzed by western blotting with specific antibodies against phospho-p38, JNK, ERK and β-actin. The data are representative of three independent experiments.
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fig2: Functions of IL-20 in mouse podocytes. (a–c) Mouse podocytes were treated with hydrogen peroxide (0.5 mM), glucose (25 mM) and TGF-β1 (20 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR using primers specific for IL-20 to amplify the transcripts. GAPDH served as an input control. *P<0.05, **P<0.01 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (d) Mouse podocytes were treated with IL-20 (100, 200 or 400 ng ml−1) and glucose (25 mM) for 72 h. The cells were fixed with ethanol, stained with propidium iodide (PI) and analyzed by flow cytometry. The percentages of dead cells (M1 region) are shown. *P<0.05 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (e) Mouse podocytes were treated with glucose (25 mM) and IL-20 (200 ng ml−1) for 24 h. The cells were stained with the TUNEL agent. The data are representative of three independent experiments. Magnification: × 200. Scale bars, 30 μm. (f) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with IL-20 (400 ng ml−1) for 24 h. Total cell lysates were analyzed by western blotting with specific antibodies against caspase-8 and β-actin. The data are representative of three independent experiments. (g) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with mIL-20 (200 ng ml−1) for the indicated time periods. Total cell lysates were analyzed by western blotting with specific antibodies against phospho-p38, JNK, ERK and β-actin. The data are representative of three independent experiments.

Mentions: Other studies have reported that glucose, ROS and TGF-β1 can activate podocytes.3, 7 Therefore, we examined whether IL-20 expression could be induced in mouse podocytes in the presence of these stimuli. RT-qPCR showed that hydrogen peroxide markedly upregulated the IL-20 transcripts in podocytes (Figure 2a). In addition, IL-20 expression was also significantly upregulated in podocytes after glucose and TGF-β1 treatment (Figure 2b and c). NAC (N-acetyl-L-cysteine) is a potent free-radical scavenger that is known to inhibit ROS-induced apoptosis. To assess whether NAC affects H2O2-induced IL-20 expression in podocytes, mouse podocytes were preincubated with 5–20 mM of NAC for 1 h and then treated with H2O2 for another 8 h. RT-qPCR showed that NAC dose-dependently inhibited H2O2-induced IL-20 expression in podocytes (Supplementary Figure S2).


Interleukin-20 targets podocytes and is upregulated in experimental murine diabetic nephropathy
Functions of IL-20 in mouse podocytes. (a–c) Mouse podocytes were treated with hydrogen peroxide (0.5 mM), glucose (25 mM) and TGF-β1 (20 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR using primers specific for IL-20 to amplify the transcripts. GAPDH served as an input control. *P<0.05, **P<0.01 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (d) Mouse podocytes were treated with IL-20 (100, 200 or 400 ng ml−1) and glucose (25 mM) for 72 h. The cells were fixed with ethanol, stained with propidium iodide (PI) and analyzed by flow cytometry. The percentages of dead cells (M1 region) are shown. *P<0.05 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (e) Mouse podocytes were treated with glucose (25 mM) and IL-20 (200 ng ml−1) for 24 h. The cells were stained with the TUNEL agent. The data are representative of three independent experiments. Magnification: × 200. Scale bars, 30 μm. (f) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with IL-20 (400 ng ml−1) for 24 h. Total cell lysates were analyzed by western blotting with specific antibodies against caspase-8 and β-actin. The data are representative of three independent experiments. (g) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with mIL-20 (200 ng ml−1) for the indicated time periods. Total cell lysates were analyzed by western blotting with specific antibodies against phospho-p38, JNK, ERK and β-actin. The data are representative of three independent experiments.
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fig2: Functions of IL-20 in mouse podocytes. (a–c) Mouse podocytes were treated with hydrogen peroxide (0.5 mM), glucose (25 mM) and TGF-β1 (20 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR using primers specific for IL-20 to amplify the transcripts. GAPDH served as an input control. *P<0.05, **P<0.01 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (d) Mouse podocytes were treated with IL-20 (100, 200 or 400 ng ml−1) and glucose (25 mM) for 72 h. The cells were fixed with ethanol, stained with propidium iodide (PI) and analyzed by flow cytometry. The percentages of dead cells (M1 region) are shown. *P<0.05 compared with the untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate. (e) Mouse podocytes were treated with glucose (25 mM) and IL-20 (200 ng ml−1) for 24 h. The cells were stained with the TUNEL agent. The data are representative of three independent experiments. Magnification: × 200. Scale bars, 30 μm. (f) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with IL-20 (400 ng ml−1) for 24 h. Total cell lysates were analyzed by western blotting with specific antibodies against caspase-8 and β-actin. The data are representative of three independent experiments. (g) Mouse podocytes were starved in serum-free medium for 24 h and then incubated with mIL-20 (200 ng ml−1) for the indicated time periods. Total cell lysates were analyzed by western blotting with specific antibodies against phospho-p38, JNK, ERK and β-actin. The data are representative of three independent experiments.
Mentions: Other studies have reported that glucose, ROS and TGF-β1 can activate podocytes.3, 7 Therefore, we examined whether IL-20 expression could be induced in mouse podocytes in the presence of these stimuli. RT-qPCR showed that hydrogen peroxide markedly upregulated the IL-20 transcripts in podocytes (Figure 2a). In addition, IL-20 expression was also significantly upregulated in podocytes after glucose and TGF-β1 treatment (Figure 2b and c). NAC (N-acetyl-L-cysteine) is a potent free-radical scavenger that is known to inhibit ROS-induced apoptosis. To assess whether NAC affects H2O2-induced IL-20 expression in podocytes, mouse podocytes were preincubated with 5–20 mM of NAC for 1 h and then treated with H2O2 for another 8 h. RT-qPCR showed that NAC dose-dependently inhibited H2O2-induced IL-20 expression in podocytes (Supplementary Figure S2).

View Article: PubMed Central - PubMed

ABSTRACT

Interleukin (IL)-20, a proinflammatory cytokine of the IL-10 family, is involved in acute and chronic renal failure. The aim of this study was to elucidate the role of IL-20 during diabetic nephropathy development. We found that IL-20 and its receptor IL-20R1 were upregulated in the kidneys of mice and rats with STZ-induced diabetes. In vitro, IL-20 induced MMP-9, MCP-1, TGF-&beta;1 and VEGF expression in podocytes. IL-20 was upregulated by hydrogen peroxide, high-dose glucose and TGF-&beta;1. In addition, IL-20 induced apoptosis in podocytes by activating caspase-8. In STZ-induced early diabetic nephropathy, IL-20R1-deficient mice had lower blood glucose and serum BUN levels and a smaller glomerular area than did wild-type controls. Anti-IL-20 monoclonal antibody (7E) treatment reduced blood glucose and the glomerular area and improved renal functions in mice in the early stage of STZ-induced diabetic nephropathy. ELISA showed that the serum IL-20 level was higher in patients with diabetes mellitus than in healthy controls. The findings of this study suggest that IL-20 induces cell apoptosis of podocytes and plays a role in the pathogenesis of early diabetic nephropathy.

No MeSH data available.