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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.


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IL-20 was upregulated in the renal podocytes of mice with STZ-induced early diabetic nephropathy and induced fibrogenic gene expression. (a–e) Mice (n=3 per group) were killed at the indicated times after STZ injection. Control mice were killed 8 weeks after they had been injected with buffer. (a) The blood glucose levels of mice treated with or without STZ were analyzed. **P<0.01 compared with controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (b) Renal tissues from mice with STZ-induced diabetic nephropathy were collected, mRNA was isolated, and the transcripts of IL-20, IL-20R1, IL-20R2 and IL-22R1 were measured by RT-qPCR. GAPDH served as an internal control. *P<0.05, compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (c) Levels of IL-20 in kidney from control mice and mice injected with STZ were measured by ELISA (n=3 per group). Control mice were killed 8 weeks after they had been injected with buffer. **P<0.01 compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (d) Mice were killed 8 weeks after STZ treatment. Paraffin sections of kidney tissue were stained with anti-IL-20 mAb 7E. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). IL-20 stained strongly in podocytes (arrows). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 5 μm. (e) Paraffin sections of kidney tissue were stained with 7E (green) and nephrin antibody as a marker of podocytes (red). Nuclei were stained with DAPI (blue). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 25 μm. (f) Mouse podocytes were stained to evaluate IL-20, IL-20R1, IL-20R2 and IL-22R1 expression using the indicated monoclonal antibodies. Mouse (m) IgG served as a negative control. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). The data are representative of two independent experiments. Magnification: × 200. Scale bars, 100 μm. (g) Mouse podocytes were treated with or without IL-20 (200 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR analysis with primers specific for MMP-9, MCP-1, TGF-β1 and VEGF to amplify the transcripts. GAPDH was an input control. *P<0.05, **P<0.01 compared with untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate.
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fig1: IL-20 was upregulated in the renal podocytes of mice with STZ-induced early diabetic nephropathy and induced fibrogenic gene expression. (a–e) Mice (n=3 per group) were killed at the indicated times after STZ injection. Control mice were killed 8 weeks after they had been injected with buffer. (a) The blood glucose levels of mice treated with or without STZ were analyzed. **P<0.01 compared with controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (b) Renal tissues from mice with STZ-induced diabetic nephropathy were collected, mRNA was isolated, and the transcripts of IL-20, IL-20R1, IL-20R2 and IL-22R1 were measured by RT-qPCR. GAPDH served as an internal control. *P<0.05, compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (c) Levels of IL-20 in kidney from control mice and mice injected with STZ were measured by ELISA (n=3 per group). Control mice were killed 8 weeks after they had been injected with buffer. **P<0.01 compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (d) Mice were killed 8 weeks after STZ treatment. Paraffin sections of kidney tissue were stained with anti-IL-20 mAb 7E. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). IL-20 stained strongly in podocytes (arrows). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 5 μm. (e) Paraffin sections of kidney tissue were stained with 7E (green) and nephrin antibody as a marker of podocytes (red). Nuclei were stained with DAPI (blue). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 25 μm. (f) Mouse podocytes were stained to evaluate IL-20, IL-20R1, IL-20R2 and IL-22R1 expression using the indicated monoclonal antibodies. Mouse (m) IgG served as a negative control. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). The data are representative of two independent experiments. Magnification: × 200. Scale bars, 100 μm. (g) Mouse podocytes were treated with or without IL-20 (200 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR analysis with primers specific for MMP-9, MCP-1, TGF-β1 and VEGF to amplify the transcripts. GAPDH was an input control. *P<0.05, **P<0.01 compared with untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate.

Mentions: We used a mouse model of STZ-induced diabetes22 to examine whether IL-20 is involved in diabetic nephropathy. The blood glucose of the mice was analyzed from 2 to 8 weeks after they had been injected with STZ. The blood glucose levels in STZ-treated mice were significantly higher than those in control mice (Figure 1a). To analyze whether IL-20 is involved in the pathogenesis of diabetic nephropathy, we measured the expression levels of IL-20 and its receptors (IL-20R1, IL-20R2 and IL-22R1) at different time points in STZ-induced diabetic mice. Real-time quantitative polymerase chain reaction (RT-qPCR) showed that the transcripts of IL-20 and IL-20R1 were upregulated in the kidneys of diabetic mice, whereas the IL-20R2 and IL-22R1 transcripts were not altered (Figure 1b). ELISA showed that the levels of IL-20 protein were significantly increased in kidney tissues from 4 to 8 weeks after STZ treatment (Figure 1c). Moreover, a rat model of STZ-induced diabetes was used to confirm the role of IL-20 in diabetic nephropathy with similar results (Supplementary Figure S1A–D).


Interleukin-20 targets podocytes and is upregulated in experimental murine diabetic nephropathy
IL-20 was upregulated in the renal podocytes of mice with STZ-induced early diabetic nephropathy and induced fibrogenic gene expression. (a–e) Mice (n=3 per group) were killed at the indicated times after STZ injection. Control mice were killed 8 weeks after they had been injected with buffer. (a) The blood glucose levels of mice treated with or without STZ were analyzed. **P<0.01 compared with controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (b) Renal tissues from mice with STZ-induced diabetic nephropathy were collected, mRNA was isolated, and the transcripts of IL-20, IL-20R1, IL-20R2 and IL-22R1 were measured by RT-qPCR. GAPDH served as an internal control. *P<0.05, compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (c) Levels of IL-20 in kidney from control mice and mice injected with STZ were measured by ELISA (n=3 per group). Control mice were killed 8 weeks after they had been injected with buffer. **P<0.01 compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (d) Mice were killed 8 weeks after STZ treatment. Paraffin sections of kidney tissue were stained with anti-IL-20 mAb 7E. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). IL-20 stained strongly in podocytes (arrows). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 5 μm. (e) Paraffin sections of kidney tissue were stained with 7E (green) and nephrin antibody as a marker of podocytes (red). Nuclei were stained with DAPI (blue). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 25 μm. (f) Mouse podocytes were stained to evaluate IL-20, IL-20R1, IL-20R2 and IL-22R1 expression using the indicated monoclonal antibodies. Mouse (m) IgG served as a negative control. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). The data are representative of two independent experiments. Magnification: × 200. Scale bars, 100 μm. (g) Mouse podocytes were treated with or without IL-20 (200 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR analysis with primers specific for MMP-9, MCP-1, TGF-β1 and VEGF to amplify the transcripts. GAPDH was an input control. *P<0.05, **P<0.01 compared with untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate.
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fig1: IL-20 was upregulated in the renal podocytes of mice with STZ-induced early diabetic nephropathy and induced fibrogenic gene expression. (a–e) Mice (n=3 per group) were killed at the indicated times after STZ injection. Control mice were killed 8 weeks after they had been injected with buffer. (a) The blood glucose levels of mice treated with or without STZ were analyzed. **P<0.01 compared with controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (b) Renal tissues from mice with STZ-induced diabetic nephropathy were collected, mRNA was isolated, and the transcripts of IL-20, IL-20R1, IL-20R2 and IL-22R1 were measured by RT-qPCR. GAPDH served as an internal control. *P<0.05, compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (c) Levels of IL-20 in kidney from control mice and mice injected with STZ were measured by ELISA (n=3 per group). Control mice were killed 8 weeks after they had been injected with buffer. **P<0.01 compared with the controls. The data are expressed as the mean±s.d. of three independent experiments, each performed in triplicate. (d) Mice were killed 8 weeks after STZ treatment. Paraffin sections of kidney tissue were stained with anti-IL-20 mAb 7E. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). IL-20 stained strongly in podocytes (arrows). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 5 μm. (e) Paraffin sections of kidney tissue were stained with 7E (green) and nephrin antibody as a marker of podocytes (red). Nuclei were stained with DAPI (blue). The figures represent similar patterns in three individual mice. Magnification: × 200. Scale bars, 25 μm. (f) Mouse podocytes were stained to evaluate IL-20, IL-20R1, IL-20R2 and IL-22R1 expression using the indicated monoclonal antibodies. Mouse (m) IgG served as a negative control. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). The data are representative of two independent experiments. Magnification: × 200. Scale bars, 100 μm. (g) Mouse podocytes were treated with or without IL-20 (200 ng ml−1) for the indicated times. Total RNA was isolated for RT-qPCR analysis with primers specific for MMP-9, MCP-1, TGF-β1 and VEGF to amplify the transcripts. GAPDH was an input control. *P<0.05, **P<0.01 compared with untreated controls. The data are expressed as the mean±s.e.m. of three independent experiments, each performed in triplicate.
Mentions: We used a mouse model of STZ-induced diabetes22 to examine whether IL-20 is involved in diabetic nephropathy. The blood glucose of the mice was analyzed from 2 to 8 weeks after they had been injected with STZ. The blood glucose levels in STZ-treated mice were significantly higher than those in control mice (Figure 1a). To analyze whether IL-20 is involved in the pathogenesis of diabetic nephropathy, we measured the expression levels of IL-20 and its receptors (IL-20R1, IL-20R2 and IL-22R1) at different time points in STZ-induced diabetic mice. Real-time quantitative polymerase chain reaction (RT-qPCR) showed that the transcripts of IL-20 and IL-20R1 were upregulated in the kidneys of diabetic mice, whereas the IL-20R2 and IL-22R1 transcripts were not altered (Figure 1b). ELISA showed that the levels of IL-20 protein were significantly increased in kidney tissues from 4 to 8 weeks after STZ treatment (Figure 1c). Moreover, a rat model of STZ-induced diabetes was used to confirm the role of IL-20 in diabetic nephropathy with similar results (Supplementary Figure S1A–D).

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.


Related in: MedlinePlus