High glucose/ChREBP-induced Hif-1α transcriptional activation in CD4+ T cells reduces the risk of diabetic kidney disease by inhibiting the Th1 response

Figures

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  Fig. 1. T cell-specific Hif-1α inactivation accelerates the progression of DKD. (a) Mice were rendered diabetic by one intraperitoneal injection of STZ (140 mg/kg). Normal saline (154 mmol/l NaCl) was injected in controls. Blood glucose levels in Hif-1α−/− mice and Hif-1α+/+ WT littermates. p>0.05 between Hif-1α−/− and WT groups at the same time point. (b) Survival of Hif-1α−/− and WT mice after STZ injection. All mice survived longer than 12 weeks in saline groups and data are not shown. Knockout of Hif-1α led to a significant survival disadvantage in diabetic mice by Kaplan–Meier analysis (logrank test, p<0.05). (c) Body weight of Hif-1α−/− and WT mice after STZ or saline injection. p<0.05 between Hif-1α−/− and WT diabetic mice at the same time point. No statistical difference was observed between Hif-1α−/− and WT mice without STZ injection. (d) Kidney weight/body weight ratio, (e) Cr and (f) BUN of Hif-1α−/− mice and WT littermates at 12 weeks after STZ or saline injection. *p<0.05 between Hif-1α−/− and WT diabetic mice. (g) Urinary albumin/creatinine ratio (ACR) of Hif-1α−/− and WT mice after STZ or saline injection. *p<0.05, **p<0.01 for Hif-1α−/− diabetic mice vs WT diabetic mice.…

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  Fig. 2. Hif-1α−/− CD4+ T cells exacerbate diabetic kidney injury. (a) Immunohistofluorescence staining of CD3 (red), CD4 (green) and DAPI (blue) in renal samples from Hif-1α−/− and WT diabetic mice harvested at 12 weeks after STZ injection. 20 μm scale bar for immunohistofluorescence staining (b) CD4+ T cells isolated from splenocytes of Hif-1α−/− or WT mice were adoptively transferred into WT diabetic mice once every 5 days until euthanasia. Urine samples were collected at different points after the first adoptive transfer and albumin/creatinine ratio (ACR) results are shown. *p<0.05, **p<0.01. (c) Cr and (d) BUN at 12 weeks after the adoptive transfer. *p<0.05. (e) Survival of WT diabetic mice after the adoptive transfer of Hif-1α−/− or WT CD4+ T cells. p<0.05 between the two groups throughout the experimental process. (f) Representative PAS and Masson’s trichrome staining of renal samples harvested at 12 weeks after the adoptive transfer. Scale bars, 50 μm for PAS and Masson’s trichrome staining. (g) CD4+CD62L+ cells, (h) CD4+CD25+ cells and (i) CD4+NK1.1+ NKT cells were isolated from spleens of Hif-1α−/− mice and WT littermates, and then were adoptively transferred into WT diabetic…

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  Fig. 3. High glucose triggers more IFN-γ and TNF-α production in Hif-1α−/− than in WT CD4+ T cells, and IFN-γ is responsible for the aggravated diabetic kidney injury. (a) CD4+ T cells were isolated from spleens of Hif-1α−/− mice and WT controls, and cultured under the indicated conditions for 6 h. Then the expression levels of Ifn-γ in these cells were determined by real-time reverse transcriptase PCR and normalised to β-actin. *p<0.05. (b) Flow cytometry analysis showing the proportion of IFN-γ-producing CD4+ T cells. (c) Lck-Cre+Hif-1α−/− mice were crossed with Ifn-γ knockout mice to generate Hif-1α−/−Ifn-γ−/− double-knockout mice. Urinary albumin/creatinine ratio (ACR) in Hif-1α−/−Ifn-γ−/− mice and controls after STZ injection. *p<0.05, **p<0.01 for Hif-1α−/−Ifn-γ+/+ mice vs other groups. No statistical difference was observed between Hif-1α−/−Ifn-γ−/− mice and Hif-1α+/+ mice. (d) Representative PAS (purple) and Masson’s trichrome (pink) staining of sections of renal samples harvested at 12 weeks after STZ injection. Scale bars, 50 μm for PAS and Masson’s trichrome staining. (e) CD4+ T cells from Hif-1α−/− mice and WT controls were cultured under the indicated conditions for 6 h.…

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  Fig. 4. High glucose activates T cell Hif-1α transcription via ChREBP, which also plays a part in Th1 restriction and DKD protection. CD4+ T cells were isolated from spleens of Chrebp−/− mice and WT controls, and cultured under the indicated conditions for 6 h. Then the expression levels of Hif-1α (a) and Ifn-γ (b) mRNA in these cells were determined by real-time reverse transcriptase PCR and normalised to β-actin. *p<0.05, **p<0.01. (c) CD4+ T cells were isolated from spleens of Chrebp−/− mice and WT controls, and cultured under the indicated conditions for 6 h. DMOG was added to stabilise Hif-1α protein under normoxia, as described in the Methods. Nuclear Hif-1α expression was evaluated by western blot analysis and co-detection of TBP was performed to assess equal loading (n=4). (d) HIF-1α protein bands were quantified and normalised to TBP. Data are expressed as mean ± SD. *p<0.05. (e) Spleen CD4+ T cells isolated from Chrebp−/− mice, Hif-1α−/− mice or WT controls were adoptively transferred into WT diabetic mice once every 5 days until euthanasia. Urine samples were collected at different points after the adoptive transfer and urinary albumin/creatinine ratio (ACR) results are shown.…

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  Fig. 5. ChREBP directly binds to Hif-1α promoter and activates Hif-1α expression under high glucose. (a) Scheme of the 2000 bp Hif-1α promoter-driven luciferase reporter system. (b) 293T cells were transfected with either a WT Hif-1α promoter–pGL3 recombinant reporter construct (WT) or a mutant construct (MT), with or without Chrebp plasmid overexpression, and treated with low or high glucose. Then luciferase assays were performed and relative luciferase activities are shown. (c) The putative binding site of ChREBP protein in the mouse Hif-1α promoter region. (d) Sorted CD4+ T cells were cultured with low glucose or high glucose for 6 h. Then chromatin was isolated and fragmented. Chromatin immunoprecipitation (ChIP) was performed with control IgG or anti-ChREBP antibody. Validated primers for Hif-1α were used for quantitative real-time PCR. The data are presented as fold increase for the signal from other groups relative to the control IgG/low-glucose group. Results are presented as mean ± SD; n=3 × 3 times. *p<0.05 and **p<0.01. Ab, antibody; HG, high glucose; LG, low glucose; TSS, transcription start site