Research ArticleKidney Disease

Yin Yang 1 protein ameliorates diabetic nephropathy pathology through transcriptional repression of TGFβ1

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Science Translational Medicine  18 Sep 2019:
Vol. 11, Issue 510, eaaw2050
DOI: 10.1126/scitranslmed.aaw2050
  • Fig. 1 YY1 negatively regulates TGFB1 transcription through direct binding to the TGFB1 promoter.

    (A) Representative images of immunohistochemical (IHC) staining and Masson’s trichrome staining of clinical renal glomeruli healthy controls (Cntr) (n = 3) and patients with mild DN symptoms (eGFR over 90 ml/min per 1.73 m2; n = 20) termed “Mild DN” and severe DN symptoms (eGFR < 90 ml/min per 1.73 m2; n = 40) termed “Severe DN.” (B) Schematic of procedures for mass spectrometry (MS) screening in low glucose–treated (5.5 mM) and high glucose–treated (30 mM) human renal mesangial cells (HRMCs). Strep., streptavidin. (C) Screening identified YY1 bound to the TGFB1 promoter. m/z, mass/charge ratio. (D and E) Protein (D) and mRNA expression (E) of indicated genes in HRMCs transfected with a YY1-expressing plasmid. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F and G) Protein (F) and mRNA expression (G) of indicated genes in HRMCs transfected with siYY1. siNC, no target siRNA control. (H) Luciferase reporter activity of TGFB1 and protein abundance in HRMCs transfected with increasing doses of YY1-expressing plasmid. (I) Luciferase reporter assays of TGFB1 promoter fragments fused with a luciferase reporter gene (Luc1 to Luc6) in YY1-transfected HRMCs. (J) Chromatin immunoprecipitation (ChIP) assay–qPCR analysis in the region −3258/−2854 of the TGFB1 promoter. IgG, immunoglobulin G. (K) Three putative YY1 binding sites in the TGFB1 promoter. (L) Electrophoretic mobility shift assay (EMSA) of YY1 binding to the TGFB1 promoter with three probes. (M) An EMSA was performed on the TGFB1 promoter. Ab, antibody. (N) A point mutation of the YY1 binding site on the TGFB1 promoter (−3123/−3115) was constructed (TGFB1 YY1-mut). (O) Reporter activity of wild-type TGFB1 (TGFB1 wt) or mutated TGFB1 (TGFB1 YY1-mut) with YY1 overexpression. Data are means ± SEM. *P < 0.05, one-way ANOVA.

  • Fig. 2 YY1 expression in glomeruli is negatively associated with TGFβ1 in db/db mice.

    (A to F) Kidney samples of db/db mice were collected at indicated time points. Control (Cntr): C57BLKS/J db/dm mice at 8 weeks (n = 3 for each group). (A) Immunoblotting assays of YY1, TGFβ1, and FN in db/db mice of different ages and control mice. (B) Quantification of (A). ***P < 0.001 versus TGFβ1 and versus FN. (C) mRNA expression of Yy1 and Tgfb1 in db/db mice. ***P < 0.001 versus Tgfb1. (D) Representative images of IHC staining of db/db mice of different ages. (E) Densitometric quantitative results of indicated proteins in (D). ***P < 0.001 versus TGFβ1, versus FN, and versus p-Smad2. (F) Representative images of H&E, PAS, and Masson’s trichrome staining of db/db mice of different ages. (G to M) Kidneys of 12-week-old STZ-induced type 1 diabetic mice. Control (Cntr): C57BL/6J mice (n = 10 for each group). (G to K) YY1 protein expression (G), Yy1 mRNA expression (H), blood glucose (I), proteinuria (J), and representative images of PAS and Masson’s trichrome staining (K) of control and STZ-injected mice. (L and M) Densitometric quantification of PAS (L) and Masson’s trichrome staining (M) in (K). Data are means ± SEM. Two-way ANOVA was used for the db/db mouse model (B, C, and E), and Wilcoxon signed-rank test (H to M) was used for STZ-injected mice. *P < 0.05 and ***P < 0.001.

  • Fig. 3 YY1 is increased in mesangial cells from kidneys of DN mice but remains unchanged in 5/6 Nx mice.

    (A to I) Eight-week-old male C57BL/6J mice were used for a 5/6 Nx mouse model (control group, n = 6 and 5/6 Nx group, n = 7). Control (Cntr): Sham-operated mice. (A) Histopathological staining. (B) Quantification of PAS and (C) Masson’s trichrome staining in (A). (D) Representative images of IHC staining. (E) Quantitative assessment of (D). (F) YY1 and TGFβ1 protein expression and (G) mRNA expression in the kidneys. (H) Tight-slit pore density (I) thickness of the glomerular basal membrane (GBM) from transmission electron microscopy analysis. (J to L) Immunofluorescence analysis of YY1 and specific markers for (J) mesangial cells (α-SMA), (K) podocytes (WT1), and (L) tubular epithelial cells (AQP-1) in kidneys from db/dm (Cntr) and db/db mice (16 weeks). DAPI, 4′,6-diamidino-2-phenylindole. (M) Quantification of immunofluorescence staining in (J). (N to P) mRNA expression in (N) HRMCs, (O) mouse renal podocytes (MPCs), and (P) rat tubular epithelial cells (NRKs) treated with high glucose (30 mM). (Q to S) Protein expression in high glucose (HG)–treated HRMCs (Q), MPCs (R), and NRKs (S). Data are means ± SEM. Wilcoxon signed-rank test (B, C, and I) and Student’s t test (H and M) were used for 5/6 Nx mice, or two-way ANOVA was used for in vitro analysis (N). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 Nrf2 interacts with and up-regulates YY1.

    (A) YY1 and Nrf2 protein expression in HRMCs treated with high glucose (30 mM) for indicated periods. (B) Nrf2 and YY1 in the presence or absence of Nrf2 small interfering RNA (siRNA) in HG-treated HRMCs. (C and D) Proteins in HRMCs transfected with Nrf2 (C) or siNrf2 (D). (E and F) mRNA expression in HRMCs transfected with (E) Nrf2 or (F) siNrf2. HO-1 (heme oxygenase 1) was used as control. (G) Cytoplasmic and nuclear Nrf2 and YY1 from the kidneys of db/db mice at different ages (n = 3). Control (Cntr): db/dm mice, 8 weeks. (H) ChIP-qPCR and ChIP-PCR analysis for binding of Nrf2 and YY1 to the TGFB1 promoter in HRMCs. (I) Immunoblotting assays after immunoprecipitation of YY1 from HRMCs. LG, low glucose. (J) Immunoblotting assays after immunoprecipitation of Nrf2 from HRMCs. (K) qPCR and PCR analysis of ChIP-reChIP assays for binding of Nrf2 and YY1 to the TGFB1 promoter in HRMCs. (L) Reporter activity and TGFβ1 expression were determined in HRMCs. Data are means ± SEM. Significance was determined by one-way ANOVA. *P < 0.05. HSP90, heat shock protein 90.

  • Fig. 5 Knockdown of YY1 exacerbates diabetic kidney lesions.

    (A to P) An HFD/STZ diabetic model was induced in YY1flox/flox mice. Animals were subsequently in situ renal injected with AAV2-Cre (YY1-KD group, n = 6) or AAV2-GFP [control (Cntr) group, n = 7]. (A) Expression of Yy1, Tgfb1, and Fn in kidneys. (B) Immunoblot analysis of YY1 and TGFβ1 in kidneys. (C) Immunofluorescence of YY1 in renal glomeruli. (D) Body weight and (E) blood glucose of YY1-KD and control mice. (F) Representative images of H&E, PAS, and Masson’s trichrome staining of glomeruli. (G and H) Quantitative analysis of mesangial expansion (G) and mesangial fibrosis (H) in glomeruli in (F). (I to K) Urea albumin creatinine ratio (UACR) (I), urine cystatin C (J) in the urea, and blood urea nitrogen (BUN) (K) in the blood of YY1-KD and control mice. (L) Transmission electron microscopy analysis of glomerular lesions. (M and N) Quantitative analysis of tight-slit pore density (M) and thickness of the glomerular basal membrane (GBM) (N) in (L). Foot process fusion was estimated by measuring the density of tight-slit pore. (O) IHC staining of indicated proteins in renal glomeruli. (P) Quantitative analysis of the IHC staining in (O). Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, two-way ANOVA (D and E), Wilcoxon signed-rank test (G, M, and N), and Student’s t test (H to K).

  • Fig. 6 AAV2-mediated YY1 overexpression ameliorates diabetic glomerulosclerosis.

    (A to O) An HFD/STZ diabetic model was induced in C57BL/6J mice. Mice were then in situ renal injected with AAV2-GFP [control (Cntr) group] or AAV2-YY1 [YY1-overexpressing (YY1-OE) group] (n = 10 for each group). (A) Relative mRNA expression of Yy1, Tgfb1, and Fn in kidneys. (B) Representative immunoblot analysis of indicated proteins in kidneys. (C) Immunofluorescence of YY1 in renal glomeruli. (D) Body weight, (E) blood glucose, (F) UACR, (G) urine cystatin C, (H) BUN, and (I) kidney/body weight ratios in YY1-OE and control mice. (J) Representative images of H&E, PAS, and Masson’s trichrome staining of glomeruli. (K and L) Quantitative analysis of mesangial expansion (K) and mesangial fibrosis (L) in glomeruli in (J). (M) Transmission electron microscopy analysis of glomerular lesions. (N) Quantification of tight-slit pore density in (M). Foot process fusion was assessed by the density of tight-slit pore. (O) Quantification of thickness of GBM in (M). (P) IHC staining of indicated proteins in renal glomeruli. (Q) Quantitative results of the IHC staining in (P). Data are presented as means ± SEM. One-way ANOVA (A and Q), Wilcoxon signed-rank test (F to I, K, L, N, and O), and two-way ANOVA (D and E). *P < 0.05 and **P < 0.01. NS, not significant.

  • Fig. 7 Expression of YY1 in renal biopsies of DN patients correlates with DN progression.

    (A) IHC staining of YY1, TGFβ1, FN, and COL4 in control patients with minimal change disease [control (Cntr), n = 3], mild DN (n = 20), and severe DN (n = 40). (B) Quantitative results of YY1, TGFβ1, p-Smad2, FN, and COL4 in IHC staining in (A). (C) H&E, PAS, and Masson’s trichrome staining of the indicated human renal biopsies. (D and E) Quantitative analysis of PAS staining (D) and Masson’s trichrome staining (E) of the control and DN individuals in (C). (F to H) Correlation of YY1 expression in human renal biopsies with various parameters, including eGFR (F), proteinuria (G), p-Smad2 (H), and FN expression (I). Red dots represent patients with severe DN; black dots represent patients with mild DN. Data are means ± SEM. *P < 0.05, one-way ANOVA.

  • Fig. 8 Eudesmin treatment protects mice from diabetic kidney lesions by activating YY1.

    (A) Heat map representing the screening hits obtained from a luciferase reporter screening system. (B and C) mRNA (B) and protein expression (C) of YY1, TGFβ1, and FN from HRMCs treated with eudesmin (EDN; 1 μg/ml) in the presence or absence of high glucose (30 mM). (D to K) HFD/STZ-induced diabetic mice were injected with saline (Cntr) or EDN for 8 weeks (n = 10 in each group). (D and E) Representative images (D) and quantitative results (E) of IHC staining of control and EDN-injected mice. (F to H) Immunoblotting (F) and qRT-PCR (G) analysis and H&E, PAS, and Masson’s trichrome staining (H) of control and EDN-injected mice. (I to K) Zand BUN (K) of control and EDN-injected mice. (L to S) HFD/STZ-induced mice were injected with saline (Cntr) or EDN for 8 weeks in the presence or absence of YY1-KD (n = 10 in each group). (L) H&E, PAS, and Masson’s trichrome staining of control or EDN-treated mice with or without YY1-KD. (M to P) UACR (M), urine cystatin C (N), BUN (K), and representative images of IHC staining (P) in control or EDN-injected mice with or without YY1 depletion. (Q) Quantitative results of (P). (R and S) qRT-PCR (R) and representative immunoblotting (S) analysis in kidney samples of control or EDN-injected mice with or without YY1 depletion. Results are means ± SEM. Wilcoxon signed-rank test (I to K), one-way ANOVA (B, E, G, M to O, Q, and R). *P < 0.05.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/510/eaaw2050/DC1

    Materials and Methods

    Fig. S1. Transcription factors binding to the promoter of TGFB1 identified by mass spectrometry–based DNA-protein interaction screening.

    Fig. S2. Expression of YY1 in male and female db/db mice at different time periods.

    Fig. S3. Transmission electron microscopy analysis of glomerular lesions in the kidneys of control and 5/6 Nx mice.

    Fig. S4. The effects of siNrf2 alone or in combination with siYY1 on the transcriptional activity of TGFB1 in HRMCs.

    Fig. S5. Pharmacological activation of endogenous YY1 protects mouse kidneys from diabetic lesion development.

    Fig. S6. Off-target effects and toxicity analysis of EDN in the regulation of DN progression.

    Fig. S7. Intervention of EDN in db/db mice ameliorates DN progression.

    Fig. S8. A graphical abstract of this study.

    Table S1. Clinical characteristics of DN in biopsy samples.

    Table S2. List of the compounds that increased the luciferase activity of YY1 but decreased that of TGFB1.

    Table S3. The sequences of siRNAs used.

    Table S4. Primers used in DNA pull-down assays.

    Table S5. Primers used in qRT-PCR.

    Table S6. Primers used in ChIP assays and site-directed mutagenesis assays.

    Table S7. Primers used in EMSA.

    Data file S1. Raw data from mass spectrometry analysis.

    Data file S2. Data plotted in figures.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Transcription factors binding to the promoter of TGFB1 identified by mass spectrometry–based DNA-protein interaction screening.
    • Fig. S2. Expression of YY1 in male and female db/db mice at different time periods.
    • Fig. S3. Transmission electron microscopy analysis of glomerular lesions in the kidneys of control and 5/6 Nx mice.
    • Fig. S4. The effects of siNrf2 alone or in combination with siYY1 on the transcriptional activity of TGFB1 in HRMCs.
    • Fig. S5. Pharmacological activation of endogenous YY1 protects mouse kidneys from diabetic lesion development.
    • Fig. S6. Off-target effects and toxicity analysis of EDN in the regulation of DN progression.
    • Fig. S7. Intervention of EDN in db/db mice ameliorates DN progression.
    • Fig. S8. A graphical abstract of this study.
    • Table S1. Clinical characteristics of DN in biopsy samples.
    • Table S2. List of the compounds that increased the luciferase activity of YY1 but decreased that of TGFB1.
    • Table S3. The sequences of siRNAs used.
    • Table S4. Primers used in DNA pull-down assays.
    • Table S5. Primers used in qRT-PCR.
    • Table S6. Primers used in ChIP assays and site-directed mutagenesis assays.
    • Table S7. Primers used in EMSA.
    • Legends for data files S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data from mass spectrometry analysis.
    • Data file S2 (Microsoft Excel format). Data plotted in figures.

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