Fig 1: PKCα promotes CUX1 degradation under Klotho‐deficient conditions. (A) Co‐IF staining and quantitative analysis of PKC, Nephrin, and WT1 in glomeruli of control and diabetic mice (n = 6). Scale bar, 20 µm. (B, C) Western blot and quantification analysis of PKC expression in isolated glomeruli of diabetic mice (B, n = 6) and in podocytes under HG (C, n = 3). (D) Co‐IF staining and quantitative analysis of PKC, Nephrin, and WT1 in kidney sections from WT, KL +/−, and TgKL diabetic mice (n = 6). Scale bar, 20 µm. (E, F) IF staining of PKCα, Nephrin, and WT1 and quantitative analysis of PKCα in podocytes in indicated mouse groups (n = 6). Scale bar, 20 µm. (G, H) Protein levels and quantification of PKCα, PKCβ, and PKCγ in mouse isolated glomeruli and podocytes (n = 6). (I) Representative images and quantification of PKCα and Phalloidin in podocytes pretreated with recombinant Klotho before HG exposure (n = 3). Scale bar, 25 µm. (J, K) Western blot analysis and quantification of CUX1 and SPARC expression in podocytes following PKCα knockdown (J) or overexpression (K) (n = 3). (L) Western blot and quantification analysis of oxidative stress‐ and mitochondrial‐related proteins (GPX4, SLC7A11, Parkin, Mitofilin, COXIV, VDAC1, P53, 4‐HNE, ACSL4) and CUX1/SPARC/PKCα in podocytes with PKCα overexpression ± CUX1 co‐expression (n = 3). (M) PKCα overexpression reduced GSH and SOD levels, similar to HG treatment, an effect reversed by CUX1 overexpression (n = 3). (N) Recombinant CUX1 was incubated with PKCα, ATP, and Gö 6976 as indicated. Phosphorylation (pSer/Thr) and total CUX1 (loading control) were detected by immunoblotting. (O–Q) Podocytes were treated with PKCα modulator, HG, CHX, and either MG132 or CQ as indicated, then CUX1 protein stability was assessed (n = 3). (R) Podocytes were transfected with oe‐PKCα and Ub as indicated. Co‐IP with anti‐CUX1 antibody followed by anti‐Ub blotting showed that PKCα overexpression increased the ubiquitination of CUX1. Data are presented as mean ± SD; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA with Dunnett's test or Tukey's test (D, F–J, L–M), Two‐way ANOVA with Dunnett's test or Tukey's test (O–Q), unpaired Student's t‐test (A–C, E and K). IF, Immunofluorescence; HG, high glucose; WT, Wild‐Type; CQ, chloroquine; Ub, Ubiquitin; CHX, Cycloheximide.
Fig 2: Klotho alleviates mitochondrial damage‐mediated ferroptosis by inhibiting SPARC in DKD. (A) Blood urea nitrogen (BUN), serum creatinine, and urinary albumin‐to‐creatinine ratio (UACR) were measured in WT and Sparc −/− diabetic mice (n = 6‐9). (B) Representative HE and PAS staining of kidney sections from WT and SPARC −/− diabetic mice (n = 6). Scale bar, 50 µm. (C) Western blot and quantification of MFN1, GPX4, P53, SLC7A11, PINK1, Beclin1, Mitofilin, DRP1, VDAC1, 4‐HNE, ACSL4, and Parkin in glomerular tissue from WT and SPARC −/− diabetic mice (n = 6). (D) IF staining of GPX4, 4‐HNE, Nephrin, and WT1 and quantitative analysis of GPX4 and 4‐HNE in podocytes in indicated mouse groups (n = 6). Scale bar, 20 µm. (E) Western blot and quantitative analysis of mitochondrial and ferroptosis‐related proteins (COXIV, VDAC1, Mitofilin, Parkin, 4‐HNE, P53, GPX4, SLC7A11, SPARC, ACSL4) in podocytes subjected to various treatments (n = 3). (F–H) Suppression of SPARC under HG conditions significantly increased GSH levels (F), SOD activity (G), and ATP levels (H) (n = 3). (I) FerroOrange fluorescence intensity reflected the relative content of ferrous ions in each group (n = 3). Scale bar, 25 µm. (J) Western blot shows the expression of COXIV, Parkin, VDAC1, Mitofilin, GPX4, SLC7A11, 4‐HNE, and P53, along with quantitative analysis, in podocytes treated with HG or recombinant SPARC protein (n = 3). (K–M) SPARC significantly decreased SOD activity (K), GSH levels (L), and ATP levels (M) in experimental groups (n = 3). (N–R) IF detection of ferrous ion, C11‐BODIPY581/591, ferroptosis regulators (GPX4, SLC7A11) and mitochondrial markers (MitoTracker, JC‐1, MitoSOX) in podocytes under indicated treatments (n = 3). Scale bar, 25 µm. Data are presented as mean ± SD; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA with Dunnett's test or Tukey's test (E–H, J–M, O–R), unpaired Student's t‐test (A, C, D). IF, Immunofluorescence; HG, high glucose; WT, Wild‐Type.
Fig 3: CUX1 acts as a transcriptional repressor of SPARC in podocytes. (A) Heatmap and hierarchical clustering of differentially expressed proteins in podocytes treated with HG ± recombinant Klotho. (B) Top 10 significantly enriched pathways among proteins downregulated by HG; this downregulation was reversed by Klotho pretreatment. (C) Venn diagram showing overlap between SPARC DNA pull‐down‐associated proteins and Cluster 1 differentially expressed proteins identified by mass spectrometry. (D) Top three most abundant proteins in the overlapping set, ranked by spectral intensity. (E, F) Prediction of CUX1 binding sites in the SPARC promoter using JASPAR, validated by dual‐luciferase reporter (n = 6) and CUT‐tag analysis (n = 3). (G) The inhibitory effect was completely abrogated by mutation of the core binding site, as demonstrated by dual‐luciferase assay (n = 6). (H, I) Co‐IF staining for CUX1, Nephrin, and WT1 and quantitative analysis of CUX1 in podocytes in kidney sections from DKD patients (n = 10) and indicated mouse groups (n = 6). Scale bar, 50 µm (H), 20 µm (I). (J, K) Western blot and quantification analysis of CUX1 protein levels in corresponding mouse (J, n = 6) and cellular models (K, n = 3). (L) Knockdown of CUX1 increased SPARC expression, confirmed by Western blot (n = 3). (M) Combined CUX1 knockdown and SPARC silencing significantly restored SOD activity, GSH levels, and ATP levels in podocytes under HG (n = 3). (N) CUX1 overexpression reduced SPARC protein levels in podocytes (n = 3). (O) CUX1 overexpression exacerbated the HG‐induced decline in GSH and SOD levels (n = 3). (P‐R) IF staining (P, n = 6) and Western blot (Q, n = 6, R, n = 3) analyses reveal a positive correlation between CUX1 and Klotho expression levels. Scale bar, 20 µm (P). Data are presented as mean ± SD; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, One‐way ANOVA with Dunnett's test or Tukey's test (F, M‐R), unpaired Student's t‐test (E, G‐L). IF, Immunofluorescence; HG, high glucose; WT, Wild‐Type.
Fig 4: Klotho regulates SPARC expression in podocytes. (A) Venn diagram showing the overlap between differentially expressed genes identified by tandem mass spectrometry and ferroptosis‐related genes, followed by STRING‐based protein–protein interaction network analysis. (B, C) Glomerular co‐IF staining for SPARC, Nephrin, and WT1 and quantitative analysis of SPARC in podocytes in renal biopsy specimens from DKD patients (B, n = 13) and in glomeruli of diabetic mice (C, n = 6). Correlation analysis between the expression of SPARC in podocytes and eGFR (n = 13). Scale bar, 50 µm (B), 20 µm (C). (D, E) Glomerular SPARC protein levels assessed by IHC staining (D) and Western blot (E) across mouse groups. (n = 6). Scale bar, 50 µm. (F) Serum SPARC concentrations in control versus diabetic mice, measured by ELISA (n = 6). (G) Western blot and quantification of SPARC in podocytes exposed to HG (n = 3). (H, I) Representative images and quantitative analysis of glomerular SPARC protein levels assessed by IHC (H) and IF (I) staining in WT, KL +/−, and TgKL diabetic mice (n = 6). Scale bar, 50 µm (H), 20 µm (I). (J) Glomerular SPARC protein levels in the different diabetic mouse groups by Western blot (n = 6). (K) Serum SPARC levels in WT, KL +/−, and TgKL diabetic mice, measured by ELISA (n = 6). (L, M) Co‐IF staining (L) and Western blot (M) of SPARC in podocytes treated with HG ± Klotho (n = 3). Scale bar, 25 µm. (N) qPCR analysis of SPARC mRNA levels in podocytes under the indicated treatments (n = 3). Data are mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA with Dunnett's test or Tukey's test (H–N), Pearson's correlation test (B), unpaired Student's t‐test (B–G). IF, Immunofluorescence; ELISA, Enzyme‐Linked Immunosorbent Assay; IHC, Immunohistochemistry; HG, high glucose; WT, Wild‐Type.
Fig 5: Klotho inhibits SPARC/TGFβ‐RII/Smad‐driven ferroptosis in podocytes. (A, B) Glomerular co‐IF staining for TGFβ‐RII, Nephrin, and WT1 and quantitative analysis of TGFβ‐RII in podocytes in DKD patients (A, n = 11) and diabetic mice (B, n = 6). Scale bar, 50 µm (A), 20 µm (B). (C, D) Glomerular TGFβ‐RII protein levels assessed by IHC staining (C), and Western blot (D) across experimental mouse groups (n = 6). Scale bar, 50 µm (C). (E) TGFβ‐RII expression and quantification in podocytes exposed to HG (n = 3). (F) Representative IF and IHC staining combined with IHC staining and quantitative analysis of TGFβ‐RII in WT, KL +/−, and TgKL diabetic mice (n = 6). Scale bar, 20 or 50 µm. (G) Western blot and quantification of TGFβ‐RII, phosphorylated (p‐) and total Smad2/3 in isolated glomeruli of WT, KL +/−, and TgKL diabetic mice (n = 6). (H–J) Expression of TGFβ‐RII, p‐Smad2/3, and total Smad2/3 in podocytes under indicated treatments, analyzed by Western blot (n = 3). (K) The levels of GSH and the activity of SOD were determined in podocytes among different treatment groups (n = 3). (L) The protein levels of p‐Smad2/3, and total Smad2/3, mitochondrial functional markers (Mitofilin, Parkin, VDAC1) and ferroptosis‐related regulators (GPX4, SLC7A11, P53) were determined by Western blot in podocytes of each group (n = 3). (M) Co‐immunoprecipitation (Co‐IP) assay and Proximity Ligation Assay confirming the physical interaction between SPARC and TGFβ‐RII in podocytes. Scale bar, 10 µm. Data are presented as mean ± SD; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA with Dunnett's test or Tukey's test (F–L), unpaired Student's t‐test (A–E). IF, Immunofluorescence; IHC, Immunohistochemistry; HG, high glucose; WT, Wild‐Type.
Supplier Page from MedChemExpress for SPARC Protein, Human