Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: High glucose– (6 or 24 hours, 30 mM) induced csGRP78 expression, assessed by biotinylation, was increased in ( A ) PTEC ( n = 6) and ( B ) renal fibroblasts ( n = 3). Production of α2M (24 and 48 hours) by PTEC ( C ) and renal fibroblasts ( D ) was increased by high glucose (30 mM, n = 5 and 8, respectively). Similar results were observed for α2M activation ( E and F , respectively) (high glucose 48 hours, 30 mM, n = 5 and 4, respectively). Inhibition of csGRP78 interaction with α2M* using the GRP78-targeting antibody C38 prevented high glucose– (30 mM, 48 hours) induced fibronectin and collagen IV production in both ( G ) PTEC ( n = 4–6) and ( H ) renal fibroblasts ( n = 3–5). Similarly, α2M* inhibition with the Fα2M antibody attenuated matrix protein production in high glucose (30 mM, 48 hours, n = 5–6 PTEC and 4 renal fibroblasts ( I = PTEC and J = renal fibroblasts). Peptide inhibition of the csGRP78/α2M* interaction also prevented matrix protein production in high glucose (30 mM, 48 hours) by ( K ) PTEC ( n = 4–9) and ( L ) renal fibroblasts ( n = 3–6) (* P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001).

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Activation Assay, Inhibition

Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: TGF-β1 (5 ng/mL, 6 or 24 hours) increased localization of GRP78 to the surface of both PTEC and renal fibroblasts, assessed by biotinylation ( A and B , respectively) ( n = 4). Similarly, TGF-β1– (5 ng/mL) induced α2M production (24 and 48 hours) and activation (48 hours) were increased in PTEC ( n = 6 production and 10 activation) ( C and E ) and renal fibroblasts ( n = 8–9 production and 7 activation) ( D and F ). TGF-β1– (5 ng/mL, 48 hours induced fibronectin and collagen IV production were attenuated by csGRP78 inhibition ( G and H for PTEC and renal fibroblasts, respectively) ( n = 4 for both). Similarly, α2M* inhibition in PTEC and renal fibroblasts prevented TGF-β1-induced matrix protein production ( I and J ) (5 ng/mL, 48 hours, n = 4 and 6) (* P < 0.05, ** P < 0.01, *** P < 0.005; Kruskal-Wallis test used for α2M in D ).

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Inhibition

Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: High glucose– (30 mM, 48 hours) induced activation of Smad3 (measured as phosphorylation at Ser473/475) was prevented by csGRP78 inhibition in PTEC ( n = 3–4) ( A ) and renal fibroblasts ( n = 5) ( B ). Similarly, α2M* inhibition attenuated Smad3 activation by high glucose with either neutralizing antibody ( n = 6 PTEC and 4 renal fibroblasts) ( C = PTEC and D = renal fibroblasts) or inhibitory peptide ( n = 4–5 PTEC and 3–4 renal fibroblasts) ( E = PTEC and F = renal fibroblasts). In both PTEC and renal fibroblasts, csGRP78 (C38, 10 μg) did not prevent TGF-β1– (5 ng/mL, 48 hours) induced Smad3 activation ( n = 4 and 6) ( G and J , respectively). TGF-β1–induced Smad3 activation was also not prevented by α2M* inhibition (Fα2M, 10 μg) in PTEC or renal fibroblasts ( n = 6 for both) ( H and K , respectively). We confirmed these results using the Smad3-mediated reporter CAGA 12 -luciferase. TGF-β1-induced luciferase activation was not prevented by csGRP78 inhibition in either PTEC or renal fibroblasts ( n = 8 for both) ( I and L , respectively). Similarly, inhibition of α2M* did not prevent activation by TGF-β1 ( I and L , respectively) (0.05 ng/mL, 24 hours, n = 8 for both) (* P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001; Kruskal-Wallis test used for CAGA 12 -luciferase in K ).

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Phospho-proteomics, Inhibition, Luciferase

Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: Increased YAP and TAZ in response to TGF-β1 (5 ng/mL, 48 hours) were prevented by inhibition of csGRP78 ( n = 4–6 PTEC and 6 renal fibroblasts) ( A = PTEC and B = renal fibroblasts) and α2M* ( n = 4–6 PTEC and 6–8 renal fibroblasts) ( C = PTEC and D = renal fibroblasts). Using the TEAD-luciferase reporter construct, we confirmed that inhibition of both csGRP78 and α2M* in PTEC ( n = 8–10) ( E ) and renal fibroblasts ( n = 7–8) ( F ) prevented YAP/TAZ signaling in response to TGF-β1. High glucose– (30 mM, 48 hours) induced YAP and TAZ expression were also attenuated by csGRP78 ( n = 4 PTEC and 4–6 renal fibroblasts) ( G = PTEC and H = renal fibroblasts) and α2M* ( n = 4–6 PTEC and 4 renal fibroblasts) ( I = PTEC and J = renal fibroblasts) inhibition, as well as by the peptide inhibitor of csGRP78/α2M* interaction ( n = 3–4, PTEC, K ; and n = 4, renal fibroblasts, L ). (* P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001.)

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Inhibition, Luciferase, Construct, Expressing

Journal: iScience

Article Title: ATF6 ameliorates renal warm ischemia-reperfusion injury through FHL2-mediated NF-κB signaling pathway

doi: 10.1016/j.isci.2026.115173

Figure Lengend Snippet: Renal warm I/R injury in mice increases the expression of ATF6 (A) RT-qPCR analysis of ATF6 expression in mouse kidney tissues. (B–C) Representative Western blot images and quantification of ATF6 and activated ATF6 (cATF6) expression in mouse kidney tissues subjected to Sham and ischemia 1h/reperfusion 6h (I/R). (D–E) Representative IHC images and quantification of ATF6 expression in kidney tissues (Scale bars, 100 μm and 50 μm). (F–G) ATF6 expression in cultured HK-2 in response to normoxia and hypoxia/reoxygenation (H/R) treatment was determined by RT-qPCR and Western blot. (H) Quantitative statistical analysis of ATF6 protein. Data represent mean ± SDs, and statistical analysis was performed using t test. (∗ p < 0.05). T-vs-C means IR-vs-Sham.

Article Snippet: For PTECs-specific ATF6 knockdown, adeno-associated viruses (AAV)-based delivery system with pAAV9-MCS-miR30shRNA (Atf6)-WPRE or pAAV9-MCS-miR30shRNA (NC)-WPRE (0.5 × 1012 vg, OBiO Technology, Shanghai, China) were injected by tail vein injection to C57BL/6 mice.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: iScience

Article Title: ATF6 ameliorates renal warm ischemia-reperfusion injury through FHL2-mediated NF-κB signaling pathway

doi: 10.1016/j.isci.2026.115173

Figure Lengend Snippet: ATF6 plays a protective role in renal ischemia-reperfusion injury (A–B) The levels of serum Cr and BUN in mice. (C–D) The levels of serum IL-6 and TNF-α in mice. (E–F) The representative images and kidney injury assessment score of HE staining of the kidney. Ceapin-A7 (10 μM) is an ATF6 inhibitor, and AA147 (10 μM) is an ATF6 agonist. Data represent mean ± SDs, and statistical analysis was performed using one-way ANOVA. (∗ p < 0.05).

Article Snippet: For PTECs-specific ATF6 knockdown, adeno-associated viruses (AAV)-based delivery system with pAAV9-MCS-miR30shRNA (Atf6)-WPRE or pAAV9-MCS-miR30shRNA (NC)-WPRE (0.5 × 1012 vg, OBiO Technology, Shanghai, China) were injected by tail vein injection to C57BL/6 mice.

Techniques: Staining

Journal: iScience

Article Title: ATF6 ameliorates renal warm ischemia-reperfusion injury through FHL2-mediated NF-κB signaling pathway

doi: 10.1016/j.isci.2026.115173

Figure Lengend Snippet: ATF6 plays a protective role in renal ischemia-reperfusion injury (A–B) The levels of serum Cr and BUN in mice. (C–D) The levels of serum IL-6 and TNF-α in mice. (E–F) The representative images and kidney injury assessment score of HE staining of the kidney. (G–H) RT-qPCR analysis of TNF-α and IL-6 expression in cultured HK-2 in response to H/R treatment. (I–J) RT-qPCR analysis of IL-6 and TNF-α expression in cultured HK-2 in response to H/R using siATF6. Data represent mean ± SDs, and statistical analysis was performed using one-way ANOVA. (∗ p < 0.05).

Article Snippet: For PTECs-specific ATF6 knockdown, adeno-associated viruses (AAV)-based delivery system with pAAV9-MCS-miR30shRNA (Atf6)-WPRE or pAAV9-MCS-miR30shRNA (NC)-WPRE (0.5 × 1012 vg, OBiO Technology, Shanghai, China) were injected by tail vein injection to C57BL/6 mice.

Techniques: Staining, Quantitative RT-PCR, Expressing, Cell Culture

Journal: iScience

Article Title: ATF6 ameliorates renal warm ischemia-reperfusion injury through FHL2-mediated NF-κB signaling pathway

doi: 10.1016/j.isci.2026.115173

Figure Lengend Snippet: ATF6 inhibits the activation of the NF-κB pathway (A) Western blot detection of the expression of NF-κB pathway-related proteins in renal tissue; GAPDH was used as a loading control. (B) Quantitative analysis of protein expression, data represent mean ± SDs, and statistical analysis was performed using t test. (∗ p < 0.05). (C) After hypoxia/reoxygenation in the WT group and knockdown group, the expression of NF-κB pathway-related proteins was detected by Western blot, GAPDH was used as a loading control. (D) Quantitative analysis of protein expression. (E) After hypoxia/reoxygenation in the WT group and the ATF6 overexpression group, the expression of NF-κB pathway-related proteins was detected by Western blot, GAPDH was used as a loading control. (F) Quantitative analysis of protein expression. Data represent mean ± SDs, and statistical analysis was performed using one-way ANOVA. (∗ p < 0.05).

Article Snippet: For PTECs-specific ATF6 knockdown, adeno-associated viruses (AAV)-based delivery system with pAAV9-MCS-miR30shRNA (Atf6)-WPRE or pAAV9-MCS-miR30shRNA (NC)-WPRE (0.5 × 1012 vg, OBiO Technology, Shanghai, China) were injected by tail vein injection to C57BL/6 mice.

Techniques: Activation Assay, Western Blot, Expressing, Control, Knockdown, Over Expression

Journal: iScience

Article Title: ATF6 ameliorates renal warm ischemia-reperfusion injury through FHL2-mediated NF-κB signaling pathway

doi: 10.1016/j.isci.2026.115173

Figure Lengend Snippet: The expression of FHL2 is transcriptionally regulated by ATF6 (A) Expression of FHL2 in RNA-seq after using siATF6. (B) RT-qPCR analysis of FHL2 expression in mouse kidney tissues. (C) Representative Western blot images and quantification of FHL2 expression in mouse kidney tissues (GAPDH as the loading control). (D–E) Representative IHC images and quantification of FHL2 expression in kidney tissues (Scale bars, 100 μm). (F–G) FHL2 expression in cultured HK-2 in response to normoxia and hypoxia/reoxygenation (H/R) treatment was determined by Western blot. GAPDH was used as a loading control. (H–I) The expression of FHL2 and ATF6 in HK-2 cells after ATF6 knockdown was examined by Western blot. GAPDH was used as a loading control; (J-K) The expression of FHL2 and ATF6 in HK-2 cell lines after ATF6 overexpression was examined by Western blot. GAPDH was used as a loading control. (L) The input of ATF6 and IgG binding the FHL2 promoters in HK-2 after hypoxia/physoxia treatment by CHIP-qPCR. (M) Luciferase activity of transfected HEK-293T targeting FHL2 and its mutant after H/R using the dual-luciferase reporter assay. (N) The putative ATF6-binding sites in the FHL2 promoter and the nucleotide sequences representing the predicted binding sequences with the red capital letters indicating core binding elements; Data represent mean ± SDs, and statistical analysis was performed using one-way ANOVA. (∗ p < 0.05).

Article Snippet: For PTECs-specific ATF6 knockdown, adeno-associated viruses (AAV)-based delivery system with pAAV9-MCS-miR30shRNA (Atf6)-WPRE or pAAV9-MCS-miR30shRNA (NC)-WPRE (0.5 × 1012 vg, OBiO Technology, Shanghai, China) were injected by tail vein injection to C57BL/6 mice.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Control, Cell Culture, Knockdown, Over Expression, Binding Assay, ChIP-qPCR, Luciferase, Activity Assay, Transfection, Mutagenesis, Reporter Assay

Journal: The American Journal of Pathology

Article Title: Injured Proximal Tubular Epithelial Cells Lose Hepatocyte Nuclear Factor 4α Expression Crucial for Brush Border Formation and Transport

doi: 10.1016/j.ajpath.2025.01.011

Figure Lengend Snippet: Primary proximal tubular epithelial cells (PTECs) lose expression of proximal tubular markers during culture. PTECs harvested directly following tissue dissociation, preseeding (P0), and at consecutive passages (P1 to P4), followed by immunohistochemical staining, show a rapid reduction of hepatocyte nuclear factor 4α (HNF4A) expression. Expression of the proximal tubular marker CD10 is also down-regulated, but this is more protracted in time. In contrast, the scattered tubular cell marker vimentin (VIM) is rapidly and highly induced in cultured PTECs. n = 3. Scale bars = 100 μm.

Article Snippet: Renal PTECs (RPTECs), purchased from Lonza, harvested at passage 2 to 5 (P2 to P5), and subjected to immunohistochemical staining, show an expression pattern consistent with that in the current cultured primary PTECs [ie, reduced expression of hepatocyte nuclear factor 4α (HNF4A) and CD10, and high vimentin (VIM) expression].

Techniques: Expressing, Immunohistochemical staining, Staining, Marker, Cell Culture

Journal: The American Journal of Pathology

Article Title: Injured Proximal Tubular Epithelial Cells Lose Hepatocyte Nuclear Factor 4α Expression Crucial for Brush Border Formation and Transport

doi: 10.1016/j.ajpath.2025.01.011

Figure Lengend Snippet: The proximal tubular phenotype is partially restored in primary proximal tubular epithelial cells (PTECs) after hepatocyte nuclear factor 4α (HNF4A) transduction. A: Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) following HNF4A transduction of PTECs using Gene Ontology biological process displays overrepresentation of gene sets associated with transport and absorption. B: GSEA using Gene Ontology cellular component (GO-CC) shows overrepresentation of gene sets linked to brush border. C and D: Enrichment plots of GO-CC gene sets apical part of cell and brush border, respectively, demonstrate significant enrichment. E and F: Gene sets of proximal tubule S1 to S2 and S3 segments, respectively, from single-nucleus RNA sequencing of adult human kidney were shown to be significantly enriched among DEGs in HNF4A-transduced cells (figures from our data analysis). False discovery rate (FDR) < 0.25 is considered statistically significant. NES, normalized enrichment score.

Article Snippet: Renal PTECs (RPTECs), purchased from Lonza, harvested at passage 2 to 5 (P2 to P5), and subjected to immunohistochemical staining, show an expression pattern consistent with that in the current cultured primary PTECs [ie, reduced expression of hepatocyte nuclear factor 4α (HNF4A) and CD10, and high vimentin (VIM) expression].

Techniques: Transduction, RNA Sequencing