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: 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

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: Purchased renal PTECs from Lonza exhibited a staining pattern that matched serially passaged PTECs, and a negligible fraction of renal PTECs displayed HNF4A positivity ( ).

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: Purchased renal PTECs from Lonza exhibited a staining pattern that matched serially passaged PTECs, and a negligible fraction of renal PTECs displayed HNF4A positivity ( ).

Techniques: Transduction, RNA Sequencing