Journal: Scientific Reports

Article Title: Downregulation of Akt induces proximal tubule epithelial cell apoptosis via FOXO and BIM pathway in proteinuric States

doi: 10.1038/s41598-025-21498-1

Figure Lengend Snippet: Albumin overload in PTECs cause downregulation of Akt activity and phosphorylation of its downstream targets Foxo1 and Foxo3 : Fig-4a-c-HKC-8 cells are incubated with endotoxin free human albumin (10 mg/ml) for 6, 16 and 24 h and probed to investigate phosphorylation of Foxo1 and Foxo3. Protein expression at 6, 16 and 24 hour levels were compared to time 0 . Independent samples t-test was used for statistical comparison between two groups. Phosphorylation of Foxo1 at Ser24 and Foxo3 at Ser 253, both representing Akt phosphorylation sites were diminished at 16 and 24 hours in association with proapoptotic BCL-2 family protein, BIM expression by Mann-Whitney U test. (n=5). * = p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HKC-8 cells were transfected by CMV-Constitutively active (CA) PKB/Akt (courtesy of David Cook, University of Melbourne), Foxo1 (pcDNA3 Flag FKHR AAA mutant was a gift from Kunliang Guan, Addgene plasmid # 13508) and Foxo3 plasmids with mutations at the Akt phosphorylation sites (FLAG-FOXO3 6 A was a gift from Anne Brunet, Addgene plasmid # 24382) plasmid using Lipofectamine – .

Techniques: Activity Assay, Phospho-proteomics, Incubation, Expressing, Comparison, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Downregulation of Akt induces proximal tubule epithelial cell apoptosis via FOXO and BIM pathway in proteinuric States

doi: 10.1038/s41598-025-21498-1

Figure Lengend Snippet: Inhibition of phosphorylation by Akt and nuclear translocation of Foxo1 promotes apoptosis in association with increased BIM transcription in PTECs in response to albumin overload : Fig. 5a-HKC-8 cell transfected with plasmids possessing mutations at Akt phosphorylation sites of Foxo1 and Foxo3 were subjected to albumin overload for 24 h ( n = 5) Inhibition of Foxo1phosphorylation by Akt augmented PTEC apoptosis induced by albumin overload. Independent samples T test was used for comparison of two groups. * p < 0.05, ** p < 0.01. Figure 5b-Nuclear and cytosolic Foxo1 and Foxo3 expression in response to albumin overload was examined by western blotting. Cytosol and nuclei were isolated after 24 h of albumin overload in PTECs. Foxo1 but not Foxo3 translocated to nuclei in association with albumin induced apoptosis ( n = 6). Figure 5c-Proximal tubule epithelial cells transfected with GFP-Foxo1 displayed nuclear translocation of Foxo1 and apoptosis evidenced by nuclear condensation and rounded up cells ( n = 3). Figure 5d-We demonstrated nuclear translocation of Foxo1 in Akt1/2 lox/lox SGLT2 cre mice kidneys subjected to albumin overload. In CHIP experiments, we investigated DNA-protein interactions. Nuclear extracts of PTEC subjected to albumin overload were immunoprecipitated with Foxo1 antibody. Figure 5e-The immunoprecipitated were subjected to PCR using primers against BIM promoters( n = 3). GAPDH and Histon3 was utilized as housekeeping gene for cytosol and nuclei respectively. Independent samples T test was used for comparison of two groups.

Article Snippet: HKC-8 cells were transfected by CMV-Constitutively active (CA) PKB/Akt (courtesy of David Cook, University of Melbourne), Foxo1 (pcDNA3 Flag FKHR AAA mutant was a gift from Kunliang Guan, Addgene plasmid # 13508) and Foxo3 plasmids with mutations at the Akt phosphorylation sites (FLAG-FOXO3 6 A was a gift from Anne Brunet, Addgene plasmid # 24382) plasmid using Lipofectamine – .

Techniques: Inhibition, Phospho-proteomics, Translocation Assay, Transfection, Comparison, Expressing, Western Blot, Isolation, Immunoprecipitation

Journal: Scientific Reports

Article Title: Downregulation of Akt induces proximal tubule epithelial cell apoptosis via FOXO and BIM pathway in proteinuric States

doi: 10.1038/s41598-025-21498-1

Figure Lengend Snippet: Proposed pathway to proteinuria induced PTEC apoptosis: In proteinuric states, high concentrations of albumin in the glomerular ultrafiltrate downregulates phosphorylation and activation of Akt resulting in dephosphorylation of Foxo1 by Akt. Increased activation and translocation of Foxo1 to the nucleus triggers transcription of Bcl-2 family BH3 protein BIM. Activation of BIM and Bax induces mitochondrial pore formation and translocation of cytochrome c to the cytoplasm causing apoptosis via caspase-9 in PTECs.

Article Snippet: HKC-8 cells were transfected by CMV-Constitutively active (CA) PKB/Akt (courtesy of David Cook, University of Melbourne), Foxo1 (pcDNA3 Flag FKHR AAA mutant was a gift from Kunliang Guan, Addgene plasmid # 13508) and Foxo3 plasmids with mutations at the Akt phosphorylation sites (FLAG-FOXO3 6 A was a gift from Anne Brunet, Addgene plasmid # 24382) plasmid using Lipofectamine – .

Techniques: Phospho-proteomics, Activation Assay, De-Phosphorylation Assay, Translocation Assay