control vector aav2 hsyn dio mcherry  (Addgene inc)

Journal: bioRxiv

Article Title: Calcineurin inhibition deactivates pyruvate dehydrogenase and induces proximal tubule cell metabolic dysfunction, causing profibrotic phenotype

doi: 10.1101/2024.11.20.624584

Figure Lengend Snippet: (A) mRNA expression of genes associated with cellular senescence measured by quantitative PCR in primary human renal proximal tubule epithelial cells (RPTECs) cultured with vehicle or cyclosporin A (CsA) for 24 h (n = 6 per group). (B) Western blot of p21 WAF1/Cip1 and actin in primary human RPTECs cultured with DMSO or CsA for 5 days. (C and D) Flow cytometric assay for quantification of senescence-associated β-galactosidase activity in primary human RPTECs cultured with DMSO, CsA or doxorubicin for 24 h (C). Mean fluorescence intensity is plotted in (D). RPTECs cultured with doxorubicin were used as a positive control. (E) Western blot of PDP1, PDHA1 phosphorylation at Ser293, total PDHA1 and actin in primary human RPTECs transduced with lentiviral vectors encoding the gene of either a control protein (mCherry) or PDP1 and cultured with DMSO or CsA for 12 h. PDHA1, pyruvate dehydrogenase E1 subunit alpha 1; PDP1, pyruvate dehydrogenase phosphatase 1. (F and G) Oxygen consumption rate (OCR) normalized to total protein levels in primary human RPTECs transduced with lentiviral vectors encoding the gene of either a control protein (mCherry) or PDP1 and cultured with DMSO or CsA for 24 h (F). Maximum mitochondrial respiration is calculated and plotted in (G). n = 15 per group. (H) mRNA expression of genes associated with cellular senescence measured by quantitative PCR in primary human RPTECs transduced with lentiviral vectors encoding the gene of either a control protein (mCherry) or PDP1 and cultured with DMSO or CsA for 24 h (n = 3 per group). (I) Schematic illustration of proposed mechanism of chronic calcineurin inhibitor nephrotoxicity. Inhibition of calcineurin causes deactivation of pyruvate dehydrogenase. This results in a decreased flux of TCA cycle metabolites and decreased mitochondrial respiration. Cellular metabolic dysfunction causes proinflammatory, profibrotic phenotypes associated with cellular senescence and induce kidney fibrosis. Data are presented as mean ± 1.96 SE (A, F and H) or mean ± SD (D and G). Unpaired two-tailed Welch’s t test was used. Holm’s correction for multiple comparisons was used in (G and H). n represents the number of biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s., not significant. See also Figure S4.

Article Snippet: Lentiviral vectors that encoded PDP1 or a control protein (mCherry) were purchased from VectorBuilder Japan (Kanagawa, Japan).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Flow Cytometry, Activity Assay, Fluorescence, Positive Control, Transduction, Control, Inhibition, Two Tailed Test