Abstract: FR-PO990
Transcription Factor PKNOX2 in Myofibroblasts and Tubular Epithelial Cell Contributes to Their Function and Survival During Kidney Fibrosis Progression
Session Information
- CKD: Pathobiology - I
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2203 CKD (Non-Dialysis): Mechanisms
Authors
- Obana, Masanori, Osaka Daigaku, Suita, Osaka, Japan
- Yamamoto, Ayaha, Osaka Daigaku, Suita, Osaka, Japan
- Fujio, Yasushi, Osaka Daigaku, Suita, Osaka, Japan
Background
PBX/Knotted Homeobox 2 (PKNOX2), a nuclear transcription factor, belongs to the Three Amino acid Loop Extension (TALE) class of homeodomain proteins. We found that PKNOX2 was increased in murine fibrotic kidneys using microarrays. However, the pathophysiological roles of PKNOX2 in kidneys remain unclear. The aim of this study is to elucidate the role of PKNOX2 in fibrotic kidneys.
Methods
To assess the relevance of PKNOX2 to kidney fibrosis, murine unilateral ureteral obstruction (UUO) model was induced and the spaciotemporal expression of PKNOX2 was examined using quantitative PCR, western blotting and immunofluorescence. To examine the function of PKNOX2, lentiviral shRNA knockdown system was performed in myofibroblast cell line NRK49F cells and tubular cell HK-2 cells. After NRK49F cells were treated with TGF-β1, cell migration was examined using scratch assay. The viability of myofibroblasts and tubular cells was analyzed using CellTiter Blue assay and TUNEL staining.
Results
The mRNA and protein expression of PKNOX2 was increased after UUO in a time-dependent manner. Immunofluorescence revealed that the number of PKNOX2-expressing myofibroblasts was increased, whereas the expression of PKNOX2 was decreased in tubular epithelial cells after UUO. PKNOX2 was upregulated by TGF-β1 in NRK49F cells. PKNOX2 knockdown reduced TGF-β1-induced migration of NRK49F cells and differentiation of fibroblasts into myofibroblasts. The viability was suppressed in PKNOX2-knockdown NRK49F cells either in the presence or absence of TGF-β1 (Cell viability vs control (%): shcontrol-TGFβ1+; 139.8±46.9, shPknox2#1-TGFβ1+; 34.0±10.5, shPknox2#2-TGFβ1+; 34.0±12.3, n=6). Interestingly, knockdown of PKNOX2 also decreased the viability and increased apoptosis of HK-2 cells (Cell viability (%): shcontrol; 100.0±3.6, shPKNOX2#1; 84.8±6.0, shPKNOX2#2; 63.9±12.7, n=6).
Conclusion
PKNOX2 regulates (myo)fibroblast functions and tubular cell survival during kidney fibrosis progression.