Abstract: TH-PO1102
Cell Cycle Arrest and CCN2 Expression in Proximal Tubular Epithelial Cells in a Mouse CKD Model
Session Information
- CKD: Mechanisms - 1
October 24, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Kosakai, Wakako, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
- Sato, Tetsuya, Saitama Ika Daigaku, Hidaka-shi, Saitama, Japan
- Inoue, Tsutomu, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
- Fukaya, Daichi, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
- Amano, Hiroaki, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
- Okada, Hirokazu, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
Background
Proximal tubular epithelial cells (PTECs) enter the M phase in response to injury, after which a subset of the cells undergo G2/M arrest and acquire a pro-fibrotic phenotype (PFP), expressing CCN2 and TGF-β. Alternatively, other reports indicate G1/S arrest resulting in a PFP. To investigate the relationship between the cell cycle of PTECs and the PFP, we used a unilateral renal ischemia-reperfusion injury (IRI) model.
Methods
We reanalyzed the publicly available data for scRNA-seq from IRI, extracting only the PTEC data (cont. 7,315 cells; IRI 1, 3, 14 days; 2,689 cells: Nat. Commun. 13 (1): 4018). Furthermore, we performed studies in IRI model established using F1 mice generated by crossing Fucci2aR mice with γ-glutamyltransferase-Cre mice, allowing visualization of the cell cycle by fluorescence (G1 = Cherry, S/G2/M = mVenus). We also used cultured renal tubular epithelial cells (HK-2 cells).
Results
Reanalysis of scRNA-seq data revealed that the proportion of PTECs in the G2/M phase in fibrotic kidneys was similar to that in normal kidneys, with a minor increase in the acute phase (10.3% normal vs. 12.2% at 3 days, 8.7% at 14 days post-fibrosis). In F1 mice, mVenus-positive cells increased sharply after IRI, but declined from a peak at 3 days (2.3 ± 2.1%) to less than 1% by day 7 (0.8 ± 1.1%) with no further increase observed, despite progressive fibrosis. Mimicking IRI in HK-2 cells by transiently inducing G1/G0 arrest via a hypoxia/glucose-free medium followed by re-oxygenation and the addition of growth media led to transient cyclin-dependent kinase 1 upregulation and progression into the S/G2 phase. However, as the cell density increased, p21/p27 expression increased, with the proportion of G1/G0 PTECs increasing again by 72 h. These re-arrested G1/G0 cells exhibited increased TGF-β and CCN2 expression. Higher cell densities tended to further enhance CCN2 expression.
Conclusion
While G2/M arrest has been highlighted in the PFP, our findings suggest that G1/G0-arrested PTECs can also acquire this phenotype. In injured kidneys, the proportion of G1/G0-arrested PTECs exceeded that observed in the G2/M phase. Although the importance of PTECs in chronic kidney disease progression is undisputed, further studies should consider cells in all cell cycle states rather than limiting their focus to particular phases.
Funding
- Government Support – Non-U.S.