Kidney Week

Abstract: FR-PO990

A Novel Method to Identify and Visualize Senescent Cells In Vivo with High Sensitivity

Session Information

• CKD Mechanisms: From Mendel to Mars
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

• 2303 CKD (Non-Dialysis): Mechanisms

Authors

• Yamada, Ryo, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan

Ryo Yamada,

• Morinishi, Takuya, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan

Takuya Morinishi,

• Iwashige, Yohei, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan

Yohei Iwashige,

• Muro, Koji, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan

Koji Muro,

• Yamamoto, Shigenori, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan

Shigenori Yamamoto,

• Kitamura, Toshio, Koeki Zaidan Hojin Kobe Iryo Sangyo Toshi Suishin Kiko, Kobe, Hyogo, Japan

Toshio Kitamura,

• Yanagita, Motoko, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan

Motoko Yanagita,

Group or Team Name

• Cell Cycle Group.

Background

Cellular senescence occurs in response to repeated passages, persistent DNA damage, and oxidative stress and is characterized by permanent growth arrest. Recently, it has been noticed that senescent cells accumulate in vivo and correlate with organ dysfunction, but a highly sensitive method to identify senescent cells in vivo has not yet been established.

Methods

The cell cycle of proximal tubular cells was evaluated in mice expressing the proximal tubule-specific G0 marker (Ndrg1CreER^T2:R26-mVenus-p27K^-). The G0 marker is mVenus-p27K^-, a fusion protein that combines the fluorescent protein mVenus with CDK inhibitor p27^Kip1 mutant p27K^- (p27 protein without CDK inhibitory function), and is usually used to identify cells in quiescent phase (G0 phase). Using this mouse line, we analyzed cell cycle of proximal tubular cells after DNA damage caused by aristolochic acid and cisplatin nephropathy models. We also performed spatial transcriptomics of G0 marker-positive cells using photo-isolation chemistry (PIC)-based system.

Results

In G0 marker mice, almost all proximal tubular cells remained in the G0 phase under physiological conditions. In the chronic phase of kidney injury, most proximal tubular cells were G0 marker-positive, but some G0 marker-positive cells exhibited nuclear abnormalities, such as nuclear enlargement. Surprisingly, immunohistochemical analysis revealed the G0 marker-positive cells with nuclear abnormalities expressed high levels of cyclin D1 along with the G0 marker (G0⁺CycD1^high), which was inconsistent with normal cell cycle. The PIC-based RNAseq study showed that G0⁺CycD1^high cells exhibited the characteristic features of “Injured PTC” and “Senescence” compared with G0⁺ cells in the vehicle group. We also isolated primary proximal tubular cells from G0 marker mice and induced senescence. Time-lapse imaging assays revealed that these senescent cells continuously expressed G0 marker. Furthermore, most of the senescent cells with G0 marker expressed Cyclin D1 and had enlarged nuclei.

Conclusion

Our data suggest that G0 marker mice can be a useful tool for identifying senescent proximal tubular cells both in vivo and in vitro, in combination with cyclin D1 expression. This system can be used to identify new markers of cellular senescence in vivo and evaluate the efficacy of senolytic treatments.