Kidney Week

Abstract: FR-PO993

Lineage-Tracing Experiments of Senescent Cell Marker p16INK4a-Positive Cells Show that Kidney Aging Proceeds on a Nephron-by-Nephron Basis

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

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

Koji Muro,

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

Ryo Yamada,

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

Takuya Morinishi,

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

Yohei Iwashige,

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

Shigenori Yamamoto,

• Kimura, Tomonori, Kokuritsu Kenkyu Kaihatsu Hojin Iyaku Kiban Kenko Eiyo Kenkyujo, Ibaragi, Osaka, Japan

Tomonori Kimura,

• Nakanishi, Makoto, Tokyo Daigaku Ikagaku Kenkyujo, Minato-ku, Tokyo, Japan

Makoto Nakanishi,

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

Motoko Yanagita,

Background

Senescent cells in in vitro experiments are the cells that have permanently arrested the cell cycle and acquired senescence-associated secretory phenotypes. p16^INK4a is a known marker for senescent cells. However, the lack of good antibodies has made it difficult to identify p16^INK4a-positive cells in vivo and to elucidate their distribution and dynamics in tissues.

Methods

We lineage-traced senescent cells using p16Cre^ERT2:R26-tdTomato mice, where tdTomato (tdT) was induced by tamoxifen in the cells with p16^INK4a activity. We analyzed the localization of tdT+ cells in aged mice and young mice with kidney injury models, specifically focusing on the proximal tubules (PTs). Using the novel photo-isolation chemistry (PIC) technology, which allows us to obtain RNA from a single cell in tissue sections, we compared PIC-RNAseq in three groups: tdT+ proximal tubular cells (PTCs), tdT- PTCs located in PTs with or without tdT+ PTCs.

Results

Among the other segments, the PTs had the highest number of tdT+ cells. Interestingly, the number of tdT+ PTCs was significantly higher in old female mice than that in young female mice, whereas there was no difference between young and old male mice. Furthermore, tdT+ PTCs were significantly increased in female mice with ovariectomy as well as in male mice that received irradiation. Tissue clearing and three-dimensional imaging of the kidneys of aged female mice revealed a unique localization of tdT+ PTC, which clustered and contiguous within the same nephron. PIC-RNAseq showed shared gene expression patterns between tdT+ PTCs and tdT- PTCs located in the same PTs, and genes involved in ER stress, stress response and post-translational modifications were heterogeneously enhanced in these cell groups compared with tdT- PTCs in PTs without tdT+ PTCs. These results were further confirmed by immunostaining.

Conclusion

We found that senescent cells clustered in the same proximal tubules in aged kidneys and that senescent cells and non-senescent cells that reside in the same tubule shared common characteristics. These results indicated that kidney aging occurs at the nephron level, which is important for future therapeutic strategies.