IN VIVO TRACKING UNCOVERS AGE-DEPENDENT DIVERGENCE IN TUBULAR CELL CYCLE RESPONSE FOLLOWING KIDNEY INJURY

A substantial proportion of kidney proximal tubular cells (PTCs) initiate the cell cycle following kidney injury. Previous research has indicated that cell cycle progression during the acute phase may be associated with the severity of tissue damage in the later phase. However, conventional methods for evaluating cell cycle progression rely on “snapshot” images of fixed tissue samples, making it impossible to monitor cell cycle progression over time after kidney injury. Furthermore, the impact of aging on post-injury cell cycle entry remains unclear.

We utilized proximal tubule-specific hKi67p-Gaussia luciferase (Gluc) transgenic mice (Nature Communications, 2023), wherein Gluc is transcribed under the human Ki67 promoter, and serum Gluc activities serves as an indicator of PTCs-specific Mki67 transcription through its secretion into the circulation. Unilateral ischemia-reperfusion injury (IRI) was induced in young (8 weeks old) and aged (12 months old) transgenic male mice, followed by multiple blood collections for luminescence assays up to post-IRI day 14. We analyzed the association between the serum Gluc activities and kidney fibrosis in the chronic phase.

First, we conducted quantitative PCR 2 days post injury, which showed serum Gluc activity was positively correlated with Mki67, confirming Gluc activity works as an indicator of PTC proliferation in these transgenic mice. In a 30-minute ischemia model using young mice, Gluc activity increased during the acute phase, peaking on day 2 and returning to baseline by day 5. In contrast, in the 45-minute ischemia model, Gluc peak activity was significantly higher and occurred later compared to the 30-minute model. Furthermore, recovery of serum Gluc activity to baseline levels was delayed until day 14. In both 30-minute and 45-minute ischemia models using young mice, Gluc peak activities were positively correlated with kidney fibrosis in the chronic phase. 

In aged mice, Gluc activity peaked on day 3 in the 30-minute ischemia model and recovered to near baseline levels by day 14. When compared to the same model in young mice, Gluc peak activities showed no significant difference between the two groups, whereas kidney fibrosis was markedly more severe in the aged mice. 

In a milder injury model, 23-minute IRI, in aged mice, Gluc peak activities was observed earlier on day 2, compared to a 30-minute model. Conversely, the extent of fibrosis on day 14 showed no significant difference between 23-minute and 30-minute ischemia groups. Additionally, unlike in young mice, Gluc peak activities in aged mice did not correlate with kidney fibrosis in the chronic phase.

Using this novel in vivo tracking method, we, for the first time, enabled the ex vivo diagnosis of PTC proliferation during kidney injury. Furthermore, we found that in young mice, PTC proliferation during the acute phase correlated with kidney fibrosis in the chronic phase. In contrast, in aged mice, the degree of acute-phase PTC proliferation did not significantly differ from that in young mice, whereas the association with chronic-phase fibrosis was lost. This technique is an extremely useful tool for clarifying the relationship between PTC proliferation and the progression of kidney disease, which had previously been inferred from snapshot observations.

This work was first presented at ASN Kidney Week 2025, and re-submission is permitted by ASN.