Direct Evidence of Spontaneous Transition of Residual Healthy Tubules in Injured Tissue to Proinflammatory Failed Repair Proximal Tubular Cells

Background: Recent studies have shown that proinflammatory failed-repair proximal tubular cells (FR-PTCs) emerge during the chronic phase after kidney injury and play a role in the transition from acute kidney injury (AKI) to chronic kidney disease (CKD). Interestingly, FR-PTCs are also present in the kidneys of CKD patients with no prior history of AKI, leaving their origin unclear. We hypothesized that residual normal tubular cells in injured kidneys might convert into FR-PTCs even in the absence of new injury, driven by compensatory functional overload. Methods: We conducted lineage tracing of residual healthy tubules in injured kidneys using mice carrying a tamoxifen-inducible tdTomato reporter specific to normal proximal tubules (Slc34a1GCE/R26tdTomato). Tamoxifen was administered 28 days after ischemia-reperfusion injury (IRI) to genetically label surviving normal tubules. The fate of the labeled cells was analyzed two months later. Additionally, we performed transcriptomic profiling of the labeled tubules to identify contributing molecular changes. Results: Immunostaining confirmed accurate labeling of healthy residual tubules in the injured kidneys, as tdTomato+ cells were LTL-positive and KIM1-negative. After two months, despite no further injury, a subset of tdTomato+ cells expressed VCAM1, indicating their transition into FR-PTCs. To explore mechanisms driving this change, we conducted RNA sequencing on the isolated tdTomato+ cells and found enhanced fatty acid metabolism in healthy tubules, suggesting increased energy demand. Immunostaining and ELISA revealed that lipid peroxidation product 4-hydroxynonenal (4HNE) was, mainly elevated in residual healthy tubules. In vitro, 4HNE exposure induced DNA damage, cell cycle arrest, and VCAM1 expression in tubular epithelial cells. Conclusion: These findings from lineage tracing indicate that healthy tubules in injured kidneys can spontaneously transform into FR-PTCs during the chronic phase post-injury. This transformation is driven by elevated energy needs, leading to increased fatty acid metabolism and lipid peroxide generation, which in turn triggers tubular injury and the emergence of FR-PTCs. Our results suggest that controlling functional overload in surviving normal tubules is essential to preserve kidney integrity in CKD.