Nrf2 signalling promotes ex vivo tubular epithelial cell survival and regeneration via murine double minute (MDM)-2.

ABSTRACT

BACKGROUND: Tubular repair upon injury involves regeneration from either surviving tubular epithelial cells or from their surviving local progenitor cells; hence, compound screening with cell lines may be inadequate. Here, we demonstrate that the renal cell isolation procedure and subsequent outgrowth of tubular cells can mimic the renal injury phase and tubular cell regeneration from whichever surviving renal cells. METHODS: We set up assays to systematically screen and identify mediators of tubular survival and repair. RESULTS: Forty-eight hours after plating total kidney isolates from C57BL/6 mice, 69% of cells survived when prepared from 2-week-old pups, but only 4% of cells from 8-week-old mice, respectively. This poor survival was not modulated by co-incubation with any of 24 cytokines and growth factors, except for the Nrf2 agonist sulforaphane. In addition, only sulforaphane enhanced the regenerative outgrowth of tubular epithelial cells from the mixed population. Furthermore, sulforaphane enhanced wound closure upon scratching tubular epithelial cell monolayers in a dose-dependent manner. This process was associated with the induction of the tested Nrf2 target genes HO-1, NQO1 and murine-double minute 2 (MDM2). MDM2 blockade with nutlin-3a completely blocked the protective effects of sulforaphane on renal cell survival, outgrowth and wound closure. CONCLUSIONS: Together, renal cell isolation is a model of acute kidney injury (AKI). Primary tubular epithelial cell outgrowth represents a model of tubular regeneration. Nrf2 activation can enhance renal cell survival and tubular repair by inducing the cell cycle regulator MDM2.