Background: We previously reported that rats that had recovered from mild proximal tubule (PT) injury induced by a sub-toxic dose of uranyl acetate (UA) showed partial resistance to a subsequent nephrotoxic dose of UA in association with reduced renal dysfunction and accelerated PT proliferation. We demonstrated that this resistance may involve hepatocyte growth factor (HGF)/c-Met signaling. Here, we examined whether primary cultured tubular cells derived from this model had acquired sensitivity to HGF.
Methods: Tubular cells were isolated by collagenase digestion from rat kidneys after recovery from UA-induced mild PT injury and were cultured for 48 h. Their survival and proliferation were examined using the MTS assay/5-bromo-2'-deoxyuridine labeling or MTS assay, respectively, and their migration was assayed using wound-healing and cell scattering assays, with/without HGF. HGF/c-Met signaling was assayed using phospho-specific antibodies.
Results: HGF-stimulated cultured tubular cells from UA-treated rats showed better survival after UA exposure and higher proliferation and migration than cells from vehicle-treated rats. Furthermore, HGF induced higher phosphorylation of c-Met (Tyr1234/1235) and of its major downstream signals (AKT and extracellular signal-regulated kinase 1/2) with maintained dephosphorylation of Ser985 as a negative regulator of HGF/c-Met signaling in the tubular cells of UA-treated rats compared to those of vehicle-treated rats. Immunohistochemically, dephosphorylated Ser985 was confirmed in PT cells in vivo.
Conclusions: These results suggest that elevated sensitivity to HGF, via dephosphorylated Ser985 of c-Met of tubular cells that had recovered from mild tubular injury, may be associated with cytoprotection, accelerated proliferation and migration.