Renal cell regeneration following oxidant exposure: inhibition by TGF-beta1 and stimulation by ascorbic acid

Renal proximal tubular cell (RPTC) monolayers exposed to the model oxidant tert-butylhydroperoxide (TBHP; 0.8 mM) for 1.5 hrs were 33 and 31% confluent after 1 and 4 days, respectively. Control monolayers remained 100% confluent throughout the experiment. Exogenous TGF-beta1 promoted monolayer deterioration by potentiating cellular death and suppressed EGF-stimulated regeneration of the RPTC monolayer. Net TGF-beta1 production in injured RPTC increased 1.7- and 3.2-fold on Days 1 and 2, respectively, and returned to control levels 4 days following TBHP treatment. An anti-TGF-beta antibody increased monolayer confluence to 50% and DNA content 1.3-fold 4 days after TBHP exposure. L-Ascorbic acid 2-phosphate (AscP) present only during the recovery period increased monolayer confluence to 67% but had no effect on RPTC proliferation, suggesting that AscP promoted monolayer regeneration by cellular migration/spreading. AscP present continuously had no effect on the extent of TBHP-induced injury but promoted regeneration of RPTC with increased monolayer confluence (1.8-fold) and DNA content (1.8-fold) and decreased cellular lysis by 52% 4 days following TBHP exposure. The results demonstrate that TBHP-induced injury increases net TGF-beta1 production in RPTC and that autocrine TGF-beta1 inhibits regeneration of the monolayer by potentiating cellular injury and monolayer deterioration. The data also show that AscP is not cytoprotective during TBHP exposure but promotes RPTC regeneration by stimulating proliferation and migration/spreading and decreasing cellular death during the recovery period.