Albumin-stimulated DNA synthesis is mediated by Ca 2 (cid:1) /PKC as well as EGF receptor-dependent p44/42 MAPK and NF- (cid:2) B signal pathways in renal proximal tubule cells

Albumin-stimulated DNA synthesis is mediated by Ca 2 (cid:1) /PKC as well as EGF receptor-dependent p44/42 MAPK and NF- (cid:2) B signal pathways in renal proximal tubule cells. Am J Physiol Renal Physiol F534–F541, is now recognized that signiﬁcant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [ 3 H]thymidine incorporation in a dose ( (cid:1) 3 mg/ml)- and time ( (cid:1) 3 h)-dependent manner, intracellular Ca 2 (cid:1) concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca 2 (cid:1) chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N (cid:3) ,N (cid:3) -tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca 2 (cid:1) chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor- (cid:2) B (NF- (cid:2) B) and inhibitor of NF- (cid:2) B (I (cid:2) B) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca 2 (cid:1) , PKC, EGFR, p44/42 MAPK, or NF- (cid:2) B signal pathways blocked the BSA-induced incorporation of [ 3 H]thymidine. Consequently, the inhibition of Ca 2 (cid:1) , PKC, EGFR, p44/42 MAPKs, or NF- (cid:2) B blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21 WAF/Cip1 and p27 Kip1 expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca 2 (cid:1) /PKC as well as the EGFR-dependent p44/42 MAPK and NF- (cid:2) B signal pathways in PTCs. before the medium was used. The rabbit were perfused through the renal artery, ﬁrst with phosphate-buffered saline (PBS) and then with a medium containing 0.5% iron oxide. Renal cortical slices were prepared and homogenized. The homogenate was poured ﬁrst through a 253- (cid:5) m-mesh ﬁlter and then through an 83- (cid:5) m-mesh ﬁlter. The tubules and glomeruli on the top of the 83- (cid:5) m ﬁlter were transferred to a sterile medium. The glomeruli (containing iron oxide) were removed with a magnetic stir bar. The remaining proximal tubules were incubated brieﬂy in a medium containing collagenase (0.125 mg/ml) and 0.025% soybean trypsin inhibitor. The tubules were then washed by centrifugation, resuspended in a medium containing the three supplements, and transferred into tissue culture dishes. The medium was changed 1 day after plating and every 2 days thereafter. Primary cultured rabbit PTCs were maintained at 37°C in a 5% CO 2 humidiﬁed environment in a serum-free basal medium that was supplemented with the three growth supplements.