Na,K-ATPase expression regulates via PKC , ERK1/2 and ZEB activation.

<p><b>A.</b> Cells were cultured, incubated with kinase inhibitors and analyzed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. Experiments were performed at least 5 times and representative Western blot or EMSA images are shown. GAPDH protein was used as a loading control for total cell lysate. <b>B.</b> Effect of siRNA- mediated silencing of ZEB on ZEB and Na,K-ATPase α₁-subunit protein expression in HRTC. GAPDH protein was used as a loading control. Experiments were performed at least 4 times and representative images are shown.</p

C-peptide in concentration of 1 nM stimulates ERK transocation into the nucleus and the kinase activatory phosphorylation.

<p>Cells were cultured as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. Total cell lysate and nuclear extracts were subject to Western blot analysis to determine ERK1/2 abundance and phosphorylation. A representative Western blot image is shown in the upper panel of each graph. <b>A.</b> The total ERK1 expression. <b>B.</b> ERK1 nuclear abundance . <b>C.</b> Total ERK1/2 phosphorylation. <b>D.</b> Nuclear ERK1/2 phosphorylation . GAPDH and histone H3 proteins were used as loading controls for total cell lysate and nuclear extracts, respectively. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide.</p

Culturing of HRTCs with 1 nM of C-peptide resulted in increase in PKCε phosphorylation, while phosphorylation of PKCs α/β increased by high glucose concentrations.

<p>Cells were cultured as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. Total cell lysates were subject to Western blot analysis to determine PKCs phosphorylation. A representative Western blot image is shown in the upper panel of each graph. <b>A.</b> Phospho PKCα/β. <b>B.</b> Phospho PKCδ, <b>C.</b> Phospho PKCε. GAPDH protein was used as a loading control. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide.</p

Effects of different C-peptide and glucose concentrations on Na,K-ATPase expression and activity in HRTC.

<p>Cells were cultured and analyzed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. <b>A.</b> C-peptide at 1 nM concentration stimulates Na,K-ATPase protein expression. A representative Western blot image is shown in the upper panel. GAPDH protein was used as a loading control. <b>B.</b> Ouabain-sensitive ⁸⁶Rb⁺ uptake in intact HRTC. <b>C.</b> Na,K-ATPase α₁-subunit abundance in basolateral membrane fractions isolated from HRTC. A representative Western blot image is shown in the upper panel. GLUT2 protein was used as a loading control. <b>D.</b> Na,K-ATPase enzymatic activity in basolateral membrane fractions isolated from HRTC. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide. † P<0.05 versus 5 mM glucose with 1 nM of C-peptide.</p

C-peptide induced transcription factor ZEB DNA binding and Na,K-ATPase α₁-subunit mRNA expression.

<p><b>A.</b> Exposure to 1 nM of C-peptide increased DNA binding activity of ZEB detected by EMSA. <b>B.</b> The electrophoretic mobility supershift assay with specific antibodies against ZEB. A–B. Experiments were performed at least 5 times and representative images are shown. <b>C.</b> mRNA expression of Na,K-ATPase α₁-subunit. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide.</p

Schematic representations of intracellular signaling pathways for regulation of the sodium pump activity by C- peptide in human renal tubular cells.

<p>C- peptide binds specifically to a membrane structure, most likely a G-protein coupled receptor, with subsequent activation of PLC, isoforms of both classic and novel PKC, Rho A, MEK1/2 and ERK1/2. The latter elicits activation of ZEB (AREB6) and regulation of the gene expression for Na,K-ATPase α₁-subunit.</p