C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86)Rb(+)) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1)-subunit protein expression, accompanied with increase in (86)Rb(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase α(1)-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1)-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect

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

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

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

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

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