C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells

PLoS One. 2011;6(12):e28294. doi: 10.1371/journal.pone.0028294. Epub 2011 Dec 5.


Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • C-Peptide / chemistry*
  • Cell Nucleus / metabolism
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Gene Expression Regulation, Enzymologic*
  • Gene Silencing
  • Homeodomain Proteins / biosynthesis*
  • Humans
  • Hyperglycemia / metabolism
  • Kidney Tubules / metabolism*
  • MAP Kinase Signaling System*
  • Models, Biological
  • Ouabain / pharmacology
  • Peptides / chemistry
  • Phosphorylation
  • Protein Isoforms
  • Protein Kinase C / metabolism*
  • Protein Kinase C-alpha / metabolism
  • Protein Kinase C-delta / metabolism
  • Protein Kinase C-epsilon / metabolism
  • Signal Transduction
  • Sodium / chemistry
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Transcription Factors / biosynthesis*
  • Zinc Finger E-box-Binding Homeobox 1


  • C-Peptide
  • Homeodomain Proteins
  • Peptides
  • Protein Isoforms
  • Transcription Factors
  • ZEB1 protein, human
  • Zinc Finger E-box-Binding Homeobox 1
  • Ouabain
  • Sodium
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Protein Kinase C-delta
  • Protein Kinase C-epsilon
  • Extracellular Signal-Regulated MAP Kinases
  • Sodium-Potassium-Exchanging ATPase