Protein Phosphatase 1 Inhibitor-1 Mediates the cAMP-Dependent Stimulation of the Renal NaCl Cotransporter

J Am Soc Nephrol. 2019 May;30(5):737-750. doi: 10.1681/ASN.2018050540. Epub 2019 Mar 22.


Background: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT.

Methods: We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions.

Results: Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)-dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC.

Conclusions: Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.

Keywords: Cell & Transport Physiology; Na transport; NaCl cotransporter; cyclic AMP; distal tubule; signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Animals
  • Biological Transport / drug effects*
  • Biological Transport / genetics
  • Colforsin / pharmacology*
  • Humans
  • Immunoblotting
  • In Vitro Techniques
  • Kidney Tubules, Distal / metabolism*
  • Mice
  • Mice, Knockout
  • Phosphorylation / drug effects
  • Protein Phosphatase 1 / antagonists & inhibitors*
  • Proteins / pharmacology*
  • Signal Transduction / genetics
  • Sodium Chloride / metabolism
  • Solute Carrier Family 12, Member 3 / metabolism


  • Proteins
  • Solute Carrier Family 12, Member 3
  • phosphoprotein phosphatase inhibitor 1
  • Colforsin
  • Sodium Chloride
  • Protein Phosphatase 1