BASIC RESEARCH www.jasn.org Protein Phosphatase 1 Inhibitor–1 Mediates the cAMP-Dependent Stimulation of the Renal NaCl Cotransporter David Penton,1,2 Sandra Moser,1 Agnieszka Wengi,1 Jan Czogalla,1,2 Lena Lindtoft Rosenbaek,3,4 Fritz Rigendinger,1 Nourdine Faresse,1,2 Joana R. Martins ,1,2 Robert A. Fenton,3 Dominique Loffing-Cueni,1 and Johannes Loffing1,2 1Institute of Anatomy, University of Zurich, Zurich, Switzerland; 2Swiss National Centre for Competence in Research “Kidney Control of Homeostasis,” Zurich, Switzerland; 3Department of Biomedicine, Aarhus University, Aarhus, Denmark; and 4Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark ABSTRACT 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’srole 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’seffectsonNCC,andconductedyeast 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, nor- epinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stim- ulation 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 in- creased 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 path- way might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hor- mones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity. J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018050540 Received May 22, 2018. Accepted February 6, 2019. The thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) is cru- S.M. and A.W. contributed equally to this study. + cial for the fine-tuning of renal sodium (Na ) Published online ahead of print. Publication date available at reabsorption and hence for the control of BP. www.jasn.org. NCC and the DCT are also critically involved in Correspondence: Prof. Johannes Loffing, University of Zurich, the renal control of potassium (K+), magnesium Institute of Anatomy, Winterthurerstrasse 190, CH-8057 Zurich, (Mg2+), calcium (Ca2+), and acid/base homeosta- Switzerland. Email: johannes.loffi[email protected] sis.1 The crucial role of NCC is evidenced by genetic Copyright © 2019 by the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3005-ccc 1 BASIC RESEARCH www.jasn.org diseases in which loss-of-function mutations of NCC cause Significance Statement Gitelman syndrome featuring hypokalemic alkalosis, hypo- magnesemia, hypocalciuria, and lowered arterial BP.2 Con- Stimuli that elevate cAMP, including b-adrenergic agonists and versely, enhanced NCC activity due to mutations in its regulating parathyroid hormone, increase phosphorylation (and hence activ- kinases, namely the with no lysine (K) (WNK) WNK1 ity) of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule. The protein phosphatase 1 (PP1) modulates and WNK4, causes familial hyperkalemic hypertension with NCC phosphorylation, but its role and the mechanism regulating hypermagnesemia and hypercalciuria.3 Moreover, mutations its function are obscure. The authors used in vitro and ex vivo ap- in ubiquitin-ligase complex proteins such as kelch-like-3 proaches to demonstrate that a PP1 inhibitor, protein phosphatase (KLHL3) and cullin-3 (CUL3), which control WNK4 stability, 1 inhibitor–1 (I1), mediates the effects of cAMP-elevating hormones 4,5 on NCC. They propose a novel signaling pathway in which protein are also causative of familial hyperkalemic hypertension. The – – kinase A dependent phosphorylation of I1 inhibits the PP1- WNK kinases control NCC activity via the STE20/SPS 1 related dependent dephosphorylation of NCC. Given NCC’s critical role in proline- and alanine-rich kinase (SPAK) and the oxidative renal control of ion homeostasis and BP, this pathway may con- stress–response kinase 1 (OSR1). SPAKand OSR1 directly phos- tribute to the physiologic regulation of NCC and the development phorylate NCC at several serine and threonine residues located of arterial hypertension in the context of abnormal hormonal within the N-terminal tail of the cotransporter.6,7 stimulation. The activity of the WNK-SPAK kinase pathway and NCC is regulated by various factors including the renin-angiotensin- Interestingly, both the catalytic activity and the substrate aldosterone system.8 Although the DCTexpresses the cognate specificity of phosphatases are often modulated by the inter- receptors for angiotensin II and aldosterone, recent work sug- actionwith specific regulatory subunits. Werecently found that gests that the effect of these hormones on NCC is indirectly the endogenous inhibitor 1 (I1) of PP1 is highly expressed in + + mediated via changes in plasma K concentration ([K ]).9,10 the DCT with strong effects on NCC phosphorylation and + Plasma [K ] is proposed to modulate WNK4 activity through arterial BP.22 I1 is a small, 171–amino acid cytosolic protein 2 changes in DCTmembrane voltage and intracellular Cl con- encoded by the Ppp1r1a gene.23 It is expressed in many organs centration.11 Other NCC stimulators such as the b-adrenergic including the brain, skeletal muscle, and the heart, where it is agonist isoproterenol as well as the parathyroid hormone thought to contribute to neuronal plasticity,24 muscle glyco- (PTH) are thought to mediate their effects via intracellular gen metabolism,25 and cardiac contractility and excitabil- cAMP.12–14 Recently, the cAMP-dependent protein kinase ity.23,26 Moreover, I1 was implicated in the control of the (protein kinase A [PKA]) was implicated in the regulation of activity of the Na-K-ATPase in the heart,27 whereas PP1 was WNK4, suggesting that cAMP may also act via the WNK/ found to modulate the inhibitory effect of WNK4 on ROMK SPAK kinase pathway.13 However, these studies were mainly in the kidney.28 PKA phosphorylates I1 at a threonine residue performed in heterologous expression systems and it in position 35 (T35), which activates I1 and makes it a strong 29 remained unclear whether this and/or additional pathways and very specific inhibitor of PP1 with an IC50 value of 1 nM. contribute to the cAMP-dependent regulation of NCC in the Dephosphorylation of T35 by phosphatases such as PP2A and native DCT. Some studies suggested that the kinase OSR1 and calcineurin (PP3) terminates the inhibitory action of I1.26 In- the extracellular signal–regulated kinase (ERK)1/2 mitogen- terestingly, I1 is critically involved in b-adrenergic and cAMP- activated protein kinase (MAPK) are also involved in the activation dependent signaling in skeletal and heart muscle,23,26 and of NCC by catecholamines15 and PTH,16 respectively. I1-deficient mice are partially protected from isoprenaline- Despite the progress on the elucidation of the role and reg- induced cardiac remodeling and arrhythmia.30 ulation of the WNK/SPAK/OSR1 kinase pathway, little is Here,wetestedthehypothesisthatI1isalsocriticallyinvolvedin known about the phosphatases that counterbalance the ac- the cAMP/PKA-dependent stimulation of NCC phosphorylation. tion of these kinases. As yet, three protein phosphatases are Using a variety of ex vivo approaches, we propose a novel signal suggested to modulate NCC phosphorylation: protein phos- transduction pathway in which cAMP-dependent phosphoryla- phatase–1 (PP1), PP3 (calcineurin), and PP4. In Xenopus tion and activation of I1 mediates the effect of cAMP-elevating laevis oocytes, heterologous coexpression of NCC with hormones on NCC phosphorylation and hence activity. PP4 lowered NCC phosphorylation.17 Likewise, pharmaco- logic inhibition of PP1 with calyculin A 18 and of PP3 with tacrolimus19,20 increased NCC phosphorylation in various METHODS experimental settings. The stimulatory effect of PP3 inhibi- tion on NCC may have important clinical implications. In Reagents, Cells, and Antibodies fact, a common side effect of calcineurin-inhibitor treat- Unless otherwise stated, reagents were purchased from Sigma ment is renal Na+ retention and arterial hypertension, which Aldrich (Buchs, Switzerland). Calyculin Awas purchased from correlates with an enhanced urinary excretion of phosphor- Cell Signaling Technologies (Danvers, MA). 8-Br-cAMP; PKI ylated NCC.18,21 Nevertheless, the physiologic role of the 14–22 amide, myristoylated; and H-89 were purchased from different phosphatases in the DCTand the underlying mech- Tocris