Protein Phosphatase 1 Inhibitor–1 Mediates the Camp-Dependent Stimulation of the Renal Nacl Cotransporter

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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, 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.

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 inhibition 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 Bioscience (Bristol, UK). Endothall was purchased anism regulating their function are unclear. from EMD Millipore (Billerica, MA).

2 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

tNCC, pT53NCC, pT58NCC, and pS71NCC antibodies were Electron microscopy confirmed that the structural integrity previously described.21,22,31 I1 antibody was purchased from Ep- of DCT cells was preserved under the ex vivo incubation of itomics (catalog no. 1747–1; Burlingame, CA.). The phosphosite- the tissue slices (Supplemental Figure 2). Further experi- specific antibody recognizing pT35I1 was obtained via affinity mental details can be found in the Supplemental Material purification of serum from rabbits immunized with the phos- and elsewhere.18 pho-peptide NH2-CRRRP(pT)PATL-CONH2 corresponding to mouse I1 (Pineda, Berlin, Germany). The specificity of the anti- Immunoblotting body was confirmed by immunohistochemistry (Supplemental Immunoblotting was performed as previously described.18 Figure 1). b-actin antibody was purchased from Sigma (catalog no. A5316; Buchs, Switzerland). Rabbit anti-FLAG antibody was Statistics purchased from GenScript (catalog no. A01868; Piscataway, NJ). Unpaired Student's t-test was used to compare two groups. For Rabbit anti-AQP1 antibody was previously described.32 tNCC multiple comparison, one-way or two-way ANOVA followed and pT58NCC antibodies used to detect calyculin A and endothall by Tukey’s multiple comparison post-test was performed. stimulation of NCC in MDCK type I cells were previously de- Experimental details of the following methods: yeast two- scribed (33,34 respectively). PP1c antibody was purchased from hybrid, immunoprecipitation, immunofluorescence staining Abcam (catalog no. 53315; Cambridge, UK). Phospho-PKA sub- and fluorescence quantification, isolated perfused mouse kid- strate antibody was purchased from Cell Signaling Technologies ney, electron microscopy, and automated DCT isolation are (catalog no. 9624; Danvers, MA). OSR1 antibody was purchased included in the Supplemental Material for this manuscript. from Abcam (catalog no. 125468; Cambridge, UK). MDCK type Icells with tetracycline-inducible FLAG-tagged NCC were previously characterized.35 RESULTS

Animals cAMP-Dependent Stimulation of NCC Phosphorylation All animal experiments were conducted according to Swiss Is Mediated by PKA Laws and approved by the veterinary administration of the First, weinvestigatedwhether an increasein intracellularcAMP Canton of Zurich, Switzerland (license numbers: 213/2015 levels stimulates NCC phosphorylation in native DCTs via a and 185/2017). Experiments were conducted in male and PKA-dependent pathway. We isolated EGFP-positive early female I1-deficient mice (I1-KO)24 or wild type (WT). For DCT fragments (DCT1) from transgenic mice expressing automated DCT isolation, mice expressing the enhanced EGFP under the control of the parvalbumin promoter green fluorescent protein (EGFP) in the early segment of the (PV-EGFP).22 The isolated DCTs were incubated with a cock- DCT (DCT1) under the parvalbumin promoter (PV-EGFP) tail of the adenylate cyclase stimulator FSK (10 mmol/L) and were used.22 Both transgenic lines and WT animals were kept the phosphodiesterase inhibitor IBMX (100 mmol/L) in the in a homogenetic C57Bl/6 background. Mice were maintained presence or absence of the PKA inhibitor PKI 14–22. Stimu- in a 12-hour light/dark cycle and had access to standard chow type lation with FSK and IBMX triggered a significant increase in 3430 purchased from Provimi-Kliba (Kaiseraugst, Switzerland) the phosphorylation of NCC accompanied with a mild in- and water ad libitum. Animals were age, weight, and sex matched crease in pSPAK-pOSR1 and a marked activation of PKA for each experimental series. as monitored using a phospho-PKA substrate antibody (Figure 1). NCC phosphorylation and the anti–phospho-PKA Kidney Slices substrate signal substantially diminished upon coincubation Sex-, age-, and weight-matched mice were used for the prep- with the PKA inhibitor PKI 14–22 (1 mmol/L). Under these aration of kidney slices as described previously.18 To avoid conditions, the phosphorylation of SPAK and OSR1 remained confounding effects on NCC phosphorylation due to unequal almost unchanged compared with FSK/IBMX stimulation dietary intake of K+, all mice were food deprived 16 hours alone. Incubation of DCTs with 10 mMPKI14–22 abolished before the experiment. Slices 280 mm thick were incubated the phosphorylation of NCC and of the SPAK-OSR1 kinases, in Ringer-type solution for 30 minutes at 30.5°C for equili- indicating an overinhibition of the kinase pathway. Similar to bration. The [K+] of the buffer was always 3 mmol/L. Stock PKI 14–22, the PKA inhibitor (H-89) blocked the stimulatory solutions of isoproterenol; 3-isobutyl-1-methylxanthine effect of FSK/IBMX on NCC phosphorylation. Surprisingly, and (IBMX); calyculin A; PKI 14–22 amide, myristoylated; and for unclear reasons, the effect of H-89 was even more pro- forskolin (FSK) were prepared in DMSO. Stock solutions of nounced in cAMP-stimulated DCTs than in unstimulated 8-Bromo-cAMP, Na+ salt, PTH, NE, and H-89 were prepared DCTs (Supplemental Figure 3). in H2O. Equal volumes of either DMSO or H2O were added as control vehicle when needed. After 30-minutes incubation Genetic Ablation of I1 Attenuates the cAMP- with the drugs or vehicle solutions, slices were snap frozen Dependent Stimulation of NCC Phosphorylation in liquid nitrogen or immersion fixed with 3% PFA and PKA phosphorylates I1 at position T35 in vitro, which renders processed for immunoblotting and histology, respectively. I1 a potent and highly selective PP1 inhibitor.29,36 To test

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vehicle FSK/IBMXFSK/IBMXFSK/IBMX + PKI 1 +μM PKI 10μM

tNCC 300 300 130 *

200 200 pT53NCC 130 100 100 70 tSPAK 55 pT53NCC / tNCC (4) (4) (4) (4) (4) (4)

0 pSPAK-pOSR1 / tSPAK 0 70 normalized band intensity (%) pSPAK-pOSR1 normalized band intensity (%) 55

FSK/IBMX FSK/IBMX phospho-PKA 150 substrate 130 100 FSK/IBMX + PKI 1 μM FSK/IBMX + PKI 1 μM FSK/IBMX + PKI 10 μM FSK/IBMX + PKI 10 μM β-actin 43

Figure 1. PKA activation stimulates NCC phosphorylation in native DCTs. Left panel represents a typical immunoblot of isolated DCTs treated with vehicle, 10 mmol/L FSK+100 mmol/L IBMX, FSK/IBMX+1 mmol/L PKI 14–22, and FSK/IBMX+10 mmol/L PKI 14–22. The activity of PKA was monitored using a phospho-PKA substrate antibody. Each lane corresponds to 400 DCT fragments. On the right, the densitometric analysis of pT53NCC/tNCC and pSPAK-pOSR1/tSPAK from four independent experiments (n in brackets) normalized to control vehicle group (red line) is represented. Error bars represent the SEM. *P,0.05 compared with control vehicle condition and assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. whether I1 is critical for the cAMP-dependent stimulation of dephosphorylate a synthetic peptide corresponding to the NCC, we analyzed kidney slices from WT and I1-KO mice. N-terminal tail of mouse NCC with a phosphorylated threo- Slices were incubated with FSK, IBMX, or the PKA-specific nine at the position 58 (Figure 3B). activator 8-Br-cAMP. All agonists strongly increased NCC To confirm the functional relevance of PP1 for the regula- phosphorylation at T53 (Figure 2, A–C) and at T58 and S71 tion of NCC, NCC-expressing MDCK cells were also treated (Supplemental Figure 4) in kidney slices from WT mice. In with PP1 and PP2A inhibitors. Although the inhibition of PP1 contrast, FSK, IBMX, and 8-Br-cAMP had only a small effect with calyculin Aprofoundly stimulated NCC phosphorylation, on NCC phosphorylation in kidney slices from I1-KO mice the specific inhibition of PP2A with endothall did not change (Figure 2, A–C, Supplemental Figure 4). the phosphorylation of NCC (Figure 3C). Likewise, calyculin A increased NCC phosphorylation in kidney slices from both PP1 Interacts with and Dephosphorylates NCC WTand I1-KO mice to the same extent (Figure 3D), indicating Previous studies by us and others showed that all isoforms of that the effect of calyculin A is downstream of the regulatory the catalytic subunit of PP1 (Ppp1ca, Ppp1cb, and Ppp1cc) action of I1. are highly expressed in mouse22 and rat37 DCTs. Using a yeast two-hybrid screen on a mouse total kidney library, we found NE and PTH Stimulate NCC Phosphorylation in a Dose- that an NCC fragment comprising the first 133 amino acids of and I1- Dependent Manner rat NCC interacts with PP1 (Ppp1cb, GI number 161484667) It has been previously proposed that NE stimulates the phos- in addition to other known interacting partners (e.g.,OSR1,38 phorylation of NCC via a PKA-dependent mechanism.41 SPAK,39 and Hsp4040). To further confirm that PP1 interacts Moreover, PTH was shown to activate the adenylate cyclase with NCC, coimmunoprecipitation experiments were per- in the human and rat DCT promoting a strong increase in formed using lysates from MDCK type I cells stably transfec- intracellular cAMP.12,42 Using kidney slices from WT and ted with a tetracycline-inducible FLAG-tagged NCC.35 As I1-KO mice, we tested whether these two hormones di- shown in Figure 3A, endogenous PP1 was detected in samples rectly stimulate the phosphorylation of NCC in native immunoprecipitated with an anti-FLAG antibody but not DCTs and whether this effect depends on I1. As shown in samples immunoprecipitated with an anti-AQP1 antibody in Figure 4, A and B, both hormones promote a dose- or in the absence of antibody. Moreover, in vitro experi- dependent increase in the phosphorylation of NCC in ments showed that the PP1 catalytic subunit a is able to kidney slices from WT animals. The effect of NE on NCC

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A WT I1-KO WT I1-KO [IBMX] (μmol/L) 0 100 0 100 300 *** **** tNCC 200 130 * pT53NCC 100 130

pT53NCC / tNCC (9) (9) (9)(9) β-actin 43 0 normalized band intensity (%) vehicleIBMX vehicleIBMX B WT I1-KO WT I1-KO [FSK] (μmol/L) 0100 10 300 *** **** tNCC 130 200 *

pT53NCC 100 130

pT53NCC / tNCC (9) (9) (9) (9) β-actin 43 0 normalized band intensity (%) vehicle vehicle forskolin forskolin C WT I1-KO WT I1-KO [8-Br-cAMP] (μmol/L) 0100 10 300 * *** tNCC 200 130 **

pT53NCC 100 130 (6) (6) (6) (6) β-actin 43 pT53NCC / tNCC 0 normalized band intensity (%) vehicle vehicle 8-Br-cAMP 8-Br-cAMP

Figure 2. PKA stimulation of NCC phosphorylation is impaired in I1-KO mice. Representative immunoblots showing the effect of (A) IBMX (100 mmol/L), (B) FSK (10 mmol/L), and (C) 8-Br-cAMP (10 mmol/L) on NCC phosphorylation at position T53 in WT and I1-KO kidney slices. Bar charts represent the densitometric quantification of pT53NCC/tNCC normalized to control vehicle of each genotype from 6–9slices(n in brackets) from 2–3 mice. Error bars represent the SEM. *P,0.05, **P,0.01, ***P,0.001, ****P,0.0001 assessed by two-way ANOVA followed by Tukey’s multiple comparison post-test. phosphorylation was completely abolished in I1-KO mice test this hypothesis, kidney slices from WT and I1-KO mice (Figure 4A). On the other hand, PTH still triggered a re- were incubated with the b-adrenergic agonist isoprotere- sidual phosphorylation of NCC in kidney slices from I1-KO nol. Isoproterenol caused a significant rise in NCC phos- animals, although substantially weaker than in WT slices phorylation in kidney slices from WT mice (Figure 5A) in (Figure 4B). agreement with previous observations by us18 and others.15 However, this stimulatory effect was blunted in kidney The b-Adrenergic Stimulation of NCC Phosphorylation slices from I1-KO mice, confirming the results with NE Is Mediated by I1 stimulation. Similar results were obtained using another Terker and coworkers suggested that b-adrenergic receptors ex vivo model, namely the isolated perfused mouse kidney are instrumental for the stimulation of NCC phosphorylation (Figure 5B). by catecholamines.15 We hypothesized that the b-adrenergic Surprisingly, we did not observe any significant increase stimulation of NCC phosphorylation is mediated via I1. To in the phosphorylation of SPAK-OSR1 upon stimulation

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A +Ab -Ab +Ab (AQP1) +Ab -Ab +Ab (AQP1) B IP:FLAG IP:FLAG 150 37 tNCC PP1c PP1 10uPP1 15uno PP1

pT58NCC Input Input 150 37 tNCC PP1c Streptavidin

C vehicle endothall calyculin A 250 * 200 tNCC 150 150 100 pT58NCC 150 50 (6) (6) (6) β-actin 43 pT58NCC / tNCC 0 normalized band intensity (%)

vehicle endothall calyculin A

D WT I1-KO

vehicle calyculin A vehicle calyculin A WT I1-KO tNCC 500 NS 130 400 **** **** pT53NCC 300 130 200

pT58NCC 130 100 pT53NCC / tNCC (6) (6) (6) (6) 0 β-actin 43 normalized band intensity (%)

vehicle vehicle calyculin A calyculin A

Figure 3. PP1 interacts with NCC and dephosphorylates it. (A) FLAG IP pull down of NCC (left) and the catalytic subunit of PP1 (PPIc) (right). Unrelated anti-AQP1 antibody as well as no antibody were used as negative control. (B) In vitro dephosphorylation of bio- tinylated pT58-mNCC peptide by PP1. (C) Changes in NCC phosphorylation at T58 in MDCK type I cells with tetracycline-inducible FLAG-tagged NCC expression upon treatment with calyculin A (left panel) or specific PP2A inhibitor endothall (right panel). Graph represents the densitometric quantification of immunoblots from two independent experiments. *P,0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Changes in NCC phosphorylation upon treatment of WT and I1-KO mouse kidney slices with calyculin A (20 nmol/L). Bar charts represent the densitometric quantification of pT53NCC/tNCC from six slices (in brackets) normalized to control vehicle of each genotype (two mice per group). Error bars represent the SEM. ****P,0.0001 assessed by two-way ANOVA followed by Tukey’s multiple comparison post-test. with isoproterenol (Figure 5A). Moreover, when SPAK expression of SPAK (Figure 5A) nor of OSR1 (Supplemental phosphorylation and activity were clamped at high levels Figure 6) shows any difference between WTand I1-KO kid- 2 by incubating the kidney slices in a low Cl solution neys. These findings suggest that, at least in our experimen- (5 mmol/L),18 the stimulatory effect of isoproterenol on tal settings, the b-adrenergic stimulation of NCC is largely NCC was preserved and clearly additive to the effect of independentfromanactivationoftheWNK/SPAK-OSR1 2 low Cl (Supplemental Figure 5). Moreover, neither the pathway.

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A WT [NE] 0 0.1 1 10 100 (nmol/L)

tNCC 130 WT 400 pT53NCC I1-KO * 130 **** β-actin 43 300 *

I1-KO 200 * [NE] 100 0 0.1 1 10 100 (3) (3) (3) (3)

(nmol/L) pT53NCC / tNCC 0 tNCC 0.1 1 10 100 130 normalized band intensity (%) [NE] (nmol/L)

pT53NCC 130 β-actin 43

B WT [PTH] 0 0.1 1 10 100 (nmol/L)

tNCC 130 400 pT53NCC WT I1-KO 130 300 ** β-actin 43 * 200 I1-KO 100 [PTH] (3) (6) (6) (6)

0 0.1 1 10 100 pT53NCC / tNCC (nmol/L) 0 0.1 1 10 100 tNCC normalized band intensity (%) [PTH] (nmol/L) 130

pT53NCC 130 β-actin 43

Figure 4. I1 mediates the effect of cAMP-increasing hormones on NCC phosphorylation. Dose-response effect of (A) NE and (B) PTH on NCC phosphorylation (T53) in WT and I1-KO mouse kidney slices. Graphs represent the densitometric quantification of pT53NCC/tNCC normalized to control vehicle of each genotype (red line). The numbers of slices assayed from one (NE) or two (PTH) mice per genotype are in brackets. Error bars represent the SEM. Stars denote statistically significant differences (*P,0.05, **P,0.01, ****P,0.0001) between the two genotypes for the same hormone concentration assessed by unpaired t test. cAMP Promotes I1 Phosphorylation at T35 in Native immunohistochemistry. Consecutive cryosections obtained DCTs from kidney slices incubated ex vivo either with vehicle or iso- To assess whether the activation of PKA promotes I1 phos- proterenol were stained with antibodies against total I1 (tI1), phorylation in native DCTs,we developed a phosphoform-specific pT35I1, tNCC, and pT53NCC (Figure 6), and the staining inten- antibody against the PKA-phosphorylation site. This new pT35I1 sities in DCTs were then quantified using ImageJ software as de- antibody recognizes specifically the phosphorylated form of I1 as scribed in the Methods section. As previously reported,22 I1 demonstrated by peptide competition experiments in immuno- protein was found to be highly abundant in DCTs and in thick fluorescent studies (Supplemental Figure 1). Unfortunately, ascending limbs of Henle’s loop (Figure 6A). In contrast to the antibody works only for immunofluorescent studies. total I1, pT35I1 was barely detectable in DCTs in vehicle- Therefore, we assessed the phosphorylation levels of I1 by treated kidney slices. However, the signal for pT35I1 and

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A Mouse Kidney Slices WT [isoproterenol] (nmol/L) 0 0.1 1 100

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400 200 WT WT I1-KO I1-KO 300 **** 150 ** 200 * * 100 (6) (6) (3) (6) 100 50

pT53NCC / tNCC (6) (6) (3) (15)

0 pSPAK-pOSR1 / tSPAK 0 normalized band intensity (%) 0.1 110 100 normalized band intensity (%) 0.11 10 100 [isoproterenol] (nmol/L) [isoproterenol] (nmol/L)

Figure 5. The b-adrenergic stimulation of NCC phosphorylation is impaired in I1-KO mice. (A) Dose-response effect of b-adrenergic agonist isoproterenol on the phosphorylation of NCC (T53), SPAK, and OSR1 in WT and I1-KO kidney slices. Graphs represent the densitometric quantification of the phosphorylation of pT53NCC/tNCC and pSPAK-pOSR1/tSPAK normalized to vehicle control of each genotype from 3–15 slices (in brackets) (1–5 mice). Stars represent statistical significance (*P,0.05, **P,0.01, ****P,0.0001) of the comparison between the two genotypes for the same concentration of isoproterenol assessed by unpaired t test. (B) b-adrenergic stimulation of NCC phosphorylation in isolated perfuse mouse kidneys. Bar charts represent the densitometric quantification of pT53NCC/tNCC normalized to control vehicle of each genotype in 4–5 mice per experiment (n in brackets). **P,0.01 assessed by two- way ANOVA followed by Tukey’s multiple comparison post-test. Error bars represent the SEM. also pT53NCC significantly increased in DCTs in kidney slices correlation (Figure 6B). Of note, both the total and the phos- stimulated with isoproterenol (Figure 6, A and B). Strikingly, phorylated form of I1 were mainly seen at the apical cell surface the phosphorylation of I1 and NCC showed a strong linear of DCTs and hence in proximity to NCC (Figure 6, A and C).

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B Isolated Perfused Mouse Kidney WT I1-KO

vehicleiso vehicle iso

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WT I1-KO 400 ** 300 ns 200

100 pT53NCC / tNCC (4) (4) (4) (5) 0 normalized band intensity (%)

vehicle vehicle

isoproterenol isoproterenol

Figure 5. Continued.

DISCUSSION degrade PTH to inactive metabolites. Independent from these possible technical hurdles, the data clearly indicate that both The DCT is the target for several cAMP-elevating hormones hormones are able to elicit a graded response of NCC phosphor- including b-adrenergic agonists, PTH, and vasopressin.12 ylation. At least for NE, this graded response occurs in the range These hormones are known to activate the DCT-specific of physiologic and pathophysiologic plasma NE variations and NCC but the involved signal transduction pathways remained may hence contribute to altered renal Na+ reabsorption in re- poorly defined. In this study, we used a set of ex vivo ap- sponse to changed sympathetic tone. proaches to reveal a complete signal transduction pathway Several studies have already suggested that cAMP-dependent by which cAMP-elevating hormones, via PKA, I1, and PP1, activation of PKA contributes to the hormonal regulation of control NCC phosphorylation and hence activity. NCC.13,15,41,47–49 Nevertheless, as pointed out by Mutig In our studies, we tested the effects of two physiologically et al.,48 direct experimental support for the role of cAMP and relevant hormones, namely NE and PTH. Both hormones PKA for the control of NCC activity in native DCTs had been strongly stimulated NCC phosphorylation in a dose-dependent limited, probably due to the difficulty to establish readily avail- manner in a range from 0.1 to 100 nmol/L. For NE, this range able and suitable DCT cell models. Now, using isolated mouse matches well with the reference range of normal plasma NE DCTs and kidney slices incubated ex vivo with FSK and IBMX in concentrations in humans (i.e., 0.83–10 nmol/L).43 For PTH, the absence or presence of the PKA inhibitors PKI 14–22 and this range is above physiologic levels (20–65 ng/L, approximately H-89, we provide compelling evidence that cAMP and PKA 2–6.5 pmol/L).44 However, the PTH applied to native tissue modify NCC activity in the native DCT. I1 is a direct target for elicited effects on NCC already at concentrations that were far PKA, which phosphorylates I1 at a threonine in position 53 below those reported in the literature (100 nmol/L) to activate (pT35I1) and converts it into a potent and selective inhibitor NCC and TRPV516,45 and to downregulate NaPi2a46 in cell sys- of PP1.29 Consistent with an activation of the cAMP/PKA path- tems and tissue slices, respectively. It is also important to con- way in native DCTs, we observed a profound stimulation of I1 sider that PTH is a peptide hormone that likely penetrates less phosphorylation at the consensus PKA site T35 in response to efficiently into the tissue slices than the much smaller catechol- isoproterenol stimulation. The I1 phosphorylation strongly amines. Moreover, the high levels of peptidases in the kidney correlated with the level of NCC phosphorylation, suggesting slices (e.g., in the brush border of proximal tubules) may rapidly that they are functionally linked. Interestingly, I1 appears to

J Am Soc Nephrol 30: ccc–ccc, 2019 I1 Mediates cAMP Stimulation of NCC 9 BASIC RESEARCH www.jasn.org

A tI1 pT35l1 tNCC pT53NCC vehicle isoproterenol

B 4 8 vehicle ** 10 * isoproterenol 3 6 8

6 2 4 4 R2=0.85 1 2 2

pT35l1 intensity (a.u.) (7) (6) (7) (6) pT53NCC intensity (a.u.) 0 0 pT53NCC intensity (a.u.) 0 012345 pT35I1 intensity (a.u.) vehicle vehicle

isoproterenol isoproterenol

C vehicle isoproterenol pT35l1

Figure 6. b-adrenergic stimulation promotes the phosphorylation of I1 at T35 in native DCTs. (A) Representative immunofluorescence stainings of tI1, pT35I1, tNCC, and pT53NCC in consecutive sections of kidney slices from WT mice are shown. Slices were treated with isoproterenol 100 nmol/L or vehicle (scale bar, 25 mm). (B) The graph represents the relative mean intensity of pT35I1 staining (left panel) or pT53NCC (middle panel) in 6–7 slices (in brackets) from three mice (see Supplemental Material for details). Error bars represent the SEM. *P,0.05, **P,0.01 assessed by unpaired t test. Right panel represents the linear correlation between I1 (pT35) and NCC (pT53) phosphorylation. (C) Higher magnification of typical pT35I1 staining in kidney slices treated with vehicle (left panel) and isoproterenol (right panel), highlighting its marked apical accumulation (scale bar, 25 mm). be barely phosphorylated at T35 in DCTs of vehicle-treated In vitro kinase assays and experiments in heterologous expres- kidney slices, suggesting that I1-dependent NCC regulation sion systems suggested that PKA exerts its effects on NCC via the plays a significant role in response to hormonal stimuli but classic KLHL3-WNK4-SPAK pathway. PKA-mediated phos- not under resting conditions. phorylation of KLHL3 at S433 decreases KLHL3-dependent

10 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH ubiquitination and degradation of WNK4.47 Likewise, PKA SPAK, and OSR1 kinase pathways.15,35,48,53 This study adds phosphorylates WNK4 at multiple sites including S64 and I1 as an important additional regulator and suggests that, in S1169, which finally promotes WNK4-dependent phosphoryla- addition to the NCC-controlling kinases, also the phosphatases tion and activation of SPAK.13 PP1 was shown to modulate the are tightly regulated. Figure 7 shows the proposed signaling phosphorylation levels of both WNK428 and SPAK.50 Studies on model that we believe best explains the current observations in the regulation of the Na-K-2Cl cotransporter NKCC1, which is the context of prior knowledge. structurally related to NCC, suggested that PP1 binds to the Aside from these novel insights into the molecular mech- N-terminal tail of NKCC1 in direct proximity to SPAK to de- anism controlling NCC function, our findings may have some phosphorylate both SPAK and NKCC1.50 NCC lacks the amino clinical implications. Inappropriately high sympathetic activ- acid motif mediating the binding of PP1 to NKCC1.50 Neverthe- ity is thought to contribute to cardiovascular diseases including less, our yeast two-hybrid and coimmunoprecipitation data cardiac arrhythmia, cardiac failure, and arterial hyperten- suggest that PP1 and NCC do also interact and might be linked sion.54 Previous studies already implicated I1 in the b-adrenergic in a signaling complex that may also involve WNK4 and SPAK/ response of the heart modulating cardiac contractility,55 OSR1. Therefore, it is conceivable that I1 and PP1 may control excitibality,22 and remodeling.23 This study extends these NCC activity directly via NCC dephosphorylation and/or indi- observations to the kidney and shows that I1 is also critically rectly via controlling WNK4 and SPAK/OSR1 phosphorylation. involved in the renal response to catecholamines. Our data in- However, neither in this nor in our previous studies did we ob- dicate that catecholamines increase the phosphorylation of I1 at serve significant evidence for an involvement of I1 in SPAK/OSR1 the PKA consensus site T35, which converts I1 into a potent regulation. The abundance and subcellular localization of total- inhibitor of PP1-dependent NCC dephosphorylation. This finally SPAK, total-OSR1 (this study), and pSPAK/pOSR1 were similar increases NCC phosphorylation as observed in this and several in the kidneys and DCTs of WT and I1-KO mice.22 Moreover, other,15,41,56 but not all,57 previous studies and may contribute FSK/IBMX and isoproterenol had no significant effects on SPAK/ to NE-induced salt-sensitive hypertension.58 Interestingly, the OSR1 phosphorylation in isolated DCTs (Figure 1) and in kidney pT35 site of I1 is dephosphorylated by PP3 (calcineurin).23 Im- slices (Figure 5), respectively. Likewise, incubation of the kidney munosuppressive therapy with calcineurin inhibitors such as 2 slices in a low-Cl solution, which clamps SPAK/OSR1 activity at tacrolimus and cyclosporine A is often complicated by the devel- high levels,18 did not block the stimulatory effect of isoproterenol opment of arterial hypertension, which was suggested to be on NCC phosphorylation (Supplemental Figure 5). Nevertheless, linked to renal Na+ retention due to an activation of NCC.19 these negative results do not formally exclude some activation of It is tempting to speculate that at least parts of these effects are also the WNK4/SPAK/OSR1 pathway. In fact, we consistently ob- mediated via I1. Future in vivo studieswillhavetoaddressthe served slight, but never statistically significant, increases in relevance of renal I1 for catecholamine- and calcineurin-induced pSPAK/OSR1 immunoreactivities in tissue samples treated with arterial hypertension. FSK/IBMX and isoproterenol. Moreover, previous studies on In summary, this study identified the I1 of PP1 as a central heterologous expression systems linked SPAK to the cAMP/ regulatory element in the signal transduction cascade that me- PKA-dependent NCC regulation,13 whereas studies on knock- diates the stimulatory effects of cAMPon the thiazide-sensitive out mouse models implicated OSR1 (but not SPAK) in the NCC. I1 may represent an interesting point of convergence for catecholamine-dependent control of NCC.15 Parts of these dis- different kinase and phosphatase pathways in the DCT con- crepancies may reflect the different experimental settings (e.g., tributing to the regulation of NCC in health and disease. Given in vitro, ex vivo, in vivo) and compounds used, but they may its relevance for both the cardiac and renal response to also indicate some redundancies in the signaling pathways b-adrenergic stimulation, I1 might also be an interesting by which cAMP-elevating hormones stimulate NCC activity. drug target for the treatment of cardiovascular diseases, includ- In line with this view, we consistently observed some residual ing arterial hypertension. cAMP-dependent stimulation of NCC phosphorylation in kidneys of I1-KO mice in response to FSK/IBMX and in par- ticular in response to PTH. In contrast, the effect of catechol- ACKNOWLEDGMENTS amines (isoproterenol and NE) on NCC phosphorylation appeared to depend almost exclusively on the presence of The authors thank Monique Carrel, Michèle Heidemeyer, Shunmugam I1. A possible explanation for these differences might be a Nagarajan, and Tina Drejer for their excellent technical assistance and compartmentalization of the cAMP signaling pathways. In Adisa Trnjanin-Hadzic and Erich Brunner for their help with immu- fact, cAMP reporter assays provided evidence for a spatial noprecipitations and biosorting, respectively. and temporal control of cAMP dynamics in local microdomains The authors thank the Center of Microscopy and Image Analysis with proteins producing and degrading cAMP,51 which allow (ZMB) from the University of Zurich for excellent technical support. circumscribed effects independent from cAMP levels outside Dr. Hannah Monyer and Dr. Paul Greengard provided the PV-EGFP of these microdomains.52 Thus, the hormone-induced cAMP/ transgenic and the I1-deficient mice, respectively. PKA-dependent activation of NCC may involve several redun- D. Penton is a fellow of the Program on Integrative Kidney dant pathways including the previously characterized WNK4, Physiology and Pathophysiology (IKPP2) funded by the European

J Am Soc Nephrol 30: ccc–ccc, 2019 I1 Mediates cAMP Stimulation of NCC 11 BASIC RESEARCH www.jasn.org

Nonstimulated Stimulated

p NCC NCC I1 I1

PP1 PP1 PKA Na+ Na+

NCC p NCC p Cl- Cl- SPAK SPAK cAMP OSR1 OSR1

NCC NCC WNK

Figure 7. Model of the stimulation of NCC phosphorylation by cAMP-increasing hormones. The cAMP-dependent activation of the WNK/SPAK/ OSR1 pathway was shown in previous studies.13,15,53 The cAMP-dependent activation of the I1/PP1 pathway is presented in this study.

2 Union’s Seventh Framework Program for research, technologic Supplemental Figure 5. Effect of low Cl on the stimulation of development, and demonstration under grant agreement no. NCC and SPAK-OSR1 phosphorylation by isoproterenol in kidney 608847. J. L Loffing is supported by research funds from the Swiss slices of WT mouse. National Centre for Competence in Research “Kidney.CH,” and by Supplemental Figure 6. Expression of OSR1 in WTand I1-KO mice. project grants from the Swiss National Science Foundation Supplemental Table 1. (310030_143929/1). Funding to R.A. Fenton is provided by the Supplemental Table 2. Danish Medical Research Foundation, the Lundbeck Foundation, the Novo Nordisk Foundation, and Aarhus University Research Foundation. REFERENCES D. Penton, R.A. Fenton, D. Loffing-Cueni, and J. Loffing conceived and designed the study. D. Penton, A. Wengi, S. Moser, J. Czogalla, L.L. 1. Subramanya AR, Ellison DH: Distal convoluted tubule. Clin J Am Soc Rosenbaek, F. Rigendinger, J.R. Martins, N. Faresse, R.A. Fenton, Nephrol 9: 2147–2163, 2014 and D. Loffing-Cueni collected, analyzed, and interpreted the 2. Riveira-Munoz E, Chang Q, Bindels RJ, Devuyst O: Gitelman’s syn- data. D. Penton, R.A. Fenton, and J. Loffing wrote the manuscript. drome: Towards genotype-phenotype correlations? Pediatr Nephrol 22: 326–332, 2007 All authors approved the final version of the manuscript. 3. Murthy M, Kurz T, O’Shaughnessy KM: WNK signalling pathways in blood pressure regulation. Cell Mol Life Sci 74: 1261–1280, 2017 4. Shibata S, Zhang J, Puthumana J, Stone KL, Lifton RP: Kelch-like 3 and DISCLOSURES Cullin 3 regulate electrolyte homeostasis via ubiquitination and deg- None. radation of WNK4. Proc Natl Acad Sci U S A 110: 7838–7843, 2013 5. Boyden LM, Choi M, Choate KA, Nelson-Williams CJ, Farhi A, Toka HR, et al.: Mutations in kelch-like 3 and cullin 3 cause hypertension and – SUPPLEMENTAL MATERIAL TABLE OF CONTENT electrolyte abnormalities. Nature 482: 98 102, 2012 6. Yang S-S, Fang Y-W, Tseng M-H, Chu P-Y, Yu I-S, Wu H-C, et al.: Phosphorylation regulates NCC stability and transporter activity in vivo. This article contains the following supplemental material online at J Am Soc Nephrol 24: 1587–1597, 2013 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018050540/-/ 7. Hadchouel J, Ellison DH, Gamba G: Regulation of renal electrolyte – DCSupplemental transport by WNK and SPAK-OSR1 kinases. Annu Rev Physiol 78: 367 389, 2016 Detailed methods 8. Rojas-Vega L, Gamba G: Mini-review: Regulation of the renal NaCl fi Supplemental Figure 1. Assessment of the speci city of the pT35I1 cotransporter by hormones. Am J Physiol Renal Physiol 310: F10–F14, antibody by immunohistochemistry. 2016 Supplemental Figure 2. Assessment of DCT integrity in kidney 9. Terker AS, Yarbrough B, Ferdaus MZ, Lazelle RA, Erspamer KJ, slices via electron microscopy. Meermeier NP, et al.: Direct and indirect mineralocorticoid effects – Supplemental Figure 3. Inhibition of cAMP-dependent phos- determine distal salt transport. J Am Soc Nephrol 27: 2436 2445, 2016 10. Czogalla J, Vohra T, Penton D, Kirschmann M, Craigie E, Loffing J: phorylation of NCC by the PKA inhibitor H-89. The mineralocorticoid receptor (MR) regulates ENaC but not Supplemental Figure 4. Stimulation of NCC phosphorylation at NCCinmicewithrandomMRdeletion.Pflugers Arch 468: 849–858, T58 and S71 with cAMP-elevating agents. 2016

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