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

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.


Introduction
The thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) is crucial for the fine-tuning of renal sodium (Na + ) reabsorption and hence for the control of blood pressure. NCC and the DCT are also critically involved in the renal control of potassium (K + ), magnesium (Mg 2+ ), calcium (Ca 2+ ) and acid/base homeostasis 1 . The crucial role of NCC is evidenced by genetic diseases in which loss of function mutations of NCC cause Gitelman syndrome featuring hypokalemic alkalosis, hypomagnesemia, hypocalciuria and lowered arterial blood pressure 2 .
The activity of the WNK-SPAK kinase pathway and NCC is regulated by various factors including the renin-angiotensin-aldosterone (RAAS) system 8 . Although the DCT expresses the cognate receptors for angiotensin II and aldosterone, recent work suggests that the effect of these hormones on NCC is indirectly mediated via changes in plasma K + concentration ([K + ]) 9,10 . Plasma [K + ] is proposed to modulate WNK4 activity through changes in DCT membrane voltage and intracellular Clconcentration 11 . Other NCC stimulators such as the β-adrenergic agonist isoproterenol as well as the parathyroid hormone (PTH) are thought to mediate their effects via intracellular cAMP [12][13][14] . Recently, the cAMP-dependent protein kinase (PKA) was implicated in the regulation of WNK4, suggesting that cAMP may also act via the WNK/SPAK kinase pathway 13 . However, these studies were mainly performed in heterologous expression systems and it remained unclear whether this and/or additional pathways contribute to the cAMP-dependent regulation of NCC in the native DCT. Some studies suggested that the kinase OSR1 and the extracellular signalregulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) are also involved in the activation of NCC by catecholamines 15 and PTH 16 , respectively.

I1 mediates cAMP stimulation of NCC
Despite the progress on the elucidation of the role and regulation of the WNK/SPAK/OSR1 kinase pathway, little is known about the phosphatases that counterbalance the action of these kinases. As yet, three protein phosphatases (PP) were suggested to modulate NCC phosphorylation; PP1, PP3 (calcineurin) and PP4.
In Xenopus laevis oocytes, heterologous co-expression of NCC with PP4 lowered NCC phosphorylation 17 . Likewise, pharmacological inhibition of PP1 with calyculin A 18 and of PP3 with tacrolimus 19,20 increased NCC phosphorylation in various experimental settings. The stimulatory effect of PP3 inhibition on NCC may have important clinical implications. In fact, a common side effect of calcineurin-inhibitor treatment is renal sodium retention and arterial hypertension, which correlates with an enhanced urinary excretion of phosphorylated NCC 18,21 . Nevertheless, the physiological role of the different phosphatases in the DCT and of the underlying mechanism regulating their function are unclear.
Interestingly, both the catalytic activity and the substrate-specificity of phosphatases are often modulated by the interaction with specific regulatory subunits. We recently found that the endogenous inhibitor 1 (I1) of protein phosphatase 1 is highly expressed in the DCT with strong effects on NCC phosphorylation and arterial blood pressure 22 .
I1 is a small 171 amino acid cytosolic protein encoded by the Ppp1r1a gene 23 . It is expressed in many organs including the brain, skeletal muscle, and the heart, where it is thought to contribute to neuronal plasticity 24 , muscle glycogen metabolism 25 , and cardiac contractility and excitability 23,26 . Moreover, I1 was implicated in the control of the activity of the Na-K-ATPase in the heart 27 , while PP1 was found to modulate the inhibitory effect of WNK4 on ROMK in the kidney 28 . Protein kinase A (PKA) phosphorylates I1 at a threonine residue in position 35 (T35), which activates I1 and makes it a strong and very specific inhibitor of PP1 with an IC50 value of 1nM 29 .
Dephosphorylation of Thr35 by phosphatases such as PP2A and calcineurin (PP3) terminate the inhibitory action of I1 26 . Interestingly, I1 is critically involved in βadrenergic and cAMP-dependent signaling in skeletal and heart muscle 23,26 and I1 deficient mice are partially protected from isoprenaline induced cardiac remodeling and arrhythmia 30 .
Here, we tested the hypothesis that I1 is also critically involved in the cAMP/PKAdependent stimulation of NCC phosphorylation. Using a variety of ex vivo approaches, we propose a novel signal transduction pathway in which cAMP-dependent I1 mediates cAMP stimulation of NCC phosphorylation and activation of I1 mediates the effect of cAMP-elevating hormones on NCC phosphorylation and hence activity.

Reagents, cells and antibodies
Unless otherwise stated, reagents were purchased from Sigma Aldrich (Buchs, Switzerland MDCK type I cells with tetracycline inducible FLAG-tagged NCC were previously characterized 35 .

Animals
All animal experiments were conducted according to Swiss Laws and approved by the

I1 mediates cAMP stimulation of NCC
Mice were maintained in a 12/12 h light/dark cycle and had access to standard chow type 3430 purchased from Provimi-Kliba (Kaiseraugst, Switzerland) and water ad libitum. Animals were age, weight and sex matched for each experimental series.

Kidney slices
Sex, age, and weight matched mice were used for the preparation of kidney slices as described previously 18 . To avoid confounding effects on NCC phosphorylation due to unequal dietary intake of K + , all mice were food deprived 16 hours prior to the experiment. 280 μm thick slices were incubated in Ringer-type solution for 30 minutes at 30,5°C for equilibration. The K + concentration of the buffer was always 3 mmol/L.

Immunoblotting
Immunoblotting was performed as previously described 18 .

Statistics
Unpaired Student's t-test was used to compare two groups. For multiple comparison, one-way or two-way ANOVA followed by Tukey's multiple comparison post-test was performed.
Experimental details of the following methods: yeast two hybrid, immunoprecipitation, immunofluorescence staining and fluorescence quantification, isolated perfused mouse kidney, electron microscopy and automated DCT isolation are included as supplementary material of this manuscript.

cAMP-dependent stimulation of NCC phosphorylation is mediated by PKA
First, we investigated whether an increase in intracellular cAMP levels stimulates NCC phosphorylation in native DCTs via a PKA-dependent pathway. We isolated EGFPpositive early DCT fragments (DCT1) from transgenic mice expressing EGFP under the control of the parvalbumin promoter (PV-EGFP) 22

Genetic ablation of I1 attenuates the cAMP-dependent stimulation of NCC phosphorylation
PKA phosphorylates I1 at position T35 in vitro, which renders I1 a potent and highly selective PP1 inhibitor 29,36 . To test whether I1 is critical for the cAMP-dependent stimulation of NCC, we analyzed kidney slices from WT and I1 deficient mice (I1-KO).
Slices were incubated with either FSK, IBMX or the PKA-specific activator 8-Br-cAMP.
All agonists strongly increased NCC phosphorylation at Thr 53 (

Protein phosphatase 1 interacts with and dephosphorylates NCC
Previous studies by us and others showed that all isoforms of the catalytic subunit of PP1 (Ppp1ca, Ppp1cb and Ppp1cc) are highly expressed in mouse 22 and rat 37 DCTs.
Using a yeast-two-hybrid screen on a mouse total kidney library, we found that a NCC fragment comprising the first 133 amino acids of rat NCC interacts with PP1 (Ppp1cb, GI number 161484667) in addition to other known interacting partners (e.g. OSR1 38 , SPAK 39 and Hsp40 40 ). To further confirm that PP1 interacts with NCC, coimmunoprecipitation experiments were performed using lysates from MDCK type I cells stably transfected with a tetracycline-inducible FLAG-tagged NCC 35 . As shown in figure 3A, endogenous PP1 was detected in samples immunoprecipitated with an anti-FLAG antibody but not in samples immunoprecipitated with an anti-AQP1 antibody or in the absence of antibody. Moreover, in vitro experiments showed that the PP1 catalytic subunit α is able to dephosphorylate a synthetic peptide corresponding to the N-terminal tail of mouse NCC with a phosphorylated threonine at the position T58 ( Fig 3B).
To confirm the functional relevance of PP1 for the regulation of NCC, NCC-expressing MDCK cells were also treated with PP1 and PP2A inhibitors. While the inhibition of PP1 with calyculin A profoundly stimulated NCC phosphorylation, the specific inhibition of PP2A with endothall did not change the phosphorylation of NCC ( Fig 3C).
Likewise, calyculin A increased NCC phosphorylation in kidney slices from both WT and I1-KO mice to the same extent ( Fig 3D), indicating that the effect of calyculin A is downstream of the regulatory action of I1.

Norepinephrin and PTH stimulate NCC phosphorylation in a dose-and I1dependent manner
It has been previously proposed that norepinephrine (NE) stimulates the phosphorylation of NCC via a PKA-dependent mechanism 41 . Moreover, parathyroid hormone (PTH) was shown to activate the adenylate cyclase in the human and rat DCT promoting a strong increase in intracellular cAMP 12,42 . Using kidney slices from WT and I1-KO mice, we tested whether these two hormones directly stimulate the phosphorylation of NCC in native DCTs and whether this effect depends on I1. As shown in figure 4 A and B, both hormones promote a dose-dependent increase in the phosphorylation of NCC in kidney slices from WT animals. The effect of NE on NCC I1 mediates cAMP stimulation of NCC phosphorylation was completely abolished in I1-KO mice ( Fig 4A). On the other hand, PTH still triggered a residual phosphorylation of NCC in kidney slices from I1-KO animals, although substantially weaker than in WT slices ( Fig 4B).

The β-adrenergic stimulation of NCC phosphorylation is mediated by I1
Terker and coworkers suggested that β-adrenergic receptors are instrumental for the stimulation of NCC phosphorylation by catecholamines 15 . We hypothesized that the β-adrenergic stimulation of NCC phosphorylation is mediated via I1. To test this hypothesis, kidney slices from WT and I1-KO mice were incubated with the βadrenergic agonist isoproterenol. Isoproterenol caused a significant rise in NCC phosphorylation in kidney slices from WT mice (Fig 5A) in agreement with previous observations by us 18 and others 15 . However, this stimulatory effect was blunted in kidney slices from I1-KO mice, confirming the results with NE stimulation. Similar results were obtained using another ex-vivo model, namely the isolated perfused mouse kidney (Fig 5B).
Surprisingly, we did not observe any significant increase in the phosphorylation of SPAK-OSR1 upon stimulation with isoproterenol ( Fig 5A). Moreover, when SPAK phosphorylation and activity was clamped at high levels by incubating the kidney slices in a low Clsolution (5 mmol/L) 18 , the stimulatory effect of isoproterenol on NCC was preserved and clearly additive to the effect of low Cl -(supplementary figure 5).
Moreover, neither the expression of SPAK (Fig 5A) nor of OSR1 (Supplementary figure 6) show any difference between WT and I1-KO kidneys. These findings suggest that at least in our experimental settings, the β-adrenergic stimulation of NCC is largely independent from an activation of the WNK/SPAK-OSR1 pathway.

cAMP promotes I1 phosphorylation at threonine 35 (T35) in native DCTs
To assess whether the activation of PKA promotes I1 phosphorylation in native DCTs, we developed a phosphoform-specific antibody against the PKA-phosphorylation site.
This new pT35-I1 antibody recognizes specifically the phosphorylated form of I1 as demonstrated by peptide competition experiments in immunofluorescent studies (supplementary figure 1). Unfortunately, the antibody works only for immunofluorescent studies. Therefore, we assessed the phosphorylation levels of I1 by immunohistochemistry. Consecutive cryosections obtained from kidney slices incubated ex vivo either with vehicle or isoproterenol were stained with antibodies I1 mediates cAMP stimulation of NCC against total I1 (tI1), pT35I1, tNCC and pT53NCC (Fig 6) and the staining intensities in DCTs were then quantified using ImageJ software as described in the material and method section. As previously reported 22 , I1 protein was found to be highly abundant in DCTs and in thick ascending limbs of Henle's loop (TAL) (Fig 6A). In contrast to total I1, pT35I1 was barely detectable in DCTs in vehicle treated kidney slices.
However, the signal for pT35I1 and also pT53NCC significantly increased in DCTs in kidney slices stimulated with isoproterenol (Fig 6A and B). Strikingly, the phosphorylation of I1 and NCC showed a strong linear correlation (Fig 6B). Of notice, both the total and the phosphorylated form of I1 were mainly seen at the apical cell surface of DCTs and hence in proximity to NCC (Fig 6A and C).

Discussion
The DCT is the target for several cAMP-elevating hormones including β-adrenergic agonists, PTH and vasopressin 12 . These hormones are known to activate the DCTspecific NaCl cotransporter NCC but the involved signal transduction pathways remained poorly defined. In the present study, we used a set of ex vivo approaches to reveal a complete signal transduction pathway by which cAMP-elevating hormones, via PKA, I1 and PP1 control NCC phosphorylation and hence activity.
In our studies, we tested the effect of two physiologically relevant hormones, namely norepinephrine and PTH. Both hormones strongly stimulated NCC phosphorylation in a dose-dependent manner in a range from 0.1 nmol/L to 100 nmol/L. For norepinephrine, this range matches well with the reference range of normal plasma norepinephrine concentrations in humans (i.e. 0.83-10 nmol/L) 43 . For PTH, this range is above physiological levels (20-65 ng/L, ~2-6.5 pmol/L) 44 . However, the PTH applied to native tissue elicited effects on NCC already at concentrations that were far below those reported in the literature (100 nmol/L) to activate NCC and TRPV5 16,45 and to downregulate NaPi2a 46 in cell systems and tissue slices, respectively. It is also important to consider that PTH is a peptide hormone that likely penetrates less efficiently into the tissue slices than the much smaller catecholamines. Moreover, the high levels of peptidases in the kidney slices (e.g. in the brush border of proximal tubules) may rapidly degrade PTH to inactive metabolites. Independent, from these possible technical hurdles, the data clearly indicate that both hormones are able to elicit a graded response of NCC phosphorylation. At least for norepinephrine, this In vitro kinase assays and experiments in heterologous expression systems suggested that PKA exerts its effects on NCC via the classical KHL3-WNK4-SPAK pathway.
PKA-mediated phosphorylation of KLH3 at S433 decreases KLH3-dependent ubiquitination and degradation of WNK4 47 . Likewise, PKA phosphorylates WNK4 at multiple sites including S64 and S1169, which finally promotes WNK4-dependent phosphorylation and activation of SPAK 13 . PP1 was shown to modulate the phosphorylation levels of both WNK4 28 and SPAK 50 . Studies on the regulation of the Na-K-2Cl cotransporter NKCC1, which is structurally related to NCC, suggested that PP1 binds to the N-terminal tail of NKCC1 in direct proximity to SPAK to dephosphorylate both SPAK and NKCC1 50 . NCC lacks the amino acid motif mediating the binding of PP1 to NKCC1 50 . Nevertheless, our yeast-two-hybrid and coimmunoprecipitation data suggest that PP1 and NCC do also interact and might be linked in a signaling complex that may involve also WNK4 and SPAK/OSR1. Therefore, it is conceivable that I1 and PP1 may control NCC activity directly via NCCdephosphorylation and/or indirectly via controlling WNK4 and SPAK/OSR1 phosphorylation. However, neither in the current nor in our previous studies, we observed significant evidence for an involvement of I1 in SPAK/OSR1 regulation. The abundance and subcellular localization of total-SPAK, total-OSR1 (this study) and pSPAK/pOSR1 were similar in the kidneys and DCTs of wildtype and I1-KO mice 22 .
Likewise, incubation of the kidney slices in a low Cl --solution, which clamps SPAK/OSR1 activity at high levels 18 did not block the stimulatory effect of isoproterenol on NCC phosphorylation (suppl. Fig 5). Nevertheless, these negative results do not I1 mediates cAMP stimulation of NCC formally exclude some activation of the WNK4/SPAK/OSR1 pathway. In fact, we consistently observed slight, but never statistically significant, increases in pSPAK/OSR1 immunoreactivities in tissue samples treated with FSK/IBMX and isoproterenol. Moreover, previous studies on heterologous expression systems linked SPAK to the cAMP/PKA-dependent NCC regulation 13 , while studies on knockout mouse models implicated OSR1 (but not SPAK) in the catecholamine-dependent control of NCC 15 . Part of these discrepancies may reflect the different experimental settings (e.g. in vitro, ex vivo, in vivo) and compounds used, but they may also indicate some redundancies in the signaling pathways, by which cAMP-elevating hormones stimulate NCC activity. In line with this view, we consistently observed some residual cAMP-dependent stimulation of NCC phosphorylation in kidneys of I1-KO mice in response to forskolin/IBMX and in particular in response to PTH. In contrast, the effect of catecholamines (isoproterenol and norepinephrine) on NCC phosphorylation appeared to depend almost exclusively on the presence of I1. A possible explanation for these differences might be a compartmentalization of the cAMP signaling pathways.
In fact, cAMP reporter assays provided evidence for a spatial and temporal control of cAMP dynamics due to the presence of local micro-domains with proteins producing and degrading cAMP 51 , which allow circumscribed effects independent from cAMP levels outside these microdomains 52 . Thus, the hormone-induced cAMP/PKAdependent activation of NCC may involve several redundant pathways including the previously characterized WNK4, SPAK and OSR1 kinase pathways 15,35,48,53 . The current study adds I1 as an important additional regulator and suggests that in addition to the NCC-controlling kinases also the phosphatases are tightly regulated. Figure 7 shows the proposed signaling model that we believe best explains the current observations in the context of prior knowledge.
Aside from these novel insights into the molecular mechanism controlling NCC function, our findings may have some cIinical implications. Inappropriately high sympathetic activity is thought to contribute to cardiovascular diseases including cardiac arrhythmia, cardiac failure, and arterial hypertension 54 . Previous studies already implicated I1 in the β-adrenergic response of the heart modulating cardiac contractility 55 , excitibality 22 , and remodeling 23 . The present study extends these observations to the kidney and shows that I1 is also critically involved in the renal response to catecholamines. Our data indicate that catecholamines increase the  23 . Immunosuppressive therapy with calcineurin inhibitors such as tacrolimus and cyclosporine A is often complicated by the development of arterial hypertension, which was suggested to be linked to renal Na + retention due to an activation of NCC 19 . It is tempting to speculate that at least part of these effects are also mediated via I1. Future in vivo studies, will have to address the relevance of renal I1 for catecholamine-and calcineurin-induced arterial hypertension.
In summary, the present study identified the inhibitor 1 (I1) of protein phosphatase 1 as a central regulatory element in the signal transduction cascade that mediates the stimulatory effects of cAMP on the thiazide-sensitive NaCl cotransporter. I1 may represent an interesting point of convergence for different kinase and phosphatase pathways in the DCT contributing to the regulation of NCC in health and disease.
Given its relevance for both the cardiac and renal response to β-adrenergic stimulation, I1 might also be an interesting drug target for the treatment of cardiovascular diseases, including arterial hypertension.  averaged to obtain one value per slice which is represented in figure 6.

Isolated perfused mouse kidney
Isolated mouse kidney perfusion was performed at 37°C in a small animal perfusion system (Hugo Sachs Elektronik, Germany) as previously described 2 . To reduce the scattering of NCC activation between animals, the Renin-Angiotensin-Aldosterone System (RAAS) was suppressed 2 days prior to the experiment by feeding age and weight matched male WT and I1 deficient mice with 8% NaCl diet. In all experiments, kidneys were perfused for 40 minutes with control buffer before isoproterenol was added to a final concentration of 100 nmol/L. Kidneys were further perfused for another 40 minutes and finally snap frozen in liquid nitrogen for western blot analysis.

Electron Microscopy
Kidney slices were cut with a vibratome as described before and incubated in control

Automated DCT isolation
To sort single renal DCT1 fragments, mice expressing EGFP under the control of the parvalbumin promoter (PV-EGFP) 3