SIRT7 modulates the stability and activity of the renal K‐Cl cotransporter KCC4 through deacetylation
2021; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.15252/embr.202050766
ISSN1469-3178
AutoresLilia G. Noriega, Zesergio Melo, Renuga Devi Rajaram, Adriana Mercado, Armando R. Tovar, Laura A. Velázquez‐Villegas, María Castañeda‐Bueno, Yazmín Reyes-López, Dongryeol Ryu, Lorena Rojas‐Vega, Germán Ricardo Magaña-Ávila, Adriana M. López‐Barradas, Mariana Sánchez-Hernández, Anne Debonneville, Alain Doucet, Lydie Cheval, Nimbe Torres, Johan Auwerx, Olivier Staub, Gerardo Gamba,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoReport22 March 2021free access Source DataTransparent process SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation Lilia G Noriega Corresponding Author Lilia G Noriega [email protected] orcid.org/0000-0003-2156-9872 Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, MexicoThese authors contributed equally to this work Search for more papers by this author Zesergio Melo Zesergio Melo orcid.org/0000-0002-6702-204X Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico CONACYT-Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, MexicoThese authors contributed equally to this work Search for more papers by this author Renuga D Rajaram Renuga D Rajaram Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland National Centre of Competence in Research, "Kidney.ch", Zurich, SwitzerlandThese authors contributed equally to this work Search for more papers by this author Adriana Mercado Adriana Mercado orcid.org/0000-0002-6881-0160 Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, MexicoThese authors contributed equally to this work Search for more papers by this author Armando R Tovar Armando R Tovar Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Laura A Velazquez-Villegas Laura A Velazquez-Villegas Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author María Castañeda-Bueno María Castañeda-Bueno Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Yazmín Reyes-López Yazmín Reyes-López Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Dongryeol Ryu Dongryeol Ryu Laboratory of Integrative and Systems Physiology (LISP), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Lorena Rojas-Vega Lorena Rojas-Vega Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author German Magaña-Avila German Magaña-Avila Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Adriana M López-Barradas Adriana M López-Barradas Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Mariana Sánchez-Hernández Mariana Sánchez-Hernández Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico Search for more papers by this author Anne Debonneville Anne Debonneville Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland National Centre of Competence in Research, "Kidney.ch", Zurich, Switzerland Search for more papers by this author Alain Doucet Alain Doucet Centre de Recherche des Cordeliers, INSERM, Sorbonne Universités, USPC, Université Paris Descartes, Université Paris Diderot, Physiologie Rénale et Tubulopathies, CNRS ERL 8228, Paris, France Search for more papers by this author Lydie Cheval Lydie Cheval Centre de Recherche des Cordeliers, INSERM, Sorbonne Universités, USPC, Université Paris Descartes, Université Paris Diderot, Physiologie Rénale et Tubulopathies, CNRS ERL 8228, Paris, France Search for more papers by this author Nimbe Torres Nimbe Torres Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Johan Auwerx Johan Auwerx orcid.org/0000-0002-5065-5393 Laboratory of Integrative and Systems Physiology (LISP), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Olivier Staub Olivier Staub Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland National Centre of Competence in Research, "Kidney.ch", Zurich, Switzerland Search for more papers by this author Gerardo Gamba Corresponding Author Gerardo Gamba [email protected] orcid.org/0000-0002-4378-9043 Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico Search for more papers by this author Lilia G Noriega Corresponding Author Lilia G Noriega [email protected] orcid.org/0000-0003-2156-9872 Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, MexicoThese authors contributed equally to this work Search for more papers by this author Zesergio Melo Zesergio Melo orcid.org/0000-0002-6702-204X Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico CONACYT-Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, MexicoThese authors contributed equally to this work Search for more papers by this author Renuga D Rajaram Renuga D Rajaram Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland National Centre of Competence in Research, "Kidney.ch", Zurich, SwitzerlandThese authors contributed equally to this work Search for more papers by this author Adriana Mercado Adriana Mercado orcid.org/0000-0002-6881-0160 Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, MexicoThese authors contributed equally to this work Search for more papers by this author Armando R Tovar Armando R Tovar Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Laura A Velazquez-Villegas Laura A Velazquez-Villegas Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author María Castañeda-Bueno María Castañeda-Bueno Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Yazmín Reyes-López Yazmín Reyes-López Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Dongryeol Ryu Dongryeol Ryu Laboratory of Integrative and Systems Physiology (LISP), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Lorena Rojas-Vega Lorena Rojas-Vega Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author German Magaña-Avila German Magaña-Avila Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Adriana M López-Barradas Adriana M López-Barradas Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Mariana Sánchez-Hernández Mariana Sánchez-Hernández Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico Search for more papers by this author Anne Debonneville Anne Debonneville Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland National Centre of Competence in Research, "Kidney.ch", Zurich, Switzerland Search for more papers by this author Alain Doucet Alain Doucet Centre de Recherche des Cordeliers, INSERM, Sorbonne Universités, USPC, Université Paris Descartes, Université Paris Diderot, Physiologie Rénale et Tubulopathies, CNRS ERL 8228, Paris, France Search for more papers by this author Lydie Cheval Lydie Cheval Centre de Recherche des Cordeliers, INSERM, Sorbonne Universités, USPC, Université Paris Descartes, Université Paris Diderot, Physiologie Rénale et Tubulopathies, CNRS ERL 8228, Paris, France Search for more papers by this author Nimbe Torres Nimbe Torres Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Search for more papers by this author Johan Auwerx Johan Auwerx orcid.org/0000-0002-5065-5393 Laboratory of Integrative and Systems Physiology (LISP), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Olivier Staub Olivier Staub Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland National Centre of Competence in Research, "Kidney.ch", Zurich, Switzerland Search for more papers by this author Gerardo Gamba Corresponding Author Gerardo Gamba [email protected] orcid.org/0000-0002-4378-9043 Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico Search for more papers by this author Author Information Lilia G Noriega *,1, Zesergio Melo2,3, Renuga D Rajaram4,5, Adriana Mercado6, Armando R Tovar1, Laura A Velazquez-Villegas1, María Castañeda-Bueno2, Yazmín Reyes-López1, Dongryeol Ryu7,10, Lorena Rojas-Vega2, German Magaña-Avila2, Adriana M López-Barradas1, Mariana Sánchez-Hernández6, Anne Debonneville4,5, Alain Doucet8, Lydie Cheval8, Nimbe Torres1, Johan Auwerx7, Olivier Staub4,5 and Gerardo Gamba *,2,9 1Department of Nutrition Physiology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico 2Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico 3CONACYT-Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico 4Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland 5National Centre of Competence in Research, "Kidney.ch", Zurich, Switzerland 6Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico 7Laboratory of Integrative and Systems Physiology (LISP), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 8Centre de Recherche des Cordeliers, INSERM, Sorbonne Universités, USPC, Université Paris Descartes, Université Paris Diderot, Physiologie Rénale et Tubulopathies, CNRS ERL 8228, Paris, France 9Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico 10Present address: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea *Corresponding author. Tel: +52 55 54870900 ext. 2802 or 6107; E-mail: [email protected] *Corresponding author. Tel: +52 55 54870900 ext. 2802 or 6107; E-mail: [email protected] EMBO Reports (2021)22:e50766https://doi.org/10.15252/embr.202050766 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract SIRT7 is a NAD+-dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+-dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology. SYNOPSIS The molecular targets and functions of SIRT7 in the kidney are currently unknown. This study demonstrates that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology. KCC4 protein levels and activity are modulated depending on its acetylation status. SIRT7 interacts with KCC4 and increases its stability and activity through deacetylation. Metabolic acidosis increased renal SIRT7 expression, while SIRT7-deficient mice presented lower renal KCC4 expression. Introduction Sirtuins (SIRT1-7) are a conserved family of NAD+-dependent deacetylases that differ in tissue expression, intracellular localization, enzymatic activity, and target proteins (Sauve et al,2001; Houtkooper et al,2012). Among the target proteins of the sirtuins are histones, several transcription factors, and a variety of enzymes involved in different cellular processes, such as proliferation, DNA repair, antioxidant activity, mitochondrial function, and metabolism (Baur et al,2012). The role of sirtuins in renal physiology has begun to be evaluated (Hao & Haase, 2010; Hershberger et al 2017; Morigi et al,2018). For instance, SIRT1 decreases sodium reabsorption and thus regulates sodium and water handling (Zhang et al,2009). Moreover, an increase in SIRT1 activity, through a boost on NAD+ production, enhances mitochondrial function and protects the kidney from acute injury (Katsyuba et al,2018). Similarly, SIRT3 and SIRT6 protects against acute kidney injury (Morigi et al,2015) and kidney fibrosis (Cai et al,2020), respectively. Although the kidney is one of the tissues that express the highest levels of Sirt7 mRNA and protein (Ford et al,2006), no role of SIRT7 has been described in renal physiology. SIRT7 regulates transcription through activation of RNA polymerase I (Ford et al,2006), deacetylates lysine 18 of histone H3 (Barber et al,2012), and desuccinylates lysine 122 of histone H3 promoting chromatin condensation and DNA double-strand breaks repair (Li et al,2016). SIRT7-deficient mice exhibit cardiac hypertrophy and inflammatory cardiomyopathy due to hyperacetylation of p53 (Vakhrusheva et al,2008) and a multisystemic mitochondrial dysfunction (Ryu et al,2014). Furthermore, recent studies have described a role for SIRT7 in adipogenesis (Cioffi et al,2015; Fang et al,2017), bone formation (Fukuda et al,2018), and lipid metabolism (Shin et al,2013; Ryu et al,2014; Yoshizawa et al,2014). In fact, SIRT7 modulates mitochondrial function through the deacetylation of the transcription factor GABPβ, a key regulator of the expression of mitochondrial proteins (Ryu et al,2014). Notably, SIRT7-deficient mice display age-dependent hearing loss, suggesting a malfunction in ion transport in the ear (Ryu et al,2014). The hearing loss phenotype resembles that observed in mice lacking the K-Cl cotransporter KCC4 (Boettger et al,2002). KCC4 belongs to the SLC12 family of electroneutral cation-chloride-coupled cotransporters that are critical for several physiological processes, such as cell volume regulation, modulation of intracellular Cl− concentration, and transepithelial ion flux, which is critical for blood pressure and acid–base homeostasis (Arroyo et al,2013). These transporters are regulated by phosphorylation/dephosphorylation (Pacheco-Alvarez et al,2006; Richardson et al,2008; Glover et al,2010) and ubiquitylation mechanisms (Ko et al,2010; Arroyo et al,2011; Gamba, 2012), but thus far, none have been shown to be regulated by acetylation/deacetylation processes. In the kidney, KCC4 is expressed in the proximal convoluted tubule, and the thick ascending limb of Henle's loop (TAL), where it constitutes a pathway for cellular exit of K+ and Cl−, and in the α-intercalated cells of the collecting duct (Melo et al,2013a), where its activity is important for proton secretion to the tubular fluid (urine), thereby maintaining the acid–base balance (Gamba, 2005). Accordingly, KCC4 expression is increased during metabolic acidosis (Melo et al,2013a), and mice deficient in KCC4 develop renal tubular acidosis (Boettger et al,2002). Given the similar deafness phenotype observed in the SIRT7- and KCC4-deficient mice, and the fact that both proteins are highly expressed in the kidney, we hypothesized that SIRT7 might be involved in KCC4 regulation. Here, using Xenopus laevis oocytes, HEK293 cells, a mouse model of metabolic acidosis, and total and renal tubular-specific SIRT7-deficient mice, we demonstrate that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and therefore reveal a role for SIRT7 in renal physiology. Results and Discussion KCC4 acetylation decreases KCC4 protein abundance and activity in X. laevis oocytes As a first approach, we performed an in silico analysis of the GeneNetwork database (www.genenetwork.org) in the kidneys of 41 recombinant inbred mouse strains of the BXD genetic reference population (Andreux et al,2012), to evaluate whether Sirt7 mRNA abundance correlates with that of Slc12a7. We confirmed that the kidney is one of the tissues that exhibit the highest levels of Sirt7 mRNA expression (Fig 1A). Interestingly, Sirt7 expression significantly correlated (Pearson's r = 0.597, P = 4.6E-05) with Slc12a7 expression (Fig 1B), suggesting that the proteins encoded by these transcripts may have a functional interaction. Interestingly, KCC4 has been found to be acetylated at the putative acetylation site lysine 114 (K114) by high throughput proteomic analysis (Lundby et al,2012), reinforcing the hypothesis that KCC4 activity could be modulated by acetylation. Thus, to evaluate whether acetylation is a post-translational modification that affects KCC4, X. laevis oocytes injected with KCC4 were incubated with NAM, a deacetylase inhibitor, or NAD+, a sirtuin co-substrate and co-activator. KCC4 was acetylated after 4 h of incubation with NAM and remained deacetylated when incubated with NAD+ (Fig 1C). We then measured KCC4 protein levels after different incubation times with NAM and observed that KCC4 protein levels decreased more than 70% after 48 h of incubation with NAM (Fig 1D, upper panel). The decrease in KCC4 protein levels was paralleled by changes in KCC4 activity since incubation with NAM decreased KCC4 activity in a time-dependent manner, reaching an 88% reduction after 48 h (Fig 1D, lower panel). The decrease in KCC4 protein levels was not attributed to a gradual decline in the expression system, as KCC4 expression increased in a time-dependent manner in untreated oocytes (Fig 1E). Unfortunately, we were unable to confirm the acetylation site of KCC4 by mass spectrometry since the amount of immunoprecipitated KCC4 was not enough to identify a sufficient number of peptides. The difficulty to evaluate acetylation in membrane proteins has been previously reported (Griffin & Schnitzeer, 2011). However, the expression of a mutant KCC4 protein, in which the K114 lysine residue was replaced by glutamine (KCC4 K114Q) to mimic the charge of the hyperacetylated KCC4, was lower than wild-type KCC4 and exhibited lower activity. In contrast, a second mutant, in which the K114 lysine residue was replaced by an arginine (KCC4 K114R) to mimic the hypoacetylated KCC4, exhibited higher expression and activity (Fig 1F). These results demonstrate that the activity of heterologously expressed KCC4 in X. laevis oocytes is modulated through acetylation and confirm that K114 is a key acetylation site. Figure 1. KCC4 expression correlates with SIRT7 expression, and KCC4 activity is modulated by acetylation In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA. Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org). Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS–PAGE/immunoblotting using an anti-acetylated–lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control. Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS–PAGE/immunoblotting or Cl−-dependent 86Rb+ uptake under hypotonic conditions. Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS–PAGE/immunoblotting. Oocytes were injected with either WT, K114R, or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in (D). Data information: Data are presented as mean ± SEM. RU: relative units. Depicted blots are representative of three independent biological replicates and values shown are the mean of the three experiments. To evaluate KCC4 activity, we performed three independent biological experiments with oocytes obtained from different frogs and each experiment included at least 4 replicates. * Indicates a significant difference versus time 0, at P < 0.05 by Student's t-test. Source data are available online for this figure. Source Data for Figure 1 [embr202050766-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Several studies using in vitro and in vivo models have demonstrated that the activity of electroneutral cation-chloride cotransporters from the SLC12 family is regulated by post-translational modifications such as phosphorylation and ubiquitylation (Gamba, 2012). On the one hand, phosphorylation mediated by with-no-lysine (WNK) serine/threonine kinases family in conjunction with the downstream Ste20-related proline/alanine-rich kinase (SPAK) or oxidative stress response 1 (OSR1) kinase plays a key role in the regulation of these cotransporters. Specifically, WNKs/SPAK phosphorylate and activate the Na+:Cl−-coupled (NCC) members of the SLC12 family, NCC and NKCC1-2 (Kahle et al,2005; Rinehart et al,2005; Chavez-Canales et al,2014), and inhibit the K+:Cl−-coupled members, KCC1-4 (de Los Heros et al,2006; Garzon-Muvdi et al,2007; Rinehart et al,2011). On the other hand, NCC and NKCC2 have been shown to be modulated by ubiquitylation through the homologous to E6-AP C-terminal (HECT)-ligase neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4)-2 (Arroyo et al,2011; Ronzaud et al,2013; Wu et al,2013). To our knowledge, this is the first description of acetylation as a post-translational modification that modulates the activity of an SLC12 cotransporter member, and our data suggest that SIRT7 may modulate this process. SIRT7 interacts with and increases KCC4 activity and protein levels in X. laevis oocytes and HEK293 cells To evaluate whether SIRT7 modulates KCC4 activity, Xenopus oocytes were coinjected with KCC4 and SIRT7 cRNA and incubated with NAD+. KCC4 activity increased in a time-dependent manner when the oocytes were incubated with NAD+ in the presence of SIRT7, reaching a 30% increase in activity at 48 h (Fig 2A). The change in KCC4 activity was associated with an increase in KCC4 protein levels (Fig 2B), which suggests that SIRT7 may increase KCC4 stability or synthesis. Notably, a SIRT7 mutant, in which the catalytic histidine H188 was replaced by tyrosine (SIRT7 H188Y), had no effect on KCC4 activity (Fig 2C). Neither 24-h incubation with NAD+ modified the expression of KCC4 K114R or K114Q mutants (Fig 2D). We then tested the capability of SIRT7 to deacetylate the K114 lysine residue of KCC4 by injecting oocytes with the cRNA of KCC4 WT, KCC4 K114R, or KCC4 K114Q mutants, with or without SIRT7, and incubating with NAM for 4 h after 48 h of KCC4 cRNA injection. Interestingly, SIRT7 reduced global acetylation of oocytes incubated with NAM (Fig EV1), and most importantly, when we evaluated the acetylation status of KCC4 by immunoprecipitation, KCC4 WT was remarkably acetylated after incubation with NAM and remained deacetylated in the presence of SIRT7 (Fig 2E). Furthermore, the KCC4 K114R and KCC4 K114Q mutants were barely acetylated, and neither their acetylation status nor their expression was modified by the presence of SIRT7. These results confirm that K114 is the key acetylation site of KCC4 and that SIRT7 is able to deacetylate this site. To evaluate whether the effect of SIRT7 on KCC4 is conserved in mammalian cells, we co-transfected SIRT7 and FLAG-KCC4 in HEK293 cells and evaluated KCC4 protein levels. KCC4 protein levels increased by 50% after 24 h of incubation with NAD+ (Fig 2F), and decreased 80% after incubation with NAM (Fig 2G). Immunoprecipitation analysis using a FLAG antibody to immunoprecipitate KCC4 revealed that SIRT7 and KCC4 interact with each other after 24 h of incubation with NAD+, and the interaction was reduced after 4 h of incubation with NAM (Fig 2H). We assayed KCC4 stability to understand how SIRT7 increases KCC4 protein levels. For this purpose, we transfected KCC4 plasmids in the presence or absence of SIRT7 in HEK293 cells and blocked protein synthesis with cycloheximide 24 h after transfection. In the presence of SIRT7, KCC4 levels remained constant when cells were incubated with cycloheximide. However, when we used a shRNA against SIRT7, KCC4 stability was significantly reduced in the presence of cycloheximide when compared with cells transfected with a scramble shRNA (Fig 2I). Altogether, these observations suggest that SIRT7 interacts with and stabilizes KCC4 in a NAD+-dependent fashion, increasing KCC4 protein levels and thus activity. Notably, this is the first report implicating a sirtuin in the regulation of a renal cotransporter activity, which may have implications in the modulation of acid–base homeostasis. Previously, it was demonstrated that SIRT1 might modulate Na+ balance by transcriptional repression of the epithelial Na+ channel α-subunit (α-ENaC) in the apical membrane of collecting duct principal cells (Zhang et al,2009). However, the inhibition of α-ENaC by SIRT1 is independent of its deacetylase activity (Zhang et al,2009), which implies a different mechanism that the one exerted by SIRT7. Our results suggest that the mechanism by which SIRT7 increases KCC4 protein levels is through enhanced KCC4 stability. Proteins are degraded in the cell by two processes: a general process that involves lysosomal proteolysis (Sakamoto, 2002) and a selective process that eliminates specific proteins in an ubiquitin-dependent manner by the proteasome (De Strooper et al,2010). Although further research is required to evaluate whether the SIRT7-mediated deacetylation of KCC4 directly modifies its ubiquitination, a recent report shows that SIRT7 modulates the ubiquitin-proteasome pathway by binding the ubiquitin ligase complex, thereby inhibiting the ubiquitination and degradation of other proteins, such as the testicular orphan nuclear receptor, TR4 (Yoshizawa et al,2014). Consistent with our observations of the role of protein acetylation and protein stability in KCC4 regulation, a previous report also linked the deacetylation of the SAM pointed domain-containing ETS transcription factor (SPDEF) by SIRT1, with an increase in SPDEF stability (Lo Sasso et al,2014). Figure 2. SIRT7 interacts with, stabilizes, and increases KCC4 activity and protein levels A, B. KCC4 activity (A), and protein levels (B) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD+. Non-injected oocytes (UI) were used as a control. C. KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild-ty
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