Cardiac Oxidative Signaling and Physiological Hypertrophy in the Na/K-ATPase α1s/sα2s/s Mouse Model of High Affinity for Cardiotonic Steroids.

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2, and ß1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3-6 months, increased α1 sensitivity to CTS resulted in a significant increase in cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio accompanied by an increase in the myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

The Na/K-ATPase α1/Src interaction regulates metabolic reserve and Western diet intolerance.

AIM: Highly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K-ATPase (NKA)-mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically. METHODS: Metabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC-PK1-derived cells expressing Src binding rat NKA α1, non-Src-binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain-of-function mutant rat NKA α2. Mice with skeletal muscle-specific ablation of NKA α1 (skα1-/-) were generated using a MyoD:Cre-Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell-permeable peptide designed by mapping one of the sites of NKA α1/Src interaction. RESULTS: Metabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1-/- mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1-/- mice were resistant to Western diet-induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1-mediated Src regulation with pNaKtide. CONCLUSIONS: These results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.

Biased Effect of Cardiotonic Steroids on Na/K-ATPase-Mediated Signal Transduction.

Recent studies have revealed that Na/K-ATPase (NKA) can transmit signals through ion-pumping-independent activation of pathways relayed by distinct intracellular protein/lipid kinases, and endocytosis challenges the traditional definition that cardiotonic steroids (CTS) are NKA inhibitors. Although additional effects of CTS have long been suspected, revealing its agonist impact through the NKA receptor could be a novel mechanism in understanding the basic biology of NKA. In this study, we tested whether different structural CTS could trigger different sets of NKA/effector interactions, resulting in biased signaling responses without compromising ion-pumping capacity. Using purified NKA, we found that ouabain, digitoxigenin, and somalin cause comparable levels of NKA inhibition. However, although endogenous ouabain stimulates both protein kinases and NKA endocytosis, digitoxigenin and somalin bias to protein kinases and endocytosis, respectively, in LLC-PK1 cells. The positive inotropic effects of CTS are traditionally regarded as NKA inhibitors. However, CTS-induced signaling occurs at concentrations at least one order of magnitude lower than that of inotropy, which eliminates their well known toxic actions on the heart. The current study adds a novel mechanism that CTS could exert its biased signaling properties through the NKA signal transducer. SIGNIFICANCE STATEMENT: Although it is now well accepted that NKA has an ion-pumping-independent signaling function, it is still debated whether direct and conformation-dependent NKA/effector interaction is a key to this function. Therefore, this investigation is significant in advancing our understanding of the basic biology of NKA-mediated signal transduction and gaining molecular insight into the structural elements that are important for cardiotonic steroid's biased action.

A caveolin binding motif in Na/K-ATPase is required for stem cell differentiation and organogenesis in mammals and C. elegans.

Several signaling events have been recognized as essential for regulating cell lineage specification and organogenesis in animals. We find that the gain of an amino-terminal caveolin binding motif (CBM) in the α subunit of the Na/K-adenosine triphosphatase (ATPase) (NKA) is required for the early stages of organogenesis in both mice and Caenorhabditis elegans. The evolutionary gain of the CBM occurred at the same time as the acquisition of the binding sites for Na+/K+. Loss of this CBM does not affect cell lineage specification or the initiation of organogenesis, but arrests further organ development. Mechanistically, this CBM is essential for the dynamic operation of Wnt and the timely up-regulation of transcriptional factors during organogenesis. These results indicate that the NKA was evolved as a dual functional protein that works in concert with Wnt as a hitherto unrecognized common mechanism to enable stem cell differentiation and organogenesis in multicellular organisms within the animal kingdom.

Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma.

Background: Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. Methods: We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. Results: We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Conclusions: Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis.

Circulating CD40 and sCD40L Predict Changes in Renal Function in Subjects with Chronic Kidney Disease.

Soluble CD40 ligand (sCD40L) has been implicated in the development of renal injury. The CD40 receptor exists in a soluble form, sCD40R, and has been shown to function as a competitive antagonist against CD40 activation. We analyzed whether plasma levels of sCD40L and sCD40R predict changes in renal function in an all-cause chronic kidney disease (CKD) cohort. Stratification of subjects based on sCD40L and sCD40R individually, as well as in combination, demonstrated that sCD40L was directly associated with declines in estimated glomerular filtration rate (eGFR). sCD40R was negatively associated with declines in eGFR. Baseline characteristics following stratification, including systolic blood pressure, history of diabetes mellitus or peripheral vascular disease, primary renal disease classification, and angiotensin converting enzyme inhibitor or angiotensin receptor blocker usage were not significantly different. High sCD40L and low sCD40R were both found to be independent predictors of a decline in eGFR at 1-year follow-up (-7.57%, p = 0.014; -6.39%, p = 0.044). Our data suggest that circulating levels of sCD40L and sCD40R are associated with changes in renal function in patients with CKD. The CD40 decoy receptor, sCD40R, may serve as a potential therapeutic target to attenuate renal function decline.

MicroRNA profiling in kidney disease: Plasma versus plasma-derived exosomes.

Liquid biopsies have advanced rapidly in recent years for use in diagnostic and prognostic applications. One important aspect of this advancement is the growth in our understanding of microRNA (miRNA) biology. The measurement of miRNAs packaged within exosomes, which are constantly released into the blood stream, may reflect pathological changes within the body. The current study performed miRNA profiling using plasma and plasma-derived exosome samples from two animal models of kidney disease, the 5/6th partial nephrectomy (PNx) and two-kidney-one-clip (2K1C) models. The RT-qPCR-based profiling results revealed that the overall miRNA expression level was much higher in plasma than in plasma-derived exosomes. With 200µl of either plasma or exosomes derived from the same volume of plasma, 629 out of 665 total miRNAs analyzed were detectable in plasma samples from sham-operated rats, while only 403 were detectable in exosomes with a cutoff value set at 35cycles. Moreover, the average miRNA expression level in plasma was about 16-fold higher than that in exosomes. We also found a select subset of miRNAs that were enriched within exosomes. The number of detectable miRNAs from plasma-derived exosomes was increased in rats subjected to PNx or 2K1C surgery compared to sham-operated animals. Importantly, we found that the changes of individual miRNAs measured in plasma had very poor concordance with that measured in plasma-derived exosomes in both animal models, suggesting that miRNAs in plasma and plasma-derived exosomes are differentially regulated in these disease conditions. Interestingly, PNx and 2K1C surgeries induced similar changes in miRNA expression, implying that common pathways were activated in these two disease models. Pathway analyses using DIANA-miRPath v3.0 showed that significantly changed exosomal miRNAs were associated with extracellular matrix (ECM) receptor interaction and mucin type-O-glycan synthesis pathways, which are related with tissue fibrosis and kidney injury, respectively. In conclusion, our results demonstrated that due to the differential changes in miRNAs, the measurement of exosomal miRNAs cannot be replaced by the measurement of miRNAs in plasma, or vice versa. We also showed that a set of miRNAs related with kidney injury and organ fibrosis were dysregulated in plasma-derived exosomes from animal models of kidney disease.

Cigarette smoking causes epigenetic changes associated with cardiorenal fibrosis.

Clinical studies indicate that smoking combustible cigarettes promotes progression of renal and cardiac injury, leading to functional decline in the setting of chronic kidney disease (CKD). However, basic studies using in vivo small animal models that mimic clinical pathology of CKD are lacking. To address this issue, we evaluated renal and cardiac injury progression and functional changes induced by 4 wk of daily combustible cigarette smoke exposure in the 5/6th partial nephrectomy (PNx) CKD model. Molecular evaluations revealed that cigarette smoke significantly (P < 0.05) decreased renal and cardiac expression of the antifibrotic microRNA miR-29b-3 and increased expression of molecular fibrosis markers. In terms of cardiac and renal organ structure and function, exposure to cigarette smoke led to significantly increased systolic blood pressure, cardiac hypertrophy, cardiac and renal fibrosis, and decreased renal function. These data indicate that decreased expression of miR-29b-3p is a novel mechanism wherein cigarette smoke promotes accelerated cardiac and renal tissue injury in CKD. (155 words).

Na/K-ATPase signaling regulates collagen synthesis through microRNA-29b-3p in cardiac fibroblasts.

Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

Involvement of Na/K-ATPase in hydrogen peroxide-induced activation of the Src/ERK pathway in LLC-PK1 cells.

We have shown that Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H2O2-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H2O2 generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPases and in ouabain-sensitive Rb(+) uptake. These effects of H2O2 seem similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H2O2 on Src and ERK1/2 were inhibited in α1 Na/K-ATPase-knockdown PY-17 cells. Whereas expression of wild-type α1 or the A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1, and not the A420P mutant, was able to restore the H2O2-induced activation of protein kinases. Consistent with this, disrupting the formation of the Na/K-ATPase/Src complex with pNaKtide attenuated the effects of H2O2 on the kinases. Moreover, a direct effect of H2O2 on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H2O2 reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggest that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H2O2 to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.

Regulation of renal function and structure by the signaling Na/K-ATPase.

The Na/K-ATPase as an essential ion pump was discovered more than 50 years ago (Skou (1989) Biochim. Biophys. Acta 1000, 439-446; Feraille and Doucet (2001) Physiol. Rev. 81, 345-418). The signaling function of Na/K-ATPase has been gradually appreciated over the last 20 years, first from the studies of regulatory effects of ouabain on cardiac cell growth. Several reviews on this topic have been written during the last few years (Schoner and Scheiner-Bobis (2007) Am. J. Physiol. Cell. Physiol. 293, C509-C536; Xie and Cai (2003) Mol. Interv. 3, 157 - 168; Bagrov et al. (2009) Pharmacol. Rev. 61, 9-38; Tian and Xie (2008) Physiology 23, 205-211; Fontana et al. (2013) FEBS J. 280, 5450-5455; Blanco and Wallace (2013) Am. J. Physiol. Renal Physiol. 305, F797-F812). This article will focus on the molecular mechanism of Na/K-ATPase-mediated signal transduction and its potential regulatory role in renal physiology and diseases.

Identification of a potential receptor that couples ion transport to protein kinase activity.

In our previous studies, we have demonstrated that the Src-coupled α1 Na/K-ATPase works as a receptor for cardiotonic steroids, such as ouabain, to regulate cellular protein kinase cascades. Here, we explore further the structural determinants of the interaction between the α1 Na/K-ATPase and Src and demonstrate that the Src-coupled α1 Na/K-ATPase allows the cell to decode the transmembrane transport activity of the Na/K-ATPase to turn on/off protein kinases. The α1 Na/K-ATPase undergoes E1/E2 conformational transition during an ion pumping cycle. The amount of E1 and E2 Na/K-ATPase is regulated by extracellular K(+) and intracellular Na(+). Using purified enzyme preparations we find that the E1 Na/K-ATPase can bind both the Src SH2 and kinase domains simultaneously and keep Src in an inactive state. Conversely, the E1 to E2 transition releases the kinase domain and activates the associated Src. Moreover, we demonstrate that changes in E1/E2 Na/K-ATPase by either Na(+) or K(+) are capable of regulating Src and Src effectors in live cells. Together, the data suggest that the Src-coupled α1 Na/K-ATPase may act as a Na(+)/K(+) receptor, allowing salt to regulate cellular function through Src and Src effectors.