Ouabain enhances renal cyst growth in a slowly progressive mouse model of autosomal dominant polycystic kidney disease.

Renal cyst progression in autosomal dominant polycystic kidney disease (ADPKD) is highly dependent on agents circulating in blood. We have previously shown, using different in vitro models, that one of these agents is the hormone ouabain. By binding to Na+-K+-ATPase (NKA), ouabain triggers a cascade of signal transduction events that enhance ADPKD cyst progression by stimulating cell proliferation, fluid secretion, and dedifferentiation of the renal tubular epithelial cells. Here, we determined the effects of ouabain in vivo. We show that daily administration of ouabain to Pkd1RC/RC ADPKD mice for 1-5 mo, at physiological levels, augmented kidney cyst area and number compared with saline-injected controls. Also, ouabain favored renal fibrosis; however, renal function was not significantly altered as determined by blood urea nitrogen levels. Ouabain did not have a sex preferential effect, with male and female mice being affected equally. By contrast, ouabain had no significant effect on wild-type mice. In addition, the actions of ouabain on Pkd1RC/RC mice were exacerbated when another mutation that increased the affinity of NKA for ouabain was introduced to the mice (Pkd1RC/RCNKAα1OS/OS mice). Altogether, this work highlights the role of ouabain as a procystogenic factor in the development of ADPKD in vivo, that the ouabain affinity site on NKA is critical for this effect, and that circulating ouabain is an epigenetic factor that worsens the ADPKD phenotype.NEW & NOTEWORTHY This work shows that the hormone ouabain enhances the progression of autosomal dominant polycystic kidney disease (ADPKD) in vivo. Ouabain augments the size and number of renal cysts, the kidney weight to body weight ratio, and kidney fibrosis in an ADPKD mouse model. The Na+-K+-ATPase affinity for ouabain plays a critical role in these effects. In addition, these outcomes are independent of the sex of the mice.

Temporal Changes in Clinical Outcomes after Minimally Invasive Surgery for Hallux Valgus Correction in Women without Postoperative Complications.

Minimally invasive surgery (MIS) is currently used to correct hallux valgus deformities. Most studies reporting on MIS techniques to correct hallux valgus deformities included patients with postoperative complications. These reported complications, with an average rate of 23%, had significant negative effects on the clinical outcomes in this patient population. In the present study, a cohort of 63 women who underwent MIS hallux valgus correction was assessed preoperatively and at a mean follow-up of 1.0, 4.7, and 6.5 years using the American Orthopaedic Foot and Ankle Society (AOFAS) scale and the Manchester Oxford Foot Questionnaire (MOXFQ). The main criterion for inclusion in this cohort was a lack of complications during the entire follow-up period. The results showed significant improvements in both AOFAS and MOXFQ scores between the preoperative and 1-year follow-up assessments. By contrast, clinically small and nonsignificant changes were observed among postoperative follow-up values. The number of enrolled patients needs to be increased in future studies, with different surgeons and techniques included. Nevertheless, our study findings will inform patients about the outcomes they can expect over the years if no complications occur.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

Pre- and Post-Operative Relationship between Radiological Measures and Clinical Outcomes in Women with Hallux Valgus.

The surgical correction of a hallux valgus (HV) deformity improves radiological parameters and clinical outcomes. However, it is not known how these improvements are related between themselves. In this retrospective study, 73 women were assessed preoperatively and 60 months after HV surgical correction. Several radiological parameters were measured: the hallux valgus angle (HVA), I−II intermetatarsal angle (IMA) and sesamoid position. The functional outcomes were assessed using the American Orthopaedic Foot and Ankle Society (AOFAS) Hallux Metatarsophalangeal-Interphalangeal (HMI) scale, and patient-reported outcomes (PROMs) were recorded with the Manchester−Oxford Foot Questionnaire (MOXFQ). A pre−post-surgery comparison of radiological and clinical values was performed, the correlation among them was studied and the differences pre−post-surgery in the radiological measurements compared with those for the clinical outcomes were studied. The results show that all the radiological parameters, functional outcomes and PROMs improved significantly from their pre-operative values to the follow-up values. Multivariate regression analysis showed a significant relationship (p < 0.001) between the differential pre−post-surgery AOFAS scoring only with two sesamoid position differential pre−post-surgery measures: position of medial sesamoid (PMS) and translation of the first metatarsal head (TMH). However, no significant association was observed between the pre−post-surgery radiological differences and the pre−post-surgery MOXFQ scoring.

The sodium-glucose cotransporter isoform 1 (SGLT-1) is important for sperm energetics, motility, and fertility†.

Glucose is a key substrate for supporting sperm energy production and function. Previous studies have demonstrated that sperm glucose uptake is facilitated by several isoforms of the glucose transporters (GLUT). Here, we report that sperm also expresses the Na+-dependent sodium glucose cotransporter (SGLT). This was first suggested by our observation that genetic deletion of the testis-specific Na,K-ATPase α4, which impairs the sperm plasma membrane Na+ gradient, reduces glucose uptake and ATP production. Immunoblot analysis revealed the presence of an SGLT in sperm, with specific expression of isoform 1 (SGLT-1), but not of isoform 2 (SGLT-2). Immunocytochemistry identified SGLT-1 in the mid- and principal piece of the sperm flagellum. Inhibition of SGLT-1 with the isotype-selective inhibitor phlorizin significantly reduced glucose uptake, glycolytic activity, and ATP production in noncapacitated and capacitated sperm from wild-type mice. Phlorizin also decreased total sperm motility, as well as other parameters of sperm movement. In contrast, inhibition of SGLT-1 had no significant effect on sperm hyperactivation, protein tyrosine phosphorylation, or acrosomal reaction. Importantly, phlorizin treatment impaired the fertilizing capacity of sperm. Altogether, these results demonstrate that mouse sperm express a functional SGLT transport system that is important for supporting sperm energy production, motility, and fertility.

Correlation among Knee Muscle Strength and Self-Reported Outcomes Score, Anterior Tibial Displacement, and Time Post-Injury in Non-Coper Anterior Cruciate Ligament Deficient Patients: A Cross-Sectional Study.

Little attention has been paid to knee muscle strength after ACL rupture and its effect on prognostic outcomes and treatment decisions. We studied hamstrings (H) and quadriceps (Q) strength correlation with a patient-reported outcome measures score (International Knee Documentation Committee, IKDC), anterior tibial translation (ATT), and time post-injury in 194 anterior cruciate ligament deficient patients (ACLD) who required surgery after a failed rehabilitation program (non-copers). The correlation between knee muscle strength and ATT was also studied in 53 non-injured controls. ACLD patients showed decreased knee muscle strength of both the injured and non-injured limbs. The median (interquartile range) values of the H/Q ratio were 0.61 (0.52-0.81) for patients' injured side and 0.65 (0.57-0.8) for the non-injured side (p = 0.010). The median H/Q ratio for the controls was 0.52 (0.45-0.66) on both knees (p < 0.001, compared with the non-injured side of patients). The H/Q, ATT, and time post-injury were not significantly correlated with the IKDC score. ATT was significantly correlated with the H/Q of the injured and non-injured knees of patients, but not in the knees of the controls. Quadriceps strength and H/Q ratio were significantly correlated with ATT for both limbs of the patients. IKDC score correlated significantly with the quadriceps and hamstrings strengths of the injured limb but not with the H/Q ratio, ATT or time passed after injury.

Reversión del bypass gástrico: indicaciones y resultados quirúrgicos / Gastric bypass reversal: surgical indications and results

Resumen Introducción: Debido a su eficacia en el tratamiento de la obesidad mórbida, el bypass gástrico (BPG) sigue siendo una intervención realizada frecuentemente. Sin embargo, un grupo reducido de pacientes puede desarrollar complicaciones nutricionales y metabólicas que no logran controlarse con un tratamiento médico óptimo. En estos casos, puede ser necesario reestablecer la continuidad del tracto gastrointestinal por medio de la reversión del BPG (R-BPG). Objetivo: Presentar las indicaciones y resultados obtenidos en una serie de pacientes sometidos a una R-BPG. Materiales y Método: Identificación y evaluación retrospectiva de todos los pacientes sometidos a una R-BPG en nuestra institución de manera consecutiva. Se registraron las características demográficas y antropométricas de la cirugía original y al momento de la reversión. Las complicaciones se registraron de acuerdo con la clasificación de Clavien-Dindo. Resultados: Se identificaron 7 pacientes en los cuales se realizó una R-BPG. En 2 casos la reversión fue por síndrome de intestino corto, en 3 casos por hipoglicemias severas refractarias a manejo médico y en 2 casos por diarrea crónica. La mediana de edad al momento de la reversión fue de 55 años. La mediana de tiempo desde la cirugía original hasta el momento de la reversión fue de 77 meses. La mediana de estadía hospitalaria fue de 6 días. No hubo complicaciones Clavien-Dindo ≥ III. La R-BPG logró revertir en todos los casos las complicaciones nutricionales y metabólicas. Conclusión: La restauración de la continuidad del tracto gastrointestinal permite el control de las complicaciones nutricionales y metabólicas.

Introduction: Due to its efficacy in the treatment of morbid obesity, roux-en-y gastric bypass (RYGB) continues to be a frequently performed intervention. However, a small group of patients may develop nutritional and metabolic complications that cannot be controlled with optimal medical treatment. In these cases, it may be necessary to reestablish the continuity of the gastrointestinal tract by reversing the RYGB (R-RYGB). Aim: To present the indications and results obtained in a series of patients who underwent to R-RYGB. Materials and Method: Identification and retrospective evaluation of all patients who underwent consecutive R-RYGB in our institution. Demographic and anthropometric characteristics of the original surgery and at the time of the reversal were recorded. Complications were classified according to Clavien-Dindo classification. Results: Seven patients were identified in whom an R-RYGB was performed. In 2 cases the reversal was due to short bowel syndrome, in 3 cases due to severe hypoglycemia refractory to medical treatment and in 2 cases due to chronic diarrhea. The median age at the time of the reversal was 55 years. The median time from primary surgery to reversal was 77 months. The median hospital stay was 6 days. There were no Clavien-Dindo complications ≥ III. The R-RYGB was able to reverse nutritional and metabolic complications in all cases. Conclusion: Restoring the continuity of the gastrointestinal tract allows control of nutritional and metabolic complications.

Na,K-ATPase α4, and Not Na,K-ATPase α1, is the Main Contributor to Sperm Motility, But its High Ouabain Binding Affinity Site is Not Required for Male Fertility in Mice.

Mammalian sperm express two Na,K-ATPase (NKA) isoforms, Na,K-ATPase α4 (NKAα4) and Na,K-ATPase α1 (NKAα1). While NKAα4 is critical to sperm motility, the role of NKAα1 in sperm movement remains unknown. We determined this here using a genetic and pharmacological approach, modifying the affinity of NKAα1 and NKAα4 for the inhibitor ouabain to selectively block the function of each isoform. Sperm from wild-type (WT) mice (naturally containing ouabain-resistant NKAα1 and ouabain-sensitive NKAα4) and three newly generated mouse lines, expressing both NKAα1 and NKAα4 ouabain resistant (OR), ouabain sensitive (OS), and with their ouabain affinity switched (SW) were used. All mouse lines produced normal sperm numbers and were fertile. All sperm types showed NKAα isoform expression levels and activity comparable to WT, and kinetics for ouabain inhibition confirming the expected changes in ouabain affinity for each NKA isoform. Ouabain at 1 µM, which only block ouabain-sensitive NKA, significantly inhibited total, progressive, and hyperactivated sperm motility in WT and OS, but had no significant effect on OR or SW sperm. Higher ouabain (1 mM), which inhibits both ouabain-sensitive and ouabain-resistant NKA, had little additional effect on sperm motility in all mouse lines, including the OR and SW. A similar pattern was found for the effect of ouabain on sperm intracellular sodium ([Na+]i). These results indicate that NKAα4, but not NKAα1 is the main contributor to sperm motility and that the ouabain affinity site in NKA is not an essential requirement for male fertility.

Na,K-ATPase Atp1a4 isoform is important for maintaining sperm flagellar shape.

PURPOSE: The aim of this study is to investigate the mechanisms by which the testis specific Na,K-ATPase ion transport system (Atp1a4) controls sperm morphology and shape. METHODS: Sperm from wild-type (WT) and Atp1a4 knockout (Atp1a4 KO) mice were analyzed morphologically, using light, transmission, and scanning electron microscopy; and functionally, applying sperm osmotic challenge and viability tests. In addition, a sperm proteomic study was performed. RESULTS: Light microscopy confirmed that sperm lacking Atp1a4 present a bend at the junction of the mid- and principal piece of the flagellum. This bend had different degrees of angulation, reaching occasionally a complete flagellar retroflexion. The defect appeared in sperm collected from the cauda epididymis, but not the epididymal caput or the testis. Transmission and scanning electron microscopy revealed a dilation of the cytoplasm at the site of the bend, with fusion of the plasma membrane in overlapping segments of the flagellum. This was accompanied by defects in the axoneme and peri-axonemal structures. Sperm from Atp1a4 KO mice showed an abnormal response to hypoosmotic challenge with decreased viability, suggesting reduced capacity for volume regulation. Exposure to Triton X-100 only partially recovered the flagellar bend of Atp1a4 KO sperm, showing that factors other than osmotic regulation contribute to the flagellar defect. Interestingly, several key sperm structural proteins were expressed in lower amounts in Atp1a4 KO sperm, with no changes in their localization. CONCLUSIONS: Altogether, our results show that Atp1a4 plays an important role in maintaining the proper shape of the sperm flagellum through both osmotic control and structurally related mechanisms.

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.

Anterior tibial translation and patient-reported outcomes after anterior cruciate ligament reconstruction with a tape locking screw: A 5-year follow-up study.

BACKGROUND: The Tape Locking Screw system (TLS) is a recognised technique used in anterior cruciate ligament reconstruction (ACLR). However, only a few previous studies have reported associated outcomes, all of which had been examined over a short-term period. The aim of this study was to assess the time-dependent changes in the objective and patient-reported outcome measures (PROM) in a group of patients with anterior cruciate ligament deficiency who have been operated on with this technique. HYPOTHESIS: Previously reported satisfactory short-term outcomes following TLS persist for several years after the operation. PATIENTS AND METHODS: This study was a retrospective observational study including 26 patients, who were followed after unilateral ACLR with TLS. Anterior tibial translation (ATT) was measured in both knees using the KT-1000 arthrometer and two PROMs: International Knee Documentation Committee (IKDC) and Lysholm subjective form scores were examined preoperatively, 6 months postoperatively, and annually for 5 years thereafter in all patients. RESULTS: One patient suffered a rupture of the graft, and one patient had a screw loosening. Two patients were lost for follow-up, so 22 patients were the final study group. Median (25-75%) ATT side-to-side differences between the injured and uninjured sides were 4 (3,5-4)mm preoperatively, 0,75 (0-1)mm 1 year postoperatively, and 0,75 (0-1)mm 5 years after the operation (P<0.001). Median (25-75%) IKDC scores were 44.25 (35.6-55.15), 92.55 (87.08-96.6), and 95.4 (90.8-97.7) points preoperatively and 1 year (P<0.001) and 5 years postoperatively, respectively. Median (25-75%) Lysholm scores were 52 (38.75-64.5), 95.5 (94.75-99.25), and 97.5 (95-99) points preoperatively and 1 year (P<0.001) and 5 years postoperatively, respectively. DISCUSSION: ACLR with TLS might already achieve favourable outcomes 1 year postoperatively, when measured objectively (ATT) and with PROMs. These outcomes persist 5 year postoperatively. LEVEL OF EVIDENCE IV: retrospective cohort study.

The Na+ and K+ transport system of sperm (ATP1A4) is essential for male fertility and an attractive target for male contraception†.

One of the mechanisms that cells have developed to fulfil their specialized tasks is to express different molecular variants of a particular protein that has unique functional properties. Na,K-ATPase (NKA), the ion transport mechanism that maintains the transmembrane Na+ and K+ concentrations across the plasma membrane of cells, is one of such protein systems that shows high molecular and functional heterogeneity. Four different isoforms of the NKA catalytic subunit are expressed in mammalian cells (NKAα1, NKAα2, NKAα3, and NKAα4). NKAα4 (ATP1A4) is the isoform with the most restricted pattern of expression, being solely produced in male germ cells of the testis. NKAα4 is abundant in spermatozoa, where it is required for sperm motility and hyperactivation. This review discusses the expression, functional properties, mechanism of action of NKAα4 in sperm physiology, and its role in male fertility. In addition, we describe the use of NKAα4 as a target for male contraception and a potential approach to pharmacologically block its ion transport function to interfere with male fertility.

21-Benzylidene digoxin decreases proliferation by inhibiting the EGFR/ERK signaling pathway and induces apoptosis in HeLa cells.

Cardiotonic steroids (CTS) are agents traditionally known for their capacity to bind to the Na,K-ATPase (NKA), affecting the ion transport and the contraction of the heart. Natural CTS have been shown to also have effects on cell signaling pathways. With the goal of developing a new CTS derivative, we synthesized a new digoxin derivative, 21-benzylidene digoxin (21-BD). Previously, we have shown that this compound binds to NKA and has cytotoxic actions on cancer, but not on normal cells. Here, we further studied the mechanisms of actions of 21-BD. Working with HeLa cells, we found that 21-BD decreases the basal, as well as the insulin stimulated proliferation. 21-BD reduces phosphorylation of the epidermal growth factor receptor (EGFR) and extracellular-regulated kinase (ERK), which are involved in pathways that stimulate cell proliferation. In addition, 21-BD promotes apoptosis, which is mediated by the translocation of Bax from the cytosol to mitochondria and the release of mitochondrial cytochrome c to the cytosol. 21-BD also activated caspases-8, -9 and -3, and induced the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). Altogether, these results show that the new compound that we have synthesized exerts cytotoxic actions on HeLa cells by inhibition of cell proliferation and the activation of both the extrinsic and intrinsic apoptotic pathways. These results support the relevance of the cardiotonic steroid scaffold as modulators of cell signaling pathways and potential agents for their use in cancer.

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception.

Na,K-ATPase α4 is a testis-specific plasma membrane Na+ and K+ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca2+, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach.

Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.

On the Many Actions of Ouabain: Pro-Cystogenic Effects in Autosomal Dominant Polycystic Kidney Disease.

Ouabain and other cardenolides are steroidal compounds originally discovered in plants. Cardenolides were first used as poisons, but after finding their beneficial cardiotonic effects, they were rapidly included in the medical pharmacopeia. The use of cardenolides to treat congestive heart failure remained empirical for centuries and only relatively recently, their mechanisms of action became better understood. A breakthrough came with the discovery that ouabain and other cardenolides exist as endogenous compounds that circulate in the bloodstream of mammals. This elevated these compounds to the category of hormones and opened new lines of investigation directed to further study their biological role. Another important discovery was the finding that the effect of ouabain was mediated not only by inhibition of the activity of the Na,K-ATPase (NKA), but by the unexpected role of NKA as a receptor and a signal transducer, which activates a complex cascade of intracellular second messengers in the cell. This broadened the interest for ouabain and showed that it exerts actions that go beyond its cardiotonic effect. It is now clear that ouabain regulates multiple cell functions, including cell proliferation and hypertrophy, apoptosis, cell adhesion, cell migration, and cell metabolism in a cell and tissue type specific manner. This review article focuses on the cardenolide ouabain and discusses its various in vitro and in vivo effects, its role as an endogenous compound, its mechanisms of action, and its potential use as a therapeutic agent; placing especial emphasis on our findings of ouabain as a pro-cystogenic agent in autosomal dominant polycystic kidney disease (ADPKD).

Ouabain promotes partial epithelial to mesenchymal transition (EMT) changes in human autosomal dominant polycystic kidney disease (ADPKD) cells.

The hormone ouabain has been shown to enhance the cystic phenotype of autosomal dominant polycystic kidney disease (ADPKD). Among other characteristics, the ADPKD phenotype includes cell de-differentiation and epithelial to mesenchymal transition (EMT). Here, we determined whether physiological concentrations of ouabain induces EMT in human renal epithelial cells from patients with ADPKD. We found that ADPKD cells respond to ouabain with a decrease in expression of the epithelial marker E-cadherin and increase in the expression of the mesenchymal markers N-cadherin, α smooth muscle actin (αSMA) and collagen-I; and the tight junction protein occludin and claudin-1. Other adhesion molecules, such as ZO-1, ß-catenin and vinculin were not significantly modified by ouabain. At the cellular level, ouabain stimulated ADPKD cell migration, reduced cell-cell interaction, and the ability of ADPKD cells to form aggregates. Moreover, ouabain increased the transepithelial electrical resistance of ADPKD cell monolayers, suggesting that the paracellular transport pathway was preserved in the cells. These effects of ouabain were not observed in normal human kidney (NHK) cells. Altogether these results show a novel role for ouabain in ADPKD, inducing changes that lead to a partial EMT phenotype in the cells. These effects further support the key role that ouabain has as a factor that promotes the cystic characteristics of ADPKD cells.

Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway.

Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key "executioner" caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression.

Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1 (m1Bei) mice, but had no effect on metanephroi from Pkd1 (m1Bei) mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl(-) efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and up-regulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na(+) transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD.