Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

Background: It remains unclear why vasopressin induces greater antidiuresis through V2R than does oxytocin. Results: Vasopressin sustains cAMP signaling during V2R internalization, a process promoted by ␤-arrestins, and is halted by the retromer complex. Conclusion: This new noncanonical model of GPCR signaling differentiates the actions of vasopressin and oxytocin.Significance: This emerging model may explain the physiological bias between ligands.

The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl -channel and epithelial Na 1 channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-b-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I sc ), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I sc in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.Keywords: metformin; cystic fibrosis transmembrane conductance regulator; ENaC; airway surface liquid; inflammationThe serious genetic disease cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) Cl -channel expressed at the apical membrane in a variety of epithelial tissues, including the lung, where CF lung disease causes considerable morbidity and mortality (1). Defective CFTR function prevents airway epithelium from adequately regulating the airway surface liquid (ASL) volume, which has an adverse effect on mucus clearance from the lung. The CF airway invariably becomes colonized by bacteria such as Pseudomonas aeruginosa and develops excessive inflammation, which can cause extensive damage to lung architecture and eventuate in respiratory failure (2). There is growing evidence that a lack of functional CFTR promotes an exaggerated or prolonged inflammatory response to bacterial and other insults, although the underlying pathophysiologic mechanisms are unclear (3-5). Both up-regulation of proinflammatory mediators (e.g., IL-8, IL-6, TNF-a, and GM-CSF) and down-regulation of anti-inflammatory mediators (e.g., IL-10 and inducible nitric oxide synthase) in the CF airway may play an important role in this process (6-8).Excessive activity of the epithelial sodium channel (ENaC) on the luminal airway surface is a key factor involved in th...

The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca(2+)-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20-40 independent Ca(2+)-binding sites on synaptic vesicles, only a fraction of which need to bind Ca(2+) to trigger fusion, are sufficient to predict physiological release. Our excess-Ca(2+)-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca(2+)-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca(2+) dynamics during synapse activation.

This information about liver transplant recipients is important for researchers and clinicians. Researchers can develop guidelines by using stable but modifiable characteristics of patients to identify transplant candidates at risk of nonadherence. Such guidelines would enable clinicians to prepare patients better to manage the posttransplant regimen.

Context-Little is known about patients' contribution to health outcomes after liver transplantation. Yet, in other transplant recipients, nonadherent behavior is directly related to the leading causes of morbidity and mortality in liver transplant recipients.Objective-To examine patient and environmental factors in relation to all aspects of adherence to the posttransplantation regimen and health outcomes in the first 6 months after transplantation.Design-A descriptive analysis of individual and environmental factors in relation to adherence and health outcomes at 6 months after liver transplantation.Participants, Setting-One hundred fifty-two adult liver transplant recipients at the University of Pittsburgh Medical Center.Main Outcome Measures-Adherence to medication taking, appointment keeping, lifestyle changes, mood, quality of life, and clinical markers of liver function.Results-Nonadherence was prevalent (47% with appointments, 73% with medication); relapse to drug/alcohol use occurred among a few recipients (5.6%), all with a history of substance abuse before transplantation. Patterns of coping, decision making, attitude, and social support were correlated with adherence, clinical markers, and psychological function (r = 0.22-0.45). Avoidant coping, affective dysregulation, and caregiver support emerged as robust predictors of negative clinical and mental health outcomes (β = .224-.363).Conclusion-This information about liver transplant recipients is important for researchers and clinicians. Researchers can develop guidelines by using stable but modifiable characteristics of patients to identify transplant candidates at risk of nonadherence. Such guidelines would enable clinicians to prepare patients better to manage the posttransplant regimen.Posttransplant adherence for liver recipients involves taking medications on a regular basis, keeping appointments, and making and sustaining recommended lifestyle changes related to smoking, drinking, drug use, and other high-risk behaviors. Much of the research to date has been focused primarily on 1 facet of adherence in a subgroup of liver transplant recipients, namely, return to drinking among recipients who received a transplant because of alcoholic liver disease. Little is known about the contribution of patient and environmental factors to adherence or health outcomes in the general population of liver transplant recipients. Yet Liver transplant patients vary widely in terms of psychiatric, behavioral, and medical history. Approximately half of the population receives a transplant because of hepatitis C and/or alcoholic cirrhosis, contracted through substance abuse and/or other risky activities; most other causes of end-stage liver disease are unrelated to behavior. The range of psychopathology and behavioral history unique to this population 5,6 suggests that liver transplant recipients may differ from other transplant groups with respect to patterns of behavior that affect adherence and health outcomes.Rates and risk factors for nonadherence to the...

The debate over the status of the physician workforce seems to be concluded. It now is clear that a shortage of physicians exists and is likely to worsen. In retrospect it seems obvious that a static annual production of physicians, coupled with a population growth of 25 million persons each decade, would result in a progressively lower physician to population ratio. Moreover, Cooper has demonstrated convincingly that the robust economy of the past 50 years correlates with demand for physician services. The aging physician workforce is an additional problem: one third of physicians are over 55 years of age, and the population over the age of 65 years is expected to double by 2030. Signs of a physician and surgeon shortage are becoming apparent. The largest organization of physicians in the world (119,000 members), the American College of Physicians, published a white paper in 2006 titled, "The Impending Collapse of Primary Care Medicine and Its Implications for the State of the Nation's Health Care" [37]. The American College of Surgeons, the largest organization of surgeons, has published an article on access to emergency surgery [38], and the Institute of Medicine of the National Academies of Science has published a book on the future of emergency care (Fig. 10). The reports document diminished involvement and availability of emergency care by general surgeons, neurologic surgeons, orthopedists, hand surgeons, plastic surgeons, and others. The emergency room has become the primary care physician after 5 PM for much of the population. A survey done by the Commonwealth Fund revealed that less than half of primary care practices have an on-call arrangement for after-hours care. Other evidence of evolving shortage are reports of long wait times for appointments, the hospitalist movement, and others. The policies for the future should move beyond dispute over whether or not a shortage exists. The immediate need is for the United States, as a society, to commit to workforce self sufficiency in health care. The reliance on international graduates for more than 25% of the nation's physicians is a transnational problem. Reliance on IMGs, nurses and other health professions for the United States workforce is an issue of international distributive justice. Wealthy, developed countries, such as the United States, should be able to educate sufficient health professionals without relying on a less fortunate country's educated health workers. The 2000 Report of the Chair of the AAMC, the accrediting agency for United States and Canadian medical schools through the LCME, recommended expansion of medical school class sizes and expansion of medical schools [41]. For the past 25 years, the AAMC has supported a no-growth policy and the goal that 50% of USMGs be primary care physicians. In 2003, the AAMC developed a workforce center,-led by Edward Salsberg. The workforce center has provided valuable data and monitoring of the evolving workforce graduating from medical and and osteopathic schools in the United States. The NRMP, also...