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Caloric restriction extends healthy lifespan in multiple species 1 . Intermittent fasting, an alternative form of dietary restriction, is potentially more sustainable in humans, but its effectiveness remains largely unexplored 2-8 . Identifying the most efficacious forms of dietary restriction is key for developing interventions to improve human health and longevity 9 . Here we performed an extensive assessment of graded levels of caloric restriction (20% and 40%) and intermittent fasting (1 and 2 days fasting per week) on the health and survival of 960 genetically diverse female mice. We show that caloric restriction and intermittent fasting both resulted in lifespan extension in proportion to the degree of restriction. Lifespan was heritable and genetics had a larger influence on lifespan than dietary restriction. The strongest trait associations with lifespan included retention of body weight through periods of handling-an indicator of stress resilience, high lymphocyte proportion, low red blood cell distribution width and high adiposity in late life. Health effects differed between interventions and exhibited inconsistent relationships with lifespan extension. 40% caloric restriction had the strongest lifespan extension effect but led to a loss of lean mass and changes in the immune repertoire that could confer susceptibility to infections. Intermittent fasting did not extend the lifespan of mice with high pre-intervention body weight, and two-day intermittent fasting was associated with disruption of erythroid cell populations. Metabolic responses to dietary restriction, including reduced adiposity and lower fasting glucose, were not associated with increased lifespan, suggesting that dietary restriction does more than just counteract the negative effects of obesity. Our findings indicate that improving health and extending lifespan are not synonymous and raise questions about which end points are the most relevant for evaluating aging interventions in preclinical models and clinical trials., (© 2024. The Author(s).)

In response to decreasing numbers of individuals entering into nephrology fellowships, the American Society of Nephrology launched Kidney Tutored Research and Education for Kidney Scholars (TREKS) to stimulate interest in nephrology among medical students, graduate students, and postdoctoral fellows. The program combines a 1-week intensive exposure to kidney physiology with a longitudinal mentorship program at the participants' home institutions. Ten years in, an analysis was conducted to assess its effectiveness. We surveyed participants to assess their opinions regarding nephrology before and after the course and followed them longitudinally to determine their career choices. TREKS applicants who were not selected to participate were used as a comparison group. Three hundred eighty-one people participated in the program, and 242 completed the survey. After TREKS, both medical students and graduate students showed increased interest in nephrology, with rank scores of 5.6±0.2 before to 7.5±0.1 after the course for medical students (mean±SD, n =189, P = 0.001) and 7.3±0.3 to 8.7±0.3 ( n =53, P = 0.001) for graduate students. In long-term follow-up, TREKS medical students chose a nephrology pipeline residency at a higher rate than medical students overall (57% versus 31%, P = 0.01) and TREKS applicants who did not participate (47% versus 31%, P = 0.04). Nephrology fellowship rates for these groups exceeded the general population but did not significantly differ between TREKS participants and applicants. Doctor of Philosophy students and postdoctoral TREKS participants had a higher rate of participation in nephrology research compared with TREKS applicants (66% versus 30%, P = 0.01). In summary, the American Society of Nephrology Kidney TREKS program has demonstrated that it can increase interest in nephrology in the short term and increase the number of individuals going into nephrology careers. This long-term effect is most evident in Doctor of Philosophy students and postdoctoral participants. Further study is needed to assess the impact of TREKS on enrollment in nephrology fellowship programs., (Copyright © 2024 by the American Society of Nephrology.)

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data., (© 2024. The Author(s).)

Purinergic signaling plays an important role in regulating bladder contractility and voiding. Abnormal purinergic signaling is associated with lower urinary tract symptoms (LUTS). Ecto-5'-nucleotidase (NT5E) catalyzes dephosphorylation of extracellular AMP to adenosine, which in turn promotes adenosine-A2b receptor signaling to relax bladder smooth muscle (BSM). The functional importance of this mechanism was investigated using Nt5e knockout (Nt5eKO) mice. Increased voiding frequency of small voids revealed by voiding spot assay was corroborated by urodynamic studies showing shortened voiding intervals and decreased bladder compliance. Myography indicated reduced contractility of Nt5eKO BSM. These data support a role for NT5E in regulating bladder function through modulation of BSM contraction and relaxation. However, the abnormal bladder phenotype of Nt5eKO mice is much milder than we previously reported in A2b receptor knockout (A2bKO) mice, suggesting compensatory response(s) in Nt5eKO mouse bladder. To better understand this compensatory mechanism, we analyzed changes in purinergic and other receptors controlling BSM contraction and relaxation in the Nt5eKO bladder. We found that the relative abundance of muscarinic CHRM3 (cholinergic receptor muscarinic 3), purinergic P2X1, and A2b receptors was unchanged, whereas P2Y12 receptor was significantly downregulated, suggesting a negative feedback response to elevated ADP signaling. Further studies of additional ecto-nucleotidases indicated significant upregulation of the nonspecific urothelial alkaline phosphatase ALPL, which might mitigate the degree of voiding dysfunction by compensating for Nt5e deletion. These data suggest a mechanistic complexity of the purinergic signaling network in bladder and imply a paracrine mechanism in which urothelium-released ATP and its rapidly produced metabolites coordinately regulate BSM contraction and relaxation., (© 2024 Federation of American Societies for Experimental Biology.)

Diabetic bladder dysfunction (DBD) is the most common complication in diabetes. Myogenic abnormalities are common in DBD; however, the underlying mechanisms leading to these remain unclear. To understand the importance of smooth muscle insulin receptor (IR)-mediated signaling in the pathogenesis of DBD, we conditionally deleted it to achieve either heterozygous (SMIR+/-) or homozygous (SMIR-/-) deletion in smooth muscle cells. Despite impaired glucose and insulin tolerance seen with SMIR-/- mice, both SMIR+/- and SMIR-/- mice exhibited normal blood glucose and plasma insulin levels. Interestingly, these mice had abnormal voiding phenotypes, that included urinary frequency and small voids, and bladder smooth muscle (BSM) had significantly diminished contraction force. Morphology revealed a dilated bladder with thinner BSM layer, and BSM bundles were disorganized with penetrating interstitial tissue. Deletion of IR elevated FoxO and decreased mTOR protein expression, which further decreased the expression of Chrm3, P2x1, Sm22, and Cav1.2, crucial functional proteins for BSM contraction. Furthermore, we determined the expression of adiponectin in BSM, and deletion of IR in BSM inhibited adiponectin-mediated signaling. In summary, disruption of IR-mediated signaling in BSM caused abnormalities in proliferation and differentiation, leading to diminished BSM contractility and a voiding dysfunction phenotype that recapitulates human DBD., (© 2022 by the American Diabetes Association.)

The bladder undergoes large shape changes as it fills and empties and experiences complex mechanical forces. These forces become abnormal in diseases of the lower urinary tract such as overactive bladder, neurogenic bladder, and urinary retention. As the primary mechanosensors linking the actin cytoskeleton to the extracellular matrix (ECM), integrins are likely to play vital roles in maintaining bladder smooth muscle (BSM) homeostasis. In a tamoxifen-inducible smooth muscle conditional knockout of β1-integrin, there was concomitant loss of α1- and α3-integrins from BSM and upregulation of αV- and β3-integrins. Masson's staining showed a reduction in smooth muscle with an increase in collagenous ECM. Functionally, mice exhibited a changing pattern of urination by voiding spot assay up to 8 wk after tamoxifen. By 8 wk, there was increased frequency with reductions in voided volume, consistent with overactivity. Cystometrograms confirmed that there was a significant reduction in intercontractile interval with reduced maximal bladder pressure. Muscle strip myography revealed a loss of contraction force in response to electrical field stimulation, that was entirely due to the loss of muscarinic contractility. Quantitative western blotting showed a loss of M3 receptor and no change in P2X 1 . qPCR on ECM and interstitial genes revealed loss of Ntpd2, a marker of an interstitial cell subpopulation; and an upregulation of S100A4, which is often associated with fibroblasts. Collectively, the data show that the loss of appropriate mechanosensation through integrins results in cellular and extracellular remodeling, and concomitant bladder dysfunction that resembles lower urinary tract symptoms seen in older people., Competing Interests: The authors have no conflicts of interest to disclose., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Few generalists engage in basic science research or feel comfortable teaching physiology at the bedside. This may reflect a lack of understanding or confidence teaching physiologic principles., Aim: To inspire general internists to relearn and teach physiology in clinical practice., Setting: An active biomedical research laboratory., Participants: We educated 67 faculty participants (4 primary care, 59 hospitalists, and 4 other specialties) from 24 medical centers, representing 17 states., Program Description: The 5-day course was structured around re-learning basic physiology principles and developing teaching skills. Participants engaged in hands-on experiments through 4 modules using aquatic species, each paired with a physiology content primer. Participants also developed teaching scripts based on their experiments., Program Evaluation: Post-course surveys revealed that 97% felt confident teaching physiology at the bedside, 100% felt the course enhanced their understanding of the mechanisms of disease, and there was a significant improvement in self-reported teaching ability., Discussion: An immersive, hands-on faculty development course that integrated physiology with clinical decision-making increased participants' comfort level and self-rated ability to teach and incorporate physiology in their clinical work. We believe faculty development is one potential solution to the growing chasm between clinicians and scientists in general medicine., (© 2021. Society of General Internal Medicine.)

Acute urinary retention (AUR) is a common urological emergency and affects a significant patient population. The inability to eliminate urine may lead to permanent damage to the bladder's structure and functioning. However, we know little about the underlying molecular sequelae to the urine retention. To closely mirror the potential high pressures that patients with AUR could experience, we catheterized anesthetized female mice via the urethra and filled the bladder by pumping saline (25 µL/min) into the bladder lumen to 50 cm or 80 cm water pressure. A water column with designated height (50 or 80 cm) was then adjusted to maintain constant pressure in the bladder lumen for 30 minutes. Functional and morphological evaluations were performed from 0 to 24 hours after AUR treatment. Mice exhibited incontinence and overactivity with diminished voiding pressure. Significant injury was confirmed which revealed bladders with disrupted urothelial barrier, edematous lamina propria, and distorted muscle bundles. Bladder smooth muscle (BSM) from pressure-treated mice have significantly diminished contraction force, suggesting that bladder voiding dysfunction can be attributed to impaired BSM contractility. Indeed, dysregulation of acetylcholine and purinergic signaling pathways were demonstrated, indicating that reduced efficacy of these pathways contributes to impaired BSM contractility. Finally, altered expression of β1-integrin and extracellular matrix mediated mechanotransduction pathways were detected, suggesting a profound remodeling process. These data demonstrated an easy to perform, quantifiable, and reproducible AUR mouse model, which mimics well the characteristics of human AUR patients, and our data generate new insights into the molecular mechanisms that occur following AUR., (© 2021 Federation of American Societies for Experimental Biology.)

Background: AKI is a significant complication of coronavirus disease 2019 (COVID-19), with no effective therapy. Niacinamide, a vitamin B3 analogue, has some evidence of efficacy in non-COVID-19-related AKI. The objective of this study is to evaluate the association between niacinamide therapy and outcomes in patients with COVID-19-related AKI., Methods: We implemented a quasi-experimental design with nonrandom, prospective allocation of niacinamide in 201 hospitalized adult patients, excluding those with baseline eGFR <15 ml/min per 1.73 m 2 on or off dialysis, with COVID-19-related AKI by Kidney Disease Improving Global Outcomes (KDIGO) criteria, in two hospitals with identical COVID-19 care algorithms, one of which additionally implemented treatment with niacinamide for COVID-19-related AKI. Patients on the niacinamide protocol (B3 patients) were compared against patients at the same institution before protocol commencement and contemporaneous patients at the non-niacinamide hospital (collectively, non-B3 patients). The primary outcome was a composite of death or RRT., Results: A total of 38 out of 90 B3 patients and 62 out of 111 non-B3 patients died or received RRT. Using multivariable Cox proportional hazard modeling, niacinamide was associated with a lower risk of RRT or death (HR, 0.64; 95% CI, 0.40 to 1.00; P =0.05), an association driven by patients with KDIGO stage-2/3 AKI (HR, 0.29; 95% CI, 0.13 to 0.65; P =0.03; P interaction with KDIGO stage=0.03). Total mortality also followed this pattern (HR, 0.17; 95% CI, 0.05 to 0.52; in patients with KDIGO stage-2/3 AKI, P =0.002). Serum creatinine after AKI increased by 0.20 (SEM, 0.08) mg/dl per day among non-B3 patients with KDIGO stage-2/3 AKI, but was stable among comparable B3 patients (+0.01 [SEM, 0.06] mg/dl per day; P interaction=0.03)., Conclusions: Niacinamide was associated with lower risk of RRT/death and improved creatinine trajectory among patients with severe COVID-19-related AKI. Larger randomized studies are necessary to establish a causal relationship., Competing Interests: A. Asnani reports being a scientific advisor or members of Sarnoff Cardiovascular Research Foundation, and receiving honoraria from UpToDate. R.S. Brown received royalties from Börm Bruckmeier Publishing LLC for a book and two applications, Nephrology Pocket and Acid Base and Electrolytes, on smartphones, with the second edition of Nephrology Pocket to be published by Elsevier, Inc. In R.S. Brown’s role at Harvard Medical Faculty Physicians (HMFP) at Beth Israel Deaconess Medical Center, he serves as an associate medical director of DaVita Dialysis Center (Brookline, MA). All income paid by this facility goes directly to HMFP, from which he receives a fixed salary. He also reports receiving honoraria from Harvard Medical School Department of Continuing Education, Beth Israel Deaconess Medical Center; having ownership interest in Innovative Wellness Systems, Inc.; being a member of the medical advisory board of the National Kidney Foundation of New England; and being on the board of directors and president of The Organization for Renal Care in Haiti (TORCH) Inc., a nonprofit, charitable corporation in Massachusetts. No income is received from any of these entities. K. Mukamal reports having other interests/relationships with US Highbush Blueberry Council and Wolters Kluwer. S.M. Parikh is listed as an inventor on patent filings from Beth Israel Deaconess Medical Center related to NAD+. S.M. Parikh reports having consultancy agreements with Aerpio, Alkermes, Astellas, Cytokinetics, Hope Pharmaceuticals, Janssen, Leerink Swann, Mission Therapeutics, and Mitobridge; being a scientific advisor for or member of Aerpio, JASN, Kidney360, and Raksana; receiving consulting fees in the last 3 years from Alkermes, Astellas, Cytokinetics, Daiichi Sankyo, Janssen, and Mission Therapeutics; receiving honoraria from American Society of Nephrology; receiving research funding from Baxter; and having ownership interest in Eunoia and Raksana. Work in S.M. Parikh’s laboratory is supported by National Heart, Lung, and Blood Institute grant R35-HL139424, National Institute of Diabetes and Digestive and Kidney Diseases grant R01-DK095072, and National Institute on Aging grant R01-AG027002. A. Poyan Mehr reports receiving research funding from ASN Carl W. Gottschalk Research Scholar Grant (2018) and Retrophin; being the site principle investigator for the DUPLEX Study (a phase-3 randomized trial in primary FSGS); having industry-sponsored clinical trial agreements with OMEROS, Roche, and Vertex; being the director of the GlomCon Project, an initiative to enhance education about glomerular disorders. This project is collaborating closely with the NephCure Foundation, which has provided financial and in-kind support for a continuing medical education–accredited conference series focused on clinical trials. A. Poyan Mehr has additional educational collaborations with the Renal Pathology Society and the European Renal Association. A. Poyan Mehr’s employer does not permit serving on any speaker bureau, advisory board, perform consultancy, or receive industry honoraria. N.H. Raines reports being a scientific advisor for or member of La Isla Network. J. Schlondorff reports being a scientific advisor for or member of the Alport Syndrome Foundation Medical Advisory Committee and having patents and inventions with Partners Healthcare. T.I. Steinman reports being a reviewer for CJASN and a member of the editorial board for Nephrology News and Issues; receiving research funding from Kadmon, Reata, and Retrophin; receiving honoraria from Mallinkrodt and Otsuka; being on the medical advisory board for the National Kidney Foundation of Massachusetts, Rhode Island, New Hampshire, and Vermont; and having other interests/relationships with National Kidney Foundation and Polycystic Kidney Foundation. M.L. Zeidel reports being a scientific advisor for or member of the Beth Israel Deaconess Medical Center and Hebrew Senior Life. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

The general anesthetic ketamine has been repurposed by physicians as an anti-depressant and by the public as a recreational drug. However, ketamine use can cause extensive pathological changes, including ketamine cystitis. The mechanisms of ketamine's anti-depressant and adverse effects remain poorly understood. Here we present evidence that ketamine is an effective L-type Ca 2+ channel (Cav1.2) antagonist that directly inhibits calcium influx and smooth muscle contractility, leading to voiding dysfunction. Ketamine prevents Cav1.2-mediated induction of immediate early genes and transcription factors, and inactivation of Cav1.2 in smooth muscle mimics the ketamine cystitis phenotype. Our results demonstrate that ketamine inhibition of Cav1.2 signaling is an important pathway mediating ketamine cystitis. In contrast, Cav1.2 agonist Bay k8644 abrogates ketamine-induced smooth muscle dysfunction. Indeed, Cav1.2 activation by Bay k8644 decreases voiding frequency while increasing void volume, indicating Cav1.2 agonists might be effective drugs for treatment of bladder dysfunction.

Background: Our understanding of the neural circuits controlling micturition and continence is constrained by a paucity of techniques for measuring voiding in awake, behaving mice., New Method: To facilitate progress in this area, we developed a new, non-invasive assay, micturition video thermography (MVT), using a down-facing thermal camera above mice on a filter paper floor., Results: Most C57B6/J mice void infrequently, with a stereotyped behavioral sequence, and usually in a corner. The timing of each void is indicated by the warm thermal contrast of freshly voided urine. Over the following 10-15 min, urine cools to ∼3 °C below the ambient temperature and spreads radially in the filter paper. By measuring the area of cool contrast comprising this "thermal void spot," we can derive the initially voided volume. Thermal videos also reveal mouse behaviors including a home-corner preference apart from void spots, and a stereotyped, seconds-long pause while voiding., Comparison With Existing Methods and Conclusions: MVT is a robust, non-invasive method for measuring the timing, volume, and location of voiding. It improves on an existing technique, the void spot assay, by adding timing information, and unlike the cystometrogram preparation, MVT does not require surgical catheterization. Combining MVT with current neuroscience techniques will improve our understanding of the neural circuits that control continence, which is important for addressing the growing number of patients with urinary incontinence as the population ages., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diabetic bladder dysfunction is a frequent complication of diabetes. Although many mouse models of diabetes now exist, there has been little systematic effort to characterize them for the timing of onset and severity of bladder dysfunction. We monitored metabolic status and tested bladder function by void spot assay and limited anesthetized cystometry in both male and female mice of three models of obesity and diabetes: a type 1 diabetes model (the Akita mouse) and two type 2 diabetes models [the diet-induced obese (DIO) model and the ob/ob mouse]. Akita mice had insulin pellets implanted subcutaneously every 3 mo to mimic poorly controlled type 1 diabetes in humans. Mice were hyperglycemic by 48 days after implants. Female mice exhibited no bladder dysfunction at any age up to 20 mo and gained weight normally. In contrast, by 7 mo, male Akita mice developed a profound polyuria and failed to show normal weight gain. There were no observable signs of bladder dysfunction in either sex. DIO mice on high/low-fat diets for 16 mo exhibited mild hyperglycemia in female mice (not in male mice), mild weight gain, and no evidence of bladder dysfunction. Ob/ob mice were followed for 8 mo and became extremely obese. Male and female mice were glucose intolerant, insulin intolerant, and hyperinsulinemic at 4 mo. By 8 mo, their metabolic status had improved but was still abnormal. Urine volume increased in male mice but not in female mice. Bladder dysfunction was observed in the spotting patterns of female mice at 4 and 6 mo of age, resolving by 8 mo. We conclude there are dramatic sex-related differences in lower urinary tract function in these models. Male Akita mice may be a good model for polyuria-related bladder remodeling, whereas female ob/ob mice may better mimic storage problems related to loss of outlet control in a setting of type 2 diabetes complicated by obesity.

Lower urinary tract symptoms (LUTS) are exceptionally common and debilitating, and they are likely caused or exacerbated by dysfunction of neural circuits controlling bladder function. An incomplete understanding of neural control of bladder function limits our ability to clinically address LUTS. Barrington's nucleus (Bar) provides descending control of bladder and sphincter function, and its glutamatergic neurons expressing corticotropin releasing hormone (Bar Crh/Vglut2 ) are implicated in bladder control. However, it remains unclear whether this subset of Bar neurons is necessary for voiding, and the broader circuitry providing input to this control center remains largely unknown. Here, we examine the contribution to micturition behavior of Bar Crh/Vglut2 neurons relative to the overall Bar Vglut2 population. First, we identify robust, excitatory synaptic input to Bar. Glutamatergic axons from the periaqueductal gray (PAG) and lateral hypothalamic area (LHA) intensely innervate and are functionally connected to Bar, and optogenetic stimulation of these axon terminals reliably provokes voiding. Similarly, optogenetic stimulation of Bar Vglut2 neurons triggers voiding, whereas stimulating the Bar Crh/Vglut2 subpopulation causes bladder contraction, typically without voiding. Next, we genetically ablate either Bar Vglut2 or Bar Crh/Vglut2 neurons and found that only Bar Vglut2 ablation replicates the profound urinary retention produced by conventional lesions in this region. Fiber photometry recordings reveal that Bar Vglut2 neuron activity precedes increased bladder pressure, while activity of Bar Crh/Vglut2 is phase delayed. Finally, deleting Crh from Bar neurons has no effect on voiding and related bladder physiology. Our results help identify the circuitry that modulates Bar neuron activity and identify subtypes that may serve different roles in micturition., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Abnormalities in purine availability or purinergic receptor density are commonly seen in patients with lower urinary tract symptoms (LUTS), but the underlying mechanisms relating altered receptor function to LUTS are unknown. Here we provide extensive evidence for the reciprocal interplay of multiple receptors responding to ATP, ADP (adenosine diphosphate), and adenosine, agonists that regulate bladder function significantly. ADP stimulated P2Y12 receptors, causing bladder smooth muscle (BSM) contraction, whereas adenosine signaling through potentially newly defined A2b receptors, actively inhibited BSM purinergic contractility. The modulation of adenylyl cyclase-cAMP signaling via A2b and P2Y12 interaction actively regulated bladder contractility by modulating intracellular calcium levels. KO mice lacking the receptors display diametrically opposed bladder phenotypes, with P2Y12-KO mice exhibiting an underactive bladder (UAB) phenotype with increased bladder capacity and reduced voiding frequency, whereas A2b-KO mice have an overactive bladder (OAB), with decreased capacity and increased voiding frequency. The opposing phenotypes in P2Y12-KO and A2b-KO mice not only resulted from dysregulated BSM contractility, but also from abnormal BSM cell growth. Finally, we demonstrate that i.p. administration of drugs targeting P2Y12 or A2b receptor rescues these abnormal phenotypes in both KO mice. These findings strongly indicate that P2Y12 and A2b receptors are attractive therapeutic targets for human patients with LUTS.

Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted for study of lower urinary tract physiology. The spontaneous void spot assay (VSA), a popular micturition assessment tool, involves placing a mouse in an enclosure lined by filter paper and quantifying the resulting urine spot pattern. The VSA has advantages of being inexpensive and noninvasive, but some investigators challenge its ability to distinguish lower urinary tract function from behavioral voiding. A consensus group of investigators who regularly use the VSA was established by the National Institutes of Health in 2015 to address the strengths and weaknesses of the assay, determine whether it can be standardized across laboratories, and determine whether it can be used as a surrogate for evaluating urinary function. Here we leverage experience from the consensus group to review the history of the VSA and its uses, summarize experiments to optimize assay design for urinary physiology assessment, and make best practice recommendations for performing the assay and analyzing its results.

Purinergic signalling plays an important role in the regulation of bladder smooth muscle (BSM) contractility, and P2X 4 receptor is expressed in the bladder wall, where it may act by forming heteromeric receptors with P2X 1 , the major purinergic force-generating muscle receptor. To test this hypothesis, we examined mouse BSM contractile properties in the absence and presence of selective P2X 1 (NF449 & NF279) and P2X 4 antagonists (5-BDBD). These drugs inhibited BSM purinergic contraction only partially, suggesting the possibility of a heteromeric receptor. However, carefully controlled co-immunoprecipitation experiments indicated that P2X 1 and P2X 4 do not form physically linked heteromers. Furthermore, immunofluorescence staining showed that P2X 4 is not present in mouse BSM per se, but in an unknown cellular structure among BSM bundles. To investigate whether deletion of P2X 4 could impact voiding function in vivo, P2X 4 null mice were characterized. P2X 4 null mice had normal bladder weight and morphology, normal voiding spot size and number by voiding spot assay, normal voiding interval, pressure and compliance by cystometrogram, and normal BSM contractility by myography. In conclusion, these data strongly suggest that P2X 4 is not present in mouse BSM cells, does not affect smooth muscle contractility and that mice null for P2X 4 exhibit normal voiding function.

Barrington's nucleus (Bar) is thought to contain neurons that trigger voiding and thereby function as the "pontine micturition center." Lacking detailed information on this region in mice, we examined gene and protein markers to characterize Bar and the neurons surrounding it. Like rats and cats, mice have an ovoid core of medium-sized Bar neurons located medial to the locus coeruleus (LC). Bar neurons express a GFP reporter for Vglut2, develop from a Math1/Atoh1 lineage, and exhibit immunoreactivity for NeuN. Many neurons in and around this core cluster express a reporter for corticotrophin-releasing hormone (Bar CRH ). Axons from Bar CRH neurons project to the lumbosacral spinal cord and ramify extensively in two regions: the dorsal gray commissural and intermediolateral nuclei. Bar CRH neurons have unexpectedly long dendrites, which may receive synaptic input from the cerebral cortex and other brain regions beyond the core afferents identified previously. Finally, at least five populations of neurons surround Bar: rostral-dorsomedial cholinergic neurons in the laterodorsal tegmental nucleus; lateral noradrenergic neurons in the LC; medial GABAergic neurons in the pontine central gray; ventromedial, small GABAergic neurons that express FoxP2; and dorsolateral glutamatergic neurons that express FoxP2 in the pLC and form a wedge dividing Bar from the dorsal LC. We discuss the implications of this new information for interpreting existing data and future experiments targeting Bar CRH neurons and their synaptic afferents to study micturition and other pelvic functions., (© 2017 Wiley Periodicals, Inc.)

Background: Patient handoffs between units can introduce risk and time delays. Verbal communication is the most common mode of handoff, but requires coordination between different parties., Objective: We present an asynchronous patient handoff process supported by a structured electronic signout for admissions from the emergency department (ED) to the inpatient medicine service., Methods: A retrospective review of patients admitted to the medical service from July 1, 2011 to June 30, 2015 at a tertiary referral center with 520 inpatient beds and 57,000 ED visits annually. We developed a model for structured electronic, asynchronous signout that includes an option to request verbal communication after review of the electronic handoff information., Results: During the 2010 academic year (AY) all admissions used verbal communication for signout. The following academic year, electronic signout was implemented and 77.5% of admissions were accepted with electronic signout. The rate increased to 87.3% by AY 2014. The rate of transfer from floor to an intensive care unit within 24 h for the year before and 4 years after implementation of the electronic signout system was collected and calculated with 95% confidence interval. There was no statistically significant difference between the year prior and the years after the implementation., Conclusions: Our handoff model sought to maximize the opportunity for asynchronous signout while still providing the opportunity for verbal signout when deemed necessary. The process was rapidly adopted with the majority of patients being accepted electronically., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It has long been viewed that the maintenance of osmotic balance in response to high salt intake is a passive process that is mediated largely by increased water consumption to balance the salt load. Two studies in this issue of the JCI challenge this notion and demonstrate that osmotic balance in response to high salt intake involves a complex regulatory process that is influenced by hormone fluctuation, metabolism, food consumption, water intake, and renal salt and water excretion. Rakova et al. report the unexpected observation that long-term high salt intake did not increase water consumption in humans but instead increased water retention. Moreover, salt and water balance was influenced by glucocorticoid and mineralocorticoid fluctuations. Kitada et al. extend upon these findings in mouse models and determined that increased urea and a corresponding increase in urea transporters in the renal medulla as the result of increased protein intake promote the water retention that is needed to achieve osmotic homeostasis. Together, the results of these two studies lay the groundwork for future studies to determine how, in the face of chronic changes in salt intake, humans maintain volume and osmotic homeostasis.

Academic clinical departments have the opportunity and responsibility to improve the quality and value of care and patient safety by supporting effective quality improvement activities. The pressure to provide high-value care while further developing academic programs has increased the complexity of decision making and change management in academic health systems. Overcoming these challenges will require faculty engagement and leadership; however, most academic departments do not have a sufficient number of individuals with expertise and experience in quality improvement and patient safety (QI/PS). Accordingly, the authors of this article advocate for a targeted and proactive approach to developing faculty working in QI/PS. They propose a strategy predicated on the identification of QI/PS as a strategic priority for academic departments, the creation of enabling resources in QI/PS, and the expansion of rigorous training programs in change management and in improvement and implementation sciences. Professional organizations, health systems, medical schools, and academic departments should recognize successful QI/PS work with awards and promotions. Individual faculty members should expand their collaborative networks, consider the generalizability and scholarly impact of their efforts when designing QI/PS initiatives, and benchmark the outcomes of their performance. Appointments and promotions committees should work proactively with department and QI/PS leaders to ensure that outstanding achievement in QI/PS is defined and recognized. As with the development of physician-investigators and clinician-educators, departments and health systems need a comprehensive approach to support and recognize the contributions of faculty working in QI/PS to meet the considerable needs and opportunities in health care.

The coming decades will see enormous changes in how kidney disease is diagnosed and treated. We can only predict a small proportion of the discoveries that will catalyze these changes. It is exhilarating to imagine how such discoveries might soon translate into improved medical care for millions of people.

Health care costs in the US have grown rapidly over the past several decades. As a result, there has been increasing interest in strategies to contain health care costs, without sacrificing the quality of medical care. While many factors contribute to rising costs, one major contributor to health care expenditure is diagnostic imaging. In this article, we address the growth of diagnostic imaging, the financial and clinical adverse effects of over-utilization, and discuss a variety of strategies to encourage appropriate use of diagnostic testing., (Copyright © 2016 Alliance for Academic Internal Medicine. Published by Elsevier Inc. All rights reserved.)

Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Kidney Tutored Research and Education for Kidney Students (TREKS) Program is a product of the American Society of Nephrology (ASN) Workforce Committee that seeks to connect medical and graduate students to nephrology. This program starts with a weeklong camp-like course introducing participants to renal physiology through classic and modern experiments. Next, each student is matched with a nephrology mentor at his or her home institution to foster a better understanding of a nephrology career. Lastly, the students are encouraged to participate in scholarly activities and attend the ASN Kidney Week. Now in its third year, with a total of 84 participants, survey data suggest early success of the program, with a self-reported 40% increased interest in nephrology fellowship and/or research careers. In addition, students give high ratings to the course components and mentorship pairings. Continued student tracking will be necessary to determine the long-term program effect., (Copyright © 2016 by the American Society of Nephrology.)

Void spot assays (VSA) and cystometry are two of the most common tests performed in mice to assess lower urinary tract function. Assay protocols and methodology vary greatly among laboratories, and little is known about reproducibility of results generated by different laboratories. We performed VSA in four mouse strains, comparing males with females and comparing results between two independent laboratories. Unique aspects of the current study include direct comparison of results of VSA performed in a similar manner in two locations and comparison of cystometry performed using two different rates of infusion in these two laboratories. Both assays were performed in male and female 129S1/SvImJ, C57BL/6J, NOD/ShiLtJ, and CAST/EiJ mice, and cystometry was performed under urethane anesthesia (10/group). Assays were performed and results analyzed as previously described. Results obtained in female mice were compared with previously reported values. Results of lower urinary tract function testing in mice vary in a consistent manner with strain and sex. Variables in husbandry, testing techniques, and analysis of results can significantly affect conclusions, particularly those obtained by cystometry. Although VSA results were remarkably similar between the two laboratories, consistent methods for performing lower urinary tract function testing in mice are required to compare results among studies with confidence., (Copyright © 2015 the American Physiological Society.)

Alterations in water homeostasis can disturb cell size and function. Although most cells can internally regulate cell volume in response to osmolar stress, neurons are particularly at risk given a combination of complex cell function and space restriction within the calvarium. Thus, regulating water balance is fundamental to survival. Through specialized neuronal "osmoreceptors" that sense changes in plasma osmolality, vasopressin release and thirst are titrated in order to achieve water balance. Fine-tuning of water absorption occurs along the collecting duct, and depends on unique structural modifications of renal tubular epithelium that confer a wide range of water permeability. In this article, we review the mechanisms that ensure water homeostasis as well as the fundamentals of disorders of water balance., (Copyright © 2015 by the American Society of Nephrology.)

Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The concept of homeostasis has been inextricably linked to the function of the kidneys for more than a century when it was recognized that the kidneys had the ability to maintain the "internal milieu" and allow organisms the "physiologic freedom" to move into varying environments and take in varying diets and fluids. Early ingenious, albeit rudimentary, experiments unlocked a wealth of secrets on the mechanisms involved in the formation of urine and renal handling of the gamut of electrolytes, as well as that of water, acid, and protein. Recent scientific advances have confirmed these prescient postulates such that the modern clinician is the beneficiary of a rich understanding of the nephron and the kidney's critical role in homeostasis down to the molecular level. This review summarizes those early achievements and provides a framework and introduction for the new CJASN series on renal physiology., (Copyright © 2014 by the American Society of Nephrology.)

Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations., (Copyright © 2014 the American Physiological Society.)

The Atlantic killifish (Fundulus heteroclitus) is a model environmental organism that has an extremely low assimilation rate of environmental arsenic. As a first step in elucidating the mechanism behind this phenomenon, we used quantitative real-time PCR to identify aquaglyceroporins (AQPs), which are arsenite transporters, in the killifish gill. A novel homolog killifish AQP3 (kfAQP3a) was cloned from the killifish gill, and a second homolog was identified as the consensus from a transcriptome database (kfAQP3b). The two were 99% homologous to each other, 98% homologous to a previously identified killifish AQP3 from embryos (kfAQP3ts), and 78% homologous to hAQP3. Expression of kfAQP3a in Xenopus oocytes significantly enhanced water, glycerol, and urea transport. However, kfAQP3a expressed in HEK293T cells did not transport significant amounts of arsenic. All sequence motifs thought to confer the ability of AQP3 to transport solutes were conserved in kfAQP3a, kfAQP3b, and kfAQP3ts; however, the C-terminal amino acids were different in kfAQP3a versus the other two homologs. Replacement of the three C-terminal amino acids of kfAQP3 (GKS) with the three C-terminal amino acids of kfAQP3b and kfAQP3ts (ANC) was sufficient to enable kfAQP3a to robustly transport arsenic. Thus, the C-terminus of kfAQP3b and kfAQP3ts confers arsenic selectivity in kfAQP3. Moreover, kfAQP3a, the only AQP expressed in killifish gill, is the first aquaglyceroporin identified that does not transport arsenic, which may explain, in part, why killifish poorly assimilate arsenic and are highly tolerant to environmental arsenic.

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (∼1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (∼7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (∼ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.

With more clinical information for trainees to master in the face of increasing time pressures, discussions about underlying physiology and the mechanisms of disease seem to have been de-emphasized during residency training. This reduced focus on pathophysiology and basic science may weaken trainees' clinical effectiveness and reduce their interest in pursuing research careers.In response, the authors helped to develop in 2006 a one-week immersive comparative physiology course for second- and third-year internal medicine residents at Beth Israel Deaconess Medical Center. The course, held at the Mount Desert Island Biological Laboratory, includes four modules (hematology, vascular physiology, secretory physiology, and salt and water homeostasis), each composed of basic science experiments using aquatic species and accompanied by clinical correlation discussions and group presentations.From 2007 to 2010, 72 residents rotated through the course. Most reported that it enhanced their understanding of the mechanisms of disease in their patients. After the course, residents reported that physiology played a more prominent role in their teaching and clinical decision making during both ward and intensive care unit rotations. They also reported being more likely than before the course to read about the pathophysiology of disease when faced with a clinical problem.Although cost-intensive and geographically unique, this model for teaching the mechanisms of disease could be applied elsewhere with the help of physician-scientists and clinician-educators. In this article, the authors describe the development of the course, share preliminary data evaluating progress toward its goals, and discuss future directions and lessons learned.

Effective scheduling of and ready access to doctor appointments affect ambulatory patient care quality, but these are often sacrificed by patients seeking care from physicians at academic medical centers. At one center, Beth Israel Deaconess Medical Center, the authors developed interventions to improve the scheduling of appointments and to reduce the access time between telephone call and first offered appointment. Improvements to scheduling included no redirection to voicemail, prompt telephone pickup, courteous service, complete registration, and effective scheduling. Reduced access time meant being offered an appointment with a physician in the appropriate specialty within three working days of the telephone call. Scheduling and access were assessed using monthly "mystery shopper" calls. Mystery shoppers collected data using standardized forms, rated the quality of service, and transcribed their interactions with schedulers. Monthly results were tabulated and discussed with clinical leaders; leaders and frontline staff then developed solutions to detected problems. Eighteen months after the beginning of the intervention (in June 2007), which is ongoing, schedulers had gone from using 60% of their registration skills to over 90%, customer service scores had risen from 2.6 to 4.9 (on a 5-point scale), and average access time had fallen from 12 days to 6 days. The program costs $50,000 per year and has been associated with a 35% increase in ambulatory volume across three years. The authors conclude that academic medical centers can markedly improve the scheduling process and access to care and that these improvements may result in increased ambulatory care volume.

Background: Interstitial cells of Cajal (ICC) have been identified in urinary bladder of several species, but their presence in mice remains uncertain. Meanwhile, dozens of reports indicate that dysregulation of connexin 43 plays an important role in bladder overactivity, but its localization has not been clearly defined, with reports of expression in either the smooth muscle or in myofibroblasts. We recently identified a population of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) positive cells that resemble ICC and are distinct from smooth muscle, fibroblasts, myofibroblasts and neurons. Thus we sought to define more clearly the molecular signature of ICC and in doing so resolve some of these uncertainties., Principle Findings: Immunofluorescent localization revealed that NTPDase2-positive cells lie closely adjacent to smooth muscle but are separate from them. NTPDase2 positive cells exhibited co-localization with the widely accepted ICC marker - c-kit. They were further shown to co-localize with other ICC markers CD34 and Ano1, but not with mast cell marker tryptase. Significantly, they show convincing co-localization with connexin 43, which was not present in smooth muscle. The identity of these cells as ICC was further confirmed by the presence of three mesenchymal markers - vimentin, desmin, and PDGFβ receptor, which indicates their mesenchymal origin. Finally, we observed for the first time, the presence of merlin/neurofibromin 2 in ICC. Normally considered a neuronal protein, the presence of merlin suggests ICC in bladder may have a role in neurotransmission., Conclusions: NTPDase2 positive cells in mice bladder are ICC, which can be defined by the presence of c-Kit, CD34, Ano1, NTPDase2, connexin 43, vimentin, desmin, PDGFβ receptor and merlin/NF2. These data establish a definitive molecular expression profile, which can be used to assist in explorations of their functional roles, and the presence of NTPDase2 suggests that purinergic signaling plays a role in regulation of ICC function.

As a major component of homeostasis, all organisms regulate the water composition of various compartments. Through the selective use of barrier membranes and surface glycoproteins, as well as aquaporin water channels, organisms ranging from Archaebacteria to humans can vary water permeabilities across their cell membranes by 4 to 5 orders of magnitude. In barrier epithelia the outer, or exofacial, leaflet acts as the main resistor to water flow; this leaflet restricts water flow by minimizing the surface area of lipid molecules which is not covered by phosphate headgroups and by packing hydrocarbon chains at maximal density. Cells may enhance the barrier by expressing glycoproteins that augment the "thickness" of unstirred layers at their surfaces, reducing osmotic gradients at the lipid bilayer surface. Aquaporins markedly and highly selectively accelerate water flux and are "switched on" either by deployment into membranes or gating. This review summarizes these mechanisms in many species, and indicates potential roles for manipulating water permeabilities in treating disease.

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this brief review, written from the perspective of a physician-leader who has fostered the development of comprehensive quality improvement efforts at two academic medical centers, I review the need for improvement, some conceptual barriers that must be overcome, the goals of a comprehensive quality improvement (QI) effort, some of the results we have obtained, and some observations on how to develop a culture of continuous improvement in an academic medical center. The mandate for quality improvement is clear; current healthcare is wasteful and error-prone, leading to excessive morbidity and mortality and unsustainably high costs. Successful quality improvement requires the abandonment of two paradigms: the craft model of medical practice and the notion that many forms of harm to patients are not preventable. I will describe how dramatic improvement has been achieved in reducing, by up to 10-fold, rates of central line infections, ventilator-associated pneumonias, peritonitis in peritoneal dialysis patients, and mortality due to cardiac arrest in hospital. I will describe as well how these methods can improve access to out-patient clinics dramatically and enhance the reliability and safety of hand-offs between covering physicians. To develop and maintain systematic quality improvement in all phases of medical care we must articulate a culture in which: everyone working at the medical center makes improvements every day; front-line staff, who know best how the work is done, are empowered to improve the processes of care; and multidisciplinary teams create the protocols that reduce variation that is due to physician preference, leaving only the variation required by the individual needs of patients. I will review as well the crucial elements of education of trainees and faculty members needed to guide and sustain a culture of quality. Finally, I will add some observations on how oversight boards and medical center leaders can help create systematic quality improvement in their medical centers.

Objective: Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBCs). The aim of this study was to address whether this process affects the biophysical properties of RBCs., Methods: Serum and RBCs were isolated from patients with SLE and healthy controls. RBCs from healthy universal donors (type O, Rh negative) were incubated with SLE or control serum. We used flow cytometry to assess complement fragment deposition on RBCs. RBC membrane deformability was measured using 2-dimensional microchannel arrays. Protein phosphorylation levels were quantified by Western blotting., Results: Incubation of healthy universal donor RBCs with sera from patients with SLE, but not with control sera, led to deposition of C4d fragments on the RBCs. Complement-decorated RBCs exhibited significant decreases in both membrane deformability and flickering. Sera from SLE patients triggered a transitory Ca(++) influx in RBCs that was associated with decreased phosphorylation of β-spectrin and with increased phosphorylation of band 3, two key proteins of RBC cytoskeleton. Finally, incubation with SLE sera led to the production of nitric oxide by RBCs, whereas this did not occur with control sera., Conclusion: Our data suggest that complement activation in patients with SLE leads to calcium-dependent cytosketeletal changes in RBCs that render them less deformable, probably impairing their flow through capillaries. This phenomenon may negatively affect the delivery of oxygen to the tissues., (Copyright © 2011 by the American College of Rheumatology.)

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Objective: To review the neural and renal mechanisms of osmotic homeostasis, provide a rationale for the sensitivity of the central nervous system to hyponatremia, and outline modern approaches to therapy of acute and chronic hyponatremia., Methods: Review of relevant literature with focus on physiologic mechanisms., Results: With careful monitoring, acute hyponatremia can be managed, while minimizing risks both of continued hyponatremia and the osmotic demyelination that can occur with overly rapid correction of severe hyponatremia. Chronic hyponatremia due to disorders of volume regulation (congestive heart failure or cirrhosis) or to syndrome of inappropriate antidiuretic hormone release can be managed effectively with vasopressin V2 receptor antagonists, but there is no evidence that controlling the hyponatremia enhances survival associated with the underlying diseases., Conclusions: Therapy in the acute setting balances the risk of the osmotic disturbance with the risk of overly rapid correction. The V2 receptor antagonist tolvaptan has enhanced our ability to improve chronic hyponatremia in conditions such as congestive heart failure, cirrhosis, and syndrome of inappropriate antidiuretic hormone hypersecretion.

We located fully sequenced putative genes of the plasma membrane intrinsic proteins (PIPs) family in the Populus trichocarpa (Torr. Gray), genome. Of 23 gene candidates, we assigned eight genes to the PIP2 subfamily. All eight putative genes were expressed in vegetative tissues (roots, leaves, bark and wood), and all of them showed water channel activity after being expressed in Xenopus oocytes. Six of eight proteins were affected by mercury ions. No proteins were affected by the presence of nickel or tungsten ions, or by lowering the pH of bathing external solution from 7.4 to 6.5. The presence of copper ions caused seven of eight PIP2 proteins to increase their water transport capacity by as much as 50%. This systematic study of the PIP2 subfamily of proteins in P. trichocarpa provides a basic overview of their activity as water channels and will be a useful reference for future physiological studies of plant water relations that use P. trichocarpa as a model system.