Factors Associated with the Decision to Complete Bariatric Metabolic Surgery among a Racially and Ethnically Diverse Sample of Adults: A Classification and Regression Tree Analysis.

BACKGROUND: Less than 50% of eligible candidates who are referred complete Bariatric Metabolic Surgery (BMS). The factors influencing the decision to complete BMS, particularly how these factors vary across different racial and ethnic groups, remain largely unexplored. METHODS: This prospective cohort study included adult patients referred to a bariatric surgeon or obesity medicine program between July 2019-September 2022. Sociodemographic characteristics, body mass index (BMI), anxiety, depression, body appreciation, and patient-physician relationship information were collected via survey and electronic health records. The association between BMS completion and potential decision-driving factors was examined using Classification and Regression Tree (CART) analysis. RESULTS: A total of 406 BMS -eligible patients participated in the study (mean [SD] age: 47.5 [11.6] years; 87.2% women; 18.0% Hispanic, 39% non-Hispanic Black [NHB], and 39% non-Hispanic White [NHW]; mean [SD] BMI: 45.9 [10.1] kg/m2). A total of 147 participants (36.2%) completed BMS. Overall, the most influential factor driving the decision to complete BMS was younger age (< 68.4 years), higher patient satisfaction, and BMI (≥ 38.0 kg/m2). Hispanic participants prioritized age (< 55.4 years), female sex, and body appreciation. For NHB participants, the highest ranked factors were age < 56.3 years, BMI ≥ 35.8 kg/m2, and higher patient satisfaction. For NHW patients, the most influential factors were age (39.1 to 68.6 years) and higher body appreciation. CONCLUSION: These findings highlight racial and ethnic group differences in the factors motivating individuals to complete BMS. By acknowledging these differences, healthcare providers can support patients from different backgrounds more effectively in their decision-making process regarding BMS.

Impact of employment status and work setting on body weight and health behaviors among people with obesity.

BACKGROUND: The impact of employment and the work-from-home (WFH) setting on body weight among people with obesity is unknown. OBJECTIVE: This study examined the relationship between employment status, WFH setting, and change in body weight among adults with obesity during the COVID-19 pandemic. METHODS: Patients from an obesity medicine clinic completed an online survey in 2022 to assess health, work, and lifestyle behaviors. Multiple regression models examined the association between WFH status and change in body weight. RESULTS: The analytical sample included 380 patients (81.9% female, mean age 52.5 years, mean body mass index 43.25, 50.0% non-Hispanic White, 25.7% non-Hispanic Black, 18.7% Hispanic, 5.7% other ethnicity). During the pandemic, 28.7% were employed in a WFH setting (73.4% of this subgroup were in a WFH setting only), 36.1% were employed but not in a WFH setting, 11.8% were unemployed, and 23.4% were retired. Mean body weight change for participants who partially WFH during the pandemic was +5.4 (±7.2) % compared to those completely WFH -0.3 (±9.8) % (P = 0.006). After adjusting for key demographics, participants who were partially WFH gained 7.8% more weight compared to those completely WFH (ß= 7.28, SE = 1.91, p <  0.001). The most significant predictors for weight gain after adjusting for WFH frequency (partially vs. completely) included overeating (aOR 11.07, 95% CI 3.33-36.77), more consumption of fast food (aOR 7.59, 95% 2.41-23.91), and depression (aOR 6.07, 95% CI 1.97-18.68). CONCLUSION: These results show that during the COVID-19 pandemic, the WFH hybrid (combination of office and WFH) setting is associated with greater weight gain compared to those who completely WFH in people with obesity. Risk factors include overeating, higher fast food consumption, and depression.

Depression and Anxiety as Predictors of Metabolic and Bariatric Surgery Completion Among Ethnically Diverse Patients.

BACKGROUND: Mental health conditions including depression and anxiety are often prevalent among metabolic and bariatric surgery (MBS) patients, but it is not known if these conditions predict the decision to complete the procedure and if this varies by race and ethnicity. This study aimed to determine if depression and anxiety are associated with MBS completion among a race/ethnically diverse sample of patients. METHODS: This prospective cohort study included participants who were referred to an obesity program or two MBS practices between August 2019 and October 2022. Participants completed the Mini International Neuropsychiatric Interview (MINI) instrument to determine history of anxiety and/or depression, as well as MBS completion status (Y/N). Multivariable logistic regression models determined the odds of MBS completion by depression and anxiety status adjusting for age, sex, body mass index, and race/ethnicity. RESULTS: The sample consisted of 413 study participants (87 % women, 40% non-Hispanic White, 39% non-Hispanic Black, and 18% Hispanic). Participants with a history of anxiety were less likely to complete MBS (aOR = 0.52, 95% CI = 0.30-0.90, p = 0.020). Women had increased odds of a history of anxiety (aOR = 5.65, 95% CI = 1.64-19.49, p = 0.006) and of concurrent anxiety and depression (aOR = 3.07, 95% CI = 1.39-6.79, p = 0.005) compared to men. CONCLUSIONS: Results showed that participants with anxiety were 48% less likely to complete MBS compared to those without anxiety. Additionally, women were more likely to report a history of anxiety with and without depression versus men. These findings can inform pre-MBS programs about risk factors for non-completion.

Effectiveness of semaglutide versus liraglutide for treating post-metabolic and bariatric surgery weight recurrence.

OBJECTIVE: The aim of this study was to compare the effectiveness of semaglutide versus liraglutide for treating post-metabolic and bariatric surgery (MBS) weight recurrence. METHODS: A retrospective analysis of 207 adults with post-MBS weight recurrence treated with semaglutide 1.0 mg weekly (n = 115) or liraglutide 3.0 mg daily (n = 92) at an academic center from January 1, 2015, through April 1, 2021, was conducted. The primary end point was percentage body weight change at 12 months of treatment with regimens containing semaglutide or liraglutide. RESULTS: The mean sample age was 55.2 years; mean BMI was 40.4 kg/m2 ; 89.9% were female; and 50% completed sleeve gastrectomy, 29% completed Roux-en-Y gastric bypass, and 21% completed adjustable gastric banding. Least-squares mean weight change at 12 months was -12.92% versus -8.77% in the semaglutide and liraglutide groups, respectively (p < 0.001). The adjusted odds ratios were 2.34 (95% CI: 1.28-4.29) for ≥10% weight loss and 2.55 (95% CI: 1.22-5.36) for ≥15% weight loss over 12 months in the semaglutide group versus liraglutide group, respectively. Weight-loss efficacy of semaglutide (vs. liraglutide) did not differ by subgroups explored, including age, sex, and MBS procedure. CONCLUSIONS: These results show that treatment regimens including semaglutide 1.0 mg weekly lead to superior weight loss compared with liraglutide 3.0 mg daily for treating post-MBS weight recurrence, regardless of procedure type or the magnitude of weight recurrence.

Telehealth utilization among multi-ethnic patients with obesity during the COVID-19 pandemic.

INTRODUCTION: The science of telemedicine has shown great advances over the past decade. However, the field needs to better understand if a change in care delivery from in-person to telehealth as a result of the COVID-19 pandemic will yield durable patient engagement and health outcomes for patients with obesity. The objective of this study was to examine the association of mode of healthcare utilization (telehealth versus in-person) and sociodemographic factors among patients with obesity during the COVID-19 pandemic. METHODS: A retrospective medical chart review identified patients with obesity from a university outpatient obesity medicine clinic and a community bariatric surgery practice. Patients completed an online survey (1 June 2020-24 September 2020) to assess changes in healthcare utilization modality during subsequent changes in infection rates in the geographic area. Logistic regression analysis examined the association of mode of healthcare utilization and key sociodemographic characteristics. RESULTS: A total of 583 patients (87% female, mean age 51.2 years (standard deviation 13.0), mean body mass index 40.2 (standard deviation 6.7), 49.2% non-Hispanic white, 28.7% non-Hispanic black, 16.4% Hispanic, 7% other ethnicity, 33.1% completed bariatric surgery) were included. Adjusted logistic regression models showed older age was inversely associated with telehealth use (adjusted odds ratio = 0.58, 95% confidence interval 0.34-0.98) and non-Hispanic black were more likely to use telehealth compared to non-Hispanic white (adjusted odds ratio = 1.72, 95% confidence interval 1.05-2.81). CONCLUSIONS: The COVID-19 pandemic is impacting access to healthcare among patients with obesity. Telehealth is an emerging modality that can maintain healthcare access during the pandemic, but utilization varies by age and ethnicity in this high-risk population.

Association Between Patient Satisfaction With Their Patient-Physician Relationship and Completion of Bariatric Surgery by Race and Ethnicity Among US Adults.

Importance: Metabolic and bariatric surgery (MBS) is an effective and safe treatment for obesity and its comorbidities, but less than 50% of those who are eligible and referred for MBS complete the procedure. The patient-physician relationship could be a decisive factor in the decision to complete MBS; however, this relationship has not been explored, particularly among racially and ethnically diverse populations. Objective: To examine the association between patient-reported satisfaction with their patient-physician relationship and MBS completion by self-reported racial and ethnic group. Design, Setting, and Participants: This prospective cohort study included 408 patients who were referred to a bariatric surgeon or obesity medicine program between July 24, 2019, and May 19, 2022. Exposure: Patient satisfaction with their physician was measured by 7 dimensions (general satisfaction, technical quality, interpersonal manner, communication, financial aspects, time spent with physician, and accessibility and convenience) using the Patient Satisfaction Questionnaire Short Form. Main Outcomes and Measures: Completion status for MBS (yes or no). Results: A total of 408 patients (mean [SD] age, 47.3 [11.6] years; among 366 with data available, 317 [86.6%] women and 49 [13.4%] men) were included in the study. Of 363 patients with data available on race and ethnicity, 66 were Hispanic/Latinx (18.2%), 136 (37.5%) were non-Hispanic Black, 146 (40.2%) were non-Hispanic White, and 15 (4.1%) were other race or ethnicity. A total of 124 patients (30.4%) completed MBS. Overall, the mean (SD) patient satisfaction score was significantly greater in MBS completers vs noncompleters (3.86 [0.56] vs 3.61 [0.64]; P < .001). Multivariable logistic regression analysis showed technical quality was the most significant factor for MBS completion (adjusted odds ratio [aOR], 1.99 [95% CI, 1.24-3.19]), followed by communication (aOR, 1.78 [95% CI, 1.16-2.72]) and accessibility and convenience (aOR, 1.61 [95% CI, 1.03-2.53]). The interaction between racial and ethnic groups and patient satisfaction was not significant (eg, mean [SD] score for Hispanic/Latinx completers, 3.95 [0.55] vs 3.77 [0.60] for non-Hispanic White completers; P = .46 for interaction). Conclusions and Relevance: These findings suggest that there is an association between patient satisfaction with the patient-physician relationship and the decision to complete MBS regardless of race and ethnicity. These findings have important implications for strategies to improve the proportion of qualified patients who complete MBS to achieve improved health outcomes.

Changes in body weight, health behaviors, and mental health in adults with obesity during the COVID-19 pandemic.

OBJECTIVE: The aim of this study was to examine the relationships between body weight changes, health behaviors, and mental health in adults with obesity during the second year of the COVID-19 pandemic. METHODS: Between March 1, 2021, and November 30, 2021, adults from three obesity practices completed an online survey. The primary outcomes were ≥ 5% of body weight change since March 2020 and associated health behaviors and mental health factors. RESULTS: The sample (n = 404) was 82.6% female (mean age 52.5 years, mean BMI 43.3 kg/m2 ). Mean weight change was + 4.3%. Weight gain ≥ 5% was reported by 30% of the sample, whereas 19% reported ≥ 5% body weight loss. The degree of both weight gain and weight loss correlated positively with baseline BMI. Eighty percent of the sample reported difficulties with body weight regulation. Those who gained ≥ 5% versus those who lost ≥ 5% body weight were more likely to report higher levels of stress, anxiety, and depression; less sleep and exercise; less healthy eating and home-cooked meals; and more takeout foods, comfort foods, fast foods, overeating, and binge eating. CONCLUSIONS: Weight gain in adults with obesity during the COVID-19 pandemic is associated with higher baseline BMI, deteriorations in mental health, maladaptive eating behaviors, and less physical activity and sleep. Further research is needed to identify effective interventions for healthier minds, behaviors, and body weight as the pandemic continues.

Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade.

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies.1,2 Circadian variation in the intracellular chloride concentration of mammalian pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels.3 However, results have been contradictory,4-9 and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response.10 Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLNv) pacemaker neurons. In control flies, intracellular chloride increases in sLNvs over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations.11Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLNv pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

Substance Use, Mental Health, and Weight-Related Behaviors During the COVID-19 Pandemic Among Metabolic and Bariatric Surgery Patients.

PURPOSE: The impact of the COVID-19 pandemic on behavioral issues among those who have completed bariatric surgery (BS) is not well described in ethnically diverse populations. The aim of this study was to compare the impact of COVID-19 lockdown orders and after lockdown orders were lifted on substance use, mental health, and weight-related behaviors among a sample of post-BS adults. MATERIALS AND METHODS: A retrospective medical chart review identified BS patients from one university-based obesity medicine clinic and two BS practices. An online non-anonymous survey was implemented in two phases: during lockdown (April 1-May 31, 2020) and after lockdown orders were lifted (June 1, 2020-September 30, 2020) to obtain information about the COVID-19 pandemic's impact on BS patients. RESULTS: A total of 189 (during lockdown=39, post-lockdown=150) participants (90.4% female, mean age 52.4 years, SD 11.1, 49.8% non-Hispanic White, 30.6% non-Hispanic Black, 16.1% Hispanic) participated. Lockdown participants were more likely to have sleep problems (74.3% vs. 56.1%, P=.039) and feel anxious (82.0% vs. 63.0%, P=.024) versus post-lockdown participants. A majority (83.4%) reported depression in both lockdown/post-lockdown. Post-lockdown participants were more than 20 times more likely to report substance use compared those in lockdown (aOR 20.56, 95% CI 2.66-158.4). CONCLUSIONS AND RELEVANCE: The COVID-19 pandemic is having a substantial negative impact on substance use, mental health, and weight-related health behaviors in diverse BS patients. These findings have important implications for post-BS patient care teams and may suggest the integration of screening tools to identify those at high risk for behavioral health issues.

Substance use, mental health and weight-related behaviours during the COVID-19 pandemic in people with obesity.

Studies have shown the negative impact of COVID-19 lockdown orders on mental health and substance use in the general population. The aim of this study was to examine the impact of the COVID-19 pandemic onsubstance use, mental health and weight-related behaviors in a sample of adults with obesity after lockdown orders were lifted (June-September 2020). A retrospective medical chart review identified patients with obesity from one university-based obesity medicine clinic, and two metabolic and bariatric surgery (MBS) practices. Patients who completed an online survey from June 1, 2020 to September 30, 2020 were included. The primary outcome measure was substance use (various drugs, alcohol, tobacco). Substance use and mental health survey questions were based on standardized, validated instruments. A total of 589 patients (83.3% female, mean age 53.6 years [SD 12.8], mean BMI 35.4 [SD 9.1], 54.5% Non-Hispanic white, 22.3% post-MBS) were included. Seventeen patients (2.9%) tested positive for SARS-CoV-2 and 13.5% reported symptoms. Nearly half (48.4%) of the sample reported recreational substance use and 9.8% reported increased use since the start of the pandemic. There was substantial drug use reported (24.3% opioids, 9.5% sedative/tranquilizers, 3.6% marijuana, and 1% stimulants). Patients who reported stockpiling food more (adjusted Odds Ratio [aOR] 1.50, 95% CI 1.03-2.18), healthy eating more challenging (aOR 1.47, 95% CI 1.01-2.16), difficulty falling asleep (aOR 1.64, 95% CI 1.14-2.34), and anxiety (aOR 1.47, 95% CI 1.01-2.14) were more likely to report substance use versus non-users. Results here show that the COVID-19 pandemic is having a deleterious impact on substance use, mental health and weight-related health behaviors in people with obesity regardless of infection status.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

Impact of COVID-19 stay-at-home orders on weight-related behaviours among patients with obesity.

How the impact of the COVID-19 stay-at-home orders is influencing physical, mental and financial health among vulnerable populations, including those with obesity is unknown. The aim of the current study was to explore the health implications of COVID-19 among a sample of adults with obesity. A retrospective medical chart review identified patients with obesity from an obesity medicine clinic and a bariatric surgery (MBS) practice. Patients completed an online survey from April 15, 2020 to May 31, 2020 to assess COVID-19 status and health behaviours during stay-at-home orders. Logistic regression models examined the impact of these orders on anxiety and depression by ethnic group. A total of 123 patients (87% female, mean age 51.2 years [SD 13.0]), mean BMI 40.2 [SD 6.7], 49.2% non-Hispanic white (NHW), 28.7% non-Hispanic black, 16.4% Hispanic, 7% other ethnicity and 33.1% completed MBS were included. Two patients tested positive for severe acute respiratory syndrome coronavirus 2 and 14.6% reported symptoms. Then, 72.8% reported increased anxiety and 83.6% increased depression since stay-at-home orders were initiated. Also 69.6% reported more difficultly in achieving weight loss goals, less exercise time (47.9%) and intensity (55.8%), increased stockpiling of food (49.6%) and stress eating (61.2%). Hispanics were less likely to report anxiety vs NHWs (adjusted odds ratios 0.16; 95% CI, 0.05-0.49; P = .009). Results here showed the COVID-19 pandemic is having a significant impact on patients with obesity regardless of infection status. These results can inform clinicians and healthcare professionals about effective strategies to minimize COVID-19 negative outcomes for this vulnerable population now and in post-COVID-19 recovery efforts.

The glial sodium-potassium-2-chloride cotransporter is required for synaptic transmission in the Drosophila visual system.

The Drosophila Ncc69 gene encodes a Na+-K+-2Cl--cotransporter (NKCC) that is critical for regulating intra- and extracellular ionic conditions in different tissues. Here, we show that the Ncc69 transporter is necessary for fly vision and that its expression is required non-autonomously in glia to maintain visual synaptic transmission. Flies mutant for Ncc69 exhibit normal photoreceptor depolarization in response to a light pulse but lack the ON and OFF-transients characteristic of postsynaptic responses of lamina neurons, indicating a failure in synaptic transmission. We also find that synaptic transmission requires the Ncc69 regulatory kinases WNK and Fray in glia. The ERG phenotype is associated with a defect in the recycling of the histamine neurotransmitter. Ncc69 mutants exhibit higher levels of the transport metabolite carcinine in lamina cartridges, with its accumulation most intense in the extracellular space. Our work reveals a novel role of glial NKCC transporters in synaptic transmission, possibly through regulating extracellular ionic conditions.

Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule.

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

Trans-ethnic Fine Mapping Highlights Kidney-Function Genes Linked to Salt Sensitivity.

We analyzed genome-wide association studies (GWASs), including data from 71,638 individuals from four ancestries, for estimated glomerular filtration rate (eGFR), a measure of kidney function used to define chronic kidney disease (CKD). We identified 20 loci attaining genome-wide-significant evidence of association (p < 5 × 10(-8)) with kidney function and highlighted that allelic effects on eGFR at lead SNPs are homogeneous across ancestries. We leveraged differences in the pattern of linkage disequilibrium between diverse populations to fine-map the 20 loci through construction of "credible sets" of variants driving eGFR association signals. Credible variants at the 20 eGFR loci were enriched for DNase I hypersensitivity sites (DHSs) in human kidney cells. DHS credible variants were expression quantitative trait loci for NFATC1 and RGS14 (at the SLC34A1 locus) in multiple tissues. Loss-of-function mutations in ancestral orthologs of both genes in Drosophila melanogaster were associated with altered sensitivity to salt stress. Renal mRNA expression of Nfatc1 and Rgs14 in a salt-sensitive mouse model was also reduced after exposure to a high-salt diet or induced CKD. Our study (1) demonstrates the utility of trans-ethnic fine mapping through integration of GWASs involving diverse populations with genomic annotation from relevant tissues to define molecular mechanisms by which association signals exert their effect and (2) suggests that salt sensitivity might be an important marker for biological processes that affect kidney function and CKD in humans.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule.

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Hypotonicity stimulates potassium flux through the WNK-SPAK/OSR1 kinase cascade and the Ncc69 sodium-potassium-2-chloride cotransporter in the Drosophila renal tubule.

The ability to osmoregulate is fundamental to life. Adult Drosophila melanogaster maintain hemolymph osmolarity within a narrow range. Osmolarity modulates transepithelial ion and water flux in the Malpighian (renal) tubules of the fly, which are in direct contact with hemolymph in vivo, but the mechanisms causing increased transepithelial flux in response to hypotonicity are unknown. Fly renal tubules secrete a KCl-rich fluid. We have previously demonstrated a requirement for Ncc69, the fly sodium-potassium-2-chloride cotransporter (NKCC), in tubule K(+) secretion. Mammalian NKCCs are regulated by a kinase cascade consisting of the with-no-lysine (WNK) and Ste20-related proline/alanine-rich (SPAK)/oxidative stress response (OSR1) kinases. Here, we show that decreasing Drosophila WNK activity causes a reduction in K(+) flux. Similarly, knocking down the SPAK/OSR1 homolog fray also decreases K(+) flux. We demonstrate that a hierarchical WNK-Fray signaling cascade regulates K(+) flux through Ncc69, because (i) a constitutively active Fray mutant rescues the wnk knockdown phenotype, (ii) Fray directly phosphorylates Ncc69 in vitro, and (iii) the effect of wnk and fray knockdown is abolished in Ncc69 mutants. The stimulatory effect of hypotonicity on K(+) flux is absent in wnk, fray, or Ncc69 mutant tubules, suggesting that the Drosophila WNK-SPAK/OSR1-NKCC cascade is an essential molecular pathway for osmoregulation, through its effect on transepithelial ion flux and fluid generation by the renal tubule.