Identifying genetic loci for metabolic disorders affecting the renal tract

Introduction and Objectives: Nephrolithiasis is one of the most common renal diseases, but with poorly understood pathophysiology. The current understanding of how genetic, environmental, and metabolic factors act singly or in concert to trigger stone formation remains patchy, and the progress of medical therapy has been very modest. Vertebrate models for nephrolithiasis are limited in their ability to rapidly screen multiple and varied interventions that modulate urinary stone formation. Therefore, we hypothesis that the basic research directed at model systems that elucidate the pathophysiology of stone disease is the best hope for advancing the field and leading to the development of new therapeutic approaches that have the potential to reduce the morbidity, mortality, and cost associated with this disease. Further, most studies have confounded that age and temperature share some contribution in precipitation of kidney stones. We also hypothesise that change in temperature (from colder to warmer, low temperature to higher temperature) may have a greater impact on the formation rate of nephrolithiasis. Methods: A short lifespan, rapid deployment of transgenic lines and conception of rapid stone formation makes Drosophila melanogaster an ideal system to screen large numbers of interventions to assess their effect on kidney stone formation. Knockdown of gene candidates for their ability to vary the formation of urinary stones was employed utilising Drosophila. Microdissection, imaging and quantification facilitate identification and collection of fly stones within the lumen of the Drosophila Malpighian tubules (the functional equivalent of the human renal tubule). Further, UAS-RNAi knockdown efficiency was validated by qPCR . The genes which upon knockdown modulated the concentration of accumulated stones were further studied using various genetic, immunostaining and molecular techniques. In addition to that I also identified role of temperature and age in kidney stone formation by rearing flies at 18, 22, 26 and 29 for different time frame (day 7, day 14, day 21 and day 28) and quantifying accumulated stones during that period. The stones obtained were further validated by genetic, temperature switch, colorimetric and biochemical assays. Results: Calcium oxalate stone formation was associated with the stones accumulated intraluminally in parental lines and gene knockdown or mutant panel of genes fed with sodium oxalate (0.2% for 2 days). An unbiased knockdown screen of more than 20 RNAi lines was performed utilising the GAL4-UAS RNAi system and identified 8 genes that rescued kidney stone accumulation compared to parental lines. Microdissection and microscopy confirmed that five genes decreased stone formation in the fly, including genes Waterwitch (Wtrw), Serine Pyruvate Amino Transferase (Spat), Cinnamon (Cin), Na+-dependent inorganic Phosphate Co-transporter (NaPi-T), Sarcoplasmic Calcium-binding Protein 2 (Scp2) and Secretory Pathway Ca2+/Mn2+-ATPase (SPoCK). From the hits, I showed that mutation of SRY interacting protein (Sip1) in Drosophila MTs Stellate cells resulted in the accumulation of uric acid stones with a particularly notable interaction with Moesin and NHE2. I also demonstrated a combined impact of age and temperature in stone formation. Critically, I confirmed that they cause an accumulation of stones due to variation in expression of phosphate transporters NaPi-T and Picot. Our work also provides more precise insight into the impact of age and temperature in stone formation, given that we have shown that stone accumulation increases with age and in flies reared at high temperature compared to flies reared at a lower temperature. Conclusions: A Drosophila urinary stone model was leveraged to perform large scale genetic screens to identify novel genes that modulate calculi formation. Our hits may now be screened as a candidate gene in future cases with a clinical presentation. Our study documents the first case of uric-acid stone formation caused by mutations of a gene using Drosophila as a model organism. Confirmation of these mutations as a causative factor and that the stones accumulated were uric acid stones was done by genetic, molecular and physiological experiments

Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster

Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, we describe here the macros “Autophagoquant” and “Exoquant” to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ.Code available at: https://github.com/IreneSaMi/Exoquant-AutophagoquantIdEx Bordeau

Targeted renal knockdown of Na+/H+ exchanger regulatory factor Sip1 produces uric acid nephrolithiasis in Drosophila

Nephrolithiasis is one of the most common kidney diseases with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophilamelanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. Here, we demonstrate the function of Sip1 (SRY-interacting protein 1) gene, an orthologue of human NHERF1 in Drosophila MTs, and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNAi Sip1 knockdowns in otherwise normal flies, confirming a renal aetiology. This phenotype was abolished in rosy flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to co-locate with Na+/H+ exchanger NHE2, and with moesin, in stellate cells; and so a model was developed in which Sip1 normally regulates NHE2 activity and thus luminal pH. Drosophila renal tubule thus offers a useful model for urate nephrolithiasis

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4. As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments

Medication adherence perspectives in haemodialysis patients: a qualitative study

Background: End-stage kidney disease patients undergoing haemodialysis are prescribed with multiple complex regimens and are predisposed to high risk of medication nonadherence. The aims of this study were to explore factors associated with medication adherence, and, to examine the differential perspectives on medication-taking behaviour shown by adherent and nonadherent haemodialysis patients. Methods: A qualitative exploratory design was used. One-on-one semi-structured interviews were conducted with 30 haemodialysis patients at the outpatient dialysis facility in Hobart, Australia. Patient self-reported adherence was measured using 4-item Morisky Green Levine scale. Interview transcripts were thematically analysed and mapped against the World Health Organization (WHO) determinants of medication adherence. Results: Participants were 44–84 years old, and were prescribed with 4–19 medications daily. More than half of the participants were nonadherent to their medications based on self-reported measure (56.7%, n = 17). Themes mapped against WHO adherence model comprised of patient-related (knowledge, awareness, attitude, self-efficacy, action control, and facilitation); health system/ healthcare team related (quality of interaction, and mistrust and collateral arrangements); therapy-related (physical characteristics of medicines, packaging, and side effects); condition-related (symptom severity); and social/ economic factors (access to medicines, and relative affordability). Conclusions: Patients expressed a number of concerns that led to nonadherence behaviour. Many of the issues identified were patient-related and potentially modifiable by using psycho-educational or cognitive-behavioural interventions. Healthcare professionals should be more vigilant towards identifying these concerns to address adherence issues. Future research should be aimed at understanding healthcare professionals’ perceptions and practices of assessing medication adherence in dialysis patients that may guide intervention to resolve this significant issue of medication non-adherence

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

The global burden of cancer attributable to risk factors, 2010-19 : a systematic analysis for the Global Burden of Disease Study 2019

Background Understanding the magnitude of cancer burden attributable to potentially modifiable risk factors is crucial for development of effective prevention and mitigation strategies. We analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 to inform cancer control planning efforts globally. Methods The GBD 2019 comparative risk assessment framework was used to estimate cancer burden attributable to behavioural, environmental and occupational, and metabolic risk factors. A total of 82 risk-outcome pairs were included on the basis of the World Cancer Research Fund criteria. Estimated cancer deaths and disability-adjusted life-years (DALYs) in 2019 and change in these measures between 2010 and 2019 are presented. Findings Globally, in 2019, the risk factors included in this analysis accounted for 4.45 million (95% uncertainty interval 4.01-4.94) deaths and 105 million (95.0-116) DALYs for both sexes combined, representing 44.4% (41.3-48.4) of all cancer deaths and 42.0% (39.1-45.6) of all DALYs. There were 2.88 million (2.60-3.18) risk-attributable cancer deaths in males (50.6% [47.8-54.1] of all male cancer deaths) and 1.58 million (1.36-1.84) risk-attributable cancer deaths in females (36.3% [32.5-41.3] of all female cancer deaths). The leading risk factors at the most detailed level globally for risk-attributable cancer deaths and DALYs in 2019 for both sexes combined were smoking, followed by alcohol use and high BMI. Risk-attributable cancer burden varied by world region and Socio-demographic Index (SDI), with smoking, unsafe sex, and alcohol use being the three leading risk factors for risk-attributable cancer DALYs in low SDI locations in 2019, whereas DALYs in high SDI locations mirrored the top three global risk factor rankings. From 2010 to 2019, global risk-attributable cancer deaths increased by 20.4% (12.6-28.4) and DALYs by 16.8% (8.8-25.0), with the greatest percentage increase in metabolic risks (34.7% [27.9-42.8] and 33.3% [25.8-42.0]). Interpretation The leading risk factors contributing to global cancer burden in 2019 were behavioural, whereas metabolic risk factors saw the largest increases between 2010 and 2019. Reducing exposure to these modifiable risk factors would decrease cancer mortality and DALY rates worldwide, and policies should be tailored appropriately to local cancer risk factor burden. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.Peer reviewe

Quantifying the impact of nitrogen levels on spring maize varieties (Zea mays L.) in Kanchanpur, Nepal

Maize has tremendous potential for increasing productivity, profitability, and sustainability in agriculture. A crucial obstacle to maximizing yield of the suitable maize variety with appropriate nitrogen (N) doses. In order to assess the impact of varying N levels on the growth and yield of spring maize varieties, a two-factorial study was conducted in the farmer’s field at Kanchanpur, Nepal. Conducted from February to July 2022, the experiment design was randomized complete block with two factors; where hybrid varieties Bioseed 9220 and Arun-2 were the first factors and four different levels of nitrogen (0, 60, 120, and 180 kg N ha-1) were the second factors. Results showed that 180 kg N ha-1 and Bioseed 9220 treatment produced significantly higher plant height, kernel row-1, kernel row cob-1, cob plant-1, and thousand-grain weight as compared to other doses and Arun-2, respectively. Similarly, Bioseed 9220 and 180 kg N ha-1 treatment had significantly longer days for tasseling and silking. The grain yield of Bioseed 9220 (5.48 t ha-1) was significantly greater than Arun-2 (4.15 t ha-1) and the N level of 180 kg ha-1 had a higher yield (5.11 t ha-1) compared to 120 kg ha-1 (4.8 t ha-1), 60 kg ha-1 (4.78 t ha-1) and least in 0 kg ha-1 (4.56 t ha-1). The result indicates that Bioseed 9220 performs better than Open Pollinated Variety (OPV) Arun-2 in growth and yield attributes. N enrichment also boosts yield and yield-attributing characteristics. Bioseed 9220 with 180 kg N ha-1 should be prioritized among farmers to increase the productivity and yield of maize

Enhanced Locomotor Activity Is Required to Exert Dietary Restriction-Dependent Increase of Stress Resistance in Drosophila

Dietary restriction (DR) is known to be one of the most effective interventions to increase stress resistance, yet the mechanisms remain elusive. One of the most obvious DR-induced changes in phenotype is an increase in locomotor activity. Although it is conceptually perceivable that nutritional scarcity should prompt enhanced foraging behavior to garner additional dietary resources, the significance of enhanced movement activity has not been associated with the DR-dependent increase of stress resistance. In this study, we confirmed that flies raised on DR exhibited enhanced locomotive activity and increased stress resistance. Excision of fly wings minimized the DR-induced increase in locomotive activity, which resulted in attenuation of the DR-dependent increase of stress resistance. The possibility that wing clipping counteracts the DR by coercing flies to have more intake was ruled out since it did not induce any weight gain. Rather it was found that elimination of reactive oxygen species (ROS) that is enhanced by DR-induced upregulation of expression of antioxidant genes was significantly reduced by wing clipping. Collectively, our data suggests that DR increased stress resistance by increasing the locomotor activity, which upregulated expression of protective genes including, but not limited to, ROS scavenger system