MAGED2: the gene associated with Bartter syndrome type 5 is a key regulator of Gαs signalling in the developing kidney

Pathogenic variants in the Melanoma-Associated Antigen D2 (MAGED2) gene have been identified as the cause of the severe but transient antenatal Bartter syndrome type 5 (BS5) accounting for approximately 10% of all Bartter syndrome cases. The disorder is characterized by severe polyhydramnios leading to premature delivery and still birth as well as postnatal polyuria that declines after a few weeks or months followed by normal further development. The underlying molecular mechanisms of BS5 and especially the function of MAGED2 in kidney physiology had been largely unknown so far. In this study, we show that Maged2 expression in mouse embryonic kidneys declines during development. As MAGED2 interacts with G-protein alpha subunit Gαs (GNAS), it was inferred to play a role in G-protein coupled receptor (GPCR) signalling. Accordingly, we showed that knockdown of MAGED2 in HEK293T cells induces major changes in protein phosphorylation but not in protein abundance. In the murine collecting duct cell line mpkCCD, Maged2 knockdown modulated vasopressin type 2 receptor (V2R)-induced phosphorylation-dependent signalling in terms of cAMP kinetics and weakened phosphorylation on downstream targets like cAMP response element-binding protein (CREB). Unexpectedly, Maged2 knockdown resulted in vitro in a marked increase of the water channel aquaporin-2 (AQP2) abundance upon long-term V2R activation, which was mediated transcriptionally. To further analyse Maged2 function in vivo, we generated a knock-in mouse model of the human mutation p.R446C via CRISPR/Cas9. Mendelian ratio of Maged2R446C/Y mice was clearly skewed towards the wildtype genotype. Affected male mice also showed impaired embryonic development in form of general paleness as well as slightly decreased body and kidney weight. On the proteome level MAGED2 loss in P0 kidneys led to significant alterations of abundance of 307 proteins, with 112 proteins showing increased and 195 proteins showing decreased expression. Remarkably, Gαs was significantly upregulated, while the Thiazide-Sensitive Sodium-Chloride Cotransporter (NCC) was significantly downregulated. Localization studies in kidneys from P0 Maged2R446C/Y mice could not show an aberrant localization of targets defective in other types of BS like the cotransporters Na-K-2Cl Cotransporter 2 (NKCC2), NCC and the Potassium Inwardly Rectifying Channel Subfamily J Member 1 (ROMK), while the apical localization of AQP2 and also its phosphorylated form pAQP2 were clearly impaired. A similar diffuse instead of apical AQP2 distribution was observed in the only available kidney sections from a stillborn BS5 infant. Taken together, MAGED2 modulates GPCR-signalling at a specific period of time and acts as a desensitizer of V2R in vitro. Moreover, we demonstrate that Maged2 loss in vivo impairs the abundance and the targeting of numerous renal proteins which could explain the overall severe character of the disease in comparison to other forms of Bartter syndrome. The studies presented here serve as a basis for further research on BS5, potentially allowing the development of targeted treatments for this disease. Especially the mouse model will help us to dissect the complex effects of MAGED2 in health and disease and integrate the discovery in the context of tubulopathies and general fluid homeostasis. Ultimately, backtracking the reductive cascade of MADED2 functions will yield a more precise understanding of fluid and electrolyte homeostasis during embryonic development and beyond

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations

The Role of Aquaporin 3 (AQP3) in Breast Cancer

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention

Is the endothelial nitric oxide synthase (eNOS) gene a susceptibility gene for coronary artery disease, hypertension and type 2 diabetes among North Indian populations?

Coronary artery disease (CAD), Hypertension (Ht) and Type 2 Diabetes Mellitus (T2DM) are all global health problems. This is particularly evident amongst South Asian population groups. The conventional risk factors do not fully explain the higher prevalence of these diseases among South Asians. The endothelial Nitric Oxide Synthase (eNOS) gene is responsible for the production of Nitric Oxide (NO), which may contribute to the physiology of all three disease states. Endothelial dysfunction (which is characterised by a reduction in basal NO) has been shown to be present in, or prior to all three diseases. Numerous variations exist within the eNOS gene, of these variations three have been shown to have a possible functional effect. The first is the Glu298Asp polymorphism within the exon region of the gene, resulting in an amino acid substitution of Glutamate (Glu) to Aspartate (Asp). The second, known as the T-786C polymorphism, is a thymine to cytosine mutation at position -786 in the promoter region. Finally a VNTR polymorphism in Intron 4 causes either a 4 27bp repeat or a 5 27bp repeat. It is hypothesised that these variations could have an effect on the ability of eNOS to produce NO and thus may increase the risk or contribute to the development of the diseases. Previous studies on these variants have shown conflicting results and further studies are warranted to understand and confirm the role of eNOS gene polymorphisms in cardio-metabolic diseases. There is very limited research into the distributions of these genetic variants and their interaction in diseases processes in North Indian populations. Objectives: 1. To analyse through a case control study three different polymorphisms of the eNOS gene for possible association with Coronary Artery Disease (CAD), Hypertension (Ht) and Type 2 Diabetes Mellitus (T2DM) in North Indian population groups. 2.To statistically evaluate descriptive statistics including; age, gender, smoking, dietary behaviours and lipid parameters for possible influence on disease and potential interaction with genetic polymorphisms. 3.To evaluate linkage disequilibrium between the three eNOS variants and carryout haplotype analysis to work out haplotype risk in different diseases. 4.To analyse through a case control study the deletion variant of the Angiotensin-converting enzyme (ACE) gene for possible association with Coronary Artery Disease (CAD), Hypertension (Ht) and Type 2 Diabetes Mellitus (T2DM) in North Indian population groups. 5.To determine a possible interactive effect of the eNOS polymorphisms with the ACE polymorphism. Subjects and Methods: The Glu298Asp and Intron 4 variants were genotyped using a PCR-RFLP technique, the T-786C variant was genotyped using a real time-PCR technique. The ACE deletion variant was also genotyped using a standard PCR technique. The genotyping was undertaken in a total of 457 CAD patients and 220 matched controls from Lucknow, Uttar Pradesh in North India, 319 T2DM patients and 307 matched controls from Punjab, North India and 210 Ht and 162 matched controls, also from Punjab, North India. Results: CAD: The Glu298Asp was significantly associated with CAD among smokers (TT+GT vs. GG OR=2.84 (CI: 1.61-5.0), p<0.001). The Intron 4 variant was also significantly associated with CAD in a smoking dependent manner (4aa+4ab vs. 4bb OR=0.56 (CI: 0.33-0.96). The T-786C variant showed no overall influence on CAD risk. There was also evidence for both synergistic and haplotypic effects of the eNOS gene on CAD status (haplotype G-C-4b OR=4.76 (CI: 1.43-15.78), p<0.001). The ACE genetic variant was confirmed to be a strong independent risk factor for CAD under a dominant model (OR=2.18 (CI: 1.46-3.25), p<0.001). There was no evidence for an interactive effect between the ACE deletion and any of the three eNOS variants incorporated in the current study. Ht: The Glu298Asp variant was not shown to increase Ht risk, with a reduced risk association found under a recessive model (OR=0.316 (CI:0.089-1.116), p=0.061). The T-786C variant s role in disease remained unclear with the findings showing a non significant increased risk. The Intron 4 variant was also shown to increase Ht risk, in a non significant manner. Sufficiently powered studies would be required to clarify these possible associations. The combined analysis, using logistic regression and haplotype analysis revealed no significant associations, but there was a possible protective effect of the T-C-4b haplotype (OR=0.46 (CI: 0.21-1.01), p=0.054). The ACE gene variant was confirmed to be a strong independent risk factor for Ht under a recessive model (OR=1.81 (CI: 1.20-2.74), p=0.01). Again there was no evidence for an interactive effect between the ACE deletion and any of the three eNOS variants in hypertension. T2DM: The Glu298Asp variant was found to be associated with T2DM under a dominant model, the protective effect remained significant following adjustment for conventional risk factors and other gene variants (OR=0.407 (CI: 0.231-0.717), p=0.002). The T-786C variant showed no overall influence on T2DM risk. The Intron 4 variant also found no overall influence. Haplotype analysis found the T-T-4b was found to be significantly protective for T2DM (OR=0.41 (CI: 0.26-0.65), p=0.0002). Finally the ACE gene variant was confirmed to be a risk factor for T2DM under a dominant model (OR=2.62 (CI: 1.51-4.54), p=0.001). Overall Conclusions: To conclude, this study successfully identified the frequency of three eNOS gene variants and the ACE deletion variant in three complex diseases within north Indian populations. There is a clear role of the eNOS gene in all three diseases and consequently the genetic variants have susceptible/protective associations. The association with disease was found to be present at an individual level, in association with risk factors and at a haplotypic level. These findings warrant further studies to confirm and untangle the genetics of complex diseases and genetic risk profiles calculations which will contribute to the field of medical genomics/personalised medicare and interventions among North Indian populations

Mécanisme(s) d'action de l'insuline dans la prévention de l'hypertension et la progression de la tubulopathie dans le diabète : rôle de hnRNP F, Nrf2 et Bmf

Le diabète sucré est un trouble métabolique complexe qui se caractérise par une homéostasie anormale du glucose résultant en une concentration plasmatique trop élevée en glucose et due à un déficit absolu ou relatif de la production de l’insuline ou de son action. Les patients souffrant de diabète sont plus à risque de développer diverses complications comme la néphropathie diabétique (DN), qui demeure la principale cause de maladie rénale en phase terminale (ESRD) et est associée à une morbidité et mortalité cardiovasculaire accrue. Bien que les diabètes de type I et de type II (T1D et T2D) se développent par le biais de mécanismes différents, il n’existe pas de différences pathophysiologiques majeures entre la progression de la DN et ESRD pour les deux types de diabète. La tubulopathie, qui comprend à la fois l’apoptose/atrophie tubulaire et la fibrose tubulo-interstitielle, est déjà bien acceptée comme marqueur final de la progression de la DN. Et quoique l’hyperglycémie et le stress oxydant soient tous deux associés à l’hypertension et aux lésions tubulaires, leurs mécanismes moléculaires précis d’action demeurent incertains. Pour les patients T1D, le traitement intensif à l’insuline par le biais d’injections quotidiennes demeure la thérapie la plus efficace mais est associé à de nombreux inconvénients, dont l’hypoglycémie. Le but de cette thèse est d’identifier des gènes ou molécules en aval de l’action de l’insuline comme nouvelles cibles thérapeutiques pour contrer la progression de la DN. Dans un premier temps, nous avons examiné si l’insuline peut affecter l’expression rénale de Nrf2 dans le T1D et étudié les mécanismes sous-jacents. Le traitement avec l’insuline chez les souris Akita a permis de normaliser l’hyperglycémie, l’hypertension, le stress oxydant et les dommages rénaux; l’inhibition de l’expression rénale de Nrf2 et Agt et l’augmentation de l’expression de hnRNP F/K (ribonucléoprotéines nucléaires hétérogènes F et K) ont également été demontrées. In vitro en condition HG, l’insuline réprime la transcription de Nrf2 et Agt, mais stimule celle de hnRNP F/K via la signalisation p44/42 MAPK (p44/42 mitogen-activated protein kinase) dans les RPTCs. L’inhibition de p44/42 MAPK, hnRNP F ou hnRNP K au moyen de siRNA permet de renverser l’inhibition de la transcription de Nrf2 par l’insuline. Un élément de réponse a l’insuline (IRE) a également été identifié dans le promoteur du gène Nrf2 de rat auquel peuvent se lier hnRNP F/K. Dans des études réalisées sur des souris hyperinsulinémiques-euglycémiques, l’expression de Nrf2 et Agt était diminuée alors que celle de hnRNP F/K était augmentée, indiquant que les effets de l’insuline sur l’expression de Nrf2 et Agt le sont principalement indépendamment de son effet hypoglycémiant. Finalement, les mécanismes sous-jacents de l’action de l’insuline sur la prévention de l’apoptose des RPTCs ont été élucidés. Nous avons créé une souris Tg surexprimant le gène Bcl2-modifying factor (Bmf) humain, spécifiquement dans les RPTCs, et caractérisé ce modèle. Les mécanismes moléculaires de l’action de l’insuline dans la prévention de l’apoptose des RPTCs induite par Bmf et la perte des RPTCs dans des souris diabétiques ont également été étudiés. Afin de démontrer l’effet de l’insuline sur l’expression de Bmf, des souris Akita traitées avec des implants d’insuline, ainsi que des souris surexprimant hnRNP F spécifiquement au niveau des RPTCs ont été utilisées. Les souris Bmf-Tg présentent une augmentation de la pression systolique (SBP), du ratio albumine-créatinine urinaire (ACR), de l’apoptose des RPTCs et un plus grand nombre de RPTCs urinaires que les souris non-Tg. Le traitement avec l’insuline et la surexpression de hnRNP F dans les souris Akita permet de supprimer l’expression de Bmf des RPTCs et leur apoptose. In vitro dans les RPTCs en culture, l’insuline inhibe l’expression du gène Bmf induite par le HG via la voie de signalisation p44/42 MAPK. La transfection de siRNA contre hnRNP F prévient l’inhibition de la transcription de Bmf par l’insuline. HnRNP F inhibe la transcription de Bmf via un élément sensible à hnRNP F localisé dans le promoteur du gène Bmf. En résumé, ces études ont permis de démontrer que l’activation chronique de Nrf2 par l’hyperglycémie aggrave la dysfonction rénale par le biais de l’augmentation de l’expression intrarénale de l’Agt et l’activation du système rénine-angiotensine dans le diabète. Nous avons montré que l’insuline stimule l’expression de hnRNP F et hnRNP K dans les RPTCs afin d’inhiber l’expression de Agt, Nrf2 et Bmf, et ultérieurement atténue l’hypertension et les dommages rénaux chez les souris diabétiques Akita. Les travaux présentés dans cette thèse ont donc permis d’identifier hnRNP F/K, Nrf2 et Bmf comme cibles potentielles pour le traitement de l’hypertension et de la maladie rénale dans le diabète.Diabetes mellitus is a complex metabolic disorder characterized by abnormal glucose homeostasis, resulting in higher plasma glucose due to an absolute or relative deficit in insulin production or action. People with diabetes have an increased risk of developing complications including diabetic nephropathy (DN), which is the major cause of developing end stage renal disease (ESRD) and is associated with increased cardiovascular morbidity and mortality. Although, type I and type II diabetes (T1D and T2D) are developed by different mechanisms, there is no major pathophysiological difference between nephropathy progression and ESRD in both diabetes. Tubulopathy including tubular apoptosis/atrophy and tubule-interstitial fibrosis is known to be final marker for DN progression. Hyperglycemia and oxidative stress are associated with hypertension and tubular injury; their precise molecular mechanisms remain unclear. Intensive insulin treatment for T1D patients, including daily insulin injections is the most effective therapy but is associated with drawbacks such as hypoglycemia. The aim of this thesis is to identify downstream target genes or molecules of insulin action as potential therapeutic targets to counter DN progression. Firstly, we investigated whether insulin affects renal Nrf2 expression in T1D and studied its underlying mechanism and reported that insulin treatment normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and Agt gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F/K) expression in Akita mice. In vitro, insulin suppressed Nrf2 and Agt but stimulated hnRNP F/K gene transcription in HG via p44/42 mitogen-activated protein kinase (p44/42 MAPK) signalling in RPTCs. Inhibition with siRNAs of p44/42 MAPK, hnRNP F, or hnRNP K, reversed insulin inhibition of Nrf2 gene transcription. We further identified an insulin-responsive element (IRE) in rat Nrf2 promoter that binds to hnRNP F/K. In hyperinsulinemic-euglycemic clamp studies, renal Nrf2 and Agt expression were downregulated, whereas hnRNP F/K expression was upregulated, indicating insulin-mediated effects on Nrf2 and Agt expression largely occur independently of its glucose-lowering effect. Secondly, the underlying mechanism of insulin action on preventing RPTC apoptosis was studied. In the present study, a Tg mouse overexpressing human Bcl2-modifying factor (Bmf) in RPTs was created and characterized. Furthermore, the molecular mechanism(s) of insulin action on preventing Bmf-induced RPTC apoptosis and loss in diabetic mice were investigated. To study the effect of insulin on Bmf expression, Akita mice implanted with insulin, specifically those overexpressing hnRNP F in their RPTCs, were used. Bmf-Tg mice exhibited higher systolic blood pressure (SBP), urinary albumin-creatinine ratio (ACR), RPTC apoptosis and more urinary RPTCs than non-Tg mice. Insulin treatment and hnRNP F-overexpression in Akita mice suppressed RPTC Bmf expression and apoptosis. In vitro, insulin inhibited HG-induced Bmf gene expression in RPTCs via p44/42 MAPK signaling. Transfection of hnRNP F siRNA prevented insulin inhibition of Bmf transcription. HnRNP F inhibited Bmf transcription via hnRNP F-responsive element in the Bmf promoter. In summary, this thesis demonstrated that chronic Nrf2 activation by hyperglycemia aggravates renal dysfunction via enhanced intrarenal Agt expression and RAS activation in diabetes. It was demonstrated that insulin inhibits Agt, Nrf2, and Bmf expression in RPTCs via hnRNP F and hnRNP K expression and, subsequently, attenuates hypertension and kidney injury in Akita mice. This study identifies renal hnRNP F/K, Nrf2 and Bmf as potential targets for the treatment of hypertension and kidney injury in diabetes

Amyloidosis

Amyloidoses are a heterogeneous group of diverse etiology diseases. They are characterized by an endogenous production of abnormal proteins called amyloid proteins, which are not hydrosoluble, form depots in various organs and tissue of animals and humans and cause dysfunctions. Despite many decades of research, the origin of the pathogenesis and the molecular determinants involved in amyloid diseases has remained elusive. At present, there is not an effective treatment to prevent protein misfolding in these amyloid diseases. The aim of this book is to present an overview of different aspects of amyloidoses from basic mechanisms and diagnosis to latest advancements in treatment

Proceedings of the 2016 Berry Summer Thesis Institute

Thanks to a gift from the Berry Family Foundation and the Berry family, the University Honors Program launched the Berry Summer Thesis Institute in 2012. The institute introduces students in the University Honors Program to intensive research, scholarship opportunities and professional development. Each student pursues a 12-week summer thesis research project under the guidance of a UD faculty mentor. This contains the product of the students\u27 research