NIHRDataInsights/HRCSData huggingface dataset:
Title: Development of a cytotopically-modified antithrombotic agent for prevention of acute intra-graft thrombosis in transplantation
Funder: Wellcome Trust

Abstract: "Dr Richard Smith and Professor Anthony Darling of the MRC Centre for Transplantation at King's College London have received a Translation Award to develop a peptide-based anti-thrombin agent that is capable of rendering a transplanted organ resistant to thrombosis without using systemic anticoagulation . This is achieved by perfusing the organ with a membraneinteractive ("cytotopic") agent (which sticks to the organ vasculature) and then flushing out excess agent prior to transplantation. At least 25% of patients currently on the waiting list for renal transplantation are there because they have pre-existing antibodies against potential donor tissue. Attempts to transplant organs into such patients result in thrombosis within the organ which is difficult to treat with conventional anticoagulants and can lead to severe graft damage. The translation programme will refine processes for the manufacture of the lead cytotopic agent ("Thrombalexin') and will include safety studies to enable a firstin- man study in patients at high risk of intra-graft thrombosis following renal transplantation."


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: RENAL AND UROGENITAL
Title: Advancing renal tissue engineering: equipping tissue-engineered kidneys with a ureter.
Funder: Medical Research Council

Abstract: This proposal addresses the need for renal organoids to have ureters. We have already solved and published the problem of making the epithelial part of the ureter, attached to a collecting duct, but the muscular and stromal parts of the ureter have a different embryonic origin. We propose to use mouse ex-fetu progenitor cells to validate fully our pilot data indicating that transplanting Tbx18+, tailbud-derived peri-Wolffian mesenchyme (PWM) next to induced urothelial tubes will result in correct ureter mesenchyme development. This will be shown first in organoids made from ex-fetu renogenic cells and then in organoids made from mouse ES cells. We will then adapt existing mouse ES cell differentiation protocols to make PWM from ES cells, and verify they behave like ex-fetu ones in organoids of both types just mentioned. Having constructed all-ES-derived renal organoids complete with ureters, we will finally adapt (concentrations, timing) the technique to human iPS cells to make an all-human renal organoid complete with a ureter. The outcome will be important to the science of tissue engineering, to discovery science directly through its data and through providing more realistic human-derived renal organoids accessible to experiment, to disease modelling, and to testing of potential nephrotoxicants by pharma. It will also be an important step towards the long-term goal of making transplantable kidneys from human induced pluripotent stem cells (iPSC).


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: RENAL AND UROGENITAL
Title: Does activation of the endothelin system promote the transition of acute kidney injury to chronic kidney disease?
Funder: Medical Research Council

Abstract: Acute kidney injury (AKI) is a major health problem. It has a high mortality and is expensive to manage. More recently, it has been recognised that patients surviving AKI have an increased risk of developing chronic kidney disease (CKD) and cardiovascular disease. This risk is further enhanced in the elderly. Our preliminary studies in patients surviving AKI show that they have higher levels of the peptide endothelin-1 in their blood and urine than do healthy controls. We modelled this in mice using 50 minutes of renal ischemia followed by reperfusion to induce AKI. Transient renal ischaemia is the most common cause of AKI in humans; in mice it also caused activation of the endothelin system. We found that chronic kidney damage and inflammation were prevented in mice by treatment with an endothelin receptor antagonist. This project builds on these preliminary studies to find out how endothelin receptor antagonism preserves renal vascular function and reduces inflammation. This study in mice is coupled with a clinical study measuring the activity of the endothelin system in 100 patients across a broad spectrum of AKI. If the outcome of this project is positive, it provides a scientific rationale for a future interventional clinical trial with endothelin receptor blockers, which are already approved for use in humans.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: RENAL AND UROGENITAL
Title: Elucidating the genetic architecture of cystic kidney disease using whole-genome sequencing
Funder: Medical Research Council

Abstract: Cystic Kidney Diseases are a group of monogenic disorders that make a major contribution to kidney failure worldwide, with the commonest cause, Polycystic Kidney Disease, alone accounting for 10% of kidney failure. However, in many cases the genetic cause for the disease has not been identified and the full spectrum of involved genes and mutations responsible yet to be defined. The 100,000 Genomes (100KG) Project is a national initiative to undertake whole genome sequencing (WGS) in large numbers of UK families, including over 1200 with Cystic Kidney Disease. We will study data from these families, alongside that from many unaffected people, to answer the following: 1. Can WGS identify the cause of Cystic Kidney Disease? 2. What are the changes in known genes that cause Cystic Kidney Disease? 3. Are there additional causative genes that have not been reported before? 4. Are patients accurately diagnosed using conventional clinical and imaging criteria? 5. How does the gene and type of mutation relate to severity? 6. Does genetic variation in other genes affect severity or clinical manifestations? A range of genetic analysis techniques, including association, variance and burden testing will be used, with the large sample size (combined with very large number of unaffected individuals sequenced on the same pipeline) providing significant power to detect previously unrecognised causes and contributors to disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Gonadotropin action in the polycystic ovary
Funder: Medical Research Council

Abstract: Polycystic ovary syndrome (PCOS) is the commonest endocrine disorder in women, affecting between 5-10% of women of reproductive age. It is a major cause of infertility, acne and hirsutism that all contribute to a negative impact on quality of life. PCOS is also associated with risks to long-term health, including a 3-fold increase in the risk of developing type 2 diabetes. The major biochemical hallmark of PCOS is hypersecretion of androgen which, in turn, impacts on reproductive and metabolic abnormalities. Genetic factors play a major role in development of PCOS but the aetiology remains unclear. Recent data from the genetic (GWAS) studies, together with our own studies of ovarian tissue, suggest that abnormalities of gonadotropin action play a key role in pathogenesis of PCOS. Gonadotropin action is mediated by the G-protein-coupled receptors (GPCRs) LHR and FSHR. Our recent studies have highlighted the functional importance of distinct signalling pathways associated with GPCRs and focused on novel aspects of LH/FSH receptor function, particularly intracellular trafficking of the receptor and endomembrane signalling. Our hypothesis is that there are intrinsic differences between normal and polycystic ovaries in gonadotropin receptor function, affecting the mechanism of anovulation, glucose metabolism and steroidogenesis. Using granulosa and theca cells from women with and without PCOS, our primary aims are to investigate 1) the mechanisms underlying enhanced LH activity in PCOS at the level of LH receptor (LHR), including the role of endomembrane receptor signalling; 2) cross-talk between LHR and the FSHR in human ovarian cells; 3) differences in LH/FSH-mediated effects on metabolism and steroidogenesis between PCOS and control subjects; 4) the impact of androgens on gonadotropin receptor function. Our findings will inform development of specific biomarkers for PCOS and to help identify pathways susceptible to novel targeted pharmaceutical intervention.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Understanding the Properdin Paradox using C3 D1115N (gain-of-function) knock-in mice
Funder: Medical Research Council

Abstract: Recent published data suggests that blockade of properdin (FP), a key positive regulator of the alternative pathway of complement (AP), can prevent thrombotic aHUS. However, previous studies in the related AP driven disease, C3 glomerulopathy, suggested FP blockade exacerbated disease. We have recently generated a C3 gain-of-function mouse model of aHUS based on the C3 D1115N change found in the NRCTC patient cohort. Homozygous C3 D1115N mice develop aHUS by post-partum day 21 (as assessed by all biochemical and histological criteria) which can be reversed or blocked through either use of BB5.1 or genetic deletion of C5. Interestingly, our pilot data indicates that FP blockade is ineffective in preventing disease in the C3 D1115N model. Herein, we wish to establish if FP blockade is a valid treatment for aHUS (as a result of C3 variance) by: 1. Treating homozygous C3 D1115N mice with H4 (anti-FP) either after disease onset (proteinuria and haematuria present) or prophylactically from post partum day 14. Increased mouse survival, improved biochemical and cellular characteristics with reduced kidney pathology would be anticipated if anti-FP works in C3 variant driven aHUS. 2. Backcross onto the FP-/- knockout mice followed by comprehensive analysis as above. 3. Using SPR (Biacore) to assess changes in binding and interaction of FP to recombinant or ex vivo purified and generated C3bBb in the presence of C3 variants. Furthermore, the ability of recombinant Factor H (FH) variants influence FP function and vice versa would also be analysed to gain a much clear picture of why modulation of FP can have disparate effects in murine models of aHUS and C3G. 4. Establish the optimal complement therapeutic regime to treat individuals with C3 variants. We will compare the effectiveness of murine versions of complement therapeutics i.e. Homodimeric minimal FH, anti-FP, anti-Factor B to that of BB5.1 in the model to assess the most efficacious treatment regime.


Research activity codes: 5.7 PHYSICAL
Health categories: RENAL AND UROGENITAL
Title: Defining functional impacts of macrophages in the formation of surgical adhesions
Funder: Medical Research Council

Abstract: We hypothesise that specific macrophage subpopulations (resident vs recruited) and IL4/13 receptor signalling are critical players in modulating adhesion formation. Macrophages play a crucial role in orchestrating effective tissue repair and display a dynamic switch in phenotype during this process, with IL4 and IL13 as essential switch factors. Critically, dysregulation in the recruitment, local expansion and IL4Ralpha activation of macrophages is strongly implicated in tissue fibrosis. Our project comprises three technical objectives: 1. To define the macrophage subpopulations involved in adhesion formation we will use a robust mouse surgical model that generates adhesions which phenocopy the human disease scenario. Furthermore, we will use mouse strains with different susceptibilities to adhesion development. Combining this approach with macrophage depletion protocols and specific gene-deficient mice that show an altered macrophage recruitment profile, we will define the possible contribution of these cells to a fibrotic outcome post-surgery. 2. Having defined the major macrophage subpopulations involved, we will specifically address the contribution of IL4/IL13 in adhesion formation in mice lacking IL4Ralpha. Next, we will limit receptor deficiency to just myeloid cells or tissue resident stromal cells using radiation chimeras. This restricted receptor approach will allow us to unravel which cell type requires IL4Ralpha signalling for adhesion formation and/or healthy repair. 3. To gain a mechanistic insight to the interplay between distinct macrophage subpopulations and peritoneal stromal cells, we will exploit our expertise in peritoneal cell biology and use a 3D co-culture adhesion model and induce peritoneal fibrosis in specific macrophage gene-deficient mice. Our experimental strategy will not only illuminate the pathobiology of adhesion formation, but also identify novel biological therapies to prevent adhesions forming post-surgery.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Determining the role of coagulation proteases in the development of renal fibrosis.
Funder: Medical Research Council

Abstract: The cellular and molecular basis of fibrogenesis, particularly in the kidneys, is incompletely understood, though has become an exciting field in recent years. There is a growing body of evidence for coagulation protease involvement in fibrogenesis in a number of tissues, but the origin of the cellular component, the myofibroblast, remains elusive. All the proposed myofibroblast precursors express either Tissue Factor (TF) or protease-activated receptors, consistent with the concept that coagulation proteases could be influencing any of these putative precursors. During my ACF, I refined a protocol to induce fibrosis in C57BL/6 mice following repeated doses of a prevalent tubular toxin, aristolochic acid (AA). I demonstrated this was associated with upregulation of TF, and that transgenic mice expressing a membrane-tethered inhibitor of coagulation proteases, human Tissue Factor Pathway Inhibitor (hTFPI) on an alphaSMA promoter (alpha-TFPI-Tg) were more resistant to fibrosis than wild-type C57BL/6 mice. My first objective is to describe the specific patterns of TF expression and innate immune activation seen during fibrogenesis in C57BL/6 mice and compare these patterns to those seen in alpha-TFPI-Tg mice. I will then explore, using reciprocal bone marrow transplants and adoptive transfer of labelled monocytes, whether the resistance to fibrosis conferred by the transgene is conferred by renal parenchymal cells (e.g. resident fibroblasts or pericytes), or bone marrow-derived cells (e.g. monocytes or circulating fibrocytes). In the third objective I will isolate and examine in vitro the cells implicated by the experiments above, investigating the pathways involved in transducing coagulation protease-mediated signals to fibrogenesis. This will be done by assessing for Ras/ERK activation and using PAR 1,2 and 4 inhibitors, k-Ras antisense oligonucleotides and siRNA to evaluate their impact on myofibroblast formation, proliferation and collagen production.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Is Diabetic Nephropathy the Result of a Podocyte Tauopathy?
Funder: Medical Research Council

Abstract: Diabetic Nephropathy (DN) is the leading cause of kidney failure in the world. Its natural history is dominated by progressive albuminuria suggesting that the glomerular podocyte may be an early target cell in this condition. We have evidence that accumulation and phosphorylation of microtubular associated protein Tau (MAPT) occurs in podocytes rendered diabetic. We believe this may be an important early initiator in the pathogenesis of DN and also be a mechanistic explanation as to why patients with diabetes and/or proteinuric kidney disease are significantly more likely to develop Alzheimer's dementia. We hypothesize that in diabetes hyperphosphorylated MAPT accumulates in the podocyte causing cellular dysfunction initiating kidney disease. To address this hypothesis we have 3 aims to: 1. Define the physiological importance of increased human MAPT in the podocyte in the perfused glomerulus. This will be achieved by generating a podocyte specific human MAPT over expressing mouse model, which will be compared with an MAPT knockout mouse in diabetic and non-diabetic conditions. 2. Assess the molecular actions of MAPT in the human podocyte. MAPT over expressing, wild-type and knockout cell lines will be generated and an array of cellular functions assessed in diabetic and non-diabetic conditions. 3. Define the importance of MAPT hyper-phosphorylation for podocyte function in diabetic and non-diabetic conditions. This will be achieved by using a set of constitutively phospho-active and phospho-null MAPT constructs stably transfected into human podocytes. Collectively this project will define if podocyte MAPT levels and its phosphorylation status are important in the pathogenesis of DN. If true it will open up many new therapeutic avenues for this condition.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: The impact of primary and secondary epithelial senescence on renal function and fibrosis
Funder: Medical Research Council

Abstract: In this fellowship, I will address the hypothesis that secondary senescence is critical for kidney fibrosis, dysfunction and leukocyte recruitment after injury. I will use our system of transgenic conditional primary (1ry) renal proximal tubular epithelial cell (PTEC) senescence induction to answer 2 questions: i) Is 1ry PTEC senescence in the absence of renal injury sufficient to induce 2ry PTEC senescence and fibrosis in vitro and in the murine kidney in vivo? I will use our validated in-house transgenic models of conditional genetic senescent cell (SC) induction and fluorescent labelling in PTECs with either normal TGFb1 expression or with SC TGFb1 knockout to test the effect of 1ry senescence on 2ry PTEC senescence transmission / fibroblast activation in vitro and in vivo. I will: a) Determine the transcriptome and secretome of 1ry / 2ry PTEC SCs in vitro b) Assess 2ry SC induction via cell contact, media transfer and TGFb1 signalling, and the ability of TGFb signalling blockade to inhibit these pathways in vitro c) Test the impact of 1ry PTEC senescence on fibroblast activation in vitro/in vivo d) Characterise 2ry PTEC senescence, leukocyte kinetics and fibrosis in response to targeted SC induction in vivo and the necessity for TGFb1 release by 1ry SCs ii) Can early/late depletion / inhibition of SCs prevent fibrosis in vivo? Using our transgenic model of 1ry SC generation and renal fibrosis, I will test the ability of senolytic therapy with ABT-263 or TGFb1 inhibition with Galunisertib to: a) Establish if SC killing is sufficient to prevent fibrosis b) Quantify the therapeutic window where senolytic treatment is effective c) Test the ability of early TGFbetaRI inhibition to inhibit 2ry senescence and fibrosis (representing a translational target in kidney disease) d) Sort, single cell sequence and identify 1ry and 2ry SCs in vivo in the kidney e) Determine the transcriptional trajectories of 1ry and 2ry SCs after injury/therapy


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Defining the oxidative stress-related mechanisms by which activation of the transcription factor Nrf2 arrests and resolves liver fibrosis
Funder: Medical Research Council

Abstract: A 6-week carbon tetrachloride (CCl4) exposure period will be used to produce liver fibrosis in various transgenic mouse lines. The ability of Nrf2 activation to inhibit fibrogenesis once initiated will be studied by treating the mice with TBE-31 (to pharmacologically activate Nrf2) or tamoxifen (to genetically activate Nrf2) over the last 3 weeks of CCl4 exposure. The ability of Nrf2 activation to accelerate resolution of liver fibrosis once injury has been curtailed will be studied by treating mice with TBE-31 or tamoxifen immediately after CCl4 exposure. In the first case, the effects of Nrf2 on fibrogenesis will focus on hepatic stellate cells (HSCs) by using the Pdgfrb gene promoter to allow both knockout and genetic activation of Nrf2. In the second case, the effects of Nrf2 on resolution of fibrosis will focus on liver monocyte-derived macrophages by using the Lys2 gene promoter. Upon sacrifice, HSCs or monocytes/macrophages will be prepared from the livers of these mice and determination of their trans-differentiation status and pro-inflammatory/resolution phenotypes measured by RNA-seq, using an NGS platform, to monitor expression of relevant genes. Thereafter, the HSCs and liver monocytes/macrophages will be cultured and the effects of Nrf2 on TGF-beta/PDGF signalling and the Nur-77 transcription factor in HSCs and monocytes/macrophages, respectively, examined by western blotting and by mass spectrometry (on an Agilent 1200 nanoLC-Orbitrap) to monitor oxidation of active site Cys residues in PTPs, PTEN and TXN1. In translational experiments, we will examine whether in macrophages collected from patients with cirrhosis, in vitro activation of Nrf2 stimulates a pro-resolving phenotype using RNA-seq to follow gene expression and MSD V-plex immunoassay to measure cytokines production. Also, we will examine whether in HSCs collected from cirrhotic livers, in vitro activation of Nrf2 can cause reversal of the myofibroblast phenotype using qRT-PCR assays.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Preclinical gene therapy for genetic urinary bladder disease
Funder: Medical Research Council

Abstract: Heterozygous Hpse2 or Lrig2 parent mice are mated to generate neonatal litters with wild-type, heterozygous and homozygous mutants in a 1:2:1 ratio. Neonatal litters will be intravenously administered AAV9 viral vectors and followed for six weeks, covering the period of bladder functional maturation to young adulthood. Our Objectives are: 1. To significantly improve urination defects in mutant mice. If successful, this outcome will be most relevant to treating the crippling symptoms of this disease. Here, we will use the voided stain on paper method to assess urination frequency and volumes, respectively increased and decreased in UFS compared with wild-type mice. 2. To document effects on bladder physiology, bladder nerves, and expression of genes in pelvic ganglia. These results will help us better understand the pathobiology of UFS. Cystometry under terminal anaesthesia will be used to measure pressures inside bladders (increased in UFS) and seek vesicoureteric reflux of urine (present in UFS). We will quantify bladder nerves using peripherin, tyrosine hydroxylase and nNOS immunostaining because we reported aberrant patterns in untreated UFS mice. We will quantify Hpse2 and Lrig2 by in situ hybridisation, and neuronal associated genes (e.g. Sox10, Cntnap2, Capza1 and Wnt4) that we found were deregulated in untreated UFS mutant mouse bladders 3. To compare the efficacy of a neural-specific (SYN1) promoter with the broad action CAG (compound CMV/ACTB) promoter, as being used in human gene transfer studies. We reason that the neural-specific promoter will be most relevant to UFS, given that it appears to have a neurogenic basis. 4. To provide safety information, in addition to urinary tract analyses, we will measure blood levels of liver enzymes and full blood count and undertake histology of extra-renal organs. As well as assessing mutants, we will compare virus administered with vehicle-only administered wild-type mice.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: RENAL AND UROGENITAL