NIHRDataInsights/HRCSData huggingface dataset:
Title: Functional significance of VEGF regulation by SRPK1
Funder: Medical Research Council

Abstract: Altered blood vessel growth and permeability by altered VEGF-A signalling underpin many diseases of the western world. VEGF-A is generated as two different isoforms families - pro-angiogenic, pro-permeability VEGF-A165a, and anti-angiogenic, anti-permeabiity, cytoprotective VEGF-A165b - by alternative splicing. We recently identified splice factors that regulate this alternative splicing, including the protein kinase SRPK1, which acts by phosphorylating SRSF1 in kidney and retinal epithelial cells to switch splicing from VEGF165b to VEGF165a. Mutations in WT1 that result in the proteinuric kidney disease Denys Drash Syndrome have increased SRPK1 expression in the kidney, and this prevents splicing to VEGF-A165b. Moreover, small molecular weight inhibitors of SRPK1 can prevent angiogenesis in the eye. Thus SRPK1 appears to be linked to disorders of both permeability (glomerular permeability results in proteinuria), and angiogenesis. However, the quantitative and qualitative contribution of SRPK1 to VEGF mediated permeability and angiogenesis is not known. We will define this contribution using minigenes that we have recently generated that can track the splicing events in cells in culture. We also intend to investigate the contribution of SRPK1 in controlling VEGF splicing in health and disease by generating tissue specific knockout and over-expressing mice in which SRPK1 expression is inhibited or activated. We will bring in LoxP-SRPK1 mice from Xian-Dong Fu at University of California at San Diego and generate a TetO-SRPK1 mouse. We will cross our existing inducible kidney specific (Pod-rtTA/TetO-Cre) and eye specific (MCT-3-Cre from Steve Moss, UCL) mouse lines with these lines to result in podocyte and eye specific inducible knockout and over-expressing mice, which will be used for angiogenesis assays and glomerular permeability assays and cross the podocyte specific mice with WT1 mutant mice to determine the role of SRPK1 in Denys Drash Syndrome.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Genetic and Acquired Complement Abnormalities in Idiopathic Membranoproliferative Glomerulonephritis
Funder: Medical Research Council

Abstract: Membranoproliferative glomerulonephritis (MPGN) accounts for ~10% of biopsy proven glomerulonephritis and of the primary glomerulonephritides, is the third commonest cause of End Stage Renal Disease (ESRD). It is well established that MPGN is associated with activation of the alternative pathway (AP) of complement. Preliminary data from our lab and others suggests that autoantibodies and mutations affecting the complement system may predispose to disease. The aims of this project are to:- 1) Establish the prevalence of inherited and acquired complement abnormalities in MPGN. 2) Analyse their functional consequences. 3) Define clinical and pathological correlates with specific complement abnormalities. In both the Newcastle cohort of MPGN patients (currently n=40) and the MPGN cohort of RaDaR (renal Rare Disease Registry), I will screen the genes encoding factor H, factor I, membrane cofactor protein, factor B and C3 for mutations. I will also screen serum samples for autoantibodies against factor H, factor I, membrane cofactor protein, and the complement factor H related proteins. I will assess the functional significance of identified genetic variants in CFH using recombinantly produced protein in C3b binding assays (surface plasmon resonance (SPR)), decay acceleration assays (cell surface; SPR) and in cofactor assays (fluid phase; cell surface). Complement haemolytic assays will be used to assess the functional consequences of autoantibodies to complement components. In these patients with complement abnormalities I will determine if there are clinical and pathological correlates. Similar studies in Newcastle on the role of the AP in pathogenesis of atypical haemolytic uraemic syndrome (aHUS) led to successful clinical trials of the complement inhibitor Eculizumab. Given the similarities with aHUS, a characterisation of the role of complement in MPGN, is likely to lead to similar translation benefits.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: RENAL AND UROGENITAL
Title: Investigating the role of pharmacological preconditioning of organs from brain dead donors to improve the outcomes of kidney transplantation
Funder: Medical Research Council

Abstract: Aims and objectives: Exp1: To determine the effects of brain death (BD) on HIF and HIF downstream effector production in the donor kidney. Exp2: To evaluate whether dimethyloxaylglycine (DMOG) administration can up-regulate the production of HIF downstream effectors in the BD setting. Establishing the optimal dose of DMOG (Exp 2.1) and time point for administration (Exp 2.2) in the BD rat. The pharmacodynamics of the agent will be further evaluated (Exp 2.3). Exp3: To determine whether DMOG administration to the BD donor improves the outcomes of isogeneic rat renal transplantation? Methodology: Surgical preparation: BD is induced in anaesthetised adult male Fischer rats (250-300g) using slow inflation of an epidural catheter. After confirmation of BD, anaesthesia is terminated but ventilation continued. 4 hours after BD induction samples including kidney biopsies are obtained. In Exp 2 left kidneys will be explanted for evaluation on an isolated kidney perfusion apparatus. In Exp 3 isogeneic renal transplantation will be performed on bilaterally nephrectomised rats using kidneys from BD rats after cold storage (20 hours). Blood samples will be taken on post-operative days 0, 3, 7 and 14. Transplant nephrectomy will be performed on day 14. Drugs: DMOG (40/200mg/kg i.v.) will be administered prior to BD induction (Exp 2.1). Further experiments will elucidate the optimal time point for administration (30min prior to BD induction, post BD induction: 30, 60 and 120 min after BD induction, Exp 2.2) and the pharmacodynamics of DMOG in the BD setting (Exp 2.3). Primary outcome measures: Exp 1 and Exp 2 HIF target genes (rtPCR: HO-1, AngPTL4, Glut-4, VEGF, EPO, GLUT1, NFkB, MAPK, E-selectin, VCAM). Exp 3 Day 3 post operative creatinine Scientific and medical opportunities: This project aims to increase our understanding of the role of the HIF pathway in protecting against kidney injury following BD and the effect of HIF up-regulation using DMOG.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: RENAL AND UROGENITAL
Title: Characterisation of human and murine renal myeloid immune sentinels and investigation of their role in sterile inflammation
Funder: Medical Research Council

Abstract: Aims and objectives: Parts 1-3: To characterise human kidney myeloid cells and assess their response to tissue damage. Part 4: To examine the dynamic behaviour of murine renal dendritic cells (DCs) in sterile inflammation. Methodology: 1: Surface markers and anatomical location. We will: a. Use a panel of surface markers (including CD11c, CD14, CD16, CD32A/B, CD68, BDCA1, BDCA2, HLA-dr, CD123, CD141, DC-SIGN, DC-LAMP) in flow cytometric studies and compare subsets to peripheral blood DCs/monocytes. b. Perform gene expression profiling using microarray analysis on renal DCs/macrophages and compare expression patterns with those observed in peripheral blood DCs/monocytes. c. Determine the anatomical location of human renal DCs and macrophages in the cortex and medulla using immunohistochemistry and confocal microscopy. 2: Functional studies The large volume of renal tissue available will allow us to isolate sufficient numbers of cells to perform functional studies on renal DCs/macrophages. We will determine: a. The response to a panel of DAMPs (including HMGB-1, HSP, ATP, uric acid, formyl peptides). We will also assess whether other immune signals can modulate inflammasome activation, particularly IgG immune complexes. b. The effect of DAMPs on DC chemotaxis. We wish to determine if DAMPs can prime DCs to migrate from kidneys to draining lymph nodes by assessing chemokine receptor expression (CCR7) in renal DCs and to utilise a novel 3-D chemotaxis assay with a defined chemokine (CCL19) gradient. 3: Ex vivo assessment of human kidney myeloid cells Kidneys donated for transplant (but unsuitable for use) will be reperfused using a normothermic perfusion circuit. Core biopsies will be processed for histology/confocal microscopy and flow cytometry. 4: Assess the dynamic behaviour of renal DCs in sterile inflammation using 2-photon microscopy and CD11cEYFP mice. Scientific potential:our work will generate a unique dataset on human renal DCs.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: The Role of Follicular T Cells in the Alloimmune Response and Alloantibody Formation
Funder: Medical Research Council

Abstract: Aims and objectives: To examine the roles of follicular T cells in the formation of alloantibody in humans and mice undergoing transplantation, the development of humoral rejection in humans and mice undergoing transplantation and the development of autoantibodies in humans undergoing transplantation. Human work: 1.Flow cytometry: Identifying cells of interest using a panel of surface and intracellular markers to compare numbers and phenotype between the lymphoid tissue and peripheral blood. Markers will include: T cells: CD3, CD4, CD8, CD45RA, CCR7, CD25, HLA-dr, CD127, CXCR5, CCR6, CXCR3, IL-23R, CD57, Bcl6, FoxP3. B cells: CD19, CD20, CD24, CD27, CD10, CD38, CD138, IgM, IgD, CD71, Bcl6. NK/NKT cells: CD16, CD56, NKG2D, CD57, Va24, CD3, CD4, CD8. Mononuclear cells: CD14, CD16, CD11c, CD11b, CCR2, CX3CR1, CCR7, HLA-dr, CCR5, CCR1 2.Immunohistology: Using sections of lymph node (fixed and frozen) or biopsy (fixed and paraffin embedded) we will use a more limited panel of antibodies to identify B cell follicles (IgD), germinal centres (CD57) and T cells within these follicles (CD4, FoxP3, Bcl6) 3.Functional analysis: Using flow sorting, we will separate cell subtypes and perform functional studies looking at the B helper and suppressive functions of different cell subtypes. 4.Cytokine analysis: Using serum taken at the time of blood sampling we will assess for antibody levels and proinflammatory cytokines including IL-21 Mouse work: 1.We will use two models of transplantation to explore the roles of follicular CD4 T cells, using a model antigen system to look at responses to OVA and HEL. We will adoptively transfer in cells with the congenic marker CD45.1 to allow us to track antigen specific cells throughout the response to the transplanted tissue. 2.By manipulating this model, we will be able to adjust both inflammatory stimuli and follicular T cell numbers to see the effects of different conditions on the development of antibodies in the model systems


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: The role of heat shock protein 90 inhibition as a novel means of modulating ischemia/reperfusion injury in the kidney
Funder: Medical Research Council

Abstract: BACKGROUND Ischemia/reperfusion injury (IRI) contributes to delayed graft function (DGF) and poorer outcomes in kidney transplantation. Inhibition of heat shock protein 90 (Hsp90) has been shown by this research group to reduce kidney injury in a murine IRI model. We are currently investigating an Hsp90 inhibitor with a low toxicity profile in human studies (AT13387). However, the mechanism of protection conferred remains unclear. If delineated, this drug could lead to improved outcomes following transplantation and a better understanding of IRI/inflammatory processes in general. Macrophages are key cells in both the initiation and resolution of renal injury, while T lymphocytes and renal tubular cells participate actively in renal IRI. This study will investigate the specific influence of AT13387 upon these cells. METHODS In an established renal IRI model, mice will be pre-treated with AT13387 prior to left renal artery pedicle clamping/contra-lateral nephrectomy. 24 h later, kidney function will determined by serum urea/creatinine and cytokine levels measured. Immunohistochemistry will be used to score morphological injury and establish patterns of cellular infiltration. Flow cytometry of enzymatically dissociated kidneys will assess both cell numbers and phenotype. To study whether AT13387 attenuates renal IRI by modulation of macrophage infiltration and phenotype, CD11b-DTR mice with macrophage-specific expression of human diphtheria toxin receptor (DTR) will be treated with DT 24 h prior to surgery to induce monocyte/macrophage ablation. The severe combined immune-deficient mouse will be used to establish whether AT13387 attenuates renal IRI by modulation of lymphocyte activation and function. In vitro, murine bone marrow derived macrophages will be prepared and co-cultured with proximal tubular epithelial cells. This model will assess whether AT13387 can attenuate macrophage-mediated cytotoxicity or augment tubular cell modulation of macrophage phenotype.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: RENAL AND UROGENITAL
Title: Translational and Mechanistic Studies of Hemearginate-mediated Protection of Human Kidney Cells from Injury
Funder: Medical Research Council

Abstract: Acute kidney injury is a common condition that has no specific therapy except supportive care. Following a significant period of preclinical research using experimental murine models of acute kidney injury, we are now developing a translational research programme of relevance to patients and this project will explore the effect of hemearginate in patients about to undergo cardiac surgery. In addition, this project will use purified human blood cells and well validated kidney tubular cells to dissect the protective effects of hemearginate upon cells starved of oxygen in cell culture studies as this may yield further novel potential therapies. The endpoint of this project will establish the safety and efficacy of hemearginate in upregulating the protective anti-inflammatory enzyme hemeoxygenase-1 that has been demonstrated by us and other investigators to effectively limit acute kidney injury. It is envisaged that the legacy of this work will be to lay the foundation for establishing a large UK multi-centre study to test the effects of pre-operative hemearginate administration on the incidence of acute kidney injury and mortality rates in high-risk patients. This could lead to a change in practice and improved patient outcomes in the future.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Signalling pathways to Proteinuria
Funder: Medical Research Council

Abstract: The leading cause of acquired end-stage renal disease (ESRD) in children is Steroid Resistant Nephrotic Syndrome (SRNS). This is as devastating disease which usually leads to end stage renal failure despite the use of prolonged and toxic immunosuppression. The recurrence of the disease in transplanted patients (often within minutes or hours of the graft being perfused), the fact that immunosuppressive drug therapy and plasma exchange have proven to be useful in treating the recurrence of SRNS and the case of transient nephrotic syndrome in a newborn whose mother had SRNS has led to the the 'circulating factor hypothesis' in the pathogenesis of the disease with the speculation of a toxic 'SRNS factor' that has deleterious effects on the kidney podocyte . Observations by ourselves and others are shedding light on novel disease mechanisms linking the activation of protease receptors, especially PAR1, and signalling to the podocyte cytoskeleton, which results in changes in podocyte motility and in breakdown of filtration. We wish to demonstrate the role of proteases and PAR1 activation in the pathogenesis of SRNS, characterize the cellular effects of PAR1 activation on podocyte biology and identify the downstream signalling pathways involved with particular emphasis on the role of the cation channel TRPC6 and the small GTPase RhoA. We propose to investigate this pathway comprehensively by (1) mapping the protease substrate specificities of our relapse and remission patient samples, (2) generating a transgenic mouse expressing constitutively active PAR1, inducibly, in a podocyte specific distribution (3) using unique patient samples, TRPC6 knockout podocytes, constitutively active and dominant-negative RhoA constructs and Rho kinase inhibitors to delineate signalling pathways activated by diseased plasma. Understanding the cellular mechanisms underlying podocyte damage in SRNS is a critical step in designing and developing new therapeutic approaches.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Autologous cell engineered bladder augmentation
Funder: Medical Research Council

Abstract: 1. Sourcing of autologous cells. Bladder mucosa will be harvested from exstrophy babies undergoing primary repair according to ethical approval. Buccal mucosa epithelium will be obtained from discarded tissue during hypospadias repair. Cells will be isolated and expanded using established techniques and tested for their capacity to express differentiation-associated markers and for functional barrier properties by measuring trans-epithelial electrical resistance and permeability to urea and water. The harvest of differentiated sheets and suitable carrier for transport and delivery will be also refined. 2. Refine the procedures to allow completion of GMP standard operating. Appropriate standard operating procedures and batch manufacturing records will be based on cell culture optimization to define a robust and reproducible process able to be tested in a pre-GMP setting. All critical reagents will be identified, sourced and, if necessary, validated for GMP compliance. Human urothelial, exstrophy and buccal epithelial GMP-compliant cells will be produced/banked for transplantation into pigs. 3. Establish the porcine experimental surgical model of composite cystoplasty. A vascularised, de-epithelialized, seromuscular colonic segment will be developed in a total of 24 immunosuppressed pigs. After isolation, the patch will receive the tissue-engineered epithelial sheet in complex with the carrier (e.g. Vicryl mesh) to facilitate epithelial attachment. The pigs will be divided into 3 groups (8 animals each): one set will receive a bladder augment with a patch built with tissue derived from healthy urothelium; the second set from exstrophy-derived urothelium, and finally the third set will undergo augmentation using tissue from buccal mucosa epithelium. The animals will be sacrificed at 3 and 6 months post-augment. The assessment will include voiding behaviour records and post-mortem immunohistological analysis of both the augmented and native mucosa.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: RENAL AND UROGENITAL
Title: Structural mechanisms of regulation and assembly in the nephronophthisis INVS-NPHP3-NEK8-ANKS6 module
Funder: Medical Research Council

Abstract: There is an urgent need to characterize the molecular pathways that underlie ciliopathies, such as the cystic kidney disease nephronophthisis (NPHP), in order to identify new therapeutic targets. Proteins that are mutated in NPHP can be grouped into modules based on their localization, interactions and functional relationships. One module is formed from the protein kinase NEK8, with INVS, ANKS6 and NPHP3. Disease-associated mutations in NEK8 disrupt its localization, which is dependent on INVS. We have now shown that INVS induces NEK8 to self-activate, and found that the region responsible is disrupted in INVS mutations that cause disease. Here, through structural, molecular and cell biology approaches, we will investigate the hypothesis that the INVS-NPHP3-NEK8-ANKS6 module acts as a protein complex that localizes NEK8 and modulates its activity and that disease mutations in this module lead to dysregulation of NEK8. Work Package 1 Regulation of NEK8 catalytic activity by INVS The mechanism of NEK8 autophosphorylation will be assigned using a kinetic test using an automated kinase assay system. The structures of NEK8 alone and bound to INVS will be determined using X-ray crystallography. Based on the structures, the mechanisms that regulate NEK8 will be probed using site-specific mutagenesis, kinase assays and cell-based assays that address their function in ciliary signalling. Work Package 2 Molecular interactions within the INVS-NPHP3-NEK8-ANKS6 module We will characterize the molecular interactions within the module with respect to binding and regulation of NEK8. The four module proteins will be co-expressed in mammalian cells and complex formation assessed using co-immunoprecipitation, co-purification and SEC-MALLS, and NEK8 activity measure using kinase assays. The interactions within NEK8-containing complexes will be dissected by co-expression of module proteins lacking individual domains, having only single domains or harbouring disease mutations.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL
Title: Development of in vitro human and rat proximal tubule cell models as a platform for drug transporter and drug-drug interaction studies
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Accurate prediction of human drug toxicity is a major challenge in drug development. It is estimated that around 50% of the pre clinical, in vivo screening of new potential drug molecules in rat fail to predict subsequent human toxicity. Regulatory authorities recognise the poor performance of current in vivo screening and have called for the development of alternative in vitro cell based screening assays to reduce and replace current animal testing in pre clinical screening. In this project, we aim to characterise and validate primary renal proximal tubule models from human and rat kidney as in vitro platform for drug safety studies. We will measure species differences in the handling of key molecules between rat kidney proximal tubule cells and human kidney proximal tubule cells to predict human toxicity. To do this we will generate in vitro – in viivo correlations for the handling of a wide range of drug molecules of diverse chemistries and renal clearance rates in both models. With these models in place we will gain clear unambiguous transport data and be able to compare, at an early stage, the handling of molecules in very similar in-vitro models derived from rat kidney and human kidney. With these 2 screening platforms in place, not only would there be an in vitro platform to investigate drug handling in either species, but crucially, direct comparison of the renal handling of a molecule between rat and human kidney would flag up differences in handling that might impact on the progress of a candidate drug molecule into a pre clinical animal study. Acceptance of our models would result in the replacement of significant numbers of animals in drug testing with a more predictive in vitro assay.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: RENAL AND UROGENITAL