Prolonged renal warm ischaemia (WI) has functional implications both in transplantation and urological practice. Critical limits of WI are unknown. This study assesses biochemical changes during WI and functional limits of WI using microdialysis, cold pulsatile machine perfusion (CPMP) parameters, histological assessment, serum creatinine and estimated glomerular filtration (eGFR) rate in a porcine model. MATERIALS & METHODS: The left renal artery of female large-white pigs (45-75 kg) was clamped for 60, 90, 120, 150 or 180 minutes to simulate clinical WI. Microdialysis sampling was performed every 15 minutes before, during and after clamping. Samples were analysed for glucose, pyruvate, lactate and glycerol concentrations (CMA 600 Analyser, Lab Model, Sweden). After identical WI simulation on the right kidney a right nephrectomy was performed on day 7 and CPMP parameters measured for 2 hours. Histological assessment was performed at the end of WI (left and right kidneys), after one hour ofreperfusion (left kidneys), after 2 hours CPMP (right kidneys) and 4 weeks after left renal WI (left kidneys). "Recovery" was defined as return of serum creatinine concentration to within +50% of baseline by 4 weeks or eGFR to within 60ml/min. (License: PPL 70/6580-UK) Microdialysis markers, CPMP parameters, histological assessment, serum creatinine and eGFR were unable to stratify the duration of renal WI time suffered. Kidneys exposed to WI of 120 minutes or less achieved "recovery" but those exposed to over 150 minutes did not. A calculated projection suggests that all kidneys may have reached recovery at 7 weeks. All pigs survived the study period. CONCLUSIONS: This study suggests the critical limits lies between 120 and 150 minutes of WI although extrapolations from animal data should be made with caution. Microdialysis markers, whilst able to detect ischaemia, are not suitable for use as criteria for viability.

INTRODUCTION: Podocytes are specialised cells integral to the normal functioning kidney. As a consequence of diabetes podocyte dysfunction, phenotype impairment, and podocytopaenia occur. The ERK5 MAPK is involved in cell survival, proliferation, differentiation, and motility; processes central to the pathologies of diabetic nephropathy. AIMS: To establish an in vitro cellular model for studying the MEK5-ERK5 pathway in human podocytes and its regulation by diabetes-associated stimuli. METHODS: Conditionally immortalised human podocytes were treated with EGF (1.25-10 ng/ml), TGF B1 (1.25-10 ng/ml), high o-glucose (15, 30 mM), BSA and AGE-BSA (50,200 Ilg/ml). MEK5 was inhibited with BIX02188 (10 IlM), EGFR with AG1478 (10 IlM), ALK5 with SB431542 (10 IlM), Ras signalling with farnesylthiosalicylic acid (10 IlM), p38 with SB202190 (2 .5 IlM), proteasome action with MG132 (10 IlM). Proteins investigated by western blotting and immunopre,cipitation; gene expression by qPCR; phenotype and protein subcellular localisation by immun ~fl uo rescence; cell number by MTS proliferation assay; motility by scratch assay; barrier function by electric cell -substrate impedance sensing; apoptosis by flow cytometry. RESULTS: Podocytes expressed ERK5 which wa~ predominantly nuclear. EGF-induced ERK5 phosphorylation was AG1478- and BIX02188-sensitive. TGF - ~l- induced ERK5 phosphorylation was SB431542-, AG1478- and BIX02188-sensitive. EGF reduced synaptopodin immunofluorescence; TGF- ~l decreased nuclear P-cadherin and increased a-SMA; BIX02188 prevented these changes. EGF- and TGF-~l- induced a BIX02188-sensitive increase in cell number. Podocyte motility decreased with BIX02188 and TGF- ~1. BIX02188 reduced transcellular impedance which was alleviated with EGF, but prevented the TGF - ~l-induced reduction. Although high o-glucose increased p-ERK5, ERK5 protein was reduced. AGE-BSA decreased p-ERK5 and induced ERK5 SUMOylation. TGF- ~l in a SB202190-sensitive manner, BIX02188, high o-glucose, and BSA increased apoptosis. CONCLUSION: This work describes the novel expression and altered phosphorylat ion of ERK5 within human podocytes in a diabetic environment. A MEK5 inhibitor prevented certain growth factor-induced phenotypic alterations. Diabetic insults dysregulated ERK5 and along with a MEK5 inhibitor induced apoptosis.

Granulomatosis with polyangiitis (GPA, formerly known as Wegener’s granulomatosis) is a rare and sometimes fatal systemic autoimmune disease. Anti-neutrophil cytoplasmic antibodies (ANCAs) specific for proteinase 3 (PR3) are associated with GPA. However, the pathogenesis of GPA is not yet clear. Our aim was to investigate the local autoimmune response, circulating immune modulatory cells and cells expressing the immune suppressor molecules programmed death 1 (PD-1) and its ligands in GPA. In mucosa from GPA patients, activated B cells were observed located alongside PR3 expressing cells and B cell survival factors BAFF and APRIL, which was produced by the granulomas and giant cells. B cells were proliferating and persistent in biopsies. However no evidence of B cell clones from the mucosal biopsies circulating in peripheral blood was observed in GPA. An increased frequency of circulating TFH cells and a reduced frequency of Treg cells was observed in peripheral blood from GPA patients on conventional therapies compared to healthy controls. No such difference was found in GPA patients treated with rituximab. The frequency of circulating TFH and Treg cells was found to be inversely correlated in human peripheral blood. No difference in the relative quantity of mRNA encoding PD-1 in lymphocytes and monocytes was found in GPA patients compared with healthy controls. Lower percentage of CD14+ monocytes expressing PD-1 was observed in GPA patients. Lower relative quantity of mRNA encoding PD-1 ligands PD-L1 and PD-L2 in T cells and monocytes was observed in GPA patients. In conclusion, data in this thesis identifies activated B cells alongside auto-antigens and B cell survival factors in the mucosa in GPA. A negative correlation between TFH and Treg cells is observed that implies the balance between T cell subsets and its B cell dependence are associated with disease activity in GPA. The deficiency of PD-L1 and PD-L2 mRNA in lymphocytes and monocytes may contribute to the pathogenesis of GPA.

Aberrant proliferation of human mesangial cells (MC) is a critical step in the pathogenesis of mesangioproliferative renal diseases. The T-type calcium channel (T-CaCN) has been proposed to play an important role in the proliferation of a number of non-excitable cell types, but T-CaCN expression and functional significance in MC is not known. The aim of this thesis was to investigate the hypothesis that T-CaCN may play an important role in the proliferation of MC in primary culture. Expression of mRNA encoding the α1H isoform (Cav3.2 clone) (but not the α1G nor α1I T-CaCN isoforms) was demonstrated in human MC by RT-PCR. Expression of α1H T-CaCN protein was difficult to assess directly due to the lack of a highly sensitive and specific antibody, despite attempts at upregulation of the protein. Electrophysiological studies of MC demonstrated an inward calcium current in a proportion of cells with characteristics consistent with T-CaCN. Culture of MC with the T-CaCN antagonists mibefradil, Ni2+ and TTL-1177 resulted in a significant reduction in the growth velocity of MC. This effect was not seen upon incubation with verapamil, an L-type calcium channel antagonist. DNA synthesis in MC treated with each of the T-CaCN antagonists was significantly reduced by up to 50% as shown by BrdU incorporation. This anti-proliferative effect was not associated with direct drug-induced cytotoxicity or apoptosis. FACS analysis of MC incubated with T-CaCN antagonists illustrated an increased proportion of cells remaining in G1 and not progressing into S-phase. Treatment of cultured MC with TTL-1177 resulted in a significant reduction in the signalling protein p-ERK within 30 minutes, an effect not seen with verapamil, suggesting a possible mechanism needing further investigation. MC transfection with siRNA targeting the α1H isoform resulted in significant knockdown of T-CaCN α1H mRNA and a reduction in the growth velocity of cultured MC of approximately 50% compared to control siRNA transfection, with an associated 37% reduction in DNA synthesis. These results demonstrate evidence for an important role for T-type calcium channels in the proliferation of human mesangial cells, justifying further study in in greater detail. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.

Aims: Vascular endothelial growth factor (VEGF-A) is differentially spliced to give two functionally different isoform families, the pro-angiogenic, pro-permeability VEGFxxx and the anti-angiogenic, anti-permeability VEGFxxxb families. Evidence suggests that the balance of pro- and anti-angiogenic isoforms of VEGF-A is key to maintaining normal glomerular function. A switch in splicing of VEGF-A to favour pro-angiogenic, pro-permeability VEGFxxx expression has been documented in many diseases such as cancer and diabetes. Furthermore, VEGF165b has been shown to protect against increases in albuminuria in mouse models of diabetic nephropathy, reduce glomerular water permeability (LpAlVi) in vivo, and be cyto-protective on podocytes in culture. This PhD aimed to investigate the manipulation of VEGF mRNA splicing in models of glomerular injury and test the general hypothesis that constitutive over-expression of VEGF165b under a nephrin promoter is able to rescue the injury phenotype seen in two models of glomerular disease the inducible podocyte-specific VEGF-A knock-out (KO) mouse and the podocyte expressing human diphtheria toxin receptor (Pod-DTR) mouse. The mechanism of action of VEGF16sb in the glomerulus with regards to VEGFR-2 expressed by glomerular endothelial cells (GEnCs) both in vivo and in vitro was also investigated. Results: The inducible podocyte-specific VEGF-A KO mouse had a significant reduction in glomerular VEGF-A mRNA 10 weeks after induction with doxycycline, when they became albuminuric and had an increased glomerular LpAlVi, when compared to wildtype (WT) littermate controls. Constitutive podocyte over-expression of human VEGF165b rescued the inducible podocyte-specific VEGF-A KO phenotype, resulting in reductions in albuminuria and glomerular LpAlVi. Furthermore, VEGF16sb resulted in a reduction in glomerular basement membrane (GBM) thickening observed in the VEGF-A KO mouse after 14 weeks. previous characterization, Pod-DTR mice did not develop albuminuria, podocyte loss or any major glomerular structural abnormalities in our hands, upon treatment with diphtheria toxin (DT) at a range of doses. However, glomerular LpA/Vi was significantly elevated over WT littermate controls, and the pattern of nephrin expression to be altered in these mice. Constitutive podocyte over-expression of human VEGF165b rescued the increased glomerular LpA/Vi and prevented the changes in nephrin expression observed in the Pod-DTR model. VEGF165b was found to increase both the expression and phosphorylation of VEGFR- 2 on GEnCs in vivo and in cultured GEnCs. Conclusions Podocyte VEGF165b over-expression rescues the podocyte-specific VEGF-A KO model and the Pod-DTR model of glomerular disease, suggesting that VEGF165b plays a protective role and that manipulation of the VEGF-A isoform ratio may have therapeutic potential. Podocyte-expressed VEGF16sb is probably acting as a weakipaltial, qualitatively specific agonist to VEGFR-2, expressed on the GEnCs.

Acute kidney injury (AKI) following cardiac surgelY is common, is associated with a 4-fold increased risk of in-hospital death, a doubling of healthcare costs and prolonged hospital stay. The aim of this thesis was to develop novel renoprotective strategies by systematically reviewing current preventative sh·ategies, developing a large animal model of post-cardiopulmonalY bypass (CPB) AKI and assessing the interaction of red blood cell (RBC) transfusion and CPB in the pathogenesis of AKI. This thesis has found that cUlTently there are no effective renoprotective strategies for the prevention of post-CPB AKI. By developing a novel large animal porcine recovery model, we have shown that post-CPB AKI is characterised by endothelial injury, activation and dysfunction, nitric oxide depletion, inflammation, tubular epithelial cell stress and apoptosis with no evidence of acute tubular necrosis. Targeting endothelial pathways using Sitaxsentan sodium, an endothelin-A receptor antagonist, and Sildenafil citrate, a phosphodiesterase-5 inhibitor given intravenously at the start of CPB prevented AKI. Contrary to observational studies, we found that allogeneic porcine RBC transfusion did not cause AKI. Instead RBC transfusion prevented post-CPB AKI by reversing haemodilutional anaemia and improving systemic haemodynamics in the porcine model. However, RBC transfusion did cause Transfusion-Related Acute Lung Injury (TRALI). The severity of TRALI was storage dependent and therefore older RBCs caused greater injUly than younger RBCs. RBC transfusion in the presence of CPB exacerbated TRALI. TRALI was characterised by endothelial activation, inflammation, platelet activation and dysfunction, and coagulopathy. Washing of old, stored RBCs ameliorated TRALI. This thesis has contributed two novel large animal recovery models: a model of postCPB AKI and a model ofTRALI. These models have been utilised as platforms for the development of novel organ protection strategies. This thesis has identified three such strategies that can be rapidly translated into clinical trials, namely, endothelin-A receptor antagonism using Sitaxsentan sodium, phosphodiesterase-5 inhibition using sildenafil citrate and washing of stored human RBCs prior to transfusion.

Teashirt genes encode putative transcription factors, and were first identified in Drosophila melanogaster. Teashirt genes regulate various aspects of Drosophila and vertebrate embryonic development, and there is evidence of functional conservation of Teashirts between species. Since Teashirt genes are expressed during (and may play a role in) the development of the Drosophila renal system, I hypothesised that Teashirt genes play a role in the development of the mammalian renal system/urinary tract. To investigate this possibility I sought the expression of the mouse Teashirt genes (mTshzl, mTshz2 and mTshz3) in the kidneys of mice at various stages of development using polymerase chain reaction based methods. I found that all mouse Teashirt genes were expressed in metanephric kidneys from inception and through their maturation, findings consistent with my hypothesis. In order to develop reagents to further define the expression patterns of Teashirt genes in urinary tracts (including kidneys) of embryonic and adult mice I had polyclonal antibodies designed and generated to specifically recognise proteins encoded by mTshzl, mTshz2 and mTshz3, and tested their reactivity and specificity. Secondly, I used a mutant mouse (in which mTshz3 had been inactivated by replacing the majority of its coding sequence with a lacZ reporter construct) to investigate the expression and function of mlshz3 in the urinary tract. By following lacZ expression in heterozygous transgenic mice, I found that mTshz3 was expressed in kidney medullary stroma, and in mesenchymal cells of the ureters and urinary bladder. Homozygous (mTshz3 null) transgenic mice displayed a striking, bilateral hydronephrosis and hydroureter phenotype. I showed that this phenotype develops due to a failure of ureteric smooth muscle differentiation, leading to defective peristalsis, and therefore urine accumulates in the proximal ureter and renal pelvis. Thus mTshz3 is required during mouse urinary tract development for the differentiation of ureteric smooth muscle.

Ras monomeric GTPases are key molecules in the signalling pathway of renal fibrogenesis and exist in three isoforms (Kirsten, Harvey and Neural). The aim of this thesis was to determine whether inhibition of Ki-Ras expression by ASOs could ameliorate the process of renal fibrosis. All isoforms of Ras were expressed in rat renal fibroblasts (primary culture and cell line (NRK-49F)) and in a rat epithelial cell line (NRK-52E). In vitro administration of Ki-Ras ASOs resulted in a specific knockdown of the isoform mRNA in all cells. An associated rise in Ha-Ras mRNA was noted in fibroblast cells but absent in epithelial cells. ASOs administration in non-diseased male Wistar rats resulted in specific renal Ki- Ras mRNA knockdown and an associated decrease in Ras protein expression. Oligo accumulation was seen in the renal proximal tubular cells with extra-renal deposition seen in the liver, heart and wound-tissue. Significant hepatic Ki-Ras mRNA knockdown and inflammation was also demonstrated in ASO treated subjects. In a rat model of unilateral ureteric obstruction (UUO) a significant rise in both Ki- Ras and N-Ras mRNA expression in the obstructed kidneys compared to nonobstructed kidney samples was demonstrated. Ki-Ras ASOs administration to models of UUO significantly reduced the isoform mRNA expression, when compared to vehicle-only and scrambled-oligos (SO), and resulted in a significant decrease in renal fibrosis scores, based on trichrome and picrosirius red staining, and interstitial collagen I and !-SMA expression, based on immunohistological staining and western blotting. ASO administration resulted in an increase in interstitial cell expression of FSP-1, Ki- 67 and ED-1. These findings were absent in vehicle-only and control oligo groups. In summary, administration of ASOs specifically and significantly reduce Ki-Ras mRNA expression and ameliorate fibrosis in a rat model of renal fibrosis in the presence of increased interstitial cell proliferation and inflammation.

Introduction & Hypothesis Recurrent urinary tract infection (rUTI) is a debilitating problem affecting 5% of women. Current treatment using intermittent or long-term antibiotics gives limited symptomatic benefit and encourages bacterial resistance. The aetiology of rUTI is unclear but may involve altered innate defence mechanisms in susceptible individuals. Colonisation of vaginal mucosa by uropathogenic Escherichia coli (UPEC) is the initiating event for UTI, with subsequent migration up the urethra and attachment to bladder epithelium. Protective innate immunological mechanisms include epithelial synthesis of cationic antimicrobial peptides (AMPs) such as the beta defensins. These may be expressed constitutively or induced via Toll-like-receptor (TLR) activation by pathogen associated molecular patterns (PAMPs). This study investigates the hypothesis that women suffering from rUTI have altered tolerance to infecting bacteria related to differences in expression of endogenous AMPs and that identification of such deficiency gives a potentially useful opportunity for novel preventive therapy. Methods and Patients A synergistic methodological approach of in vitro modelling with validation in clinical samples from the relevant patient group and controls was used. In vitro, cell culture was carried out using RT4 immortalised urothelial-cells, VK2 E6/E7 immortalised vaginal cells and finite primary culture of normal human urothelium. Cells were challenged with E. coli and PAMPs. Assays for Beta defensin AMP gene expression, secretion and antimicrobial activity were carried out. Clinically, 98 women (60 rUTI, 38 controls) were recruited with ethical approval. All subjects provided symptom and health state questionnaires; blood for single nucleotide polymorphism (SNP) analysis; vaginal and bladder biopsies for AMP gene expression analysis; plus vaginal washings and overnight urine samples for AMP peptide secretion assays. Results In vitro, cell culture experiments demonstrated that beta defensin 1 (BD1) was constitutively expressed and secreted in urothelial and vaginal cells. Beta defensin 2 (BD2) expression and secretion was induced by E. coli flagellin and is a potent antimicrobial against UPEC. In vaginal cells, BD2 expression and secretion was enhanced by estrogen. Clinically, women with rUTI were identified as having significantly lower basal levels of vaginal BD2 expression and secretion than controls but no difference in BD1 expression. Postmenopausal women had significantly lower BD2 levels than pre-menopausal women. 392Stop During active UTI, women with history of rUTI and the TLR5 SNP showed significantly lower BD2 expression and secretion in both the bladder and vagina than women with wild type TLR5 gene and rUTI. Discussion This study identifies flagellin induced BD2 expression as a novel and important urogenital innate immune response against invading E. coli, which is reduced in a significant proportion of women with rUTI particularly those with the TLR5 392Stop SNP. Observations in vitro on the BD2 inducing effect of estrogen, and clinically in pre- and post-menopausal women, raise the possibility that BD2 expression can be modulated by exogenous factors.

Emerging evidence suggests that abnormal stem cells play a critical role in cancer development and progression. The aim of the thesis was to identify and characterise benign and malignant stem-like cells from the prostate gland. The central hypothesis tested in this study was that prostate stem-like cells (PSLCs) from benign and malignant prostate glands are identified by cell surface marker expression. Culture systems were compared to establish the conditions for mouse PSLC (mPSLC) culture. A rare stem cell-like subpopulation (0.2-0.8% of total cells) capable of clonal proliferation, self-renewal, and gland-like structure formation were identified within monolayer and spheroid-colony forming cultures, but not in suspension culture. Mouse prostate cells sorted according to expression of one or more candidate cell surface markers showed CD49f+Sca-1+ cells to contain the highest proportion of mPSLCs. mPSLC properties were also investigated in a model of prostate cancer. Cancer-derived cells produced larger spheroid colonies and contained higher proportions of CD49f+Sca-1+ cells than benign controls, suggesting dysregulated PSLC function. Stem-like cells were also detected in the human prostate (hPSLCs) representing 0.5% of total cells. CD49f+ selection resulted in significant enrichment and high recovery of hPSLCs, whereas CD44+ or CD133+ selection led to hPSLC depletion compared to unsorted cells. By immunohistochemistry, CD49f expression was detected in the basal layer, consistent with the predicted niche of stem cells. Using an optimised protocol of cell isolation, hPSLCs were isolated from needle biopsy tissue containing advanced prostate cancer cells. Cancer-derived hPSLCs were best isolated by CD49f+ selection rather than by CD44+ or CD133+ selection, similar to benign PSLCs. The study identified an efficient marker for stem-like cells of the human and mouse prostate. Future studies will identify differences between stem-like cells from cancer and benign tissues as well as novel therapeutic approaches for PSLC targeting in cancer.