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International audience; Excessive type I interferon (IFNα/β) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/β, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/β signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/β signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/β activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.

Background Kidney transplantation in older people has increased, however older transplant recipients experience mixed outcomes that invariably impacts on their quality of life. The increased vulnerability of older end stage kidney disease patients to frailty and cognitive impairment, may partially explain the differences in outcomes observed. The Kidney Transplantation in Older People (KTOP): impact of frailty on clinical outcomes study is an active clinical study aiming to explore the experience of older people waiting for and undergoing transplantation. In this manuscript we present the study protocol, the study cohort, and the prevalence of frailty and cognitive impairment identified at recruitment. Methods The KTOP study is a single centre, prospective, mixed methods, observational study. Recruitment began in October 2019. All patients aged 60 or above either active on the deceased donor waitlist or undergoing live donor transplantation were eligible for recruitment. Recruited participants completed a series of questionnaires assessing frailty, cognition, and quality of life, which are repeated at defined time points whilst on the waitlist and post-transplant. Clinical data was concurrently collected. Any participants identified as frail or vulnerable were also eligible for enrolment into the qualitative sub-study. Results Two hundred eight participants have been recruited (age 60–78). Baseline Montreal Cognitive Assessments were available for 173 participants, with 63 (36.4%) participants identified as having scores below normal (score Conclusion In the KTOP study cohort we have identified a prevalence of 36.4% of participants with MoCA scores suggestive of cognitive impairment, and a prevalence of frailty of 15.8% at recruitment. A further 20.1% were vulnerable. As formal testing for cognition and frailty is not routinely incorporated into the work up of older people across many units, the presence and significance of these conditions is likely not known. Ultimately the KTOP study will report on how these parameters evolve over time and following a transplant, and describe their impact on quality of life and clinical outcomes.

Renal sodium balance is the main regulator of blood pressure homeostasis. Sodium balance is regulated by aldosterone (ALDO) and, according to recent studies, also by glucocorticoids. Abnormal hormonal regulation of sodium equilibrium results in kidney disease and hypertension. In previous studies ALDO and the glucocorticoid corticosterone (CORT) and its inactive metabolite 11-dehydrocorticosterone (11DHC) have been quantified in plasma and urine samples but their sites of actions within the kidney remain unknown. Glucocorticoids were recently localised in brain and adrenal tissue using mass spectrometry imaging (MSI). MSI has the potential, therefore, to map CORT, 11DHC and ALDO to defined regions of the kidney. The hypothesis of this study was that the proportions of active and inactive corticosteroid change along the different kidney histological zones. The major aim was to optimise MSI technology for application to the kidney and use this approach to assess the distribution of steroids under control, high and low salt diets. In the second chapter corticosteroid amounts were assessed in kidney subregions using liquid chromatography tandem mass spectrometry (LC-MS/MS). Kidneys were physically separated into cortex and medulla with CORT levels being higher compared to 11DHC in both histological areas. Western blots were used to validate cortex-medulla separation indicating that following manual dissection, cortical contamination remained in medullary samples and vice versa. Therefore, MSI was explored as an alternative approach. Optimisation of a MSI method to assess renal steroids distribution is described in Chapter 3, adapting a method previously reported to assess brain steroids. Detection of corticosteroids derivatised with Girard-T (GirT) was achieved in kidney using matrix assisted laser desorption/ionisation (MALDI), which proved more successful than desorption electrospray ionisation. Subsequent experiments utilised MALDI coupled to a 12T FT-ICR instrument. Several different parameters such as cutting temperature, humidity level, matrix density and spatial resolution were optimised and the final approach validated. In the following chapter, the validated method was applied to kidneys from healthy male mice (age 10 weeks) to develop an approach for co-registration of MSI maps with histology images. MSI maps of corticosteroids were generated superimposed and used to define steroid localisation along the kidney sections. CORT was localised along papilla, medulla and inner cortex, while 11DHC was highly concentrated only in medulla. High levels of ALDO were found in medulla and outer cortex. Molecular markers of histology within the MSI datasets were screened and putatively identified. Data imputation was also implemented for later comparison of steroid signal intensity between experimental groups. In the last data chapter the effect of the varying dietary salt intake on the amounts and distribution of CORT, 11DHC, and ALDO in murine kidney was explored; low (0.03%), normal (0.3%), and the high (3%) salt diets were compared. The pattern of distribution of active and inactive glucocorticoids as well as ALDO remained the same, as described above, across the different salt diets. Quantitation of signal intensity showed that both CORT and 11DHC were an order of magnitude lower than ALDO and in all three diets ALDO remained the same. To conclude, the outcome of this thesis was to successfully develop approaches to allow MSI of corticosteroids within the mouse kidney. The major findings were: 1) Active and inactive corticosteroids were localised in different areas in mice that underwent normal salt diet, 2) Molecular markers were found in MSI datasets and used for definition of kidney areas and 3) Quantitation of corticosteroids in plasma and tissue samples both in normal and abnormal salt intake conditions showed that CORT is higher than ALDO in plasma but ALDO is higher in kidney regardless of the type of diet. MSI holds great promise to dissect corticosteroid signalling in functional zones of the kidney and thus will be a new tool to help uncover the mechanisms whereby the kidney contributes to high blood pressure and why this differs between individuals.

P2RX7, an ionotropic receptor for extracellular ATP, is expressed on immune cells, including macrophages, monocytes and dendritic cells and is up-regulated on non-immune cells following injury. P2RX7 plays a role in many biological processes, including production of pro-inflammatory cytokines such as IL-1β via the canonical inflammasome pathway. P2RX7 has been shown to be important in inflammation and fibrosis and may also play a role in autoimmunity. We have developed and phenotyped a novel P2RX7 knock-out (KO) inbred rat strain and taking advantage of the human-resembling unique histopathological features of rat models of glomerulonephritis, we induced three models of disease: nephrotoxic nephritis, experimental autoimmune glomerulonephritis, and experimental autoimmune vasculitis. We found that deletion of P2RX7 does not protect rats from models of experimental glomerulonephritis or the development of autoimmunity. Notably, treatment with A-438079, a P2RX7 antagonist, was equally protective in WKY WT and P2RX7 KO rats, revealing its 'off-target' properties. We identify a novel ATP/P2RX7/K+ efflux-independent and caspase-1/8-dependent pathway for production of IL-1β in rat dendritic cells, which was absent in macrophages. Taken together, these results comprehensively establish that inflammation and autoimmunity in glomerulonephritis is independent of P2RX7 and reveals the off-target properties of drugs previously known as selective P2RX7 antagonists. Rat mononuclear phagocytes may be able to utilise an 'alternative inflammasome' pathway to produce IL-1β independently of P2RX7, which may account for the susceptibility of P2RX7 KO rats to inflammation and autoimmunity in glomerulonephritis. This article is protected by copyright. All rights reserved.

Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. Although a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney as well as in the proximal nephron tubules in neonatal mice. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild-type kidney. Transmission electron microscopy of Tshz3+/lacZ glomeruli revealed a reduced thickness of the glomerular basement membrane and a larger foot process width. Compared to wild type, Tshz3+/lacZ mice showed lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild-type (control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Background/Aims: Necrotizing glomerular lesions are a feature of severe glomerulonephritis. Unlike apoptosis, cellular necrosis has the potential to release damage-associated proteins into the microenvironment, thereby potentiating inflammation. Until recently necrosis was thought to be an unregulated cellular response to injury. However, recent evidence suggests that under certain circumstances receptor mediated necrosis occurs in response to death ligand signalling, one form of which is termed necroptosis. RIPK3, a receptor interacting protein, is a limiting step in the intracellular signalling pathway of necroptosis. A non-redundant role for RIPK3 has been implicated in mouse models of renal ischaemia reperfusion injury and toxic renal injury. The aim of this study was to investigate the role of RIPK3 in nephrotoxic nephritis (NTN), a model of immune complex glomerulonephritis in mice. Methods: We induced NTN in RIPK3-/- and WT mice, comparing histology and renal function in both groups. Results: There was no improvement in urinary albumin creatinine ratio, serum urea, glomerular thrombosis or glomerular macrophage infiltration in the RIPK3-/- mice compared to WT. There was also no difference in number of apoptotic cells in glomeruli as measured by TUNEL staining between the RIPK3-/- and WT mice. Conclusion: The data suggests that RIPK3 is not on a critical pathway in the pathogenesis of nephrotoxic nephritis.

IgA nephropathy (IgAN) is a common cause of chronic kidney disease and end-stage renal failure, especially in young people. Due to a wide range of clinical outcomes and difficulty in predicting response to immunosuppression, we need to understand why and identify which patients with IgAN will develop progressive renal impairment. A deletion polymorphism affecting the genes encoding the complement factor H-related protein (FHR)-1 and FHR-3 is robustly associated with protection against IgAN. Some FHR proteins, including FHR-1 and FHR-5, antagonize the ability of complement factor H (fH), the major negative regulator of the complement alternative pathway, to inhibit complement activation on surfaces, a process termed fH deregulation. From a large cohort of patients, we demonstrated that plasma FHR-1 and the FHR-1/fH ratio were elevated in IgAN and associated with progressive disease. Plasma FHR-1 negatively correlated with eGFR but remained elevated in patients with IgAN with normal eGFR. Serum FHR5 was slightly elevated in IgAN but did not correlate with eGFR. Neither FHR5 levels nor the FHR-5/fH ratio was associated with progressive disease. However, higher serum FHR-5 levels were associated with a lack of response to immunosuppression, the presence of endocapillary hypercellularity, and histology scores of disease severity (the Oxford Classification MEST score). Thus, FHR-1 and FHR-5 have a role in IgAN disease progression.

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions.

Endoglin (CD105) is an auxiliary receptor for members of the TFG-β superfamily. Whereas it has been demonstrated that the deficiency of endoglin leads to minor and defective angiogenesis, little is known about the effect of its increased expression, characteristic of several types of cancer. Angiogenesis is essential for tumor growth, so high levels of proangiogenic molecules, such as endoglin, are supposed to be related to greater tumor growth leading to a poor cancer prognosis. However, we demonstrate here that endoglin overexpression do not stimulate sprouting or vascularization in several in vitro and in vivo models. Instead, steady endoglin overexpression keep endothelial cells in an active phenotype that results in an impairment of the correct stabilization of the endothelium and the recruitment of mural cells. In a context of continuous enhanced angiogenesis, such as in tumors, endoglin overexpression gives rise to altered vessels with an incomplete mural coverage that permit the extravasation of blood. Moreover, these alterations allow the intravasation of tumor cells, the subsequent development of metastases and, thus, a worse cancer prognosis., This work was supported by the Ministerio de Economía y Competitividad of Spain (SAF2010-15881 AND SAF2013-45784-R), the Junta de Castilla y León (GR100), the Fundación Renal Iñigo Álvarez de Toledo, the Kidney Research Network REDINREN (RD06/0016/0013, RD12/0021/0032 and RD016/0009/0025) and the Instituto de Salud Carlos III (PI16/00460 and co-funded by FEDER). Furthermore, CO-I and EN-G are supported by a contract from the Ministerio de Economía y Competitividad of Spain, and CE-T is supported by a fellowship from the Fundación Miguel Casado San José.

The mechanisms that link complement activation and glomerular injury are not understood. Recently, novel mechanisms of complement dysregulation mediated by factor H related protein (FHR)1 and FHR5 have been described. IgA nephropathy (IgAN) is important and poorly understood. Exciting recent evidence suggests FHR proteins and lectin complement pathways contribute to IgAN pathogenesis. C3 glomerulopathy (C3G) demonstrates the potential for alternative complement pathway dysregulation to drive glomerular injury. However, in the majority of C3G cases, precise pathogenesis is not understood. I hypothesised IgAN and C3G pathogenesis were dependent on imbalances of FHR1, FHR5 and lectin complement pathway proteins. I demonstrated circulating FHR1 levels and the ratio of FHR1 to factor H (FH) were increased in patients with IgAN compared to healthy controls and in patients with progressive compared to stable IgAN. I found IgAN patients had higher circulating FHR5 levels than healthy controls and higher circulating FHR5 levels associated with histology markers of IgAN severity. Glomerular FHR5 deposition associated with both clinical and histologic markers of IgAN severity and immunohistolgical markers of complement activation. Glomerular FHR5 deposition also associated with clinical and histology markers of C3G severity and co-localised with glomerular complement (C)3 deposits in C3G. Glomerular FHR5 deposition co-localised with markers of both ongoing (C3b/iC3b/C3c) and previous (C3dg) alternative complement activation in C3G and IgAN. I detected FHR5 in Immunoglobulin (Ig)A containing immune complexes. With regards to the lectin complement pathway, I found IgAN patients had higher circulating levels of M-ficolin, L-ficolin, mannan binding lectin (MBL) associated serine protease (MASP)-1 and MBL associated protein (MAp)19 than healthy controls, and lower circulating levels of MASP-3 than healthy controls. Lower circulating MASP-3 levels also associated with markers of IgAN severity. My research addresses fundamental gaps in our understanding of glomerular complement activation and IgAN pathogenesis. The results are limited by a number of important confounding factors that need to be addressed. However, they justify researching the mechanisms by which FHR5 contributes to complement activation and disease severity in IgAN and C3G and could contribute to novel and exciting diagnostic tools and therapeutic targets for IgAN, C3G and other complement dependent glomerulopathies. Open Access

Sudden Cardiac Death (SCD) is the leading cause of cardiovascular death in dialysis patients. This review discusses potential underlying arrhythmic mechanisms of SCD in the dialysis population. It examines recent evidence from studies using implantable loop recorders and from electrophysiological studies in experimental animal models of chronic kidney disease. The review summarizes advances in the field of non-invasive electrophysiology for risk prediction in dialysis patients focusing on the predictive value of the QRS-T angle and of the assessments of autonomic imbalance by means of heart rate variability analysis. Future research directions in non-invasive electrophysiology are identified to advance the understanding of the arrhythmic mechanisms. A suggestion is made of incorporation of non-invasive electrophysiology procedures into clinical practice.Key Concepts:– Large prospective studies in dialysis patients with continuous ECG monitoring are required to clarify the underlying arrhythmic mechanisms of SCD in dialysis patients.– Obstructive sleep apnoea may be associated with brady-arrhythmias in dialysis patients. Studies are needed to elucidate the burden and impact of sleeping disorders on arrhythmic complications in dialysis patients.– The QRS-T angle has the potential to be used as a descriptor of uremic cardiomyopathy.– The QRS-T angle can be calculated from routine collected surface ECGs. Multicenter collaboration is required to establish best methodological approach and normal values.– Heart Rate Variability provides indirect assessment of cardiac modulation that may be relevant for cardiac risk prediction in dialysis patients. Short-term recordings with autonomic provocations are likely to overcome the limitations of out of hospital 24-h recordings and should be prospectively assessed.

Aims:Hypertension is common. Recent data suggest that macrophages (Mφ) contribute to, and protect from, hypertension. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor with additional pro-inflammatory properties. We investigated the role of the ET system in experimental and clinical hypertension by modifying Mφ number and phenotype.Methods and results:In vitro, Mφ ET receptor function was explored using pharmacological, gene silencing, and knockout approaches. Using the CD11b-DTR mouse and novel mice with myeloid cell-specific endothelin-B (ETB) receptor deficiency (LysMETB−/−), we explored the effects of modifying Mφ number and phenotype on the hypertensive effects of ET-1, angiotensin II (ANG II), a model that is ET-1 dependent, and salt. In patients with small vessel vasculitis, the impacts of Mφ depleting and non-depleting therapies on blood pressure (BP) and endothelial function were examined. Mouse and human Mφ expressed both endothelin-A and ETB receptors and displayed chemokinesis to ET-1. However, stimulation of Mφ with exogenous ET-1 did not polarize Mφ phenotype. Interestingly, both mouse and human Mφ cleared ET-1 through ETB receptor mediated, and dynamin-dependent, endocytosis. Mφ depletion resulted in an augmented chronic hypertensive response to both ET-1 and salt. LysMETB−/− mice displayed an exaggerated hypertensive response to both ET-1 and ANG II. Finally, in patients who received Mφ depleting immunotherapy BP was higher and endothelial function worse than in those receiving non-depleting therapies.Conclusion:Mφ and ET-1 may play an important role in BP control and potentially have a critical role as a therapeutic target in hypertension.

Background: Cardiovascular disease is the commonest cause of death in hemodialysis (HD) patients but accurate risk prediction is lacking. The spatial QRS – T angle is a promising electrophysiological marker for sudden cardiac death risk stratification. The aim of this study was to assess the prognostic value of spatial QRS-T angle derived from standard 12 lead electrocardiograms (ECG) and its association with echocardiographic parameters in HD patients. Methods: This prospective study of 178 prevalent HD patients (aged 67 ± 14 years, 72% men) collected ECG and echocardiographic data on an annual basis. Baseline echocardiograms at study entry were used for cross-sectional comparisons with ECGs. Study endpoints were all-cause mortality, cardiovascular mortality, and major adverse cardiac events (MACE). The QRS – T angle was calculated from standard 10-s ECG as the total cosine R to T (TCRT) using singular value decomposition and expressed in degrees. TCRT above 100∘ was defined as abnormal. Results: During a follow-up period of 36 ± 19 months, 74 patients died, including 17 cardiac deaths, and 54 suffered from MACE. In multivariate Cox regression analysis, QRS-T angle by TCRT at baseline was associated with increased cardiovascular mortality both as a continuous value and dichotomized below or above 100∘ (HR 1.016, p = 0.029, CI: 1.002–1.030 and HR 3.506, CI: 1.118–10.995, p = 0.031 respectively) and with MACE dichotomized at 100∘ (HR 1.902, CI: 1.046–3.459; p = 0.035). In multivariate regression analysis including baseline parameters, echocardiographic global longitudinal strain (GLS) was significantly correlated with TCRT (F 9.648, r2 = 0.192, standardized β = 0.331, unstandardized β = 3.567, t = 4.4429, CI: 1.976–5.157, p < 0.001). Conclusion: TCRT correlates with GLS and is independently associated with cardiac deaths and MACE in HD patients.

Objective When molecular drivers of healthy adipogenesis are perturbed, this can cause hepatic steatosis. The role of arachidonic acid (AA) and its downstream enzymatic cascades, such as cyclooxygenase, in adipogenesis is well established. The exact contribution of the P450 epoxygenase pathway, however, remains to be established. Enzymes belonging to this pathway are mainly encoded by the CYP2J locus which shows extensive allelic expansion in mice. Here we aimed to establish the role of endogenous epoxygenase during adipogenesis under homeostatic and metabolic stress conditions. Methods We took advantage of the simpler genetic architecture of the Cyp2j locus in the rat and used a Cyp2j4 (orthologue of human CYP2J2) knockout rat in two models of metabolic dysfunction: physiological aging and cafeteria diet (CAF). The phenotyping of Cyp2j4−/− rats under CAF was integrated with proteomics (LC-MS/MS) and lipidomics (LC-MS) analyses in the liver and the adipose tissue. Results We report that Cyp2j4 deletion causes adipocyte dysfunction under metabolic challenges. This is characterized by (i) down-regulation of white adipose tissue (WAT) PPARγ and C/EBPα, (ii) adipocyte hypertrophy, (iii) extracellular matrix remodeling, and (iv) alternative usage of AA pathway. Specifically, in Cyp2j4−/− rats treated with a cafeteria diet, the dysfunctional adipogenesis is accompanied by exacerbated weight gain, hepatic lipid accumulation, and dysregulated gluconeogenesis. Conclusion These results suggest that AA epoxygenases are essential regulators of healthy adipogenesis. Our results uncover their synergistic role in fine-tuning AA pathway in obesity-mediated hepatic steatosis., Highlights • Cyp2j4 deletion in rats offers a simplified genetic landscape as opposed to the mice locus, which is under significant allelic expansion. • Under different metabolic stresses (aging, cafeteria diet), Cyp2j4−/− rats show an accelerated adipogenesis, which progress towards adipocyte dysfunction. • Adipocyte dysfunction in Cyp2j4−/− rats under cafeteria diet (CAF) is characterised by down-regulation of white adipose tissue (WAT) PPARγ and C/EBPα, adipocyte hypertrophy and extracellular matrix remodeling. • Cyp2j4−/− rats treated with CAF display exacerbated weight gain, insulin resistance, hepatic lipid accumulation and dysregulated gluconeogenesis. • Cyp2j4−/− rats display alternative arachidonic acid pathway usage in their adipose tissue upon CAF and aging.

IgA nephropathy is the most common type of primary glomerulonephritis worldwide. At least 25% of patients may progress to kidney failure requiring dialysis or transplantation. Treatment of IgA nephropathy using generalised immunosuppression is controversial, with concerns regarding the balance of safety and efficacy in a non-specific approach. The aim of this review is to describe the recent scientific evidence, and a current clinical trial, investigating whether spleen tyrosine kinase (SYK) may be a novel and selective therapeutic target for IgA nephropathy. SYK, a cytoplasmic tyrosine kinase, has a pivotal role as an early intermediate in intracellular signal transduction cascades for the B cell receptor and the immunoglobulin Fc receptor, and thus is critical for B cell proliferation, differentiation and activation, and for mediating pro-inflammatory responses following Fc receptor engagement in various cell types. In renal biopsies of patients with IgA nephropathy, increased expression and phosphorylation of SYK were detected, and this correlated with the histological features of mesangial and endocapillary proliferation. In cell culture studies, patient-derived IgA1 stimulated mesangial cell SYK activation, cell proliferation and cytokine production, and these responses were attenuated by pharmacological or molecular inhibition of SYK. A global randomised, double-blind, placebo-controlled trial investigating the safety and efficacy of fostamatinib (an oral pro-drug SYK inhibitor) in the treatment of patients with IgA nephropathy is ongoing, which may provide important evidence of the safety and efficacy of targeting this pathway in clinical disease.

et al., Insulin signaling to the glomerular podocyte is important for normal kidney function and is implicated in the pathogenesis of diabetic nephropathy (DN). This study determined the role of the insulin receptor substrate 2 (IRS2) in this system. Conditionally immortalized murine podocytes were generated from wild-type (WT) and insulin receptor substrate 2-deficient mice (Irs2-/-). Insulin signaling, glucose transport, cellular motility and cytoskeleton rearrangement were then analyzed. Within the glomerulus IRS2 is enriched in the podocyte and is preferentially phosphorylated by insulin in comparison to IRS1. Irs2-/- podocytes are significantly insulin resistant in respect to AKT signaling, insulin-stimulated GLUT4-mediated glucose uptake, filamentous actin (F-actin) cytoskeleton remodeling and cell motility. Mechanistically, we discovered that Irs2 deficiency causes insulin resistance through up-regulation of the phosphatase and tensin homolog (PTEN). Importantly, suppressing PTEN in Irs2-/- podocytes rescued insulin sensitivity. In conclusion, this study has identified for the first time IRS2 as a critical molecule for sensitizing the podocyte to insulin actions through its ability to modulate PTEN expression. This finding reveals two potential molecular targets in the podocyte for modulating insulin sensitivity and treating DN., This work was supported by Ministerio de Economia y Competitividad (SAF2012-33283, Spain), Comunidad de Madrid (S2010/BMD-2423, Spain) and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Spain) to A.M.V. B.S was supported by Sara Borrell Programme (ISCIII, Spain). R.J.C and his team have support from the Medical Research Council (MR/K010492/1) and Kidney Research UK. D.B. was supported by Diabetes UK, Biotechnology and Biological Sciences Research Council, the Northern Ireland Kidney Research Fund (NIKRF) and DEL Northern Ireland. R.C. was supported by an Irish government IRCSET PhD studentship. AO was supported by Instituto de Salud Carlos III (REDINREN 012/0021, Spain).

The complement–mediated renal diseases C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) strongly associate with inherited and acquired abnormalities in the regulation of the complement alternative pathway (AP). The major negative regulator of the AP is the plasma protein complement factor H (FH). Abnormalities in FH result in uncontrolled activation of C3 through the AP and associate with susceptibility to both C3G and aHUS. Although previously developed FH–deficient animal models have provided important insights into the mechanisms underlying susceptibility to these unique phenotypes, these models do not entirely reproduce the clinical observations. FH is predominantly synthesized in the liver. We generated mice with hepatocyte–specific FH deficiency and showed that these animals have reduced plasma FH levels with secondary reduction in plasma C3. Unlike mice with complete FH deficiency, hepatocyte–specific FH–deficient animals developed neither plasma C5 depletion nor accumulation of C3 along the glomerular basement membrane. In contrast, subtotal FH deficiency associated with mesangial C3 accumulation consistent with C3G. Although there was no evidence of spontaneous thrombotic microangiopathy, the hepatocyte–specific FH–deficient animals developed severe C5–dependent thrombotic microangiopathy after induction of complement activation within the kidney by accelerated serum nephrotoxic nephritis. Taken together, our data indicate that subtotal FH deficiency can give rise to either spontaneous C3G or aHUS after a complement-activating trigger within the kidney and that the latter is C5 dependent.

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, exhibiting a variable clinical and pathological course and significantly contributing to the global burden of chronic kidney disease and end-stage renal disease. Current standards of care focus on optimization of antihypertensive and antiproteinuric therapies (typically renin- angiotensin system blockade) to reduce disease progression. Much recent attention has focused on whether additional immunosuppression confers better outcomes than supportive management alone, and indeed, several trials have demonstrated renoprotective effects following the use of oral corticosteroids. However, results have been inconsistent, and perceived benefits must be balanced against risks and adverse effects associated with generalized immunosuppression, as highlighted by the high-profile STOP-IgAN and TESTING clinical trials. Recent translational research in vitro and animal models of IgAN have generated greater insight into potential therapeutic targets for this complex autoimmune disease. Deeper understanding of the roles of the mucosal immune barrier, complement activation and deposition, T-cell dependent and independent mechanisms of B cell activation, and of the deposition and downstream inflammatory signaling pathways of nephritogenic polymeric IgA1 complexes (e.g., signaling of immune receptors via spleen tyrosine kinase) has formed the rationale for the development of novel agents and clinical trials of more targeted therapies. However, translating findings into clinical practice is challenging, with many immunopathological features of IgAN specific to humans. Recent comprehensive reviews outline current understanding of mechanisms of IgAN as well as ongoing and future clinical trials; it is not our aim to replicate this here. Instead, we take a mechanistic approach to current treatment strategies, outlining advantages and limitations of each before exploring ongoing research with potential translation into future targeted therapies for this complex disease.

Genetic studies in yeast and Drosophila led to identification of cyclin-dependent kinases (CDKs), Polo-like kinases (PLKs) and Aurora kinases as essential regulators of mitosis. These enzymes have since been found in the majority of eukaryotes and their cell cycle-related functions characterized in great detail. However, genetic studies in another fungal species, Aspergillus nidulans, identified a distinct family of protein kinases, the NEKs, that are also widely conserved and have key roles in the cell cycle, but which remain less well studied. Nevertheless, it is now clear that multiple NEK family members act in networks to regulate specific events of mitosis, including centrosome separation, spindle assembly and cytokinesis. Here, we describe our current understanding of how the NEK kinases contribute to these processes, particularly through targeted phosphorylation of proteins associated with the microtubule cytoskeleton. We also present the latest findings on molecular events that control the activation state of the NEKs and how these are revealing novel modes of enzymatic regulation relevant not only to other kinases but also to pathological mechanisms of disease, AF acknowledges support from Worldwide Cancer Research, Cancer Research UK, The Wellcome Trust, the BBSRC and Kidney Research UK. RB acknowledges support from Cancer Research UK, MRC and BBSRC. JR acknowledges support from the Ministerio de Economía, Industria y Competitividad (MINECO) from Spain, through the Plan Nacional de I+D grant BFU2014-58422.

People with metabolic syndrome and obesity are at an increased risk of mortality from cardiovascular disease. Over the past decade, there has been intensive research into new ways to stimulate weight loss to lower this risk. One of the key tissues is the metabolically active brown adipose tissue (BAT). Given that this is a thermogenic tissue, it can be stimulated by cold and is suggested by some as a new frontier in weight loss and improvements in other aspects of human health. But what does this mean for the health of our arteries? In the current issue of Circulation Research , Williams et al,1 reveal that ambient cool temperature may not correlate to benefit in our blood vessels. By comparing atherosclerotic lesion development over a series of temperatures and in 2 atherosclerotic prone models, they reveal stark differences in plaque size, such that larger lesions are found in animals under cold conditions. Their studies reveal a novel temperature-sensitive modulation of monocyte release from the bone marrow that ultimately impacts atherogenesis. Importantly, Williams et al1 have also uncoupled the metabolic protective role of the heat-generating UCP1 (uncoupling protein 1) from atherosclerosis. Article, see p 662 Cooler temperatures have long been associated with an increased mortality because of cardiovascular disease; however, the causality has been indirect, rationalized by changes in traditional risk factors (eg, lipids and blood pressure).2 In line with this, Dong et al3 originally found that cold housing (4°C) compared with thermoneutrality (the temperature at which energy expenditure is not needed to maintain body temperature) stimulated BAT and the browning of white adipose tissue. Cold temperature through UCP1 caused lipolysis and resulted in elevated levels of atherogenic lipoproteins. However, the role of ambient temperature and cholesterol seems controversial, with the current study reporting no changes.1 …

Crescentic glomerulonephritis (Crgn) is a complex disorder where macrophage activity and infiltration are significant effector causes. In previous linkage studies using the uniquely susceptible Wistar Kyoto (WKY) rat strain, we have identified multiple crescentic glomerulonephritis QTL (Crgn) and positionally cloned genes underlying Crgn1 and Crgn2, which accounted for 40% of total variance in glomerular inflammation. Here, we have generated a backcross (BC) population (n = 166) where Crgn1 and Crgn2 were genetically fixed and found significant linkage to glomerular crescents on chromosome 2 (Crgn8, LOD = 3.8). Fine mapping analysis by integration with genome-wide expression QTLs (eQTLs) from the same BC population identified ceruloplasmin (Cp) as a positional eQTL in macrophages but not in serum. Liquid chromatography-tandem mass spectrometry confirmed Cp as a protein QTL in rat macrophages. WKY macrophages overexpress Cp and its downregulation by RNA interference decreases markers of glomerular proinflammatory macrophage activation. Similarly, short incubation with Cp results in a strain-dependent macrophage polarization in the rat. These results suggest that genetically determined Cp levels can alter susceptibility to Crgn through macrophage function and propose a new role for Cp in early macrophage activation.

C3 glomerulopathy refers to a disease process in which abnormal control of complement activation, degradation or deposition results in predominant C3 fragment deposition within the glomerulus and glomerular damage. Recent studies have improved our understanding of its pathogenesis. The key abnormality is uncontrolled C3b amplification in the circulation and/or along the glomerular basement membrane. Family studies in which disease segregates with structurally abnormal complement factor H-related (CFHR) proteins demonstrate that abnormal CFHR proteins are important in some types of C3 glomerulopathy. This is currently thought to be due to the ability of these proteins to antagonize the major negative regulator of C3 activation, complement factor H (CFH), a process termed ‘CFH de-regulation’. Recent clinicopathological cohort studies have led to further refinements in case definition, culminating in a 2013 consensus report, which provides recommendations regarding investigation and treatment. Early clinical experience with complement-targeted therapeutics, notably C5 inhibitors, has also now been published. Here, we summarize the latest developments in C3 glomerulopathy.

et al., Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged. © 2013 the American Physiological Society., This work was supported by the Medical Research Council, Kidney Research UK, above and beyond funding from the University Hospitals Bristol Trust UK and SAF2012-33283 (MINECO, Spain).

ACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development. ispartof: American Journal of Human Genetics vol:101 issue:6 pages:1021-1033 ispartof: location:United States status: published

Obesity has reached pandemic levels worldwide. The current models of dietinduced obesity in rodents use predominantly high-fat based diets that do not take into account the consumption of variety of highly palatable, energy dense foods that are prevalent in Western society. We and others have shown that cafeteria diet (CAF) is a robust and reproducible model of human metabolic syndrome with tissue inflammation in the rat. We have previously shown that inbred rat strains such as Wistar Kyoto (WKY) and Lewis (LEW) show different susceptibilities to CAF diets with distinct metabolic and morphometric profiles. Here we show a difference in plasma MCP-1 levels and investigate the effect of CAF diet on peripheral blood monocyte transcriptome as powerful stress-sensing immune cells in WKY and LEW rats. We found that 75.5% of the differentially expressed transcripts under CAF diet were up-regulated in WKY rats and were functionally related to the activation of the immune response. Using a gene co-expression network constructed from the genes differentially expressed between CAF diet-fed LEW and WKY rats, we identified the Acyl-CoA synthetase short-chain family member 2 (Acss2) as a hub gene for a nutrient sensing cluster of transcripts in monocytes. Acss2 genomic region is significantly enriched for previously established metabolism quantitative trait loci in the rat. Notably, monocyte expression levels of Acss2 significantly correlated with plasma glucose, triglyceride, leptin and NEFA levels as well as morphometric measurements such as body weight and the total fat following CAF in the rat. These results show the importance of the genetic background in nutritional genomics and identify inbred rat strains as potential models for CAF-induced obesity.

Spleen tyrosine kinase (Syk), a 72 kDa cytoplasmic non-receptor protein-tyrosine kinase, plays an important role in signal transduction in a variety of cell types. Ever since its discovery in the early 1990s, there has been accumulating evidence to suggest a pathogenic role of Syk in various allergic disorders, autoimmune diseases and malignancies. Additionally, there is emerging data from both pre-clinical and clinical studies that Syk is implicated in the pathogenesis of proliferative glomerulonephritis, including anti-glomerular basement membrane (anti-GBM) disease, ANCA-associated glomerulonephritis (AAGN), lupus nephritis (LN), and immunoglobulin A nephropathy (IgAN). Moreover, recent animal studies have shed light on the importance of Syk in mediating acute renal allograft rejection, Epstein Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) and kidney fibrosis. Fostamatinib, an oral Syk inhibitor, has undergone clinical testing in rheumatoid arthritis (RA), refractory immune thrombocytopenic purpura (ITP), leukemia, and lymphoma. The recent STOP-IgAN trial showed that the addition of non-selective immunosuppressive therapy to intensive supportive care did not improve clinical outcomes in high-risk IgAN patients. A Syk-targeted approach may be beneficial and is currently being evaluated in a phase II randomized controlled trial. In this review, we will discuss the pathogenic role of Syk and potential use of Syk inhibitor in a variety of renal diseases.

WOS: 000367338700040, PubMed ID: 26381917, Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathics., (C) 2015 The Authors. Published by Elsevier B.V., European Cooperation in Science and Technology (COST Action)European Cooperation in Science and Technology (COST) [BM1203/EU-ROS]; Kidney Research UKKidney Research UK (KRUK) [PDF3/2014], Several authors of this review were supported by the European Cooperation in Science and Technology (COST Action BM1203/EU-ROS). HKID is funded by Kidney Research UK (PDF3/2014).

Dense deposit disease (DDD) is a rare, progressive and incurable kidney disease characterized by complement C3 accumulation along the glomerular basement membrane (GBM). It is the prototypical form of C3 glomerulopathy, which comprises renal disorders due to excessive C3 activation via the alternative pathway (AP). Human and murine studies indicate that renal inflammation in DDD is initiated by the specific C3 activation product iC3b. I have assessed the role of iC3b in DDD using the uniquely informative experimental model of C3 glomerulopathy in factor H (FH)-deficient mice. I demonstrate that coexisting deficiency of CD11b, the specific leukocyte receptor for iC3b, exacerbates the spontaneous renal phenotype in FH-deficient mice. This suggests that the iC3b-CD11b interaction may mediate partial renal protection in DDD. I also show that CD11b deficiency produces hypersensitivity to experimentally triggered glomerular injury in both FH-deficient and FH-sufficient mice. My second experimental approach in vivo and in vitro was to assess whether C3 activation in the circulation or on the GBM surface is the predominant cause of iC3b accumulation in DDD. This included experiments in which administration of a novel engineered human FH protein fragment reduced glomerular C3 staining in FH-deficient mice. Open Access

The aim of my research was to define the roles of local and hepatic-derived factor H, in particular the contribution of extra-hepatic factor H to plasma C3 regulation and spontaneous renal disease associated with factor H deficiency. Factor H is the major soluble regulator of the alternative pathway of complement activation, synthesised predominantly by the liver. To investigate the role of extra-hepatic factor H, I generated mice with hepatocyte-specific factor H deficiency (hepatocyte-Cfh-/-) by intercrossing conditional factor H-deficient animals with mice expressing Cre recombinase under the control of the murine albumin promoter. Plasma factor H levels in hepatocyte-Cfh-/- mice were 19% of wild-type levels and were associated with a secondary reduction in plasma C3 levels. Higher plasma C3 levels than in Cfh-/- animals demonstrated that extra-hepatic factor H contributes to plasma C3 regulation, confirmed by tubulointerstitial C3 staining in hepatocyte-Cfh-/- mice. Extra-hepatic factor H prevented the GBM C3 deposition typical of complete factor H deficiency and was associated with spontaneous mesangial C3 deposition. To test the hypothesis that underlying dysregulation of the alternative pathway predisposes to exaggerated renal injury in the presence of additional complement activation, I assessed the response of hepatocyte-Cfh-/- mice to accelerated serum nephrotoxic nephritis. Injection of nephrotoxic serum led to the rapid onset of haemolytic uraemic syndrome. Subtotal deficiency of factor H in hepatocyte-Cfh-/- mice therefore provides a novel model of either C3 glomerulopathy or atypical haemolytic uraemic syndrome depending on the environmental milieu. This mouse model will enable the pathophysiological mechanisms involved to be further defined and new therapeutics investigated. I also examined the association between abnormalities in factor H-related protein 5 and C3 glomerulopathy. Minimal expression of normal factor H-related protein 5 within the kidney could not prevent CFHR5 nephropathy. Serum factor H-related protein 5 levels may be a marker of renal complement deposition. Open Access

The lymphatic system plays an important role in both tissue homeostasis and inflammation. During the surgical procedure there is complete disruption of lymphatic drainage of the allograft kidney. The time course and nature of lymphatic reconnection following transplantation is poorly understood. In addition to the extra-renal lymphangiogenesis required for lymphatic reconnection, some patients may develop de novo lymphatic vessels within the renal parenchyma during acute rejection or chronic allograft damage. This work sought to examine the time course and mechanism of lymphangiogenesis and the role of macrophages in this process. Injection of carbon black and Evan’s blue into the rat kidney resulted in rapid transit to the draining hilar renal lymph node. Surgical disruption of the lymphatic drainage of the kidney prevented trafficking of carbon black to the renal lymph node at 24 hours. At day 6 there was macroscopic and microscopic evidence of carbon black localisation in the renal lymph node suggesting functional reconnection. Careful histological analysis of hilar renal tissue indicated that the large lymphatic trunks were replaced by a network of small proliferating lymphatic vessels. Assessment of intra-renal lymphangiogenesis was undertaken in 2 distinct experimental models of renal transplantation. In a murine model of acute allograft rejection there was no evidence of increased lymphatic vessel number at day 7. In a collaboration with Sheffield University, tissue from a rat model of interstitial fibrosis and tubular atrophy was examined. The rat tissue exhibited a prominent macrophage and T-cell infiltration at 12 months but there was no difference in the number of perivascular lymphatic vessels. In contrast, there were numerous lymphatic vessels evident in the interstitium that were absent in control isograft tissue. Interestingly, the number of lymphatic vessels correlated with the extent of fibrosis. Analysis of vascular endothelial cell growth factor-C (VEGF-C) mRNA expression did not show any increase in allografts. The model of unilateral ureteric obstruction (UUO) was employed as a model of rapidly progressive inflammatory fibrosis. UUO was associated with rapid and prominent interstitial lymphangiogenesis. This was associated with a marked increase in macrophage and T-lymphocyte infiltration and increased whole kidney mRNA expression of VEGF-C. The role of macrophages in lymphangiogenesis was explored by administration of macrophage depleting liposomal clodronate. No effect upon lymphangiogenesis was found but liposomal clodronate failed to deplete ED-1 positive macrophages in the kidney. A macrophage isolation strategy was thus employed using the myeloid CD11b marker cells and flow cytometric cell sorting and immunomagnetic bead sorting. Although gene expression studies demonstrated increased ED1 mRNA expression by CD11b enriched cells, no difference in VEGF-C mRNA expression between CD11b cells obtained from obstructed kidneys versus cells from sham controls was evident. Lastly, despite extensive efforts, immunostaining for VEGF-C was unsuccessful. In summary, lymphangiogenesis can reconstitute the lymphatic drainage of the kidney and is prominent in both chronic allograft injury and the acutely obstructed kidney in the rat. Although VEGF-C is the likely driver of lymphangiogenesis direct evidence of macrophage VEGF-C production was not found.

Currently, older people predominantly begin treatment on haemodialysis (HD), with the proportion of people starting on peritoneal dialysis (PD) steadily declining over recent years despite survival and some quality of life (QOL) indicators appearing similar between those on PD and HD. This thesis explores whether PD is under-utilised by older people in the UK through three cross-sectional, multicentre studies focusing on QOL and the modality decision making process. The first study aims to extend knowledge of HD and PD QOL outcomes. Seventy older patients on PD were matched to patients on HD by age, sex, time on dialysis, ethnicity and index of deprivation. QOL assessments included the SF-12 and Illness Intrusiveness Ratings Scale. Regression analyses, adjusted for multiple variables including comorbidities, found that patients on PD experienced less illness and treatment intrusion than those on HD, with other QOL outcomes found to be similar between the two groups. The second study investigates the involvement that 65 older patients, new to HD, had in their modality selection. Data was collected using a questionnaire designed for the purpose. Only 52% of the sample perceived having had some involvement in their modality decision and 33% expressed a preference for greater involvement than that experienced. In the final study, experiences of modality decision making and life on dialysis were explored through qualitative analysis of narratives from 30 older patients on HD and PD. Findings demonstrate that decisions are influenced by patients’ prior experiences, as well as their medical and social context. Quality of education can impact on modality selection and the integration of dialysis into patients’ lives. In conclusion, a lack of involvement in modality selection can have a detrimental impact on older patients’ QOL. Healthcare professionals should, therefore, strive to implement effective shared decision making in the selection of a dialysis modality.

INTRODUCTION: Podocytes are terminally differentiated, highly specialized glomerular cells that form the final barrier to protein loss. Podocyte injury is characterised by proteinuria. Proteinuria is an important prognostic marker in kidney diseases, and lowering proteinuria has become a principal clinical goal. Compelling evidence supports the notion that continuing loss of podocytes plays a major role in the initiation and progression of glomerular diseases. It is my hypothesis that interventions that reduce the disruption by rescuing susceptible podocytes next to injured ones are potential therapies to restore podocyte phenotype and filtration behaviour, thereby protecting the kidney from progressive deterioration. Prevention of this damage, or ways to aid its recovery, could therefore be important to improving the management of human kidney diseases. METHODS: Transgenic mice expressing the human diphtheria toxin receptor on podocytes had been previously generated in our laboratory. Characterization of two lines showed that graded specific podocyte injury could be induced by single intraperitoneal injection of diphtheria toxin. Eight-week intervention studies involved administration of oral drug in water or food from 24h after toxin injection. Two control groups received no drug or were non-transgenic (wild-type) littermates. Primary endpoints were glomerulosclerosis and kidney function (serum creatinine). Other readouts included blood pressure, albuminuria, serum albumin, podocyte quantification and collagen staining of kidney. The angiotensin converting enzyme inhibitor (ACEi) captopril was tested because of its proven protective effect on renal function in patients with proteinuria. Subsequently another proteinuria-reducing drug, the endothelin receptor A antagonist sitaxsentan was tested alone and in combination with captopril. RESULTS: Captopril reduced proteinuria and ameliorated scarring, with matrix accumulation and glomerulosclerosis falling almost to baseline. Podocyte counts were reduced after toxin administration and showed no significant recovery irrespective of captopril treatment. In the following sitaxsentan and captopril combined intervention study, glomerular scarring was significantly reduced in all drug-treated groups either alone or in combination, but only combination drug treatment reduced glomerular damage to levels comparable to wild-type controls, demonstrating a synergistic effect of the two agents. Similarly, serum creatinine was lowered further in combined but not single drug-treated groups. Blood pressure of all drug treated mice was lowered compared to the placebo group. Surprisingly in this second study there were no significant differences in proteinuria between treated and untreated groups. CONCLUSION: These results support the hypothesis that continuing podocyte dysfunction is a key abnormality in proteinuric disease, and plays a major role in progressive glomerulosclerosis. Both captopril and sitaxsentan alone or in combination provided protection without substantial preservation or restoration of podocyte numbers at the degree of injury induced in these experiments. Combined therapy showed a synergistic effect in protecting the kidney from progressive damage. These results suggest that protection may be at least partly due to change in podocyte phenotype. The model is ideal for studying strategies to protect the kidney from progressive damage following specific podocyte injury. Further elucidations on the mechanism of action of the drugs may aid development of superior future therapeutic treatments in the field of renal diseases.

Ischaemia/Reperfusion Injury (IRI) is the most common cause of acute kidney injury- a devastating clinical problem lacking any specific treatments to promote renal recovery. Macrophages (Mφ) are pleiotropic cells of the innate immune system, with roles spanning host defence, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Mφ are also known to be important mediators of renal injury in other experimental models of renal disease including transplantation, obstruction and glomerulonephritis. This work sought to examine the role of Mφ in mediating renal IRI. Conditional renal Mφ and monocyte depletion prior to experimental IRI was achieved by administering diphtheria toxin to the CD11b-DTR transgenic animal. This had no impact on either renal function or structural injury. In contrast liposomal clodronate mediated Mφ depletion provided functional and structural protection from injury. Administration of exogenous apoptotic cells also protected renal function if delivered 24h prior to IRI. Immunodeficient SCID mice exhibited a protected injury phenotype after IRI, however derived no additional protection from the administration of either liposomal clodronate or i.v. apoptotic cells. These findings suggest that the protective phenotype must involve either lymphocyte populations or circulating antibody. Preliminary work demonstrates that SCID mice lack IgM natural antibody which deposits in the kidney in the first 30 minutes after IRI. It was also demonstrated that apoptotic cells bind IgM natural antibody present within the circulation. The potential for the key antioxidant enzyme Heme oxygenase-1 (HO-1) to protect renal function was also examined in aged mice using hemearginate (HA) - a potent HO-1 inducer. Echoing epidemiological studies in humans aged mice had increased susceptibility to IRI, whilst failing to induce medullary HO-1. The main site of medullary HO-1 induction by HA was in medullary Mφ, and the protective phenotype was abolished by Mφ ablation, implicating Mφ as the key mediators of HA induced protection in renal IRI. Final studies employed adenoviral transduction to overexpress HO-1 within bone marrow derived Mφ, leading to a modified phenotype with increased IL- 10 and phagocytosis, and reduced TNFα and NO production. When these were introduced in vivo after IRI renal function was improved, potentially due to accelerated clearance of renal platelet deposition.

Renal tubulointerstitial fibrosis is a major predictor of progressive glomerular disease. It occurs as a result of persistent inflammation and is characterised by excessive deposition of extracellular matrix (ECM) proteins, including accumulation of type I collagen, the most abundant protein of the ECM. Type I collagen is encoded by two genes, COL1A1 and COL1A2, that are tightly regulated at a transcriptional level. A key aim of this study was to use the previously identified COL1A2 promoter and enhancer sequences in order to identify novel regulatory cis-acting elements and the relevant transcription factors that regulate collagen transcription in cells derived from healthy or diseased kidneys. Moreover, the effects of hypoxia and transforming growth factor beta (TGFβ), which are both profibrotic stimuli, on collagen transcription were studied. TGFβ is known to activate CDP/cux transcription factors which can suppress gene activation; based on this finding the role of CDP/cux in COL1A2 transcriptional regulation was assessed. In conclusion,the work presented in this thesis provides an insight into the complex control mechanisms that regulate collagen transcription in both physiological and pathological conditions.

The role of coagulation proteins (CPs) in systems other than haemostasis is now recognised. Many of these cellular effects are through protease activated receptors (PARs). This project investigates how CPs influence the adaptive immune response, firstly through the expression of tissue factor (TF) on DCs; secondly through the action of PARs on dendritic cells (DCs) and T cells; and thirdly by examining the direct effect of anti-thrombin (AT) on DCs. This work identified for the first time a subset of mouse DCs that expresses TF. The form of TF changed from cryptic to pro-coagulant as DCs matured. In addition it was found that blocking TF on immature but not mature DCs enhanced their stimulatory capacity, possibly through PAR-2 signalling. In vivo studies supported this finding and suggest that inhibiting TF breaks T cell tolerance. Thrombin enhanced the T cell response through an effect on DCs but not T cells. Both primary and secondary responses increased but there was no change when stimulated T cells were rechallenged with thrombin-incubated DCs. This mechanism was not through changes in MHCII, co-stimulatory molecules or cytokine production. Although DCs expressed PAR, individual PAR-1 or PAR-4 activation did not affect DC stimulatory capacity. Anti-thrombin was found to reduce T cell activation in vivo. When DCs were treated with AT alone there was no change in T cell response, whereas treatment with AT before LPS led to an increase in IL-4 and IL-10 from T cells suggesting induction of tolerance. Anergy and T cell suppressor assays, however, failed to demonstrate a tolerant phenotype. Overall the findings demonstrate that DCs have the ability to generate and respond to CPs and thus influence the T cell response. This work identifies potential new targets in the innate system which may be used to influence the adaptive immune response.

C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh(-/-)), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh(-/-) mice. This effect was found to be dependent on CR3 expression on bone marrow-derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b-CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial.