The Canadian Glomerulonephritis Registry (CGNR) and Translational Research Initiative: Rationale and Clinical Research Protocol.

Background: Glomerulonephritis (GN) is a leading cause of kidney failure and accounts for 20% of incident cases of end-stage kidney disease (ESKD) in Canada annually. Reversal of kidney injury and prevention of progression to kidney failure is possible; however, limited knowledge of underlying disease mechanisms and lack of noninvasive biomarkers and therapeutic targets are major barriers to successful therapeutic intervention. Multicenter approaches that link longitudinal clinical and outcomes data with serial biologic specimen collection would help bridge this gap. Objective: To establish a national, patient-centered, multidimensional web-based clinical database and federated virtual biobank to conduct human-based molecular and clinical research in GN in Canada. Design: Multicenter, prospective observational registry, starting in 2019. Setting: Nine participating Canadian tertiary care centers. Patients: Adult patients with a histopathologic pattern of injury consistent with IgA nephropathy, focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy, C3 glomerulopathy, and membranoproliferative GN recruited within 24 months of biopsy. Measurements: Initial visits include detailed clinical, histopathological, and laboratory data collection, blood, urine, and tonsil swab biospecimen collection, and a self-administered quality of life questionnaire. Follow-up clinical and laboratory data collection, biospecimen collection, and questionnaires are obtained every 6 months thereafter. Methods: Patients receive care as defined by their physician, with study visits scheduled every 6 months. Patients are followed until death, dialysis, transplantation, or withdrawal from the study. Key outcomes include a composite of ESKD or a 40% decline in estimated glomerular filtration rate (eGFR) at 2 years, rate of kidney function decline, and remission of proteinuria. Clinical and molecular phenotypical data will be analyzed by GN subtype to identify disease predictors and discover therapeutic targets. Limitations: Given the relative rarity of individual glomerular diseases, one of the major challenges is patient recruitment. Initial registry studies may be underpowered to detect small differences in clinically meaningful outcomes such as ESKD or death due to small sample sizes and short duration of follow-up in the initial 2-year phase of the study. Conclusions: The Canadian Glomerulonephritis Registry (CGNR) supports national collaborative efforts to study glomerular disease patients and their outcomes. Trial registration: NCT03460054.

Contexte: Les glomérulonéphrites (GN) sont des causes importantes d'insuffisance rénale; elles représentent 20 % des cas incidents d'insuffisance rénale terminale (IRT) au Canada chaque année. Inverser la néphropathie et prévenir la progression vers l'insuffisance rénale est possible, mais deux obstacles majeurs freinent la réussite de l'intervention thérapeutique: une compréhension limitée des mécanismes sous-jacents de la maladie, de même que l'absence de biomarqueurs non invasifs et de cibles thérapeutiques. Les approches multicentriques reliant les données cliniques longitudinales et les résultats de santé à la collecte d'échantillons biologiques en série permettraient de combler cette lacune. Objectif: Créer une base de données cliniques nationale en ligne, multidimensionnelle et axée sur le patient, de même qu'une biobanque virtuelle fédérée pour permettre de mener des recherches moléculaires et cliniques humaines sur les GN au Canada. Type d'étude: Registre d'observation prospectif multicentrique débuté en 2019. Cadre: Neuf centres de soins tertiaires canadiens. Sujets: Des patients adultes recrutés dans les 24 mois suivant la biopsie et présentant un profil histopathologique de lésion compatible avec une néphropathie à IgA, une hyalinose segmentaire et focale, une maladie à changement minime, une glomérulonéphrite extra-membraneuse, une glomérulopathie à C3 et une glomérulonéphrite membranoproliférative. Mesures: La première visite comporte une collecte détaillée des données cliniques, histopathologiques et de laboratoire, la collecte d'échantillons biologiques (sang, urine et écouvillonnage des amygdales), ainsi qu'un questionnaire autoadministré sur la qualité de vie. Pour le suivi, la collecte des données cliniques et de laboratoire, la collecte des échantillons biologiques et les questionnaires s'effectuent tous les six mois. Méthodologie: Les patients reçoivent des soins comme établi par leur médecin, et les visites d'étude sont programmées tous les six mois. Les patients sont suivis jusqu'au décès ou jusqu'à la dialyse, à la transplantation ou au retrait de l'étude. Un critère de jugement combiné (IRT, ou diminution de 40 % du débit de filtration glomérulaire estimé après deux ans), ainsi que le taux de déclin de la fonction rénale et la rémission de la protéinurie sont les principaux critères de jugement. Les données phénotypiques cliniques et moléculaires seront analysées par sous-types de GN afin d'identifier les prédicteurs de la maladie et de découvrir de nouvelles cibles thérapeutiques. Limites: Le recrutement des sujets demeure un des principaux défis puisque les maladies glomérulaires prises individuellement sont relativement rares. La faible taille des échantillons et la courte durée du suivi pendant les deux ans de la phase initiale de l'étude pourraient faire en sorte que les études initiales issues du registre ne soient pas assez puissantes pour détecter de légères différences dans les résultats cliniquement significatifs comme l'IRT ou le décès. Conclusion: Le Canadian Glomerulonephritis Registry (CGNR) appuie les efforts de collaboration nationale visant à étudier les patients atteints de maladies glomérulaires et leur évolution clinique. Enregistrement de l'essai: NCT03460054.

Multivalent nephrin-Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis.

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.

Hepatitis B virus-related hepatocellular carcinoma in young adults: Efficacy of nationwide selective vaccination.

AIM: Hepatitis B vaccination in infancy was carried out in Japan only when the mother was persistently infected from 1986 to 2016. The aim of the present study was to elucidate the results of vaccination for the prevention of hepatocellular carcinoma in young adults. METHODS: We studied the number of patients who had liver cancer and died from 1976 to 2017 using a national database. Furthermore, we carried out a nationwide survey focusing on patients with hepatitis B virus-related hepatocellular carcinoma who were diagnosed when aged <40 years from 2007 to 2016. RESULTS: The national database showed that the number of deaths of patients aged <40 years decreased from 337 in 1986 to 61 in 2016. Among the 122 patients with hepatocellular carcinoma (HCC) who were registered in the survey, just three patients were born after the start of the vaccination in 1986. Liver cirrhosis, defined by a high Fib-4 index (≥3.25), was found in just 12.5% of the patients at the time of the survey. HCC was incidentally diagnosed in 85 of the 122 (69%) patients. More than 60% of the patients (54/88) were dead at the time of the study, which may be attributed to the delay in diagnosis. CONCLUSIONS: Selective vaccination was effective for the prevention of hepatitis B virus-related HCC. In contrast, many young adults who missed the chance of hepatitis B vaccination and HCC surveillance developed HCC and died. Hepatitis B virus screening in young adults and careful follow up of infected patients are important to prevent HCC development.

Targeting the mTOR pathway uncouples the efficacy and toxicity of PD-1 blockade in renal transplantation.

Immune checkpoint inhibitor (ICI) use remains a challenge in patients with solid organ allografts as most would undergo rejection. In a melanoma patient in whom programmed-death 1 (PD-1) blockade resulted in organ rejection and colitis, the addition of the mTOR inhibitor sirolimus resulted in ongoing anti-tumor efficacy while promoting allograft tolerance. Strong granzyme B+, interferon (IFN)-γ+ CD8+ cytotoxic T cell and circulating regulatory T (Treg) cell responses were noted during allograft rejection, along with significant eosinophilia and elevated serum IL-5 and eotaxin levels. Co-treatment with sirolimus abated cytotoxic T cell numbers and eosinophilia, while elevated Treg cell numbers in the peripheral blood were maintained. Interestingly, numbers of IFN-γ+ CD4+ T cells and serum IFN-γ levels increased with the addition of sirolimus treatment likely promoting ongoing anti-PD-1 efficacy. Thus, our results indicate that sirolimus has the potential to uncouple anti-PD-1 therapy toxicity and efficacy.

The Canadian childhood nephrotic syndrome (CHILDNEPH) study: report on mid-study feasibility, recruitment and main measures.

BACKGROUND: To assess reasons for continuing practice variation in the management of childhood nephrotic syndrome despite expert reviews and guidelines, we are conducting a longitudinal cohort study in children with glucocorticoid sensitive nephrotic syndrome. Objectives of this mid-study report are to describe patient and physician recruitment characteristics, glucocorticoid prescriptions, use of second line agents, biopsy practices, and adherence to study protocol. METHODS: Children with new onset nephrotic syndrome and providers are being recruited from all 12 pediatric nephrology centres across Canada with > 2½ years follow-up. Data collection points of observation are over a minimum 36 months. Details of prescribed glucocorticoids and of all second line agents used during treatment are being collected. All relapses are being recorded with time to urinary remission of proteinuria. RESULTS: To date, 243 patients (57.1% male) from 12 centres were included. Median number of patients per centre was 29 (range 2-45), and median age of cohort was 7.3 (IQR 4.2) at enrollment. Forty-eight physicians were recruited, median 5 (range 2-8) per site. Median number of relapses per patient year of follow-up was 2.1 (IQR 4). Cumulative dose variability of glucocorticoids prescribed per episode of proteinuria and length of treatment was observed between participating centres. CONCLUSION: The Canadian pediatric nephrology community established a longitudinal childhood nephrotic syndrome cohort study that confirms ongoing practice variability. The study will help to evaluate its impact on patient outcomes, and facilitate clinical trial implementation in nephrotic syndrome.

ShcA Adaptor Protein Promotes Nephrin Endocytosis and Is Upregulated in Proteinuric Nephropathies.

Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, via its SH2 domain, was associated with several phosphorylated tyrosine residues on nephrin. Overexpression of ShcA promoted nephrin tyrosine phosphorylation and reduced nephrin signaling and cell surface expression in vitro In a rat model of reversible podocyte injury and proteinuria, phosphorylated nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. In vivo biotinylation assays confirmed that nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb nephrin phosphosignaling dynamics, leading to aberrant nephrin turnover and slit diaphragm disassembly.

Epidemiologic features of 348 children with hepatitis C virus infection over a 30-year period: a nationwide survey in Japan.

BACKGROUND: Although the epidemiology of hepatitis C virus (HCV) infection among children may be rapidly changing, few reports have characterized large nationwide cohorts of children with HCV infection. We, therefore, sought to clarify the epidemiology and natural history of HCV infection in Japanese children born over the last three decades. METHODS: Sixty-five pediatric centers retrospectively and prospectively recruited consecutive, otherwise-healthy HCV-infected children born during 1986 to 2015. RESULTS: Entry criteria were met by 348 children. Age at initial diagnosis of infection has decreased significantly in recent years. Cirrhosis and hepatocellular carcinoma were not identified. Prevalence of spontaneous clearance and of interferon treatment with/without ribavirin were 9 and 54%, respectively. Maternal transmission has increased significantly, representing over 99% of cases in the last decade. No transfusion-related cases have been seen after 1994. HCV genotype 2 has increased to become the most prevalent in Japanese children. Histopathology examination of liver specimens showed no or mild fibrosis in most children with chronic hepatitis C; none showed cirrhosis. CONCLUSIONS: This largest nationwide cohort study of Asian children with HCV infection spanned the last three decades. None of these Japanese children developed cirrhosis or hepatocellular carcinoma. Maternal transmission increased to account for 99% of cases during the last decade. Genotype 2 now is most prevalent in these children. Histopathologically, most children with chronic hepatitis C showed mild fibrosis or none.

AT2 R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression.

Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFß1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Changing Paradigms in the Management of Rejection in Kidney Transplantation: Evolving From Protocol-Based Care to the Era of P4 Medicine.

PURPOSE OF REVIEW: P4 medicine denotes an evolving field of medicine encompassing predictive, preventive, personalized, and participatory medicine. Using the example of kidney allograft rejection because of donor-recipient incompatibility in human leukocyte antigens, this review outlines P4 medicine's relevance to the various stages of the kidney transplant cycle. SOURCES OF INFORMATION: A search for English articles was conducted in Medline via OvidSP (up to August 18, 2016) using a combination of subject headings (MeSH) and free text in titles, abstracts, and author keywords for the concepts kidney transplantation and P4 medicine. The electronic database search was expanded further on particular subject headings. FINDINGS: Available histocompatibility methods exemplify current applications of the predictive and preventive domains of P4 medicine in kidney transplant recipients' care. Pharmacogenomics are discussed as means to facilitate personalized immunosuppression regimens and promotion of active patient participation as a means to improve adherence. LIMITATIONS: For simplicity, this review focuses on rejection. P4 medicine, however, should more broadly address health concerns in kidney transplant recipients, including competing outcomes such as infections, malignancies, and cardiovascular disease. This review highlights how biomarkers to evaluate these competing outcomes warrant validation and standardization prior to their incorporation into clinical practice. IMPLICATIONS: Consideration of all 4 domains of the P4 medicine framework when caring for and/or studying kidney transplant recipients has the potential of increasing therapeutic efficiency, minimizing adverse effects, decreasing health care costs, and maximizing wellness. Technologies to gauge immune competency, immunosuppression requirements, and early/reversible immune-mediated injuries are required to optimize kidney transplant care.

OBJECTIF DE LA REVUE: La médecine des 4P constitue une approche évolutive qui englobe la médecine prédictive, préventive, personnalisée et participative. En prenant l'exemple du rejet du greffon pour cause d'incompatibilité des antigènes HLA entre le donneur et le receveur en transplantation rénale, cette revue fait état de la pertinence de faire intervenir la médecine des 4P dans les différentes étapes du processus menant à la greffe. SOURCES: Une recherche a été effectuée sur Medline via OvidSP pour répertorier les articles publiés en anglais avant le 18 août 2016 au sujet de la transplantation de rein et de la médecine des 4P. Les articles mentionnant ces concepts dans la rubrique des objectifs de l'étude, dans le titre, dans l'abrégé ou dans les mots-clés listés par les auteurs ont été retenus. La recherche sur la base de données électronique a été davantage élargie pour certains sujets de rubriques. CONSTATATIONS: Les méthodes d'histocompatibilité disponibles illustrent bien les applications actuelles des branches prédictive et préventive de la médecine des 4P du côté des soins prodigués au receveur de la greffe de rein. La pharmacogénomique est pressentie comme moyen de mieux personnaliser le protocole d'immunosuppression du receveur et de favoriser la participation active du patient pour améliorer son adhérence. LIMITES: Pour simplifier la recherche, la revue s'est concentrée sur les rejets de greffon. La médecine des 4P devrait toutefois tenir compte des préoccupations de santé pour les receveurs de greffe de manière plus globale en prenant également en compte les risques concurrents tels que les infections, le développement de tumeurs malignes ou les maladies cardiovasculaires. Cette revue met en lumière la manière dont les biomarqueurs, pour évaluer ces risques, justifient une validation et de la standardisation avant leur intégration aux pratiques cliniques. CONCLUSIONS: Le fait de tenir compte des 4 domaines de la médecine des 4P au moment de prodiguer des soins ou d'étudier le cas des receveurs de greffe de rein a le potentiel d'améliorer l'efficacité thérapeutique, de minimiser les effets indésirables, de réduire les coûts des soins de santé et de maximiser le bien-être des patients. Afin d'optimiser les soins en transplantation rénale, des méthodes permettant d'évaluer la compétence immune, les exigences en immunosuppression ainsi que les lésions à médiation immunitaire précoces ou réversibles sont nécessaires.

Natural history of chronic hepatitis B virus infection in children in Japan: a comparison of mother-to-child transmission with horizontal transmission.

BACKGROUND: It is necessary to evaluate the natural history of children with hepatitis B virus (HBV) infection in each country to consider their long-term management. METHODS: A multi-center observational study of children with chronic HBV infection who were diagnosed at age ≤15 years was carried out in 18 hospitals in Japan. RESULTS: We reviewed children with HBV infection including 381 with mother-to-child transmission (MTCT) and 154 with horizontal transmission, genotype C being the most prevalent virus genotype (83%). Children with horizontal transmission were more frequently infected with HBV genotype A or B and more likely to receive interferon therapy than those infected by MTCT. The HBeAg seroconversion rate at 15 years of age was 42% in the MTCT group and 38% in the horizontal group. It was lower in children with genotype C infection than in those infected with other genotypes (33 versus 45%). Hepatitis developed at any age but before 4 years of age the incidence was high in the horizontal group. At 3 years after the onset of the hepatitis, 26% of children with MTCT and 30% of those with horizontal transmission became inactive carriers. The incidences of hepatocellular carcinoma (HCC) at 30 years of age were 6% in the MTCT group and 11% in the horizontal group. CONCLUSIONS: Patients with childhood-onset HBV infection with MTCT and horizontal transmission developed hepatitis and seroconverted to anti-HBe at any age and had a lifetime risk of developing HCC.

Hepatocellular carcinoma in children and young patients with chronic HBV infection and the usefulness of alpha-fetoprotein assessment.

The aims of the study were to elucidate the clinical characteristics of patients who developed hepatocellular carcinoma (HCC) related to persistent HBV infection since childhood and to investigate usefulness of assessing alpha-fetoprotein (AFP) in this population. A nationwide multicenter survey of children with chronic HBV infection was performed. Among 548 patients, 15 patients developed HCC at the median age of 15 years (range 9-36), including 13 males and 2 females. A case-control comparison showed that HBeAg seroconversion and liver cirrhosis were associated with the occurrence of HCC. Of the 15 HCC patients, 5 were treated with interferon and none of them responded to interferon therapy as compared with 12 of the 17 responders in the control group. Of the 15 patients, 10 died and 9 of the 10 who died never visited any medical facilities until diagnosis of HCC, while the remaining 5 surviving patients never stopped their clinic visits. The usefulness of AFP assessment was shown by the findings that AFP levels were elevated in all HCC cases, that elevations in AFP levels were detected prior to the diagnosis in the surviving patients, and that sensitivity of AFP as a diagnostic test for HCC was very high among 40 patients including our 14 and an additional 26 collected from the literature. HBeAg seroconversion and liver cirrhosis are associated with the occurrence of HCC. Regular measurement of AFP might be helpful to watch for the occurrence of HCC when following children and young patients with chronic HBV infection since childhood.

A Point Mutation in p190A RhoGAP Affects Ciliogenesis and Leads to Glomerulocystic Kidney Defects.

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.

Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.

Acute kidney injury (AKI) is associated with prolonged hospitalization and high mortality, and it predisposes individuals to chronic kidney disease. To date, no effective AKI treatments have been established. Here we show that the apoptosis inhibitor of macrophage (AIM) protein on intraluminal debris interacts with kidney injury molecule (KIM)-1 and promotes recovery from AKI. During AKI, the concentration of AIM increases in the urine, and AIM accumulates on necrotic cell debris within the kidney proximal tubules. The AIM present in this cellular debris binds to KIM-1, which is expressed on injured tubular epithelial cells, and enhances the phagocytic removal of the debris by the epithelial cells, thus contributing to kidney tissue repair. When subjected to ischemia-reperfusion (IR)-induced AKI, AIM-deficient mice exhibited abrogated debris clearance and persistent renal inflammation, resulting in higher mortality than wild-type (WT) mice due to progressive renal dysfunction. Treatment of mice with IR-induced AKI using recombinant AIM resulted in the removal of the debris, thereby ameliorating renal pathology. We observed this effect in both AIM-deficient and WT mice, but not in KIM-1-deficient mice. Our findings provide a basis for the development of potentially novel therapies for AKI.

Increased SHP-1 protein expression by high glucose levels reduces nephrin phosphorylation in podocytes.

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr(1127) and Tyr(1152) are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2(+/C96Y)) compared with control littermate mice (Ins2(+/+)), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.

Complement-mediated glomerular injury is reduced by inhibition of protein-tyrosine phosphatase 1B.

The unfolded protein response and endoplasmic reticulum-associated degradation (ERAD) contribute to injury in renal glomerular diseases, including those mediated by complement C5b-9. In the present study, we address the role of protein-tyrosine phosphatase 1B (PTP1B) in complement-mediated glomerular injury and ERAD. In glomerular epithelial cells (GECs)/podocytes and PTP1B-deficient mouse embryonic fibroblasts exposed to complement, inhibition/deletion of PTP1B reduced ERAD, as monitored by the ERAD reporter CD3δ. Overexpression of PTP1B produced an effect similar to PTP1B deficiency on ERAD in complement-treated GECs. Complement-mediated cytotoxicity was reduced after PTP1B overexpression and tended to be reduced after PTP1B inhibition. PTP1B enhanced the induction of certain ERAD components via the inositol-requiring-1α branch of the unfolded protein response. PTP1B knockout mice with anti-glomerular basement membrane glomerulonephritis had decreased proteinuria and showed less podocyte loss and endoplasmic reticulum dysfunction compared with wild-type littermates. These results imply that endogenous levels of PTP1B are tightly regulated and that both overexpression and inhibition can affect ERAD. The cytoprotective effects of PTP1B deletion in cultured cells and in anti-glomerular basement membrane nephritis suggest that PTP1B may potentially be a therapeutic target in complement-mediated diseases.

Role of guanine nucleotide exchange factor-H1 in complement-mediated RhoA activation in glomerular epithelial cells.

Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.

M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified. METHODS: We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets. RESULTS: The mAChRs were detected on the crypts (K d = 163.2 ± 32.3 pM, B max = 47.3 ± 2.6 fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8 ± 0.4 % of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium. CONCLUSIONS: These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80 %) and M3 (20 %). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

Severe Legionnaires' disease with pneumonia and biopsy-confirmed myocarditis most likely caused by Legionella pneumophila serogroup 6.

We herein describe the successful treatment of a patient with possible Legionella pneumophila serogroup 6 infection complicated by pneumonia and myocarditis. A 32-year-old man presented with a five-day history of cough, dyspnea and chest pain. Chest radiography revealed patchy opacities in both lungs suggestive of bilateral pneumonia, and a urinary antigen test for Legionella pneumophila was positive. After admission, the patient developed congestive heart failure due to pathologically confirmed myocarditis. He was successfully treated with minocycline, macrolide, steroids and noninvasive positive-pressure ventilation (NPPV). He eventually recovered with a normalized cardiac function. L. pneumophila serogroup 6 was isolated from the bathwater in the patient's home.

New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag".

Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.

Endoplasmic reticulum stress in glomerular epithelial cell injury.

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in specific podocyte proteins. This study addresses mediation of GEC injury, focusing on endoplasmic reticulum (ER) stress. We studied signaling in cultured GECs in the presence or absence of the extracellular matrix (ECM). Adhesion to collagen supports cell survival, but adhesion to plastic (loss of contact with ECM) leads to apoptosis. Compared with collagen-adherent cells, GECs on plastic showed increased protein misfolding in the ER, and an adaptive-protective ER stress response, including increased expression of ER chaperones, increased phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), and a reduction in protein synthesis. Activation of these ER stress pathways counteracted apoptosis. However, tunicamycin (a potent stimulator of ER stress) changed the ER stress response from protective to cytotoxic, as tunicamycin induced the proapoptotic ER stress gene, C/EBP homologous protein-10, and exacerbated apoptosis in GECs adherent to plastic, but not collagen. In GECs adherent to plastic, adaptive ER stress was associated with an increase in polyubiquitinated proteins and "choking" of the proteasome. Furthermore, pharmacological inhibition of the proteasome induced ER stress in GECs. Finally, we show that ER stress (induction of ER chaperones and eIF2α phosphorylation) was evident in experimental FSGS in vivo. Thus interactions of GECs with ECM may regulate protein folding and induction of the ER stress response. FSGS is associated with induction of ER stress. Enhancing protective aspects of the ER stress response may reduce apoptosis and possibly glomerulosclerosis.