A multi-institutional study found a possible role of anti-nephrin antibodies in post-transplant focal segmental glomerulosclerosis recurrence.

Possible roles of anti-nephrin antibodies in post-transplant recurrent focal segmental glomerulosclerosis (FSGS) have been reported recently. To confirm these preliminary results, we performed a multi-institutional study of 22 Japanese pediatric kidney transplant recipients with FSGS including eight genetic FSGS and 14 non-genetic (presumed primary) FSGS. Eleven of the 14 non-genetic FSGS patients had post-transplant recurrent FSGS. Median (interquartile range) plasma levels of anti-nephrin antibodies in post-transplant recurrent FSGS measured using ELISA were markedly high at 899 (831, 1292) U/mL (cutoff 231 U/mL) before transplantation or during recurrence. Graft biopsies during recurrence showed punctate IgG deposition co-localized with nephrin that had altered localization with increased nephrin tyrosine phosphorylation and Src homology and collagen homology A expressions. Graft biopsies after remission showed no signals for IgG and a normal expression pattern of nephrin. Anti-nephrin antibody levels decreased to 155 (53, 367) U/mL in five patients with samples available after remission. In patients with genetic FSGS as in those with non-genetic FSGS without recurrence, anti-nephrin antibody levels were comparable to those of 30 control individuals, and graft biopsies had no signals for IgG and a normal expression pattern of nephrin. Thus, our results suggest that circulating anti-nephrin antibodies are a possible candidate for circulating factors involved in the pathogenesis of post-transplant recurrent FSGS and that this may be mediated by nephrin phosphorylation. Larger studies including other ethnicities are required to confirm this finding.

The extrafollicular B cell response is a hallmark of childhood idiopathic nephrotic syndrome.

The efficacy of the B cell-targeting drug rituximab (RTX) in childhood idiopathic nephrotic syndrome (INS) suggests that B cells may be implicated in disease pathogenesis. However, B cell characterization in children with INS remains limited. Here, using single-cell RNA sequencing, we demonstrate that a B cell transcriptional program poised for effector functions represents the major immune perturbation in blood samples from children with active INS. This transcriptional profile was associated with an extrafollicular B cell response marked by the expansion of atypical B cells (atBCs), marginal zone-like B cells, and antibody-secreting cells (ASCs). Flow cytometry of blood from 13 children with active INS and 24 healthy donors confirmed the presence of an extrafollicular B cell response denoted by the expansion of proliferating RTX-sensitive extrafollicular (CXCR5-) CD21low T-bet+ CD11c+ atBCs and short-lived T-bet+ ASCs in INS. Together, our study provides evidence for an extrafollicular origin for humoral immunity in active INS.

Multiplex In Situ Hybridization in the Study of Acute Kidney Injury : Multiplex In Situ Hybridization in AKI.

Recently developed in situ hybridization (ISH) methods, such as RNAscope™, have greatly expanded the accessibility and usefulness of ISH in biomedical research. Among many other advantages over traditional ISH, these newer methods enable the simultaneous use of multiple probes, including combination with antibody or lectin staining. We herein illustrate the application of RNAscope™ multiplex ISH in the study of the adapter protein Dok-4 in acute kidney injury (AKI). Specifically, we used multiplex ISH to define the expression of Dok-4 and some of its putative binding partners, together with nephron segment markers, as well as markers of proliferation and tubular injury. We also illustrate the use of QuPath image analysis software to perform quantitative analyses of multiplex ISH. Furthermore, we describe how these analyses can exploit the uncoupling of mRNA and protein expression in a knockout (KO) mouse created by CRISPR/CAS9-mediated frame shift to carry out highly focused molecular phenotyping studies at the single-cell level.

Monitoring of Rho GTPase Activity in Podocytes.

The glomerulus is the filtration unit of the kidney, where the first step of urine formation occurs. Podocytes are characterized by their actin-based projections called foot processes. Podocyte foot processes play a critical role in the permselective filtration barrier, along with fenestrated endothelial cells and the glomerular basement membrane. The Rho family of small GTPases (Rho GTPases) is the master regulators of the actin cytoskeleton and functions as molecular switches. Recent studies have shown that disruption of Rho GTPase activity and subsequent changes in foot process structure cause proteinuria. Here, we describe an effector pull-down assay using GST-fusion proteins to monitor the activity of RhoA, Rac1, and Cdc42, which are prototypical Rho GTPases in podocytes.

The impact of treatment on the psychological burden of mothers of children with chronic hepatitis C virus infection: a multicenter, questionnaire survey.

Mothers of children with chronic hepatitis C virus (HCV) infection experience anxiety about the health of their children. In this study we assessed an impact of treating children with chronic HCV infection on the psychological burden of their mothers. This was a multicenter, questionnaire survey conducted at six institutions in Japan. A newly-developed questionnaire for this study was used to assess changes in the mothers' various concerns regarding HCV infection and thoughts about their child's HCV infection. Responses at the time of diagnosis and at the time of the survey were compared between mothers of children who had received treatment and those without treatment. Responses were received from 36 of 37 eligible mothers (11 and 25, non-treatment and treatment groups, respectively). All children in treatment group had successfully eliminated the virus. Mothers in both groups were psychologically stressed in various ways, including concern about their child's health in the present and future at the time of diagnosis, concern about school, employment, and marriage, concern about the behavior of others towards them and infecting others with HCV, and feelings of guilt regarding their child. These concerns were significantly lower in the present compared to at the time of diagnosis in treatment group, and the rate of decrease was significantly higher in treatment group compared to non-treatment group. Successful treatment greatly reduced mothers' concerns about their children's HCV infection, indicating that treatment during childhood is beneficial from the perspective of the mothers' psychological burden.

Advancing Discovery Research in Nephrology in Canada: A Conference Report From the 2021 Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit.

Purpose of conference: New discoveries arising from investigations into fundamental aspects of kidney development and function in health and disease are critical to advancing kidney care. Scientific meetings focused specifically on fundamental biology of the kidney can facilitate interactions, support the development of collaborative groups, and accelerate translation of key findings. The Canadian fundamental kidney researcher community has lacked such a forum. On December 3 to 4, 2021, the first Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit was held to address this gap with the goal of advancing fundamental kidney research nationally. The meeting was held virtually and was supported by a planning and dissemination grant from the Canadian Institutes of Health Research. Attendees included PhD scientists, nephrology clinician scientists, engineers, industry representatives, graduate students, medical residents, and fellows. Sources of information: This report was prepared from the scientific program, registration numbers, and details obtained from the online platform WHOVA, and summaries written by organizers and participants of the 2021 meeting. Methods: A 21-person team, consisting of the organizing committee members and participants from the meeting, was assembled. Key highlights of the meeting and future directions were identified and the team jointly assembled this report. Key findings: Participation in the meeting was strong, with more than 140 attendees across a range of disciplines. The program featured state-of-the-art presentations on diabetic nephropathy, the immune system, kidney development, and fibrosis, and was heavily focused on trainee presentations. The moderated "Investigator Summit" identified key barriers to research advancement and discussed strategies for overcoming them. These included establishment of a pan-Canadian fundamental kidney research network, development of key resources, cross-pollination with clinical nephrology, better reintegration into the Canadian Society of Nephrology, and further establishment of identity and knowledge translation. Limitations and implications: The 2021 M3K meeting represented a key first step in uniting fundamental kidney researchers in Canada. However, it was universally agreed that regular meetings were necessary to sustain this momentum. The proceedings of this meeting and future actions to sustain the M3K Scientific Meeting and Investigator Summit are presented in this article.

Objectif de la conférence: De nouvelles découvertes découlant des enquêtes sur les aspects fondamentaux du développement et de la fonction des reins en santé ou malades sont essentielles pour faire progresser les soins rénaux. Les réunions scientifiques axées spécifiquement sur la biologie fondamentale du rein peuvent faciliter les interactions, appuyer le développement de groupes de collaboration et accélérer l'application des principaux résultats. La communauté canadienne des chercheurs fondamentaux en néphrologie a manqué d'un tel forum. Les 3 et 4 décembre 2021, le premier Sommet des chercheurs et la réunion scientifique M3K (Molecules and Mechanisms Mediating Kidney Health and Disease) sur les molécules et les médiateurs de la santé et des maladies rénales ont eu lieu pour combler cette lacune; l'objectif était de faire progresser la recherche fondamentale en néphrologie à l'échelle nationale. La réunion s'est tenue virtuellement et était financée par une subvention de planification et de diffusion des Instituts de recherche en santé du Canada. Des doctorants, cliniciens-chercheurs en néphrologie, ingénieurs, représentants de l'industrie, étudiants diplômés, résidents en médecine et en surspécialisation figuraient parmi les participants. Sources: Ce rapport a été préparé à partir du program scientifique, des informations et des numéros d'inscription tirés de la plateforme en ligne WHOVA, et des résumés rédigés par les organisateurs et les participants à la réunion de 2021. Méthodologie: Une équipe de 20 personnes composée de membres du comité organisateur et de participants à la réunion a été formée. Les principaux points saillants de la réunion et les orientations futures ont été déterminés, puis l'équipe a rédigé conjointement le présent rapport. Principaux résultats: La réunion s'est avérée un succès; plus de 140 personnes provenant d'un large éventail de disciplines y ont participé. Le program comprenait des présentations de pointe sur la néphropathie diabétique, le système immunitaire, le développement des reins et la fibrose, et était fortement axé sur des présentations par des stagiaires. Le « Sommet des chercheurs ¼, animé par un modérateur, a permis de déterminer les principaux obstacles à l'avancement de la recherche et de discuter des stratégies pour les surmonter. Ces dernières incluent notamment la création d'un réseau pancanadien de recherche fondamentale en néphrologie, le développement de ressources clés, la pollinisation croisée avec la néphrologie clinique, une « meilleure réintégration dans la Société canadienne de néphrologie ¼ et la poursuite de l'établissement de l'identité et de l'application des connaissances. Limites et implications: La réunion M3K de 2021 a constitué une première étape clé dans l'unification des chercheurs fondamentaux en néphrologie au Canada. On a cependant universellement convenu que des réunions régulières étaient nécessaires pour maintenir cet élan. Le compte rendu de cette réunion ainsi que les actions futures pour soutenir la réunion scientifique M3K et le Sommet des chercheurs sont présentés dans le présent article.

Involving Patient Partners in the KRESCENT Peer Review: Intent, Process, Challenges, and Opportunities.

Purpose of review: The Kidney Research Scientist Core Education and National Training (KRESCENT) is a national Canadian training program for kidney scientists, funded by the Kidney Foundation of Canada (KFOC), the Canadian Institutes of Health Research (CIHR), and the Canadian Society of Nephrology (CSN). We describe our first year of incorporating patient partners into a scientific peer-review committee, the 2017 committee to select senior research trainees and early-career kidney researchers for funding and training, in the hope that it will be helpful to others who wish to integrate the perspective of people with lived experience into the peer-review process. Sources of information: Other peer-review committees, websites, journal articles, patient partners, Kidney Foundation of Canada Research Council, Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (Can-SOLVE CKD) Patient Council, participants in the 2017 Kidney Foundation of Canada KRESCENT peer-review panel. Methods: We describe our motivation, rationale, guiding principles, plans, feedback, implementation, and response. Key findings: We disseminated a "call for patient partners" 8 weeks before the meeting, seeking patients or their care givers to partner with the KRESCENT peer-review panel; we defined these people with lived experience of kidney disease as patient partners. Eight patient partners came forward and all participated as reviewers. Patient partners first participated in a webinar to learn about the function, structure, and processes of a peer-review committee. They practiced reviewing plain language summaries and giving feedback. In a subsequent teleconference, they shared and discussed their reviews. Plain language summaries were scored, overall, on the same 0-5 quality scale used by scientific reviewers. Three patient reviewers participated in some or all of the 6-hour meeting, which was conducted as usual, for this panel, by teleconference (initially audio only; from 2020 onwards by videoconference). In the meeting, the 2 assigned scientific reviewers first gave their scores, followed by the patient reviewers giving their scores, and discussion (mostly scientific, and conducted in usual scientific language). Scientific reviewers then negotiated a consensus score based on their initial scores, the discussion, patient reviewers' scores and statements, and the scientific officer's notes. Patient reviewers, scientific reviewers, and the Kidney Foundation of Canada (KFOC) were generally positive about the process. The increased length of the meeting (estimated at 1 hour) was generally thought to be acceptable. Patient reviewers also provided feedback on the methods used to incorporate patients into the research under review. These comments were concrete, insightful, and helpful. The patients did not uniformly recommend that basic scientists involve patients in their work. We did not detect bias against preclinical science, work that did not involve patients, or rarer diseases. Some patients found participation inspiring and enlightening. All participants appreciated the idea of patient partners as community witnesses to a group process committed to fairness and supportiveness. We discussed assigning formal meaningful weight to patient reviewers' assessments. Most, but not all, patients thought that the scientific reviewers were ultimately the best judges of the allocation of scarce research resources. Limitations: Patient participants tended to be Caucasian, middle class, and well educated. Because of the difficulties of travel for some people living with or supporting those living with kidney disease, our findings may not generalize fully to peer-review meetings that are conducted face to face. This is explicitly a supportive panel, committed to reviewing junior scientists with kindness as well as rigor; our findings may not generalize to panels conducted differently. We did not use formal qualitative methodology. Implications: Inclusion of patient partners as patient reviewers for the KRESCENT program peer-review panel was feasible, added value for scientific and patient reviewers, and for the funding stakeholders (CIHR, KFOC, and CSN). We were glad that we had taken this step and continue to refine the process with each successive competition.

Motif de la revue: Le KRESCENT (Kidney Research Scientist Core Education and National Training) est un programme national de formation pour les chercheurs en santé rénale financé par la Fondation canadienne du rein (FCR), les Instituts de recherche en santé du Canada (IRSC) et la Société canadienne de néphrologie (SCN). Nous décrivons notre première année d'intégration de partenaires patients dans un comité d'examen scientifique par les pairs, le comité de 2017, visant la sélection de stagiaires de recherche et de chercheurs en santé rénale en début de carrière pour le financement et la formation, dans l'espoir que cela sera utile à ceux qui souhaitent intégrer la perspective des personnes ayant une expérience vécue au processus d'examen par les pairs. Sources: Autres comités d'examen par les pairs, sites Web, articles de revues, partenaires patients, Conseil de recherche de la Fondation canadienne du rein, conseil des patients de Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (CAN-SOLVE CKD), participants au comité d'examen par les pairs de la Fondation canadienne du rein de 2017. Méthodologie: Nous décrivons ce qui a motivé cette étude, notre raisonnement, nos principes directeurs, nos plans, la rétroaction, la mise en œuvre et les réponses. Principaux résultats: Nous avons diffusé un « appel à des partenaires patients ¼ huit semaines avant la réunion pour trouver des patients ou des soignants prêts à collaborer avec le comité d'examen par les pairs de KRESCENT; nous avons défini comme partenaires patients les personnes ayant une expérience vécue de maladie rénale. Huit partenaires patients ont répondu à l'appel et tous ont participé en tant qu'examinateurs. Les partenaires patients ont d'abord participé à un webinaire pour en apprendre davantage sur la fonction, la structure et les processus d'un comité d'examen par les pairs. Ils se sont ensuite entraînés à examiner des résumés en langage simple et à donner des commentaires. Lors d'une téléconférence ultérieure, ils ont partagé et discuté de leurs examens respectifs. Les résumés en langage clair ont été notés, dans l'ensemble, sur la même échelle de qualité de 0 à 5 utilisée par les examinateurs scientifiques. Trois patients examinateurs ont participé à une partie ou à la totalité de la réunion de 6 heures, qui s'est tenue comme d'habitude, pour ce panel, par téléconférence (initialement en audio seulement; par vidéoconférence à partir de 2020). Au cours de la réunion, les deux examinateurs scientifiques désignés ont d'abord donné leurs notes, puis les patients examinateurs ont donné leurs notes, et une discussion a suivi (principalement scientifique, et menée dans le langage scientifique habituel). Les examinateurs scientifiques ont ensuite négocié pour établir une note consensuelle en fonction de leurs notes initiales, de la discussion, des notes et des commentaires des patients examinateurs et des notes de l'agent scientifique.Les patients examinateurs, les examinateurs scientifiques et la Fondation canadienne du rein étaient généralement positifs à l'égard du processus. La durée accrue de la réunion (estimée à 1 heure) a généralement été jugée acceptable. Les patients examinateurs ont également fourni des commentaires sur les méthodes utilisées pour intégrer les patients à la recherche à l'étude. Ces commentaires étaient concrets, pertinents et utiles. Les patients ne recommandent pas uniformément que les scientifiques en recherche fondamentale impliquent les patients dans leur travail. Nous n'avons pas détecté de biais contre la science préclinique, les études qui n'impliquent pas de patients ou les maladies plus rares. Certains patients ont trouvé la participation inspirante et instructive. Tous les participants ont aimé l'idée des partenaires patients comme témoins communautaires d'un processus de groupe engagé dans l'équité et le soutien.Nous avons discuté de l'attribution d'un poids formel significatif aux évaluations des patients examinateurs. La plupart des patients, mais pas tous, étaient d'avis que les examinateurs scientifiques étaient en fin de compte les meilleurs juges de l'allocation des ressources limitées de la recherche. Limites: Les patients participants étaient pour la plupart de race blanche, de classe moyenne et bien éduqués. En raison des difficultés de déplacement pour certaines personnes vivant avec ou soutenant les personnes vivant avec une maladie rénale, nos résultats peuvent ne pas se généraliser entièrement aux réunions d'examen par les pairs menées en personne. Il s'agit essentiellement d'un groupe de soutien, qui s'est engagé à examiner les jeunes chercheurs avec bienveillance et rigueur; nos conclusions peuvent ne pas se généraliser à des groupes de travail menés différemment. Nous n'avons pas utilisé de méthodologie qualitative officielle. Résultats: L'inclusion de partenaires patients comme examinateurs dans un comité d'examen par les pairs du programme KRESCENT s'est avérée réalisable, et une valeur ajoutée pour les examinateurs scientifiques, les patients examinateurs et les parties responsables du financement (IRSC, FCR et SCN). Nous sommes heureux d'avoir franchi cette étape, nous continuons de raffiner le processus à chaque concours successif.

CdGAP maintains podocyte function and modulates focal adhesions in a Src kinase-dependent manner.

Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and ß-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with ß-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired ß-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury.

A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

The role of serum Wisteria floribunda agglutinin-positive Mac-2 binding protein in the assessment of fibrosis in children with chronic hepatitis C.

At present, noninvasive fibrosis markers are not available for the assessment of liver fibrosis in children with chronic hepatitis C. Sixty-three children with chronic hepatitis C were included. Changes in Wisteria floribunda agglutinin-positive Mac-2 binding protein (M2BPGi) levels were evaluated in l3 of 27 treatment-naive patients during the natural course of disease (median 4, range 3-6 years). Changes during treatment were evaluated in 27 of 36 patients for 4 (2-9) years of posttreatment follow-up. There were significant differences in the levels of M2BPGi between control group and HCV F0 group (P = 0.002) and between control group and HCV F1 group (P < 0.001). Receiver operating characteristic curve analysis showed that to discriminate stage F1 fibrosis from F0, the cut-off value was 0.95 for M2BPGi with a sensitivity of 52%, specificity of 90%, and area under the curve of 0.687. A substantial decrease in M2BPGi levels by treatment was shown from 0.98 ± 0.57 at pretreatment to 0.42 ± 0.15 at posttreatment (P < 0.001) in the 27 treated patients. Our study shows new findings that M2BPGi may be useful to predict the presence of a mild degree of fibrosis in children with chronic hepatitis C, and such mild fibrosis may be quickly resolved by treatment.

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.

Clinical practice guidelines for the management of children with mother-to-child transmitted hepatitis C virus infection.

BACKGROUND: The first guidelines for care of pregnant women carrying the hepatitis C virus (HCV) and their infants were published in 2005 in Japan. Since then, evidence has gradually accumulated worldwide regarding the natural course and treatment of this condition and, especially in recent years, treatment for chronic hepatitis C in adult patients has made great progress. However, the clinical practice policy for children has not been standardized, and new clinical practice guidelines for children with mother-to-child (MTC) transmitted HCV infection have become necessary. METHODS: In the development of the current guideline, we requested cooperation from The Japanese Society for Pediatric Infectious Diseases, The Japan Society of Hepatology, and the Japan Society of Obstetrics and Gynecology. The committee members were recommended and approved by each society to participate in developing the guidelines. The guideline was also created in accordance with the Minds Guide for Practice Guideline Development. The statements were prepared by consensus-building using the Delphi method, based on the comprehensively searched academic papers and guidelines. These articles were retrieved through searching the PubMed, Cochrane Library, and the Igaku Chuo Zasshi databases. RESULTS: Eight clinical questions (CQs) with clinical statements were developed regarding etiology (CQs 1-3), diagnosis (CQs 4 and 5), and treatment (two CQs 6 and 7). In each statement, the consensus rate, evidence level, and recommendation level were determined. CONCLUSION: The guidelines will be helpful in the management of children with hepatitis C MTC transmission.

Role of Rho GTPase Interacting Proteins in Subcellular Compartments of Podocytes.

The first step of urine formation is the selective filtration of the plasma into the urinary space at the kidney structure called the glomerulus. The filtration barrier of the glomerulus allows blood cells and large proteins such as albumin to be retained while eliminating the waste products of the body. The filtration barrier consists of three layers: fenestrated endothelial cells, glomerular basement membrane, and podocytes. Podocytes are specialized epithelial cells featured by numerous, actin-based projections called foot processes. Proteins on the foot process membrane are connected to the well-organized intracellular actin network. The Rho family of small GTPases (Rho GTPases) act as intracellular molecular switches. They tightly regulate actin dynamics and subsequent diverse cellular functions such as adhesion, migration, and spreading. Previous studies using podocyte-specific transgenic or knockout animal models have established that Rho GTPases are crucial for the podocyte health and barrier function. However, little attention has been paid regarding subcellular locations where distinct Rho GTPases contribute to specific functions. In the current review, we discuss cellular events involving the prototypical Rho GTPases (RhoA, Rac1, and Cdc42) in podocytes, with particular focus on the subcellular compartments where the signaling events occur. We also provide our synthesized views of the current understanding and propose future research directions.

Rho GTPase regulatory proteins in podocytes.

The Rho family of small GTPases (Rho GTPases) are the master regulators of the actin cytoskeleton and consist of 22 members. Previous studies implicated dysregulation of Rho GTPases in podocytes in the pathogenesis of proteinuric glomerular diseases. Rho GTPases are primarily regulated by the three families of proteins; guanine nucleotide exchange factors (GEFs; 82 members), GTPase-activating proteins (GAPs; 69 members), and GDP dissociation inhibitors (GDIs; 3 members). Since the regulatory proteins far outnumber their substrate Rho GTPases and act in concert in a cell/context-dependent manner, the upstream regulatory mechanism directing Rho GTPases in podocytes is largely unknown. In this review, we summarize recent advances in the understanding of the role of Rho GTPase regulatory proteins in podocytes, including the known mutations of these proteins that cause proteinuria in humans. We also provide critical appraisal of the in vivo and in vitro studies and identify the knowledge gap in the field that will require further studies.

ARHGEF7 (ß-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function.

BACKGROUND: Previous studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown. METHODS: We used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange factors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specific ARHGEF7 (commonly known as ß-PIX) knockout mice by crossing ß-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with ß-PIX knockdown and their controls. RESULTS: We identified ß-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific ß-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific ß-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific ß-PIX knockout mice and cultured mouse podocytes with ß-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of ß-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated protein. CONCLUSIONS: These findings indicate that ß-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho-guanine nucleotide exchange factor plays a critical role in podocytes.

Association between fruit and vegetable allergies and pollen-food allergy syndrome in Japanese children: a multicenter cross-sectional case series.

BACKGROUND: Recently, the prevalence of food allergies during childhood is increasing, with fruits being common allergens. However, data on allergens that cause fruit and vegetable allergies and pollen-food allergy syndrome (PFAS) in childhood are relatively few. This study aimed to examine the allergens in fruit and vegetable allergies in pediatric patients and to determine the association between fruit and vegetable allergies and PFAS. OBJECTIVE: This study aimed to examine the current status of fruit and vegetable allergies in Japanese children. METHODS: This was a multicenter case series observational study. The participants included children aged <15 years who developed allergic symptoms after eating fruits and vegetables and subsequently received treatment in the Pediatric Department of 6 hospitals in the Osaka Prefecture in Japan during the study period from August 2016 to July 2017. Participants' information was obtained using a questionnaire, and data were obtained by performing several types of allergy tests using blood samples. RESULTS: A total of 97 children (median age, 9 years; 56 males) were included in the study. Apple was the most common allergen, followed by peach, kiwi, cantaloupe, and watermelon. A total of 74 participants (76%) exhibited allergic symptoms due to PFAS; moreover, pathogenesis-related protein-10 (PR-10) was the most common allergen superfamily. On the contrary, in the group where neither PR-10 nor profilin was sensitized, kiwi and banana were the most common allergens, and the age of onset was lower than that in the PFAS group. Specific antibody titer was significantly associated with Birch for Bet v1 and latex for Bet v2 (r = 0.99 and r = 0.89). CONCLUSION: When we examine patients with fruit and vegetable allergies, we should first consider PFAS even in childhood specifically for children greater than 4 years old.

Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3+ Regulatory T Cells.

Regulatory T (Treg) cells integrate diverse environmental signals to modulate their function for optimal suppression. Translational regulation represents a favorable mechanism for Treg cell environmental sensing and adaptation. In this study, we carry out an unbiased screen of the Treg cell translatome and identify serum/glucocorticoid-regulated kinase 1 (SGK1), a known salt sensor in T cells, as being preferentially translated in activated Treg cells. We show that high salt (HS) drives thymic Treg cells to adopt a T helper type 17 (Th17)-like phenotype and enhances generation of Th17-like induced Treg cells in a SGK1-dependent manner, all the while maintaining suppressive function. Salt-mediated Th17-like differentiation of Treg cells was evident in mice fed with HS diet or injected with HS-preconditioned T cells. Overall, SGK1 enables Treg cells to adapt their function in response to environmental cues. By understanding these environmental-sensing mechanisms, we envision targeted approaches to fine-tune Treg cell function for better control of inflammation.

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.

Mammalian target of rapamycin is activated in the kidneys of patients with scleroderma renal crisis.

Objectives: Scleroderma renal crisis is a rare but serious complication affecting 2%-15% of patients with systemic sclerosis. Despite treatment with angiotensin-converting enzyme inhibitors, outcomes for scleroderma renal crisis patients are still poor. The cellular signaling mechanisms in scleroderma renal crisis are not yet known. Mammalian target of rapamycin, comprised of the subunits mTORC1 and mTORC2, has been shown to be activated in vascular lesions of renal transplant patients with anti-phospholipid antibody syndrome. Given the similarities between the pathophysiology of scleroderma renal crisis and anti-phospholipid antibody syndrome, we hypothesized that the mammalian target of rapamycin pathway would also be activated in the renal vasculature of patients with scleroderma renal crisis. Methods: We retrospectively analyzed renal biopsies of five patients with scleroderma renal crisis in the Canadian Scleroderma Research Group cohort. Immunostaining was performed using anti-P-S6RP antibodies to evaluate the phosphorylation of mTORC1, and anti-Rictor and anti-S473 to determine activation of mTORC2. Results: Four of the five patients showed mTORC1 activation in arteriolar endothelial cells, and three of the five patients showed mTORC1 activation in the arterial endothelial cells. Two of four samples showed Rictor expression in the arteriolar and arterial endothelial cells, showing mTORC2 activation. There was no expression of mTORC1 or mTORC2 in samples from two healthy controls. Conclusion: We demonstrate that both mTORC1 and mTORC2 are activated in renal biopsies with typical histologic features of scleroderma renal crisis. Dual mammalian target of rapamycin inhibitors are currently available and in development. These findings could inform further research into novel treatment targets for scleroderma renal crisis.