A Population-Based Analysis of the Risk of Glomerular Disease Relapse after COVID-19 Vaccination.

BACKGROUND: Although case reports have described relapses of glomerular disease after COVID-19 vaccination, evidence of a true association is lacking. In this population-level analysis, we sought to determine relative and absolute risks of glomerular disease relapse after COVID-19 vaccination. METHODS: In this retrospective population-level cohort study, we used a centralized clinical and pathology registry (2000-2020) to identify 1105 adult patients in British Columbia, Canada, with biopsy-proven glomerular disease that was stable on December 14, 2020 (when COVID-19 vaccines first became available). The primary outcome was disease relapse, on the basis of changes in kidney function, proteinuria, or both. Vaccination was modeled as a 30-day time-varying exposure in extended Cox regression models, stratified on disease type. RESULTS: During 281 days of follow-up, 134 (12.1%) patients experienced a relapse. Although a first vaccine dose was not associated with relapse risk (hazard ratio [HR]=0.67; 95% confidence interval [95% CI], 0.33 to 1.36), exposure to a second or third dose was associated with a two-fold risk of relapse (HR=2.23; 95% CI, 1.06 to 4.71). The pattern of relative risk was similar across glomerular diseases. The absolute increase in 30-day relapse risk associated with a second or third vaccine dose varied from 1%-2% in ANCA-related glomerulonephritis, minimal change disease, membranous nephropathy, or FSGS to 3%-5% in IgA nephropathy or lupus nephritis. Among 24 patients experiencing a vaccine-associated relapse, 4 (17%) had a change in immunosuppression, and none required a biopsy. CONCLUSIONS: In a population-level cohort of patients with glomerular disease, a second or third dose of COVID-19 vaccine was associated with higher relative risk but low absolute increased risk of relapse.

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6.

AIMS/HYPOTHESIS: Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function. METHODS: Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed. RESULTS: Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1ß secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression. CONCLUSIONS/INTERPRETATION: We conclude that islet macrophages are major contributors to islet IL-1ß secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1ß- and IL-6-mediated decreased insulin gene expression.

Adapting Nephrology Training Curriculum in the Era of COVID-19.

PURPOSE OF REVIEW: The COVID-19 pandemic has widespread implications not only for clinical practice but also for academic medicine and postgraduate training. The need to promote physical distancing and flexibility within our department has generated important revisions to the core curriculum for the Adult Nephrology Training Program in Vancouver, Canada. SOURCES OF INFORMATION: We reviewed available educational resources and objectives to develop curricular adaptations informed by staff and trainee feedback. METHODS: Many facets of the program including clinical rotations, scholarly activities, evaluation, and wellness have been impacted, and thus revised for online delivery where possible. Trainees have personalized a learning plan based on individual goals and supplemented by a list of internet-based resources for independent review. Changes in learning objectives and methods for specific rotations have occurred and are described. Ongoing evaluation will be undertaken. KEY FINDINGS: Curriculum adaptation in the era of COVID-19 is necessary to ensure ongoing high-quality education for future nephrologists. We describe existing changes to formal training in British Columbia (BC), which will be tailored as the pandemic evolves, and anticipate them to have lasting impact on the way we structure training programs in the future. Standardization and harmonization of modified curriculum may be possible across Canada with sharing of these learnings. LIMITATIONS: Formal evaluation of these changes in terms of knowledge acquisition and examination performance has not yet been undertaken. Next steps will include assessing and documenting the impact of this curricular transformation to further optimize scheduling, educational yield, and trainee wellness.

Trends in Biopsy-Based Diagnosis of Kidney Disease: A Population Study.

BACKGROUND: Kidney biopsy is considered the gold standard for diagnosis of renal disease. It is increasingly performed in cases of diagnostic uncertainty, including in patients with coexistent diabetes and hypertension, for which a presumptive clinical diagnosis can be made. Little is known about the incidence and distribution of biopsy-proven kidney diseases. Changes in the distribution of biopsy diagnoses over time may have significant implications for resource allocation and future research. OBJECTIVE: We studied the relative frequency of kidney diseases in Southern Alberta over the past 30 years, to determine whether the population-standardized annual biopsy rate and incidence of selected diagnostic categories have changed. We hypothesized an increasing incidence of renal biopsies and a growing proportion of nonglomerular diseases (eg, tubulointerstitial disorders) likely due to evolving indications for biopsy. Given the rise in obesity, diabetes, and aging population with chronic kidney disease (CKD), we anticipated a rise in nephroangiosclerosis and diabetic nephropathy over time. DESIGN: Retrospective population-based cohort study using the Biobank for the Molecular Classification of Kidney Disease (BMCKD). SETTING: Southern Alberta, Canada. PATIENTS: All patients who underwent renal biopsy between 1985 and 2015 in our database. MEASUREMENTS: We used descriptive and quantitative analysis to characterize demographics and biopsy-based diagnoses. METHODS: We conducted a retrospective population-based cohort study to analyze all consecutive patients who underwent at least one kidney biopsy over a 30-year period in Southern Alberta (1985-2015). We considered the first adequate biopsy. We described the annual standardized incidence of biopsy-proven kidney diseases over time and summarized associated patient characteristics. We assumed a Poisson distribution for biopsy counts and used provincial demographic information to standardize rates. RESULTS: During the study period, 6434 people (58% male; mean age: 47.9 years) underwent a kidney biopsy. The population-standardized annual biopsy rate increased from 10.8 biopsies per 100 000 person-years in the first 5 years of the study (1985-1989) to 18.2 biopsies per 100 000 person-years in the last 5 years (2010-2014). The mean age at the time of biopsy increased from 42.5 years (1985-1989) to 51.4 years (2010-2014). Glomerular diseases remained the most prevalent histopathological group, with a growing representation of diabetic kidney disease from 3.69% to 16.18%, and a relative decrease in the proportion of other glomerular diseases from 72.32% to 62.92% of glomerular diagnoses. Tubulointerstitial diseases increased from 5.87% to 7.36% of total diagnoses. LIMITATIONS: Classification schemes have changed over time, so recently recognized conditions may have been misclassified in earlier data. There was a changing group of pathologists and nephrologists over this period. Variations in interpretation and application of biopsy indications by physician may influence recorded prevalence of certain diagnoses. We do not yet have complete information on indications or patient outcomes linked to the database. CONCLUSIONS: In Southern Alberta, kidney biopsy is being utilized more frequently and in older people. Diabetic nephropathy is increasingly diagnosed, which may reflect either or both changes in the prevalence of causative factors and local biopsy practices.

CONTEXTE: La biopsie rénale est considérée comme la méthode par excellence pour diagnostiquer des néphropathies. Elle est de plus en plus pratiquée dans les cas de diagnostics incertains, notamment chez les patients souffrant également de diabète et d'hypertension, et pour lesquels un diagnostic clinique présomptif peut être posé. On en sait encore peu sur l'incidence et la distribution statistique des néphropathies avérées par biopsie. Des variations dans la distribution statistique des diagnostics par biopsie au fil du temps pourraient avoir des répercussions significatives sur l'affectation des ressources et sur les recherches futures. OBJECTIFS: Nous voulions savoir si le taux de biopsies annuel normalisé selon la population et l'incidence des catégories de diagnostic sélectionnées avaient varié. Nous avions émis l'hypothèse d'une incidence croissante des biopsies rénales et d'une proportion croissante d'affections non glomérulaires (notamment les troubles tubulo-interstitiels) susceptibles d'être attribuables à une demande croissante pour des biopsies. Compte tenu de l'augmentation des cas d'obésité et de diabète, et du vieillissement de la population atteinte de néphropathies chroniques, nous avions anticipé une augmentation des cas de néphroangiosclérose et de néphropathie diabétique au fil du temps. TYPE D'ÉTUDE: Une étude de cohorte rétrospective sur des données provenant de la biobanque BMCKD (Biobank for the Molecular Classification of Kidney Disease). CADRE DE L'ÉTUDE: Le sud de la province de l'Alberta, au Canada. SUJETS: Tous les patients répertoriés dans notre base de données en raison d'une biopsie rénale subie entre 1985 et 2015. MESURES: Nous avons employé l'analyse descriptive et quantitative pour caractériser les données démographiques des patients et le diagnostic posé à la suite d'une biopsie. MÉTHODOLOGIE: Nous avons mené une étude de cohorte rétrospective pour examiner tous les patients consécutifs ayant subi au moins une biopsie rénale sur une période de 30 ans (1985 à 2015) dans le sud de l'Alberta. Nous avons tenu compte de la première biopsie satisfaisante. Nous avons mesuré l'incidence annuelle normalisée des néphropathies avérées par biopsie au fil du temps et nous avons résumé les caractéristiques des patients qui y étaient associés. Une distribution de Poisson a permis d'établir le nombre de biopsies, alors que les données démographiques provinciales ont servi à la normalisation des taux. RÉSULTATS: Au cours de la période étudiée, 6 434 personnes (âge moyen : 47,9 ans), dont 58 % étaient des hommes, ont subi une biopsie rénale. Entre les cinq premières années (1985-1989) et les cinq dernières années de l'étude (2010-2014), le taux de biopsies annuel normalisé selon la population est passé de 10,8 par 100 000 années-personnes à 12,8 par 100 000 années-personnes; l'âge moyen au moment de subir la biopsie est passé de 42,5 ans à 51,4 ans. Le groupe histopathologique des atteintes glomérulaires est demeuré le plus prévalent des diagnostics glomérulaires avec une représentation croissante des cas de néphropathies diabétiques (de 3,69 % à 16,18 %) et une diminution relative de la proportion des autres atteintes glomérulaires (de 72,32 % à 62,92 %). Quant aux affections tubulo-insterstitielles, elles sont passées de 5,87 % à 7,36 % de tous les diagnostics posés au cours de la période étudiée. LIMITES: Plusieurs variables ont évolué au cours de la période étudiée : les équipes de pathologistes et de néphrologues ont changé, et les systèmes de classification ont été modifiés; de sorte que certaines affections diagnostiquées récemment pourraient avoir été mal classées dans les données antérieures. De plus, des différences dans l'interprétation et l'application des biopsies de la part des médecins pourraient avoir influencé la prévalence consignée pour certains diagnostics. Nous ne disposons toujours pas d'informations complètes sur les indications et les résultats des patients liés à la base de données. CONCLUSION: Dans le sud de l'Alberta, la biopsie rénale est de plus pratiquée, et ce, chez des patients plus âgés. La néphropathie diabétique est de plus en plus diagnostiquée; ce qui pourrait indiquer des variations dans la prévalence des facteurs en cause et/ou des changements dans les pratiques locales quant à l'usage de la biopsie.

Retrovirus-mediated RNA interference. Targeting hTERT through stable expression of short-hairpin RNA.

RNA interference (RNAi) has recently emerged as a reliable tool for studying the effects of knocking down or ablating the expression of specific genes. It is hoped that progress made in the laboratory toward in vitro down regulation of gene expression may be carried over into the clinic for treatment of diseases in which the expression of a specific gene is associated with initiation or progression of that disease. Such is the case with telomerase, an exciting drug target that has been the focus of numerous investigations with a wide variety of inhibitors. This chapter describes the use of retrovirally introduced short-hairpin RNA as an effector of stable, long-term RNAi in human cells.

Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1- or heregulin beta1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors.

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.

Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase.

The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv strain genomic DNA. The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter. Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1). Mass spectrometry analysis confirmed the expected mass of 17.6 kDa. Gel filtration of the enzyme indicated that it was a monomer. Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined. Methotrexate and trimethoprim inhibited the enzyme.

Potent orally bioavailable purine nucleoside phosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lymphocytes--a novel treatment approach for autoimmune diseases, organ transplantation and hematologic malignancies.

The profound suppression of T-cell immunity seen in purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficient patients supports potential application of inhibitors of PNP in the therapy of T-cell mediated diseases. BCX-4208 is a novel potent transition state analog inhibitor of human PNP with an IC(50) of 0.5 nM. PNP inhibition leads to elevation of dGuo which is converted to dGTP mainly in lymphocytes causing imbalance in deoxynucleotide (dNTP) pools and cell apoptosis. In in vitro studies, neither BCX-4208 nor dGuo alone inhibits proliferation of lymphocytes. BCX-4208 in the presence of 10 microM deoxyguanosine (dGuo) inhibits lymphocyte proliferation induced by MLR, IL-2 or Con A with IC(50)s of 0.159, 0.26 and 0.73 microM, respectively. The IC(50) for dGuo in the presence of 1 microM BCX-4208 for the IL-2 stimulated lymphocytes was 3.12 microM. dGTP in human lymphocytes is elevated and a 3-5 fold increase in dGTP results in 50% inhibition after in vitro exposure to BCX-4208 and dGuo. Flow cytometric analyses of human lymphocytes using annexin V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis in T-cells (CD3+), B-cells (CD20+, CD19+) and NK (CD56+) cells. BCX-4208 is orally bioavailable in mice and elevates plasma dGuo levels to 3.7 microM (predose levels<0.004 microM), similar to levels seen in PNP-deficient patients and levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of T-cell and B-cell mediated diseases. BCX-4208 is currently undergoing early clinical investigation in psoriasis and gout.

Evidence of extra-telomeric effects of hTERT and its regulation involving a feedback loop.

The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the enzyme telomerase which is responsible for telomeric maintenance and extension. Using RNA interference to knock down hTERT mRNA expression, we provide evidence that hTERT exerts extra-telomeric effects on the cell cycle and on its own regulatory proteins, specifically: p53 and p21. We tested our hypothesis that hTERT regulates its own expression through effects on upstream regulatory genes using transformed human embryonic kidney (HEK 293) cells, p53 and p16(INK4a) null human ovarian cancer SKOV-3 cells, and p53-null MDA-MB-157 human mammary cancer cells. In HEK 293 cells, hTERT knockdown resulted in elevated p53 and p21 transcription and a decrease in cellular proliferation. Similar results were observed in the MDA-MB-157 cell line where p21 was upregulated, correlating with cell growth inhibition. In contrast, we observed a decrease in expression of p21 in SKOV-3 cells with hTERT knockdown and cell growth appeared to be unaffected. These findings suggest that hTERT may be involved in a feedback loop system, thereby playing a role in its own regulation.