Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis.

Introduction: Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted. Methods: We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies. Results and Conclusion: Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings.

Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

Multiscale genetic architecture of donor-recipient differences reveals intronic LIMS1 mismatches associated with kidney transplant survival.

Donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLAs) contribute to kidney allograft loss, but the mechanisms remain unclear, specifically for intronic mismatches. We quantified non-HLA mismatches at variant-, gene-, and genome-wide scales from single nucleotide polymorphism (SNP) data of D-Rs from 2 well-phenotyped transplant cohorts: Genomics of Chronic Allograft Rejection (GoCAR; n = 385) and Clinical Trials in Organ Transplantation-01/17 (CTOT-01/17; n = 146). Unbiased gene-level screening in GoCAR uncovered the LIMS1 locus as the top-ranked gene where D-R mismatches associated with death-censored graft loss (DCGL). A previously unreported, intronic, LIMS1 haplotype of 30 SNPs independently associated with DCGL in both cohorts. Haplotype mismatches showed a dosage effect, and minor-allele introduction to major-allele-carrying recipients showed greater hazard of DCGL. The LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) in immune cells for GCC2 (not LIMS1), which encodes a protein involved in mannose-6-phosphase receptor (M6PR) recycling. Peripheral blood and T cell transcriptome analyses associated the GCC2 gene and LIMS1 SNPs with the TGF-ß1/SMAD pathway, suggesting a regulatory effect. In vitro GCC2 modulation impacted M6PR-dependent regulation of active TGF-ß1 and downstream signaling in T cells. Together, our data link LIMS1 locus D-R mismatches to DCGL via GCC2 eQTLs that modulate TGF-ß1-dependent effects on T cells.

Nonpodocyte Roles of APOL1 Variants: An Evolving Paradigm.

Since the seminal discovery of the trypanolytic, exonic variants in apolipoprotein L1 (APOL1) and their association with kidney disease in individuals of recent African ancestry, a wide body of research has emerged offering key insights into the mechanisms of disease. Importantly, the podocyte has become a focal point for our understanding of how risk genotype leads to disease, with activation of putative signaling pathways within the podocyte identified as playing a causal role in podocytopathy, FSGS, and progressive renal failure. However, the complete mechanism of genotype-to-phenotype progression remains incompletely understood in APOL1-risk individuals. An emerging body of evidence reports more than podocyte-intrinsic expression of APOL1 risk variants is needed for disease to manifest. This article reviews the seminal data and reports which placed the podocyte at the center of our understanding of APOL1-FSGS, as well as the evident shortcomings of this podocentric paradigm. We examine existing evidence for environmental and genetic factors that may influence disease, drawing from both clinical data and APOL1's fundamental role as an immune response gene. We also review the current body of data for APOL1's impact on nonpodocyte cells, including endothelial cells, the placenta, and immune cells in both a transplant and native setting. Finally, we discuss the implications of these emerging data and how the paradigm of disease might evolve as a result.

Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal segmental glomerulosclerosis and normal glomeruli.

Morphometric estimates of mean or individual glomerular volume (MGV, IGV) have biological implications, over and above qualitative histologic data. However, morphometry is time-consuming and requires expertise limiting its utility in clinical cases. We evaluated MGV and IGV using plastic- and paraffin-embedded tissue from 10 control and 10 focal segmental glomerulosclerosis (FSGS) mice (aging and 5/6th nephrectomy models) using the gold standard Cavalieri (Cav) method versus the 2-profile and Weibel-Gomez (WG) methods and a novel 3-profile method. We compared accuracy, bias and precision, and quantified results obtained when sampling differing numbers of glomeruli. In both FSGS and controls, we identified an acceptable precision for MGV of 10-glomerular sampling versus 20-glomerular sampling using the Cav method, while 5-glomerular sampling was less precise. In plastic tissue, 2- or 3-profile MGVs showed greater concordance with MGV when using Cav, versus MGV with WG. IGV comparisons using the same glomeruli reported a consistent underestimation bias with both 2- or 3-profile methods versus the Cav method. FSGS glomeruli showed wider variations in bias estimation than controls. Our 3-profile method offered incremental benefit to the 2-profile method in both IGV and MGV estimation (improved correlation coefficient, Lin's concordance and reduced bias). In our control animals, we quantified a shrinkage artifact of 52% from tissue processed for paraffin-embedded versus plastic-embedded tissue. FSGS glomeruli showed overall reduced shrinkage albeit with variable artifact signifying periglomerular/glomerular fibrosis. A novel 3-profile method offers slightly improved concordance with reduced bias versus 2-profile. Our findings have implications for future studies using glomerular morphometry.

Blood Transcriptomes of SARS-CoV-2-Infected Kidney Transplant Recipients Associated with Immune Insufficiency Proportionate to Severity.

BACKGROUND: Among patients with COVID-19, kidney transplant recipients (KTRs) have poor outcomes compared with non-KTRs. To provide insight into management of immunosuppression during acute illness, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort. METHODS: We ascertained clinical data by chart review. A single sample of blood was collected for transcriptome analysis. Total RNA was poly-A selected and RNA was sequenced to evaluate transcriptome changes. We also measured cytokines and chemokines of serum samples collected during acute infection. RESULTS: A total of 64 patients with COVID-19 in KTRs were enrolled, including 31 with acute COVID-19 (<4 weeks from diagnosis) and 33 with post-acute COVID-19 (>4 weeks postdiagnosis). In the blood transcriptome of acute cases, we identified genes in positive or negative association with COVID-19 severity scores. Functional enrichment analyses showed upregulation of neutrophil and innate immune pathways but downregulation of T cell and adaptive immune activation pathways. This finding was independent of lymphocyte count, despite reduced immunosuppressant use in most KTRs. Compared with acute cases, post-acute cases showed "normalization" of these enriched pathways after 4 weeks, suggesting recovery of adaptive immune system activation despite reinstitution of immunosuppression. Analysis of the non-KTR cohort with COVID-19 showed significant overlap with KTRs in these functions. Serum inflammatory cytokines followed an opposite trend (i.e., increased with disease severity), indicating that blood lymphocytes are not the primary source. CONCLUSIONS: The blood transcriptome of KTRs affected by COVID-19 shows decreases in T cell and adaptive immune activation pathways during acute disease that, despite reduced immunosuppressant use, associate with severity. These pathways show recovery after acute illness.

Blood transcriptomes of SARS-CoV-2 infected kidney transplant recipients demonstrate immune insufficiency.

BACKGROUND: Kidney transplant recipients (KTRs) with COVID-19 have poor outcomes compared to non-KTRs. To provide insight into management of immunosuppression during acute illness, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort.□. METHODS: Clinical data were collected by chart review. PAXgene blood RNA was poly-A selected and RNA sequencing was performed to evaluate transcriptome changes. RESULTS: A total of 64 cases of COVID-19 in KTRs were enrolled, including 31 acute cases (< 4 weeks from diagnosis) and 33 post-acute cases (>4 weeks). In the blood transcriptome of acute cases, we identified differentially expressed genes (DEGs) in positive or negative association COVID-19 severity scores. Functional enrichment analyses showed upregulation of neutrophil and innate immune pathways, but downregulation of T-cell and adaptive immune-activation pathways proportional to severity score. This finding was independent of lymphocyte count and despite reduction in immunosuppression (IS) in most KTRs. Comparison with post-acute cases showed "normalization" of these enriched pathways after >4 weeks, suggesting recovery of adaptive immune system activation despite reinstitution of IS. The latter analysis was adjusted for COVID-19 severity score and lymphocyte count. DEGs associated with worsening disease severity in a non-KTR cohort with COVID-19 (GSE152418) showed significant overlap with KTRs in these identified enriched pathways. CONCLUSION: Blood transcriptome of KTRs affected by COVID-19 shows decrease in T-cell and adaptive immune activation pathways during acute disease that associate with severity despite IS reduction and show recovery after acute illness. SIGNIFICANCE STATEMENT: Kidney transplant recipients (KTRs) are reported to have worse outcomes with COVID-19, and empiric reduction of maintenance immunosuppression is pursued. Surprisingly, reported rates of acute rejection have been low despite reduced immunosuppression. We evaluated the peripheral blood transcriptome of 64 KTRs either during or after acute COVID-19. We identified transcriptomic signatures consistent with suppression of adaptive T-cell responses which significantly associated with disease severity and showed evidence of recovery after acute disease, even after adjustment for lymphocyte number. Our transcriptomic findings of immune-insufficiency during acute COVID-19 provide an explanation for the low rates of acute rejection in KTRs despite reduced immunosuppression. Our data support the approach of temporarily reducing T -cell-directed immunosuppression in KTRs with acute COVID-19.

Electromyographic Analyses of Trunk Musculature after Stroke: An Integrative Review.

BACKGROUND: Observational and intervention studies examining trunk electromyographic (EMG) activity following stroke are underpowered and fail criteria for systematic reviews of randomized control trials. Objective: To systematically evaluate and summarize evidence about trunk muscle activation after stroke during ADL and with diagnostic and therapeutic interventions. METHODS: Search databases were Medline Complete, CINAHL and Health Sources: Nursing Academic Edition. Studies written in English after 1989 included these search terms: stroke, muscle activity, and trunk including abdominal or back muscles. Systematic reviews, single case studies, dissertations, or letters to the editor were excluded. Reviewers used Covidence to screen relevant research and extract information after title, abstract, and full-text screening. Information extracted related to stroke severity, time since onset, specific muscles and EMG analysis technique, and study limitations. Articles were classified as observational, intervention or device-related. RESULTS: The electronic search yielded 188 articles and a hand search found 3. Title and abstract screening yielded 102 articles for full text screening. Ultimately, 45 articles were extracted. Trunk muscle recruitment during function and movement demonstrated significant differences in trunk EMG recruitment timing, magnitude, and symmetry after stroke when compared with healthy participants. Individuals with stroke demonstrated significant differences when comparing paretic to non-paretic side trunk EMG measures. Intervention studies showed some effect on improving trunk muscle activation but they had small sample sizes and methodological issues. CONCLUSIONS: Trunk muscle activation after stroke can be monitored with EMG during rehabilitation programs which challenge functional recovery.

The effect of exposure to long working hours on stroke: A systematic review and meta-analysis from the WHO/ILO Joint Estimates of the Work-related Burden of Disease and Injury.

BACKGROUND: The World Health Organization (WHO) and the International Labour Organization (ILO) are developing joint estimates of the work-related burden of disease and injury (WHO/ILO Joint Estimates), with contributions from a large network of individual experts. Evidence from mechanistic data and prior studies suggests that exposure to long working hours may cause stroke. In this paper, we present a systematic review and meta-analysis of parameters for estimating the number of deaths and disability-adjusted life years from stroke that are attributable to exposure to long working hours, for the development of the WHO/ILO Joint Estimates. OBJECTIVES: We aimed to systematically review and meta-analyse estimates of the effect of exposure to long working hours (three categories: 41-48, 49-54 and ≥55 h/week), compared with exposure to standard working hours (35-40 h/week), on stroke (three outcomes: prevalence, incidence, and mortality). DATA SOURCES: A protocol was developed and published, applying the Navigation Guide to systematic reviews as an organizing framework where feasible. We searched electronic databases for potentially relevant records from published and unpublished studies, including Ovid MEDLINE, PubMed, EMBASE, Scopus, Web of Science, CISDOC, PsycINFO, and WHO ICTRP. We also searched grey literature databases, Internet search engines, and organizational websites; hand-searched reference lists of previous systematic reviews; and consulted additional experts. STUDY ELIGIBILITY AND CRITERIA: We included working-age (≥15 years) individuals in the formal and informal economy in any WHO and/or ILO Member State but excluded children (aged < 15 years) and unpaid domestic workers. We included randomized controlled trials, cohort studies, case-control studies and other non-randomized intervention studies with an estimate of the effect of exposure to long working hours (41-48, 49-54 and ≥55 h/week), compared with exposure to standard working hours (35-40 h/week), on stroke (prevalence, incidence or mortality). STUDY APPRAISAL AND SYNTHESIS METHODS: At least two review authors independently screened titles and abstracts against the eligibility criteria at a first review stage and full texts of potentially eligible records at a second stage, followed by extraction of data from qualifying studies. Missing data were requested from principal study authors. We combined relative risks using random-effects meta-analysis. Two or more review authors assessed the risk of bias, quality of evidence and strength of evidence, using the Navigation Guide and GRADE tools and approaches adapted to this project. RESULTS: Twenty-two studies (20 cohort studies, 2 case-control studies) met the inclusion criteria, comprising a total of 839,680 participants (364,616 females) in eight countries from three WHO regions (Americas, Europe, and Western Pacific). The exposure was measured using self-reports in all studies, and the outcome was assessed with administrative health records (13 studies), self-reported physician diagnosis (7 studies), direct diagnosis by a physician (1 study) or during a medical interview (1 study). The outcome was defined as an incident non-fatal stroke event in nine studies (7 cohort studies, 2 case-control studies), incident fatal stroke event in one cohort study and incident non-fatal or fatal ("mixed") event in 12 studies (all cohort studies). Cohort studies were judged to have a relatively low risk of bias; therefore, we prioritized evidence from these studies, but synthesised evidence from case-control studies as supporting evidence. For the bodies of evidence for both outcomes with any eligible studies (i.e. stroke incidence and mortality), we did not have serious concerns for risk of bias (at least for the cohort studies). Eligible studies were found on the effects of long working hours on stroke incidence and mortality, but not prevalence. Compared with working 35-40 h/week, we were uncertain about the effect on incidence of stroke due to working 41-48 h/week (relative risk (RR) 1.04, 95% confidence interval (CI) 0.94-1.14, 18 studies, 277,202 participants, I2 0%, low quality of evidence). There may have been an increased risk for acquiring stroke when working 49-54 h/week compared with 35-40 h/week (RR 1.13, 95% CI 1.00-1.28, 17 studies, 275,181participants, I2 0%, p 0.04, moderate quality of evidence). Compared with working 35-40 h/week, working ≥55 h/week may have led to a moderate, clinically meaningful increase in the risk of acquiring stroke, when followed up between one year and 20 years (RR 1.35, 95% CI 1.13 to 1.61, 7 studies, 162,644 participants, I2 3%, moderate quality of evidence). Compared with working 35-40 h/week, we were very uncertain about the effect on dying (mortality) of stroke due to working 41-48 h/week (RR 1.01, 95% CI 0.91-1.12, 12 studies, 265,937 participants, I2 0%, low quality of evidence), 49-54 h/week (RR 1.13, 95% CI 0.99-1.29, 11 studies, 256,129 participants, I2 0%, low quality of evidence) and 55 h/week (RR 1.08, 95% CI 0.89-1.31, 10 studies, 664,647 participants, I2 20%, low quality of evidence). Subgroup analyses found no evidence for differences by WHO region, age, sex, socioeconomic status and type of stroke. Sensitivity analyses found no differences by outcome definition (exclusively non-fatal or fatal versus "mixed") except for the comparison working ≥55 h/week versus 35-40 h/week for stroke incidence (p for subgroup differences: 0.05), risk of bias ("high"/"probably high" ratings in any domain versus "low"/"probably low" in all domains), effect estimate measures (risk versus hazard versus odds ratios) and comparator (exact versus approximate definition). CONCLUSIONS: We judged the existing bodies of evidence for human evidence as "inadequate evidence for harmfulness" for all exposure categories for stroke prevalence and mortality and for exposure to 41-48 h/week for stroke incidence. Evidence on exposure to 48-54 h/week and ≥55 h/week was judged as "limited evidence for harmfulness" and "sufficient evidence for harmfulness" for stroke incidence, respectively. Producing estimates for the burden of stroke attributable to exposures to working 48-54 and ≥55 h/week appears evidence-based, and the pooled effect estimates presented in this systematic review could be used as input data for the WHO/ILO Joint Estimates. PROTOCOL IDENTIFIER: https://doi.org/10.1016/j.envint.2018.06.016. PROSPERO REGISTRATION NUMBER: CRD42017060124.

WHO/ILO work-related burden of disease and injury: Protocol for systematic reviews of exposure to long working hours and of the effect of exposure to long working hours on stroke.

BACKGROUND: The World Health Organization (WHO) and the International Labour Organization (ILO) are developing a joint methodology for estimating the national and global work-related burden of disease and injury (WHO/ILO joint methodology), with contributions from a large network of experts. In this paper, we present the protocol for two systematic reviews of parameters for estimating the number of deaths and disability-adjusted life years from stroke attributable to exposure to long working hours, to inform the development of the WHO/ILO joint methodology. OBJECTIVES: We aim to systematically review studies on occupational exposure to long working hours (called Systematic Review 1 in the protocol) and systematically review and meta-analyse estimates of the effect of long working hours on stroke (called Systematic Review 2), applying the Navigation Guide systematic review methodology as an organizing framework, conducting both systematic reviews in tandem and in a harmonized way. DATA SOURCES: Separately for Systematic Reviews 1 and 2, we will search electronic academic databases for potentially relevant records from published and unpublished studies, including Medline, EMBASE, Web of Science, CISDOC and PsychINFO. We will also search electronic grey literature databases, Internet search engines and organizational websites; hand-search reference list of previous systematic reviews and included study records; and consult additional experts. STUDY ELIGIBILITY AND CRITERIA: We will include working-age (≥15 years) workers in the formal and informal economy in any WHO and/or ILO Member State, but exclude children (<15 years) and unpaid domestic workers. For Systematic Review 1, we will include quantitative prevalence studies of relevant levels of occupational exposure to long working hours (i.e. 35-40, 41-48, 49-54 and ≥55 h/week) stratified by country, sex, age and industrial sector or occupation, in the years 2005-2018. For Systematic Review 2, we will include randomized controlled trials, cohort studies, case-control studies and other non-randomized intervention studies with an estimate of the relative effect of a relevant level of long working hours on the incidence of or mortality due to stroke, compared with the theoretical minimum risk exposure level (i.e. 35-40 h/week). STUDY APPRAISAL AND SYNTHESIS METHODS: At least two review authors will independently screen titles and abstracts against the eligibility criteria at a first stage and full texts of potentially eligible records at a second stage, followed by extraction of data from qualifying studies. At least two review authors will assess risk of bias and the quality of evidence, using the most suited tools currently available. For Systematic Review 2, if feasible, we will combine relative risks using meta-analysis. We will report results using the guidelines for accurate and transparent health estimates reporting (GATHER) for Systematic Review 1 and the preferred reporting items for systematic reviews and meta-analyses guidelines (PRISMA) for Systematic Review 2. PROSPERO registration number: CRD42017060124.