Current anemia therapies for patients with non-dialysis-dependent CKD may require injection and medical visits. Roxadustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor, stimulates ...erythropoiesis and improves iron homeostasis.
In this double-blind phase 3 study, we randomized patients with non-dialysis-dependent CKD stages 3-5 and hemoglobin <10.0 g/dl (1:1) to thrice-weekly 70-mg oral roxadustat or placebo. Doses were titrated throughout the study based on hemoglobin levels. The primary efficacy end point was mean change from baseline in hemoglobin averaged over weeks 28-52 versus placebo, irrespective of rescue therapy use. We assessed patients for adverse events.
The study included 2781 patients, 1393 who received roxadustat and 1388 who received placebo. Mean baseline hemoglobin was 9.1 g/dl for both groups. The mean change in hemoglobin from baseline was 1.75 g/dl (95% confidence interval 95% CI, 1.68 to 1.81) with roxadustat versus 0.40 g/dl (95% CI, 0.33 to 0.47) with placebo, (
<0.001). Among 411 patients with baseline elevated high-sensitivity C-reactive protein, mean change in hemoglobin from baseline was 1.75 g/dl (95% CI, 1.58 to 1.92) with roxadustat versus 0.62 g/dl (95% CI, 0.44 to 0.80) with placebo, (
<0.001). Roxadustat reduced the risk of red blood cell transfusion by 63% (hazard ratio, 0.37; 95% CI, 0.30 to 0.44). The most common adverse events with roxadustat and placebo, respectively, were ESKD (21.0% versus 20.5%), urinary tract infection (12.8% versus 8.0%), pneumonia (11.9% versus 9.4%), and hypertension (11.5% versus 9.1%).
Roxadustat effectively increased hemoglobin in patients with non-dialysis-dependent CKD and reduced the need for red blood cell transfusion, with an adverse event profile comparable to that of placebo.
Safety and Efficacy Study of Roxadustat to Treat Anemia in Patients With CKD, Not on Dialysis, NCT02174627.
Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder associated with vascular dysfunction and fibrotic changes in the skin, vasculature and internal organs. Although serologic ...abnormalities are an important diagnostic tool for SSc, little is known about whether autoantibodies precede clinical diagnosis. Here we investigated the presence of autoantibodies before SSc diagnosis and assessed whether certain autoantibodies might associate with the future onset of scleroderma renal crisis (SRC), a potentially fatal complication of the disease. Using the Department of Defense Serum Repository, autoantibodies were analyzed from archived, prospectively collected, longitudinal serum samples from sixteen individuals with SRC (SSc/SRC) and thirty cases of SSc without SRC (SSc/no SRC), matched for age, sex, and race. Seventy five percent (12/16) of the SSc/SRC and 40% (12/30) of the SSc/no SRC were seropositive for at least one autoantibody prior to clinical diagnosis (up to 27.1 years earlier, mean = -7.4 years). Although both disease groups demonstrated a heterogeneous immunoreactivity profile against the autoantigen panel, the SSc/SRC subjects showed two enriched clusters with one featuring elevated levels of autoantibodies against Ro52 and/or Ro60 and another with high levels of immunoreactivity against the RNA polymerase complex. Consistent with larger spectrum of immunoreactivity and the elevated levels of autoantibodies in SSc/SRC, the total response against the autoantigen panel from the last time point of the seropositive subjects revealed that the SSc/SRC cohort harbored higher antibody levels (p = 0.02) compared to SSc/no SRC. Overall, our findings demonstrate that relevant seropositive autoantibodies often precede the clinical diagnosis of SSc/no SRC and SSc/SRC.
The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer ...processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.
Enteropathogenic Escherichia coli (EPEC) use a needle-like injection apparatus known as the type III secretion system (T3SS) to deliver protein effectors into host cells. Effector translocation is ...highly stratified in EPEC with the translocated intimin receptor (Tir) being the first effector delivered into the host. CesT is a multi-cargo chaperone that is required for the secretion of Tir and at least 9 other effectors. However, the structural and mechanistic basis for differential effector recognition by CesT remains unclear. Here, we delineated the minimal CesT-binding region on Tir to residues 35-77 and determined the 2.74 Å structure of CesT bound to an N-terminal fragment of Tir. Our structure revealed that the CesT-binding region in the N-terminus of Tir contains an additional conserved sequence, distinct from the known chaperone-binding β-motif, that we termed the CesT-extension motif because it extends the β-sheet core of CesT. This motif is also present in the C-terminus of Tir that we confirmed to be a unique second CesT-binding region. Point mutations that disrupt CesT-binding to the N- or C-terminus of Tir revealed that the newly identified carboxy-terminal CesT-binding region was required for efficient Tir translocation into HeLa cells and pedestal formation. Furthermore, the CesT-extension motif was identified in the N-terminal region of NleH1, NleH2, and EspZ, and mutations that disrupt this motif reduced translocation of these effectors, and in some cases, overall effector stability, thus validating the universality of this CesT-extension motif. The presence of two CesT-binding regions in Tir, along with the presence of the CesT-extension motif in other highly translocated effectors, may contribute to differential cargo recognition by CesT.
Circulating serum autoantibodies against the M-type phospholipase A2 receptor (PLA2R-AB) are a key biomarker in the diagnosis and monitoring of primary membranous nephropathy (MN). However, little is ...known about the appearance and trajectory of PLA2R-AB before the clinical diagnosis of MN.
Using the Department of Defense Serum Repository, we analyzed PLA2R-AB in multiple, 1054 longitudinal serum samples collected before diagnosis of MN from 134 individuals with primary MN, 35 individuals with secondary MN, and 134 healthy volunteers. We evaluated the presence and timing of non-nephrotic range proteinuria (NNRP) and serum albumin measurements in relation to PLA2R-AB status.
Analysis of PLA2R-AB in longitudinal serum samples revealed seropositivity in 44% (59 out of 134) of primary MN cases, 3% (one out of 35) of secondary MN cases, and in 0% of healthy controls. Among patients with MN, PLA2R-AB were detectable at a median of 274 days before renal biopsy diagnosis (interquartile range, 71-821 days). Approximately one third of the participants became seropositive within 3 months of MN diagnosis. Of the 21 individuals with documented prediagnostic NNRP, 43% (nine out of 21) were seropositive before NNRP was first documented and 28.5% (six out of 21) were seropositive at the same time as NNRP; 66% (39 out of 59) of those seropositive for PLA2R-AB had hypoalbuminemia present at the time antibody was initially detected. Twelve participants (20%) were seropositive before hypoalbuminemia became apparent, and eight participants (14%) were seropositive after hypoalbuminemia became apparent.
Circulating PLA2R-AB are detectable months to years before documented NNRP and biopsy-proven diagnosis in patients with MN.
Iron deficiency is common in heart failure and associated with worse outcomes. We examined the prevalence and consequences of iron deficiency in the DAPA-HF trial (Dapagliflozin and Prevention of ...Adverse-Outcomes in Heart Failure) and the effect of dapagliflozin on markers of iron metabolism. We also analyzed the effect of dapagliflozin on outcomes, according to iron status at baseline.
Iron deficiency was defined as a ferritin level <100 ng/mL or a transferrin saturation <20% and a ferritin level 100 to 299 ng/mL. Additional biomarkers of iron metabolism, including soluble transferrin receptor, erythropoietin, and hepcidin were measured at baseline and 12 months after randomization. The primary outcome was a composite of worsening heart failure (hospitalization or urgent visit requiring intravenous therapy) or cardiovascular death.
Of the 4744 patients randomized in DAPA-HF, 3009 had ferritin and transferrin saturation measurements available at baseline, and 1314 of these participants (43.7%) were iron deficient. The rate of the primary outcome was higher in patients with iron deficiency (16.6 per 100 person-years) compared with those without (10.4 per 100 person-years;
<0.0001). The effect of dapagliflozin on the primary outcome was consistent in iron-deficient compared with iron-replete patients (hazard ratio, 0.74 95% CI, 0.58-0.92 versus 0.81 95% CI, 0.63-1.03;
-interaction=0.59). Similar findings were observed for cardiovascular death, heart failure hospitalization, and all-cause mortality. Transferrin saturation, ferritin, and hepcidin were reduced and total iron-binding capacity and soluble transferrin receptor increased with dapagliflozin compared with placebo.
Iron deficiency was common in DAPA-HF and associated with worse outcomes. Dapagliflozin appeared to increase iron use but improved outcomes, irrespective of iron status at baseline.
URL: https://www.
gov; Unique identifier: NCT03036124.
Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require ...the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.
Glomerular filtration rate (GFR) decline is used as surrogate endpoint for kidney failure. Interventions that reduce chronic kidney disease (CKD) progression often exert acute GFR reductions which ...differ from their long-term benefits and complicate the estimation of long-term benefit. Here, we assessed the utility of two alternative trial designs (wash-out design and active run-in randomized withdrawal design) that attempt to exclude the impact of acute effects. Post-hoc analyses of two clinical trials that characterized the effect of an intervention with acute reductions in GFR were conducted. The two trials included a wash-out period (EMPA-REG Outcome testing empagliflozin vs placebo) or an active run-in period with a randomized withdrawal (SONAR testing atrasentan vs placebo). We compared the drug effect on GFR decline calculated from the first on-treatment visit to the end of treatment (chronic slope in a standard randomized trial design) with GFR change calculated from randomization to end of wash out, or GFR change from treatment-specific baseline GFR values (GFR at start-of-run-in for placebo and end-of-run-in for atrasentan) until end-of-treatment. The effect of empagliflozin versus placebo on chronic GFR slope was 1.72 (95% confidence interval 1.49-1.94) mL/min/1.73 m2/year, similar to total GFR decline from baseline to the end of wash-out period using a linear mixed model 1.64 (1.44-1.85) mL/min/1.73 m2/year). The effect of atrasentan versus placebo on chronic GFR slope was 0.72 (0.32-1.11) mL/min/1.73 m2/year, similar to total slope from a single slope model when estimated from treatment specific baseline GFR values 0.77 (0.39-1.14) mL/min/1.73 m2/year). Statistical power of the two designs outperformed the standard randomized design. Thus, wash-out and active-run-in randomized-withdrawal trial designs are appropriate models to compute treatment effects on GFR decline.
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Introduction
Limitations in physical functioning are common in patients with late-stage chronic kidney disease (CKD) and can greatly affect their lives. Using patient interviews, this study reports ...experiences associated with physical functioning limitations for patients with late-stage CKD.
Methods
A preliminary conceptual model on concepts relevant to physical functioning limitations in patients with CKD was developed using data from a targeted literature review (patients with CKD stages IV–V) and previous interviews (patients with CKD stages IIIa–IIIb). The preliminary conceptual model informed a semi-structured interview guide designed to capture experiences of physical functioning limitations in patients with CKD. Patients with CKD stages IV–V who were not receiving dialysis were interviewed; their responses were used to develop a comprehensive conceptual model summarizing their experiences associated with physical functioning limitations.
Results
A total of 25 patients with CKD stage IV (
n
= 19) or V (
n
= 6) were interviewed. Based on patient responses, the reported concepts were grouped into one of six categories: physical functioning limitations/difficulties, behavioural impacts, activity participation restrictions, symptoms attributed to physical functioning limitations, impacts on sleep and emotional functioning impacts related to physical functioning limitations. Twenty-three patients reported concepts associated with physical functioning limitations, most frequently ‘walking up and down stairs’ (83%) and ‘walking distances’ (74%). All 23 patients also reported behavioural impacts, including ‘need to rest/subsequent periods of rest’ (100%) and ‘participation in fewer activities’ (91%). As well as summarizing the reported concepts, the comprehensive conceptual model shows how concepts may relate to one another; for example, challenging symptoms or difficulty completing tasks can lead to changes in patient behaviour such as purposely reducing or avoiding activities.
Conclusions
This study found that patients with late-stage CKD not receiving dialysis who experience physical functioning limitations report a range of impacts on their daily lives. The comprehensive conceptual model summarizes the concepts reported and the relationships between them, providing a holistic understanding of how patients with late-stage CKD are affected by physical functioning limitations.
Infographic available for this article.
Infographic
The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This ...modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.
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