A therapeutic target for CKD: activin A facilitates TGFß1 profibrotic signaling.

BACKGROUND: TGFß1 is a major profibrotic mediator in chronic kidney disease (CKD). Its direct inhibition, however, is limited by adverse effects. Inhibition of activins, also members of the TGFß superfamily, blocks TGFß1 profibrotic effects, but the mechanism underlying this and the specific activin(s) involved are unknown. METHODS: Cells were treated with TGFß1 or activins A/B. Activins were inhibited generally with follistatin, or specifically with neutralizing antibodies or type I receptor downregulation. Cytokine levels, signaling and profibrotic responses were assessed with ELISA, immunofluorescence, immunoblotting and promoter luciferase reporters. Wild-type or TGFß1-overexpressing mice with unilateral ureteral obstruction (UUO) were treated with an activin A neutralizing antibody. RESULTS: In primary mesangial cells, TGFß1 induces secretion primarily of activin A, which enables longer-term profibrotic effects by enhancing Smad3 phosphorylation and transcriptional activity. This results from lack of cell refractoriness to activin A, unlike that for TGFß1, and promotion of TGFß type II receptor expression. Activin A also supports transcription through regulating non-canonical MRTF-A activation. TGFß1 additionally induces secretion of activin A, but not B, from tubular cells, and activin A neutralization prevents the TGFß1 profibrotic response in renal fibroblasts. Fibrosis induced by UUO is inhibited by activin A neutralization in wild-type mice. Worsened fibrosis in TGFß1-overexpressing mice is associated with increased renal activin A expression and is inhibited to wild-type levels with activin A neutralization. CONCLUSIONS: Activin A facilitates TGFß1 profibrotic effects through regulation of both canonical (Smad3) and non-canonical (MRTF-A) signaling, suggesting it may be a novel therapeutic target for preventing fibrosis in CKD.

Patient values and preferences in pulmonary embolism testing in the emergency department.

INTRODUCTION: Patient-centered care is concordant with patient values and preferences. There is a lack of research on patient values and preferences for pulmonary embolism (PE) testing in the emergency department (ED), and a poor physician understanding of patient-specific goals. Our aim was to map patient-specific values, preferences, and expectations regarding PE testing in the ED. METHOD: This qualitative study used constructivist grounded theory to identify patient values and expectations around PE testing in the ED. We conducted semi-structured interviews with ED patients who were being tested for PE in two EDs. Patients who were waiting for PE imaging or D-dimer results were approached and consented to take part in a 30-minute audio-recorded interview. Each interview was transcribed verbatim and analyzed using constant comparative coding. The interview script was modified to maximize information on emerging themes. Major themes and subthemes were derived, each representing an opportunity, barrier, or value to address with patient-centered PE testing. RESULTS: From 30 patient interviews, we mapped four major themes: patient satisfaction comes from addressing the patient's primary concern (for example, their pain); patients expect individualized care; patients prefer imaging over clinical examination for PE testing; and patients expect 100% confidence from their emergency physician when given a diagnosis. Subthemes included symptomatic relief, finding a diagnosis, receiving tests, rapid progression through their care, perception of highly accurate CT scans, willingness to seek a second opinion, direct physician communication, and expectation of case-specific testing with cognitive reassurance. CONCLUSION: Addressing each of these four themes by realigning ED processes could provide patient-centered PE testing.

Reliability of patient-reported outcome measures: Hemorrhage, anticoagulant, antiplatelet medication use.

BACKGROUND: Most antithrombotic medication users are older adults. Patient-reported outcome measures are commonly used in clinical research on antithrombotic medication, such as the diagnosis of intracranial hemorrhage. OBJECTIVES: To determine the reliability of patient-reported intracranial hemorrhage, anticoagulant and platelet aggregation inhibitor use in the older adult population. PATIENTS/METHODS: We conducted a secondary analysis of a prospective, observational cohort study of older adults who presented to the emergency department with a fall. The primary outcome was diagnosis of intracranial bleeding. We compared patient-reported intracranial bleeding to structured chart review with adjudication. We also compared patient-reported use of antiplatelet and anticoagulant medication to physician-reported medication use supplemented with structured chart review. We calculated the diagnostic accuracy of the patient-reported outcomes using our comparators as the reference standard. RESULTS: Exact agreement for patient-reported intracranial bleeds was 95%, with a Cohen's kappa of 0.30 (95% confidence interval [CI], 0.15-0.45). The sensitivity was 36.7% (95% CI, 20.6%-56.1%) and specificity 97.2% (95% CI, 95.8%-98.1%). For anticoagulant medication use, exact agreement was 87%, Cohen's kappa 0.66 (95% CI, 0.63-0.72), sensitivity 84.0% (95% CI, 79.3%-83.8%), and specificity 87.6% (95% CI, 85.1%-89.7%). For antiplatelet medication use, exact agreement was 77%, Cohen's kappa 0.50 (95% CI, 0.44-0.55), sensitivity 68.7% (95% CI, 64.0%-73.1%), and specificity 81.2% (95% CI, 78.0-83.8%). CONCLUSIONS: Patient-reported outcome and exposure data were unreliable in this study. Our findings have a bearing on future research study design.

Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.

miR299a-5p promotes renal fibrosis by suppressing the antifibrotic actions of follistatin.

Caveolin-1 (cav-1), an integral protein of the membrane microdomains caveolae, is required for synthesis of matrix proteins by glomerular mesangial cells (MC). Previously, we demonstrated that the antifibrotic protein follistatin (FST) is transcriptionally upregulated in cav-1 knockout MC and that its administration is protective against renal fibrosis. Here, we screened cav-1 wild-type and knockout MC for FST-targeting microRNAs in order to identity novel antifibrotic therapeutic targets. We identified that miR299a-5p was significantly suppressed in cav-1 knockout MC, and this was associated with stabilization of the FST 3'UTR. Overexpression and inhibition studies confirmed the role of miR299a-5p in regulating FST expression. Furthermore, the profibrotic cytokine TGFß1 was found to stimulate the expression of miR299a-5p and, in turn, downregulate FST. Through inhibition of FST, miR299a-5p overexpression augmented, while miR299a-5p inhibition diminished TGFß1 profibrotic responses, whereas miR299a-5p overexpression re-enabled cav-1 knockout MC to respond to TGFß1. In vivo, miR299a-5p was upregulated in the kidneys of mice with chronic kidney disease (CKD). miR299a-5p inhibition protected these mice against renal fibrosis and CKD severity. Our data demonstrate that miR299a-5p is an important post-transcriptional regulator of FST, with its upregulation an important pathogenic contributor to renal fibrosis. Thus, miR299a-5p inhibition offers a potential novel therapeutic approach for CKD.

Giant axonal neuropathy alters the structure of keratin intermediate filaments in human hair.

Giant axonal neuropathy (GAN) follows an autosomal recessive genetic inheritance and impedes the peripheral and central nervous system due to axonal swellings that are packed with neurofilaments. The patients display a number of phenotypes, including hypotonia, muscle weakness, decreased reflexes, ataxia, seizures, intellectual disability, pale skin and often curled hair. We used X-ray diffraction and tensile testing to determine potential changes to the structure of keratin intermediate filaments (IFs) in the hair of patients with GAN. A statistically significant decrease in the 47 and the 27 Å diffraction signals were observed. Tensile tests determined that the hair was slightly stiffer, stronger and more extensible in GAN patients. These results suggest that the structure of keratin IFs in hair is altered in GAN, and the findings are compatible with an increased positional disorder of the keratin tetramers within the hair fibres.

Structural Abnormalities in the Hair of a Patient with a Novel Ribosomopathy.

We report the biophysical characterization of hair from a patient with a de novo ribosomopathy. The patient was diagnosed with a mutation on gene RPS23, which codes for a protein which comprises part of the 40S subunit of the ribosome. The patient presents with a number of phenotypes, including hypotonia, autism, extra teeth, elastic skin, and thin/brittle hair. We combined optical microscopy, tensile tests, and X-ray diffraction experiments on hair samples obtained from the scalp of the patient to a multi-scale characterization of the hair from macroscopic to molecular length scales and observe distinct differences in the biophysical properties in the patient's hair when compared to hair from other family members. While no differences were observed in the coiled-coil structure of the keratin proteins or the structure of the intermediate filaments, the patient's hair was 22% thinner, while the Young's modulus remained roughly constant. The X-ray diffraction results give evidence that the amount of lipids in the cell membrane complex is reduced by 20%, which well accounts for the other observations. The pathologies characterized by these techniques may be used to inform the diagnosis of similar de novo mutations in the future.

Effect of shampoo, conditioner and permanent waving on the molecular structure of human hair.

The hair is a filamentous biomaterial consisting of the cuticle, the cortex and the medulla, all held together by the cell membrane complex. The cortex mostly consists of helical keratin proteins that spiral together to form coiled-coil dimers, intermediate filaments, micro-fibrils and macro-fibrils. We used X-ray diffraction to study hair structure on the molecular level, at length scales between ∼3-90 Å, in hopes of developing a diagnostic method for diseases affecting hair structure allowing for fast and noninvasive screening. However, such an approach can only be successful if common hair treatments do not affect molecular hair structure. We found that a single use of shampoo and conditioner has no effect on packing of keratin molecules, structure of the intermediate filaments or internal lipid composition of the membrane complex. Permanent waving treatments are known to break and reform disulfide linkages in the hair. Single application of a perming product was found to deeply penetrate the hair and reduce the number of keratin coiled-coils and change the structure of the intermediate filaments. Signals related to the coiled-coil structure of the α-keratin molecules at 5 and 9.5 Å were found to be decreased while a signal associated with the organization of the intermediate filaments at 47 Å was significantly elevated in permed hair. Both these observations are related to breaking of the bonds between two coiled-coil keratin dimers.