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Hypoxia-inducible factor activation reprograms glucose metabolism and leads to glycogen accumulation in multiple cell types. In this issue of Kidney International, Ito and colleagues demonstrate that pharmacologic inhibition of hypoxia-inducible factor-prolyl hydroxylase domain oxygen sensors in renal epithelial cells enhances glycogen synthesis and protects from subsequent hypoxia and glucose deprivation. In vivo studies advance the concept that renal glycogen metabolism contributes to cytoprotection afforded by pre-ischemic hypoxia-inducible factor-prolyl hydroxylase domain inhibition.
Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.
BACKGROUND - The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors.
METHODS - To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation.
RESULTS - We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and -actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis.
CONCLUSIONS - These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.
Copyright © 2019 by the American Society of Nephrology.
BACKGROUND - Plasminogen activator inhibitor-1 (PAI-1) expression increases extracellular matrix deposition and contributes to interstitial fibrosis in the kidney after injury. While PAI-1 is ubiquitously expressed in the kidney, we hypothesized that interstitial fibrosis is strongly dependent on fibroblast-specific PAI-1 (fbPAI-1).
METHODS - Tenascin C Cre (TNC Cre) and fbPAI-1 knockdown (KD) mice with green fluorescent protein (GFP) expressed within the TNC construct underwent unilateral ureteral obstruction and were sacrificed 10 days later.
RESULTS - GFP+ cells in fbPAI-1 KD mice showed significantly reduced PAI-1 expression. Interstitial fibrosis, measured by Sirius red staining and collagen I western blot, was significantly decreased in fbPAI-1 KD compared with TNC Cre mice. There was no significant difference in transforming growth factor β (TGF-β) expression or its activation between the two groups. However, GFP+ cells from fbPAI-1 KD mice had lower TGF β and connective tissue growth factor (CTGF) expression. The number of fibroblasts was decreased in fbPAI-1 KD compared with TNC Cre mice, correlating with decreased alpha smooth muscle actin (α-SMA) expression and less fibroblast cell proliferation. TNC Cre mice had decreased E-cadherin, a marker of differentiated tubular epithelium, in contrast to preserved expression in fbPAI-1 KD. F4/80-expressing cells, mostly CD11c+/F4/80+ cells, were increased while M1 macrophage markers were decreased in fbPAI-1 KD compared with TNC Cre mice.
CONCLUSION - These findings indicate that fbPAI-1 depletion ameliorates interstitial fibrosis by decreasing fibroblast proliferation in the renal interstitium, with resulting decreased collagen I. This is linked to decreased M1 macrophages and preserved tubular epithelium.
© The Author(s) 2019. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
Basement membranes are an ancient form of animal extracellular matrix. As important structural and functional components of tissues, basement membranes are subject to environmental damage and must be repaired while maintaining functions. Little is known about how basement membranes get repaired. This paucity stems from a lack of suitable models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult DSS becomes incorporated into the basement membrane, promoting its expansion while decreasing its stiffness, which causes morphological changes to the underlying muscles. Remarkably, two days after withdrawal of DSS, the basement membrane is repaired by all measures of analysis. We used this new damage model to determine that repair requires collagen crosslinking and replacement of damaged components. Genetic and biochemical evidence indicates that crosslinking is required to stabilize the newly incorporated repaired Collagen IV rather than to stabilize the damaged Collagen IV. These results suggest that basement membranes are surprisingly dynamic.
© 2019. Published by The Company of Biologists Ltd.
A fiber optic probe-based Raman spectroscopy system using a single laser module with two excitation wavelengths, at 680 and 785 nm, has been developed for measuring the fingerprint and high wavenumber regions using a single detector. This system is simpler and less expensive than previously reported configurations of combined fingerprint and high wavenumber Raman systems, and its probe-based implementation facilitates numerous in vivo applications. The high wavenumber region of the Raman spectrum ranges from 2800-3800 cm-1 and contains valuable information corresponding to the molecular vibrations of proteins, lipids, and water, which is complimentary to the biochemical signatures found in the fingerprint region (800-1800 cm-1), which probes DNA, lipids, and proteins. The efficacy of the system is demonstrated by tracking changes in water content in tissue-mimicking phantoms, where Voigtian decomposition of the high wavenumber water peak revealed a correlation between the water content and type of water-tissue interactions in the samples. This dual wavelength system was then used for in vivo assessment of cervical remodeling during mouse pregnancy, a physiologic process with known changes in tissue hydration. The system shows that Raman spectroscopy is sensitive to changes in collagen content in the fingerprint region and hydration state in the high wavenumber region, which was verified using an ex vivo comparison of wet and dry weight. Simultaneous fingerprint and high wavenumber Raman spectroscopy will allow precise in vivo quantification of tissue water content in the high wavenumber region, paired with the high biochemical specificity of the fingerprint region.
Greater understanding of the determinants of skeletal fragility is highly sought due to the great burden that bone affecting diseases and fractures have on economies, societies and health care systems. Being a complex, hierarchical composite of collagen type-I and non-stoichiometric substituted hydroxyapatite, bone derives toughness from its organic phase. In this study, we tested whether early observations that a strong correlation between bone collagen integrity measured by thermomechanical methods and work to fracture exist in a more general and heterogeneous sampling of the population. Neighboring uniform specimens from an established, highly characterized and previously published collection of human cortical bone samples (femur mid-shaft) were decalcified in EDTA. Fifty-four of the original 62 donors were included (26 male and 28 females; ages 21-101 years; aging, osteoporosis, diabetes and cancer). Following decalcification, bone collagen was tested using hydrothermal isometric tension (HIT) testing in order to measure the collagen's thermal stability (denaturation temperature, T) and network connectivity (maximum rate of isometric tension generation; Max.Slope). We used linear regression and general linear models (GLMs) with several explanatory variables to determine whether relationships between HIT parameters and generally accepted bone quality factors (e.g., cortical porosity, pentosidine content [pen], pyridinoline content [pyd]), age, and measures of fracture toughness (crack initiation fracture toughness, K, and total energy release/dissipation rate evaluated at the point of unstable fast fracture, J-int) were significant. Bone collagen connectivity (Max.Slope) correlated well with the measures of fracture toughness (R = 24-35%), and to a lesser degree with bound water fraction (BW; R = 7.9%) and pore water fraction (PW; R = 9.1%). Significant correlations with age, apparent volumetric bone mineral density (vBMD), and mature enzymatic [pyd] and non-enzymatic collagen crosslinks [pen] were not detected. GLMs found that Max.Slope and vBMD (or BW), with or without age as additional covariate, all significantly explained the variance in Kinit (adjusted-R = 36.7-49.0%). Also, the best-fit model for J-int (adjusted-R = 35.7%) included only age and Max.Slope as explanatory variables with Max.Slope contributing twice as much as age. Max.Slope and BW without age were also significant predictors of J-int (adjusted-R = 35.5%). In conclusion, bone collagen integrity as measured by thermomechanical methods is a key factor in cortical bone fracture toughness. This study further demonstrates that greater attention should be paid to degradation of the overall organic phase, rather than a specific biomarker (e.g. [pen]), when seeking to understand elevated fracture rates in aging and disease.
Copyright © 2018 Elsevier Inc. All rights reserved.
During the dermal wound healing process, the mechanical rigidity of the newly deposited extracellular matrix and transforming growth factor-β1 promote the transition of fibroblasts into myofibroblasts. Myofibroblasts generate large cellular forces that contract and remodel the extracellular matrix leading to scar formation. In contrast, myofibroblasts are not detected in fetal dermal wounds which are more compliant and contain less transforming growth factor-β1 than adult wounds. Instead, fetal fibroblasts orchestrate scarless healing of dermal wounds resulting in healed tissues that resemble uninjured dermis. While these biomechanical differences suggest that the fetal wound environment promotes smaller cellular forces which enable regeneration, previous studies indicate that fetal fibroblasts have unique contractile properties that may facilitate scarless dermal repair. Therefore, we tested whether physiologic wound rigidities and transforming growth factor-β1 induce contractile forces and myofibroblast differentiation of fetal dermal fibroblasts. In comparison to their adult dermal counterparts, we found that fetal fibroblasts exhibit a deficient contractile response to rigid extracellular matrix and transforming growth factor-β1. Our data suggest that the contractile phenotype of fetal dermal fibroblasts limits their cellular force production and prevents their ability to differentiate into myofibroblasts.
© 2018 The Authors. Wound Repair and Regeneration published by Wiley Periodicals, Inc. on behalf of by the Wound Healing Society.
BACKGROUND - Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP.
METHODS - We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score.
RESULTS - We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production . The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease.
CONCLUSIONS - Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.
Copyright © 2018 by the American Society of Nephrology.
Pulmonary fibrosis is a progressive inflammatory disease with high mortality and limited therapeutic options. Previous genetic and immunologic investigations suggest common intersections between idiopathic pulmonary fibrosis (IPF), sarcoidosis, and murine models of pulmonary fibrosis. To identify immune responses that precede collagen deposition, we conducted molecular, immunohistochemical, and flow cytometric analysis of human and murine specimens. Immunohistochemistry revealed programmed cell death-1 (PD-1) up-regulation on IPF lymphocytes. PD-1CD4 T cells with reduced proliferative capacity and increased transforming growth factor-β (TGF-β)/interleukin-17A (IL-17A) expression were detected in IPF, sarcoidosis, and bleomycin CD4 T cells. PD-1 T helper 17 cells are the predominant CD4 T cell subset expressing TGF-β. Coculture of PD-1CD4 T cells with human lung fibroblasts induced collagen-1 production. Strikingly, ex vivo PD-1 pathway blockade resulted in reductions in TGF-β and IL-17A expression from CD4 T cells, with concomitant declines in collagen-1 production from fibroblasts. Molecular analysis demonstrated PD-1 regulation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Chemical blockade of STAT3, using the inhibitor STATTIC, inhibited collagen-1 production. Both bleomycin administration to PD-1 null mice or use of antibody against programmed cell death ligand 1 (PD-L1) demonstrated significantly reduced fibrosis compared to controls. This work identifies a critical, previously unrecognized role for PD-1CD4 T cells in pulmonary fibrosis, supporting the use of readily available therapeutics that directly address interstitial lung disease pathophysiology.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.