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Aim: To investigate the role of the c-Jun amino terminal kinase (JNK) signalling pathway in mouse models of Aristolochic acid (AA)-induced acute and chronic kidney injury. Background(s): AA is a toxin that damages tubular epithelial cells of the kidney and is the cause of Chinese Herb Nephropathy and Balkan Nephropathy. Exposure of cultured tubular epithelial cells to AA induces a pro-fibrotic response via the JNK pathway. Method(s): In the acute model, groups (n = 7-8) of mice received a single injection of 5 mg/kg AA and were treated with vehicle, JNK inhibitor (CC-930) at 75 mg/kg/BID or untreated from day 0 until being killed on day 3. In the chronic model, mice (n = 10) received 2 mg/kg AA every second day from day 0 to day 22, and were treated with vehicle or CC-930 for the entire period. Result(s): In the acute model, CC-930 treatment substantially inhibited JNK signalling and gave significant protection from AA-induced renal function impairment (normal 8.3 +/- 1.5; untreated 33.4 +/- 4.1; vehicle 35.0 +/- 5.8; CC- 930 23.3 +/- 2.1 mumol/L serum creatinine: P < 0.001) and tubular cell damage (KIM-1 and NGAL mRNA levels; P < 0.001). This protection was associated with reduced macrophage infiltration and reduced expression of proinflammatory molecules (TNF, IL-36A). In the chronic model, CC-930 treatment significantly inhibited JNK signalling, but did not affect AA-induced renal function impairment, tubular cell damage or renal fibrosis, despite a significant reduction in the macrophage pro-inflammatory response. Conclusion(s): Blockade of JNK signalling with CC-930 suppressed the acute effects of AA on tubular cell damage and renal function impairment, but was unable to protect the kidney against the effects of chronic AA exposure.

Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification., QC 20221102

Glycosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs). The glycan pattern can have a large impact on the immunological functions, serum half-life and stability. The medium components and cultivation parameters are known to potentially influence the glycosylation profile. Mathematical modelling provides a strategy for rational design and control of the upstream bioprocess. However, the kinetic models usually contain a very large number of unknown parameters, which limit their practical applications. In this article, we consider the metabolic network of N-linked glycosylation as a Bayesian network (BN) and calculate the fluxes of the glycosylation process as joint probability using the culture parameters as inputs. The modelling approach is validated with data of different Chinese hamster ovary cell cultures in pseudo perfusion, perfusion, and fed batch cultures, all showing very good predictive capacities. In cases where a large number of cultivation parameters is available, it is shown here that principal components analysis can efficiently be employed for a dimension reduction of the inputs compared to Pearson correlation analysis and feature importance by decision tree. The present study demonstrates that BN model can be a powerful tool in upstream process and medium development for glycoprotein productions., QC 20220221

Single cardiomyocytes are widely used for investigations of the cellular and molecular mechanisms of regulation and modulation of cardiac performance. Intact cardiomyocytes allow one to study in detail cell function avoiding the effects of extracellular matrix and neighboring cells. The most established protocols of cardiomyocyte isolation are based on the isolated heart perfusion using a Langendorff-apparatus or on intraventricular perfusion using a syringe. However, the yield of single cardiomyocytes obtained by these methods may be low due to the cell injury following non-uniform enzyme digestion of connective tissue in different heart chambers. Moreover, isolation of atrial cardiomyocytes is challenging because of their small size and complex geometric shape. Here we present a new protocol for simultaneous isolation of high quality cardiomyocytes from the atria, ventricular free walls and interventricular septum. The protocol is based on the combination of the Langendorff perfusion method with the intraventricular and intra-atrial injection technique taking into account the collagen content variation between the different heart chambers. Obtained cells demonstrate rod-shaped morphology, a clear and regular sarcomere striation pattern and rat-specific frequency-dependence of contraction and calcium transient parameters. Our protocol provides gentle cell isolation that increases the yield of single cardiomyocytes suitable for biophysical researches. © 2020 The Authors

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc, resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure. Copyright: © 2021, American Society for Clinical Investigation.

Oil and gas exploration in the Arctic can result in the release of polycyclic aromatic hydrocarbons (PAHs) into relatively pristine environments. Following the recent spill of approximately 17 500 tonnes of diesel fuel in Norilsk, Russia, May 2020, our study focussed on the effects of phenanthrene, a low molecular weight PAH found in diesel and crude oil, on the isolated atrial and ventricular myocytes from the heart of the polar teleost, the Navaga cod (Eleginus nawaga). Acute exposure to phenanthrene in navaga cardiomyocytes caused significant action potential (AP) prolongation, confirming the proarrhythmic effects of this pollutant. We show AP prolongation was due to potent inhibition of the main repolarising current, IKr, with an IC50 value of ~2 µM. We also show a potent inhibitory effect (~55%) of 1 µM phenanthrene on the transient IKr currents that protects the heart from early-after-depolarizations and arrhythmias. These data, along with more minor effects on inward sodium (INa) (~17% inhibition at 10 µM) and calcium (ICa) (~17% inhibition at 30 µM) currents, and no effects on inward rectifier (IK1 and IKAch) currents, demonstrate the cardiotoxic effects exerted by phenanthrene on the atrium and ventricle of navaga cod. Moreover, we report the first data that we are aware of on the impact of phenanthrene on atrial myocyte function in any fish species. © 2021 The Authors

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical. © 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH

The purpose of this study was to evaluate the structure and material properties of native mandibular bone and those of early regenerate bone, produced by bone transport distraction osteogenesis. Ten adult foxhounds were divided into two groups of five animals each. In all animals, a 3- to 4-cm defect was created on one side of the mandible. A bone transport reconstruction plate, consisting of a reconstruction plate with an attached intraoral transport unit, was utilized to stabilize the mandible and regenerate bone at a rate of 1 mm/day. After the distraction period was finished, the animals were killed at 6 and 12 weeks of consolidation. Micro-computed tomography was used to assess the morphometric and structural indices of regenerate bone and matching bone from the unoperated contralateral side. Significant new bone was formed within the defect in the 6- and 12-week groups. Significant differences (P = 0.05) between mandibular regenerated and native bone were found in regard to bone volume fraction, mineral density, bone surface ratio, trabecular thickness, trabecular separation, and connectivity density, which increased from 12 to 18 weeks of consolidation. We showed that regenerated bone is still mineralizing and that native bone appears denser because of a thick outer layer of cortical bone that is not yet formed in the regenerate. However, the regenerate showed a significantly higher number of thicker trabeculae. © 2011 Springer Science+Business Media, LLC.

The purpose of this study was to evaluate the structure and material properties of native mandibular bone and those of early regenerate bone, produced by bone transport distraction osteogenesis. Ten adult foxhounds were divided into two groups of five animals each. In all animals, a 3- to 4-cm defect was created on one side of the mandible. A bone transport reconstruction plate, consisting of a reconstruction plate with an attached intraoral transport unit, was utilized to stabilize the mandible and regenerate bone at a rate of 1 mm/day. After the distraction period was finished, the animals were killed at 6 and 12 weeks of consolidation. Micro-computed tomography was used to assess the morphometric and structural indices of regenerate bone and matching bone from the unoperated contralateral side. Significant new bone was formed within the defect in the 6- and 12-week groups. Significant differences (P = 0.05) between mandibular regenerated and native bone were found in regard to bone volume fraction, mineral density, bone surface ratio, trabecular thickness, trabecular separation, and connectivity density, which increased from 12 to 18 weeks of consolidation. We showed that regenerated bone is still mineralizing and that native bone appears denser because of a thick outer layer of cortical bone that is not yet formed in the regenerate. However, the regenerate showed a significantly higher number of thicker trabeculae. © 2011 Springer Science+Business Media, LLC.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of shortand middle chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD.

Spherical nanoparticles (NPs) of cadmium and lead sulfides (diameter 37 ± 5 and 24 ± 4 nm, respectively) have been found to be cytotoxic for HL-1 cardiomyocytes as evidenced by decrease in adenosine triphosphate–dependent luminescence. Cadmium sulfide (CdS)-NPs were discovered to produce a much greater cytotoxic impact than lead sulphide (PbS)-NP. Given the same dose range, CdS-NP reduced the number of calcium spikes. A similar effect was observed for small doses of PbS-NP. In addition to cell hypertrophy under the impact of certain doses of CdS-NP and PbS-NP, doses causing cardiomyocyte size reduction were identified. For these 3 outcomes, we obtained both monotonic “dose–response” functions (well approximated by the hyperbolic function) and different variants of non-monotonic ones for which we found adequate mathematical expressions by modifying certain models of hormesis available in the literature. Data analysis using a response surface linear model with a cross-term provided new support to the previously established postulate that a diversity of types of joint action characteristic of one and the same pair of damaging agents is one of the important assertions of the general theory of combined toxicity. © The Author(s) 2020.

A series of 1-(2,3-dihydro-1H-indan-1-yl)-3-aryl urea/thiourea derivatives (4a-j) have been synthesized from the reaction of 2,3-dihydro-1H-inden-1-amine (2) with various aryl isocyanates/isothiocyanates (3a-j) by using N,N-DIPEA base (Hunig's base) catalyst in THF at reflux conditions. All of them are structurally confirmed by spectral (IR, 1H & 13C NMR and MASS) and elemental analysis and screened for their in-vitro antioxidant activity against DPPH and NO free radicals and found that compounds 4b, 4i, 4h & 4g are potential antioxidants. The obtained in vitro results were compared with the molecular docking, ADMET, QSAR and bioactivity study results performed for them and identified that the recorded in silico binding affinities were observed in good correlation with the in vitro antioxidant results. The Molecular docking analysis had unveiled the strong hydrogen bonding interactions of synthesized ligands with ARG 160 residue of protein tyrosine kinase (2HCK) enzyme and plays an effective role in its inhibition. Toxicology studies have assessed the potential risks of 4a-j and inferred that all of them were in the limits of potential drugs. The conformational analysis of 4a-j inferred that the urea/thiourea spacer linking 2,3-dihydro-1H-inden-1-amino and substituted aryl units has facilitated all these molecules to effectively bind with ARG 160 amino acid residue present on the α-helix of the protein tyrosine kinase (2HCK) enzyme specifically on chain A of hemopoetic cell kinase. Collectively this study has established a relationship between the antioxidant potentiality and ligands binding with ARG 160 amino acid residue of chain A of 2HCK enzyme to inhibit its growth as well as proliferation of reactive oxygen species in vivo. © 2019 Elsevier Inc.

Background: Myelofibrosis is caused by constitutive activation of the JAK/STAT pathway. JAK inhibitors reduce spleen volume and improve symptoms, but disease progression through JAK inhibition underscores the need for therapies with distinct modes of action. Bomedemstat (IMG-7289) is an inhibitor of lysine-specific demethylase-1 (LSD1), an epigenetic regulator critical for self-renewal of malignant myeloid cells and for the differentiation of myeloid progenitors. LSD1 with GFI1b licenses maturation of progenitors to megakaryocytes and enables their normal function. In mouse models of MPNs (MplW515L, JAK2V617F), LSD1 inhibition improved peripheral cell counts, spleen volumes, inflammatory cytokines, mutant allele frequencies, and marrow fibrosis (Jutzi et al. 2018). Aim(s): This multi-center, open-label Phase 2 study evaluated the safety and pharmacodynamics of bomedemstat administered orally once-daily in adult patients with high or intermediate-2 risk MF who were resistant to or intolerant of ruxolitinib. Method(s): The primary objectives were safety, PK and spleen volume (SV) reduction. Other endpoints included changes in total symptom scores (TSS), bone marrow (BM) fibrosis and inflammatory cytokines. Key inclusion criteria included platelet count >=100K/muL. Patients in the doserange finding study (N = 18) were treated daily for 12 weeks followed by a washout period of up to 28 days. Patients in the Phase 2b study (N = 16) were treated daily for 24 weeks. Abdominal imaging to measure SV was performed after every 12 weeks of treatment. The MPN10 responses were submitted at each clinic visit. Dosing was tailored using platelet count as a marker of bomedemstat activity on megakaryocyte function. Phase 2a patients started at the presumed sub-therapeutic dose of 0.25 mg/kg/d and were up-titrated weekly until the platelet count rested between 50 and 100K/muL. Phase 2b patients started at 0.5 mg/kg/d and were up-titrated until platelet counts rested between 50 and 75K

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory molecule with both cytokine and noncytokine activity. MIF is constitutively released from multiple cell types via an unconventional secretory pathway that is not well defined. Here, we looked at MIF release from human and mouse monocytes/macrophages in response to different stimuli. While MIF release was not significantly altered in response to lipopolysaccharide or heat-killed Escherichia coli, cytotoxic stimuli strongly promoted release of MIF. MIF release was highly upregulated in cells undergoing necrosis, necroptosis and NLRP3 inflammasome-dependent pyroptosis. Our data suggest that cell death represents a major route for MIF release from myeloid cells. The functional significance of these findings and their potential importance in the context of autoimmune and inflammatory diseases warrant further investigation.Copyright © 2020 Australian and New Zealand Society for Immunology Inc.

Background & Aims: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. Method(s): C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. Result(s): hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. Conclusion(s): We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.Copyright © 2020

Macrophage-mediated renal injury promotes the development of diabetic nephropathy. Blockade of chemokine (C-C motif) receptor 2 (CCR2) inhibits kidney macrophage accumulation and early glomerular damage in diabetic animals. This study tested early and late interventions with a CCR2 antagonist (CCR2A) in a model of progressive diabetic glomerulosclerosis and determined whether CCR2A provides added benefit over conventional treatment with an angiotensin-converting enzyme inhibitor (ACEi). Diabetes was induced in hypertensive endothelial nitric oxide synthase (Nos3)-deficient mice by administration of five low-dose streptozotocin (STZ) injections daily. Groups of diabetic Nos3- /- mice received a CCR2A (30 mg.kg- 1.day- 1 PF-04634817 in chow) as an early intervention (weeks 2-15 after STZ). The late intervention (weeks 8-15 after STZ) involved PF-04634817 alone, ACEi (captopril in water 10 mg.kg- 1. day- 1) alone, or combined ACEi + CCR2A. Control diabetic and nondiabetic Nos3- /- mice received normal chow and water. Early intervention with a CCR2A inhibited kidney inflammation and glomerulosclerosis, albuminuria, podocyte loss, and renal function impairment but not hypertension in diabetic Nos3- /- mice. Late intervention with a CCR2A also inhibited kidney inflammation, glomerulosclerosis, and renal dysfunction but did not affect albuminuria. ACEi alone suppressed hypertension and albuminuria and partially reduced podocyte loss and glomerulosclerosis but did not affect renal dysfunction. Compared with ACEi alone, the combined late intervention with ACEi + CCR2A provided better protection against kidney damage (inflammation, glomerulosclerosis, and renal function impairment) but not albuminuria. In conclusion, this study demonstrates that combining CCR2A and ACEi provides broader and superior renal protection than ACEi alone in a model of established diabetic glomerulosclerosis with hypertension.Copyright © 2019 the American Physiological Society

Background: Accumulating evidence suggests that dysregulation of neutrophil extracellular traps (NETs) could be associated with the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Peptidyl arginine deiminases (PADs) are enzymes that generate citrullinated proteins, which are pro-inflammatory in both innate and adaptive immune responses. Citrullination of histone 3 (H3Cit) by PAD2 and PAD4 facilitates generation of both macrophage extracellular traps (METs) and NETs. BB-Clamidine is a second generation pan (PAD) inhibitor which reduces citrullination of H3Cit and subsequent NET formation. This study investigates the contribution of NETs in the pathogenesis of experimental anti-myeloperoxidase ANCA associated glomerulonephritis (MPO-ANCA GN) and the therapeutic possibility of BB-Cl amidine disrupting NET formation in vivo, and attenuating anti-MPO GN. Method(s): Experimental anti-MPO GN was induced in C57BL/6 mice by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin. BB-Cl-amidine treatment (1mg/kg/daily, n=6 or vehicle, n=7) was administered to disrupt NET formation after the establishment of MPO autoimmunity until termination of the experiment. Result(s): NET accumulation identified by co-localisation of extracellular DNA, H3Cit, PAD4, and MPO, was prominent in glomeruli of untreated (vehicle) mice compared to BB-Cl-amidine treated mice (2.1 +/- 0.4 vs 0.4 +/- 0.1, P=0.004). Histological assessment of kidneys demonstrated prominent glomerular segmental necrosis in the untreated group versus a reduction in mice receiving BB-Cl-amidine (88 +/- 3 vs 41 +/- 3, P=0.004). BBCl-amidine significantly attenuated glomerular leukocyte infiltration; CD4 T cells (1.2 +/- 0.3 vs 0.3 +/- 0.1, P=0.03), macrophages (1 +/- 0.1 vs 0.4 +/- 0.06, P=0.008) and neutrophils (3 +/- 0.5 vs 1.6 +/-0.3, P=0.03), compared to untreated mice. BB-Cl-amidine treated mice had increased numbers of CD

Background: Antiglomerular basement membrane (anti-GBM) disease is associated with HLA-DRB1*1501 (the major predisposing genetic factor in the disease), with alpha3127-148 as a nephritogenic T and B cell epitope. Although the cause of disease remains unclear, the association of infections with anti-GBM disease hasbeenlong suspected. Method(s): To investigate whether microbesmight activate autoreactive T and B lymphocytes via molecular mimicry in anti-GBM disease, we used bioinformatic tools, including BLAST, SYFPEITHI, and ABCpred, for peptide searching and epitope prediction. We used sera from patients with anti-GBM disease to assess peptides recognized by antibodies, and immunized WKY rats and a humanized mouse model (HLA-DR15 transgenic mice) with each of the peptide candidates to assess pathogenicity. Result(s): Onthe basis of the criticalmotif, the bioinformatic approach identified 36 microbial peptides thatmimic human alpha3127-148. Circulating antibodies in sera from patients with anti-GBM recognized nine of them. One peptide, B7, derived from Actinomyces species, induced proteinuria, linear IgG deposition on the GBM, and crescent formation when injected into WKY rats. The antibodies to B7 also targeted human and rat alpha3127-148. B7 induced T cell activation from human alpha3127-148-immunized rats. T cell responses to B7 were detected in rats immunized by Actinomyces lysate proteins or recombinant proteins. We confirmed B7's pathogenicity in HLADR15 transgenic mice that developed kidney injury similar to that observed in a3135-145-immunized mice. Conclusion(s): Sera from patients with anti-GBM disease recognizedmicrobial peptides identified through a bioinformatic approach, and a peptide fromActinomycesinduced experimental anti-GBMGNbyTandBcell crossreactivity. These studies demonstrate that anti-GBM disease may be initiated by immunization with a microbial peptide.Copyright © 2020 by the American Society of Nephrology.

Background: Marked podocyte depletion is an established key feature of glomerulosclerosis (FSGS). However, little is known about the consequences of mild podocyte loss. In addition, activation of parietal epithelial cells (PECs) has been proposed as a major effector in FSGS. This study investigates the consequences of graded podocyte depletion, the hypertrophic response of podocytes and associations with PEC activation and thereby glomerulosclerosis. Method(s): We induced selective podocyte depletion in PodCreiDTR mice by injection of diphtheria toxin (DT) at different doses. L-NAME induced hypertension was used as a second hit challenge after mild podocyte loss. The mammalian target of rapamycin (mTOR) signalling pathway was manipulated using mTOR inhibitors (RAD001 and INK128). Podocyte depletion and hypertrophy were examined by 3D analysis of whole glomeruli in optically-cleared kidney slices. Result(s): PodCreiDTR mice injected with a low dose of DT presented mild podocyte depletion, compensatory podocyte hypertrophy and reversible albuminuria without PEC activation or glomerulosclerosis, even following a second hit challenge (high blood pressure), suggesting a protective role of podocyte hypertrophy. Injection of a higher dose of DT in PodCreiDTR mice led to greater podocyte loss and hypertrophy. However, these mice showed PEC activation, glomerulosclerosis and persistent albuminuria, suggesting there is a limit for the protective role of podocyte hypertrophy. Pharmacological inhibition of mTOR during the induction of mild podocyte depletion led to persistent and exacerbated albuminuria, impairment of podocyte hypertrophy, PEC activation and glomerulosclerosis. Conclusion(s): Podocyte hypertrophy via mTOR signalling is required for the adaptive hypertrophic response of remaining podocytes after mild podocyte depletion. These results are relevant for the use of mTOR inhibitors in the context of FSGS and CKD.

Interleukin (IL)-37 is a member of the IL-1 family of cytokines. Although its broad anti-inflammatory properties are well described, the effects of IL-37 on inflammasome function remain poorly understood. Performing gene expression analyses, ASC oligomerization/speck assays and caspase-1 assays in bone marrow-derived macrophages (BMDM), and employing an in vivo endotoxemia model, we studied how IL-37 affects the expression and maturation of IL-1beta and IL-18, inflammasome activation, and pyroptosis in detail. IL-37 inhibited IL-1beta production by NLRP3 and AIM2 inflammasomes, and IL-18 production by the NLRP3 inflammasome. This inhibition was partially attributable to effects on gene expression: whereas IL-37 did not affect lipopolysaccharide (LPS)-induced mRNA expression of Il18 or inflammasome components, IL-37-transgenic BMDM displayed an up to 83% inhibition of baseline and LPS-stimulated Il1b compared to their wild-type counterparts. Importantly, we observed that IL-37 suppresses nigericin-and silica-induced ASC oligomerization/speck formation (a step in inflammasome activation and subsequent caspase-1 activation), and pyroptosis (-50%). In mice subjected to endotoxemia, IL-37 inhibited plasma IL-1beta (-78% compared to wild-type animals) and IL-18 (-61%). Thus, our study adds suppression of inflammasome activity to the portfolio of anti-inflammatory pathways employed by IL-37, highlighting this cytokine as a potential tool for treating inflammasome-driven diseases.Copyright © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Background: Anti-cytokine monoclonal antibody (mAb) therapies have been effective in many autoimmune diseases but have not been successfully trialled in MPO-ANCA associated vasculitis. This study assessed the efficacy of blocking key CD4+ Th1 subset signature effector cytokines, TNFalpha and IFNgamma in MPO-ANCA GN. Assessing therapeutic efficacy of these anti-cytokine mAbs is now complicated by our recent discovery that Th subset dominance during the development of MPO autoimmunity is biphasic with initial transient Th17 dominance followed by persistent Th1 responses. Method(s): Anti-MPO autoimmunity was induced in C57BL/6 mice by MPO immunization and GN triggered using anti-GBM Ig during early and late development of anti-MPO autoimmunity, and GN assessed 4 days later (days 20 and 32, respectively). mAb treatment began 4hrs post GN triggering. Result(s): Administration of anti-TNFalpha mAb early in anti-MPO development (day 20) had no effect on kidney injury compared with vehicle treated controls: albuminuria [5.7+/-1.7 vs 5.5+/-1.7 mg/24hr, P=0.9]; glomerular segmental necrosis [GSN: 50+/-4 vs 43+/-3, P=0.1]. Similarly, anti-IFNgamma mAb was ineffective in attenuating GN at this timepoint [GSN: 48+/-6 vs 45+/-3, P=0.53]. Failure of these treatments is concordant with our observation that early developing anti-MPO autoimmunity is Th17 dominant. In contrast, anti-TNFalpha therapy during established anti-MPO GN (day 32) markedly attenuated kidney injury [GSN: 27+/-2 vs 50+/-4, P=0.01]. TNFalpha blockade acts locally in the kidney as systemic MPO specific IFNgamma and IL-17 recall responses from lymph nodes draining MPO immunization sites were similar to vehicle treated controls. Neutralizing IFNgamma at this late timepoint induced a phenotypic switch from Th1 responses to a protective Th2 with increased in serum MPO-ANCA [1.19+/-0.25 vs 0.48+/-0.13 OD450nm, P=0.02], increased IL-4 production from MPO challenged LN cells and increased the proportion of activated M2 m

Background & Aims: Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. Method(s): We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5moKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. Result(s): Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P <.01), and correlated with gastritis severity (P <.05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P <.05). After 3 months of infection with H felis, Nlrc5mo-KO mice developed gastric hyperplasia (P <.0001), splenomegaly (P <.0001), and increased serum antibody titers (P <.01), whereas control mice did not. Nlrc5mo-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P <.0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein

Background: Tubular activation of the c-Jun amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including renal ischaemia/reperfusion (I/R) injury. Both Jnk1 and Jnk2 genes are expressed in most cells of the kidney resulting in considerable redundancy. Combined blockade of JNK1/2 is known to be protective in renal I/R injury; however, the relative contribution of each isoform is unknown. The aim of this study is to determine the relative contribution of JNK1 versus JNK2 in renal I/R injury. Method(s): Preferential pharmacological inhibition of JNK1 (CC90001) was compared to mice with global deletion of Jnk1 or Jnk2 and mice with combined global Jnk2 deletion plus Jnk1 deletion in proximal tubular cells (Jnk-PT mice). Bilateral warm renal ischaemia injury was induced with vascular clamps and animals killed 24hr after reperfusion. Controls were sham operated. Sprague-Dawley rats (n=8-10/group) were treated 3 times with CC-90001 (10mg/kg) or vehicle starting 1 hour prior to surgery. Renal I/R injury studies were also performed in Jnk1-/- (n=10), Jnk2-/-(n=8), Jnk PT (n=8) and wild type (WT) mice (n=10). Result(s): Treatment with CC-90001 provided significant protection in rat I/R injury (8+/-0.5 vs 20+/-2 fold increase in serum creatinine (sCR), drug versus vehicle, respectively; P<0.001). In a separate study, Jnk1-/- mice also showed significant protection from I/R injury compared to WT mice (4+/-0.5 vs 8+/-1 fold increase in sCr respectively; p=0.01); however, this was not replicated in Jnk2-/-mice (16+/-0.5 vs 15+/-2 fold increase in sCr, Jnk2-/-versus WT, respectively; p=NS). Jnk-PT mice exposed to renal I/R injury showed significant protection from injury compared to WT mice (5.5+/-0.7 vs 15+/-2 fold increase in sCr respectively p<0.01). CC-90001 treatment reduced tubular damage and macrophage infiltration. This protection was replicated in Jnk1-/- and Jnk-PT mice. Conclusion(s): Using complementary ap

Background & Aim: Background: Chronic liver disease is characterised by progressive hepatocyte injury, inflammation and accumulation of extracellular matrix. Liver progenitor cells (LPC) are bipotent cells induced during liver injury that can regenerate both hepatocytes and cholangiocytes. In addition, macrophages play a pivotal role in fibrogenesis and fibrosis resolution. Classically, macrophages are classified into M1 (pro-inflammatory macrophages) and M2 (pro-resolution macrophages). Recently, Ly6C expression has been used to identify distinct macrophage populations, Ly6Chi (inflammatory) and Ly6Clow (resolution). We are developing a therapy for chronic liver fibrosis using human amnion epithelial cells (hAECs). The choline-deficient ethionine-supplemented (CDE) diet is a non-invasive and quick method to induce liver injury and LPC proliferation. Here, we aimed to investigate the effect of hAECs on macrophages and LPC during liver injury induced by a CDE diet. Aim(s): Methods, Results & Conclusion Methods: C57/B16J mice were commenced on a CDE diet, then 2 or 4 million hAECs were infused intraperitoneally 24h later and the CDE diet continued. Outcomes were assessed at days 1, 3, 5, 14, 21 and 42 following hAEC administrations. Sirius red staining and immunohistochemistry were used to determine fibrosis and identify LPC in liver sections. Macrophages and LPC were further identified and characterised using flow cytometery sorting and PCR. Result(s): In CDE treated mice, hAEC significantly reduced liver fibrosis compared to untreated mice. hAEC decreased the number of proinflammatory (Ly6Chi) monocyte-derived macrophages (MDM) and increased the number of anti-inflammatory and tissue remodelling (Ly6Clow) MDM. hAEC also enhanced the expression of matrix-metalloproteinase (MMP)12 and MMP13 in Ly6CLow sorted cells. LPC numbers were reduced significantly in hAEC treated mice. Conclusion(s): Our data confirm the potential efficacy of hAECs as an effective therapy in liv

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of b2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased a3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.Copyright © 2020 by The American Association of Immunologists, Inc.

IgA nephropathy (IgAN), the most common primary glomerular disorder, has a relatively poor prognosis yet lacks a pathogenesisbased treatment. Compound K (CK) is a major absorbable intestinal bacterial metabolite of ginsenosides, which are bioactive components of ginseng. The present study revealed promising therapeutic effects of CK in two complementary IgAN models: a passively induced one developed by repeated injections of IgA immune complexes and a spontaneously occurring model of spontaneous grouped ddY mice. The potential mechanism for CK includes 1) inhibiting the activation of NLRP3 inflammasome in renal tissues, macrophages and bone marrow-derived dendritic cells, 2) enhancing the induction of autophagy through increased SIRT1 expression, and 3) eliciting autophagy-mediated NLRP3 inflammasome inhibition. The results support CK as a drug candidate for IgAN.Copyright © 2020 by The American Association of Immunologists, Inc.

Background: Podocyte depletion is a common feature of glomerulosclerosis (FSGS), but its role in crescentic nephritis remains unclear. This study combined genetic tagging of podocytes with three different optical clearing techniques to determine podocyte depletion in whole glomeruli from mice with crescentic nephritis. Method(s): Podocyte nuclei were labeled by eGFP-histone in of adult male Pod-rtTA/ H2B-eGFP mice by oral doxycycline, followed by a 7-day wash out period, and a single intra-peritoneal injection of nephrotoxic serum (NTS; 5mg/g). Experimental mice were killed 10 days after NTS injection, and compared to age-matched controls. Kidney slices were optically cleared with SCALE-A4, CLARITY and Ethyl Cinnamate (ECi). Highresolution serial optical images were obtained by confocal and two-photon microscopy. Result(s): Mean podocyte number per mouse showed very low variability within controls (1-5% variability, P>0.05) and within NTS-injected mice (1-9% variability, P>0.05) independent of the clearing technique. In NTS-injected mice, a similar degree of average podocyte depletion per mouse was identified with all clearing methods (60-63%, P<0.001). The technical (dis-)advantages of each clearing protocol were also analysed, including optimal penetration depth and resolution, compatibility with immunofluorescence, microscopy set-ups, and cost-efficiency. Importantly, total podocyte number per glomerulus showed great variability: controls (mean: 78,81; ranging from 49 to 128 podocytes per glomerulus) and in NTS-injected mice (mean: 29,99; ranging from 1 to 95 podocytes per glomerulus). Using the lowest value for podocyte number in controls as a cut-off reference, only 78% of analysed glomeruli (141 of 180) from NTS-injected mice had a certain degree of podocyte depletion. While deposition of the NTS within glomeruli occurred in a global and homogeneous fashion, podocyte loss was focal. Conclusion(s): This study has identified early focal podocyte depletion in mic

Macrophages are professional phagocytes that display remarkable plasticity, with a range of phenotypes that can be broadly characterized by the M1/M2 dichotomy. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a protein known to mediate anti-inflammatory and some pro-resolving actions, including as neutrophil apoptosis. However, the role of GILZ in key macrophage function is not well understood. Here, we investigated the role of GILZ on macrophage reprogramming and efferocytosis. Using murine bone-marrow-derived macrophages (BMDMs), we found that GILZ was expressed in naive BMDMs and exhibited increased expression in M2-like macrophages (IL4-differentiated). M1-like macrophages (IFN/LPS-differentiated) from GILZ-/- mice showed higher expression of the M1 markers CD86, MHC class II, iNOS, IL-6 and TNF-alpha, associated with increased levels of phosphorylated STAT1 and lower IL-10 levels, compared to M1-differentiated cells from WT mice. There were no changes in the M2 markers CD206 and arginase-1 in macrophages from GILZ-/- mice differentiated with IL-4, compared to cells from WT animals. Treatment of M1-like macrophages with TAT-GILZ, a cell-permeable GILZ fusion protein, decreased the levels of CD86 and MHC class II in M1-like macrophages without modifying CD206 levels in M2-like macrophages. In line with the in vitro data, increased numbers of M1-like macrophages were found into the pleural cavity of GILZ-/- mice after LPS-injection, compared to WT mice. Moreover, efferocytosis was defective in the context of GILZ deficiency, both in vitro and in vivo. Conversely, treatment of LPS-injected mice with TAT-GILZ promoted inflammation resolution, associated with lower numbers of M1-like macrophages and increased efferocytosis. Collectively, these data indicate that GILZ is a regulator of important macrophage functions, contributing to macrophage reprogramming and efferocytosis, both key steps for the resolution of inflammation.Copyright © 2020 Elsevier Ltd

Cyclophilin A (CypA) is a highly abundant protein in the cytoplasm of most mammalian cells. Beyond its homeostatic role in protein folding, CypA is a Damage-Associated Molecular Pattern which can promote inflammation during tissue injury. However, the role of CypA in kidney disease is largely unknown. This study investigates the contribution of CypA in two different types of kidney injury: acute tubular necrosis and progressive interstitial fibrosis. CypA (Ppia) gene deficient and wild type (WT) littermate controls underwent bilateral renal ischaemia/reperfusion injury (IRI) and were killed 24h later or underwent left unilateral ureteric obstruction (UUO) and were killed 7 days later. In the IRI model, CypA-/- mice showed substantial protection against the loss of renal function and from tubular cell damage and death. This was attributed to a significant reduction in neutrophil and macrophage infiltration since CypA-/- tubular cells were not protected from oxidant-induced cell death in vitro. In the UUO model, CypA-/- mice were not protected from leukocyte infiltration or renal interstitial fibrosis. In conclusion, CypA promotes inflammation and acute kidney injury in renal IRI, but does not contribute to inflammation or interstitial fibrosis in a model of progressive kidney fibrosis.Copyright © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of motor neurons. Between 12 and 20% of inherited cases and approximately 1-2% of all cases are caused by mutations in the gene encoding dismutase 1 (SOD1). Mutant SOD1 A4V (alanine to valine) induces endoplasmic reticulum (ER) stress, which is increasingly implicated as a pathway to motor neuron degeneration and death in ALS. However, it remains unclear how ER stress is induced by mutant SOD1 A4V. Previous studies have established that it is induced early in pathophysiology and it precedes the formation of mutant SOD1 inclusions. SOD1 contains four cysteine residues, two of which form an intra-subunit disulphide bond involving Cys-57 and Cys-146. The remaining two cysteines, Cys-6 and Cys-111, remain unpaired and have been implicated in mutant SOD1 aggregation. In this study, we examined the relationship between the SOD1 A4V cysteine residues and aggregation, ER stress induction and toxicity. We report here that mutation of Cys-6 and Cys-111 in mutant SOD1 A4V, but not Cys-57 or Cys-146, ameliorates ER stress, inclusion formation and apoptosis in neuronal cell lines. These results imply that protein misfolding, induced by Cys-6 and Cys-111, is required for these pathological events in neuronal cells.Copyright © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1 gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/-) or elevated activin A (Inha+/-) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII- correspondingly higher in Inha+/- testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.Copyright © 2020

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2alpha) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2alpha is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2alpha in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window. c 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.Copyright © 2020 American Association for the Advancement of Science. All rights reserved.

Background: Cyclophilin D (CypD) facilitates mitochondrial-dependant cell death during pathological conditions. CypD-/-mice are protected from acute kidney injury (AKI) following ischaemia/reperfusion injury and show reduced renal fibrosis in the unilateral ureteric obstruction model. However, the contribution of CypD in aristolochic acid (AA) induced AKI or AA-induced chronic kidney disease (CKD) is unknown. We aim to determine the role of CypD in: 1) acute AA-induced nephropathy (AAN); and 2) chronic AAN. Method(s): Groups (n=10) of CypD-/-and wild type (WT) C57BL/6J mice were used. Study 1: Mice were given an intraperitoneal (IP) injection of 5mg/kg AA and killed 3 days later. Study 2: Mice were given IP injections of 2mg/kg AA every 2nd day and killed on day 28. Controls were untreated. Result(s): Study 1: Acute high dose AA caused renal failure in WT mice (39.7+/-7.1mmol/L vs 13.5+/-2.3mmol/L serum creatinine (SCr) in controls; P<0.0001) with evidence of tubular damage and cell death on PAS sections and increased cleaved caspase-3+ cells. Acute AAN also caused inflammation with infiltrating neutrophils and T cells and up-regulation of IL-36a mRNA levels. CypD-/-mice were protected from AA-induced acute renal dysfunction (18.9+/-9.5 mmol/L SCr; P<0.0001 vs WT AAN). CypD-/-mice showed reduced tubular damage and cell death on PAS and reduced cleaved caspase-3+ cells (both P<0.001 vs WT AAN), as well as reduced neutrophil infiltration and IL-36alpha mRNA levels (both P<0.001 vs WT AAN). Study 2: Chronic AA administration caused renal impairment in WT mice (34.3+/-9.9mmol/L SCr), with evidence of chronic tubular damage (KIM-1 & a-Klotho mRNA levels), increase in tubular cell death (cleaved caspase-3+ cells), and significant renal fibrosis (increased collagen IV deposition). However, CypD-/-mice were not protected from chronic AA-induced renal dysfunction (37.0+/-14.3 mmol/L SCr; P=NS) and showed no reduction in tubular damage, cell death or renal fibrosis. Conclusio

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, alpha-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGFbeta1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-beta1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.Copyright © 2020 National Academy of Sciences. All rights reserved.

Background: Cyclophilins are a family of enzymes which regulate protein folding. Of these enzymes, cyclophilin D (CypD) facilitates mitochondrial-dependant cell death during pathological conditions. CypD-/-mice are protected from acute kidney injury (AKI) following renal ischaemia/reperfusion injury, and exhibit less renal fibrosis in the unilateral ureteric obstruction model. However, the contribution of CypD in the transition of AKI to chronic kidney disease (CKD) is not known. The aim of this study was to determine the role of CypD in promoting the transition of AKI to CKD using the aristolochic acid-induced nephropathy (AAN) model. Method(s): Groups (n=10) of wild type (WT) and CypD-/-mice on the C57BL/6J background were given intraperitoneal injections of 2mg/kg aristolochic acid every 2nd day for 28 days. Mice were killed on day 28. Controls were untreated. Result(s): AAN caused >2.5-fold rise in serum creatinine (sCr) in WT mice (34.3+/-9.9mmol/L vs 13.5+/-2.3mmol/L in controls; P<0.0001), with evidence of tubular damage (KIM-1 & alpha-Klotho mRNA levels; P<0.0001), increase in tubular cell death (cleaved caspase-3+ cells; P<0.0001), and significant renal fibrosis (collagen IV immunostaining, and collagen I & alpha-SMA mRNA levels; all P<0.0001 vs controls). CypD-/-mice were not protected from AA-induced renal dysfunction (sCr 37.0+/-14.3 mmol/L; P=N.S.), and showed no reduction in tubular damage, cell death or renal fibrosis. Further analysis showed that AAN in WT mice caused loss of peritubular CD31+ capillaries (P<0.0001), and infiltration of macrophages (CD68 & CD206 mRNA levels; P<0.0001) and T-cells (CD3 & IL-2 mRNA levels; P<0.0001). CypD-/-mice were not protected from peritubular capillary loss or macrophage infiltration in AAN. However, CypD-/-mice showed reduced T-cell infiltration and activation (CD3 & IL-2 mRNA levels; P<0.0001), including Th1 cells (T-bet mRNA levels; P<0.05). Conclusion(s): CypD does not contribute to the transition of AKI to CK

Background: Loss of tolerance to MPO in ANCA-associated glomerulonephritis (GN) is poorly understood. It is unknown whether molecular mimicry plays any role in this process. We tested whether microbial peptides homologous to a dominant pathogenic MPO CD4+ T cell epitope (MPO409-428), relevant to several MHCII and lying within an MPO epitope hot spot, would induce anti-MPO autoimmunity. Method(s): Immunity to MPO and MPO409-428 was studied in microbial peptide immunized C57BL/6 (B/6, I-Ab), BALB/c (I-Ad/Ed) and HLA-DR15 transgenic mice (proliferation, IFN-gamma and IL-17A ELISPOT). Anti-MPO GN and anti-MPO immunity in response to peptides, proteins (MPO/ovalbumin as positive/negative controls) and S.aureus strains with/without plasmids carrying the relevant 6-phosphogluconate dehydrogenase (6PGD) sequence were studied in B/6 mice (I-Ab/MPO415-428 tetramers, cytokines, MPOANCA IIF/ELISA, neutrophil ROS production, MPO-ANCA transfer). Anti-6PGD antibodies (Ab) were measured in sera of healthy humans and patients. Result(s): The 4 most homologous microbial peptides were immunogenic but did not induce anti-MPO autoreactivity. However, a plasmid-derived peptide from 6PGD (6PGDp) with similar critical binding residues for MPO409-428 in B/6, BALB/c and DR15+ mice, found in some S.aureus strains, induced expansion of MPO415-428 tetramer+CD4+ cells, anti-MPO T cell autoimmunity (to MPO408-428 and whole MPO) and bioactive MPO-ANCA in B/6 mice. 6PGDp induced anti-MPO autoreactivity in mice with different MHCII (I-Ad/Ed and DR15). Related 6PGD sequences from other S.aureus strains did not induce anti-MPO responses. Healthy human and vasculitis patient sera contained anti-6PGD Ab, demonstrating its immunogenicity in humans. 6PGDp-immunized mice developed GN when MPO was deposited in glomeruli by anti-basement membrane globulin. Immunization with S.aureus containing a plasmid with the mimic 6PGD sequence, or another S.aureus strain transformed with a different plasmid expressing t

Kidney fibrosis is characterized by expansion and activation of platelet-derived growth factor receptor-beta (PDGFR-beta)-positive mesenchymal cells. To study the consequences of PDGFR-beta activation, we developed a model of primary renal fibrosis using transgenic mice with PDGFR-beta activation specifically in renal mesenchymal cells, driving their pathological proliferation and phenotypic switch toward myofibroblasts. This resulted in progressive mesangioproliferative glomerulonephritis, mesangial sclerosis, and interstitial fibrosis with progressive anemia due to loss of erythropoietin production by fibroblasts. Fibrosis induced secondary tubular epithelial injury at later stages, coinciding with microinflammation, and aggravated the progression of hypertensive and obstructive nephropathy. Inhibition of PDGFR activation reversed fibrosis more effectively in the tubulointerstitium compared to glomeruli. Gene expression signatures in mice with PDGFR-beta activation resembled those found in patients. In conclusion, PDGFR-beta activation alone is sufficient to induce progressive renal fibrosis and failure, mimicking key aspects of chronic kidney disease in humans. Our data provide direct proof that fibrosis per se can drive chronic organ damage and establish a model of primary fibrosis allowing specific studies targeting fibrosis progression and regression.Copyright © 2020 The Authors. Published under the terms of the CC BY 4.0 license

Background: Neonatal ventilation exacerbates brain injury in lambs with fetal growth restriction (FGR), characterized by neuroinflammation and reduced blood-brain barrier integrity, which is normally maintained by the neurovascular unit. We examined whether umbilical cord blood stem cell (UCBC) treatment stabilized the neurovascular unit and reduced brain injury in preterm ventilated FGR lambs. Method(s): Surgery was performed in twin-bearing pregnant ewes at 88 days' gestation to induce FGR in one fetus. At 127 days, FGR and appropriate for gestational age (AGA) lambs were delivered, carotid artery flow probes and umbilical lines inserted, lambs intubated and commenced on gentle ventilation. Allogeneic ovine UCBCs (25 x 106 cells/kg) were administered intravenously to lambs at 1 h of life. Lambs were ventilated for 24 h and then euthanized. Result(s): FGR (n = 6) and FGR+UCBC (n = 6) lambs were growth restricted compared to AGA (n = 6) and AGA+UCBC (n = 6) lambs (combined weight, FGR 2.3 +/- 0.4 vs. AGA 3.0 +/- 0.3 kg; p = 0.0002). UCBC therapy did not alter mean arterial blood pressure or carotid blood flow but decreased cerebrovascular resistance in FGR+UCBC lambs. Circulating TNF-alpha cytokine levels were lower in FGR+UCBC vs. FGR lambs (p < 0.05). Brain histopathology showed decreased neuroinflammation and oxidative stress, increased endothelial cell proliferation, pericyte stability, and greater integrity of the neurovascular unit in FGR+UCBC vs. FGR lambs. Conclusion(s): Umbilical cord blood stem cell therapy mitigates perinatal brain injury due to FGR and ventilation, and the neuroprotective benefits may be mediated by stabilization of the neurovascular unit.Copyright © 2020 The Author(s).

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. TGF-beta1/Smad3 signalling plays a major pathological role in DN; however, the contribution of Smad4 has not been examined. Smad4 depletion in the kidney using anti-Smad4 locked nucleic acid halted progressive podocyte damage and glomerulosclerosis in mouse type 2 DN, suggesting a pathogenic role of Smad4 in podocytes. Smad4 is upregulated in human and mouse podocytes during DN. Conditional Smad4 deletion in podocytes protects mice from type 2 DN, independent of obesity. Mechanistically, hyperglycaemia induces Smad4 localization to mitochondria in podocytes, resulting in reduced glycolysis and oxidative phosphorylation and increased production of reactive oxygen species. This operates, in part, via direct binding of Smad4 to the glycolytic enzyme PKM2 and reducing the active tetrameric form of PKM2. In addition, Smad4 interacts with ATPIF1, causing a reduction in ATPIF1 degradation. In conclusion, we have discovered a mitochondrial mechanism by which Smad4 causes diabetic podocyte injury.Copyright © 2020 The Authors