Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity–mediated proteolysis of IκBα resulted in increased NF-κB p65–induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.
Xuefei Tian, Kazunori Inoue, Yan Zhang, Ying Wang, C. John Sperati, Christopher E. Pedigo, Tingting Zhao, Meihua Yan, Marwin Groener, Dennis G. Moledina, Karen Ebenezer, Wei Li, Zhenhai Zhang, Dan A. Liebermann, Lois Greene, Peter Greer, Chirag R. Parikh, Shuta Ishibe
Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type–specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl– cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress–mediated progressive renal tubulointerstitial disease. These observations establish cell type–specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin.
Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly R. Hart, Claire M. Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce A. Molitoris, Kameswaran Surendran, Indra Chandrasekar
Long noncoding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNA-Seq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INflammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDL receptor–deficient (LDLR–/–) mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1β, and COX-2, in endothelial cells (ECs), vascular smooth muscle cells, and bone marrow–derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEP domain containing 4 (DEPDC4), an evolutionary conserved human ortholog of VINAS with approximately 74% homology, showed similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated antiinflammatory effects of VINAS in human ECs. These findings reveal a potentially novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.
Viorel Simion, Haoyang Zhou, Jacob B. Pierce, Dafeng Yang, Stefan Haemmig, Yevgenia Tesmenitsky, Galina Sukhova, Peter H. Stone, Peter Libby, Mark W. Feinberg
Primary varicella-zoster virus (VZV) infection in adults is often complicated by severe pneumonia, which is difficult to treat and is associated with high morbidity and mortality. Here, the simian varicella virus (SVV) nonhuman primate (NHP) model was used to investigate the pathogenesis of varicella pneumonia. SVV infection resulted in transient fever, viremia, and robust virus replication in alveolar pneumocytes and bronchus-associated lymphoid tissue. Clearance of infectious virus from lungs coincided with robust innate immune responses, leading to recruitment of inflammatory cells, mainly neutrophils and lymphocytes, and finally severe acute lung injury. SVV infection caused neutrophil activation and formation of neutrophil extracellular traps (NETs) in vitro and in vivo. Notably, NETs were also detected in lung and blood specimens of varicella pneumonia patients. Lung pathology in the SVV NHP model was associated with dysregulated expression of alveolar epithelial cell tight junction proteins (claudin-2, claudin-10, and claudin-18) and alveolar endothelial adherens junction protein VE-cadherin. Importantly, factors released by activated neutrophils, including NETs, were sufficient to reduce claudin-18 and VE-cadherin expression in NHP lung slice cultures. Collectively, the data indicate that alveolar barrier disruption in varicella pneumonia is associated with NET formation.
Werner J.D. Ouwendijk, Henk-Jan van den Ham, Mark W. Delany, Jeroen J.A. van Kampen, Gijsbert P. van Nierop, Tamana Mehraban, Fatiha Zaaraoui-Boutahar, Wilfred F.J. van IJcken, Judith M.A. van den Brand, Rory D. de Vries, Arno C. Andeweg, Georges M.G.M. Verjans
Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre–mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
Jasmine C. Wong, Pedro A. Perez-Mancera, Tannie Q. Huang, Jangkyung Kim, Joaquim Grego-Bessa, Maria del pilar Alzamora, Scott C. Kogan, Amnon Sharir, Susan H. Keefe, Carolina E. Morales, Denny Schanze, Pau Castel, Kentaro Hirose, Guo N. Huang, Martin Zenker, Dean Sheppard, Ophir D. Klein, David A. Tuveson, Benjamin S. Braun, Kevin Shannon
Chronic kidney disease (CKD) induces the failure of arteriovenous fistulas (AVFs) and promotes the differentiation of vascular adventitial GLI1-positive mesenchymal stem cells (GMCs). However, the roles of GMCs in forming neointima in AVFs remain unknown. GMCs isolated from CKD mice showed increased potential capacity of differentiation into myofibroblast-like cells. Increased activation of expression of PDGFRA and hedgehog (HH) signaling were detected in adventitial cells of AVFs from patients with end-stage kidney disease and CKD mice. PDGFRA was translocated and accumulated in early endosome when sonic hedgehog was overexpressed. In endosome, PDGFRA-mediated activation of TGFB1/SMAD signaling promoted the differentiation of GMCs into myofibroblasts, extracellular matrix deposition, and vascular fibrosis. These responses resulted in neointima formation and AVF failure. KO of Pdgfra or inhibition of HH signaling in GMCs suppressed the differentiation of GMCs into myofibroblasts. In vivo, specific KO of Pdgfra inhibited GMC activation and vascular fibrosis, resulting in suppression of neointima formation and improvement of AVF patency despite CKD. Our findings could yield strategies for maintaining AVF functions.
Ke Song, Ying Qing, Qunying Guo, Eric K. Peden, Changyi Chen, William E. Mitch, Luan Truong, Jizhong Cheng
While the RV144 HIV vaccine trial led to moderately reduced risk of HIV acquisition, emerging data from the HVTN702 trial point to the critical need to reexamine RV144-based correlates of reduced risk of protection. While in RV144, the induction of V2-binding, non-IgA, IgG3 antibody responses with nonneutralizing functions were linked to reduced risk of infection, the interactions between these signatures remain unclear. Thus, here we comprehensively profile the humoral immune response in 300 RV144 vaccinees to decipher the relationships between humoral biomarkers of protection. We found that vaccine-specific IgG1, IgG3, and IgA were highly correlated. However, ratios of IgG1:IgG3:IgA provided insights into subclass/isotype polyclonal functional regulation. For instance, in the absence of high IgG1 levels, IgG3 antibodies exhibited limited functional activity, pointing to IgG3 as a critical contributor, but not sole driver, of effective antiviral humoral immunity. Higher IgA levels were linked to enhanced antibody effector function, including neutrophil phagocytosis (ADNP), complement deposition (ADCD), and antibody-dependent NK degranulation (CD107a), some of which were increased in infected vaccinees in a case/control data set, suggesting that IgA-driven functions compromised immunity. These data highlight the interplay between IgG1, IgG3, and IgA, pointing to the need to profile the relationships between subclass/isotype selection.
Stephanie Fischinger, Sepideh Dolatshahi, Madeleine F. Jennewein, Supachai Rerks-Ngarm, Punnee Pitisuttithum, Sorachai Nitayaphan, Nelson Michael, Sandhya Vasan, Margaret E. Ackerman, Hendrik Streeck, Galit Alter
Canagliflozin (Cana) is an FDA-approved diabetes drug that protects against cardiovascular and kidney diseases. It also inhibits the sodium glucose transporter 2 by blocking renal reuptake and intestinal absorption of glucose. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing Cana at 180 ppm at 7 months of age until their death. Cana extended median survival of male mice by 14%. Cana also increased by 9% the age for 90th percentile survival, with parallel effects seen at each of 3 test sites. Neither the distribution of inferred cause of death nor incidental pathology findings at end-of-life necropsies were altered by Cana. Moreover, although no life span benefits were seen in female mice, Cana led to lower fasting glucose and improved glucose tolerance in both sexes, diminishing fat mass in females only. Therefore, the life span benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in male mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e., slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.
Richard A. Miller, David E. Harrison, David B. Allison, Molly Bogue, Lucas Debarba, Vivian Diaz, Elizabeth Fernandez, Andrzej Galecki, W. Timothy Garvey, Hashan Jayarathne, Navasuja Kumar, Martin A. Javors, Warren C. Ladiges, Francesca Macchiarini, James Nelson, Peter Reifsnyder, Nadia A. Rosenthal, Marianna Sadagurski, Adam B. Salmon, Daniel L. Smith Jr., Jessica M. Snyder, David B. Lombard, Randy Strong
Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions, including nutrient metabolism, intestinal barrier integrity, and immune regulation. Recent studies suggest that education of intestinal immunity may be ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial, and whether microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially derived metabolites can be detected in second trimester human intestinal samples. Although we were unable to amplify bacterial DNA from fetal intestines, we report a fetal metabolomic intestinal profile with an abundance of bacterially derived and host-derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial-associated metabolites either come from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that were below our detection threshold.
Yujia Li, Jessica M. Toothaker, Shira Ben-Simon, Lital Ozeri, Ron Schweitzer, Blake T. McCourt, Collin C. McCourt, Lael Werner, Scott B. Snapper, Dror S. Shouval, Soliman Khatib, Omry Koren, Sameer Agnihorti, George Tseng, Liza Konnikova
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), 2 incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43, which loses its physiological properties, leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here, we tested the target specificity, in vivo distribution, and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43-related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-κB activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge, this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model.
Silvia Pozzi, Philippe Codron, Geneviève Soucy, Laurence Renaud, Pierre Junior Cordeau, Kallol Dutta, Christine Bareil, Jean-Pierre Julien
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