Research
Abstract
The lymphatic system maintains fluid homeostasis and orchestrates immune cell trafficking throughout tissues. While extensively studied in cancer and lymphedema, its role in non-lymphoid organs, particularly the kidney, remains an emerging area of investigation. Previous research established molecular connections between NF-κB, VEGFR-3, and PROX-1 in regulating lymphatic growth during inflammation, and studies using global knockout mice revealed that the NF-κB1 subunit (p50) influences lymphatic vessel density. However, the role of RelA—a key component of the canonical NF-κB heterodimer—in regulating lymphatic growth and kidney function following acute kidney injury (AKI) remains unexplored. Using an inducible, predominantly lymphatic-specific RelA knockout mouse model, we demonstrate that RelA expression in VEGFR-3+ cells is essential for VEGFR-3 driven lymphangiogenesis following AKI. Knockout mice exhibited significantly worse kidney function, altered histological features, impaired VEGFR-3-dependent lymphangiogenesis, and dysregulated immune cell trafficking. Compensatory upregulation of PROX-1 and podoplanin occurred despite decreased VEGFR-3 and LYVE-1 total protein expression, suggesting complex regulatory mechanisms. Our findings suggest that RelA is a critical sensor for inflammation and regulator of protective lymphangiogenesis following kidney injury and provide insights into potential therapeutic targets for improved kidney injury outcomes.
Authors
Arin L. Melkonian, Amie M. Traylor, Anna A. Zmijewska, Kyle H. Moore, Gelare Ghajar-Rahimi, Stephanie Esman, Yanlin Jiang, Hani Jang, Babak J. Mehrara, Timmy C. Lee, James F. George, Anupam Agarwal
Abstract
Type 2 diabetic nephropathy (T2DN) is a major complication of type 2 diabetes and a leading cause of chronic kidney disease. This study aimed to explore MYO1C as both a candidate biomarker and elucidate its role as a mechanistic mediator of podocyte injury in T2DN. Using urinary extracellular vesicle RNA biomarkers identified from a training and validation cohort of 33 type 2 diabetes and 40 T2DN patients, we developed a machine learning diagnostic model for T2DN. The model achieved an AUC of 0.877 in validation and performed well in an independent test cohort with an AUC of 0.824. MYO1C was identified as the most influential feature in the final model. Mechanistic investigations in vitro and in vivo revealed that high glucose and high-fat conditions induced podocyte injury, inflammation, and apoptosis, with increased MYO1C expression. MYO1C knockdown in vitro and in vivo reduced podocyte damage and inflammatory responses. MYO1C overexpression enhanced p38, p-CREB, and TNF-α levels, while p38 inhibition mitigated these effects. These findings support MYO1C not only as a potential urinary biomarker for T2DN but also as a key pathogenic driver that promotes podocyte injury via p38 MAPK signaling, thereby highlighting its therapeutic promise.
Authors
Zihao Zhao, Qianqian Yan, Sijie Zhou, Fengxun Liu, Yong Liu, Jingjing Ren, Shaokang Pan, Zhenjie Liu, Dongwei Liu, Zhangsuo Liu, Jiayu Duan
Abstract
Loss of bone mass has a devastating effect on quality of life. Higher potassium (K+) intake is positively correlated with bone health. Here, we investigated whether kidney calcium (Ca2+) and phosphate (Pi) handling mechanisms mediate dietary K+ effects. Kidney Ca2+ and Pi handling proteins were altered in abundance in mice fed a 0% K+ diet for 2 weeks. In mice fed a 0.1% K+ diet for 4 or 8 weeks, urinary Ca2+ excretion increased, plasma Ca2+ levels were lower and plasma parathyroid hormone (PTH) levels were higher relative to control 1% K+ fed mice. The 0.1% K+ fed mice had greater excretion of the bone resorption marker deoxypyridinoline, increased osteoclast number, and decreased total femoral bone mineral density. During chronic low K+ intake, major changes in renal Ca2+ and Pi transport pathways were absent, except higher abundances of the sodium-potassium-chloride co-transporter (NKCC2) and the sodium-chloride co-transporter (NCC), in line with their role in kidney Ca2+ handling. Low dietary K+ induced hypocalcemia and changes in PTH were absent in mice with constitutively active NCC, supporting its role in mediating low K+ effects on Ca2+ homeostasis. Our study provides insights into the management of bone disorders in conditions of chronic electrolyte imbalance.
Authors
Sathish K. Murali, Mariavittoria D'Acierno, Xiang Zheng, Lena K. Rosenbaek, Louise N. Odgaard, Paul Richard Grimm, Alice Ramesova, Robert Little, Judith Radloff, Paul A. Welling, Qi Wu, Reinhold G. Erben, Robert A. Fenton
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease. Emerging evidence suggests manifestations beyond the neuromuscular system. Bone alterations are part of the ALS clinical picture; it remains unclear whether they are secondary to muscle denervation or due to an autonomous process. We investigated skeletal involvement in the SOD1(G93A) mouse model at presymptomatic (P45) and symptomatic (P110) stage through biomechanical and transcriptomic approaches. Three-point bending revealed significant reductions in femoral rigidity and maximum bending force in SOD1 mutants at P45, indicating early structural deficits. Micro-CT analysis demonstrated reduced trabecular bone mineral density and thickness at P45, with progressive trabecular loss and cortical thinning by P110. Histological examination revealed marked osteoblast loss at P45 suggesting impaired bone formation as the primary early mechanism. Transcriptomics of bulk bone and cultured osteoblasts from P45 mice identified dysregulation of bone differentiation, including downregulation of osteoblast differentiation genes and upregulation of negative regulators of ossification and increased cell senescence signatures. Unfolded protein response was upregulated in SOD1 osteoblasts. Immunohistochemistry confirmed the senescence phenotype with increased p16Ink4a level in SOD1 osteoblasts. These findings suggest that bone deterioration precede overt motor symptoms and are linked to osteoblast premature senescence.
Authors
Burak Özkan, Jan-Moritz Ramge, Diana Wiesner, Jelena Scekic-Zahirovic, Stefano Antonucci, Sandra Nungeß, Dorothea Gebauer, Anita Ignatius, Jochen H. Weishaupt, Melanie Haffner-Luntzer, Francesco Roselli
Abstract
Disruptions in the integrity of the intestinal epithelium occur commonly in inflammatory bowel diseases (IBD) and critical surgical disorders, but the underlying mechanisms remain largely unknown. Here we identified long noncoding RNA GAS5 as a repressor of intestinal mucosa growth and the function of the gut epithelium barrier. The levels of tissue GAS5 increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from IBD patients. Transient and tissue-specific knockdown of GAS5 in mice using CRISPR-Cas9 enhanced the renewal of the mucosa of the small intestine, increased the levels of tight junction (TJ) proteins ZO-1, ZO-2, claudin-1, and claudin-2, and improved gut barrier function. Conversely, ectopic overexpression of GAS5 in intestinal organoids and in cultured intestinal epithelium cells decreased the levels of these TJ proteins and caused epithelial barrier dysfunction. Mechanistic studies revealed that GAS5 acted as a transcriptional enhancer of the gene encoding small noncoding vault RNAs (vtRNAs) and that GAS5 repressed TJ expression by increasing the levels of vtRNAs. Together, our results indicate that GAS5 disrupts the integrity of the intestinal epithelium by impairing mucosal growth and epithelial barrier function and that it represses TJ expression at least in part via vtRNAs.
Authors
Ting-Xi Yu, Hee Kyoung Chung, Amy VanderStoep, Bridgette Warner, Hongxia Chen, Haonan Zhao, Ana S. G. Cunnigham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang
Abstract
Growing evidence indicates that PKLR, the gene for pyruvate kinase (PK), is a genetic modifier of the sickle cell phenotype. Co-inheritance of specific PKLR variants is associated with increased pain-related hospitalization and can trigger sickle cell disease (SCD) phenotypes in asymptomatic carriers. PK deficiency disrupts RBC glycolysis, leading to ATP deficits and accumulation of 2,3-diphosphoglycerate, which exacerbates sickling in SCD. Using CRISPR-Cas9, we generated null mutations in Pklr [Pklr(13ntdel/13ntdel) or Pklr(246ntdel/246ntdel)] specific for the RBC isoform (PKR) in Townes mice that were homozygous (SS) or heterozygous (AS) for the human sickle globin gene, or homozygous for human hemoglobin A (AA, controls), to investigate the effect of PKR deficiency on the sickle phenotype in mice. PKR-deficient AA and AS mice developed severe anemia, reticulocytosis, and substantial spleen and liver iron deposits. Unlike what is observed in humans, PKR-deficiency in AS and SS mice surprisingly decreased sickling, but it was also associated with increased extramedullary hematopoiesis and mitochondrial retention in mature RBCs. These results demonstrate the differential effect of Pklr mutations on the phenotype of both AS and SS mouse models, offering new insights into the complex role of PKR deficiency in SCD pathology.
Authors
Xunde Wang, Meghann Smith, Sayuri Kamimura, Quan Li, Niharika Shah, Martha Quezado, Luis E.F. Almeida, Sebastian Vogel, Mickias B. Tegegn, Kevin Y. Sun, Rafael Villasmil, Chengyu Liu, William A. Eaton, Swee Lay Thein, Zenaide M.N. Quezado
Abstract
Anoikis resistance or evasion of cell death triggered by matrix detachment is a hallmark of cancer cell survival and metastasis. We show that repeated exposure to suspension stress followed by recovery under attached conditions leads to development of anoikis resistance. The acquisition of anoikis resistance is associated with enhanced invasion, chemoresistance, and immune evasion in vitro and distant metastasis in vivo. This acquired anoikis resistance is not genetic, persisting for a finite duration without detachment stress, but is sensitive to CDK8/19 Mediator kinase inhibition that can also reverse anoikis resistance. Transcriptomic analysis reveals that CDK8/19 kinase inhibition induces bidirectional transcriptional changes in both sensitive and resistant cells, disrupting the balanced reprogramming required for anoikis adaptation and resistance by reversing some resistance associated pathways and enhancing others. Both anoikis resistance and in vivo metastatic growth of ovarian cancers are sensitive to CDK8/19 inhibition, thereby providing a therapeutic opportunity to both prevent and suppress ovarian cancer metastasis.
Authors
Mehri Monavarian, Resha Rajkarnikar, Emily Faith Page, Asha Kumari, Liz Quintero Macias, Felipe Massicano, Nam Y. Lee, Sarthak Sahoo, Nadine Hempel, Mohit Kumar Jolly, Lara Ianov, Elizabeth Worthey, Abhyudai Singh, Igor B. Roninson, Eugenia V. Broude, Mengqian Chen, Karthikeyan Mythreye
Abstract
Commotio retinae (CR) resulting from retinal trauma can lead to focal photoreceptor degeneration and permanent vision loss. Currently no therapies exist for CR-induced retinal degeneration, in part due to a lacking large animal model that replicates human injury pathology and allows testing of therapeutics. Severe CR is clinically characterized by subretinal fluid and focal photoreceptor outer nuclear layer thinning. To develop a porcine CR model, we developed a laser-guided projectile apparatus and optimized projectile delivery procedure using porcine cadaveric eyes embedded in a 3D-printed porcine skull. Scleral and corneal impacts, resulted in retinal damage consistent with patient injury but corneal impacts also led to cornea damage and opacification, which precluded follow up imaging. In live porcine eyes, scleral impacts of 39.5 m/s induced transient blood retinal barrier breakdown evidenced by subretinal fluid on optical coherence tomography (OCT), leakage observed on fluorescein and indocyanine green angiography, and transient photoreceptor outer segment disruption seen by OCT and multifocal electroretinography. Impacts above 39.5 m/s induced longer-lasting photoreceptor degeneration, but only transient blood retinal barrier breakdown. This porcine model, combined with clinically relevant imaging and diagnostic modalities will be valuable for testing the safety and efficacy of therapies to restore vision after focal photoreceptor degeneration.
Authors
Juan Amaral, Irina Bunea, Arvydas Maminishkis, Maria M. Campos, Francesca Barone, Rohan Gupta, Mitra Farnoodian, Jonathan Newport, M. Joseph Phillips, Ruchi Sharma, David M. Gamm, Kapil Bharti, Richard J. Blanch
Abstract
Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that primarily affects the mucosal layer. Previously, we identified autoantibodies against integrin αvβ6 in patients with UC. In this study, we established monoclonal antibodies (mAbs) from patients with UC to reveal the features and functions of these anti-integrin αvβ6 autoantibodies. We identified two shared heavy chain complementarity-determining region (CDR) 3 amino acid sequences among different patients with UC. Notably, several mAbs contained the RGD sequence in their heavy chain CDR3 that mimicked the key recognition sequence of integrin αvβ6 ligands such as fibronectin. Almost all mAbs selectively reacted with integrin αvβ6 in the presence of divalent cations (Ca²⁺ and Mg²⁺) and blocked fibronectin–integrin αvβ6 binding. MAbs that shared the same heavy chain CDR3 amino acid sequence showed differences in reactivity to integrin αvβ6, indicating that the reactivity of these mAbs is also affected by the light chain. Some of the mAbs showed varying degrees of cross-reactivity with integrin αvβ3. The identification of shared CDR3 amino acid sequences in anti-integrin αvβ6 antibodies from several patients with UC suggests a common mechanism underlying their production, which may help elucidate the pathogenesis of UC.
Authors
Masahiro Shiokawa, Yoshihiro Nishikawa, Ikuhisa Takimoto, Takeshi Kuwada, Sakiko Ota, Darryl Joy C. Juntila, Takafumi Yanaidani, Kenji Sawada, Ayako Hirata, Muneji Yasuda, Koki Chikugo, Risa Nakanishi, Masataka Yokode, Yuya Muramoto, Shimpei Matsumoto, Tomoaki Matsumori, Tsutomu Chiba, Hiroshi Seno
Abstract
Cardiomyocyte growth is tightly controlled by multiple signaling pathways. Identification of master kinases in this process is essential in exploring potential targets for the treatment of pathological cardiac hypertrophy and heart failure. Here we identified the mTOR-independent activation of ribosomal protein S6 kinase b1 (Rps6kb1/S6K1) during cardiomyocyte growth. By utilizing phosphoproteomics in primary neonatal rat ventricular myocytes (NRVMs), we revealed Rps6kb1 as one of most activated kinases under growth stimulation. We further demonstrated the role of Rps6kb1 phosphorylation in pathological cardiac hypertrophy and heart failure. We showed that the phosphorylation of multiple sites at Rps6kb1, including T367 in the kinase domain and S418/T421/S424 in the C-terminal domain, is not directly regulated by the activity of mTOR, rather coupled with the activation of the MEK1-ERK axis. In mice, cardiomyocyte-specific deletion of Rps6kb1 significantly inhibited both constitutively active ERK- and pressure overload-induced cardiac hypertrophy. In contrast, cardiomyocyte-specific overexpression of wild-type Rps6kb1, rather than the phosphorylation-defective mutant, elevated cardiac hypertrophy and augmented pressure overload-induced heart failure. In conclusion, our findings reveal that the MEK-ERK axis primes Rps6kb1 activation through phosphorylation of two separate domains of Rps6kb1, which may play an essential role in cardiac hypertrophy and heart failure under hemodynamic stress.
Authors
Chao Li, Pengfei Zhang, Kai Zhang, Jane A. Cook, Weidan Song, Megan Virostek, Lily A. Slotabec, Nadiyeh Rouhi, Mohammed Hazari, Michael I. Adenawoola, Xiaofei Liu, Hao Zhang, Guangyu Zhang, Erica L Niewold, Qinfeng Li, Yong Fang, Waleed M. Elhelaly, Xue-Nan Sun, Xuejiang Guo, Andrew Lemoff, Yingfeng Deng, Thomas G. Gillette, Ji Li, Philipp E. Scherer, Zhao V. Wang
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