Chronic kidney disease (CKD) is associated with systemic phosphate elevations, called hyperphosphatemia. Translational studies have shown that hyperphosphatemia contributes to CKD-associated inflammation and injury in various tissues, including the kidney, heart, liver, and parathyroid gland. Mechanisms underlying pathologic actions of elevated phosphate on cells are not well understood but seem to involve uptake of phosphate through sodium phosphate cotransporters and phosphate-induced signaling via FGFR1 (fibroblast growth factor receptor 1). Clinical studies indicate patients with CKD are more likely to develop inflammatory and restrictive lung diseases, such as fibrotic interstitial lung diseases, and here we aimed to determine whether hyperphosphatemia can cause lung injury. We found that a mouse model of CKD and hyperphosphatemia, induced by an adenine-rich diet, develops lung fibrosis and inflammation. Elevation of systemic phosphate concentration by administration of a high-phosphate diet in a mouse model of primary lung inflammation and fibrosis, induced by bleomycin, exacerbated lung injury in the absence of kidney damage. Our in vitro studies identified increases of proinflammatory cytokines in human lung fibroblasts exposed to phosphate elevations. Phosphate activated ERK 1/2 (extracellular signal-related kinase 1/2) and PKB/AKT (protein kinase B) signaling, and pharmacological inhibition of ERK, AKT, FGFR1, or sodium phosphate cotransporters prevented phosphate-induced proinflammatory cytokine upregulation. In addition, inhibition of FGFR1 or sodium phosphate cotransporters decreased the phosphate-induced activation of ERK and AKT. Our study suggests that phosphate can directly target lung fibroblasts and induce an inflammatory response and that hyperphosphatemia in CKD and non-CKD models contributes to lung injury. Phosphate-lowering strategies might protect from CKD-associated lung injury.

Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 (Col4a3−/−). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency (kl/kl). We found that adenine, Col4a3−/−, and kl/kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis.MethodsWe characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation.ResultsWe found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition.ConclusionOGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogenactivated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

An elevation in serum phosphate-also called hyperphosphatemia-is associated with reduced kidney function in chronic kidney disease (CKD). Reports show CKD patients are more likely to develop lung disease and have poorer kidney function that positively correlates with pulmonary obstruction. However, the underlying mechanisms are not well understood. Here, we report that two murine models of CKD, which both exhibit increased serum levels of phosphate and fibroblast growth factor (FGF) 23, a regulator of phosphate homeostasis, develop concomitant airway inflammation. Our in vitro studies point towards a similar increase of phosphate-induced inflammatory markers in human bronchial epithelial cells. FGF23 stimulation alone does not induce a proinflammatory response in the non-COPD bronchial epithelium and phosphate does not cause endogenous FGF23 release. Upregulation of the phosphate-induced proinflammatory cytokines is accompanied by activation of the extracellular-signal regulated kinase (ERK) pathway. Moreover, the addition of cigarette smoke extract (CSE) during phosphate treatments exacerbates inflammation as well as ERK activation, whereas co-treatment with FGF23 attenuates both the phosphate as well as the combined phosphate- and CS-induced inflammatory response, independent of ERK activation. Together, these data demonstrate a novel pathway that potentially explains pathological kidney-lung crosstalk with phosphate as a key mediator.

Fibroblast growth factors (FGFs) and their cognate receptors (FGFRs) are important biological molecules with a wide array of pleiotropic functions [...].

Background and Objectives: Chronic inflammation due to Pseudomonas aeruginosa (PA) infection in people with cystic fibrosis (CF) remains a concerning issue in the wake of modulator therapy initiation. Given the perpetuating cycle of colonization, infection, chronic inflammation, and recurrent injury to the lung, there are increases in the risk for mortality in the CF population. We have previously shown that fibroblast growth factor (FGF) 23 can exaggerate transforming growth factor (TGF) beta-mediated bronchial inflammation in CF. Our study aims to shed light on whether FGF23 signaling also plays a role in PA infection of the CF bronchial epithelium. Materials and Methods: CF bronchial epithelial cells were pretreated with FGF23 or inhibitors for FGF receptors (FGFR) and then infected with different PA isolates. After infection, immunoblot analyses were performed on these samples to assess the levels of phosphorylated phospholipase C gamma (PLCγ), total PLCγ, phosphorylated extracellular signal-regulated kinase (ERK), and total ERK. Additionally, the expression of FGFRs and interleukins at the transcript level (RT-qPCR), as well as production of interleukin (IL)-6 and IL-8 at the protein level (ELISA) were determined. Results: Although there were decreases in isoform-specific FGFRs with increases in interleukins at the mRNA level as well as phosphorylated PLCγ and the production of IL-8 protein with PA infection, treatment with FGF23 or FGFR blockade did not alter downstream targets such as IL-6 and IL-8. Conclusions: FGF23 signaling does not seem to modulate the PA-mediated inflammatory response of the CF bronchial epithelium.

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1-6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 10(2) CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro "prolonged PA infection model" recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

Background: Despite $90 billion in preclinical research and clinical trials every year, 90% of cancer clinical trials are unsuccessful. Thus, there is considerable interest in predicting clinical efficacy of preclinical formulations early in discovery using patient derived ex vivo platforms. However, extracting adequate tissue for such models can be difficult depending on tumor type and site. Dominant mechanisms for expanding patient tissue include xenografts and organoids. However, the former imposes a large time window to establish while the latter is constrained in space by sizescales. Here, we present a novel approach for expanding TNBC tissue, specifically for use in ex vivo models, that addresses these constrains with 3D bioprinted organoid tumors (BOTs). Objective: The aim of this study is to generate TNBC BOTs that mimic core biopsy tissue for use in ex vivo precision and personalized predictive biomarkers for chemotherapies. Methods: BOTs were generated using alginate-based bioink prepared with MDA-MB-231 TNBC cells. Briefly, specially prepared fresh TNBC bioink was deposited layer-by-layer using a Cellink BIO X6 bioprinter in geometrical configurations to mimic 14- to 18- gauge tumor biopsies. These were chemically cross-linked and cured in stages to allow cells and matrix to self-assemble with limited degrees of freedom. Fully cured BOTs were loaded in our ex vivo solid tumor biopsy-on-a-chip and treated with chemotherapy agents to evaluate sensitivity and resistance, with outcomes determined using immunofluorescent live and dead cell staining methods. Results: A 3-minute crosslinking time with calcium chloride provided a stable and functional sodium alginate medium for treating cellular BOTs. Two layers of TNBC bioink was determined to be the optimal size and shape for compatibility with our ex vivo biopsy-on-a-chip predictive efficacy biomarker platform. TNBC cells were verified to be evenly distributed within the cured sodium alginate matrix using live cell nuclear stains. Successful diffusion of multiple agents to spatially distinct regions of the bioprinted tissue was verified with fluorescently labeled small molecules and nucleic acid stains up to 200 µM deep. Impact: Patient derived BOT core mimics and other configurations could be used in ex vivo breast cancer chemotherapy screening models to obtain sensitivity and resistance profiles as predictive functional biomarkers both on the bedside for personalized treatment strategy development and on the bench to uncover new therapeutic targets. Due to their potential to replicate biophysical and biochemical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine platforms can provide more accurate drug efficacy readout compared to in vitro cancer models. Citation Format: Seth Bollenbecker, Zeelu Patel, Zeenia Punjani, Areesha Charania, Heli Patel, Alyssa Abbott, Kaitlyn Kunkle, Mary Kathryn Sewell-Loftin, Gregory Grossman, Karim Budhwani. Predictive efficacy biomarker for chemotherapy agents against triple-negative breast cancer bioprinted organoid tumors (BOTs) using solid tumor biopsy-on-a-chip [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-38.

Idiopathic pulmonary fibrosis (IPF) is the most common andmost severe form of pulmonary fibrosis, a scarring disease of the lungs resulting in progressive lung stiffness and hypoxemia with a poor prognosis (1). Pulmonary fibrosis remains the most common indication for lung transplantation despite the emergence of antifibrotic medications. Both of the available antifibrotic medications used in IPF, pirfenidone and nintedanib, have been shown to include fibroblast growth factor (FGF) signaling pathways as their targets. FGFs comprise a family of 22 known growth factor ligands that act in an autocrine, paracrine, or endocrine manner to signal through four known FGF receptors (2). There is evidence that pirfenidone alters FGF2 expression in mouse lungs in response to bleomycin (3), a commonly used agent to induce pulmonary fibrosis in rodents. Nintedanib is a receptor tyrosine kinase inhibitor that primarily inhibits platelet-derived growth factor, vascular endothelial growth factor, and FGF receptors (4). While FGFs and their activated signaling pathways have been implicated in the pathogenesis of pulmonary fibrosis, their specific roles, as well as whether they act as pro or antifibrotic growth factors, remain topics of active investigation. Early preclinical data suggest that FGFs are integral to the development of pulmonary fibrosis, as inhibition of FGF receptors reduced fibrosis in preclinical models of pulmonary fibrosis (5). There has been mounting evidence, however, that FGFs may play a protective role in pulmonary fibrosis. Overexpression or direct administration of the autocrine and paracrine FGFs, including FGF1, 2, 7, 9, 10, and 18, reduce fibrotic changes in both the bleomycin and Ad-TGFb1 models of pulmonary fibrosis (6–10). The endocrine FGFs (FGF21 and FGF23) are also capable of reducing experimental pulmonary fibrosis (11, 12). In this issue of the Journal, Justet and colleagues (pp. 173–187) provide an important contribution to this topic by demonstrating that the endocrine FGF ligand, FGF19, acts as an antifibrotic growth factor in pulmonary fibrosis (13). FGF19 has recently been shown to reduce liver fibrosis (14), suggesting that it may have antifibrotic activity in the lung as well. In this report, the authors show that FGF19 expression is reduced in patients with pulmonary fibrosis and then demonstrate that adenoviral-mediated overexpression of FGF19 in mice reduces pulmonary fibrosis induced by bleomycin. Using cultured alveolar epithelial cells and lung fibroblasts, the authors show that FGF19 exerts an antiapoptotic effect in the alveolar epithelium by decreasing Bcl-2-like protein 11 expression, and it reduces myofibroblast differentiation in part by decreasing TGFb-induced c-Jun N-terminal Kinase phosphorylation. Importantly, the finding that an endocrine growth factor produced by the kidneys, intestinal tract, and liver may have an effect on pulmonary fibrosis is intriguing and warrants further investigation. Endocrine growth factors such as FGF19 may provide an important link by which the gastrointestinal tract and kidneys regulate fibrotic diseases of several organs, including the lung. Furthermore, this study supports the need for future investigations into whether FGF19 or other endocrine growth factors provide an interorgan link between the gut microbiome, metabolic syndrome, acute and chronic kidney disease, liver disease, and IPF. There are several strengths to the approach taken by Justet and colleagues. Their experimental design was timed such that adenoassociated virus-induced overexpression of FGF19 peaked after the onset of experimental lung injury induced by bleomycin and TGFb1 overexpression. As treatment of IPF and pulmonary fibrosis in humans involves treating patients with fibrosis that is already present, the experimental approach from the authors mimics a possible treatment strategy in humans. In addition, expression of FGF19 induced by their adenoviral vector primarily occurred in the liver, mimicking the endocrine effects of FGF19. This approach simulates a potential therapeutic use of systemic FGF19, which would be a more likely therapeutic approach for a small protein like FGF19 than direct administration to the lungs. Finally, the authors employed two in vivo models of pulmonary fibrosis in mice (bleomycin and Ad-TGFb), supporting the idea that the effect of FGF19 is applicable to fibrotic lung disease rather than being limited to a pathway unique to a single experimental model. The small number of human samples is a limitation of this study. Because of a potential cofounding effect of meal boluses, liver disease, and kidney disease on FGF19 expression, the authors were limited to using samples from patients without cholestasis, chronic hemodialysis, diabetes mellitus, and nonalcoholic fatty liver disease. Human samples also had to be collected after fasting because FGF19 is prone to degradation. This also limits applicability to a broader population of patients with IPF or pulmonary fibrosis, as kidney and liver disease are common comorbidities for patients with pulmonary fibrosis (15, 16). Although FGF19 signaling via the FGF receptor 4 is well established (17), the precise mechanisms by which FGF receptor signaling and the potential involvement of coreceptors such as b-klotho alters fibroblast activation and myofibroblast differentiation remain an important area of future study, as the therapeutic potential of delivering FGF ligands in a clinical setting may be limited. Small molecule activators of downstream FGF signaling that halt or reverse epithelial apoptosis and/or myofibroblast activation in pulmonary fibrosis may also have significant therapeutic potential. In summary, the article by Justet and colleagues identifies an intriguing novel mechanism by which the endocrine growth factor FGF19 may constitute a link through which the gastrointestinal tract

OBJECTIVES/GOALS: Using a cell culture model, we will determine the effects of phosphate on primary lung cell cultures and use our results to delineate a pathway through which these changes are carried out. Using animal models, we will determine the effects of phosphate on inflammatory and fibrotic lung injury, both in the presence and absence of CKD. METHODS/STUDY POPULATION: For our in vitro experiments, human lung fibroblasts were treated with concentrations of 1 to 5 mM sodium phosphate and FGFR inhibitors. Expression levels of interleukin (IL)-1beta, IL-6, and IL-8 were analyzed by qPCR and secretion of these cytokines was measured by ELISA. Phosphorylation of PLCy and ERK was measured by western blot. Using an in vivo approach, we placed C57Bl/6 mice on a high phosphate (3%) diet to elevate serum phosphate levels in the absence of kidney injury and administered bleomycin via oropharyngeal aspiration to generate an acute inflammatory response. Serum FGF23 levels were measured by ELISA and serum analysis for phosphate and renal function were obtained. Furthermore, expression of FGF23 pathway and inflammatory markers were analyzed in murine lung tissue using qPCR and western blotting. RESULTS/ANTICIPATED RESULTS: Augmented phosphate concentrations led to increased cytokine expression and secretion from human lung fibroblasts as well as a concomitant increase in PLCy and ERK phosphorylation. Inhibition of FGFR1 reversed the effects of phosphate on the inflammatory cytokines and PLCy/ERK phosphorylation. Serum FGF23 levels were significantly upregulated in mice on a high phosphate diet and further increased in mice subjected to a high phosphate diet with exposure to bleomycin. Both serum phosphate and creatinine levels were significantly elevated as well. Additionally, high phosphate and bleomycin increased local FGF23 expression in murine lung tissue, when compared to controls or each stimulus alone. DISCUSSION/SIGNIFICANCE: Phosphate has a significant impact on inflammation and fibrosis in the lung, indicating that the existence of pulmo-renal crosstalk exaggerates pulmonary injury and that there are biological pathways that may be targeted therapeutically to mediate these effects. These results could have a substantial impact on the quality of life for CKD patients.

Chronic illnesses rarely present in a vacuum, devoid of other complications, and chronic kidney disease is hardly an exception. Comorbidities associated with chronic kidney disease lead to faster disease progression, expedited dialysis dependency, and a higher mortality rate. Although chronic kidney disease is most commonly accompanied by cardiovascular diseases and diabetes, there is clear cross talk between the lungs and kidneys pH balance, phosphate metabolism, and immune system regulation. Our present understanding of the exact underlying mechanisms that contribute to chronic kidney disease-related pulmonary disease is poor. This review summarizes the current research on kidney-pulmonary interorgan cross talk in the context of chronic kidney disease, highlighting various acute and chronic pulmonary diseases that lead to further complications in patient care. Treatment options for patients presenting with chronic kidney disease and lung disease are explored by assessing activated molecular pathways and the body’s compensatory response mechanisms following homeostatic imbalance. Understanding the link between the lungs and kidneys will potentially improve health outcomes for patients and guide healthcare professionals to better understand how and when to treat each of the pulmonary comorbidities that can present with chronic kidney disease.

Chronic obstructive pulmonary disease (COPD) is a systemic disease strongly associated with cigarette smoking, airway inflammation, and acute disease exacerbations. Changes in terminal sialylation and fucosylation of asparagine (N)-linked glycans have been documented in COPD, but the role that glycosyltransferases may play in the regulation of N-linked glycans in COPD has not been fully elucidated. Recent studies suggest that modulation of ST6GAL1 (ST6 beta-galactoside alpha-2,6-sialyltransferase-1), which catalyzes terminal α2-6 sialylation of cellular proteins, may regulate inflammation and contribute to COPD phenotype(s). Interestingly, it has been previously demonstrated that ST6GAL1, a Golgi resident protein, can be proteolytically processed by BACE1 (beta-site amyloid precursor protein cleaving enzyme-1) to a circulating form that retains activity. In this study, we showed that loss of ST6GAL1 expression increased interleukin (IL)-6 expression and secretion in human bronchial epithelial cells (HBECs). Furthermore, exposure to cigarette smoke medium/extract (CSE) or BACE1 inhibition resulted in decreased ST6GAL1 secretion, reduced α2-6 sialylation, and increased IL-6 production in HBECs. Analysis of plasma ST6GAL1 levels in a small COPD patient cohort demonstrated an inverse association with prospective acute exacerbations of COPD (AECOPD), while IL-6 was positively associated. Altogether, these results suggest that reduced ST6GAL1 and α2-6 sialylation augments IL-6 expression/secretion in HBECs and is associated with poor clinical outcomes in COPD.

Chronic obstructive pulmonary disease (COPD) has become a global epidemic and is the third leading cause of death worldwide. COPD is characterized by chronic airway inflammation, loss of alveolar-capillary units, and progressive decline in lung function. Major risk factors for COPD are cigarette smoking and aging. COPD-associated pathomechanisms include multiple aging pathways such as telomere attrition, epigenetic alterations, altered nutrient sensing, mitochondrial dysfunction, cell senescence, stem cell exhaustion and chronic inflammation. In this review, we will highlight the current literature that focuses on the role of age and aging-associated signaling pathways as well as their impact on current treatment strategies in the pathogenesis of COPD. Furthermore, we will discuss established and experimental COPD treatments including senolytic and anti-aging therapies and their potential use as novel treatment strategies in COPD.

Objectives: Osseointegrated prostheses are increasingly used for amputees, however, the lengthy rehabilitation time of these prostheses remains a challenge to their implementation. The aim of this study was to investigate the ability of locally applied vibration or low-intensity pulsed ultrasound (LIPUS) to accelerate osseointegration and increase peri-implant bone volume. Methods: A 4-week and 8-week rodent study were conducted in a femoral intramedullary implant model (control. vibration, LIPUS, and combined treatment) to determine effects on healing. Osseointegration was evaluated quantitatively through mechanical, mu CT and histological evaluations. Results: Maximum pushout load at 4 weeks increased with LIPUS relative to control (37.7%, P=0.002). Histologically, LIPUS and vibration separately increased peri-implant bone formation after 4 weeks relative to control. Vibration resulted in greater peri-implant bone after 8 weeks than all other groups (25.7%, P<0.001). However, no significant group differences in pushout load were noted at 8 weeks. Conclusions: Although vibration increased bone around implants, LIPUS was superior to vibration for accelerating osseointegration and increasing bone-implant failure loads at 4 weeks. However, the LIPUS benefits on osseointegration at 4 weeks were not sustained at 8 weeks.