Transcription factor HNF4α2 promotes osteogenesis and prevents bone abnormalities in mice with renal osteodystrophy.

Renal osteodystrophy (ROD) is a disorder of bone metabolism that affects virtually all patients with chronic kidney disease (CKD) and is associated with adverse clinical outcomes including fractures, cardiovascular events, and death. In this study, we showed that hepatocyte nuclear factor 4α (HNF4α), a transcription factor mostly expressed in the liver, is also expressed in bone, and that osseous HNF4α expression was dramatically reduced in patients and mice with ROD. Osteoblast-specific deletion of Hnf4α resulted in impaired osteogenesis in cells and mice. Using multi-omics analyses of bones and cells lacking or overexpressing Hnf4α1 and Hnf4α2, we showed that HNF4α2 is the main osseous Hnf4α isoform that regulates osteogenesis, cell metabolism, and cell death. As a result, osteoblast-specific overexpression of Hnf4α2 prevented bone loss in mice with CKD. Our results showed that HNF4α2 is a transcriptional regulator of osteogenesis, implicated in the development of ROD.

Immunotherapy reverses glioma-driven dysfunction of immune system homeostasis.

BACKGROUND: Glioma-induced immune dysregulation of the hematopoietic system has been described in a limited number of studies. In this study, our group further demonstrates that gliomas interrupt the cellular differentiation programming and outcomes of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. HSPCs from glioma-bearing mice are reprogrammed and driven towards expansion of myeloid lineage precursors and myeloid-derived suppressor cells (MDSCs) in secondary lymphoid organs. However, we found this expansion is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of central nervous system (CNS) malignancies, and here we describe how glioma-induced dysfunction is reversed by this immunotherapeutic platform. METHODS: The impact of orthotopic KR158B-luc glioma on HSPCs was evaluated in an unbiased fashion using single cell RNAseq (scRNAseq) of lineage- cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor RNA-pulsed dendritic cells, tumor-reactive T cells and HSPCs isolated from glioma-bearing or non-tumor-bearing mice were used to evaluate cell fate differentiation and survival. RESULTS: Using scRNAseq, we observed an altered HSPC landscape in glioma-bearing versus non-tumor-bearing mice . In addition, an expansion of myeloid lineage subsets, including granulocyte macrophage precursors (GMPs) and MDSCs, were observed in glioma-bearing mice relative to non-tumor-bearing controls. Furthermore, MDSCs from glioma-bearing mice demonstrated increased suppressive capacity toward tumor-specific T cells as compared with MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame these suppressive properties. When HSPCs from glioma-bearing mice were transferred in the context of ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.

Lipocalin 2 stimulates bone fibroblast growth factor 23 production in chronic kidney disease.

Bone-produced fibroblast growth factor 23 (FGF23) increases in response to inflammation and iron deficiency and contributes to cardiovascular mortality in chronic kidney disease (CKD). Neutrophil gelatinase-associated lipocalin (NGAL or lipocalin 2; LCN2 the murine homolog) is a pro-inflammatory and iron-shuttling molecule that is secreted in response to kidney injury and may promote CKD progression. We investigated bone FGF23 regulation by circulating LCN2. At 23 weeks, Col4a3KO mice showed impaired kidney function, increased levels of kidney and serum LCN2, increased bone and serum FGF23, anemia, and left ventricular hypertrophy (LVH). Deletion of Lcn2 in CKD mice did not improve kidney function or anemia but prevented the development of LVH and improved survival in association with marked reductions in serum FGF23. Lcn2 deletion specifically prevented FGF23 elevations in response to inflammation, but not iron deficiency or phosphate, and administration of LCN2 increased serum FGF23 in healthy and CKD mice by stimulating Fgf23 transcription via activation of cAMP-mediated signaling in bone cells. These results show that kidney-produced LCN2 is an important mediator of increased FGF23 production by bone in response to inflammation and in CKD. LCN2 inhibition might represent a potential therapeutic approach to lower FGF23 and improve outcomes in CKD.

Precision therapeutic targets for COVID-19.

Beginning in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel pathogen that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 has infected more than 111 million people worldwide and caused over 2.47 million deaths. Individuals infected with SARS-CoV-2 show symptoms of fever, cough, dyspnea, and fatigue with severe cases that can develop into pneumonia, myocarditis, acute respiratory distress syndrome, hypercoagulability, and even multi-organ failure. Current clinical management consists largely of supportive care as commonly administered treatments, including convalescent plasma, remdesivir, and high-dose glucocorticoids. These have demonstrated modest benefits in a small subset of hospitalized patients, with only dexamethasone showing demonstrable efficacy in reducing mortality and length of hospitalization. At this time, no SARS-CoV-2-specific antiviral drugs are available, although several vaccines have been approved for use in recent months. In this review, we will evaluate the efficacy of preclinical and clinical drugs that precisely target three different, essential steps of the SARS-CoV-2 replication cycle: the spike protein during entry, main protease (MPro) during proteolytic activation, and RNA-dependent RNA polymerase (RdRp) during transcription. We will assess the advantages and limitations of drugs that precisely target evolutionarily well-conserved domains, which are less likely to mutate, and therefore less likely to escape the effects of these drugs. We propose that a multi-drug cocktail targeting precise proteins, critical to the viral replication cycle, such as spike protein, MPro, and RdRp, will be the most effective strategy of inhibiting SARS-CoV-2 replication and limiting its spread in the general population.

Immune Escape After Adoptive T-cell Therapy for Malignant Gliomas.

PURPOSE: Immunotherapy has been demonstrably effective against multiple cancers, yet tumor escape is common. It remains unclear how brain tumors escape immunotherapy and how to overcome this immune escape. EXPERIMENTAL DESIGN: We studied KR158B-luc glioma-bearing mice during treatment with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested the immunogenicity of primary and escaped tumors using T-cell restimulation assays. We used flow cytometry and RNA profiling of whole tumors to further define escape mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T cells through the use of escape variant total tumor RNA and administered these cells as ACT. In addition, programmed cell death protein-1 (PD-1) checkpoint blockade was studied in combination with ACT. RESULTS: Escape mechanisms included a shift in immunogenic tumor antigens, downregulation of MHC class I, and upregulation of checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater recognition of escaped tumors than primary tumors. When administered as ACT, these T cells prolonged median survival of escape variant-bearing mice by 60%. The rational combination of ACT with PD-1 blockade prolonged median survival of escape variant glioma-bearing mice by 110% and was dependent upon natural killer cells and T cells. CONCLUSIONS: These findings suggest that the immune landscape of brain tumors are markedly different postimmunotherapy yet can still be targeted with immunotherapy.

Ferric citrate reduces fibroblast growth factor 23 levels and improves renal and cardiac function in a mouse model of chronic kidney disease.

Iron deficiency, anemia, hyperphosphatemia, and increased fibroblast growth factor 23 (FGF23) are common and interrelated complications of chronic kidney disease (CKD) that are linked to CKD progression, cardiovascular disease and death. Ferric citrate is an oral phosphate binder that decreases dietary phosphate absorption and serum FGF23 concentrations while increasing iron stores and hemoglobin in patients with CKD. Here we compared the effects of ferric citrate administration versus a mineral sufficient control diet using the Col4a3 knockout mouse model of progressive CKD and age-matched wild-type mice. Ferric citrate was given to knockout mice for four weeks beginning at six weeks of age when they had overt CKD, or for six weeks beginning at four weeks of age when they had early CKD. Ten-week-old knockout mice on the control diet showed overt iron deficiency, anemia, hyperphosphatemia, increased serum FGF23, hypertension, decreased kidney function, and left ventricular systolic dysfunction. Ferric citrate rescued iron deficiency and anemia in knockout mice regardless of the timing of treatment initiation. Circulating levels and bone expression of FGF23 were reduced in knockout mice given ferric citrate with more pronounced reductions observed when ferric citrate was initiated in early CKD. Ferric citrate decreased serum phosphate only when it was initiated in early CKD. While ferric citrate mitigated systolic dysfunction in knockout mice regardless of timing of treatment initiation, early initiation of ferric citrate also reduced renal fibrosis and proteinuria, improved kidney function, and prolonged life span. Thus, initiation of ferric citrate treatment early in the course of murine CKD lowered FGF23, slowed CKD progression, improved cardiac function and significantly improved survival.

DMP1 prevents osteocyte alterations, FGF23 elevation and left ventricular hypertrophy in mice with chronic kidney disease.

During chronic kidney disease (CKD), alterations in bone and mineral metabolism include increased production of the hormone fibroblast growth factor 23 (FGF23) that may contribute to cardiovascular mortality. The osteocyte protein dentin matrix protein 1 (DMP1) reduces FGF23 and enhances bone mineralization, but its effects in CKD are unknown. We tested the hypothesis that DMP1 supplementation in CKD would improve bone health, prevent FGF23 elevations and minimize consequent adverse cardiovascular outcomes. We investigated DMP1 regulation and effects in wild-type (WT) mice and the Col4a3-/- mouse model of CKD. Col4a3-/- mice demonstrated impaired kidney function, reduced bone DMP1 expression, reduced bone mass, altered osteocyte morphology and connectivity, increased osteocyte apoptosis, increased serum FGF23, hyperphosphatemia, left ventricular hypertrophy (LVH), and reduced survival. Genetic or pharmacological supplementation of DMP1 in Col4a3-/- mice prevented osteocyte apoptosis, preserved osteocyte networks, corrected bone mass, partially lowered FGF23 levels by attenuating NFAT-induced FGF23 transcription, and further increased serum phosphate. Despite impaired kidney function and worsened hyperphosphatemia, DMP1 prevented development of LVH and improved Col4a3-/- survival. Our data suggest that CKD reduces DMP1 expression, whereas its restoration represents a potential therapeutic approach to lower FGF23 and improve bone and cardiac health in CKD.

Genetic background influences cardiac phenotype in murine chronic kidney disease.

Background: Levels of fibroblast growth factor 23 (FGF23) increase early in chronic kidney disease (CKD) and are independently associated with left ventricular hypertrophy (LVH), heart failure and death. Experimental models of CKD with elevated FGF23 and LVH are needed. We hypothesized that slow rates of CKD progression in the Col4a3 knockout (Col4a3KO) mouse model of CKD would promote development of LVH by prolonging exposure to elevated FGF23. Methods: We studied congenic Col4a3KO and wild-type (WT) mice with either 75% 129X1/SvJ (129Sv) or 94% C57Bl6/J (B6) genomes. Results: B6-Col4a3KO lived longer than 129Sv-Col4a3KO mice (21.4 ± 0.6 versus 11.4 ± 0.4 weeks; P < 0.05). 10-week-old 129Sv-Col4a3KO mice showed impaired renal function (blood urea nitrogen 191 ± 39 versus 34 ± 4 mg/dL), hyperphosphatemia (14.1 ± 1.4 versus 6.8 ± 0.3 mg/dL) and 33-fold higher serum FGF23 levels (P < 0.05 versus WT for each). Consistent with their slower CKD progression, 10 week-old B6-Col4a3KO mice showed milder impairment of renal function than 129Sv-Col4a3KO mice and modest FGF23 elevation without other alterations of mineral metabolism. At 20 weeks, further declines in renal function in B6-Col4a3KO mice was accompanied by hyperphosphatemia and 8-fold higher FGF23 levels (P < 0.05 versus WT for each). Only the 20-week-old B6-Col4a3KO mice developed LVH (LV mass 125 ± 3 versus 98 ± 6 mg; P < 0.05 versus WT) in association with significantly increased cardiac expression of FGF receptor 4 (FGFR4) messenger RNA and protein and markers of LVH (Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (ß-MHC); P < 0.05 versus WT for each). Conclusions: In conclusion, B6-Col4a3KO mice manifest slower CKD progression and longer survival than 129Sv-Col4a3KO mice and can serve as a novel model of cardiorenal disease.

Ironing out the cross talk between FGF23 and inflammation.

The bone-secreted hormone fibroblast growth factor 23 (FGF23) has an essential role in phosphate homeostasis by regulating expression of the kidney proximal tubule sodium-phosphate cotransporters as well as parathyroid hormone levels. Induction of FGF23 early in chronic kidney disease (CKD) helps to maintain normal phosphorous levels. However, high FGF23 levels become pathological as kidney disease progresses and are associated with an increased risk of CKD progression, cardiovascular events, and death. The factors responsible for increasing FGF23 levels early in CKD are unknown, but recent work has proposed a role for inflammation and disordered iron homeostasis. Notably, FGF23 has recently been shown to elicit an inflammatory response and to display immunomodulatory properties. Here, we will review emerging evidence on the cross talk between inflammation, iron, FGF23, and bone and mineral metabolism and discuss the relevance for CKD patients.

Inflammation regulates fibroblast growth factor 23 production.

PURPOSE OF REVIEW: Fibroblast growth factor 23 (FGF23) is a hormone secreted by osteocytes and osteoblasts that regulates phosphorus and vitamin D homeostasis. FGF23 levels increase progressively in chronic kidney disease (CKD), and FGF23 excess might be a causal factor of left ventricular hypertrophy, CKD progression and death. Therefore, understanding the molecular mechanisms that control FGF23 production is critical to design therapies to lower FGF23 levels. The present review focuses on the role of inflammatory stimuli on FGF23 regulation and summarizes recent studies that support a novel framework linking inflammation to FGF23 regulation. RECENT FINDINGS: Inflammation and iron deficiency, which are common occurrences in CKD, have emerged as novel FGF23 regulators. Recent findings show that inflammation increases FGF23 production in bone through direct and iron-related indirect mechanisms. In these settings, hypoxia-inducible factor (HIF)-1α orchestrates FGF23 transcription in response to inflammation and is primarily responsible for coordinating FGF23 production and cleavage. SUMMARY: We demonstrate that inflammation increases FGF23 production and may contribute to elevated FGF23 levels in CKD. Osseous HIF-1α may represent a therapeutic target to lower FGF23 levels in CKD patients and minimize the negative consequences associated with FGF23 excess.