Inflammation primes the murine kidney for recovery by activating AZIN1 adenosine-to-inosine editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets.

FGF23 via its coreceptor αKlotho (KL) provides critical control of phosphate metabolism, which is altered in rare and very common syndromes, however the spatial-temporal mechanisms dictating renal FGF23 functions remain poorly understood. Thus, developing approaches to modify specific FGF23-dictated pathways has proven problematic. Herein, wild type mice were injected with rFGF23 for 1, 4 and 12h and renal FGF23 bioactivity was determined at single cell resolution. Computational analysis identified distinct epithelial, endothelial, stromal, and immune cell clusters, with differential expressional analysis uniquely tracking FGF23 bioactivity at each time point. FGF23 actions were sex independent but critically relied upon constitutive KL expression mapped within proximal tubule (S1-S3) and distal tubule (DCT/CNT) cell sub-populations. Temporal KL-dependent FGF23 responses drove unique and transient cellular identities, including genes in key MAPK- and vitamin D-metabolic pathways via early- (AP-1-related) and late-phase (EIF2 signaling) transcriptional regulons. Combining ATACseq/RNAseq data from a cell line stably expressing KL with the in vivo scRNAseq pinpointed genomic accessibility changes in MAPK-dependent genes, including the identification of FGF23-dependent EGR1 distal enhancers. Finally, we isolated unexpected crosstalk between FGF23-mediated MAPK signaling and pro-inflammatory TNF receptor activation via NF-κB, which blocked FGF23 bioactivity in vitro and in vivo . Collectively, our findings have uncovered novel pathways at the single cell level that likely influence FGF23-dependent disease mechanisms. Translational statement: Inflammation and elevated FGF23 in chronic kidney disease (CKD) are both associated with poor patient outcomes and mortality. However, the links between these manifestations and the effects of inflammation on FGF23-mediated mineral metabolism within specific nephron segments remain unclear. Herein, we isolated an inflammatory pathway driven by TNF/NF-κB associated with regulating FGF23 bioactivity. The findings from this study could be important in designing future therapeutic approaches for chronic mineral diseases, including potential combination therapies or early intervention strategies. We also suggest that further studies could explore these pathways at the single cell level in CKD models, as well as test translation of our findings to interactions of chronic inflammation and elevated FGF23 in human CKD kidney datasets.

ABCG2-Expressing Clonal Repopulating Endothelial Cells Serve to Form and Maintain Blood Vessels.

BACKGROUND: Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS: We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs. RESULTS: In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs. CONCLUSIONS: These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Propranolol treatment during repetitive mild traumatic brain injuries induces transcriptomic changes in the bone marrow of mice.

Introduction: There are 1.5 million new mild traumatic brain injuries (mTBI) annually in the US, with many of the injured experiencing long-term consequences lasting months after the injury. Although the post injury mechanisms are not well understood, current knowledge indicates peripheral immune system activation as a causal link between mTBI and long-term side effects. Through a variety of mechanisms, peripheral innate immune cells are recruited to the CNS after TBI to repair and heal the injured tissue; however, the recruitment and activation of these cells leads to further inflammation. Emerging evidence suggests sympathetic nervous system (SNS) activity plays a substantial role in the recruitment of immune cells post injury. Methods: We sought to identify the peripheral innate immune response after repeated TBIs in addition to repurposing the nonselective beta blocker propranolol as a novel mTBI therapy to limit SNS activity and mTBI pathophysiology in the mouse. Mice underwent repetitive mTBI or sham injury followed by i.p. saline or propranolol. Isolated mRNA derived from femur bone marrow of mice was assayed for changes in gene expression at one day, one week, and four weeks using Nanostring nCounter® stem cell characterization panel. Results: Differential gene expression analysis for bone marrow uncovered significant changes in many genes following drug alone, mTBI alone and drug combined with mTBI. Discussion: Our data displays changes in mRNA at various timepoints, most pronounced in the mTBI propranolol group, suggesting a single dose propranolol injection as a viable future mTBI therapy in the acute setting.

miRNA and mRNA Signatures in Human Acute Kidney Injury Tissue.

Acute kidney injury (AKI) is an important contributor to the development of chronic kidney disease (CKD). There is a need to understand molecular mediators that drive either recovery or progression to CKD. In particular, the role of miRNA and its regulatory role in AKI is poorly understood. We performed miRNA and mRNA sequencing on biobanked human kidney tissues obtained in the routine clinical care of patients with the diagnoses of AKI and minimal change disease (MCD), in addition to nephrectomized (Ref) tissue from individuals without known kidney disease. Transcriptomic analysis of mRNA revealed that Ref tissues exhibited a similar injury signature to AKI, not identified in MCD samples. The transcriptomic signature of human AKI was enriched with genes in pathways involved in cell adhesion and epithelial-to-mesenchymal transition (e.g., CDH6, ITGB6, CDKN1A ). miRNA DE analysis revealed upregulation of miRNA associated with immune cell recruitment and inflammation (e.g., miR-146a, miR-155, miR-142, miR-122). These miRNA (i.e., miR-122, miR-146) are also associated with downregulation of mRNA such as DDR2 and IGFBP6 , respectively. These findings suggest integrated interactions between miRNAs and target mRNAs in AKI-related processes such as inflammation, immune cell activation and epithelial-to-mesenchymal transition. These data contribute several novel findings when describing the epigenetic regulation of AKI by miRNA, and also underscores the importance of utilizing an appropriate reference control tissue to understand canonical pathway alterations in AKI.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Origin, prospective identification, and function of circulating endothelial colony-forming cells in mice and humans.

Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony-forming cells (C-ECFCs), also known as blood outgrowth endothelial cells, with proliferative and vasculogenic activity can be cultured; however, the origin and naive function of these C-ECFCs remains obscure. Herein, detailed lineage tracing revealed murine C-ECFCs emerged in the early postnatal period, displayed high vasculogenic potential with enriched frequency of clonal proliferative cells compared with tissue-resident ECFCs, and were not committed to or derived from the BM hematopoietic system but from tissue-resident ECFCs. In humans, C-ECFCs were present in the CD34bright cord blood mononuclear subset, possessed proliferative potential and in vivo vasculogenic function in a naive or cultured state, and displayed a single cell transcriptome sharing some umbilical venous endothelial cell features, such as a higher protein C receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naive function, and antigens to prospectively isolate C-ECFCs for translational studies.

Single-cell RNAseq provides insight into altered immune cell populations in human fracture nonunions.

Nonunion describes bone fractures that fail to heal, resulting in the fracture callus failing to fully ossify or, in atrophic cases, not forming altogether. Fracture healing is regulated, in part, by the balance of proinflammatory and anti-inflammatory processes occurring within the bone marrow and surface cell populations. We sought to further understand the role of osteoimmunology (i.e., study of the close relationship between the immune system and bone) by examining immune cell gene expression via single-cell RNA sequencing of intramedullary canal tissue obtained from human patients with femoral nonunions. Intramedullary canal tissue samples obtained by reaming were collected at the time of surgical repair for femur fracture nonunion (n = 5) or from native bone controls when harvesting autologous bone graft (n = 4). Cells within the samples were isolated and analyzed using the Chromium Single-Cell System (10x Genomics Inc.) and Illumina sequencers. Twenty-three distinct cell clusters were identified, with higher cell proportions in the nonunion samples for monocytes and CD14 + dendritic cells (DCs), and lower proportions of T cells, myelocytes, and promyelocytes in nonunion samples. Gene expression differences were identified in each of the cell clusters from cell types associated with osteoimmunology, including CD14 + DC, monocytes, T cells, promyelocytes, and myelocytes. These results provide human-derived gene profiles that can further our understanding of pathways that may be a cause or a consequence of nonunion, providing the clinical rationale to focus on specific components of osteoimmunology. Clinical significance: The novel single-cell approach may lead to clinically relevant diagnostic biomarkers during earlier stages of nonunion development and/or investigation into therapeutic options.

Translation Rescue by Targeting Ppp1r15a through Its Upstream Open Reading Frame in Sepsis-Induced Acute Kidney Injury in a Murine Model.

BACKGROUND: Translation shutdown is a hallmark of late-phase, sepsis-induced kidney injury. Methods for controlling protein synthesis in the kidney are limited. Reversing translation shutdown requires dephosphorylation of the eukaryotic initiation factor 2 (eIF2) subunit eIF2 α ; this is mediated by a key regulatory molecule, protein phosphatase 1 regulatory subunit 15A (Ppp1r15a), also known as GADD34. METHODS: To study protein synthesis in the kidney in a murine endotoxemia model and investigate the feasibility of translation control in vivo by boosting the protein expression of Ppp1r15a, we combined multiple tools, including ribosome profiling (Ribo-seq), proteomics, polyribosome profiling, and antisense oligonucleotides, and a newly generated Ppp1r15a knock-in mouse model and multiple mutant cell lines. RESULTS: We report that translation shutdown in established sepsis-induced kidney injury is brought about by excessive eIF2 α phosphorylation and sustained by blunted expression of the counter-regulatory phosphatase Ppp1r15a. We determined the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic and antisense oligonucleotide approaches enabled the overexpression of Ppp1r15a, which salvaged translation and improved kidney function in an endotoxemia model. Loss of this uORF also had broad effects on the composition and phosphorylation status of the immunopeptidome-peptides associated with the MHC-that extended beyond the eIF2 α axis. CONCLUSIONS: We found Ppp1r15a is translationally repressed during late-phase sepsis because of the existence of an uORF, which is a prime therapeutic candidate for this strategic rescue of translation in late-phase sepsis. The ability to accurately control translation dynamics during sepsis may offer new paths for the development of therapies at codon-level precision. PODCAST: This article contains a podcast at.

Th1 cytokine endotype discriminates and predicts severe complications in COVID-19

Treatment of severe and critical cases of coronavirus disease 2019 (COVID-19) is still a top priority in public health. Previously, we reported distinct Th1 cytokines related to the pathophysiology of severe COVID-19 condition. In the present study, we investigated the association of Th1 and Th2 cytokine/chemokine endotypes with cell-mediated immunity via multiplex immunophenotyping, single-cell RNA-Seq analysis of peripheral blood mononuclear cells, and analysis of the clinical features of COVID-19 patients. Based on serum cytokine and systemic inflammatory markers, COVID-19 cases were classified into four clusters of increasing (I-IV) severity. Two prominent clusters were of interest and could be used as prognostic reference for a targeted treatment of severe COVID-19 cases. Cluster III reflected severe/critical pathology and was characterized by decreased in CCL17 levels and increase in IL-6, C-reactive protein CXCL9, IL-18, and IL-10 levels. The second cluster (Cluster II) showed mild to moderate pathology and was characterized by predominated CXCL9 and IL-18 levels, levels of IL-6 and CRP were relatively low. Cluster II patients received anti-inflammatory treatment in early-stage, which may have led prevent disease prognosis which is accompanied to IL-6 and CRP induction. In Cluster III, a decrease in the proportion of effector T cells with signs of T cell exhaustion was observed. This study highlights the mechanisms of endotype clustering based on specific inflammatory markers in related the clinical outcome of COVID-19.

The kidney protects against sepsis by producing systemic uromodulin.

Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. Although electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We have previously demonstrated that systemic Tamm-Horsfall protein (THP; uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we showed that the kidney increases circulating THP by enhancing the basolateral release of THP from medullary thick ascending limb cells. In patients with sepsis, changes in circulating THP were positively associated with a critical illness. THP was also found de novo in injured lungs. Genetic ablation of THP in mice led to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo led macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis, and chemotaxis, and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improved survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function, and systemic THP has therapeutic potential in sepsis.NEW & NOTEWORTHY Specific therapies to improve outcomes in sepsis with kidney injury have been limited by an unclear understanding of how kidney injury increases sepsis mortality. Here, we identified Tamm-Horsfall protein, known to protect in ischemic acute kidney injury, as protective in preclinical sepsis models. Tamm-Horsfall protein also increased in clinical sepsis without severe kidney injury and concentrated in injured organs. Further study could lead to novel sepsis therapeutics.

Alterations in Protein Translation and Carboxylic Acid Catabolic Processes in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography-mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.

A Pilot Single Cell Analysis of the Zebrafish Embryo Cellular Responses to Uropathogenic Escherichia coli Infection.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) infections are common and when they disseminate can be of high morbidity. METHODS: We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed. RESULTS: Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a (Foxo1a), Tribbles Pseudokinase 3 (Trib3), Arginase 2 (Arg2) and Polo Like Kinase 3 (Plk3). CONCLUSIONS: Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.

Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease.

Background The myocardial cytoskeleton functions as the fundamental framework critical for organelle function, bioenergetics and myocardial remodeling. To date, impairment of the myocardial cytoskeleton occurring in the failing heart in patients with advanced chronic kidney disease has been largely undescribed. Methods and Results We conducted a 3-arm cross-sectional cohort study of explanted human heart tissues from patients who are dependent on hemodialysis (n=19), hypertension (n=10) with preserved renal function, and healthy controls (n=21). Left ventricular tissues were subjected to pathologic examination and next-generation RNA sequencing. Mechanistic and interference RNA studies utilizing in vitro human cardiac fibroblast models were performed. Left ventricular tissues from patients undergoing hemodialysis exhibited increased myocardial wall thickness and significantly greater fibrosis compared with hypertension patients (P<0.05) and control (P<0.01). Transcriptomic analysis revealed that the focal adhesion pathway was significantly enriched in hearts from patients undergoing hemodialysis. Hearts from patients undergoing hemodialysis exhibited dysregulated components of the focal adhesion pathway including reduced ß-actin (P<0.01), ß-tubulin (P<0.01), vimentin (P<0.05), and increased expression of vinculin (P<0.05) compared with controls. Cytoskeletal adaptations in hearts from the hemodialysis group were associated with impaired mitochondrial bioenergetics, including dysregulated mitochondrial dynamics and fusion, and loss of cell survival pathways. Mechanistic studies revealed that cytoskeletal changes can be driven by uremic and metabolic abnormalities of chronic kidney disease, in vitro. Furthermore, focal adhesion kinase silencing via interference RNA suppressed major cytoskeletal proteins synergistically with mineral stressors found in chronic kidney disease in vitro. Conclusions Myocardial failure in advanced chronic kidney disease is characterized by impairment of the cytoskeleton involving disruption of the focal adhesion pathway, mitochondrial failure, and loss of cell survival pathways.

Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML.

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Loss of proximal tubular transcription factor Krüppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation.

Loss of fatty acid ß-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.