Chronic kidney disease (CKD) is characterized by irreversible pathological processes that result in the development of end-stage renal disease (ESRD). Accumulating evidence has emphasized the important role of chronic hypoxia in the tubulointerstitium in the final common pathway that leads to development of ESRD. The causes of chronic hypoxia in the tubulointerstitium are multifactorial and include mechanisms such as hemodynamic changes and disturbed oxygen metabolism of resident kidney cells. Epidemiological studies have revealed an association between CKD and systemically hypoxic conditions, such as chronic obstructive pulmonary disease and sleep apnea syndrome. In addition to tubulointerstitial hypoxia, glomerular hypoxia can occur and is a crucial factor in the development of glomerular disorders. Chemical compounds, polarographic sensors, and radiographical methods can be used to detect hypoxia. Therapeutic approaches that target chronic hypoxia in the kidney should be effective against a broad range of kidney diseases. Amelioration of hypoxia is one mechanism of inhibiting the renin-angiotensin system, the current gold standard of CKD therapy. Future therapeutic approaches include protection of the vascular endothelium and appropriate activation of hypoxia-inducible factor, a key transcription factor involved in adaptive responses against hypoxia.

Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1␣ and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.

Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-␣ (TNF␣) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CCCTC-binding factor CTCF), and modifications of histone H3. Activation triggers a wave of transcription that sweeps along the genes at Ϸ3.1 kbp/min; splicing occurs cotranscriptionally, a major checkpoint acts several kilobases downstream of the transcription start site to regulate polymerase transit, and Pol II tends to stall at cohesin/CTCF binding sites.endothelial cell ͉ polymerase II ͉ RNA ͉ tumor necrosis factor alpha

GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.

Diabetic kidney disease (DKD) is the major cause of end-stage kidney disease. However, only renin-angiotensin system inhibitor with multidisciplinary treatments is effective for DKD. In 2019, sodium-glucose cotransporter 2 (SGLT2) inhibitor showed efficacy against DKD in Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial, adding a new treatment option. However, the progression of DKD has not been completely controlled. The patients with transient exposure to hyperglycemia develop diabetic complications, including DKD, even after normalization of their blood glucose. Temporary hyperglycemia causes advanced glycation end product (AGE) accumulations and epigenetic changes as metabolic memory. The drugs that improve metabolic memory are awaited, and AGE inhibitors and histone modification inhibitors are the focus of clinical and basic research. In addition, incretin-related drugs showed a renoprotective ability in many clinical trials, and these trials with renal outcome as their primary endpoint are currently ongoing. Hypoxia-inducible factor prolyl hydroxylase inhibitors recently approved for renal anemia may be renoprotective since they improve tubulointerstitial hypoxia. Furthermore, NF-E2–related factor 2 activators improved the glomerular filtration rate of DKD patients in Bardoxolone Methyl Treatment: Renal Function in chronic kidney disease/Type 2 Diabetes (BEAM) trial and Phase II Study of Bardoxolone Methyl in Patients with Chronic Kidney Disease and Type 2 Diabetes (TSUBAKI) trial. Thus, following SGLT2 inhibitor, numerous novel drugs could be utilized in treating DKD. Future studies are expected to provide new insights.

Chronic kidney disease (CKD) is placing an increasing burden on patients and societies because no decisive therapy has been established. Tubulointerstitial lesions accompanied by fibrosis, inflammatory cells, and capillary rarefaction not only characterize, but also aggravate renal dysfunction in CKD. In this setting, renal cells, particularly tubular cells, suffer from hypoxia caused by the imbalance of blood perfusion and oxygen demand despite their adaptive responses represented by upregulation of hypoxia-inducible factors (HIFs). Fibrosis is a pathological state characterized by excess extracellular matrix (ECM) deposition, which is also a hallmark and causative factor of many chronic diseases including CKD. Recent studies have suggested that the dominant origin of ECM-producing myofibroblasts (MFs) may be pericytes, which are indispensable cells for maintaining proper capillary functions, as they wrap capillaries and stabilize them through a fine-tuned interplay with endothelial cells. During fibrosis, pericytes are activated and detach from capillaries before conversion into MFs, which compromises capillaries and worsens hypoxia. We also discuss how hypoxia and HIFs affect fibrogenesis. Given that hypoxia is caused by insufficient angiogenesis and that fibrosis results from pericyte loss, restoration of pericytes should be an intriguing target for overcoming both hypoxia and fibrosis. We propose the deactivation of MFs to recover lost pericytes as a promising therapy for CKD.

BackgroundSynergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated.ResultsWe present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) β/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARβ/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements.ConclusionsTo the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes.

Cytoglobin (Cygb), a novel member of the globin superfamily, is expressed by fibroblasts in various organs. However, its function remains unknown. Because of its localization, we speculated that a biological role of Cygb may be related to fibrogenesis. To clarify the role of Cygb in kidney fibrosis, we employed the remnant kidney model in rats. Immunohistochemical analysis showed an increase in Cygb expression in parallel with disease progression. To investigate the functional consequence of Cygb upregulation, we established transgenic rats overexpressing rat Cygb. Overexpression of Cygb improved histological injury, preserved renal function, and ameliorated fibrosis, as estimated by the accumulation of collagen I and IV as well as Masson trichrome staining. These protective effects of Cygb were associated with a decrease in nitrotyrosine deposition in the kidney and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) excretion as a marker of oxidative stress. We also performed in vitro studies utilizing a rat kidney fibroblast cell line transiently overexpressing Cygb, an inducible kidney cell transfected with Cygb, and primary cultured fibroblasts isolated from the kidneys of the transgenic rats. These different experimental systems consistently showed that Cygb inhibited collagen synthesis. Furthermore, mutant disruption of heme in Cygb that impaired its antioxidant properties led to the loss of antifibrotic effects, suggesting that Cygb reduces fibrosis via a radical scavenging function. In conclusion, we showed that Cygb plays an important role in protection of the kidney against fibrosis via the amelioration of oxidative stress both in vitro and in vivo. Cygb might represent a good therapeutic target in chronic kidney disease.