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Chronic kidney disease (CKD) affects kidney cancer patients’ mortality. However, the underlying mechanism remains unknown. M2-like macrophages have pro-tumor functions, also exist in injured kidney, and promote kidney fibrosis. Thus, it is suspected that M2-like macrophages in injured kidney induce the pro-tumor microenvironment leading to kidney cancer progression. We found that M2-like macrophages present in the injured kidney promoted kidney cancer progression and induced resistance to anti-PD1 antibody through its pro-tumor function and inhibition of CD8+ T cell infiltration. RNA-seq revealed Slc7a11 was upregulated in M2-like macrophages. Inhibition of Slc7a11 with sulfasalazine inhibited the pro-tumor function of M2-like macrophages and synergized with anti-PD1 antibody. Moreover, SLC7A11-positive macrophages were associated with poor prognosis among kidney cancer patients. Collectively, this study dissects the characteristic microenvironment in the injured kidney that contributed to kidney cancer progression and anti-PD1 antibody resistance. This insight offers promising combination therapy with anti-PD1 antibody and macrophage targeted therapy.

IntroductionEpidemiological studies have shown that patients who recovered from acute kidney injury (AKI) may subsequently develop chronic kidney disease (CKD). AKI is primarily caused by renal hypoxia, and it causes epigenetic alterations, known as hypoxic memory. 3-Deazaneplanocin A (Dznep), an inhibitor of histone modification, suppresses renal fibrosis and the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), a profibrotic factor, in mouse ischemia–reperfusion models. The current study investigated the epigenetic regulation of TIMP2 in tubular cells.Methods and ResultsThe expression of TIMP2 was upregulated in human kidney 2 cells under hypoxic conditions and was suppressed by Dznep. ChIP-qPCR showed that Dznep reduced the expression of H3K4me3 at the promoter region of the TIMP2 gene under hypoxic condition. Formaldehyde-assisted isolation of regulatory elements-qPCR of the TIMP2 gene showed that Dznep reduced open chromatin area. In addition, based on ChIP-qPCR of hypoxia-inducible factor 1 alpha (HIF1α), Dznep inhibited the binding of HIF1α to the TIMP2 gene under hypoxic conditions.ConclusionDznep suppresses the expression of TIMP2 under hypoxic conditions by altering the histone methylations of the TIMP2 gene, decreasing open chromatin area, and inhibiting the binding of HIF1α to the TIMP2 gene.

Epigenetic memory, which refers to the ability of cells to retain and transmit epigenetic marks to their daughter cells, maintains unique gene expression patterns. Establishing programmed epigenetic memory at each stage of development is required for cell differentiation. Moreover, accumulating evidence shows that epigenetic memory acquired in response to environmental stimuli may be associated with diverse diseases. In the field of kidney diseases, the “memory” of acute kidney injury (AKI) leads to progression to chronic kidney disease (CKD); epidemiological studies show that patients who recover from AKI are at high risk of developing CKD. The underlying pathological processes include nephron loss, maladaptive epithelial repair, inflammation, and endothelial injury with vascular rarefaction. Further, epigenetic alterations may contribute as well to the pathophysiology of this AKI-to-CKD transition. Epigenetic changes induced by AKI, which can be recorded in cells, exert long-term effects as epigenetic memory. Considering the latest findings on the molecular basis of epigenetic memory and the pathophysiology of AKI-to-CKD transition, we propose here that epigenetic memory contributing to AKI-to-CKD transition can be classified according to the presence or absence of persistent changes in the associated regulation of gene expression, which we designate “driving” memory and “priming” memory, respectively. “Driving” memory, which persistently alters the regulation of gene expression, may contribute to disease progression by activating fibrogenic genes or inhibiting renoprotective genes. This process may be involved in generating the proinflammatory and profibrotic phenotypes of maladaptively repaired tubular cells after kidney injury. “Priming” memory is stored in seemingly successfully repaired tubular cells in the absence of detectable persistent phenotypic changes, which may enhance a subsequent transcriptional response to the second stimulus. This type of memory may contribute to AKI-to-CKD transition through the cumulative effects of enhanced expression of profibrotic genes required for wound repair after recurrent AKI. Further understanding of epigenetic memory will identify therapeutic targets of future epigenetic intervention to prevent AKI-to-CKD transition.

A 21-year-old woman was referred to our hospital because of proteinuria and hematuria. She had occasional flank pain. A renal biopsy was performed and revealed a thin basement membrane. Therefore, she was diagnosed with thin basement membrane disease. However, the frequency of her flank pain increased. Since her left kidney was slightly larger than the right, nutcracker syndrome (NCS) was suspected. Renal vein ultrasonography and venography were performed, and NCS was confirmed. Her hematuria was multifactorial, and NCS can go unnoticed if there is a comorbidity that also causes hematuria.

Renal tubulointerstitial hypoxia is recognized as a final common pathway of chronic kidney disease and is considered a promising drug target. However, hypoxia in the tubules is not well examined because of limited detection methods. Here, we devised a method to visualize renal tubular oxygen tension with spatial resolution at a cellular level using the cell-penetrating phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye), and confocal phosphorescence lifetime imaging microscopy to precisely assess renal hypoxia. Imaging with BTPDM1 revealed an oxygen gradient between S1 and S2 segments in mouse kidney. We also demonstrated that our microscopy system can detect subtle changes of hypoxemia and reoxygenation, and the acquired phosphorescence lifetime can be converted to partial pressure of oxygen. This new method allows, for the first time, visualization of intravital oxygen gradients at the renal surface with high spatial resolution. Thus, the confocal phosphorescence lifetime imaging microscopy platform, combined with BTPDM1, will promote an accurate understanding of tissue hypoxia, including renal hypoxia.

Kidney aging is a multifactorial process. Using genome-wide database analysis, Rowland et al. identified testis-specific Y-encoded–like protein 5 as a candidate age-related renal gene, and reported that with aging, testis-specific Y-encoded–like protein 5 expression decreases in response to increased testis-specific Y-encoded–like protein 5 promoter methylation. Genome-wide databases are readily available, and we should carefully analyze them to find a mechanism to fight kidney aging on the experiments using cells and animals, not just in silico calculations.

Acute kidney injury (AKI) was previously thought to be a merely transient event; however, recent epidemiological evidence supports the existence of a causal relationship between AKI episodes and subsequent progression to chronic kidney disease (CKD). Although the pathophysiology of this AKI-to-CKD transition is not fully understood, it is mediated by the interplay among multiple components of the kidney including tubular epithelial cells, endothelial cells, pericytes, inflammatory cells, and myofibroblasts. Epigenetic alterations including histone modification, DNA methylation, non-coding RNAs, and chromatin conformational changes, are also expected to be largely involved in the pathophysiology as a “memory” of the initial injury that can persist and predispose to chronic progression of fibrosis. Each epigenetic modification has a great potential as a therapeutic target of AKI-to-CKD transition; timely and target-specific epigenetic interventions to the various temporal stages of AKI-to-CKD transition will be the key to future therapeutic applications in clinical practice. This review elaborates on the latest knowledge of each mechanism and the currently available therapeutic agents that target epigenetic modification in the context of AKI-to-CKD transition. Further studies will elucidate more detailed mechanisms and novel therapeutic targets of AKI-to-CKD transition.

Chronic kidney disease presents a complex and distinct pathological landscape in men and women, yet this difference is poorly understood. microRNAs are powerful molecular regulators of pathophysiology in the kidney and other organs. We previously reported a significant upregulation of miR-146b-5p in the 5/6 nephrectomy rat model of chronic kidney disease. Here we investigated the sex-specific contribution of miR-146b-5p to renocardiac pathology by generating a novel miR-146b(−/−) rat and characterized the expression of miR-146b-5p in both wild-type and knockout animals. The 5/6 nephrectomy or sham surgery was performed on rats of each genotype and sex. Renal pathology was examined through gross histology, plasma and urinary analysis of electrolytes and metabolites, and by chronic blood pressure monitoring. Cardiac pathology was monitored via echocardiography and pressure-volume analysis. The miR-146b(−/−) rats show functional knockout of miR-146b-5p in both the kidney and heart. While 5/6 nephrectomy induced tissue hypertrophy, miR-146b(−/−) female rats displayed exacerbated renal hypertrophy. Additionally, miR-146b(−/−) female rats exhibited a marked elevation of renal fibrosis and significant renal dysfunction yet lower blood pressure and less pronounced cardiac remodeling. These phenotypic differences were not exhibited in miR-146b(−/−) male rats. Ovariectomy ameliorated renal pathology and abolished genotypic differences. In vitro examination of transforming growth factor-β signaling in combination with miR-146b-5p manipulation supports a role for miR-146b-5p in mediating the protective benefit of estrogen from renal pathologies. Thus, our data highlight an important role of miR-146b-5p in modulating kidney disease progression and provide new avenues for the study of sex-specific pathology

The number of patients with kidney failure has increased in recent years. Different factors contribute to the progression of chronic kidney disease, including glomerular sclerosis, atherosclerosis of the renal arteries and tubulointerstitial fibrosis. Tubulointerstitial injury is induced by hypoxia and other inflammatory signals, leading to fibroblast activation. Technological advances using high-throughput sequencing has enabled the determination ofthe expression profile of almost all genes, revealing that gene expression is intricately regulated by DNA methylation, histone modification, changes in chromosome conformation, long non-coding RNAs and microRNAs. These epigenetic modifications are stored as cellular epigenetic memory. Epigenetic memory leads to adult-onset disease or ageing in the long term and may possibley play an important role in the kidney disease process. Herein we emphasize the importance of clarifying the molecular mechanisms underlying epigenetic modifications because this may lead to the development of new therapeutic targets in kidney disease. This article is protected by copyright. All rights reserved.

Background: Kidney injury, including chronic kidney disease and acute kidney injury, is a worldwide health problem. Hypoxia and transforming growth factor-β (TGF-β) are well-known factors that promote kidney injury. Hypoxia-inducible factor (HIF) and SMAD3 are their main downstream transcriptional factors. Hypoxia-HIF pathway and TGF-β/SMAD3 pathway play a crucial role in the progression of kidney injury. However, reports on their interactions are limited, and the global transcriptional regulation under their control is almost unknown. Methods: Kidney tubular epithelial cells were cultured and stimulated by hypoxia and TGF-β. We detected global binding sites of HIF-1α and SMAD3 in cells using chromatin immunoprecipitation-sequencing (ChIP-Seq), and measured the gene expression using RNA-sequencing (RNA-Seq). ChIP-quantitative PCR (qPCR) was used to quantitatively evaluate bindings of SMAD3. Results: ChIP-Seq revealed that 2,065 and 5,003 sites were bound by HIF-1α and SMAD3, respectively, with 614 sites co-occupied by both factors. RNA-Seq showed that hypoxia and TGF-β stimulation causes synergistic upregulation of 249 genes, including collagen type I alpha 1 (COL1A1) and serpin peptidase inhibitor, clade E, member 1, which are well-known to be involved in fibrosis. Ontology of the 249 genes implied that the interaction of HIF-1α and SMAD3 is related to biological processes such as fibrosis. ChIP-qPCR of SMAD3 at HIF-1α binding sites near COL1A1 and SERPINE1 indicated that HIF-1α promotes the bindings of SMAD3, which is induced by TGF-β. Conclusions: These findings suggest that HIF-1α induced by hypoxia activates the TGF-β/SMAD3 pathway. This mechanism may promote kidney injury, especially by upregulating genes related to fibrosis.

Renal anemia is caused by the deficiency of endogenous erythropoietin (Epo) due to renal dysfunction. We think that it is possible to slow the progression of chronic kidney disease (CKD) in case we initiate Epo early in pre-dialysis patients, especially in the non-diabetic population. Erythropoiesis stimulating agent (ESA) treatments targeting mild anemia (10-12 g/dl) can decrease the risk of occurrence of cardiovascular disease (CVD) in patients with hypertension, diabetes mellitus and congestive heart failure. As the large randomized controlled trials such as Cardiovascular Risk Reduction by Early Anemia Treatment with Epoetin Beta, Correction of Hemoglobin and Outcomes in Renal Insufficiency and Trial to Reduce Cardiovascular Events with Aranesp Thearpy in the Western countries suggested, we do not recommend high doses of ESA to achieve the target hemoglobin (Hb) level. The target Hb of >13 g/dl might lead to increase in the risk of CVD although maintaining a high Hb of >12 g/dl without ESA is not harmful for CKD patients. It is desirable to determine the target Hb in dialysis patients depending on their ages. Renal anemia should be monitored constantly to start ESA and iron replacement therapy at an appropriate time, while avoiding their excess in order to minimize the occurrence of CVD and other complications. Taken all the international guidelines and our clinical experiences together, we should consider administration of ESA when the Hb level becomes

Previously acute kidney injury (AKI) had been believed to be a transient event, and recovery from AKI had been thought to lead to no consequences. However, recent epidemiological studies have shown that even if there is complete recovery of the kidney function, AKI can eventually result in chronic kidney disease (CKD) and eventually in end-stage kidney disease in the long term. Transition of AKI to CKD is mediated by multiple mechanisms, including aberrant cell cycle arrest and hypoxia. Hypoxia of the kidney is induced by rarefaction of the peritubular capillaries after AKI episodes, and induces inflammation and fibrosis. It should also be noted that epigenetic changes are closely related to hypoxia, and epigenetic changes induced by hypoxia, called “hypoxic memory” can explain the AKI-to-CKD transition in the long term after complete recovery from the initial AKI episode. Targeting hypoxia and subsequent epigenetic changes are promising strategies to block the transition from AKI to CKD.

MicroRNA (miR) are small noncoding RNA molecules that regulate gene expression and play important roles during kidney development, homeostasis, and disease. Novel contributions to the field of cell death, cell cycle, and miR regulation in the kidney have come from the Laboratory of Dong, and in the

Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end‐stage renal disease. HIF‐1 (hypoxia‐inducible factor‐1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome‐wide analysis of HIF‐1 binding sites using high‐throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF‐1. Our aim of this study is to identify novel HIF‐1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney‐2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24–72 h. We performed RNA‐seq to clarify the expression of mRNA and long non‐coding RNA (lncRNA). We also examined ChIP‐seq to identify HIF‐1 binding sites under hypoxia. RNA‐seq identified 44 lncRNAs which are up‐regulated under hypoxic condition in both cells. ChIP‐seq analysis demonstrated that HIF‐1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS‐AS1 (aspartyl‐tRNA synthetase anti‐sense 1), is up‐regulated only under hypoxia and HIF‐1 binds to its promoter region, which includes two hypoxia‐responsive elements. Its expression is also up‐regulated with cobalt chloride exposure, while it is not under hypoxia when HIF‐1 is knocked down by siRNA. To clarify the biological roles of DARS‐AS1, we measured the activity of caspase 3/7 using anti‐sense oligo of DARS‐AS1. Knockdown of DARS‐AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF‐1 under hypoxia and clarified that DARS‐AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells.

Aim: In end-stage renal disease (ESRD) patients, left ventricular hypertrophy (LVH) is common and a risk for cardiovascular events. LVH is geometrically classified into two major groups, concentric and eccentric, and accumulating evidence suggests eccentric LVH has a more negative effect than concentric LVH on ESRD outcome. However, there have been very few studies on the cardiac findings from ESRD patient autopsy in which the relationship between LVH geometry and mortality was analyzed. Methods: An observational study was performed with the autopsy findings in 30 haemodialysis patient cases between 2001 and 2006 at Mitsui Memorial Hospital, Tokyo. Between those who died of a cardiovascular cause and those who died of non-cardiovascular causes, we compared the heart/bodyweight ratio, left ventricular dilatation, and the extent of fibrosis of the left ventricle. Results: Heart/bodyweight ratio was significantly higher (P

70歳，男性．糖尿病性腎症を原疾患とし，15年の透析歴がある．1995年頃から下血を主訴に他院で消化管精査行うも出血源特定できず．AB型Rh(－）と特殊な血液型のため製剤入手に苦慮した．精査加療目的で2006年6月当院初診．小腸内視鏡上，Treitz靭帯を越えて3mの部分の拍動性の出血性病変に対してAPC焼灼とクリップで止血を行った．その後も2006年11月，2007年8月，9月に同様のエピソードがあり，内視鏡上，別部位に出血を認めたが，同様の処置を行い止血が可能であった．本例では，頻回の再発を認め，また入手困難な血液型であったため治療に苦慮したが，小腸内視鏡による止血を得た．ESRD患者は消化管出血の頻度が高く，上部下部内視鏡上，出血源が特定できず診断，治療が困難なことも多い．小腸内視鏡はこうした例でも開腹術を行わずに止血が得られ，有効な手段である．

血液透析に不可欠のバスキュラーアクセスの必要条件は，安定した血液透析が可能で，心負荷の点で患者のQOLを損なわないことである．今回，血液透析導入時のバスキュラーアクセス選択について背景因子を検討した．2003年と2004年に当院で血液透析導入した94名において，プライマリーアクセスとして動静脈内シャント（以下AVF）は79例（85％）であった．AVF以外の15例は，心機能低下（10例），動静脈の問題（8例）のいずれかまたは両者を有していた．動脈表在化を選択した低心機能の患者は比較的長期にわたり安定した維持透析が可能であった．また，長期型バスキュラーカテーテルを選択した4例は心機能低下のほか，麻酔の侵襲が危惧される全身状態不良例であった．長期型バスキュラーカテーテル4例はいずれも，1～29か月で死亡していたが，その原因は感染ではなく，すべて原病の増悪あるいは突然死であった．アクセス選択に影響する患者因子は心機能，動脈硬化症の進行，前腕静脈の荒廃，加齢であった．

Epigenetic mechanisms have been the focus of intensive research. De Marinis et al. demonstrated that high glucose levels exert stimulatory effects on activation histone marks, leading to the upregulation of thioredoxin-interacting protein ( TXNIP ) gene expression, which is proinflammatory. They also showed that the effect was reversed by the inhibition of histone acetyltransferase, suggesting a new therapeutic approach for improving diabetic kidney disease. Epigenetic changes are memorized as epigenetic memory that could exacerbate diabetic complications.

Patients with advanced chronic kidney disease are exposed to uremic toxins. In addition to causing uremic symptoms, uremic toxins accelerate the progression of renal failure. Indoxyl sulfate (IS) increases oxygen consumption in tubules, aggravating hypoxia of the kidney, and progression of the kidney disease. IS also induces endoplasmic reticulum stress and thereby contributes the progression of cellular damages in tubular epithelial cells. Hypoxia-inducible factor (HIF) is a master transcriptional regulator of adaptive responses against hypoxia and regulates expression of erythropoietin (EPO). IS suppresses EPO expression via HIF-dependent and HIF-independent manner. IS impedes the recruitment of transcriptional coactivators to HIF via upregulation of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 through a mechanism of posttranscriptional messenger RNA stabilization. Furthermore, IS induces activating transcription factor 4 via endoplasmic reticulum stress, decreasing EPO expression. Although erythropoiesis-stimulating agent (ESA) resistance is generally defined as lack of responses to exogenous ESA administration, suppression of endogenous production of EPO under uremic conditions may aggravate ESA resistance. Uremia is associated with increased formation of advanced glycation end products (AGE). Studies of transgenic rats overexpressing glyoxalse 1 (GLO1), which detoxifies precursors of advanced glycation end products, demonstrated that glycative stress causes renal senescence and vascular endothelial dysfunction. Glycative stress also suppresses HIF activation making the kidney susceptible to hypoxia as a final common pathway to end-stage kidney disease.

Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.

Increasing evidence has demonstrated that chronic hypoxia in the tubulointerstitium results in irreversible chronic kidney diseases. Hypoxia inducible factor (HIF) is a transcriptional master regulator which takes control of gene expressions under hypoxia. Recently, HIF1 has been reported to organize a cluster of histone-modifying enzymes by binding to their promoter regions in various kinds of cell line. In order to clarify the epigenetic molecular mechanisms by HIF1, we examined the genome-wide analysis of HIF1-binding sites (ChIP-seq) in endothelial cells and HIF1 downstream target genes using DNA microarrays. ChIP-seq results demonstrated that HIF1 binds to the enhancer regions in addition to the promoter regions. We clarified that one of the HIF1 downstream genes, SLC2A3 (solute-carrier family 2A3, also known as glucose transporter 3: GLUT3), is regulated by changing chromosomal conformations under hypoxia via a cooperative combination of HIF1 and KDM3A(lysine(K) specific demethylase 3A), one of the histone demethylases. KDM3A is recruited to the SLC2A3 loci in an HIF1-dependent manner and demethylates histone repressive mark, H3K9me2, up-regulating its expression. In addition, we confirmed the interactions of HIF1 and KDM3A only under hypoxia using co-immunoprecipitation. These experimental results showed novel HIF1-dependent molecular mechanisms from an epigenetic viewpoint. It is important to elucidate the epigenetic mechanisms of chronic hypoxia in order to identify novel therapeutic approaches against chronic kidney disease.

Hypoxia-inducible factor (HIF) is a transcriptional master regulator that takes control of the gene expressions under hypoxia. Several lines of evidence have shown that chronic hypoxia in tubulointerstitium results in irreversible renal disease. Recently, HIF1 was reported to organize a cluster of histone-modifying enzymes by binding to their promoter regions in various kinds of cell lines. However, its function in renal disease remains largely unknown. We focused on the epigenetic regulation on the progression of chronic kidney disease and have reviewed the latest knowledge in this area with special emphasis on the involvement of HIF. For example, a set of HIF1 downstream target genes also were reported to be regulated by cooperative combination of HIF1 and histone demethylase. We suggest a novel epigenetic pathway that affects the final common pathway to end-stage renal disease in addition to the tubulointerstitial hypoxia. We emphasize the importance of figuring out the epigenetic mechanisms of renal failure to find the novel therapeutic approach of chronic kidney disease.

Hypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, and hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia. To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing in combination with microarray analysis and identified HIF-1 targets. We focused on one of the novel targets, sperm-associated antigen 4 (SPAG4), whose function was unknown. SPAG4, an HIF-1-specific target, is up-regulated in various cultured cells under hypoxia. Examination of SPAG4 expression using a tissue microarray consisting of 190 human renal cell carcinoma (RCC) samples revealed that SPAG4 is an independent prognostic factor of cancer-specific mortality. Live-cell imaging revealed localization of SPAG4 at the intercellular bridge in telophase. We also studied cells in which SPAG4 was knocked down. Hypoxia enhances tetraploidy, which disturbs cell proliferation, and knockdown of SPAG4 increased tetraploid formation and decreased cell proliferation under both normoxic and hypoxic conditions. Studies using deletion mutants of SPAG4 also suggested the involvement of SPAG4 in cytokinesis. Microarray analysis confirmed dysregulation of cytokinesis-related genes by knockdown of SPAG4. In conclusion, SPAG4 is an independent prognostic factor in RCC and plays a crucial role in cytokinesis to defend against hypoxia-induced tetraploid formation. This defensive mechanism may promote survival of cancer cells under hypoxic conditions, thus leading to poor prognosis.

The recent and rapid advent of next-generation sequencing (NGS) has made this technology broadly available not only to researchers in various molecular and cellular biology fields but also to those in kidney disease. In this paper, we describe the usage of ChIP-seq (chromatin immunoprecipitation with sequencing) and RNA-seq for sample preparation and interpretation of raw data in the investigation of biological phenomenon in renal diseases. ChIP-seq identifies genome-wide transcriptional DNA-binding sites as well as histone modifications, which are known to regulate gene expression, in the intragenic as well as in the intergenic regions. With regard to RNA-seq, this process analyzes not only the expression level of mRNA but also splicing variants, non-coding RNA, and microRNA on a genome-wide scale. The combination of ChIP-seq and RNA-seq allows the clarification of novel transcriptional mechanisms, which have important roles in various kinds of diseases, including chronic kidney disease. The rapid development of these techniques requires an update on the latest information and methods of NGS. In this review, we highlight the merits and characteristics of ChIP-seq and RNA-seq and discuss the use of the genome-wide analysis in kidney disease.

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 HIF1α 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.

The hypoxia response regulated primarily by hypoxia-inducible factor (HIF) influences metabolism, cell survival, and angiogenesis to maintain biological homeostasis. In addition to the traditional transcriptional regulation by HIF, recent studies have shown that epigenetic modulation such as histone methylation, acetylation, and DNA methylation could change the regulation of the response to hypoxia. Eukaryotic chromatin is known to be modified by multiple post-translational histone methylation and demethylation, which result in the chromatin conformation change to adapt to hypoxic stimuli. Interestingly, some of the histone demethylase enzymes, which have the Jumonji domain–containing family, require oxygen to function and are induced by hypoxia in an HIF-1–dependent manner. Recent studies have demonstrated that histone modifiers play important roles in the hypoxic environment such as that in cancer cells and that they may become new therapeutic targets for cancer patients. It may lead to finding a new therapy for cancer to clarify a new epigenetic mechanism by HIF and histone demethylase such as JMJD1A (KDM3A) under hypoxia. Keywords:: hypoxia, hypoxia-inducible factor (HIF), histone demethylase, JMJD1A, epigenetics

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.

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.

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.

In end-stage renal disease (ESRD) patients, left ventricular hypertrophy (LVH) is common and a risk for cardiovascular events. LVH is geometrically classified into two major groups, concentric and eccentric, and accumulating evidence suggests eccentric LVH has a more negative effect than concentric LVH on ESRD outcome. However, there have been very few studies on the cardiac findings from ESRD patient autopsy in which the relationship between LVH geometry and mortality was analyzed.An observational study was performed with the autopsy findings in 30 haemodialysis patient cases between 2001 and 2006 at Mitsui Memorial Hospital, Tokyo. Between those who died of a cardiovascular cause and those who died of non-cardiovascular causes, we compared the heart/bodyweight ratio, left ventricular dilatation, and the extent of fibrosis of the left ventricle.Heart/bodyweight ratio was significantly higher (P0.0001) in the cardiovascular mortality group (n = 11, 11.7 +/- 2.5 g/kg) compared to the non-cardiac cause of death group (n = 19, 8.05 +/- 0.7 g/kg). The dilatation of the left ventricle was significantly more frequent in the cardiovascular than the non-cardiac cause of death group (P = 0.016). Additionally, the fibrotic area of left ventricular cross-section was larger in the cardiovascular (1.63 +/- 1.6%) than the non-cardiac group (0.83 +/- 1.7%, P = 0.04).This autopsy study indicates that eccentric LVH in haemodialysis patients is closely associated with cardiovascular mortality. LVH geometry, as well as LVH severity, is worthy of consideration as a clinical predictor for cardiovascular mortality.

Background/Aims: A significant number of uremic patients develop ischemic heart disease before hemodialysis (HD) is initiated. Recently, chronic cardiorenal syndrome among predialysis patients has been recognized. However, little is known about prognostic factors in this subgroup of incident HD patients. Methods: A total of 87 incident HD patients, who were classified into cardiorenal syndrome type 4 (chronic cardiorenal syndrome), were identified at Mitsui Memorial Hospital between 1984 and 2003. The survival and risk factors for mortality were examined. Results: 25 patients died and the 5-year survival rate amounted to approximately 75%. Both all-cause mortality and the adjusted mortality for age and sex were higher in patients with a lower serum albumin level (p = 0.03) or higher serum C-reactive protein level (p = 0.02). Conclusion: The poor survival rate of incident HD patients with a medical history of ischemic heart disease was predicted by malnutrition and inflammation at the start of HD.

IgA nephropathy can occur as a paraneoplastic syndrome of renal cell carcinoma. We report 3 cases of IgA nephropathy associated with renal cell carcinoma in the elderly patients and demonstrate that infiltrating lymphocytes and plasma cells around renal cell carcinoma produce IgA that likely contributed to mesangial IgA deposition.

A 72-year-old man was admitted with acute epididymis from methicillin-resistant Staphylococcus aureus. Minocycline and vancomycin were prescribed with control of symptoms. However, he subsequently developed acute renal failure and pancytopenia, and underwent hemodialysis on the twelfth day of admission. Urinalysis demonstrated 3+ protein and 3+ hemoglobin with dysmorphic erythrocytes in the urinary sediment. Renal biopsy showed endocapillary proliferation with crescents, capillary C3 deposition, and subepithelial electron dense deposits (humps), consistent with postinfectious acute glomerulonephritis. Azan staining showed erythrocytes in the tubular lumen as well as in the cytoplasm of the distal nephron without brush border (Figure 1a). Electron microscopy revealed erythrophagocytosis by intercalated cells (characterized by a large cell body, prominent mitochondria, and apical microplicae and vesicles) (Figure 1b and c). Erythrophagocytosis has also been demonstrated in the macrophages of the bone marrow, liver, and spleen; and epithelial cells in the small intestine, placenta, urinary bladder, and thyroid. Although erythrophagocytosis in the renal tubular cells has been first described by Tisher et al. over 20 years ago, its physiologic significance is uncertain.

We reported a 67-year-old woman with unilateral laminar necrosis caused by hypoxic encephalopathy. She presented with sudden onset of dyspnea, unconsciousness and left hemiplegia. Although diffusion-weighted MRI image of the brain 14 hours after onset was indistinct, MRI on the 4th day showed right laminar necrosis. Cerebral angiography demonstrated complete obstruction of the right proximal common carotid artery and severe stenosis of the left internal carotid artery. We speculate that chronic ipsilateral obstruction of the internal carotid artery may modify the unique distribution of laminar necrosis. As shown in this case, laminar necrosis can be demonstrated only on subsequent MRI. If clinical suspicion of hypoxic encephalopathy is high, repeated MRI may be appropriate.

Background Synergistic 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. Results We 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. Conclusions To 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.