Maintenance of R-loop structures by phosphorylated hTERT preserves genome integrity.

As aberrant accumulation of RNA-DNA hybrids (R-loops) causes DNA damage and genome instability, cells express regulators of R-loop structures. Here we report that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates R-loop formation. We found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles both in telomerase-positive cells and telomerase-negative cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT did not associate with telomerase RNA component in nuclear speckles but, instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT gene in ALT cells ablated RdRP activity and impaired tumour growth. Using a genome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures and DNA damage. These findings indicate that RdRP activity of p-hTERT guards against genome instability by removing R-loop structures.

Angiotensin II type 1 receptor-associated protein deletion combined with angiotensin II stimulation accelerates the development of diabetic kidney disease in mice on a C57BL/6 strain.

The progress in the research field of diabetic kidney disease (DKD) has been disturbed by the lack of reliable animal models. Angiotensin II (Ang II) type 1 receptor (AT1R)-associated protein (ATRAP) promotes internalization of AT1R and selectively inhibits pathological AT1R signaling. In this study, we investigated whether overactivation of the renin-angiotensin system (RAS) through a combination of ATRAP deletion with Ang II stimulation developed a progressive DKD model in C57BL/6 mice, which are resistant to the development of kidney injury. Eight-week-old male systemic ATRAP-knockout mice on the C57BL/6 strain (KO) and their littermate wild-type mice (Ctrl) were divided into five groups: 1) Ctrl, 2) Ctrl-streptozotocin (STZ), 3) KO-STZ, 4) Ctrl-STZ-Ang II, and 5) KO-STZ-Ang II. Ang II was administered for 6 weeks from 4 weeks after STZ administration. At 10 weeks after STZ administration, mice were euthanized to evaluate kidney injuries. Neither ATRAP deletion alone nor Ang II stimulation alone developed a progressive DKD model in STZ-induced diabetic C57BL/6 mice. However, a combination of ATRAP deletion with Ang II stimulation accelerated the development of DKD as manifested by overt albuminuria, glomerular hypertrophy, podocyte loss, mesangial expansion, kidney interstitial fibrosis and functional insufficiency, concomitant with increased angiotensinogen and AT1R expression in the kidneys. In STZ-induced diabetic C57BL/6 mice that are resistant to the development of kidney injury, the combination of ATRAP deletion and Ang II stimulation accelerates the development of DKD, which may be associated with intrarenal RAS overactivation.

[Thoracic Endovascular Aortic Repair for Mycotic Thoracic Aortic Aneurysm After BCG Intravesical Therapy].

Bacille Calmette-Guérin( BCG) intravesical therapy is an effective and safe treatment for bladder cancer; however, mycotic aneurysms have been reported as a rare complication. Case 1:A 64-year-old man with a history of BCG intravesical therapy underwent emergent thoracic endovascular aortic repair (TEVAR) for a ruptured thoracic aortic aneurysm (TAA). He was diagnosed with BCG infection by hemosputum specimen culture five months later;then, antituberculous therapy was initiated. However, his follow-up computed tomography scan revealed stent-graft infection and new aneurysm formation. Therefore, we performed a repeated TEVAR with abdominal 4-vessel debranching. There was no recurrence of infection for six years while continuing postoperative antituberculous therapy. Case 2:A 72-year-old man who had undergone BCG intravesical therapy underwent TEVAR for a rapidly enlarging mycotic TAA. He received anti-tuberculous therapy for one year with no recurrent infection for one year. TEVAR may be an effective alternative to the open surgical procedure;however, multidisciplinary treatment including anti-tuberculous therapy and careful long-term follow up are required.

Combination of sacubitril/valsartan and blockade of the PI3K pathway enhanced kidney protection in a mouse model of cardiorenal syndrome.

Aims: Angiotensin receptor-neprilysin inhibitor (ARNI) is an established treatment for heart failure. However, whether ARNI has renoprotective effects beyond renin-angiotensin system inhibitors alone in cardiorenal syndrome (CRS) has not been fully elucidated. Here, we examined the effects of ARNI on the heart and kidneys of CRS model mice with overt albuminuria and identified the mechanisms underlying ARNI-induced kidney protection. Methods and results: C57BL6 mice were subjected to chronic angiotensin II infusion, nephrectomy, and salt loading (ANS); they developed CRS phenotypes and were divided into the vehicle treatment (ANS-vehicle), sacubitril/valsartan treatment (ANS-ARNI), and two different doses of valsartan treatment (ANS-VAL M, ANS-VAL H) groups. Four weeks after treatment, the hearts and kidneys of each group were evaluated. The ANS-vehicle group showed cardiac fibrosis, cardiac dysfunction, overt albuminuria, and kidney fibrosis. The ANS-ARNI group showed a reduction in cardiac fibrosis and cardiac dysfunction compared with the valsartan treatment groups. However, regarding the renoprotective effects characterized by albuminuria and fibrosis, ARNI was less effective than valsartan. Kidney transcriptomic analysis showed that the ANS-ARNI group exhibited a significant enhancement in the phosphoinositide 3-kinase (PI3K)-AKT signalling pathway compared with the ANS-VAL M group. Adding PI3K inhibitor treatment to ARNI ameliorated kidney injury to levels comparable with those of ANS-VAL M while preserving the superior cardioprotective effect of ARNI. Conclusion: PI3K pathway activation has been identified as a key mechanism affecting remnant kidney injury under ARNI treatment in CRS pathology, and blockading the PI3K pathway with simultaneous ARNI treatment is a potential therapeutic strategy for treating CRS with overt albuminuria.

miR-125a-5p/miR-125b-5p contributes to pathological activation of angiotensin II-AT1R in mouse distal convoluted tubule cells by the suppression of Atrap.

The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.

Angiotensin II type 1 receptor-associated protein in immune cells: a possible key factor in the pathogenesis of visceral obesity.

BACKGROUND AND AIM: Dysregulation of angiotensin II type 1 receptor-associated protein (ATRAP) expression in cardiovascular, kidney, and adipose tissues is involved in the pathology of hypertension, cardiac hypertrophy, atherosclerosis, kidney injury, and metabolic disorders. Furthermore, ATRAP is highly expressed in bone marrow-derived immune cells; however, the functional role of immune cell ATRAP in obesity-related pathology remains unclear. Thus, we sought to identify the pathophysiological significance of immune cell ATRAP in the development of visceral obesity and obesity-related metabolic disorders using a mouse model of diet-induced obesity. METHODS: Initially, we examined the effect of high-fat diet (HFD)-induced obesity on the expression of immune cell ATRAP in wild-type mice. Subsequently, we conducted bone marrow transplantation to generate two types of chimeric mice: bone marrow wild-type chimeric (BM-WT) and bone marrow ATRAP knockout chimeric (BM-KO) mice. These chimeric mice were provided an HFD to induce visceral obesity, and then the effects of immune cell ATRAP deficiency on physiological parameters and adipose tissue in the chimeric mice were investigated. RESULTS: In wild-type mice, body weight increase by HFD was associated with increased expression of immune cell ATRAP. In the bone marrow transplantation experiments, BM-KO mice exhibited amelioration of HFD-induced weight gain and visceral fat expansion with small adipocytes compared BM-WT mice. In addition, BM-KO mice on the HFD showed significant improvements in white adipose tissue metabolism, inflammation, glucose tolerance, and insulin resistance, compared with BM-WT mice on the HFD. Detailed analysis of white adipose tissue revealed significant suppression of HFD-induced activation of transforming growth factor-beta signaling, a key contributor to visceral obesity, via amelioration of CD206+ macrophage accumulation in the adipose tissue of BM-KO mice. This finding suggests a relevant mechanism for the anti-obesity phenotype in BM-KO mice on the HFD. Finally, transcriptome analysis of monocytes indicated the possibility of genetic changes, such as the enhancement of interferon-γ response at the monocyte level, affecting macrophage differentiation in BM-KO mice. CONCLUSION: Collectively, our results indicate that ATRAP in bone marrow-derived immune cells plays a role in the pathogenesis of visceral obesity. The regulation of ATRAP expression in immune cells may be a key factor against visceral adipose obesity with metabolic disorders.

Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification.

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires nascent RNA synthesis and translation. Analysis of the coordinated regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprogramming of nascent RNA synthesis and translation. Our nascent P-TRAP (nP-TRAP) method may serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

Effects of a High-Protein Diet on Kidney Injury under Conditions of Non-CKD or CKD in Mice.

Considering the prevalence of obesity and global aging, the consumption of a high-protein diet (HPD) may be advantageous. However, an HPD aggravates kidney dysfunction in patients with chronic kidney disease (CKD). Moreover, the effects of an HPD on kidney function in healthy individuals are controversial. In this study, we employed a remnant kidney mouse model as a CKD model and aimed to evaluate the effects of an HPD on kidney injury under conditions of non-CKD and CKD. Mice were divided into four groups: a sham surgery (sham) + normal diet (ND) group, a sham + HPD group, a 5/6 nephrectomy (Nx) + ND group and a 5/6 Nx + HPD group. Blood pressure, kidney function and kidney tissue injury were compared after 12 weeks of diet loading among the four groups. The 5/6 Nx groups displayed blood pressure elevation, kidney function decline, glomerular injury and tubular injury compared with the sham groups. Furthermore, an HPD exacerbated glomerular injury only in the 5/6 Nx group; however, an HPD did not cause kidney injury in the sham group. Clinical application of these results suggests that patients with CKD should follow a protein-restricted diet to prevent the exacerbation of kidney injury, while healthy individuals can maintain an HPD without worrying about the adverse effects.

An atlas of substrate specificities for the human serine/threonine kinome.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Angiotensin II type-1 receptor-associated protein interacts with transferrin receptor-1 and promotes its internalization.

Kidney fibrosis is a common pathway that leads to chronic kidney disease. Angiotensin II type-1 receptor (AT1R)-associated protein (ATRAP) was originally identified as an AT1R-binding protein. Previously, we reported that systemic knockout of ATRAP exacerbates kidney fibrosis in aged mice. Although these effects of ATRAP appeared to be AT1R-independent actions, the molecular mechanism remains poorly understood. To elucidate the molecular mechanism of ATRAP independent of AT1R, we explored novel ATRAP-interacting proteins. Mass spectrometric analysis of the immunoprecipitants of a Flag-tagged ATRAP complex revealed 376 candidate proteins that potentially interact with ATRAP. Gene ontology analysis revealed that proteins related to vesicle trafficking, membrane transport, and many membrane proteins, including transferrin receptor 1 (TfR1), were enriched. Because TfR1 promotes cellular iron uptake and iron is a key factor involved in kidney fibrosis, we focused on TfR1 and confirmed that it interacts with ATRAP. In addition, our findings revealed that enhanced ATRAP expression decreased cell-surface TfR1 expression without altering the overall cellular TfR1 expression levels. Furthermore, enhanced ATRAP expression attenuated cellular iron levels. Together, our results highlight the role of ATRAP as a suppressor of TfR1 that functions by facilitating TfR1 internalization, which affects iron metabolism and oxidative stress signaling.

Mitral valve plasty in a patient with multiple cerebral cavernous malformations: A case report.

Cerebral cavernous malformations (CCMs) are blood vessel malformations, often untreated if asymptomatic. However, upon cardiac surgery with cardiopulmonary bypass, cerebral edema/hemorrhage may occur. We successful performed mitral valve plasty for a case of severe mitral regurgitation with multiple CCMs. Preoperative head magnetic resonance imaging and strict perioperative management are important.

Takayasu Arteritis Complicated by Ischemic Colitis: A Case Report.

Takayasu arteritis is an inflammatory disease of the aorta and its major branches, which results in stenosis and aneurysm formation. Lesions of the abdominal aorta and renal arteries are common. Nevertheless, lesions of the celiac and superior mesenteric arteries are less common. Since the inferior mesenteric artery is usually preserved and functions as a collateral pathway, developing intestinal ischemia is very rare in patients with Takayasu arteritis. In this study, we report the case of a patient with Takayasu arteritis complicated by ischemic colitis. The patient was treated with surgical repair, which resolved the patient's symptoms.

Deficiency of the kidney tubular angiotensin II type1 receptor-associated protein ATRAP exacerbates streptozotocin-induced diabetic glomerular injury via reducing protective macrophage polarization.

Although activation of the renin-angiotensin system and of its glomerular components is implicated in the pathogenesis of diabetic nephropathy, the functional roles of the tubular renin-angiotensin system with AT1 receptor signaling in diabetic nephropathy are unclear. Tissue hyperactivity of the renin-angiotensin system is inhibited by the angiotensin II type 1 receptor-associated protein ATRAP, which negatively regulates receptor signaling. The highest expression of endogenous ATRAP occurs in the kidney, where it is mainly expressed by tubules but rarely in glomeruli. Here, we found that hyperactivation of angiotensin II type 1 receptor signaling in kidney tubules exacerbated diabetic glomerular injury in a mouse model of streptozotocin-induced diabetic nephropathy. These phenomena were accompanied by decreased expression of CD206, a marker of alternatively activated and tissue-reparative M2 macrophages, in the kidney tubulointerstitium. Additionally, adoptive transfer of M2- polarized macrophages into diabetic ATRAP-knockout mice ameliorated the glomerular injury. As a possible mechanism, the glomerular mRNA levels of tumor necrosis factor-α and oxidative stress components were increased in diabetic knockout mice compared to non-diabetic knockout mice, but these increases were ameliorated by adoptive transfer. Furthermore, proximal tubule-specific ATRAP downregulation reduced tubulointerstitial expression of CD206, the marker of M2 macrophages in diabetic mice. Thus, our findings indicate that tubular ATRAP-mediated functional modulation of angiotensin II type 1 receptor signaling modulates the accumulation of tubulointerstitial M2 macrophages, thus affecting glomerular manifestations of diabetic nephropathy via tubule-glomerular crosstalk.

Diagnosis of Carney complex following multiple recurrent cardiac myxomas.

Carney complex is a rare syndrome caused by a genetic mutation leading to multiple endocrine abnormalities and a variety of tumors. Here, we report a case of Carney complex diagnosed due to recurrent multiple myxomas in the right atrium of a patient 16 years after the resection of the primary left atrial myxoma. Surgical excision was performed for the multiple recurrent right atrial tumors under cardiopulmonary bypass. The patient remained complication-free after surgery and was discharged on the 14th day. He was scheduled to continue echocardiographic follow-up and periodic systemic review by an endocrinologist. This case emphasizes the fact that if cardiac myxomas tend to be multiple and recurrent at a relatively young age, the possibility of Carney complex should be considered, even in the absence of any other related feature other than cardiac tumors.

Effects of tumor necrosis factor-α inhibition on kidney fibrosis and inflammation in a mouse model of aristolochic acid nephropathy.

Tumor necrosis factor (TNF)-α is a potent mediator of inflammation and is involved in the pathophysiology of chronic kidney disease (CKD). However, the effects of TNF-α inhibition on the progression of kidney fibrosis have not been fully elucidated. We examined the effects of TNF-α inhibition by etanercept (ETN) on kidney inflammation and fibrosis in mice with aristolochic acid (AA) nephropathy as a model of kidney fibrosis. C57BL/6 J mice were administered AA for 4 weeks, followed by a 4-week remodeling period. The mice exhibited kidney fibrosis, functional decline, and albuminuria concomitant with increases in renal mRNA expression of inflammation- and fibrosis-related genes. The 8-week ETN treatment partially but significantly attenuated kidney fibrosis and ameliorated albuminuria without affecting kidney function. These findings were accompanied by significant suppression of interleukin (IL)-1ß, IL-6, and collagen types I and III mRNA expression. Moreover, ETN tended to reduce the AA-induced increase in interstitial TUNEL-positive cells with a significant reduction in Bax mRNA expression. Renal phosphorylated p38 MAPK was significantly upregulated by AA but was normalized by ETN. These findings indicate a substantial role for the TNF-α pathway in the pathogenesis of kidney fibrosis and suggest that TNF-α inhibition could become an adjunct therapeutic strategy for CKD with fibrosis.

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice.

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated ß-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.

Anastomotic stenosis of a reconstructed dissecting superior mesenteric artery aneurysm undetectable by intraoperative indocyanine green angiography: A case report.

Intraoperative evaluation of blood flow using ICG angiography revealed no significant abnormality. However, the anastomotic stenosis was revealed by postoperative CT angiography; more precise intraoperative evaluation methods need to be developed.

Application of the Konno procedure for infective endocarditis in native bicuspid aortic valve with annular abscess extending into the interventricular septum.

Annular abscess is a serious complication of infective endocarditis, which often requires complex surgery and has a very high post-operative mortality rate. The Konno procedure involves valve annuloplasty for a narrow aortic annulus or left ventricular outflow tract stenosis in children; it is also performed for various cardiac conditions in adults. Here, we report a case of the Konno procedure performed in a patient with aortic valve infective endocarditis, with an annular abscess extending into the interventricular septum (IVS). A 58-year-old man who presented to our hospital with fever was diagnosed with aortic valve infective endocarditis caused by Streptococcus saccharolyticus. On echocardiography, an annular abscess in the direction of the IVS was detected, and surgery was planned. The Konno procedure was performed to secure an adequate surgical field and to debride and reconstruct the cavity created by the interventricular septal abscess. The patient was discharged uneventfully 29 days after surgery.

Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines.

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs, DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells, that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type-specific manner. We identified additional protein components present in DNB by immunoprecipitation-mass spectrometry, some of which (DBC1 and heterogeneous nuclear ribonucleoprotein L [HNRNPL]) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.