Tubular Endogenous Erythropoietin Protects Renal Function against Ischemic Reperfusion Injury.

Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24-72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24-72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24-72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection.

Immunohistochemical analysis of the distribution of RANKL: a case of disseminated carcinomatosis of bone marrow as the first presentation of relapse in curatively resected colorectal cancer.

Poorly differentiated adenocarcinoma of colorectal carcinoma (CRC) is a rare condition with poor prognosis. In this report, we describe a case of a 69-year-old man who underwent laparoscopic low anterior resection after being diagnosed with stage IIIB CRC. At 10 months post-operation, he developed fever and loss of appetite. Laboratory examination revealed > 120.0 µg/dL fibrin degradation products and > 60.0 µg/dL D-dimer. Bone marrow (BM) examination showed malignant epithelioid infiltrate with CK20 and CDX2 expression, leading to diagnosis of disseminated carcinomatosis of BM, which is rare in CRC and indicative of widespread disease throughout the body. Furthermore, immunohistochemistry revealed high expression of receptor activator of nuclear factor κB ligand (RANKL) in tumor cells, including budding cells of CRC and BM tissues. Thus, RANKL expression, which is known to indicate metastatic behavior of cancer cells, may play a critical role in promoting osteoclast formation, which has been associated with the pathogenesis of BM lesions.

Effects of Roxadustat on Erythropoietin Production in the Rat Body.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver.

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin-angiotensin-aldosterone system (RAS).

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

Case report: A case of acute exacerbation of interstitial pneumonia associated with TAFRO syndrome.

Cytokine storm caused by the overproduction of inflammatory interleukin (IL)-6 plays a central role in the development of acute inflammation. The extremely rare disease, TAFRO syndrome, progresses quickly. Renal dysfunction, fever, reticulin fibrosis, anasarca, thrombocytopenia, and organomegaly with pathological findings such as idiopathic multicentric Castleman disease are all characteristics of TAFRO syndrome. Interstitial pneumonia (IP), which is not characteristic of this disease, is probably a complication of the inflammatory process. An 88-year-old man presented with a 3-day history of fever, dry cough, and progressive dyspnea. After he was first treated with antibiotics, he was transferred to our hospital because he showed no improvement. Data showed hemoglobin Hb 90.00 (SI) (9.0 g/dL); leukocyte count WBC 23 × 109/L (SI) [23,000/µL (neutrophils 87.5%, lymphocytes 2.5%, blast cells 0%)]; hemoglobin 90 g/L (9.0 g/dL); platelet count 101.00 × 109/L (10 100/µL); lactate dehydrogenase 4.78 µkat/L (286 U/L); serum albumin 25.00 g/L (2.5 g/dL); blood urea nitrogen 18.17 µmol/L (50.9 mg/dL); creatinine 285.53 µmol/L (3.23 mg/dL); C-reactive protein 161.50 mg/L (16.15 mg/dL); IL-61830 pg/mL; and surfactant protein D level 26.6 ng/mL. Findings from computed tomography indicated increased ground-glass opacities without traction bronchiectasis consistent with acute IP. The diagnosis was leukocytosis and progressive kidney injury. After bone marrow aspiration caused by persistent pancytopenia, mild reticulin fibrosis was identified. Because of the high IL-6 concentration, which revealed small atrophic follicles with regressed germinal centers surrounded by several lymphocytes, right inguinal lymph node biopsy was performed. Two minor and three major criteria led to diagnosis of TAFRO syndrome. Administrations of antibiotic therapy and methylprednisolone pulse therapy were ineffective. After rapid progress of respiratory failure, the patient died on day 30 of hospitalization. Autopsy of lung tissues showed diffuse alveolar damage with hyaline membranes. Based on these findings, we diagnosed acute exacerbation of IP associated with TAFRO syndrome due to IL-6 overproduction-associated cytokine storm.

A case of hemophagocytic lymphohistiocytosis after BNT162b2 COVID-19 (Comirnaty®) vaccination.

RATIONALE: Coronavirus disease (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 virus, was reported in Wuhan of China in December 2019. The world is still in a state of pandemic owing to COVID-19. COVID-19 vaccines help our bodies develop immunity against the virus that causes COVID-19 without having to get the illness. Herein, we describe a rare case of a critical disorder, hemophagocytic lymphohistiocytosis (HLH), in a patient with nephritic sclerosis associated with hypertension, following mRNA COVID-19 vaccination. HLH is a life-threatening hyperinflammatory syndrome caused by aberrantly activated macrophages and cytotoxic T cells that may rapidly progress to terminal multiple organ failure. PATIENT CONCERNS: An 85-year-old Japanese woman with chronic renal failure and hypertension was included in this study. Routine laboratory investigations provided the following results: white blood cell (WBC) count, 4.6 × 109/L; hemoglobin (Hb), 8.1 g/dL; platelet count, 27 × 109/L; blood urea nitrogen 48.9 mg/dL, and serum creatinine 3.95 mg/dL. The patient developed malaise, vomiting, and persistent high fever (up to 39.7°C) on the 12th day after receiving the second dose of the vaccine. Initial evaluation revealed neutropenia. The total WBC count was 0.40 × 109/L (Neutrophils 0, Lymphocytes 240/µ, blast 0%); Hb 9.0 g/dL, platelet count 27 × 109/L; and, C Reactive Protein 9.64 mg/dL. DIAGNOSIS: Further tests showed hyperferritinemia (serum ferritin 2284.4 µg/L). Bone marrow examination revealed haemophagocytosis. A provisional diagnosis of HLH associated with the Comirnaty® vaccination was made based on the HLH-2004 diagnostic criteria. INTERVENTIONS: The patient was treated with granulocyte colony-stimulating factor and 500 mg methylprednisolone. OUTCOMES: A significant improvement was observed in the patient's condition; the abnormal laboratory results resolved gradually, and the patient was discharged. LESSONS: This case serves to create awareness among clinicians that HLH is a rare complication of COVID-19 vaccination and should be considered, especially in patients with a history of chronic renal failure and hypertension.

Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.

Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and ß, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin ß pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin ß pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

An autopsy case of TAFRO syndrome with membranoproliferative glomerulonephritis-like lesions.

TAFRO syndrome (thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly) is an atypical manifestation of multicentric Castleman's disease. Although overproduction of interleukin-6, vascular endothelial growth factor, and other cytokines may partially explain the pathophysiology of this rare syndrome, the precise mechanisms underlying the renal dysfunction associated with the condition remain unclear. Here, we describe a case of a 69-year-old male with TAFRO syndrome. He was treated with immunosuppressive agents and his renal function improved. Tapering of immunosuppressive agents resulted in a deterioration of renal function and an elevation of C-reactive protein. After 20 months of treatment, the patient died from tuberculous peritonitis and gastrointestinal bleeding. An autopsy revealed miliary tuberculosis, mediastinal lymphadenopathy, and gastric ulcers. Renal histopathology showed a membranoproliferative glomerulonephritis-like appearance. Almost all glomeruli showed lobular formations with mesangial proliferation and duplication of glomerular capillary walls on light microscopy. Immunofluorescence showed deposition of C1q and IgM along the glomerular capillary walls. Electron microscopy showed mesangial expansion and widening of the subendothelial space with a large number of electron-dense deposits. The glomerular lesions might be characteristic of TAFRO syndrome, and were regarded as the main cause of the patient's renal dysfunction.

Convenient preparation and characterization of a monoclonal antibody for the N-linked sugar chain of a glycoprotein using a microbial endoglycosidase.

We attempted to obtain the monoclonal antibody specific for the N-linked complex-type sialo-oligosaccharide in glycoproteins. We first synthesized a chimeric immunoantigen having an N-linked complex-type of oligosaccharide of glycopeptide, which was bound to a p-formylphenyl compound and conjugated with phosphatidylethanolamine dimyristoyl using the transglycosylation activity of a microbial endoglycosidase (Endo-M) and a reductive amination reaction. This preparative method was convenient and provided a good yield. By immunizing mice with this chimeric neoglycolipid, the monoclonal antibody for the complex-type of sialo-oligosaccharide was obtained in the culture fluid of the cell line even though it was relatively unstable. The monoclonal antibody reacted with various glycoproteins having complex-type sialo-oligosaccharides, but not with those having complex-type asialo-oligosaccharides and high mannose types of oligosaccharides, or with any glycosphingolipids. One of epitopes of this monoclonal antibody seemed to be an alpha-2,6-linked sialic acid at the non-reducing end of the sialo-oligosaccharide of the glycoprotein.

A superelderly case of TAFRO syndrome treated effectively using corticosteroid hormones.

Peripheral lymphocyte subsets may be less time-consuming and are a prognostic tool for managing thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome. Here, we report a superelderly case of plasma cell type TAFRO syndrome treated effectively using corticosteroid hormones.

Edoxaban was Effective for Treating Renal Vein Thrombosis in a Patient with Nephrotic Syndrome.

A 39-year-old man with nephrotic syndrome was admitted due to right dorsal pain. Contrast-enhanced CT led to a diagnosis of renal vein thrombosis and segmental pulmonary thromboembolism. Treatment with heparin and warfarin was started. After 1 month, pulmonary thromboembolism recurred. Warfarin was switched to edoxaban, and steroid therapy was initiated, which led to the remission of nephrotic syndrome and the disappearance of renal vein thrombosis. The efficacy of edoxaban was demonstrated; however, this drug has not been routinely selected for patients with renal disease. Our results suggest that edoxaban is also effective for treating venous thrombosis patients with nephrotic syndrome.

A Case of Urachal Carcinoma of the Abdominal Wall in a Kidney Transplant Recipient.

Urachal carcinoma is an extremely rare malignant tumor arising from the urachus in the fetus. We report a patient who developed urachal carcinoma 18 years after kidney transplantation. A 59-year-old man was admitted because of abdominal pain and massive ascites. He had undergone kidney transplantation 18 years earlier and had end-stage renal disease requiring dialysis. Abdominal CT showed massive ascites and an abdominal wall cystic mass separated from the peritoneal cavity. Hemodialysis was started, and paralytic ileus was diagnosed and treated. His ileus symptoms improved temporarily, but he died of myocardial infarction. An autopsy was performed, which revealed cystadenocarcinoma in the abdominal wall mass, leading to a diagnosis of urachal carcinoma.

Measurement of volatile organic compounds (VOCs) in new residential buildings and VOCs behavior over time.

For the purpose of the investigation of characteristics of VOCs found indoors in recently constructed residential buildings, we measured the behavior of VOCs which were sampled at one-month intervals over a period of one year from the, initial occupancy date in both a detached house and an apartment in a multiple' dwelling. At the first passive sampling from the wooden detached residential building, n-hexane, n-undecane, toluene, ethylacetate, methylethylketone, alpha-pinene and (+)-limonene were present in relatively high concentrations of 10 ppb or higher in the living room. Then these VOCs showed a declining trend with time. p-Dichlorobenzene showed an extremely high concentration (approx. 320 ppb) in June, which subsequently declined with each passing month. There is a high possibility that the cause was the use of a pesticide containing p-dichlorobenzene during the period of changeover from winter to summer clothes in June. On the other hand, from the multiple dwelling, four VOCs showed values of 10 ppb or more (toluene, 1,2,4-trimethylbenzene, methylethylketone and alpha-pinene). Of these VOCs, methylethylketone concentration was in excess of 100 ppb, and then also showed a declining trend with time. Even for new residential buildings completed during the same time frame, it was shown that the types of VOC contaminants and their concentrations varied significantly.

A hepatic sclerosed hemangioma with significant morphological change over a period of 10 years: a case report.

INTRODUCTION: Liver cavernous hemangioma is the most common noncystic hepatic lesion, and a hemangioma that undergoes degeneration and fibrous replacement is called a hepatic sclerosed hemangioma. CASE PRESENTATION: A 63-year-old Japanese man was admitted for detailed investigation of a liver tumor. Tumor markers carcinoembryonic antigen, alpha-fetoprotein, and CA19-9 levels in the peripheral blood were not elevated at any time. Plain computed tomography showed an approximately 1.5 cm low density mass in the periphery of segment 8, which was marginally enhanced on contrast-enhanced dynamic computed tomography. On magnetic resonance imaging, the tumor was hypointense on T1-weighted image and hyperintense on T2-weighted image. The tumor was suspected to be an atypical hemangioma, metastatic, hepatocellular carcinoma, or cholangiocellular carcinoma. Segmental hepatectomy was performed. Histological examination of the resected tumor specimen revealed a sclerosed hemangioma with marked hyalinization and sparse stromal fibrosis. Immunochemically, the tumor cells were positive for CD34 and alpha smooth muscle actin. Electron microscopically, the residual hemangioma consisted of numerous caveolae and vesicles in endothelial cells in irregular shapes and sizes. Immunostaining for caveolin-1 showed decreased or no caveolin-1 reactivity in the hyalinized lesions of the sclerosed hemangioma, but abundant caveolin-1 reactivity in the residual cavernous hemangioma. Of interest, computed tomography images of the tumor obtained 10 years earlier at our hospital depicted a 3 cm typical cavernous hemangioma. CONCLUSIONS: Hepatic sclerosed hemangioma is a rare condition. Comparison of radiological findings of the lesion over a period of 10 years was valuable in providing insight for the evolutional process from liver cavernous hemangioma to hepatic sclerosed hemangioma.

Multiscale modeling and mechanics of filamentous actin cytoskeleton.

The adaptive structure and functional changes of the actin cytoskeleton are induced by its mechanical behavior at various temporal and spatial scales. In particular, the mechanical behaviors at different scales play important roles in the mechanical functions of various cells, and these multiscale phenomena require clarification. To establish a milestone toward achieving multiscale modeling and simulation, this paper reviews mathematical analyses and simulation methods applied to the mechanics of the filamentous actin cytoskeleton. The actin cytoskeleton demonstrates characteristic behaviors at every temporal and spatial scale, and mathematical models and simulation methods can be applied to each level of actin cytoskeletal structure ranging from the molecular to the network level. This paper considers studies on mathematical models and simulation methods based on the molecular dynamics, coarse-graining, and continuum dynamics approaches. Every temporal and spatial scale of actin cytoskeletal structure is considered, and it is expected that discrete and continuum dynamics ranging from functional expression at the molecular level to macroscopic functional expression at the whole cell level will be developed and applied to multiscale modeling and simulation.

Simulations of dynamics of actin filaments by remodeling them in shear flows.

A mechanochemical relationship between intracellular flow fields of actin and remodeling of actin filaments is important to the realization of cell motility. In this study, to investigate this mechanochemical relationship, we present a mathematical model describing motions of actin filament and remodeling of the filaments in shear flows. We perform simulations of the dynamics of actin filaments remodeled by polymerization, depolymerization, and severing, and investigate contributions of these remodeling processes to effective shear viscosities of the system.