ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a central kinase involved in energy homeostasis. Increased intracellular AMP levels result in AMPK activation through the binding of AMP to the γ-subunit of AMPK. Recently, we reported that AMP-induced AMPK activation is impaired in the kidneys in chronic kidney disease (CKD) despite an increase in the AMP/ATP ratio. However, the mechanisms by which AMP sensing is disrupted in CKD are unclear. Here, we identified mechanisms of energy homeostasis in which Unc-51-like kinase 1 (ULK1)-dependent phosphorylation of AMPKγ1 at Ser260/Thr262 promoting AMP sensitivity of AMPK. AMPK activation by AMP was impaired in Ulk1 knockout mice despite an increased AMP/ATP ratio. ULK1 expression is markedly downregulated in CKD kidneys, leading to AMP sensing failure. Additionally, MK8722, an allosteric AMPK activator, stimulated AMPK in the kidneys of a CKD mouse model (5/6th nephrectomy) via a pathway that is independent of AMP sensing. Thus, our study shows that MK8722 treatment significantly attenuates the deterioration of kidney function in CKD and may be a potential therapeutic option in CKD therapeutics.

Absence of ULK1 decreases AMPK activity in the kidney, leading to chronic kidney disease progression.

AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream ß-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.

Deletion of Alox15 improves kidney dysfunction and inhibits fibrosis by increased PGD2 in the kidney.

BACKGROUND: Lipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood. METHODS: To clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys. RESULTS: In 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15-/- mice. Alox15-/- CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15-/- CKD mice. Mediator lipidomics revealed that Alox15-/- CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-ß1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines. CONCLUSION: Increased PGD2 in Alox15-/- CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD.

Renal TNFα activates the WNK phosphorylation cascade and contributes to salt-sensitive hypertension in chronic kidney disease.

The inappropriate over-activation of the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1-SPAK-NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1-SPAK-NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1- SPAK-NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.

Failure to sense energy depletion may be a novel therapeutic target in chronic kidney disease.

The kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD.

Detection and Analysis of Targeted Biological Cells by Electrophoretic Coulter Method.

Combining the electrophoresis and conventional Coulter methods, we previously proposed the electrophoretic Coulter method (ECM), enabling simultaneous analysis of the size, number, and zeta potential of individual specimens. We validated the ECM experimentally using standard polystyrene particles and red blood cells (RBCs) from sheep; the latter was the first ECM application to biological particles in biotechnology research. However, specimens are prevented from passing through the ECM module aperture, which prevents accurate determination of the zeta potential of each specimen. This problem is caused by electro-osmotic flow (EOF) due to the high zeta potential at the ECM microchannel surfaces. To significantly improve ECM feasibility for biomedicine, we here propose a method to estimate the zeta potential at the ECM microchannel surfaces separate from the zeta potential of each specimen, by investigating the electric-field dependence of the specimen's experimental electrophoretic velocity. We minimize the zeta potential at the microchannel surfaces by applying an organic-molecule coating, and we suppress the surface zeta potential and its resultant EOF by optimizing the microchannel geometry. We demonstrate that the ECM can distinguish between different biological cells using the differences in zeta potential values and/or sizes. We also demonstrate that the ECM can determine the number of biomolecules attached to individual cells and identify whether the average cell state in an analyzed vial is alive or dead. The high-performance ECM can detect cellular morphology alterations, improve immunologic test sensitivity, and identify cell states (living, dying, and dead); this information is clinically useful for early diagnosis and its follow-up.

Pituitary pars intermedia dysfunction with pituitary gland melanotroph adenoma in a Grevy's zebra (Equus grevyi).

A 22-year and 9-month-old female Grevy's zebra (Equus grevyi) showed signs of polyuria, polydipsia, glucosuria, and muscle atrophy. Blood tests revealed hyperglycemia, hypertriglyceridemia, electrolyte imbalance, high levels of adrenocorticotropic hormone (ACTH) and cortisol, and low levels of hormones secreted by the pituitary pars distalis. Pathological examinations revealed a pituitary gland tumor and bilateral adrenal cortical hyperplasia. Pituitary tumor cells showed immunoreactivity for α-melanocyte-stimulating hormone and ACTH. The deposition of amyloid ß was observed in the parenchyma and vascular walls of the cerebrum. The zebra showed clinical signs of pituitary pars intermedia dysfunction and was histopathologically diagnosed with pituitary gland melanotroph adenoma. This case report provides insight into neoplastic and endocrine diseases associated with the aging of a zebra.

Coinfection with Orthohantavirus and Leptospira spp. in Rats Collected from Markets in Indonesia.

Background: Rats are an important reservoir animal for several zoonotic pathogens worldwide, including hantaviruses and Leptospira spp., which are the causative agents of hemorrhagic fever with renal syndrome, hantavirus cardiopulmonary syndrome, and leptospirosis. Although a previous study indicated a high frequency of antihantaviral antibodies in patients with acute fever in Indonesia, circulating hantaviruses and their reservoir animals in the country remain limited. Materials and Methods: The presence of hantavirus in rats captured in the urban area of Bogor, Indonesia, from which Leptospira spp. were isolated using PCR, followed by DNA sequencing. Immunohistochemical analyses were performed to detect hantaviral and leptospiral antigens in rat kidney tissues. Results: Seoul of Orthohantavirus seoulense (SEOV) RNA was detected from 24 of 80 Rattus norvegicus (30%). SEOV and Leptospira coinfection was detected in 10 of 80 rats (12.5%). Immunohistochemistry revealed that hantavirus antigens were positively stained in the interstitial capillaries and cells, whereas Leptospira antigens were stained in the luminal side of the renal tubules. Conclusion: This study revealed a high prevalence of SEOV and SEOV and Leptospira coinfection among rats in the urban areas of Bogor, Indonesia, indicating a potential risk of rat-borne zoonotic diseases in the area.

Impact of COVID-19 versus other pneumonia on in-hospital mortality and functional decline among Japanese dialysis patients: a retrospective cohort study.

Coronavirus disease 2019 (COVID-19) affects both life and health. However, the differentiation from other types of pneumonia and effect of kidney disease remains uncertain. This retrospective observational study investigated the risk of in-hospital death and functional decline in ≥ 20% of Barthel Index scores after COVID-19 compared to other forms of pneumonia among Japanese adults, both with and without end-stage kidney disease (ESKD). The study enrolled 123,378 patients aged 18 years and older from a national inpatient administrative claims database in Japan that covers the first three waves of the COVID-19 pandemic in 2020. After a 1:1:1:1 propensity score matching into non-COVID-19/non-dialysis, COVID-19/non-dialysis, non-COVID-19/dialysis, and COVID-19/dialysis groups, 2136 adults were included in the analyses. The multivariable logistic regression analyses revealed greater odds ratios (ORs) of death [5.92 (95% CI 3.62-9.96)] and functional decline [1.93 (95% CI 1.26-2.99)] only in the COVID-19/dialysis group versus the non-COVID-19/non-dialysis group. The COVID-19/dialysis group had a higher risk of death directly due to pneumonia (OR 6.02, 95% CI 3.50-10.8) or death due to other diseases (OR 3.00, 95% CI 1.11-8.48; versus the non-COVID-19/non-dialysis group). COVID-19 displayed a greater impact on physical function than other types of pneumonia particularly in ESKD.

Safety and Effectiveness of OnabotulinumtoxinA in Patients with Laryngeal Dystonia: Final Report of a 52-Week, Multicenter Postmarketing Surveillance Study.

This postmarketing surveillance study was conducted to evaluate the safety and effectiveness of onabotulinumtoxinA in Japanese patients with laryngeal dystonia (LD). Patients receiving onabotulinumtoxinA for the first time were enrolled and observed for up to 12 months following the first injection. Safety assessment included adverse drug reactions (ADRs), and effectiveness assessments included the Voice Handicap Index-10 (VHI-10) and physician's global assessment (PGA). ADRs were observed in 48 (5.8%) of 834 patients in the safety analysis set, including dysphonia in 43 (5.2%) patients and dysphagia in 7 (0.8%) patients. The change in total VHI-10 score (mean) in 790 patients included in the effectiveness analysis set showed that improvement in adductor LD peaked at 2 months after the first injection, while patients with abductor or mixed LD showed a gradual attenuation of effect 2-4 weeks post-injection. The change in total VHI-10 score in subsequent injections was generally similar to that following the first injection. The overall effectiveness rate according to the PGA was 93.4% (738/790 patients). The results demonstrate that onabotulinumtoxinA is a well-tolerated and effective treatment for LD in real-world clinical practice.

Rutin Concentration and Characterization of Rutinosidase in Perennial Buckwheat (Fagopyrum cymosum) and Its Application in Foods.

To evaluate the potential of perennial buckwheat (Fagopyrum cymosum; FC) as a food source, rutin concentration was investigated. FC contains more than 1% (w/w) rutin and 0.03% quercetin in the leaves, flowers, and seeds. In particular, rutin and quercetin concentrations were high in plant seeds. Therefore, FC is useful as a rutin- and quercetin-rich material. In contrast, the FC seed contained a large amount of rutinosidase. Purified rutinosidase in a homogenous mixture consisted of only one isozyme with M.W. of 58.4 KD and low Km for rutin (0.367 mM). The rutin concentration in the FC dough decreased to almost zero, 10 min after the addition of water. Parallel to the decrease in rutin, quercetin was increased, and strong bitterness was generated, whereas steam-heated flour in which rutinosidase was inactivated did not have rutin hydrolysis and bitterness. These results indicate that rutinosidase is a major cause of rutin hydrolysis and bitterness. The in vitro rutinosidase is inactivated at pH 8.0 and 65 °C. Therefore, the control of dough pH and temperature should be useful in preventing rutinosidase activity.

Chronic kidney disease causes blood-brain barrier breakdown via urea-activated matrix metalloproteinase-2 and insolubility of tau protein.

Chronic kidney disease (CKD) causes cognitive impairment and contributes to the overall global burden of dementia. However, mechanisms through which the kidneys and brain communicate are not fully understood. We established a CKD mouse model through adenine-induced tubulointerstitial fibrosis. Novel object recognition tests indicated that CKD decreased recognition memory. Sarkosyl-insoluble-proteomic analyses of the CKD mouse hippocampus revealed an accumulation of insoluble MAPT (microtubule-associated protein tau) and RNA-binding proteins such as small nuclear ribonucleoprotein U1 subunit 70 (SNRNP70). Additionally, there was an accumulation of Immunoglobulin G (IgG), indicating blood-brain barrier (BBB) breakdown. We identified that expressions of essential tight-junction protein claudin-5 and adherens-junction protein platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) were decreased in the brain endothelial cells of CKD mice. We determined urea as a major uremic solute that dose dependently decreased both claudin-5 and PECAM-1 expression in the mouse brain endothelial cell line bEnd.3 cells. Gelatin zymography indicated that the serum of CKD mice activated matrix metalloproteinase-2 (MMP2), while marimastat ameliorated the reduction of claudin-5 expression by urea in bEnd.3 cells. This study established a brain proteomic signature of CKD indicating BBB breakdown and insolubility of tau protein, which are pathologically linked to Alzheimer's disease. Urea-mediated activation of MMP2 was partly responsible for BBB breakdown in CKD.

A de novo case of minimal change disease following the first dose of the Moderna mRNA-1273 SARS-CoV-2 vaccine without relapse after the second dose.

In recent times, new onset or relapse of nephrotic syndrome following the first dose of SARS-CoV-2 vaccines has been reported. Although the vaccination could trigger nephrotic syndrome, the question of whether the same vaccine should be administered as the second dose remains unanswered. A 25-year-old woman had taken the Moderna mRNA-1273 SARS-CoV-2 vaccine (mRNA-1273) and 26 days later, she noticed facial and peripheral edema. One week later she was referred and admitted to our hospital, wherein laboratory tests revealed that her serum creatinine level, serum albumin level, and urine protein-creatinine ratio were respectively 0.79 mg/dL, 2.5 g/dL, and 7.0 g/gCr. After a thorough inpatient examination including renal biopsy, she was diagnosed with minimal change disease (MCD) and treatment with steroids was initiated. She achieved complete remission the next day and did not experience a relapse upon receiving the second mRNA-1273 dose 56 days after the first, under treatment with 35 mg/day of oral prednisolone. This case report yields insight into determining whether patients who develop de novo MCD after the first mRNA-1273 dose should receive the second dose.

Isolated adrenocorticotropin deficiency associated with delirium and takotsubo cardiomyopathy.

We report a 65-year-old woman with isolated adrenocorticotropic hormone (ACTH) deficiency. The patient was transported to the emergency outpatient department by ambulance complaining of malaise and nausea. Because her laboratory data revealed hyponatremia, we performed endocrinological examinations and diagnosed isolated ACTH deficiency. After admission, she went into a delirious state and suffered from takotsubo cardiomyopathy due to adrenal insufficiency. Replacement therapy with hydrocortisone sufficiently improved her delirium and cardiomyopathy. We conclude that her unstable mental state and myocardial dysfunction were closely related to adrenal insufficiency and suggest that adrenal crisis may cause delirium and Takotsubo cardiomyopathy.