14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte.

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3ß interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, the precise localization and differential role of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3ß in glomeruli was restricted in podocytes, and 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. Although 14-3-3ß was dominantly co-localized with FKBP12 in the foot processes, a part of 14-3-3ß was co-localized with Par3 at the slit diaphragm. 14-3-3ß interacted with Par3, and FKBP12 bound to 14-3-3ß competitively with Par3. Deletion of 14-3-3ß enhanced the interaction of Par3 with Par6 in podocytes. Gene silencing for 14-3-3ß altered the structure of actin fibers and process formation. 14-3-3ß and synaptopodin expression was decreased in podocyte injury models. In contrast, 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and synaptopodin. Gene silencing for 14-3-3σ altered the structure of vimentin fibers and process formation. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury models but was decreased in the late stage. Together, the localization of 14-3-3ß at actin cytoskeleton plays a role in maintaining the foot processes and the Par complex in podocytes. In contrast, 14-3-3σ at vimentin cytoskeleton is essential for maintaining primary processes.

COVID-19 pandemic vaccination strategies of early 2021 based on behavioral differences between residents of Tokyo and Osaka, Japan.

BACKGROUND: During the fourth COVID-19 wave in Japan, marked differences became apparent in the scale of the epidemic between metropolitan Tokyo in eastern Japan and Osaka prefecture in western Japan. METHODS: Public epidemic data were analyzed, with performance of mathematical simulations using simplified SEIR models. RESULTS: The increase in the number of infected persons per 100,000 population during the fourth wave of expansion was greater in Osaka than in Tokyo. The basic reproduction number in Osaka was greater than in Tokyo. Particularly, the number of infected people in their 20 s increased during the fourth wave: The generation-specific reproduction number for people in their 20 s was higher than for people of other generations. Both Tokyo and Osaka were found to have strong correlation between the increase in the number of infected people and the average number of people using the main downtown stations at night. Simulations showed vaccination of people in their 60 s and older reduced the number of infected people among the high-risk elderly population in the fourth wave. However, age-specific vaccination of people in their 20 s reduced the number of infected people more than vaccination of people in their 60 s and older. CONCLUSIONS: Differences in the epidemic between Tokyo and Osaka are explainable by different behaviors of the most socially active generation. When vaccine supplies are adequate, priority should be assigned to high-risk older adults, but if vaccine supplies are scarce, simulation results suggest consideration of vaccinating specific groups among whom the epidemic is spreading rapidly.

Th17 Cells Participate in Thy1.1 Glomerulonephritis Which Is Ameliorated by Tacrolimus.

INTRODUCTION: Thy1.1 glomerulonephritis (Thy1.1 GN) in rats is widely used as an experimental model of mesangial proliferative glomerulonephritis (GN). We previously reported that T-helper (Th) cells were accumulated in glomeruli from the early phase of this model and that not Th2 cells but Th1 cells play an important role in the development of glomerular alterations. Although Th17 is reported to be involved in the pathogenesis of several autoimmune diseases, the role of Th17 cells in the pathogenesis of mesangial alterations in Thy1.1 GN remains unclear. METHODS: The kinetics of the infiltration of subsets of Th cells and the expression of IL-17 in Thy1.1 GN were analyzed. Next, the localization and the cell types of IL-17 receptor (IL-17R)-positive cells and IL-6-positive cells were analyzed. Then, the effect of tacrolimus on the expressions of Th17-related cytokines in Thy1.1 GN was analyzed. RESULTS: Not only Th1 cells but also Th17 cells were recruited into glomeruli from the early phase of the disease. mRNA expression of IL-17 in glomeruli was elevated. The increased positive expression of IL-17R was detected in the mesangial area, and some of IL-17R-positive cells were co-stained with IL-6. Tacrolimus treatment ameliorated mesangial alterations by suppressing the expressions of Th17-related cytokines such as IL-17 and IL-6. CONCLUSION: Th17 cells participate in the development of Thy1.1 GN, a mimic of mesangial proliferative GN, and Th17 cells and their related cytokines are pertinent therapeutic targets.

Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton.

FKBP12 was identified as a binding protein of tacrolimus (Tac). Tac binds to FKBP12 and exhibits immunosuppressive effects in T cells. Although it is reported that Tac treatment directly ameliorates the dysfunction of the podocyte in nephrotic syndrome, the precise pharmacological mechanism of Tac is not well understood yet. It is also known that FKBP12 functions independently of Tac. However, the localization and the physiological function of FKBP12 are not well elucidated. In this study, we observed that FKBP12 is highly expressed in glomeruli, and the FKBP12 in glomeruli is restricted in podocytes. FKBP12 in cultured podocytes was expressed along the actin cytoskeleton and associated with filamentous actin (F-actin). FKBP12 interacted with the actin-associated proteins 14-3-3 and synaptopodin. RNA silencing for FKBP12 reduced 14-3-3 expression, F-actin staining, and process formation in cultured podocytes. FKBP12 expression was decreased in the nephrotic model caused by adriamycin (ADR) and the cultured podocyte treated with ADR. The process formation was deteriorated in the podocytes treated with ADR. Tac treatment ameliorated these decreases. Tac treatment to the normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation. Tac enhanced the interaction of FKBP12 with synaptopodin. These observations suggested that FKBP12 at actin cytoskeleton participates in the maintenance of processes, and Tac treatment ameliorates podocyte injury by restoring FKBP12 at actin cytoskeleton.

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction.

INTRODUCTION: Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. METHODS: The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. RESULTS: Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. CONCLUSION: Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

Nephrin-Ephrin-B1-Na+/H+ Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1-associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the nephrin-ephrin-B1 complex at the slit diaphragm. The nephrin-ephrin-B1-NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both in vitro analyses with human embryonic kidney 293 cells and in vivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1-NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of nephrin-ephrin-B1-NHERF2-ezrin-actin is a novel critical axis in the podocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.

Critical roles of Astrin in the mitosis of immature rat Sertoli cells.

Male hypogonadism (hgn/hgn) rats show testicular hypoplasia accompanied by dysplastic development of seminiferous tubules due to loss-of-function mutation of the gene encoding Astrin, which is required for mitotic progression in the division cycle of HeLa cells. In the present study, we examined the cytological base leading to the decrease of Sertoli cells in hgn/hgn testes. In hgn/hgn testes on postnatal day 3, anti-phospho-histone H3 (Ser10) (pH3)-positive mitotic phase and TUNEL-positive apoptosis increased in GATA4-positive Sertoli cells. Isolated immature Sertoli cells from hgn/hgn testes showed increased pH3-assessed mitotic index accompanied by decreased 5-bromo-2'-deoxyuridine-incorporation and increased TUNEL-positive apoptosis, suggesting mitotic delay and cell death. In the visualization of mitotic progression by nocodazole (NOC)-mediated cell cycle arrest and subsequent release, hgn/hgn rat-derived Sertoli cells failed to make the transition from prometaphase to metaphase, and the cells with micronuclei and TUNEL-positive cells gradually increased in a time-dependent manner. Western blot analysis detected ≈142 kDa protein expected as Astrin in extracts of +/+ and +/hgn testes and cultured normal Sertoli cells but not in extracts of hgn/hgn testes. CLASP1 was detected in extracts of both normal and hgn/hgn testes, whereas it was localized in kinetochore of normal mitotic Sertoli cells but diffused in cytoplasm of hgn/hgn Sertoli cells. These results indicate that Astrin is required for normal mitotic progression in immature Sertoli cells and that the most severe type of testicullar dysplasia in hgn/hgn rats is caused by mitotic cell death of immature Sertoli cells due to lack of Astrin.

Progression of renal fibrosis in congenital CKD model rats with reduced number of nephrons.

A congenital reduction in the number of nephrons is a critical risk factor for both onset of chronic kidney disease (CKD) and its progression to end-stage kidney disease (ESKD). Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and show progressive CKD. This study used an immunohistological method to assess glomerular and interstitial pathogenesis in male HPK rats aged 35-210days. CD68 positive-macrophages were found to infiltrate into glomeruli in HPK rats aged 35 and 70days and to infiltrate into interstitial tissue in rats aged 140 and 210days. HPK rats aged 35 and 70days showed glomerular hypertrophy, loss of normal linear immunostaining of podocine, and increased expression of PDGFr-ß, TGF-ß, collagens, and fibronectin, with all of these alterations gradually deteriorating with age. α-SMA-positive myofibroblasts were rarely detected in glomerular tufts, whereas α-SMA-positive glomerular parietal epithelium (GPE) cells were frequently observed along Bowman's capsular walls. The numbers of PDGFr-ß-positive fibroblasts in interstitial tissue were increased in rats aged 35days and older, whereas interstitial fibrosis, characterized by the increased expression of tubular PDGF-BB, the appearance of myofibroblasts doubly positive for PDGFr-ß and α-SMA, and increased expression of collagens and fibronectin, were observed in rats aged 70 and older. These results clearly indicate that congenital CKD with only 20% of nephrons cause renal fibrosis in rats.

Renal Function and Hematology in Rats with Congenital Renal Hypoplasia.

Renal hypoplasia due to a congenitally reduced number of nephrons progresses to chronic kidney disease and may cause renal anemia, given that the kidneys are a major source of erythropoietin in adults. Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and develop CKD. This study assessed the renal function and hematologic changes in HPK rats from 70 to 210 d of age. HPK rats demonstrated deterioration of renal excretory function, slightly macrocytic erythropenia at all days examined, age-related increases in splenic hemosiderosis accompanied by a tendency toward increased hemolysis, normal plasma erythropoietin levels associated with increased hepatic and decreased renal erythropoietin production, and maintenance of the response for erythropoietin production to hypoxic conditions, with increased interstitial fibrosis at 140 d of age. These results indicate that increases in splenic hemosiderosis and the membrane fragility of RBC might be associated with erythropenia and that hepatic production of erythropoietin might contribute to maintaining the blood Hgb concentration in HPK rats.

Simulation of infectious diseases to prevent spread.

Models of infectious diseases to prevent spread, SIR model, agent-based model and net- work model, are shortly reviewed. And, the outbreak of influenza HlN1 2009 in the suburb of metropolitan Tokyo is reported as a case study of simulation using model.

Preparedness for the spread of influenza: prohibition of traffic, school closure, and vaccination of children in the commuter towns of Tokyo.

In Greater Tokyo, many people commute by train between the suburbs and downtown Tokyo for 1 to 2 h per day. The spread of influenza in the suburbs of Tokyo should be studied, including the role of commuters and the effect of government policies on the spread of disease. We analyzed the simulated spread of influenza in commuter towns along a suburban railroad, using the individual-based Monte Carlo method, and validated this analysis using surveillance data of the infection in the Tokyo suburbs. This simulation reflects the mechanism of the real spread of influenza in commuter towns. Three measures against the spread of influenza were analyzed: prohibition of traffic, school closure, and vaccination of school children. Prohibition of traffic was not effective after the introduction of influenza into the commuter towns, but, if implemented early, it was somewhat effective in delaying the epidemic. School closure delayed the epidemic and reduced the peak of the disease, but it was not as effective in decreasing the number of infected people. Vaccination of school children decreased the numbers not only of infected children but also of infected adults in the regional communities.

Modeling on social spread from immunity.

We are now planning to make a transmission model of infectious diseases in the scale of a city. People live in the city contacting other persons with daily life. The model regards a contact as a source of infection. A person will be simulated as a simple system of differential equations. As a candidate of differential equations, we are now investigating Marchuk's simple model. We adopt Marchuk's simple model because it has formation time, i.e., latent time. As Dr. Takeuchi showed, latent time is very important. There remain problems of choosing parameters for special diseases. We are now planning to use Marquardt method to minimize residuals form clinical data to estimate parameters. As for contacts, there are many approaches. The approach of the MIDAS project is very intensive. Our approach is simple. There are about 30,000 Japanese every 15 minutes daily life data, sleeping, eating, work, study, house keeping, etc. Our approach is to make virtual families, husband, wife, children in a city and assign actions from the every 15 minutes data statistically and estimate their contacts in the companies or schools, etc.