Fundamental and higher eigenmodes of qPlus sensors with a long probe for vertical-lateral bimodal atomic force microscopy.

The detection of vertical and lateral forces at the nanoscale by atomic force microscopy (AFM) reveals various mechanical properties on surfaces. The qPlus sensor is a widely used force sensor, which is built from a quartz tuning fork (QTF) and a sharpened metal probe, capable of high-resolution imaging in viscous liquids such as lubricant oils. Although a simultaneous detection technique of vertical and lateral forces by using a qPlus sensor is required in the field of nanotribology, it has still been difficult because the torsional oscillations of QTFs cannot be detected. In this paper, we propose a method to simultaneously detect vertical and lateral force components by using a qPlus sensor with a long probe. The first three eigenmodes of the qPlus sensor with a long probe are theoretically studied by solving a set of equations of motion for the QTF prong and probe. The calculation results were in good agreement with the experimental results. It was found that the tip oscillates laterally in the second and third modes. Finally, we performed friction anisotropy measurements on a polymer film by using a bimodal AFM utilizing the qPlus sensor with a long probe to confirm the lateral force detection.

Visualization of Au Nanoparticles Buried in a Polymer Matrix by Scanning Thermal Noise Microscopy.

Several researchers have recently demonstrated visualization of subsurface features with a nanometer-scale resolution using various imaging schemes based on atomic force microscopy. Since all these subsurface imaging techniques require excitation of the oscillation of the cantilever and/or sample surface, it has been difficult to identify a key imaging mechanism. Here we demonstrate visualization of Au nanoparticles buried 300 nm into a polymer matrix by measurement of the thermal noise spectrum of a microcantilever with a tip in contact to the polymer surface. We show that the subsurface Au nanoparticles are detected as the variation in the contact stiffness and damping reflecting the viscoelastic properties of the polymer surface. The variation in the contact stiffness well agrees with the effective stiffness of a simple one-dimensional model, which is consistent with the fact that the maximum depth range of the technique is far beyond the extent of the contact stress field.

Visualization of subsurface nanoparticles in a polymer matrix using resonance tracking atomic force acoustic microscopy and contact resonance spectroscopy.

A visualization technique of subsurface features with a nanometer-scale spatial resolution is strongly demanded. Some research groups have demonstrated the visualization of subsurface features using various techniques based on atomic force microscopy. However, the imaging mechanisms have not yet been fully understood. In this study, we demonstrated the visualization of subsurface Au nanoparticles buried in a polymer matrix 900 nm from the surface using two techniques; i.e., resonance tracking atomic force acoustic microscopy and contact resonance spectroscopy. It was clarified that the subsurface features were visualized by the two techniques as the area with a higher contact resonance frequency and a higher Q-factor than those in the surrounding area, which suggests that the visualization is realized by the variation of the contact stiffness and damping of the polymer matrix due to the existence of the buried nanoparticles.

Imaging of Au nanoparticles deeply buried in polymer matrix by various atomic force microscopy techniques.

Recently, some papers reported successful imaging of subsurface features using atomic force microscopy (AFM). Some theoretical studies have also been presented, however the imaging mechanisms are not fully understood yet. In the preceeding papers, imaging of deeply buried nanometer-scale features has been successful only if they were buried in a soft matrix. In this paper, subsurface features (Au nanoparticles) buried in a soft polymer matrix were visualized. To elucidate the imaging mechanisms, various AFM techniques; heterodyne force microscopy, ultrasonic atomic force microscopy (UAFM), 2nd-harmonic UAFM and force modulation microscopy (FMM) were employed. The particles buried under 960 nm from the surface were successfully visualized which has never been achieved. The results elucidated that it is important for subsurface imaging to choose a cantilever with a suitable stiffness range for a matrix. In case of using the most suitable cantilever, the nanoparticles were visualized using every technique shown above except for FMM. The experimental results suggest that the subsurface features buried in a soft matrix with a depth of at least 1 µm can affect the local viscoelasticity (mainly viscosity) detected as the variation of the amplitude and phase of the tip oscillation on the surface. This phenomenon presumably makes it possible to visualize such deeply buried nanometer-scale features in a soft matrix.

Epithelial-mesenchymal transition as a potential explanation for podocyte depletion in diabetic nephropathy.

BACKGROUND: Depletion of glomerular podocytes is an important feature of progressive diabetic nephropathy. Although the most plausible explanation for this podocyte depletion is detachment from the glomerular basement membrane after cellular apoptosis, the mechanism is unclear. Fibroblast-specific protein 1 (FSP1; encoded by the S100A4 gene) is a member of the S100 family of calcium-binding proteins and is constitutively expressed in the cytoplasm of tissue fibroblasts or epithelial cells converted into fibroblasts by means of epithelial-mesenchymal transition. STUDY DESIGN: Retrospective cross-sectional analysis. SETTINGS & PARTICIPANTS: 109 patients with type 2 diabetes mellitus, of whom 43 (39%) underwent kidney biopsy. PREDICTOR: Clinical stage (4 categories) and histological grade (5 categories) of diabetic nephropathy. OUTCOME: FSP1 expression in podocytes in urine and glomeruli in kidney biopsy specimens. MEASUREMENTS: Immunohistochemistry, real-time polymerase chain reaction, and in situ hybridization. RESULTS: 38 of 109 patients (35%) were normoalbuminuric, 16 (15%) had microalbuminuria, 8 (7%) had macroalbuminuria, and 47 (43%) had decreased kidney function. Approximately 95% of podocytes in urine sediment were not apoptotic, and 86% expressed FSP1. The number of FSP1-positive podocytes in urine sediment was significantly larger in patients with macroalbuminuria than in those with normoalbuminuria (P = 0.03). Intraglomerular expression of FSP1 occurred almost exclusively in podocytes from patients with diabetes, and the number of FSP1-positive podocytes was larger in glomeruli showing diffuse mesangiopathy than in those showing focal mesangiopathy (P = 0.01). The number also was larger in glomeruli with nodular lesions than in those without nodular lesions (P < 0.001). FSP1-positive podocytes selectively expressed Snail1 and integrin-linked kinase, a known trigger for epithelial-mesenchymal transition. LIMITATIONS: Nonrepresentative study population. CONCLUSIONS: These results suggest that the appearance of FSP1 in podocytes of patients with diabetes is associated with more severe clinical and pathological findings of diabetic nephropathy, perhaps because of induction of podocyte detachment through epithelial-mesenchymal transition-like phenomena.

[Molecular mechanisms of tissue fibrosis].

Tissue fibrosis is a common cause of organ failure. Consequently, elucidation of the mechanisms underlying both the initiation and progression of fibrosis is an essential step toward establishing new therapeutic strategies for the treatment of organ failure. Fibroblasts are the principal effectors mediating fibrosis and their heterogeneous origins, including epithelial-mesenchymal transition (EMT), bone marrow-derived cell or fibrocyte, and endothelial-mesenchymal transition (EndMT), have been demonstrated. Chronic hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis. Recently, we reported that hypoxia induces EMT in renal tubular epithelial cells through activation of hypoxia-inducible factor-1alpha (HIF-1alpha). Using the Cre-loxP mediated gene targeting of HIF-1alpha or VHL which acts as a ubiquitin ligase to promote degradation of HIF-1alpha, we showed that HIF-1alpha plays a key role in the progression of renal fibrosis. As a large number of molecules that contribute to the induction of fibrosis have been identified, and their signal transduction pathway has been characterized, these fibrosis-related molecules have been proposed as therapeutic targets. EMT antagonists, TGF-beta signal modulator, and HIF-1alpha inhibitor could be useful for the treatment of fibrosis.

Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis.

Chronic hypoxia accelerates renal fibrosis. The chief mediator of the hypoxic response is hypoxia-inducible factor 1 (HIF-1) and its oxygen-sensitive component HIF-1alpha. HIF-1 regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. To determine the specific role of HIF-1 in renal fibrosis, we generated a knockout mouse in which tubular epithelial expression of von Hippel-Lindau tumor suppressor (VHL), which acts as a ubiquitin ligase to promote proteolysis of HIF-1alpha, was targeted. We investigated the effect of VHL deletion (i.e., stable expression of HIF-1alpha) histologically and used the anti-HIF-1alpha agent [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] (YC-1) to test whether inhibition of HIF-1alpha could represent a novel approach to treating renal fibrosis. The area of renal fibrosis was significantly increased in a 5/6 renal ablation model of VHL-/- mice and in all VHL-/- mice at least 60 wk of age. Injection of YC-1 inhibited the progression of renal fibrosis in unilateral ureteral obstruction model mice. In conclusion, HIF-1alpha appears to be a critical contributor to the progression of renal fibrosis and could be a useful target for its treatment.

Prediction of corticosteroid responsiveness based on fibroblast-specific protein 1 (FSP1) in patients with IgA nephropathy.

BACKGROUND: Corticosteroids are frequently used to treat patients with active IgA nephropathy (IgAN); however, there have been few reports describing factors that are predictive of the response to corticosteroid treatment. The purpose of this study is to determine the extent to which fibroblast-specific protein 1-positive (FSP1(+)) cells are predictive of corticosteroid responsiveness in patients with IgAN. METHODS: Fifty biopsy-proven IgAN patients who received corticosteroid therapy were enrolled and followed for 7.1 +/- 3.0 years. FSP1(+) cells were identified using an anti-FSP1 antibody. RESULTS: Twelve patients showed progression of renal impairment or no reduction of urinary protein (non-responders) after steroid therapy. In the remaining 38 patients, renal function was stable during follow-up, and their urinary protein declined to <1.0 g/day (responders). Serum creatinine, estimated GFR, severity of mesangial proliferation, percent glomerulosclerosis/total glomeruli, extent of interstitial damage and FSP1(+) cell number were all significantly higher in non-responders than in responders. Cox regression analysis using two covariates with every possible combination of factors indicated that FSP1(+) cell number was the strongest and most significant predictor of corticosteroid responsiveness. When IgAN patients had >32.6 FSP1(+) cells/HPF at diagnosis, they were the more likely to show steroid resistance. CONCLUSION: FSP1(+) cell number can serve as an excellent predictor of corticosteroid responsiveness in patients with IgAN.

Hypoxia-inducible factor signaling in the development of tissue fibrosis.

Capillary rarefaction is a hallmark of fibrotic diseases and results in reduced blood perfusion and oxygen delivery. In the kidney, tubulointerstitial fibrosis, which leads to the destruction of renal tissue and the irreversible loss of kidney function, is associated with hypoxia and the activation of Hypoxia-Inducible-Factor (HIF) signaling. HIF-1 and HIF-2 are basic-helix-loop-helix transcription factors that allow cells to survive in a low oxygen environment by regulating energy metabolism, vascular remodeling, erythropoiesis, cellular proliferation and apoptosis. Recent studies suggest that HIF activation promotes epithelial to mesenchymal transition (EMT) and renal fibrogenesis. These findings raise the possibility that the spectrum of HIF activated biological responses to hypoxic stress may differ under conditions of acute and chronic hypoxia. Here we discuss the role of HIF signaling in the pathogenesis and progression of chronic kidney disease.

Submolecular resolution viscoelastic imaging of a poly(p-toluene-sulfonate) single crystal using force modulation microscopy.

Submolecular resolution viscoelastic imaging of a poly(2,4-hexadiyne-1,6-diyl bis(p-toluene-sulfonate)) single crystal was achieved using force modulation microscopy under ambient conditions. The elastic image clearly visualized the structure of p-toluene-sulfonate side chains. The viscotic image visualized that the phase delay on the main chains was smallest, while it became largest on the toluene rings in the side chains. The result is considered to be closely related to the molecular dynamics of the crystal.

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.

ANP is cleared much faster than BNP in patients with congestive heart failure.

OBJECTIVE: To obtain a better understanding of the pharmacokinetics of atrial and brain natriuretic peptides (ANP and BNP, respectively), two peptide mediators used in the treatment of congestive heart failure (CHF). Although both peptides exert their effects by binding to a common receptor (natriuretic peptide receptor A) with about the same affinity, their respective loading and maintenance doses differ. METHODS: Sixteen CHF patients were randomized to be infused for 2 h with alpha-human ANP (0.05 microg/kg per minute) or BNP (0.01 microg/kg per minute). Plasma concentrations of both peptides were measured 0, 2, 5, 15, 30, 60 and 120 min post-infusion. The pharmacokinetic parameters were then calculated using a 1-compartment model. RESULTS: The plasma BNP concentrations in the ANP and BNP groups before infusion were 464.7 +/- 339.8 and 506.8 +/- 332.5 pg/ml, respectively. Following infusion, ANP disappeared from the circulation more rapidly than BNP: their plasma half-lives were 2.4 +/- 0.7 and 12.1 +/- 3.0 min, and their total body clearance volumes were 48.2 +/- 24.1 and 10.1 +/- 2.7 ml/min per kilogram, respectively. CONCLUSION: ANP has a shorter half-life in the plasma of CHF patients than BNP, which suggests that it controls hemodynamics more readily than BNP.

Investigation of molecular chain orientation change of polymer crystals in phase transitions by friction anisotropy measurement.

Direct observation of the molecular orientation change in polymer crystals provides us visible information for understanding their structural phase-transition mechanisms. In this letter, we successfully identified the main-chain orientation of poly(vinylidenefluoride-trifluoroethylene) (P(VDF-TrFE)) crystals over all directions using friction anisotropy measured by lateral-modulation friction force microscopy (LM-FFM). This technique made possible our investigation of molecular orientation changes caused by a ferroelectric phase transition and also a fabrication process for artificial nanometer-scale structures. These results give us visual information that is directly connected to the transition mechanisms.

Surrogate markers of insulin resistance in assessing individuals with new categories "prehypertension" and "prediabetes".

BACKGROUND: There are few data on the impact of insulin resistance on the recently defined categories of prehypertension (PHT) and prediabetes (PDM). The aim of this study was to examine associations of surrogate markers of insulin resistance with PHT/PDM. METHODS: Subjects included 554 individuals who underwent a 75-g oral glucose tolerance test (OGTT). They were classified into four groups using a severity score for high blood pressure and glucose tolerance. Insulin resistance was measured using the homeostasis model assessment of insulin resistance (HOMA-R) and three surrogate markers obtained from 75-g OGTT values (ISI-composite, Stumvoll index, and OGIS index). RESULTS: HOMA-R increased significantly, but the other three surrogate indices decreased with increasing severity score. Of these markers, the OGIS index was mostly associated with prevalent PHT/PDM and the odds ratio for insulin resistance was 3.61 (95% CI 1.68-7.76, p=0.001) for subjects with either PHT or PDM and 29.98 (12.81-70.18, p<0.001) for subjects with both PHT and PDM. CONCLUSIONS: PHT and PDM frequently coexist in relatively lean Japanese subjects. Decreased insulin sensitivity may contribute to the underlying status of PHT/PDM. Among the surrogate markers of insulin resistance, the OGIS index is the most sensitive for assessment of PHT/PDM status.

Utility of elevated 2-hour postload plasma glucose as an alternative to elevated fasting glucose as a criterion for the metabolic syndrome.

We hypothesize that many persons with postchallenge hyperglycemia (PCH) but who do not meet the National Cholesterol Education Program (NCEP) criteria characterize a phenotype that is similar to the metabolic syndrome (MS) traits. Subjects included 725 Japanese who underwent a 75-g oral glucose tolerance test. If 2-hour plasma glucose of 7.8 mmol/L or higher was present, subjects with fasting glucose of less than 6.1 mmol/L could have one component of the MS (PCH-MS). Data obtained by the 75-g oral glucose tolerance test were used to calculate 3 insulin sensitivity indexes according to formulas proposed by Matsuda and DeFronzo (insulin sensitivity index composite), Stumvoll et al (Srumvoll index), and Mari et al (oral glucose insulin sensitivity index). Based on the PCH-MS and NCEP-MS criteria, 395 had neither PCH-MS nor NCEP-MS, 85 had PCH-MS, and 245 had NCEP-MS. Subjects with PCH-MS exhibited higher systolic blood pressure and triglyceride levels, lower high-density lipoprotein cholesterol levels, and lower insulin sensitivity than those who had neither PCH-MS nor NCEP-MS. A similar profile was observed when subjects with NCEP-MS were compared with those who had neither PCH-MS nor NCEP-MS. All 3 indexes of insulin sensitivity were significantly lower in subjects with PCH-MS than in those who had neither PCH-MS nor NCEP-MS, and approximately 66% of PCH-MS was in an insulin-resistant state. On the other hand, there was no statistical difference in the values between PCH-MS and NCEP-MS. Our data support the addition of abnormal 2-hour plasma glucose as a criterion for the MS, when fasting glucose is normal.

Real-time 3-D echocardiographic evaluation of the fetal heart using instantaneous volume-rendered display.

AIM: Using new real-time 3-D fetal echocardiography with instantaneous volume-rendered display, we evaluated the heart anatomy of a number of normal fetuses during pregnancy. METHODS: Eighteen normal fetuses in 17 pregnancies (16 singletons and one twin) at 18-38 weeks' gestation were studied using a transabdominal real-time 3-D ultrasound machine. This machine proved capable of providing continuous 3-D sonographic images every 0.05 and 0.035 s without the need for an external workstation or other additional, costly equipment. For each patient, the fetal heart was first monitored using conventional 2-D echocardiography and was monitored again within 10 min using real-time 3-D echocardiography. RESULTS: Consecutive real-time 3-D images showing a four-chamber view, long-axis view, short-axis view, and right ventricular outflow tract view were obtained in 100%, 66.6%, 38.8%, and 22.2% of fetuses in the study, respectively. Morphological changes to each atrium or ventricle could be observed clearly and in detail throughout the cardiac cycle. The opening and closing of each valve were clearly visible. Moreover, these observations could be made from any direction. CONCLUSIONS: Real-time 3-D echocardiography provides a novel means for evaluation of the fetal heart in 3-D in real time in the second and third trimester of pregnancy. Real-time 3-D echocardiography may be an important modality in future fetal cardiac research and in evaluation of congenital heart disease in the fetus.

Real-time 3-D sonographic observation of fetal facial expression.

AIM: There have been a few reports about 3-D sonographic observation of fetal movements using dynamic 3-D sonography. However, dynamic 3-D sonography is not real-time, the frame rate being in the region of 4-6 frames per second depending on the size of the region of interest and the number of lines employed. Recently, a new faster 3-D sonography, which acquires up to 28 frames per second, has become available. Using this system, we studied a full range of fetal facial expressions during pregnancy. METHODS: A total of 17 normal fetuses in 16 pregnancies (15 singletons and one twin) at 20-38 weeks' gestation was studied using a transabdominal real-time 3-D ultrasound machine. This 3-D ultrasound machine proved capable of providing continuous 3-D sonographic images every 0.05 and 0.035 s. The fetal face was monitored for 15 min for each subject. RESULTS: Fetal eyelid movement (fetal blinking) was observed in three of 17 fetuses (17.6%). Double blinking was identified in one fetus at 38 weeks. Various types of mouth movement (yawning, a little opening, chewing, and subtle lip movement) could be observed in nine of 17 fetuses (52.9%). In the course of yawn-like opening of the mouth, tongue movements such as tongue thrust and tongue click were clearly shown in three fetuses (17.6%). A lingula movement was also identified in the course of tongue movement. CONCLUSION: Real-time 3-D sonography provides a novel means for evaluation of fetal movement, particularly fetal facial expression, in the second and third trimesters. Real-time 3-D sonography might be an important modality in future fetal behavior research and in evaluation of fetal well-being.

Insulin resistance and pancreatic beta-cell function in patients with hypertensive kidney disease.

BACKGROUND: Insulin resistance and hyperinsulinaemia have been reported among patients with chronic renal failure. However, little is known concerning insulin sensitivity among patients with hypertensive kidney disease (HKD), especially in those with moderate or severe renal dysfunction. METHODS: We examined and compared 30 patients with HKD, 30 normotensive patients with chronic kidney disease (CKD-NT), 30 normal controls and 30 patients with hypertension and normal renal function (HTN). Moderate and severe renal dysfunction were defined according to the K/DOQI definitions (estimated glomerular filtration rates between 15 and 59 ml/min per 1.73 m(2)). The homeostasis model assessment of insulin resistance (HOMA-R) and three surrogate indexes based on 75 g oral glucose tolerance test results were used to determine insulin sensitivity. RESULTS: A trend to higher HOMA-R values in the HTN and HKD groups than in the other groups was noted, but the difference was not statistically significant. The insulin sensitivity index (ISI) proposed by Stumvoll et al. was significantly lower in the HTN, HKD and CKD-NT groups than in controls and was significantly lower in HKD than in the HTN and CKD-NT groups. The insulin sensitivity index proposed by Gutt et al. was significantly lower in HKD than in the control and HTN groups and showed a trend to being lower in HKD than in CKD-NT. The same patterns prevailed in the oral glucose ISI. We assumed that subjects whose ISI values decreased below the mean value minus 2-SD in the control group manifest apparent insulin resistance. According to this criterion, approximately 40% of HKD subjects were in an insulin-resistant state: only <10% of HTN subjects and approximately 10-30% of CKD-NT subjects were insulin resistant. CONCLUSIONS: HKD with moderate to severe renal dysfunction is associated with insulin resistance.