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Stomach cancer is a global health concern as millions of cases are reported each year. In the present study, we developed a pH-responsive microrobot with good biocompatibility, magnetic-field controlled movements, and the ability to be visualized

Wearable biosensors have the potential for developing individualized health evaluation and detection systems owing to their ability to provide continuous real-time physiological data. Among various wearable biosensors, localized surface plasmon resonance (LSPR)-based wearable sensors can be versatile in various practical applications owing to their sensitive interactions with specific analytes. Understanding and analyzing endocrine responses to stress is particularly crucial for evaluating human performance, diagnosing stress-related diseases, and monitoring mental health, as stress takes a serious toll on physiological health and psychological well-being. Cortisol is an essential biomarker of stress because of the close relationship between cortisol concentration in the human body and stress level. In this study, a flexible LSPR biosensor was manufactured to detect cortisol levels in the human body by depositing gold nanoparticle (AuNP) layers on a 3-aminopropyltriethoxysilane (APTES)-functionalized poly (dimethylsiloxane) (PDMS) substrate. Subsequently, an aptamer was immobilized on the surface of the LSPR substrate, enabling highly sensitive and selective cortisol capture owing to its specific cortisol recognition. The biosensor exhibited excellent detection ability in cortisol solutions of various concentrations ranging from 0.1 to 1000 nM with a detection limit of 0.1 nM. The flexible LSPR biosensor also demonstrated good stability under various mechanical deformations. Furthermore, the cortisol levels of the flexible LSPR biosensor were also measured in the human epidermis before and after exercise as well as in the morning and afternoon. Our biosensors, which combine easily manufactured flexible sensors with sensitive cortisol-detecting molecules to measure human stress levels, could be versatile candidates for human-friendly products.

Microrobots that can be precisely guided to target lesions have been studied for in vivo medical applications. However, existing microrobots have challenges in vivo such as biocompatibility, biodegradability, actuation module, and intra- and postoperative imaging. This study reports microrobots visualized with real-time x-ray and magnetic resonance imaging (MRI) that can be magnetically guided to tumor feeding vessels for transcatheter liver chemoembolization in vivo. The microrobots, composed of a hydrogel-enveloped porous structure and magnetic nanoparticles, enable targeted delivery of therapeutic and imaging agents via magnetic guidance from the actuation module under real-time x-ray imaging. In addition, the microrobots can be tracked using MRI as postoperative imaging and then slowly degrade over time. The in vivo validation of microrobot system–mediated chemoembolization was demonstrated in a rat liver with a tumor model. The proposed microrobot provides an advanced medical robotic platform that can overcome the limitations of existing microrobots and current liver chemoembolization.

The use of untethered microrobots for precise synergistic anticancer drug delivery and controlled release has attracted attention over the past decade. A high surface area of the microrobot is desirable to achieve greater therapeutic effect by increasing the drug load. Therefore, various nano- or microporous microrobot structures have been developed to load more drugs. However, as most porous structures are not interconnected deep inside, the drug-loading efficiency may be reduced. Here, we propose a magnetically guided helical microrobot with a Gyroid surface for high drug-loading efficiency and precise drug delivery. All spaces inside the proposed microrobot are interconnected, thereby enabling drug loading deep inside the structure. Moreover, we introduce gold nanostars on the microrobot structure for near-infrared-induced photothermal therapy and triggering drug release. The results of this study encourage further exploration of a high loading efficiency in cell-based therapeutics, such as stem cells or immune cells, for microrobot-based drug-delivery systems.

A cylindrical shaped marine invertebrate ascidian has muscle fibers surrounding its body, which induce the contraction motion when the animal senses the external stimuli. As inspired by its cylindrical shape and the contraction motion of the ascidian, we introduce a soft robot that resembles this water animal. In this letter, we first discuss the design of the robot that can be magnetically actuated and create different motions due to different magnetic moments for each segment of the robot. The crawling motion of the robot is presented with a sinusoidal waveform of the magnetic field and we demonstrate the utility of our bio-inspired soft robot for transporting a millimeter-sized object and releasing a drug in a specific location.

Oxycodone is a common type of opioid used for the treatment of moderate to severe pain. Besides its analgesic effects on neuron cells, the effects of oxycodone on other cell types are yet to be elucidated. We previously demonstrated that oxycodone displayed both pro- and anti-cancer effects on bulk cancer cells. This work further investigated the effects of oxycodone on normal and malignant hematopoietic stem cells. Using hematopoietic CD34+ cells isolated from normal bone marrow (NBM) or patients with acute myeloid leukemia (AML), we showed that oxycodone activates hematopoietic cells regardless of cell development stage and malignant status. Oxycodone dose-dependently increases colony formation and self-renewal capacity of NBM and AML stem/progenitor cells, and promotes proliferation of AML bulk cells. NBM stem/progenitor cells are more sensitive to oxycodone than AML counterparts. In addition, oxycodone alleviates chemotherapy drug-induced toxicity in AML stem/progenitor cells. Mechanism studies demonstrate that oxycodone acts on hematopoietic cells in an opioid-receptor-independent manner. Oxycodone did not affect epithelial growth factor receptor (EGFR) signaling neither but stimulated Wnt/β-catenin signaling. Rescue studies via depleting β-catenin using genetic and pharmacological approaches confirmed that β-catenin was required for the activation of hematopoietic cells induced by oxycodone. Our work demonstrates 1) the protective role of oxycodone in malignant hematopoietic cells from chemotherapy; 2) stimulatory effects of oxycodone in normal hematopoietic stem cells; and 3) ability of oxycodone in Wnt signaling activation.

CD45 is a pan-leukocyte marker, and CD45 stain is widely used to determine the extent of inflammatory cell infiltration and its association with tissue injury. In this manuscript, we share a reliable immunohistochemistry (IHC) protocol for CD45 staining in sections of paraffin-embedded mouse kidney. A rat anti-CD45 antibody was used as primary antibody, and a mouse adsorbed biotin-conjugated goat anti-rat IgG was selected as secondary antibody. A horseradish peroxidase (HRP)-linked avidin/biotin detection system was used to amplify the signal, which was detected with 3,3′-Diaminobenzidine (DAB). With this protocol, we show that the CD45 antibody recognizes cells of hematolymphoid lineage in bone marrow, as well as monocyte/macrophages in liver and lung tissue. The utility of this protocol in pathology research was indicated by dramatically increased CD45-positive (CD45(+)) cells in the kidneys of a mouse model of diabetes. Double staining for CD45 and injury marker KIM-1 showed accumulated CD45(+) cells around injured tubular cells. CD45 and F4/80 macrophage staining on adjacent tissue sections revealed overlap of CD45(+) cells with other inflammatory cells.

Understanding the academic performance of students in colleges is an essential topic in Education research field. Educators, program coordinators and professors are interested in understanding how students are learning specific topics, how specific topics may influence the learning of other topics, how students’ grades/attendances in each course may represent important indicators to measure their performance, among other tasks. The use of data visualization and analytics is expanding in education institutions to perform a variety of tasks related to data processing and gaining into data-informed insights. In this paper, we present a visual analytic tool that combines data visualization and machine learning techniques to perform some visual analysis of students’ data from program courses. Two educational data collections were used to guide the creation of i) predictive models employing a variety of well known machine learning strategies, attempting to predict students’ future grade based on grade and attendance previous semesters and ii) a set interactive layouts that highlight the relationship between grades and attendance, also including additional variables such as gender, parents education level, among others. We performed several experiments, also using these data collections, to evaluate the layouts ability of highlighting interesting patterns, and we obtained promising results, demonstrating that such analysis may help the education experts to understand deficiencies on course structures.

We described a magnetic chitosan microscaffold tailored for applications requiring high biocompatibility, biodegradability, and monitoring by real-time imaging. Such magnetic microscaffolds exhibit adjustable pores and sizes depending on the target application and provide various functions such as magnetic actuation and enhanced cell adhesion using biomaterial-based magnetic particles. Subsequently, we fabricated the magnetic chitosan microscaffolds with optimized shape and pore properties to specific target diseases. As a versatile tool, the capability of the developed microscaffold was demonstrated through in vitro laboratory tasks and in vivo therapeutic applications for liver cancer therapy and knee cartilage regeneration. We anticipate that the optimal design and fabrication of the presented microscaffold will advance the technology of biopolymer-based microscaffolds and micro/nanorobots.

Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor–stimulated enhanced expression and secretion of NF-κB–targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.

Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low‐density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high‐quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte‐like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH‐HLCs need to be corrected. Genome editing technology clustered‐regularly‐interspaced‐short‐palindromic‐repeats/CRISPR‐associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. Conclusion: We used CRISPR/Cas9 genome editing to permanently correct a 3‐base pair homozygous deletion in LDLR exon 4 of patient‐derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR‐mediated endocytosis in FH‐HLCs and demonstrates the proof‐of‐principle that CRISPR‐mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level. (Hepatology Communications 2017;1:886–898)

Understanding the academic performance of students in colleges is an essential topic in Education research field. Educators, program coordinators and professors are interested in understanding how students are learning specific topics, how specific topics may influence the learning of other topics, how students' grades/attendances in each course may represent important indicators to measure their performance, among other tasks. In this paper, we present a visual analytic tool that combines data visualization and machine learning techniques to perform some visual analysis of students' data from program courses. Using this tool, we visually analyzed the students' performance in some Computer Science program courses, and demonstrated that the results of such analysis will help the education experts to understand deficiencies on course structures.

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age-matched OVE diabetic mice to produce bi-transgenic OVE-JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE-JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day-old OVE-JTMT mice, blood glucose and HbA1c were indistinguishable from age-matched OVE mice. However, endothelial-specific MT overexpression in OVE-JTMT mice mitigated several DN complications including significantly increased non-fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE-JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560-576, 2017. © 2016 Wiley Periodicals, Inc.

There are challenges in developing practically viable biopolymer-based actuators with ecofriendly, biocompatible, and biodegradable functionalities. Therefore, we propose a cellulose acetate (CA)-based ecofriendly soft-ionic networking actuator consisting of multifunctional additive polyvinylidene difluoride (PVDF), highly conductive ammonia-functionalized graphene nanoplatelets (AFGNPs), ionic liquids (IL), and flexible conducting polymer poly(3,4-ethylenedioxuthiopene)-polystyrene sulfonate (PEDOT:PSS) as an electrode. The proposed actuator exhibits a large bending displacement and short response time in an open-air environment, resulting from its enhanced electro-chemo-mechanical properties and strong ionic and interfacial interactions. In comparison with CA/PVDF-IL actuator, the CA/PVDF–IL–AFGNPs actuator demonstrates a considerably increased IL uptake and ion-exchange capacity of up to 71.04% and 300.6%, respectively, and an increase in the specific capacitance by over 3.64 times, which lead to bending actuation performances 2.21 and 1.87 times greater, respectively, under AC (4 V) and DC (4 V). Moreover, we demonstrate that a CA/PVDF–IL–AFGNPs actuator with a hierarchical structure shows values that are 1.32, 1.5, and 1.74 times larger than those of a planar actuator in DC (4 V), AC (3 V), and blocking force (4 V), respectively. The developed high-performance CA/PVDF–AFGNPs and the hierarchical surface texture of the patterned CA/PVDF–IL–AFGNPs actuators present these extraordinary achievements together with environmentally friendly materials, a low driving voltage, easy manufacturing, and high actuation performances. Therefore, they can be candidates for human-friendly products (e.g., biomedical devices, bioinspired robots, and soft haptic devices).

The effect of diabetes in vivo has not been examined on isolated podocytes. To achieve this, GFP was expressed constitutively in podocytes of PGFP transgenic mice which were bred to OVE mice to produce diabetic OVE-GFP mice. Viewing GFP fluorescence, foot processes of OVE-GFP podocytes were visually and measurably effaced, which did not occur with less severe STZ diabetes. Over 300,000 podocytes were purified from each PGFP mouse but only 49,000 podocytes per diabetic OVE-GFP mouse. The low yield from OVE-GFP mice appeared to be due to more fragile state of most OVE-GFP diabetic podocytes which did not survive the isolation process. Diabetic podocytes that were isolated had high levels of the lipid peroxidation product 4-HNE and they were more sensitive to death due to oxidative stress. Gene array analysis of OVE-GFP podocytes showed strong diabetes induction of genes involved in inflammation. Four CXC chemokines were induced at least 3-fold and the chemokine CXCL1 was shown for the first time to be specifically induced in podocytes by OVE, dbdb and STZ diabetes.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

In diabetic nephropathy (DN) proinflammatory chemokines and leukocyte infiltration correlate with tubulointerstitial injury and declining renal function. The atypical chemokine receptor ACKR2 is a chemokine scavenger receptor which binds and sequesters many inflammatory CC chemokines but does not transduce typical G-protein mediated signaling events. ACKR2 is known to regulate diverse inflammatory diseases but its role in DN has not been tested. In this study, we utilized ACKR2−/−mice to test whether ACKR2 elimination alters progression of diabetic kidney disease. Elimination of ACKR2 greatly reduced DN in OVE26 mice, an established DN model. Albuminuria was significantly lower at 2, 4, and 6 months of age. ACKR2 deletion did not affect diabetic blood glucose levels but significantly decreased parameters of renal inflammation including leukocyte infiltration and fibrosis. Activation of pathways that increase inflammatory gene expression was attenuated. Human biopsies stained with ACKR2 antibody revealed increased staining in diabetic kidney, especially in some tubule and interstitial cells. The results demonstrate a significant interaction between diabetes and ACKR2 protein in the kidney. Unexpectedly, ACKR2 deletion reduced renal inflammation in diabetes and the ultimate response was a high degree of protection from diabetic nephropathy.

Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-response-induced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced pro-apoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.

The objective of this essay is to provide a design project for a mobile interactive picture book that features Chinese short stories and to develop a mobile application of interactive picture books by using Unity 3D. The author attempts to discuss the design solution and development process of the mobile interactive picture book compatible with children’s cognitive characteristics by weighing up the pros and cons of interactive designs of mobile picture books both at home and abroad. In conclusion, by recognizing the importance of current mobile interactive media in children’s early education, the author puts forward the concept that education is in line with recreation, featuring seeing, hearing, entertaining and thinking combined together, and designs and develops the mobile interactive picture book from the perspectives of content, interactive approach and interface design so as to make the childhood education more interesting and boost the interaction between parents and children. Also, the author aims to feasible way of spreading and inheriting traditional Chinese culture.

Human epidemiological and animal studies have shown the beneficial effect of zinc supplementation on mitigating diabetic nephropathy. However, the mechanism by which zinc protects the kidney from diabetes remains unknown. Here we demonstrate the therapeutic effects of zinc on diabetes-induced renal pathological and functional changes. These abnormalities were found in both transgenic OVE26 and Akt2-KO diabetic mouse models, accompanied by significant changes in glucose-metabolism-related regulators. The changes included significantly decreased phosphorylation of Akt and GSK-3β, increased phosphorylation of renal glycogen synthase, decreased expression of hexokinase II and PGC-1α, and increased expression of the Akt negative regulators PTEN, PTP1B, and TRB3. All of these were significantly prevented by zinc treatment for 3 months. Furthermore, zinc-stimulated changes in glucose metabolism mediated by Akt were actually found to be metallothionein dependent, but not Akt2 dependent. These results suggest that the therapeutic effects of zinc in diabetic nephropathy are mediated, in part, by the preservation of glucose-metabolism-related pathways via the prevention of diabetes-induced upregulation of Akt negative regulators. Given that zinc deficiency is very common in diabetics, this finding implies that regularly monitoring zinc levels in diabetic patients, as well as supplementing if low, is important in mitigating the development of diabetic nephropathy.

Maleic anhydride (MAH) was grafted onto low-density polyethylene (LDPE) by a solution approach with dicumyl peroxide (DCP) as initiator and xylene as solvent. The effects of various experimental conditions on the grafting degree (GD, wt%), such as DCP concentration (A), MAH concentration (B), reaction time (C), reaction temperature (D) and the time for initiator addition (E), were investigated by an orthogonal experiment. The results demonstrated that the DCP concentration had the greatest influence on GD, while the reaction temperature had a minimal influence on GD. A copolymer with high GD (up to 4%) was obtained under the optimum conditions. It was proven that MAH was grafted onto the LDPE successfully by FT-IR. The crystallization behavior of the grafted LDPE was studied by differential scanning calorimetry (DSC) and X-ray diffraction (XRD), and the crystallization kinetic parameters of the grafted LDPE were obtained. Compounds of paraffin and grafted LDPE were investigated by XRD. The results demonst...

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension-induced athero- or arteriosclerosis. This may be due either to generalized vascular disease, tissue-based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia-driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi-transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ~3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra- and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury.

Background We previously demonstrated that cellular and extracellular components of the blood–urine barrier in renal glomeruli are susceptible to damage in OVE transgenic mice, a valuable model of human diabetic nephropathy that expresses profound albuminuria. Methods To test our hypothesis that glomerular filtration barrier damage in OVE mice may be the result of oxidative insult to podocytes, 150-day-old bi-transgenic OVENmt diabetic mice that overexpress the antioxidant metallothionein specifically in podocytes were examined by enzyme-linked immunosorbent assay for albuminuria mitigation and by unbiased transmission electron microscopy (TEM) stereometry for protection from chronic structural diabetic complications. Results Although blood glucose and HbA1c levels were indistinguishable in OVE and OVENmt animals, albuminuria was significantly reduced (average >7-fold) in OVENmt mice through 8 months of age. Interestingly, the Nmt transgene provided significant glomerular protection against diabetic nephropathic complications outside of the podocyte. Glomerular filtration barrier damage was reduced in OVENmt mice, including significantly increased area occupied by endothelial luminal fenestrations (~13%), significantly reduced glomerular basement membrane (GBM) thickening (~17%) and significantly less podocyte effacement (~18%). In addition, OVENmt mice exhibited significantly reduced glomerular volume (~50%), fewer glomerular endothelial cells (~33%), fewer mesangial cells (~57%) and fewer total glomerular cells (~40%). Conclusions These results provide evidence of oxidative damage to podocytes induces primary diabetic nephropathic features including severe and sustained albuminuria, specific glomerular filtration barrier damage and alterations in glomerular endothelial and mesangial cell number. Importantly, these diabetic complications are significantly mitigated by podocyte targeted metallothionein overexpression. Copyright © 2012 John Wiley & Sons, Ltd.

Oxidative stress is a major cause of diabetic nephropathy. Upregulation of the key antioxidative transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), was found to prevent the development of diabetic nephropathy. The present study was designed to explore the therapeutic effect of Nrf2 induced by proteasomal inhibitor MG132 at a low dose (10 μg/kg) on diabetic nephropathy. Transgenic type 1 diabetic (OVE26) mice displayed renal dysfunction with albuminuria by 3 mo of age, at which time MG132 treatment was started. After 3-mo treatment with MG132, renal function, morphology, and biochemical changes were examined with real-time PCR, Western blotting, and immunohistochemical examination. Compared with age-matched, nontreated diabetic mice, MG132-treated diabetic mice showed significant improvements in terms of renal structural and functional alterations. These therapeutic effects were associated with increased Nrf2 expression and transcriptional upregulation of Nrf2-regulated antioxidants. Mechanistic study using human renal tubular HK11 cells confirmed the role of Nrf2, as silencing the Nrf2 gene with its specific siRNA abolished MG132 prevention of high-glucose-induced profibrotic response. Furthermore, diabetes was found to significantly increase proteasomal activity in the kidney, an effect that was significantly attenuated by 3 mo of treatment with MG132. These results suggest that MG132 upregulates Nrf2 function via inhibition of diabetes-increased proteasomal activity, which can provide the basis for the therapeutic effect of MG132 on the kidney against diabetes-induced oxidative damage, inflammation, fibrosis, and eventual dysfunction.

Background/Aims: OVE26 (OVE) mice provide a valuable model of advanced diabetic nephropathy (DN), but they take 8 months to develop moderate interstitial fibrosis and reduced glomerular filtration rate (GFR). The aim of this project was to produce a more rapid and advanced model of DN. Methods: Uninephrectomy was applied to OVE and FVB mice at 2 months of age. Albuminuria, GFR, glomerulosclerosis, interstitial fibrosis, gene expression and monocyte infiltration were evaluated as a function of diabetes and uninephrectomy. Results: Albuminuria, monocyte infiltration, mesangial matrix expansion and renal fibrosis were greatly accelerated in uninephrectomized mice. DN was more advanced 10 weeks after uninephrectomy than in untreated OVE mice at 8 months of age. Uninephrectomy had almost no effect on these characteristics in non-diabetic mice. Microarray studies indicated that the accelerated fibrosis and cell infiltration in nephrectomized OVE mice were accompanied by corresponding gene expression changes in canonical pathways for fibrosis and inflammation. Conclusion: Uninephrectomy greatly accelerates all features of diabetic renal damage. This procedure provides a 10-week period after surgery to examine very large changes in the pathology of DN. The model may be particularly useful for testing new therapies and for analysis of the progression of albuminuria and fibrosis in DN.

Aim: To define renal gene expression during the development of severe albuminuria in OVE26 diabetic mice. Methods: Kidney microarray analysis was performed at 2, 4 and 8 months. Data were validated by RT-PCR, in situ hybridization and immunohistochemistry. Results: Gene expression differences between control and diabetic mice increased 10-fold from 2 to 8 months. This change was most obvious for inflammatory genes. Three inflammatory genes, complement C3, VCAM1 and CD44 were upregulated more than 4-fold. Inflammatory gene expression correlated with albuminuria and C3 and CD44 increased in tubules that accumulated albumin. VCAM1 was induced in different tubules that were neither dilated nor accumulated albumin. Six of 8 genes previously reported to be markers of human advanced diabetic nephropathy and the NF-ĸB_IFN_x promoter module were elevated in the oldest diabetic mice. Vitamin D inhibits diabetic renal inflammation. Vitamin D and mRNA for vitamin D synthetic enzyme CYP2B1 were elevated in kidneys of young OVE26 mice. Conclusions: OVE26 mice induce inflammatory genes consistent with advanced renal disease, associated with severe albuminuria and to a greater extent than reported in other diabetic models. They provide an excellent model of diabetic nephropathy to assess the effect of induction of inflammatory proteins.

OVE26 (OVE) diabetic mice on the inbred strain FVB are a valuable model of diabetic nephropathy that excretes the highest amount of urine albumin of all diabetic mouse models. Crossing of OVE mice to C57BL6 or DBA2 mice reduced albuminuria 17-fold in F1 diabetic offspring without reducing diabetes. When comparing renal histology of OVE mice on the FVB background to F1 C57BL6 crosses, we found that the F1 kidneys had significantly smaller glomeruli, much less albumin accumulation in tubules, reduced mesangial matrix expansion, and less interstitial fibrosis. A genome scan of 108 OVE-positive N2 offspring for albuminuria revealed one significant peak on chromosome 11 and nearly significant peaks on chromosomes 9, 13, and 19. Homozygosity for the FVB genotype for peaks on chromosomes 11, 13, or 19 increased albuminuria. Homozygosity for the chromosome 9 peak reduced albuminuria. Combined homozyogosity for the peaks on chromosomes 11, 13, and 19 increased albuminuria over 12-fold and accounted for >70% of the difference between OVE mice on the FVB vs. the F1 background. These loci contain sequences important to susceptibility to diabetic albuminuria.

MicroRNAs (miRs), which regulate target gene expression at the post-transcriptional level, play a crucial role in inducing biological effects upon high-dose ionizing radiation. Yet, the miR expression profiles in response to repeated low-dose radiation (LDR) in vivo have not been elucidated. This study investigated the response profiles of 11 miRs with functions involved in metabolism, DNA damage and repair, inflammation, and fibrosis in mouse liver, heart, and testis upon repeated LDR exposure for 4 months. The expression profiles were evaluated using stem-loop quantitative reverse transcription polymerase chain reaction immediately and at 2 months after LDR exposure. The expression profiles varied significantly at both time points. At the organ level, the heart was the most affected, followed by the liver and testis, in which significant miR upregulation related to DNA damage response was found. Metabolism-related miRs decreased in the liver and increased in the testis. The current results showed immediate and long-lasting alterations in the miR expression profiles in response to repeated LDR in different organs.

The aldo-keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan-AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan-AKR antibodies exhibited the immune specificities and immune sensitivities, Pan-AKR-P1 for AKR1B and AKR1C, Pan-AKR-P3 for AKR1C and Pan-AKR-P4 for all the AKR proteins. Pan-AKR-P4 antibody was employed to 2-DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune-reactive spots from each tissue. Protein identification with MS revealed that most immune-positive spots were the members of AKR superfamily. Furthermore, Pan-AKR-P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.

Adriamycin (ADR) is nephrotoxic. One component of ADR-induced nephropathy may be oxidative stress. This study used a recently developed line of transgenic mice (Nmt) on the FVB background strain, which over-express the antioxidant protein metallothionein (MT) in podocytes. Cultured podocytes from Nmt mice were resistant to H(2)O(2) injury, as judged by disruption of F-actin filaments. FVB control and transgenic mice received 11 mg/kg body weight ADR by tail vein injection and 24-h urine samples were then collected for albumin analysis. Also renal morphology was investigated by light and electron microscopy. Urine albumin analysis showed that ADR treatment significantly increased albuminuria in control mice, indicating that the FVB strain is sensitive to ADR nephropathy and Nmt mice were significantly protected from elevated albuminuria. Glomerular histopathology revealed that ADR reduced podocyte number and produced foot process effacement in FVB mice. The Nmt transgene protected podocyte numbers and podocyte foot processes from the effects of ADR. These results show that metallothionein can protect podocytes from ADR toxicity.

Background/Aims: Tubulointerstitial fibrosis (TIF) is a prominent feature of progressive diabetic nephropathy. The goal of this study was to determine if hallmarks of TIF occur in the transgenic OVE26 type 1 diabetic mouse and define signaling events associated with TIF. Methods: The expression patterns of several phenotypic markers of TIF were determined in kidneys of OVE26 diabetic and control mice by immunohistochemistry and immunoblot analysis. Results: Pathological signatures of TIF are an accumulation of myofibroblasts and excessive deposition of extracellular matrix in the tubulointerstitium. Kidneys from OVE26 diabetic animals exhibited an increase in tubulointerstitial myofibroblast marker (α-smooth muscle actin), fibronectin and collagen I staining. Abundance of the pro-fibrotic cytokine TGF-β was also enhanced in diabetic tubules. As injury involving loss of epithelial cell-cell contact promotes tissue fibrosis, we examined expression of the adhesion protein, E-cadherin. The percent of E-cadherin-stained tubules was decreased in diabetic kidneys. Prominent regulators of TGF-β signaling, glycogen synthase kinase-3 (GSK-3) α and β, were also differentially expressed. Conclusions: These results indicate that TGF-β-induced TIF occurs in OVE26 diabetic mice, providing a practical in vivo model for defining novel regulatory events and treatment strategies for diabetes-induced TIF.

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′ -kinase( PI3K )- Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

Diabetic nephropathy (DN) is a serious complication of diabetes; it initially manifests with microalbuminuria and progresses towards end-stage renal failure. Sustained diabetes-related metabolic and haemodynamic perturbations can induce subclinical low-grade renal inflammation and drive kidney from repair response to damage process, eventually to renal fibrosis. In this special issue, we include articles regarding inflammation, Chinese herbs, and systems biology to present up-to-date information on immune cells, chemokine receptor, and biomarkers in DN, displaying combined therapy in treatment of DN and highlighting the effective approach in exploring genetic susceptibility of DN. (1) Inflammation. Despite the broad themes covered by this special issue, all articles focus on a common theme: immune cells and inflammation. Hyperglycemia and oxidative stress, as well as albuminuria per se, can lead the immune and inflammatory cells to infiltrate into kidney and release proinflammatory cytokines. This inflammatory “repair process” reverts to and manifests as a “chronic unfavorable process” that eventually leads to the disease phenotype (renal fibrosis). The review article by Z. Zheng and F. Zheng summarizes the role of immune cells and inflammation in DN, highlighting the contribution of APC cells, T-helper cells, and tubular epithelial cells to the inflammation. S. Zheng et al. reported the renal expression of decoy chemokine receptor ACKR2 in DN patients and renal protection in diabetic mice with ACKR2 gene knockout, revealing the unexpected negative role of ACKR2 in diabetic kidney disease. Association of haemostatic and inflammatory biomarkers with nephropathy in type I diabetic patients is shown by C. P. Domingueti et al., indicating potential therapeutic targets for DN. (2) Chinese Medicine. Herbs are major form of therapy in traditional Chinese medicine. Their value has been illustrated by the discovery of artemisinin [1], a drug saving millions of lives from malaria, derived from an active ingredient of Chinese herb. Chinese medicine is part of the treatment used against DN in China. In review by G. Sun et al., over 20 recipes of herb medicine and 30 single herbs or monomers are summarized. These therapies have showed efficacy at improving albuminuria and inflammation in diabetic patients. Ongoing research programs focus on identifying the effective component(s) contained in the most promising herbs with the ultimate aim of developing safe and novel compounds for the treatment of DN. (3) Systems Biology. As defined by NIH, systems biology is an approach used in biomedical research to understand the “bigger picture”—be it at the level of the organism, tissue, or cell—to reconstruct the biology from huge volumes data using computational and mathematical methods. This is in stark contrast to decades of reductionist biology, which involved taking the pieces apart in order to understand the biology [2]. As technology advances, genomics, proteomics, and metabolomics become reliable, affordable, and readily available to explore the molecular profiles of human disease. F. Conserva et al. present a systems biology overview of human DN, from genetic susceptibility to posttranscriptional and posttranslational modifications. Molecules identified by genomics, transcriptome, and epigenetic studies in area of DN await to be validated. Using proteomics approach, M. Barati et al. report the influence of acute high glucose exposure on the change in protein abundance in murine glomerular mesangial cells. These discovery-based studies shed new light and new perspectives in DN research.

In an effort to test our working hypothesis that diabetes-induced renal glomerular damage may be the result of oxidative insult, JTMT transgenic mice were produced that overexpress the antioxidant ...

Diabetic cardiomyopathy is a common complication leading to heightened risk of heart failure and death. In the present report, we performed proteomic analysis on total cardiac proteins from the OVE26 mouse model of type 1 diabetes to identify protein changes that may contribute to diabetic cardiomyopathy. This analysis revealed that a surprising high proportion (12 of 20) of the altered proteins that could be identified by mass spectrometry were of mitochondrial origin. All but one of these proteins were upregulated by diabetes. Quantitative RT-PCR, performed for two of these proteins, indicated that part of the upregulation was attributed to increased messenger RNA levels. Morphological study of diabetic hearts showed significantly increased mitochondrial area and number as well as focal regions with severe damage to mitochondria. Diabetic mitochondria also showed reduced respiratory control ratio (9.63 ± 0.20 vs. 6.13 ± 0.41, P < 0.0001), apparently due to reduced state 3 rate, and diminished GSH level (5.5 ± 0.9 vs. 8.2 ± 2.5 μmol/mg protein, P < 0.05), indicating impaired mitochondrial function and increased oxidative stress. Further examination revealed increased mitochondrial DNA (1.03 ± 0.18 vs. 0.69 ± 0.13 relative copy number, P < 0.001) and a tendency to higher protein yield in OVE26 cardiac mitochondria, as well as increased mRNA level for mitochondrial transcription factor A and two mitochondrial encoded proteins. Taken together, these results show that mitochondria are a primary target in the diabetic heart, probably due to oxidative stress, and that this damage coincides with and may stimulate mitochondrial biogenesis.

Speedup is considered as the criterion of determining whether a parallel algorithm is optimal. But broadcast-class problems, existing only on parallel computer system, have no sequential algorithms at all. Speedup standard becomes invalid here. Through this research on broadcast algorithms under several typical parallel computation models, a model-independent evaluation standard minC 2 is developed, which can be not only used to determine an optimal broadcasting algorithm, but also normalized to apply to any parallel algorithm. As a new idea, minC 2 will lead to a new way in this field.

Type 1 and Type 2 diabetic patients are at high risk of developing diabetic nephropathy (DN). Renal functional decline is gradual and there is high variability between patients, though the reason for the variability is unknown. Enough diabetic patients progress to end stage renal disease to make diabetes the leading cause of renal failure. The first symptoms of DN do not appear for years or decades after the onset of diabetes. During and after the asymptomatic period structural changes develop in the diabetic kidney. Typically, but not always, the first symptom of DN is albuminuria. Loss of renal filtration rate develops later. This review examines the structural abnormalities of diabetic kidneys that are associated with and possibly the basis for advancing albuminuria and declining GFR. Mouse models of diabetes and genetic manipulations of these models have become central to research into mechanisms underlying DN. This article also looks at the value of these mouse models to understanding human DN as well as potential pitfalls in translating the mouse results to humans.

Glomerulonephritis (GN) is characterized by immune complex deposition and ensuing inflammation within the glomerulus of the kidney. Frank J. Dixon, a pioneer in immunologic kidney disease research, reported in 1965 that injection of nephrotoxic antibodies in rats and rabbits resulted in proteinuria and glomerular accumulation of neutrophils, and that depletion of circulating neutrophils reduced this immune-mediated glomerular damage and proteinuria. In the decades since, studies have confirmed a role for glomerular neutrophil accumulation in human GN, but the mechanisms for neutrophil recruitment and neutrophil-directed injury have not been resolved. We previously reported genetic variants for a NF-κB inhibitor ABIN1 as risks for GN. Others also report that glomerular ABIN1 gene expression is altered in GN in mice and humans. Here we present that transgenic disruption of ABIN1 function results in exacerbated podocyte injury, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in a mouse model of acute antibody-mediated GN. Moreover, a novel inhibitor of neutrophil granule release (SNAP23 peptide) also attenuates podocyte injury in this model. These in vivo findings are supported by in vitro experiments showing that secretome from homologous ABIN1 mutant podocytes activates neutrophil chemotaxis and granule release and neutrophil granule contents specifically disrupts cytoskeletal organization of ABIN1 mutant podocytes. These studies unfold a role for ABIN1 dysfunction in a novel neutrophil-mediated mechanism of podocyte injury in GN and presents inhibition of neutrophil granule release as a promising novel therapeutic direction for kidney inflammation.

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension-induced athero- or arteriosclerosis. This may be due either to generalized vascular disease, tissue-based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia-driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi-transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P0.05) and Mt (P0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ~3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra- and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury.

We previously demonstrated that cellular and extracellular components of the blood-urine barrier in renal glomeruli are susceptible to damage in OVE transgenic mice, a valuable model of human diabetic nephropathy that expresses profound albuminuria.To test our hypothesis that glomerular filtration barrier damage in OVE mice may be the result of oxidative insult to podocytes, 150-day-old bi-transgenic OVENmt diabetic mice that overexpress the antioxidant metallothionein specifically in podocytes were examined by enzyme-linked immunosorbent assay for albuminuria mitigation and by unbiased transmission electron microscopy (TEM) stereometry for protection from chronic structural diabetic complications.Although blood glucose and HbA(1c) levels were indistinguishable in OVE and OVENmt animals, albuminuria was significantly reduced (average7-fold) in OVENmt mice through 8 months of age. Interestingly, the Nmt transgene provided significant glomerular protection against diabetic nephropathic complications outside of the podocyte. Glomerular filtration barrier damage was reduced in OVENmt mice, including significantly increased area occupied by endothelial luminal fenestrations (~13%), significantly reduced glomerular basement membrane (GBM) thickening (~17%) and significantly less podocyte effacement (~18%). In addition, OVENmt mice exhibited significantly reduced glomerular volume (~50%), fewer glomerular endothelial cells (~33%), fewer mesangial cells (~57%) and fewer total glomerular cells (~40%).These results provide evidence of oxidative damage to podocytes induces primary diabetic nephropathic features including severe and sustained albuminuria, specific glomerular filtration barrier damage and alterations in glomerular endothelial and mesangial cell number. Importantly, these diabetic complications are significantly mitigated by podocyte targeted metallothionein overexpression.

Diabetic patients have a high risk of pulmonary disorders that are usually associated with restrictive impairment of lung function, suggesting a fibrotic process (van den Borst B, Gosker HR, Zeegers MP, Schols AM. Chest 138: 393–406, 2010; Ehrlich SF, Quesenberry CP Jr, Van Den Eeden SK, Shan J, Ferrara A. Diabetes Care 33: 55–60, 2010). The present study was undertaken to define whether and how diabetes causes lung fibrosis. Lung samples from streptozotocin-induced type 1 diabetic mice, spontaneously developed type 1 diabetic OVE26 mice, and their age-matched controls were investigated with histopathological and biochemical analysis. Signaling mechanism was investigated with cultured normal human lung fibroblasts in vitro. In both diabetes models, histological examination with Sirius red and hemotoxylin and eosin stains showed fibrosis along with massive inflammatory cell infiltration. The fibrotic and inflammatory processes were confirmed by real-time PCR and Western blotting assays for the increased fibronectin, CTGF, PAI-1, and TNFα mRNA and protein expressions. Diabetes also significantly increased NADPH oxidase (NOX) expression and protein nitration along with upregulation of angiotensin II (Ang II) and its receptor expression. In cell culture, exposure of lung fibroblasts to Ang II increased CTGF expression in a dose- and time-dependent manner, which could be abolished by inhibition of superoxide, NO, and peroxynitrite accumulation. Furthermore, chronic infusion of Ang II to normal mice at a subpressor dose induced diabetes-like lung fibrosis, and Ang II receptor AT1 blocker (losartan) abolished the lung fibrotic and inflammatory responses in diabetic mice. These results suggest that Ang II plays a critical role in diabetic lung fibrosis, which is most likely caused by NOX activation-mediated nitrosative damage.

OVE26 diabetic mice develop severe albuminuria. Immunohistochemical analysis revealed a pattern of intense albumin staining in a small subset of OVE26 tubules. Immunostaining was strikingly heterogeneous; some tubules stained intensely for albumin, but most tubules had weak or no staining. Serial sectioning showed that staining patterns were distinctive for each nephron. Electron microscopy revealed that albumin accumulated in villi and at the base of the brush border. Tubule cell injury, as shown by loss of villi, tubule dilation, and cellular protrusions into the tubule lumen, was unambiguously associated with albumin staining. Examination of albumin staining of proteinuric human kidneys also showed a heterogeneous pattern of staining. Analysis of OVE26 serial sections indicated that all glomeruli connected to albumin-positive tubules were identified by albumin-stained lesions in the tuft that adhered to Bowman's capsule, implicating this as a critical feature of heavy albumin leakage. These results indicate that albumin accumulation provides a marker of damaged nephrons, and confirm that albumin leakage produces significant tubular damage. This study shows that that formation of sclerotic glomerular adhesions is a critical step leading to severe albuminuria.

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electronmicroscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport.

Podocytes are critical components of the selective filtration barrier of the glomerulus and are susceptible to oxidative damage. For investigation of the role of oxidative stress and podocyte damage in diabetic nephropathy, transgenic mice that overexpress the antioxidant protein metallothionein (MT) specifically in podocytes (Nmt mice) were produced. MT expression was increased six- and 18-fold in glomeruli of two independent lines of Nmt mice, and podocyte-specific overexpression was confirmed. Glomerular morphology and urinary albumin excretion were normal in Nmt mice. OVE26 transgenic mice, a previously reported model of diabetic nephropathy, were crossed with Nmt mice to determine whether an antioxidant transgene targeted to podocytes could reduce diabetic nephropathy. Double-transgenic OVE26Nmt mice developed diabetes similar to OVE26 mice, but MT overexpression reduced podocyte damage, indicated by more podocytes, less glomerular cell death, and higher density of podocyte foot processes. In addition, expansion of glomerular and mesangial volume were significantly less in OVE26Nmt mice compared with OVE26 mice. Four-month-old OVE26Nmt mice had a 70 to 90% reduction in 24-h albumin excretion, but this protection does not seem to be permanent. These results provide evidence for the role of oxidative damage to the podocyte in diabetic mice and show that protection of the podocyte can reduce or delay primary features of diabetic nephropathy.

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′ -kinase(PI3K)-Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

Recent studies show that podocyte nuclear density (N(V)) and numbers of renal podocytes per glomerulus (N) are altered in experimental and spontaneous diabetes mellitus. N(V) and N are generally reduced, and it has been hypothesized that these morphological changes may relate to the loss of glomerular permselectivity in diabetic nephropathy (DN). In the current study, OVE26 transgenic diabetic mice and age-matched (FVB) controls (60, 150, or 450 days) were fixed by vascular perfusion and renal cortical tissues were prepared for morphometric analyses. ImageJ software and point counting analyses were carried out on light and transmission electron micrographs to determine glomerular volume (V(G)), N(V), and N. As expected, mean V(G) in OVE26 mice increased substantially ( approximately 134%) over the course of the study and was significantly increased over FVB mice at all ages. At 60 days, N(V) and N were not statistically distinguishable in OVE26 and control mice, while at 150 days, N(V) was significantly reduced in diabetics but not N. In 450-day-old OVE26 animals, however, N(V) and N were both significantly decreased ( approximately 231% and approximately 99%, respectively) relative to age-matched FVB mice. These data suggest that in the OVE26 model of diabetes, significant podocyte loss occurs relatively late in the course of the disease. Moreover, it seems possible that these podocytic changes could play a role in sustaining the increased permeability of the blood-urine barrier in the later stages of diabetic renal decompensation.

Diabetic nephropathy is a common diabetic complication that is associated with alterations in the expression of several renal proteins and abnormal calcium homeostasis. We performed proteomic analysis to screen for global changes of renal protein expression in diabetic kidney. Proteins extracted from the whole kidney of 120-day-old OVE26 (a transgenic model of Type 1 diabetes) and FVB (non-diabetic background strain) mice were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by SYPRO Ruby staining (n = 5 in each group). Quantitative intensity analysis revealed 41 differentially expressed proteins, of which 30 were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. One of the altered proteins with the greatest magnitude of change was the calcium-binding protein, calbindin-D28k, whose expression was increased 6.7-fold in diabetic kidney. We confirmed the increase in calbindin-D28k expression in diabetic kidney by Western blot analysis. Immunohistochemical study demonstrated that calbindin-D28k expression was markedly increased in tubular epithelial cells of distal convoluted tubules (DCT), collecting ducts (CD), and proximal convoluted tubules (PCT) in diabetic kidney. Calbindin-D28k plays a critical role in maintaining calcium homeostasis. The elevation in renal calbindin-D28k expression in our model may indicate a compensatory mechanism to overcome hypercalciuria in diabetes.