Oxycodone stimulates normal and malignant hematopoietic progenitors via opioid-receptor-independent-ß-catenin activation.

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/ß-catenin signaling. Rescue studies via depleting ß-catenin using genetic and pharmacological approaches confirmed that ß-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.

High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity.

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.

ABIN1 Determines Severity of Glomerulonephritis via Activation of Intrinsic Glomerular Inflammation.

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.

Intravenous infusions of mesenchymal stromal cells have cumulative beneficial effects in a porcine model of chronic ischemic cardiomyopathy.

AIMS: The development of cell therapy as a widely-available clinical option for ischemic cardiomyopathy is hindered by the invasive nature of current cell delivery methods. Furthermore, the rapid disappearance of cells after transplantation provides a cogent rationale for using repeated cell doses, which, however, has not been done thus far in clinical trials because it is not feasible with invasive approaches. The goal of this translational study was to test the therapeutic utility of the intravenous route for cell delivery. METHODS AND RESULTS: Pigs with chronic ischemic cardiomyopathy induced by myocardial infarction received one or three intravenous doses of allogeneic bone marrow mesenchymal stromal cells (MSCs) or placebo 35 days apart. Rigor guidelines, including blinding and randomization, were strictly followed. A comprehensive assessment of LV function was conducted with three independent methods (echocardiography, magnetic resonance imaging, and hemodynamic studies). The results demonstrate that three doses of MSCs improved both load-dependent and independent indices of left ventricular (LV) function and reduced myocardial hypertrophy and fibrosis; in contrast, one dose failed to produce most of these benefits. CONCLUSIONS: To our knowledge, this is the first study to show that intravenous infusion of a cell product improves LV function and structure in a large animal model of chronic ischemic cardiomyopathy and that repeated infusions are necessary to produce robust effects. This study, conducted in a clinically-relevant model, supports a new therapeutic strategy based on repeated intravenous infusions of allogeneic MSCs and provides a foundation for a first-in-human trial testing this strategy in patients with chronic ischemic cardiomyopathy.

Potential role for Nrf2 activation in the therapeutic effect of MG132 on diabetic nephropathy in OVE26 diabetic mice.

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.

Podocyte-specific overexpression of metallothionein mitigates diabetic complications in the glomerular filtration barrier and glomerular histoarchitecture: a transmission electron microscopy stereometric analysis.

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 HbA(1c) 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.

Bone Marrow and Wharton's Jelly Mesenchymal Stromal Cells are Ineffective for Myocardial Repair in an Immunodeficient Rat Model of Chronic Ischemic Cardiomyopathy.

BACKGROUND: Although cell therapy provides benefits for outcomes of heart failure, the most optimal cell type to be used clinically remains unknown. Most of the cell products used for therapy in humans require in vitro expansion to obtain a suitable number of cells for treatment; however, the clinical background of the donor and limited starting material may result in the impaired proliferative and reparative capacity of the cells expanded in vitro. Wharton's jelly mesenchymal cells (WJ MSCs) provide a multitude of advantages over adult tissue-derived cell products for therapy. These include large starting tissue material, superior proliferative capacity, and disease-free donors. Thus, WJ MSC if effective would be the most optimal cell source for clinical use. OBJECTIVES: This study evaluated the therapeutic efficacy of Wharton's jelly (WJ) and bone marrow (BM) mesenchymal stromal cells (MSCs) in chronic ischemic cardiomyopathy in rats. METHODS: Human WJ MSCs and BM MSCs were expanded in vitro, characterized, and evaluated for therapeutic efficacy in a immunodeficient rat model of ischemic cardiomyopathy. Cardiac function was evaluated with hemodynamics and echocardiography. The extent of cardiac fibrosis, hypertrophy, and inflammation was assessed with histological analysis. RESULTS: In vitro analysis revealed that WJ MSCs and BM MSCs are morphologically and immunophenotypically indistinguishable. Nevertheless, the functional analysis showed that WJ MSCs have a superior proliferative capacity, less senescent phenotype, and distinct transcriptomic profile compared to BM MSC. WJ MSCs and BM MSC injected in rat hearts chronically after MI produced a small, but not significant improvement in heart structure and function. Histological analysis showed no difference in the scar size, collagen content, cardiomyocyte cross-sectional area, and immune cell count. CONCLUSIONS: Human WJ and BM MSC have a small but not significant effect on cardiac structure and function when injected intramyocardially in immunodeficient rats chronically after MI.

Wearable Localized Surface Plasmon Resonance-Based Biosensor with Highly Sensitive and Direct Detection of Cortisol in Human Sweat.

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.

Magnetically controlled reversible shape-morphing microrobots with real-time X-ray imaging for stomach cancer applications.

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 via X-ray imaging. The microrobot consisted of composite resin and a pH-responsive layer. This microrobot was found to fold itself in high pH environments and unfold itself in low pH environments. In addition, the neodymium (NdFeB) magnetic nanoparticles present inside the composite resin provided the microrobot with an ability to be controlled by a magnetic field through an electromagnetic actuation system, and the monomeric triiodobenzoate-based particles were found to act as contrast agents for real-time X-ray imaging. The doxorubicin coating on the microrobot's surface resulted in a high cancer-cell killing effect. Finally, we demonstrated the proposed microrobot under an ex vivo environment using a pig's stomach. Thus, this approach can be a potential alternative to targeted drug carriers, especially for stomach cancer applications.

Microrobot with Gyroid Surface and Gold Nanostar for High Drug Loading and Near-Infrared-Triggered Chemo-Photothermal Therapy.

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.

Multifunctional microrobot with real-time visualization and magnetic resonance imaging for chemoembolization therapy of liver cancer.

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.

Angiotensin II plays a critical role in diabetic pulmonary fibrosis most likely via activation of NADPH oxidase-mediated nitrosative damage.

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.

Inhibition of inflammation by pentosan polysulfate impedes the development and progression of severe diabetic nephropathy in aging C57B6 mice.

Inflammation has a key role in diabetic nephropathy (DN) progression. Pentosan polysulfate (PPS) has been shown to decreases interstitial inflammation and glomerulosclerosis in 5/6 nephrectomized rats. Since PPS has an excellent long-term safety profile in interstitial cystitis treatment, and we recently found that old diabetic C57B6 mice develop DN characterized by extensive tubulointerstitial inflammatory lesions that mimics human DN, we examined the effect of PPS on old diabetic mice. We also examined the anti-inflammatory properties of PPS in renal cells in vitro. Diabetes was induced with streptozotocin in 18 months female (early aging) C57B6 mice. Mice were then randomized to receive oral PPS (25 mg/kg/day) or water for 4 months. The effect of PPS on NF-κB activation and on TNFα, high glucose or advanced glycation end products (AGEs) stimulated proinflammatory gene expression in renal cells was examined. We found that PPS treatment preserved renal function, significantly reduced albuminuria, and markedly decreased the severity of renal lesions, including tubulointerstitial inflammation. PPS also reduced upregulation of TNFα and proinflammatory genes in aging diabetic kidneys. Furthermore, PPS suppressed NF-κB, decreased the proinflammatory actions of TNFα, and decreased high glucose and AGEs stimulated MCP-1 production in vitro. Finally, PPS decreased TNFα-induced increase in albumin permeability in podocyte monolayers. In conclusion, PPS treatment largely prevents the development/progression of nephropathy in aging diabetic mice. As this may be mediated by suppression of TNFα, high glucose, and AGE-stimulated NF-κB activation and inflammation in vitro, the in vivo blockade of DN may be due to the anti-inflammatory properties of PPS.

Uninephrectomy of diabetic OVE26 mice greatly accelerates albuminuria, fibrosis, inflammatory cell infiltration and changes in gene expression.

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.

Inflammatory gene expression in OVE26 diabetic kidney during the development of nephropathy.

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-κB_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.

CD45 Immunohistochemistry in Mouse Kidney.

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.

Multifunctional Biodegradable Microrobot with Programmable Morphology for Biomedical Applications.

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.

FVB mouse genotype confers susceptibility to OVE26 diabetic albuminuria.

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.

Diabetic albuminuria is due to a small fraction of nephrons distinguished by albumin-stained tubules and glomerular adhesions.

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.

A new approach to monitor expression of aldo-keto reductase proteins in mouse tissues.

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.