Prevention of diabetic nephropathy in Ins2+/−AkitaJ mice by the mitochondria-targeted therapy MitoQ

Mitochondrial production of ROS (reactive oxygen species) is thought to be associated with the cellular damage resulting from chronic exposure to high glucose in long-term diabetic patients. We hypothesized that a mitochondria-targeted antioxidant would prevent kidney damage in the Ins2+/−AkitaJ mouse model (Akita mice) of Type 1 diabetes. To test this we orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period and assessed tubular and glomerular function. Fibrosis and pro-fibrotic signalling pathways were determined by immunohistochemical analysis, and mitochondria were isolated from the kidney for functional assessment. MitoQ treatment improved tubular and glomerular function in the Ins2+/−AkitaJ mice. MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Consistent with previous studies, renal mitochondrial function showed no significant change between any of the diabetic or wild-type groups. Importantly, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and β-catenin showed a nuclear accumulation in the Ins2+/−AkitaJ mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy. They also highlight a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis

Peanut (<i>Arachis hypogaea</i>)<i> </i>lectin: Use in quantitation of desialylation of glycoproteins

294-298A high affinity lectin from an easily available source, peanut (Arachis hypogaea)  agglutinin (PNA) which specifically recognizes desialylated versions of sialylated oligosaccharides is a unique tool in glycoconjugate biotechnology. By a single step affinity chromatography on cross- linked guar galactomannan, PNA was purified to homogeneity with 19 times higher hemagglutinating activity than the sample prepared by existing methods involving defatting with organic solvents. Agglutinating activity of the new preparation remained unchanged for at least 6 months while PNA prepared from defatted seed lost activity within one week. Glycoproteins desialylated to varying degrees were prepared by treating bovine fetuin with 0.1 N H2SO4 at 80°C for durations of 10 seconds and above. Enzyme-linked lectin assay of desialylation of differentially desialylated glycoproteins coated on microplates, using horse radish peroxidase (HRP) conjugate of PNA (PNA-HRP), along with sialic content assay revealed that PNA can be used as a quantitative probe for assay of desialylation in sialylated glycoproteins

Effects of venous needle turbulence during ex vivo hemodialysis on endothelial morphology and nitric oxide formation

Arteriovenous grafts used for hemodialysis frequently develop intimal hyperplasia (IH), which ultimately leads to graft failure. Although the turbulent jet from the dialysis needle may contribute to vessel wall injury, its role in the pathogenesis of IH is relatively unexplored. In the current study, using bovine aortic endothelial cells (BAEC) cultured on the inner surface of a compliant tube, we evaluated the effects of simulated hemodialysis conditions on morphology and nitric oxide (NO) production. The flows via the graft and needle were 500 ml/min (Reynolds number = 819) and 100 ml/min (Reynolds number = 954), respectively. In the presence of the needle jet for 6 h, 19.3% (±1.53%) of BAEC were sheared off, whereas no loss of BAEC was observed in the presence of graft flow alone (P < 0.05). In the presence of graft flow alone, assessment of cell orientation by the Saltykov method revealed that BAEC were oriented along the flow direction. This alignment, however, was lost in the presence of needle flow. Finally, NO production was also significantly decreased in the presence of the needle flow compared to the presence of graft flow alone (16 ± 3.1 vs 34.7 ± 1.9 nmol / 1 0 6 cells / h, P < 0.05). NO is a key player in vascular homeostasis mechanisms modulating vasomotor tone, inhibiting inflammation and smooth muscle cell proliferation. Thus, the loss of NO signaling and the loss of endothelial integrity caused by needle jet turbulence may contribute to the cascade of events leading to IH formation during hemodialysis

Glutaraldehyde cross-linking of lectins to marker enzymes: Protection of binding site by specific sugars

77-80The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site or jacalin , which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti α-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins

Mitochondrially targeted compounds and their impact on cellular bioenergetics

Mitochondria are recognized as critical sites of localized injury in a number of chronic pathologies which has led to the development of organelle directed therapeutics. One of the approaches employed to target molecules to the mitochondrion is to conjugate a delocalized cation such as triphenylphosphonium (TPP+) to various redox active compounds. Mitochondrially targeted antioxidants have also been used in numerous cell culture based studies as probes of the contribution of the mitochondrial generation of reactive oxygen species on cell signaling events. However, concentrations used in vitro are typically 10–100 times greater than those generated from oral dosing in a wide range of animal models and in humans. In the present study, we determined the effects of mitochondrial targeted antioxidants, MitoQ, MitoTempol, and MitoE on cellular bioenergetics of mesangial cells in culture and compared these to TPP+ conjugated compounds which lack the antioxidant functional group. We found that all TPP+ compounds inhibited oxidative phosphorylation to different extents independent of the antioxidant functional groups. These findings show that the TPP+ moiety can disrupt mitochondrial function at concentrations frequently observed in cell culture and this behavior is dependent on the linker group and independent of antioxidant properties. Moreover, the TPP+ moiety alone is unlikely to achieve the concentrations needed to contribute to the protective mechanisms of the mitochondrially targeted compounds that have been reported in vivo

Feasibility of cellular bioenergetics as a biomarker in porphyria patients

Porphyria is a group of metabolic disorders due to altered enzyme activities within the heme biosynthetic pathway. It is a systemic disease with multiple potential contributions to mitochondrial dysfunction and oxidative stress. Recently, it has become possible to measure mitochondrial function from cells isolated from peripheral blood (cellular bioenergetics) using the XF96 analyzer (Seahorse Bioscience). Mitochondrial respiration in these cells is measured with the addition of activators and inhibitors of respiration. The output is measured as the O2 consumption rate (OCR) at basal conditions, ATP linked, proton leak, maximal, reserve capacity, non-mitochondrial, and oxidative burst. We performed cellular bioenergetics on 22 porphyria (12 porphyria cutanea tarda (PCT), seven acute hepatic porphyria (AHP), and three erythropoietic protoporphyria (EPP)) patients and 18 age and gender matched healthy controls. Of porphyria cases, eight were active (2 PCT, 1 EPP, and 5 AHP) and 14 in biochemical remission. The OCR were decreased in patients compared to healthy controls. The bioenergetic profile was significantly lower when measuring proton leak and the non-mitochondrial associated OCR in the eight active porphyria patients when compared to 18 healthy controls. In conclusion, we demonstrate that the bioenergetic profile and mitochondrial activities assessed in porphyria patients and is different than in healthy control individuals. Further, our novel preliminary findings suggest the existence of a mitochondrial dysfunction in porphyria and this may be used as potential non-invasive biomarker for disease activity. This needs to be assessed with a systematic examination in a larger patient cohort. Studies are also suggested to examine mitochondrial metabolism as basis to understand mechanisms of these findings and deriving mitochondrial based therapies for porphyria. Keywords: PCT, AIP, AHP, Protoporphyria, Mitochondria

Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

Cellular cross-talk within the tumor microenvironment (TME) by exosomes is known to promote tumor progression. Tumor promoting macrophages with an M2 phenotype are suppressors of anti-tumor immunity. However, the impact of tumor-derived exosomes in modulating macrophage polarization in the lung TME is largely unknown. Herein, we investigated if lung tumor-derived exosomes alter transcriptional and bioenergetic signatures of M0 macrophages and polarize them to an M2 phenotype. The concentration of exosomes produced by p53 null H358 lung tumor cells was significantly reduced compared to A549 (p53 wild-type) lung tumor cells, consistent with p53-mediated regulation of exosome production. In co-culture studies, M0 macrophages internalized tumor-derived exosomes, and differentiated into M2 phenotype. Importantly, we demonstrate that tumor-derived exosomes enhance the oxygen consumption rate of macrophages, altering their bioenergetic state consistent with that of M2 macrophages. In vitro co-cultures of M0 macrophages with H358 exosomes demonstrated that exosome-induced M2 polarization may be p53 independent. Murine bone marrow cells and bone marrow-derived myeloid-derived suppressor cells (MDSCs) co-cultured with lewis lung carcinoma (LLC)-derived exosomes differentiated to M2 macrophages. Collectively, these studies provide evidence for a novel role for lung tumor-exosomes in M2 macrophage polarization, which then offers new therapeutic targets for immunotherapy of lung cancer