This study reports the consequences of knocking out NADPH oxidase 4 (Nox4) upon the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4 resulting in a loss of the ~68 kD band in Western blot analysis of renal cortical tissue of the SSNox4−/− rats. SSNox4−/− rats exhibited a significant reduction of salt-induced hypertension compared to SS rats after 21 days of 4.0% NaCl diet (134±5 vs 151±3 mmHg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3D cryoimaging revealed significantly higher redox ratios (NADH/FAD) in the kidneys of SSNox4−/− rats even when fed the 0.4% NaCl diet indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared to SS rats. Prior to the development of hypertension, RNA expression levels of NADPH oxidase subunits Nox2, p67phox, and p22phox were found to be significantly lower (p<0.05) in SSNox4−/− compared to SS rats in the renal cortex. Thus the mutation of Nox4 appears to modify transcription of a number of genes in ways that contribute to the protective effects observed in the SSNox4−/− rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SSNox4−/− rat could be the result of multiple pathways including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.
The development of smart anti-cancer drugs that can selectively kill cancer cells while sparing the surrounding healthy tissues/cells unharmed is of paramount importance for safe and effective cancer therapy. We report a novel class of bifunctional compounds based on diarylidenylpiperidone (DAP) conjugated with an N-hydroxypyrroline (NOH, a nitroxide precursor) group. We hypothesized that the DAP would have cytotoxic (anti-cancer) activity, while the NOH moiety would function as a tissue-specific modulator (anti-oxidant) of cytotoxicity. The study used four DAPs, namely H-4073 and H-4318 without NOH and HO-3867 and HO-4200 with NOH substitution. The goal of the study was to evaluate the 'proof-of-concept' anticancer-versus-antioxidant efficacy of the DAPs using a number of cancerous (breast, colon, head and neck, liver, lung, ovarian, and prostate cancer) and noncancerous (smooth muscle, aortic endothelial, and ovarian surface epithelial cells) human cell lines. Cytotoxicity was determined using an MTT-based cell viability assay. All four compounds induced significant loss of cell viability in cancer cells, while HO-3867 and HO-4200 showed significantly less cytotoxicity in noncancerous cells. EPR measurements showed a metabolic conversion of the N-hydroxylamine function to nitroxide with significantly higher levels of the metabolite and superoxide radical-scavenging (antioxidant) activity in noncancerous cells when compared to cancer cells. Western-blot analysis showed that the DAP-induced growth arrest and apoptosis in cancer cells were mediated by inhibition of STAT3 phosphorylation at Tyr705 and Ser727 residues and induction of apoptotic markers of cleaved caspase-3 and PARP. The results suggest that the antioxidant-conjugated DAPs will be useful as a safe and effective anticancer agent for cancer therapy.
Histologic examination of fixed renal tissue is widely used to assess morphology and the progression of disease. Commonly reported metrics include glomerular number and injury. However, characterization of renal histology is a time-consuming and user-dependent process. To accelerate and improve the process, we have developed a glomerular localization pipeline for trichrome-stained kidney sections using a machine learning image classification algorithm. We prepared 4-m slices of kidneys from rats of various genetic backgrounds that were subjected to different experimental protocols and mounted the slices on glass slides. All sections used in this analysis were trichrome stained and imaged in bright field at a minimum resolution of 0.92 m per pixel. The training and test datasets for the algorithm comprised 74 and 13 whole renal sections, respectively, totaling over 28,000 glomeruli manually localized. Additionally, because this localizer will be ultimately used for automated assessment of glomerular injury, we assessed bias of the localizer for preferentially identifying healthy or damaged glomeruli. Localizer performance achieved an average precision and recall of 96.94% and 96.79%, respectively, on whole kidney sections without evidence of bias for or against glomerular injury or the need for manual preprocessing. This study presents a novel and robust application of convolutional neural nets for the localization of glomeruli in healthy and damaged trichrome-stained whole-renal section mounts and lays the groundwork for automated glomerular injury scoring.
Fatty acid synthase (FAS) and focal adhesion kinase (FAK), which are overexpressed in a variety of human epithelial tumors, play a key role in the migration and invasion of cancer cells. Hence, strategies targeted at inhibiting the FAS/FAK proteins may have therapeutic potential for cancer treatment. The goal of the present study was to determine the effect of HO-3867, a synthetic compound, on the migratory ability of ovarian cancer cells and to understand the mechanistic pathways including the involvement of FAS, FAK, and associated signaling proteins. The study was done using two established human ovarian cancer cell lines, A2780 and SKOV3. Incubation with 10 μmol/L HO-3867 for 24 hours significantly inhibited the native as well as the vascular endothelial growth factor (VEGF)-mediated migration and invasion of the cells. HO-3867 significantly attenuated FAS and FAK protein levels apparently through accelerated ubiquitin-dependent degradation, as shown by a clear downregulation of isopeptidase USP2a. Exposure of cells to HO-3867 also significantly inhibited FAS activity and mRNA levels and a number of downstream proteins, including phospho-extracellular signal-regulated kinase 1/2, phospho-human epidermal growth factor receptor 1, sterol regulatory element binding protein 1, VEGF, and matrix metalloproteinase 2. Western blot and immunohistochemical analyses of A2780 xenograft tumors in mice treated with HO-3867 showed significant reduction in FAS, FAK, VEGF, and downstream protein levels when compared with the untreated control. Collectively, the results showed that HO-3867 suppressed the migration and invasion of ovarian cancer cells by inhibiting the expression or activity of FAS and FAK proteins. The study suggests that molecular targeting of FAS and FAK by HO-3867 may be a potential strategy for ovarian cancer therapy. Mol Cancer Res; 8(9); 1188-97. ©2010 AACR.
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