Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91phox) and cytosolic subunit (p47phox). Then, confocal microscopy showed that gp91phox and p47phox could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their co-localization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-β-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91phox or LR-p47phox transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91phox and p47phox were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys.
SummaryCell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.
In the present study, mice lacking gp91phox gene were used to address the role of NADPH oxidase in hyperhomocysteinemia-induced podocyte injury. It was found that the folate-free diet increased plasma homocysteine levels, but failed to increase O2.− production in the glomeruli from gp91phox gene knockout (gp91−/−) mice, compared with wild-type (gp91+/+) mice. Proteinuria and glomerular damage index (GDI) were significantly lower, while the glomerular filtration rate (GFR) was higher in gp91−/− than gp91+/+ mice when they were on the folate-free diet (Urine albumin excretion: 21.23±1.88 vs. 32.86±4.03 μg/24 h; GDI: 1.17±0.18 vs. 2.59 ±0.49; and GFR: 53.01±4.69 vs. 40.98±1.44 μL/min). Hyperhomocysteinemia-induced decrease in nephrin expression and increase in desmin expression in gp91+/+ mice were not observed in gp91−/− mice. Morphologically, foot process effacement and podocyte loss due to hyperhomocysteinemia were significantly attenuated in gp91−/− mice. In in vitro studies of podocytes, homocysteine was found to increase gp91phox expression and O2.− generation, which was substantially inhibited by gp91phox siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A (VEGF-A) production was abolished by gp91phox siRNA or diphenyleneiodonium, a NADPH oxidase inhibitor. These results suggest that the functional integrity of NADPH oxidase is essential for hyperhomocysteinemia-induced podocyte injury and glomerulosclerosis.
The Herb Rhinacanthus nasutus (L.) Kurz, which is native to Thailand and Southeast Asia, has become known for its antioxidant properties. Neuronal loss in a number of diseases including Alzheimer’s disease is thought to result, in part, from oxidative stress. Glutamate causes cell death in the mouse hippocampal cell line, HT-22, by unbalancing redox homeostasis, brought about by a reduction in glutathione levels, and amyloid-β has been shown to induce reactive oxygen species (ROS) production. Here in, we show that ethanol extracts of R. nasutus leaf and root are capable of dose dependently attenuating the neuron cell death caused by both glutamate and amyloid-β treatment. We used free radical scavenging assays to measure the extracts antioxidant activities and as well as quantifying phenolic, flavonoid and sterol content. Molecules found in R. nasutus, lupeol, stigmasterol and β-sitosterol are protective against glutamate toxicity.
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