Stabilization of Hypoxia Inducible Factor by Cobalt Chloride Can Alter Renal Transepithelial

Kidney cyst expansion, stagnant fluid accumulation, and insufficient vascular supply can result in localized chronic ischemia-hypoxia in kidney cysts, as well as in normal renal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of Hypoxia Inducible Factor 1a (HIF1a), a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular transport, transforming a normal absorptive phenotype to a secretory and paracellularly leaky phenotype, leading to cyst expansion. Using 100 µmol/L cobalt chloride (CoCl2), HIF1a was stabilized in cellular nucleus of a mouse cortical collecting duct cell line (mCCD 1296 (d)), which resulted in an increased level of erythropoietin, an effector and reporter molecule of HIF1a. The mCCD monolayers have a high transepithelial resistance (TER) value (¿ 3000 O-cm2) and around 95% amiloride-sensitive voltage value. Equivalent current was calculated to compare active ion transport. Our results showed that TER values decreased significantly after 48 and 72 hours of HIF-stabilization. The decrease of TER value was consistent with the increase in the permeability of 70 kDa FITC-dextran molecules, supporting the hypothesis that HIF-stabilization altered paracellular transport. Stabilization of HIF caused a significant decrease in the protein level of zonula occludin 1 (ZO1), which controls paracellular transport through tight junctions. Decrease in the ZO1 protein level was consistent with the decreased TER value and the increased paracellular permeability. Similarly, HIF-stabilization was found to increase paracellular permeability in Mardin-Darby Canine Kidney (MDCK) epithelial monolayers. In mCCD monolayers, HIF-stabilization for 48 hours caused loss of active sodium ion (Na+) transport, and very interestingly, 72 hours of HIF-stabilization caused a switch in the direction of net active ion transport. HIF-stabilization caused a significant decrease of protein level of sodium-potassium-ATPase (Na+/ K+-ATPase) a1 subunit, the catalytic subunit of the enzyme responsible for active Na+ transport, consistent with the loss of active transport of Na+. Our results indicate that HIF-stabilization can transform a normal absorptive epithelium to a paracellularly leaky and cyst-like secretory epithelium by reversing net Na+ transport and increasing monolayer permeability due to alterations of tight junctions, and thereby HIF-stabilization may contribute to cyst expansion

Stabilization of Hypoxia Inducible Factor by Cobalt Chloride Can Alter Renal Epithelial Transport

© 2017 The Authors. Given the importance of the transcriptional regulator hypoxia-inducible factor-1 (HIF-1) for adaptive hypoxia responses, we examined the effect of stabilized HIF-1α on renal epithelial permeability and directed sodium transport. This study was motivated by histological analysis of cystic kidneys showing increased expression levels of HIF-1α and HIF-2α. We hypothesize that compression induced localized ischemia-hypoxia of normal epithelia near a cyst leads to local stabilization of HIF-1α, leading to altered transepithelial transport that encourages cyst expansion. We found that stabilized HIF-1α alters both transcellular and paracellular transport through renal epithelial monolayers in a manner consistent with secretory behavior, indicating localized ischemia-hypoxia may lead to altered salt and water transport through kidney epithelial monolayers. A quantity of 100 µmol/L Cobalt chloride (CoCl2) was used acutely to stabilize HIF-1α in confluent cultures of mouse renal epithelia. We measured increased transepithelial permeability and decreased transepithelial resistance (TER) when HIF-1α was stabilized. Most interestingly, we measured a change in the direction of sodium current, most likely corresponding to abnormal secretory function, supporting our positive-feedback hypothesis

Detection and Antibiotic Treatment of Mycoplasma Arginini Contamination in A Mouse Epithelial Cell Line Restore Normal Cell Physiology

Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Positive identification of mycoplasma typically requires cells to be either fixed and stained for DNA or processed with PCR. We present a method to detect mycoplasma using live-cell optical microscopy typically used for routine observation of cell cultures. Images of untreated mycoplasma-infected epithelial cells alongside images of infected cells treated with Plasmocin, a commercially available antibiotic targeted to mycoplasma, are shown. We found that optical imaging is an effective screening tool for detection of mycoplasma contamination. Importantly, we found that cells regained normal function after the contamination was cleared. In conclusion, we present a technique to diagnose probable mycoplasma infections in live cultures without fixation, resulting in faster response times and decreased loss of cell material

Luminescence in Mn-doped CDS nanocrystals

We have synthesized Mn-doped CdS nanocrystals (NCs) with size ranging from 1.8-3 nm. Photoluminescence (PL) spectra of the doped NCs differ from that of the undoped NCs with an additional peak due to Mn d-d transitions. Electron paramagnetic resonance spectra along with X-ray absorption spectroscopy and PL spectra confirm the incorporation of Mn in the CdS lattice. The fact that emissions from surface states and the Mn d levels occur at two different energies, allowed us to study the PL lifetime decay behaviour of both kinds of emissions

Detection and Antibiotic Treatment of Mycoplasma arginini Contamination in a Mouse Epithelial Cell Line Restore Normal Cell Physiology

Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Positive identification of mycoplasma typically requires cells to be either fixed and stained for DNA or processed with PCR. We present a method to detect mycoplasma using live-cell optical microscopy typically used for routine observation of cell cultures. Images of untreated mycoplasma-infected epithelial cells alongside images of infected cells treated with Plasmocin, a commercially available antibiotic targeted to mycoplasma, are shown. We found that optical imaging is an effective screening tool for detection of mycoplasma contamination. Importantly, we found that cells regained normal function after the contamination was cleared. In conclusion, we present a technique to diagnose probable mycoplasma infections in live cultures without fixation, resulting in faster response times and decreased loss of cell material

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish.

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration