Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct.

The inner medullary collecting duct (IMCD) produces very high levels of endothelin-1 (ET-1) that acts as an autocrine inhibitor of IMCD Na+ and water reabsorption. Recent studies suggest that IMCD ET-1 production is enhanced by extracellular hypertonicity as can occur during high salt intake. Although NFAT5 has been implicated in the IMCD ET-1 hypertonicity response, no studies in any cell type have identified NFAT5 as a transcriptional regulator of the EDN1 gene; the current study examined this using a mouse IMCD cell line (IMCD3). Media hypertonicity increased IMCD3 ET-1 mRNA in a dose- and time-dependent manner associated with increased NFAT5 nuclear localization. Knockdown of NFAT5 using small-interfering RNA or by CRISPR/Cas9-mediated targeting of exon 4 of the NFAT5 gene reduced the ET-1 hypertonicity response. Chromatin immunoprecipitation using an NFAT5 antibody pulled down ET-1 promoter regions containing NFAT5 consensus binding sequences. Transfected ET-1 promoter reporter constructs revealed maximal hypertonicity-induced reporter activity in the proximal 1-kb region; mutation of the two NFAT5 consensus-binding sites in this region abolished hypertonicity-induced reporter activity. The 1-kb ET-1 promoter-reporter construct lost hypertonicity responsiveness when transfected in CRISPR/Cas9-induced NFAT5-deficient cells. In summary, these findings represent the first description that NFAT5 is a direct transcriptional regulator of the EDN1 gene in IMCD cells and point to a potentially important mechanism by which body Na+ homeostasis is maintained.

Role for reactive oxygen species in flow-stimulated inner medullary collecting duct endothelin-1 production.

Inner medullary collecting duct (IMCD)-derived endothelin-1 (ET-1) is stimulated by volume expansion, in part through augmented luminal flow, whereupon it can elicit natriuresis and diuresis. Since flow can alter nitric oxide (NO) and reactive oxygen species (ROS), both of which can affect collecting duct salt transport, we asked whether NO and/or ROS mediate flow-stimulated IMCD ET-1. Mouse IMCD3 cells were exposed to flow, and ET-1/GAPDH mRNA was assessed. A shear stress of 10 dyn/cm2 for 1 h increased ET-1 mRNA by fourfold compared with no flow (ET-1 flow response). Global NO synthase (NOS) inhibition [NG-nitro-l-arginine methyl ester (l-NAME)] reduced the ET-1 flow response; however, pharmacological inhibition of NOS1 or NOS2, inhibition of NOS3 siRNA, inhibition of arginase inhibition, removal of media l-Arg, or inhibition of NO-dependent signaling pathways (PKG, guanylyl cyclase, or NF-κB) did not affect the ET-1 flow response. Tempol reduced the ET-1 flow response; no further inhibition occurred with l-NAME. Superoxide dismutase, but not catalase, reduced the ET-1 flow response. Inhibition of NAPDH oxidase (NOX) (apocynin), pharmacological inhibition of NOX1/4, or NOX4 siRNA reduced the ET-1 flow response. Finally, flow increased IMCD3 ROS production and this was inhibited by apocynin, NOX1/4 inhibition, and, to a small extent, by l-NAME. Taken together, these data suggest that NOX4-derived ROS in general, and possibly superoxide in particular, are involved in flow-stimulated IMCD ET-1 production. To our knowledge, this is the first report of flow-stimulated ROS production by the CD, as well as the first report of such flow-stimulated CD ROS exerting a biological effect.

Expression profiling of human glial precursors.

BACKGROUND: We have generated gene expression databases for human glial precursors, neuronal precursors, astrocyte precursors and neural stem cells and focused on comparing the profile of glial precursors with that of other populations. RESULTS: A total of 14 samples were analyzed. Each population, previously distinguished from each other by immunocytochemical analysis of cell surface markers, expressed genes related to their key differentiation pathways. For the glial precursor cell population, we identified 458 genes that were uniquely expressed. Expression of a subset of these individual genes was validated by RT-PCR. We also report genes encoding cell surface markers that may be useful for identification and purification of human glial precursor populations. CONCLUSION: We provide gene expression profile for human glial precursors. Our data suggest several signaling pathways that are important for proliferation and differentiation of human glial precursors. Such information may be utilized to further purify glial precursor populations, optimize media formulation, or study the effects of glial differentiation.

Isolation, characterization and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders.

AIM: Glial-restricted progenitor cells (GRPs), a neural cell population that gives rise to astrocytes and oligodendrocytes both in vitro and in vivo, hold great promise as a cellular therapeutic for the treatment of demyelinating and neurodegenerative diseases of the CNS. The manufacturing and characterization protocols of human-derived GRPs (hGRPs; trade name Q-Cells) for use in a clinical setting that adhere to rigorous standards for their isolation, propagation, characterization and storage are presented. MATERIALS & METHODS: hGRPs, defined by their immunoreactivity with A2B5 antibodies, were isolated from fetal cadaver forebrain tissue of mice 17-24 weeks gestational age using Miltenyi paramagnetic bead cell separation technology. GRPs were grown in a defined xenobiotic-free medium for 6 days. At harvest, hGRPs were characterized using immunocytochemical techniques. Long-term cryopreservation and storage conditions, and viability upon freeze-thaw were determined. The phenotypic differentiation potential of hGRPs was determined by implantation experiments into the CNS of shiverer mice. RESULTS: hGRPs were isolated from over 50 neural tissues of either sex during gestational ages of 17-24 weeks. Cells expanded out to 6 days in vitro in a xenobiotic-free medium demonstrated very consistent immunocytochemical profiles. No residual antibody used in the purification process was detected after 6 days of growth in vitro. GRPs could be frozen at up to 24 million cells/ml and were over 70% viable upon freeze-thaw. Thawed hGRPs transplanted into the brain of the dysmyelinated shiverer mouse model were observed to differentiate into both glial fibrillary acidic protein-positive astrocytes and myelin basic protein-positive oligodendrocytes; no human-derived NeuN-positive neuronal cells were observed and no abnormal cell proliferation was observed. CONCLUSION: We demonstrate that hGRPs can be consistently obtained, propagated, cryopreserved and characterized using protocols that can be transferred to a good laboratory practice/good manufacturing practice setting for the manufacture of clinical-grade hGRP cellular therapeutics. Functional data demonstrate that cells manufactured under these conditions are able to differentiate into appropriate cellular phenotypes in an animal model of dysmyelination.