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We previously reported that, both in cell culture and in the renal inner medulla in vivo, elevating NaCl increased the number of DNA breaks, which persisted as long as NaCl remained high but were rapidly repaired when NaCl was lowered. Furthermore, those breaks did not induce the DNA repair protein [gamma]H2AX or cause activation of the MRN (Mre11, Rad50, Nbs1) complex. In contrast, others recently reported that high NaCl does induce [gamma]H2AX and MRN complex formation and concluded that these activities are associated with repair of the DNA (Sheen MR, Kim SW, Jung JY, Ahn JY, Rhee JG, Kwon HM, Woo SK. Am J Physiol Renal Physiol 291:F1014-F1020, 2006). The purpose of the present studies was to resolve the disparity. The important difference is that HeLa cells, which were the main subject of the later report, are much less tolerant of high NaCl than are the mIMCD3 cells, which were our main subject, mIMCD3 cells survive levels of NaCl that kill HeLa cells by apoptosis. Here we demonstrate that in both cell types raising NaCl to a level that the cells survive (higher for mIMCD3 than HeLa) increases DNA breaks without inducing [gamma]H2AX or activating the MRN complex and that the DNA breaks persist as long as NaCl remains elevated, but are rapidly repaired when it is lowered. Importantly, in both cell types, raising NaCl further to cause apoptosis activates these DNA damage response proteins and greatly fragments DNA, associated with cell death. We conclude that [gamma]H2AX induction and MRN activation in response to high NaCl are associated with apoptosis, not DNA repair. DNA damage; hypertonicity; osmotic stress; HeLa cells: mIMCD3 cells

Hypertonicity (e.g., high NaCl) activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), increasing transcription of protective genes. In the present studies, by stably expressing amino acids 1-547 of TonEBP/OREBP in HEK 293 cells and immunoprecipitating it plus associated proteins from the nuclei of cells exposed to high NaCl, we identify 14 proteins that are physically associated with TonEBP/OREBP. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp9013 and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirm identification of most of the proteins by Western analysis and also demonstrate by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we find that PARP-1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl. osmolarity; immunoprecipitation; mass spectrometry

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45[b.sup.-/-], Gadd45[g.sup.-/-], and Gadd45[bg.sup.-/-] mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. P[53.sup.-/-] Cells do not tolerate as high a concentration of NaCl (or urea) as p[53.sup.+/+] cells, but urinary concentrating ability of p[53.sup.-/-] mice is normal, as is the histology of inner medullas from p[53.sup.-/-] and Gadd45[abg.sup.-/-] mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ. high NaCl; high urea; cell survival doi:10.1152/ajprenal.00518.2005

Hypertonicity activates the transcription factor tonicity-responsive enhancer/ osmotic response element binding protein (TonEBP/OREBP), resulting in increased expression of genes involved in osmoprotective accumulation of organic osmolytes, including glycine betaine, and in increased expression of osmoprotective heat shock proteins. Our previous studies showed that high NaCI increases reactive oxygen species (ROS), which contribute to activation of TonEBP/OREBP. Mitochondria are a major source of ROS. The purpose of the present study was to examine whether mitochondria produce the ROS that contribute to activation of TonEBP/OREBP. We inhibited mitochondrial ROS production in HEK293 cells with rotenone and myxothiazol, which inhibit mitochondrial complexes I and III, respectively. Rotenone (250 nM) and myxothiazol (12 nM) reduce high NaCl induced ROS over 40%, whereas apocynin (100 [micro]M), an inhibitor of NADPH oxidase, and allopurinol (100 [micro]M), an inhibitor of xanthine oxidase, have no significant effect. Rotenone and myxothiazol reduce high NaCl-induced increases in TonEBP/OREBP transcriptional activity (ORE/TonE reporter assay) and BGTI (betaine transporter) mRNA abundance ranging from 53 to 69%. They inhibit high NaCl induced TonEBP/OREBP transactivating activity, but not its nuclear translocation. Release of ATP into the medium on hypertonic stress has been proposed to be a signal that triggers cellular osmotic responses. However, we do not detect release of ATP into the medium or inhibition of high NaCl-induced ORE/TonE reporter activity by an ATPase, apyrase (20 U/ml), indicating that high NaCl-induced activation of TonEBP/OREBP is not mediated by release of ATP. We conclude that high NaCl increases mitochondrial ROS production, which contributes to the activation of TonEBP/OREBP by increasing its transactivating activity. superoxides; rotenone: myxothiazol: transactivation; nuclear translocation; ATP release

Hypertonicity increases mRNA and protein abundance of the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), contributing to increased transcription of downstream osmoprotective genes. Previously, this was attributed to increased transcription of TonEBP/OREBP because no change was found in its mRNA stability. However, there is no direct evidence for increased transcription, and the 3'-untranslated region (UTR) of TonEBP/OREBP contains numerous adenylate/uridylate-rich elements, which can modulate RNA stability. Therefore, we have reinvestigated the effect of hypertonicity on TonEBP/OREBP mRNA stability. We find that, in mouse inner medullary collecting duct cells, raising osmolality from 300 to 500 mosmol/kg[H.sub.2]O by adding NaCl increases TonEBP/OREBP mRNA to a peak of 2.3-fold after 4 h, followed by a decline. TonEBP/OREBP protein increases to a sustained peak of 3.0-fold at 8 h. To determine the stability of TonEBP/ OREBP mRNA, we measured the rate of its decrease after inhibiting transcription with actinomycin D, finding that it is stabilized for 6 h after addition of NaCl. This stabilization is sufficient to explain the increase in mRNA without any change in transcription. To investigate how hypertonicity stabilizes TonEBP/OREBP mRNA, we tested luciferase reporters containing parts of the TonEBP/OREBP mRNA UTR. Inclusion of both the 5'- and 3'-UTR increases reporter activity, consistent with mRNA stabilization. Surprisingly, however, it is the 5'-UTR that stabilizes; the 3'-UTR, by itself, decreases reporter activity. We concluded that 1) hypertonicity stabilizes TonEBP/ OREBP mRNA, contributing to its increase, and 2) stabilization depends on the presence of the 5'-UTR. mRNA stability; hypertonicity

High NaCl activates the transcription factor tonicity-responsive enhancer/ osmotic response element binding protein (TonEBP/OREBP) by increasing its abundance and transactivation, the latter signaled by a variety of protein kinases. In addition, high NaCl causes TonEBP/ OREBP to translocate into the nucleus, but little is known about the signals directing this translocation. The result is increased transcription of protective genes, including those involved in accumulation of organic osmolytes. High NaCl also damages DNA, and DNA damage activates ataxia telangiectasia-mutated (ATM) kinase through autophosphorylation on serine 1981. We previously found that ATM is involved in the high NaCl-induced increase in TonEBP/OREBP transactivation. The purpose of the present studies was to test whether ATM is also involved in high NaCl-induced TonEBP/OREBP nuclear translocation. We quantified TonEBP/OREBP in nuclear and cytoplasmic extracts from cultured cells by Western blot analysis. In COS-7 cells, wortmannin, an inhibitor of ATM, reduces high NaCl-induced nuclear translocation of TonEBP/OREBP. We used AT cells (in which ATM is inactive) to test the specificity of this effect. Nuclear translocation of native TonEBP/OREBP and of its recombinant N[H.sub.2]-terminal rel homology domain, which contains the nuclear localization signal, is reduced in AT cells and is restored when the cells are reconstituted with functional ATM. In conclusion, activation of ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP. ataxia telangiectasia-mutated kinase; tonicity-responsive enhancer/ osmotic response element binding protein; wortmannin; AT cells

The signaling pathways leading to high NaCl-induced activation of the transcription factor tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) remain incompletely understood. High NaCl has been reported to produce oxidative stress. Reactive oxygen species (ROS), which are a component of oxidative stress, contribute to regulation of transcription factors. The present study was undertaken to test whether the high NaCl-induced increase in ROS contributes to tonicity-dependent activation of TonEBP/OREBP. Human embryonic kidney 293 cells were used as a model. We find that raising NaCl increases ROS, including superoxide. N-acetylcysteine (NAC), an antioxidant, and MnTBAP, an inhibitor of superoxide, reduce high NaCl-induced superoxide activity and suppress both high NaCl-induced increase in TonEBP/OREBP transcriptional activity and high NaCl-induced increase in expression of BGT1mRNA, a transcriptional target of TonEBP/OREBP. Catalase, which decomposes hydrogen peroxide, does not have these effects, whether applied exogenously or overexpressed within the cells. Furthermore, NAC and MnTBAP, but not catalase, blunt high NaCl-induced increase in TonEBP/ OREBP transactivation. [N.sup.G]-monomethyl-L-arginine, a general inhibitor of nitric oxide synthase, has no significant effect on either high NaCl-induced increase in superoxide or TonEBP/OREBP transcriptional activity, suggesting that the effects of ROS do not involve nitric oxide. Ouabain, an inhibitor of Na-K-ATPase, attenuates high NaCl-induced superoxide activity and inhibits TonEBP/OREBP transcriptional activity. We conclude that the high NaCl-induced increase in ROS, including superoxide, contributes to activation of TonEBP/OREBP by increasing its transactivation. superoxides; nitric oxide; ouabain; transactivation; nuclear translocation