Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo

Abstract

Acute exposure of cells in culture to high NaCl damages DNA and impairs its repair. However, after several hours of cell cycle arrest, cells multiply in the hypertonic medium. Here, we show that, although adapted cells proliferate rapidly and do not become apoptotic, they nevertheless contain numerous DNA breaks, which do not elicit a DNA damage response. Thus, in adapted cells, Mre11 exonuclease is mainly present in the cytoplasm, rather than nucleus, and histone H2AX and chk1 are not phosphorylated, as they normally would be in response to DNA damage. Also, the adapted cells are deficient in repair of luciferase reporter plasmids damaged by UV irradiation. On the other hand, the DNA damage response activates rapidly when the level of NaCl is reduced. Then, Mre11 moves into the nucleus, and H2AX and chk1 become phosphorylated. Renal inner medullary cells in vivo are normally exposed to a variable, but always high, level of NaCl. As with adapted cells in culture, inner medullary cells in normal mice exhibit numerous DNA breaks. These DNA breaks are rapidly repaired when the NaCl level is decreased by injection of the diuretic furosemide. Moreover, repair of DNA breaks induced by ionizing radiation is inhibited in the inner medulla. Histone H2AX does not become phosphorylated, and repair synthesis is not detectable in response to total body irradiation unless NaCl is lowered by furosemide. Thus, both in cell culture and in vivo, although cells adapt to high NaCl, their DNA is damaged and its repair is inhibited.

Characterization and Fate of Telomerase-expressing Epithelia during Kidney Repair

“…40 Telomerase may therefore play a role in the repair of doublestrand breaks, which have been detected almost exclusively in renal papilla. 37 This nonhomologous end-joining repair pathway involves the DNA repair heterodimer Ku86/Ku70 in IMCD cells, 41 and in other systems Ku70 directly associates with telomerase providing a biochemical link between telomerase and IMCD cell DNA repair. 33,42 Further support for this hypothesis is the observation that the deacetylase Sirt1 is selectively expressed in the inner medulla and papilla, and it also is a positive regulator of telomere length, like telomerase.…”

“…36 IMCD cells expressed high levels of mTert mRNA at baseline, compared with renal cortex and papilla ( Figure 7A). Using a hypotonic shock DNA damage model, 37 we subjected IMCD cells that were acclimated to culture in media at 520 mOsm to media at 320 mOsm. This rapidly activated the DNA damage pathway, as indicated by phosphorylation of ATM kinase ( Figure 7B).…”

“…Some cells were adapted to high osmolarity medium using NaCl in stepwise fashion, exactly as described. 37 Osmotic shock was accomplished using pre-equilabrated medium. The cells were used within the first three passages.…”

Gadd45 Proteins Induce G2/M Arrest and Modulate Apoptosis in Kidney Cells Exposed to Hyperosmotic Stress

“…This hyperosmotic cell cycle delay is likely a consequence of activation of DNA damage checkpoints because hyperosmolality causes multiple forms of DNA damage (13,15,17). However, whether any of the three Gadd45 isoforms plays a role in mediating hyperosmotic G 2 /M arrest remains unclear.…”

“…Potential Gadd45 functions during environmental stress include promotion of cell cycle arrest, apoptosis, chromatin remodeling, and DNA repair (10). All of these cell functions are altered during hyperosmolality (1,(11)(12)(13)(14)(15). To assess the mechanistic relevance of hyperosmotic Gadd45 induction, it is necessary to determine which of these cell functions are altered as a result of increased levels of each of the three mammalian Gadd45 isoforms.…”