H. Moo Kwon

Tonicity-Responsive Enhancer Binding Protein Is an Essential Regulator of Aquaporin-2 Expression in Renal Collecting Duct Principal Cells

Tonicity-responsive enhancer binding protein (TonEBP) plays a key role in protecting renal cells from hypertonic stress by stimulating transcription of specific genes. Under hypertonic conditions, TonEBP activity is enhanced via increased nuclear translocation, transactivation, and abundance. It was reported previously that hypertonicity exerted a dual, time-dependent effect on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCDcl4). Whereas AQP2 abundance decreased after 3 h of hyperosmotic challenge, it increased after 24 h of hypertonic challenge. This study investigated the role that TonEBP may play in these events by subjecting mpkCCDcl4 cells to 3 or 24 h of hypertonic challenge. Hypertonic challenge increased TonEBP mRNA and protein content and enhanced TonEBP activity as illustrated by both increased TonEBP-dependent luciferase activity and mRNA expression of several genes that are targeted by TonEBP. Irrespective of the absence or presence of vasopressin, decreased TonEBP activity in cells that were transfected with either TonEBP small interfering RNA or an inhibitory form of TonEBP strongly reduced AQP2 mRNA and protein content under iso-osmotic conditions and blunted the increase of AQP2 abundance that was induced after 24 h of hypertonic challenge. Conversely, decreased TonEBP activity did not significantly alter reduced expression of AQP2 mRNA that was induced by 3 h of hypertonic challenge. Mutation of a TonE enhancer element located 489 bp upstream of the AQP2 transcriptional start site abolished the hypertonicity-induced increase of luciferase activity in cells that expressed AQP2 promoter-luciferase plasmid constructs, indicating that TonEBP influences AQP2 transcriptional activity at least partially by acting directly on the AQP2 promoter. These findings demonstrate that in collecting duct principal cells, TonEBP plays a central role in regulating AQP2 expression by enhancing AQP2 gene transcription.

Osmoprotective Transcription Factor NFAT5/TonEBP Modulates Nuclear Factor-κB Activity

Tonicity responsive binding protein (TonEBP) is a transcription factor that plays a key role in osmoprotection. Here, we demonstrate enhanced activity of prosurvival NF-κB—at the onset of hypertonic challenge that depends on p38 kinase—and Akt-dependent formation of p65-TonEBP complexes that bind to elements of NF-κB-responsive genes.

Multiple Domains of TonEBP Cooperate to Stimulate Transcription in Response to Hypertonicity

Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, belongs to the Rel family of transcriptional activators. In the kidney medulla and thymus, TonEBP plays a major role in protecting renal cells and T cells from the deleterious effects of ambient hypertonicity. TonEBP is stimulated by hypertonicity via several pathways: increased expression of protein, nuclear translocation, and increased transactivation. In this study, we identified five domains of TonEBP involved in transactivation. The two conserved glutamine repeats were not involved in transactivation. There were three activation domains that could stimulate transcription independently. In addition, there were two modulation domains that potentiated the activity of the activation domains. One of the activation domains is unique to a splice isoform that is more active than others, indicating that alternative splicing can affect the activity of TonEBP. Another activation domain and one of the modulation domains were stimulated by hypertonicity. All the five domains acted in synergy in every combination. Although overall phosphorylation of TonEBP increased in response to hypertonicity, phosphorylation of the activation and modulation domains did not increase in isolation. In sum, TonEBP possesses far more elaborate domains involved in transactivation compared with other Rel proteins.

NF-κB Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells

Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor κB (NF-κB) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCDcl4), we found that, acting independently of vasopressin, activation of NF-κBby lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time- and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IκB kinase β decreased AQP2 expression. The LPS-induced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V2 receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative κB elements. Mutation of either κB element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to κB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-κB subunits. We additionally found that hypertonicity activated NF-κB in mpkCCDcl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-κB is an important physiological regulator of AQP2 transcription.

Adaptation of kidney medulla to hypertonicity: Role of the transcription factor tonEBP

The osmolality of the mammalian kidney medulla is very high. The high osmolality provides the driving force for water reabsorption and urinary concentration, key functions of the kidney for maintaining proper body fluid volume and blood pressure. Salt and urea are the major solutes in the renal medullary interstitium. Unfortunately, high salt (hypertonicity) causes DNA damage and cell death. In response, the renal medullary cells adapt to the hypertonicity by accumulating compatible osmolytes. A regulatory protein, tonicity-responsive enhancer binding protein (TonEBP), plays a central role in the accumulation of these compatible osmolytes by stimulating genes whose products either actively transport or synthesize the appropriate osmolytes. TonEBP is active under isotonic conditions. It responds to both an increase and a decrease in ambient tonicity, in opposite directions, which involves changes in its abundance and nucleocytoplasmic distribution. In the kidney medulla, however, nucleocytoplasmic distribution is the major site of control, under normal conditions of diuresis and antidiuresis.

Dimerization is required for phosphorylation and DNA binding of TonEBP/NFAT5.

TonEBP (NFAT5) is a newly identified member of the Rel family of transcriptional activators that include NF-kappaB and NFAT1 to NFAT4. Activated in response to hypertonicity, TonEBP stimulates transcription of transporters of organic osmolytes, certain cytokines, and a molecular chaperone. We provide biochemical data demonstrating that full-length TonEBP dimerizes via the C-terminus of the Rel-homology domain (CRHD). The two polyglutamine motifs were not involved. The dimerization was not affected by nucleocytoplasmic shifts in TonEBP in response to changes in ambient tonicity. Preventing the dimer formation by deleting the CRHD did not affect the nucleocytoplasmic shifts. On the other hand, deletion of the CRHD prevented DNA binding and eliminated the dominant negative activity of a C-terminal truncated TonEBP. Furthermore, phosphorylation was dramatically reduced especially in hypertonic conditions by deletion of the CRHD. We conclude that dimerization is required for proper phosphorylation of TonEBP as well as DNA binding.

Downregulation of Renal Sodium Transporters and Tonicity-Responsive Enhancer Binding Protein by Long-Term Treatment with Cyclosporin A

Tonicity-responsive enhancer binding protein (TonEBP) is a transcriptional activator that is regulated by ambient tonicity. TonEBP protects the renal medulla from the deleterious effects of hyperosmolality and regulates the urinary concentration by stimulating aquaporin-2 and urea transporters. The therapeutic use of cyclosporin A (CsA) is limited by nephrotoxicity that is manifested by reduced GFR, fibrosis, and tubular defects, including reduced urinary concentration. It was reported recently that long-term CsA treatment was associated with decreased renal expression of TonEBP target genes, including aquaporin-2, urea transporter, and aldose reductase. This study tested the hypothesis that long-term CsA treatment reduces the salinity/tonicity of the renal medullary interstitium as a result of inhibition of active sodium transporters, leading to downregulation of TonEBP. CsA treatment for 7 d did not affect TonEBP or renal function. Whereas expression of sodium transporters was altered, the medullary tonicity seemed unchanged. Conversely, 28 d of CsA treatment led to downregulation of TonEBP and overt nephrotoxicity. The downregulation of TonEBP involved reduced expression, cytoplasmic shift, and reduced transcription of its target genes. This was associated with reduced expression of active sodium transporters-sodium/potassium/chloride transporter type 2 (NKCC2), sodium/chloride transporter, and Na(+),K(+)-ATPase-along with increased sodium excretion and reduced urinary concentration. Infusion of vasopressin restored the expression of NKCC2 in the outer medulla as well as the expression and the activity of TonEBP. It is concluded that the downregulation of TonEBP in the setting of long-term CsA administration is secondary to the reduced tonicity of the renal medullary interstitium.

Hypertonic Stress in the Kidney: A Necessary Evil

The interstitium of the renal medulla is hypertonic, imposing deleterious effects on local cells. At the same time, the hypertonicity provides osmotic gradient for water reabsorption and is a local signal for tissue-specific gene expression and differentiation of the renal medulla, which is a critical organ for water homeostasis.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Interstitial tonicity controls TonEBP expression in the renal medulla

Cells in the hyperosmotic kidney medulla, express a transcriptional activator termed tonicity responsive enhancer binding protein (TonEBP). Genes targeted by TonEBP protect kidney cells from the deleterious effects of hyperosmolality by inducing the expression of organic osmolytes and molecular chaperones, and other genes that mediate urine concentration such as aquaporin-2 and urea transporters. We tested here the effect of hypertonicity and hyperosmotic salt in the renal medullary interstitium on the expression TonEBP. When massive water diuresis was induced in rats the medullary sodium concentrations did not change, neither did TonEBP expression. In these animals the medullary tonicity was unchanged despite the production of dilute urine. On the other hand, treatment with the loop diurectic furosemide resulted in a dose-dependent decrease in the medullary sodium concentration causing a reduction in interstitial tonicity. Here, TonEBP expression was blunted in the outer and inner medulla which was due, in part, to decreased mRNA abundance. As expected, the expression of TonEBP target genes in the renal medulla also decreased in response to furosemide. Hence TonEBP expression in the renal medulla is stimulated by interstitial hypertonicity.