AJP - Renal Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F408-F415, 2007. First published April 4, 2007; doi:10.1152/ajprenal.00502.2006
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F408    most recent
00502.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeon, U. S.
Right arrow Articles by Kwon, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeon, U. S.
Right arrow Articles by Kwon, H. M.

Downregulation of renal TonEBP in hypokalemic rats

Un Sil Jeon,1,* Ki-Hwan Han,2,* Soo-Hyun Park,1 Sang Do Lee,1 Mee Rie Sheen,1 Ju-Young Jung,3 Wan Young Kim,4 Jeff M. Sands,5 Jin Kim,4 and H. Moo Kwon1

1Department of Medicine, University of Maryland, Baltimore, Maryland; 2Department of Anatomy, Ewha Womans University, Seoul; 3Department of Veterinary Medicine, Chungnam National University, Daejeon, Korea; 4Department of Anatomy, Catholic University, Seoul, Korea; and 5Department of Medicine, Emory University, Atlanta, Georgia

Submitted 18 December 2006 ; accepted in final form 2 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypokalemia causes a significant decrease in the tonicity of the renal medullary interstitium in association with reduced expression of sodium transporters in the distal tubule. We asked whether hypokalemia caused downregulation of the tonicity-responsive enhancer binding protein (TonEBP) transcriptional activator in the renal medulla due to the reduced tonicity. We found that the abundance of TonEBP decreased significantly in the outer and inner medullas of hypokalemic rats. Underlying mechanisms appeared different in the two regions because the abundance of TonEBP mRNA was lower in the outer medulla but unchanged in the inner medulla. Immunohistochemical examination of TonEBP revealed cell type-specific differences. TonEBP expression decreased dramatically in the outer and inner medullary collecting ducts, thick ascending limbs, and interstitial cells. In the descending and ascending thin limbs, TonEBP abundance decreased modestly. In the outer medulla, TonEBP shifted to the cytoplasm in the descending thin limbs. As expected, transcription of aldose reductase, a target of TonEBP, was decreased since the abundance of mRNA and protein was reduced. Downregulation of TonEBP appeared to have also contributed to reduced expression of aquaporin-2 and UT-A urea transporters in the renal medulla. In cultured cells, expression and activity of TonEBP were not affected by reduced potassium concentrations in the medium. These data support the view that medullary tonicity regulates expression and nuclear distribution of TonEBP in the renal medulla in cell type-specific manners.

NKCC2; NCC; AQP2; UT-A; UT-B; hypertonicity

TONICITY-RESPONSIVE ENHANCER binding protein (TonEBP) is a transcriptional activator essential in the function and development of the renal medulla. TonEBP plays a critical role in protecting renal medullary cells from the deleterious effects of hypertonicity. This is achieved by promoting the cellular accumulation of organic osmolytes via stimulating transcription of genes encoding specific membrane transporters and biosynthetic enzymes (see Ref. 15 for a review), i.e., the sodium-myo-inositol cotransporter, sodium chloride-betaine cotransporter, sodium chloride/taurine transporter, aldose reductase (AR; for production of sorbitol), and a phospholipase for production of glycerophosphorylcholine (9). TonEBP also stimulates the transcription of heat shock protein 70 (HSP70), which protects cells from the harmful effects of a high concentration of urea in the renal medulla (38). TonEBP is a key regulator of urinary concentration via stimulation of transcription of aquaporin-2 (AQP2) (13) and UT-A urea transporters (30) in a manner independent of vasopressin. Finally, TonEBP is involved in the postnatal development of the renal medulla (19). Animals deficient in TonEBP in the kidney display severe renal medullary atrophy (18, 23) because cells failed to adapt to hypertonicity (10). These animals also suffer life-threatening volume depletion due to defective urinary concentrating ability.

Regulation of TonEBP has been extensively characterized in cultured cells. Stimulation of TonEBP in response to hypertonicity involves nuclear distribution (6), increased transactivation (20), and increased abundance (37). On the other hand, not much is known about the regulation of TonEBP in the kidney. Recently, we obtained indirect evidence that hypertonicity was an important stimulator of TonEBP in the renal medulla. Chronic treatment using cyclosporin A resulted in the downregulation of TonEBP in the renal medulla-reduced abundance and a cytoplasmic shift in association with reduced expression of TonEBP target genes (22). The downregulation correlates temporally with reduced abundance of active sodium transporters, i.e., sodium-potassium-chloride cotransporter type 2 (NKCC2), sodium-chloride cotransporter (NCC), and Na+-K+-ATPase, that drive medullary salinity/hypertonicity.

In this study, we examined TonEBP in hypokalemic rats because their medullary tonicity is known to be reduced (3), most likely due to reduced expression of NKCC2 and the ROMK channel in the thick ascending limb (1, 27). We find that TonEBP is downregulated in the renal medulla of hypokalemic animals, supporting the view that medullary hypertonicity stimulates TonEBP in vivo, as it does in cultured cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and experimental design. Pathogen-free male Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 200–250 g, were kept in metabolic cages with a daily 12:12-h light-dark cycle. After 3 days of acclimation in the metabolic cages, animals were divided into two groups. The first group was fed the AIN-76A diet (MP Biomedical, Irvine, CA), and the second group was fed the potassium-deficient AIN-76 diet for 14 days. Rats in both groups had free access to water and food throughout the experiment. Animals were maintained in accordance with University of Maryland animal care guidelines.

Chemistry. Animals were anesthetized with intraperitoneal ketamine (40 mg/kg body wt), blood was collected from the abdominal aorta, and the kidneys were harvested. Urine was collected for 24 h before and after the 14 days of treatment. Serum and urinary osmolality were measured using a vapor pressure osmometer (Wescor, Logan, UT). Serum and urinary chemistries were analyzed by a commercial service (IDEXX Lab, Sacramento, CA).

RNAse protection assays. After the kidneys were gently perfused with ice-cold PBS for 15 s through the abdominal aorta, the cortex, outer medulla, and inner medulla were quickly dissected for isolation of RNA using TRIzol (Invitrogen, Carlsbad, CA). The perfusion with PBS did not affect the yield and integrity of RNA from the inner medulla. An RNase protection assay (RPA) was performed to detect mRNA using a commercial kit (RPA III kit, Ambion, Austin, TX). RPA probes were synthesized as follows: NKCC2, nucleotides 1471-1818 of the NCBI locus NM_019134; NCC, nucleotides 2632-2825 of NM_019345 [GenBank] ; the {alpha}1-subunit of Na+-K+-ATPase, nucleotides 2517-2889 of NM_012504 [GenBank] ; TonEBP, nucleotides 4026-4319 of AF089825; AQP2, nucleotides 318-630 of NM_012498; UT-A1, nucleotides 1671-1981 of U77971 [GenBank] ; UT-A2, nucleotides 635-841 of U09957 [GenBank] ; UT-B, nucleotides 778-981 of NM_019346; AR, nucleotides 453-889 of NM_012498; and HSP70, nucleotides 3820-4142 of NM_212546. A probe for rat cyclophilin (Ambion) was used as the loading control to correct for RNA loading.

Immunoblot analysis. Cortex, outer medulla, and inner medulla were dissected and immediately homogenized in lysis buffer (10 mM Tris, pH 7.5, 1 mM sodium orthovanadate, and 1% SDS). The homogenate was adjusted to 60 mM Tris·HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 10% glycerol, and 0.003% bromophenol blue and boiled for 5 min. Samples of homogenates were run on 10% SDS-polyacrylamide gels and stained with Coomassie blue. The images were scanned and quantified as described below to adjust for equal loading. Equal amounts of protein were run on 7.5, 10, or 12% SDS-polyacrylamide gels and electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The blots were blocked with 5% nonfat milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5, for 30 min at room temperature and then incubated overnight at 4°C with primary antibodies: TonEBP (28) diluted 1;5,000; NKCC2 (17) 1:2,000; NCC (16) 1:1,000; the {alpha}1-subunit of Na+-K+-ATPase (Novus Biologicals, Littleton, CO) 1:10,000; AQP2 (Almone Labs, Jerusalem, Israel) 1:2,000; UT-A and UT-B (7) 1: 1,000; HSP70 (StressGen, Victoria, British Columbia, Canada) 1:1,000; and AR (21) 1:1,000. After a washing, the blots were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, Boston, MA) diluted 1:5,000 in blocking solution at room temperature. Antibody binding was visualized using the enhanced chemiluminescence system (Amersham Bioscience, Buckinghamshire, UK). Specific bands were scanned and quantified using Quantity One software (Bio-Rad, Hercules, CA).

Madin-Darby canine kidney cells. Madin-Darby canine kidney (MDCK) cells adapted to defined medium were grown to confluence as described previously (39). KCl concentration in the medium was varied from 0 to 5 mM by adding appropriate amounts of KCl to KCl-deficient medium. Hypertonic medium was made by addition of 100 mM NaCl. Cultured cells were washed in PBS and lysed in 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2 mM PMSF, 20 µg/ml aprotinin, 12.5 µg/ml pepstatin, and 12.5 µg/ml leupeptin for 1 h at 4°C. The cell lysates were diluted 1:1 with 2x SDS sample buffer (5% glycerol, 100 mM dithiothreitol, 2% SDS, 0.01% bromophenol blue, and 125 mM Tris, pH 6.8) and processed as above for immunoblotting of TonEBP and HSP70.

Immunohistochemical analysis of TonEBP. The kidneys were preserved by in vivo perfusion through the abdominal aorta. The animals were perfused with PBS as described above. This was followed by perfusion with the periodatelysine-paraformaldehyde (PLP) solution for 4 min. The kidneys were removed and cut into sagittal slices of 1- to 2-mm thickness and postfixed overnight in the PLP solution at 4°C. Fixed slices were cut along the sagittal plane on a vibratome (Technical Products International, St. Louis, MO) at a thickness of 50 µm and processed for immunohistochemistry.

Preembedding immunolabeling of TonEBP and postembedding double immunolabeling with AQP1, the beta1-subunit of Na+-K+-ATPase, and CLC-K were performed as described previously (12).

Statistical analysis. Student's t-test was performed using Excel software (Microsoft, Redmond, WA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Potassium-deficient diet induces hypokalemia and downregulation of sodium transporters. Rats fed the potassium-deficient diet for 14 days developed significant hypokalemia (2.9 ± 0.1 vs. 4.6 ± 0.2 meq/l, P < 0.01) (Table 1). Plasma concentrations of HCO3– and pH were not significantly affected (not shown). We did not control the amount of food intake, and the hypokalemic animals weighed less than control animals (214 ± 5 vs. 294 ± 7 g, P < 0.01). However, normal responses to hypokalemia were observed: elevated urine flow (25 ± 4 vs. 5 ± 1 ml/day, P < 0.01), reduced urine osmolality (572 ± 103 vs. 2,239 ± 97 mosmol/kgH2O, P < 0.01), higher serum creatinine (0.8 ± 0.05 vs. 0.6 ± 0.02 mg/dl, P < 0.01), and downregulation of AQP2 and sodium transporters (see below). The plasma aldosterone level was not significantly lower in the hypokalemic animals.


View this table:
[in this window]
[in a new window]

  		Table 1. Physiological data from animals on control or potassium-deficient diet

 
In the hypokalemic rats, expression of NCC in the renal cortex and NKCC2 in the outer medulla was dramatically reduced (Fig. 1). The abundance of mRNA also decreased significantly, indicating that reduced transcription and/or mRNA stability contributed to the downregulation, confirming a previous report (1). Expression of the {alpha}1-subunit of Na+-K+-ATPase in the outer medulla did not change. In addition, we noted that the kidneys from hypokalemic animals displayed expanded outer medullas (see Fig. 4, A and D) and hypertrophy in medullary collecting ducts (see Figs. 5 and 6). These changes are consistent with cellular hypertrophy and cellular proliferation reported previously (5, 33). As a result, RNA yield from the outer and inner medulla was 120 (P < 0.01) and 53% greater (P < 0.05), respectively, in the hypokalemic animals compared with control animals. Taken together, these data demonstrated that we successfully developed hypokalemic rats with correct physiological, biochemical, and morphological characteristics.


Figure 1
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 1. Effects of hypokalemia on the renal expression of sodium transporters. The sodium-chloride cotransporter (NCC) and its mRNA were measured from the cortex, and sodium-potassium-chloride cotransporter type 2 (NKCC2) and the {alpha}1-subunit of Na+-K+-ATPase from the outer medulla. Proteins were immunoblotted and quantified with correction for loading as described in MATERIALS AND METHODS. mRNA was quantified by RPA with correction for loading using beta-actin mRNA. Abundance of protein or mRNA in hypokalemic animals relative to control is shown in % at right. Values are means ± SE; n = 6. *P < 0.05 and @P < 0.01 compared with control.

 

Figure 4
View larger version (116K):
[in this window]
[in a new window]

  		Fig. 4. Light micrographs of 50-µm-thick sections illustrating immunostaining of TonEBP in kidneys from control (A–C) and hypokalemic animals (D–F). C and F are high-magnification views of the terminal regions of the papillae. Expansion of the outer medulla is evident in the hypokalemic animal (D vs. A). Arrows, nuclei of inner medullary collecting duct (IMCD); arrowheads, nuclei of tubular cells showing strong immunoreactivity in hypokalemia. Magnification: x15 (A and D); x50 (B and E); x370 (C and F).

 

Figure 5
View larger version (115K):
[in this window]
[in a new window]

  		Fig. 5. Light micrographs of 3 serial 1-µm-thick sections illustrating single immunostaining for TonEBP (brown, B and E), double immunostaining for TonEBP and aquaporin-1 (AQP1; blue, A and D), or TonEBP and the beta1-subunit of Na+-K+-ATPase (blue, C and F) in the inner stripe of the outer medulla. A–C are from control animals, and D–F are from hypokalemic animals. Pronounced hypertrophy of outer medullary collecting duct (OMCD) is seen in F. TAL, thick ascending limb of loop of Henle; filled stars, descending thin limb of loop of Henle (DTL); open stars, OMCD; arrows, nuclear TonEBP in DTL; open arrows, cytoplasmic TonEBP in DTL. Magnification: x600.

 

Figure 6
View larger version (81K):
[in this window]
[in a new window]

  		Fig. 6. Light micrographs of two serial 1-µm-thick sections illustrating double immunostaining for TonEBP (brown) and AQP1 (blue; to label DTL in A, C, E, and G), or TonEBP and CLC-K [blue; to label ascending thin limb of loop of Henle (ATL) in B, D, F, and H]. A, B, E, and F are from control animals, and C, D, G, and H are from hypokalemic animals in which hypertrophy of IMCD is present. The middle (A–D) and terminal portions (E–H) of the inner medulla are shown. Open stars, IMCD; asterisks, ATL; solid stars, DTL; arrowheads, interstitial cells; arrows, nuclei of IMCD. Magnification: x600.

 
Downregulation of TonEBP in the renal medulla by hypokalemia. In the hypokalemic animals, expression of TonEBP was significantly reduced in the outer and inner medulla but not in the cortex (Fig. 2). Abundance of TonEBP mRNA was reduced in the outer medulla but unchanged in the inner medulla, indicating transcriptional (or reduced mRNA stability) and posttranscriptional mechanisms of downregulation, respectively.


Figure 2
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 2. Effects of hypokalemia on the renal expression of tonicity-responsive enhancer binding protein (TonEBP). Immunoblot and RPA were performed as in Fig 1. CO, cortex; OM, outer medulla; IM, inner medulla. Abundance in hypokalemic animals relative to control is shown at right in %. Values are means ± SE; n = 6. @P < 0.01 compared with control.

 
We asked whether a reduced concentration of potassium was responsible for the downregulation of TonEBP. To answer the question, MDCK cells were cultured in 0–5 mM potassium for 1 (Fig. 3) or 3 days (not shown). Expression of TonEBP or HSP70 was not affected by the reduced potassium concentration in isotonic or hypertonic medium. Neither was the abundance of AR and BGT1 mRNA (not shown). These results demonstrate that reduced potassium concentration does not influence expression and activity of TonEBP.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. Effects of low potassium concentration on expression of TonEBP and heat shock protein (HSP) 70 in Madin-Darby canine kidney cells (MDCK) cells. Confluent MDCK cells were switched for 1 day to isotonic (I) or hypertonic medium (H; made by addition of 100 mM NaCl) with potassium concentration of 0–5 mM as indicated. The cells were immunoblotted for TonEBP, HSP70, and HSC70. Representative data from 3 independent experiments are shown.

 
We next examined TonEBP in the kidneys of hypokalemic animals using immunohistochemistry. Overall, the intensity of TonEBP immunoreactivity was reduced in the outer and inner medulla (Fig. 4, A and D). The most profound decrease was seen at the tip of the papilla (Fig. 4, E and F), where TonEBP expression was markedly reduced in the inner medullary collecting ducts and but remained high in a subset of tubular profiles.

To examine TonEBP in more detail, double immunostaining was performed on serial thin sections to identify tubular segments. In Fig. 5, descending thin limbs were labeled with AQP1 antibody and thick ascending limbs with the beta1-subunit of Na+-K+-ATPase antibody in the inner stripe of the outer medulla. In the hypokalemic animals, TonEBP immunoreactivity was reduced in thick ascending limbs and outer medullary collecting ducts. On the other hand, subcellular distribution of TonEBP changed in descending thin limbs: nuclear in control animals to cytoplasmic in hypokalemic animals (Fig. 5, B and E). In Fig. 6, descending thin limbs were labeled with AQP1 antibody and ascending thin limbs with CLC-K antibody in the middle and terminal portions of the inner medulla. The results show dramatic decreases in TonEBP expression in the inner medullary collecting ducts and interstitial cells of the hypokalemic animals. On the other hand, TonEBP expression decreased little or modestly in descending thin limbs and ascending thin limbs without cytoplasmic shift in these animals. Thus there were wide variations in the abundance and nuclear distribution of TonEBP in response to hypokalemia.

Renal expression of TonEBP target genes in hypokalemia. In view of the downregulation of TonEBP in the renal medulla of the hypokalemic animals, we asked whether expression of TonEBP target genes was affected in these animals. Expression of AR and its mRNA in the inner medulla was decreased in the hypokalemic animals (Fig. 7, top) consistent with transcriptional downregulation. On the other hand, reduced expression of HSP70 was not associated with reduced mRNA abundance (Fig. 7, bottom).


Figure 7
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 7. Effects of hypokalemia on the renal expression of TonEBP target genes aldose reductase (AR) and HSP70. AR and HSP70 and their mRNA were measured from the inner medulla by immunoblotting and RPA. Abundance in hypokalemic animals relative to control is shown at right in %. Values are means ± SE; n = 6. @P < 0.01 compared with control.

 
AQP2 expression was reduced throughout the kidney, from cortex to inner medulla (Fig. 8A). While decreased abundance of AQP2 mRNA was found in the cortex and inner medulla, the mRNA abundance did not change in the outer medulla. Since AQP2 expression is limited to the collecting ducts which were grossly hypertrophied, we suspect that mRNA and protein abundance was overestimated in the outer medulla, and also in the inner medulla to a lesser degree, although we do not have direct evidence. Similarly, UT-A2 expression was transcriptionally reduced while UT-A1 was reduced posttranscriptionally, i.e., without changes in mRNA abundance.


Figure 8
View larger version (60K):
[in this window]
[in a new window]

  		Fig. 8. Effects of hypokalemia on the renal expression of AQP2 and urea transporters. A: immunoblot and RPA for AQP2 were performed as in Fig 2. B: immunoblotting and RPA were performed for the outer medulla (urea transporters UT-A2 and UT-B) and inner medulla (UT-A1). Abundance in hypokalemic animals relative to control is shown at right in %. Values are means ± SE; n = 6. *P < 0.05 and @P < 0.01 compared with control.

 
To our surprise, UT-B expression was reduced with a concomitant decrease in mRNA abundance. It is not known whether vasopressin or TonEBP is involved in the expression of UT-B. At any rate, our data show that all the known urea transporters in the tubules and descending vasa recta are downregulated in response to hypokalemia.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of this study was to test the hypothesis that TonEBP was stimulated by hypertonicity in the renal medulla. We chose to examine hypokalemic rats because their medullary tonicity was expected to be significantly decreased due to the downregulation of NKCC2 and ROMK (1, 27) involved in the single effect, active reabsorption of sodium in the thick ascending limb. Indeed, the renal medullary interstitial tonicity was reported to be ~30% lower in hypokalemic animals (3). Although the study (3) was performed using water-deprived animals, the data are likely relevant to our study because the urine osmolality is comparable in control, i.e., normokalemic, animals, 2,548 vs. 2,239 mosmol/kgH2O (Table 1). In addition, although we could not directly demonstrate a reduction in tonicity of the renal medullary interstitium because of technical limitations of analyzing tissue homogenates (22), we did observe a clear downregulation of NKCC2, suggesting that the single effect was compromised, and therefore, the medullary tonicity was decreased.

We found that the abundance of TonEBP dropped significantly in the renal medullas of the hypokalemic animals in the medullary collecting duct, thick ascending limb, and interstitial cells, and TonEBP shifted to the cytoplasm in the descending thin limb in the outer medulla. Expression of AR and its mRNA waned significantly, indicating that the activity of TonEBP decreased as expected. Since reduced potassium concentration did not affect the abundance and activity of TonEBP in MDCK cells in culture, it is unlikely that hypokalemia itself caused the downregulation of TonEBP. Overall, the data support the hypothesis that hypertonicity stimulates expression and activity of TonEBP in the renal medulla.

It is of interest to compare the hypokalemia with water diuresis (4) and cyclosporin A-induced polyuria (22) because urine osmolality is comparable in these models, i.e., 500–600 mosmol/kgH2O. Renal medullary tonicity appears lowest in the cyclosporin A-induced polyuria because 1) Na+-K+-ATPase is downregulated in addition to NKCC2 and NCC; and 2) in addition to reduced abundance of TonEBP in the outer and inner medulla, a cytoplasmic shift of TonEBP is seen in various cell types in the outer and inner medullas (22). Water diuresis induced by the drinking of a sucrose solution appears to produce the least reduction in medullary tonicity. This is because the abundance of TonEBP or its mRNA is not reduced, and only a cytoplasmic shift of TonEBP is seen in various cell types in the outer medulla and the initial portion of the papilla (4).

A salient feature in the hypokalemic animals was the dramatic downregulation of TonEBP at the tip of the inner medulla. A simple interpretation of the data would be that the decrease in interstitial tonicity is much greater in the tip compared with the base. Although a significant decrease in interstitial tonicity at the tip in response to hypokalemia was demonstrated using electron microprobe analysis (see above), the information is not available for the base or other regions of the kidney due to technical limitations. Alternatively, factors other than tonicity might be involved. In lymphocytes, TonEBP expression is markedly induced by activation of T cell receptors (35). In human breast and colon carcinomas, TonEBP is induced by an isoform-specific integrin signaling (14). It is possible that the downregulation of TonEBP in the papillary tip represents local changes in the signaling environment in response to hypokalemia (see more below).

Another remarkable feature in the renal expression of TonEBP was the wide variations in the expression level and nuclear distribution in different cell types even when they are neighbors sharing the same interstitial fluid, e.g., thin limbs and collecting ducts in the inner medulla (Figs. 2 and 6). The variations are distinct in the three forms of diuresis discussed above: water diuresis, cyclosporine-induced nephrotoxicity, and hypokalemia. While we do not understand their basis, such variations in the level of TonEBP expression and in the response to hypertonicity have been elegantly described in the brain. In the brain, TonEBP expression is limited to neurons with little expression detected in other cell types such as astrocytes, ependymocytes, and endothelial cells (24). Furthermore, induction of TonEBP in response to hypernatremia varies greatly in various cell types in a cell size-dependent manner, more induction in larger neurons (25). It is likely that factors other than tonicity are responsible for these variations.

In MDCK cells, changes in TonEBP abundance in response to alterations in the ambient tonicity are accompanied by corresponding changes in mRNA abundance (37). While the reduced expression of TonEBP in the outer medulla of hypokalemic rat kidneys was associated with reduced mRNA abundance, the reduced expression in the inner medulla was not, i.e., posttranscriptional downregulation. In the cyclosporin A-induced polyuria, while the downregulation of TonEBP is accompanied by a decrease in mRNA abundance in both the outer and inner medulla, its recovery in response to dDAVP infusion is not (21). Thus regulation of TonEBP appears more complex in the kidney than in cultured cells.

While the reduced expression of AR in hypokalemic animals is explained by the downregulation of TonEBP, it is complicated for other TonEBP target genes, AQP2 and UT-A urea transporters. This is because ADH is a major regulatory factor for AQP2 and UT-A, and the renal responsiveness to ADH is blunted in hypokalemic animals (2, 31). Our recent data showed that, while transcriptional stimulation of AQP2 and UT-A1 in response to dDAVP infusion is vigorous in the cortex, the transcriptional stimulation is not observed in the inner medulla. Instead, TonEBP appears to have a dominant role in the inner medulla because transcription of AQP2 and UT-A is stimulated in correlation with enhanced expression of TonEBP in those animals subjected to long-term treatment with cyclosporin A (22). Hypertonicity has been suggested to be a major, ADH-independent factor for transcription of AQP2 in the renal medulla (8, 26) in which TonEBP is clearly involved (13). We suggest that reduced transcription due to the downregulation of TonEBP contributed to the downregulation of AQP2 and UT-A in the renal medulla of hypokalemic animals (see Fig. 8).

A common electrolyte disorder in clinical medicine, hypokalemia causes various types of renal damage. These includes both structural, hypertrophy of collecting ducts and hyperplasia of tubules (see above), and functional damage, including reduced glomerular filtration and polyuria. In addition to the blunted response to ADH mentioned above, other changes in the signaling environment within the kidney have been reported. They include altered expression of vasoactive agents (angiotensin II, endothelin-1, nitric oxide, and prostaglandins) (34), expression of insulin-like growth factor (32), expression of cSrc (36), production of arachidonic acid metabolites (11), and changes in membrane lipids (40). This study adds another, the downregulation of TonEBP due to reduced medullary tonicity. We propose the following sequence of events in the renal medulla in response to hypokalemia: 1) downregulation of NKCC2 and ROMK; 2) reduction in interstitial tonicity; 3) reduced expression and/or cytoplasmic shift of TonEBP; 4) downregulation of TonEBP target genes such as AR, AQP2, and UT-A; and 5) reduced accumulation of organic osmolytes (29) and exacerbation of polyuria.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-61677 to H. M. Kwon and Korea Research Foundation Grant KRF-2006-311-E00005 to K.-H. Han.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. M. Kwon, Dept. of Medicine, Univ. of Maryland, 22 South Greene St., Suite N3W143, Baltimore, MD 21201 (e-mail: mkwon{at}medicine.umaryland.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* U. S. Jeon and K.-H. Han made contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amlal H, Wang Z, Soleimani M. Potassium depletion downregulates chloride-absorbing transporters in rat kidney. J Clin Invest 101: 1045–1054, 1998.[ISI][Medline]
  2. Beck N, Webster SK. Impaired urinary concentrating ability and cyclic AMP in K+-depleted rat kidney. Am J Physiol 231: 1204–1208, 1976.[Abstract/Free Full Text]
  3. Beck FX, Müller E, Fraek ML, Dörge A, Thurau K. Inner-medullary organic osmolytes and inorganic electrolytes in K depletion. Pflügers Arch 439: 471–476, 2000.[CrossRef][ISI][Medline]
  4. Cha JH, Woo SK, Han KH, Kim YH, Handler JS, Kim J, Kwon HM. Hydration status affects nuclear distribution of transcription factor tonicity-responsive enhancer binding protein in rat kidney. J Am Soc Nephrol 12: 2221–2230, 2001.[Abstract/Free Full Text]
  5. Cheval L, van Huyen DJP, Bruneval P, Verbavatz JM, Elanouf JM, Doucet A. Plasticity of mouse renal collecting duct in response to potassium depletion. Physiol Genomics 19: 61–73, 2004.[Abstract/Free Full Text]
  6. Dahl SC, Handler JS, Kwon HM. Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP. Am J Physiol Cell Physiol 280: C248–C253, 2001.[Abstract/Free Full Text]
  7. Doran JJ, Klein JD, Kim YH, Smith TD, Kozlowski SD, Gunn RB, Sands JM. Tissue distribution of UT-A and UT-B mRNA and protein in rat. Am J Physiol Regul Integr Comp Physiol 290: R1446–R1459, 2006.[Abstract/Free Full Text]
  8. Ecelbarger CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, Verbalis JG. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 99: 1852–1863, 1997.[ISI][Medline]
  9. Gallazzini M, Ferraris JD, Kunin M, Morris RG, Burg MB. Neuropathy target esterase catalyzes osmoprotective renal synthesis of glycerophosphocholine in response to high NaCl. Proc Natl Acad Sci USA 103: 15260–15265, 2006.[Abstract/Free Full Text]
  10. Go WY, Liu X, Roti MA, Liu F, Ho SN. NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proc Natl Acad Sci USA 101: 10673–10678, 2004.[Abstract/Free Full Text]
  11. Gu R, Wei Y, Jiang H, Balazy M, Wang WH. Role of 20-HETE in mediating the effect of dietary K intake and the apical K channels in the mTAL. Am J Physiol Renal Physiol 280: F223–F230, 2001.[Abstract/Free Full Text]
  12. Han KH, Woo SK, Kim WY, Park SH, Cha JH, Kim J, Kwon HM. Maturation of TonEBP expression in developing rat kidney. Am J Physiol Renal Physiol 287: F878–F885, 2004.[Abstract/Free Full Text]
  13. Hasler U, Jeon US, Kim JA, Mordasini D, Kwon HM, Feraille E, Martin PY. Tonicity-responsive enhancer binding protein is an essential regulator of aquaporin-2 expression in renal collecting duct principal cells. J Am Soc Nephrol 17: 1521–1531, 2006.[Abstract/Free Full Text]
  14. Jauliac S, Lopez-Rogriguez C, Shaw LM, Brown LF, Rao A, Toker A. The role of NFAT transcription factors in integrin-mediated carcinoma invasion. Nat Cell Biol 4: 540–544, 2002.[CrossRef][ISI][Medline]
  15. Jeon US, Kim JA, Sheen MR, Kwon HM. How tonicity regulates genes: story of TonEBP transcriptional activator. Acta Physiol 187: 241–247, 2006.[CrossRef]
  16. Kim GH, Masilamani S, Turner R, Mitchell C, Wade JB, Knepper MA. The thiazide-sensitive Na-Cl co-transporter is an aldosterone-induced protein. Proc Natl Acad Sci USA 95: 14552–14557, 1998.[Abstract/Free Full Text]
  17. Kim GH, Ecelbarger CA, Mitchell C, Packer RK, Wade JB, Knepper MA. Vasopressin increases Na-K-2Cl co-transporter expression in thick ascending limb of Henle's loop. Am J Physiol Renal Physiol 276: F96–F103, 1999.[Abstract/Free Full Text]
  18. Lam AKM, Ko BCB, Tam S, Morris R, Yang JY, Chung SK, Chung SSM. Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism. J Biol Chem 279: 48048–48054, 2004.[Abstract/Free Full Text]
  19. Lee HW, Kim WY, Song HK, Yang CW, Han KH, Kwon HM, Kim J. Sequential expression of NKCC2, TonEBP, aldose reductase, and urea transporter-A in developing mouse kidney. Am J Physiol Renal Physiol 292: F269–F277, 2007.[Abstract/Free Full Text]
  20. Lee SD, Colla E, Sheen MR, Na KY, Kwon HM. Multiple domains of TonEBP cooperate to stimulate transcription in response to hypertonicity. J Biol Chem 278: 47571–47577, 2003.[Abstract/Free Full Text]
  21. Lim SW, Li C, Sun BK, Han KH, Kim WY, Oh YW, Lee JU, Kador PF, Knepper MA, Sands JM, Kim J, Yang CW. Long-term treatment with cyclosporine decreases aquaporins and urea transporters in the rat kidney. Am J Phyiol Renal Physiol 287: F139–F151, 2004.[CrossRef]
  22. Lim SW, Ahn KO, Sheen MR, Jeon US, Kim J, Yang CW, Kwon HM. Downregulation of renal sodium transporters and tonicity-responsive enhancer binding protein by long-term treatment with cyclosporin A. J Am Soc Nephrol 18: 421–429, 2007.[Abstract/Free Full Text]
  23. Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT, Igarashi P, Rao A, Olson EN. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci USA 101: 2392–2397, 2004.[Abstract/Free Full Text]
  24. Loyher ML, Mutin M, Woo SK, Kwon HM, Tappaz ML. Transcription factor tonicity-responsive enhancer-binding protein (TonEBP) which transactivates osmoprotective genes is expressed and upregulated following acute systemic hypertonicity in neurons in brain. Neuroscience 124: 89–104, 2004.[CrossRef][ISI][Medline]
  25. Maallem S, Mutin M, Kwon HM, Tappaz ML. Differential cellular distribution of tonicity-induced expression of transcription factor TonEBP in the rat brain following prolonged systemic hypertonicity. Neuroscience 137: 51–71, 2006.[CrossRef][ISI][Medline]
  26. Marples D, Christensen BM, Frokiaer J, Knepper MA, Nielsen S. Dehydration reverses vasopressin antagonist-induced diuresis and aquaporin-2 downregulation in rats. Am J Physiol Renal Physiol 275: F400–F409, 1998.[Abstract/Free Full Text]
  27. Mennitt PA, Frindt G, Silver RB, Palmer LG. Potassium restriction downregulates ROMK expression in rat kidney. Am J Physiol Renal Physiol 278: F916–F924, 2000.[Abstract/Free Full Text]
  28. Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM. Tonicity-responsive enhancer binding protein, a Rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 96: 2538–2542, 1999.[Abstract/Free Full Text]
  29. Nakanishi T, Takamitsu Y, Nakahama H, Sugita M. Impairment of renal medullary osmolyte accumulation in potassium-depleted rats. Am J Physiol Renal Fluid Electrolyte Physiol 267: F139–F145, 1994.[Abstract/Free Full Text]
  30. Nakayama Y, Peng T, Sands JM, Bagnasco SM. The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression. J Biol Chem 275: 38275–38280, 2000.[Abstract/Free Full Text]
  31. Raymond KH, Lifschitz MD, McKinney TD. Prostaglandins and the urinary concentrating defect in potassium-depleted rabbits. Am J Physiol Renal Fluid Electrolyte Physiol 253: F1113–F1119, 1987.[Abstract/Free Full Text]
  32. Rohan RM, Unterman TG, Liu L, Hise MK. Expression of the insulin-like growth factor system in the hypokalemic rat kidney. Am J Physiol Renal Physiol 272: F661–F667, 1997.[Abstract/Free Full Text]
  33. Stetson DL, Wade JB, Giebisch G. Morphologic alterations in the rat medullary collecting duct following potassium depletion. Kidney Int 17: 45–56, 1980.[ISI][Medline]
  34. Suga SI, Phillips MI, Ray PE, Raleigh JA, Vio CP, Kim YG, Mazzali M, Gordon KL, Hughes J, Johnson RJ. Hypokalemia induces renal injury and alterations in vasoactive mediator that favor salt sensitivity. Am J Physiol Renal Physiol 281: F620–F629, 2001.[Abstract/Free Full Text]
  35. Trama J, Lu Q, Hawley RG, Ho SN. The NFAT-related protein NFATL1 (TonEBP/NFAT5) is induced upon T cell activation in a calcineurin-dependent manner. J Immunol 165: 4884–4894, 2000.[Abstract/Free Full Text]
  36. Wei Y, Bloom P, Lin D, Gu R, Wang WH. Effect of dietary K intake on apical small-conductance K channel in CCD: role of protein tyrosine kinase. Am J Physiol Renal Physiol 281: F206–F212, 2001.[Abstract/Free Full Text]
  37. Woo SK, Dahl SC, Handler JS, Kwon HM. Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity. Am J Physiol Renal Physiol 278: F1006–F1012, 2000.[Abstract/Free Full Text]
  38. Woo SK, Lee SD, Na KY, Park WK, Kwon HM. TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity. Mol Cell Biol 22: 5753–5760, 2002.[Abstract/Free Full Text]
  39. Yamauchi A, Kwon HM, Uchida S, Preston AS, Handler JS. Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F197–F202, 1991.[Abstract/Free Full Text]
  40. Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Glycosphingolipids modulate renal phosphate transport in potassium deficiency. Kidney Int 60: 694–704, 2001.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
A. Dagan, H. M. Kwon, V. Dwarakanath, and M. Baum
Effect of renal denervation on prenatal programming of hypertension and renal tubular transporter abundance
Am J Physiol Renal Physiol, July 1, 2008; 295(1): F29 - F34.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F408    most recent
00502.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeon, U. S.
Right arrow Articles by Kwon, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeon, U. S.
Right arrow Articles by Kwon, H. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.