Aquaporin-2 Transcription Research Articles

HDAC3 Inhibition Improved Urinary Concentrating Defect in Hypokalemia by Promoting AQP2 Transcription

Hypokalemia is a common electrolyte disturbance frequently encountered in clinical medicine. Animals with hypokalemia had a significant arginine vasopressin (AVP)‐resistant polyuria with reduced expression of aquaporin 2 (AQP2) levels in the collecting ducts of the kidneys. The precise molecular mechanisms of hypokalemia‐induced nephrogenic diabetes insipidus remain unresolved. AVP induced a marked increase in histone H3K27 acetylation (H3K27ac) across the body of the AQP2 gene, in the promoter and in upstream of the AQP2 transcriptional start site in mouse mpkCCD cell line (PMID: 29572403). The purpose of the present study was to investigate whether enhanced histone acetylation prevented decreased AQP2 expression and improved urinary concentrating defect in rats with hypokalemia. Male Wistar rats were fed potassium‐free diet with or without 4‐phenylbutyric acid (4‐PBA) or a selective inhibitor of histone deacetylase 3 (HDAC3) RGFP966 for 4 days. 4‐PBA or RGFP966 markedly increased AQP2 mRNA and protein levels in kidney inner medulla of rats with hypokalemia, which was associated with decreased urine output and increased urinary osmolality. Protein expression of H3K27ac was markedly decreased in kidney inner medulla of hypokalemic rats, which was significantly prevented by 4‐PBA or RGFP966. Expression of H3K27ac was decreased in primary cultured inner medullary collecting duct cells (IMCD) or mpkCCD cells cultured in potassium‐deprivation media, in particular, H3K27ac in the promoter region of AQP2 was markedly decreased, which was associated with reduced mRNA levels of AQP2. Protein expression of HDAC3 was significantly upregulated in both mpkCCD and IMCD cells in response to potassium deprivation; the binding of HDAC3 to the promoter of AQP2 was also dramatically increased. RGFP966 significantly increased expression of H3K27ac and AQP2 protein and enhanced the binding of H3K27ac to AQP2 in mpkCCD cells. In conclusion, HDAC3 inhibition prevented downregulation of AQP2 and improved urinary concentrating defect induced by potassium deprivation, likely through enhancing acetylation of H3K27 and transcription of AQP2.

HDAC3 inhibition improves urinary-concentrating defect in hypokalaemia by promoting AQP2 transcription.

This study investigated whether enhanced histone acetylation, achieved by inhibiting histone deacetylases (HDACs), could prevent decreased aquaporin-2 (AQP2) expression during hypokalaemia. Male Wistar rats were fed a potassium-free diet with or without 4-phenylbutyric acid (4-PBA) or the selective HDAC3 inhibitor RGFP966 for 4days. Primary renal inner medullary collecting duct (IMCD) cells and immortalized mouse cortical collecting duct (mpkCCD) cells were cultured in potassium-deprivation medium with or without HDAC inhibitors. 4-PBA increased the levels of AQP2 mRNA and protein in the kidney inner medullae in hypokalaemic (HK) rats, which was associated with decreased urine output and increased urinary osmolality. The level of acetylated H3K27 (H3K27ac) protein was decreased in the inner medullae of HK rat kidneys; this decrease was mitigated by 4-PBA. The H3K27ac levels were decreased in IMCD and mpkCCD cells cultured in potassium-deprivation medium. Decreased H3K27ac in the Aqp2 promoter region was associated with reduced Aqp2 mRNA levels. HDAC3 protein expression was upregulated in mpkCCD and IMCD cells in response to potassium deprivation, and the binding of HDAC3 to the Aqp2 promoter was also increased. RGFP966 increased the levels of H3K27ac and AQP2 proteins and enhanced binding between H3K27ac and AQP2 in mpkCCD cells. Furthermore, RGFP966 reversed the hypokalaemia-induced downregulation of AQP2 and H3K27ac and alleviated polyuria in rats. RGFP966 increased interstitial osmolality in the kidney inner medullae of HK rats but did not affect urinary cAMP levels. HDAC inhibitors prevented the downregulation of AQP2 induced by potassium deprivation, probably by enhancing H3K27 acetylation.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Glucocorticoid Receptor Maintains Vasopressin‐Regulated Water Reabsorption Pathway in the Kidney Collecting Duct Cells

Aquaporin‐2 (AQP2) is a transmembrane protein expressed in the principal cells of the kidney collecting ducts, which mediate osmotic water reabsorption. AQP2 is regulated by the antidiuretic peptide hormone arginine vasopressin (AVP). When AVP binds to the vasopressin type 2 receptor (V2R) in the principal cells, it initiates a signaling network that results in 1) AQP2 trafficking to the apical plasma membrane for water transport and 2) AQP2 gene expression. In a previous study, we found that dexamethasone, a glucocorticoid receptor (GR) agonist, enhanced AVP‐induced AQP2 gene expression in the collecting duct cell model mpkCCD. Here, we showed that GR knockdown drastically decreased AVP‐induced AQP2 mRNA and protein levels, indicative of a role of GR in AVP‐induced AQP2 gene expression. We further showed that the 1,000bp region upstream to the AQP2 transcription start site was sufficient to mediate GR‐enhanced AVP‐induced promoter activity and that GR bound to the above AQP2 promoter region. RNA‐seq was used to profile the transcriptome of the GR knockdown vs. control cells. A total of 93,308 transcripts belonging to 22,025 genes were quantified. GR knockdown decreased expression of 5,266 genes and increased expression of 3,709 genes. These two gene groups respectively represent positive and negative regulators for the AVP responses. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis showed 62 pathways associated with the positive regulators and 48 pathways associated with the negative regulators. The AVP‐regulated water reabsorption pathway was among the 62 positive pathways whose gene expression was reduced upon GR knockdown. Quantitative RT‐PCR analysis showed about 60% reduction in the V2R mRNA level upon GR knockdown. Taken together, our results implicated that GR in one hand potentiates AVP‐induced AQP2 gene expression and in the other hand maintains expression of genes involved in the AVP‐regulated water reabsorption pathway in the kidney collecting duct cells.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, Taiwan (107‐2320‐B‐002‐057‐MY3).

Steviol slows renal cyst growth by reducing AQP2 expression and promoting AQP2 degradation

Overexpression of aquaporin 2 (AQP2) was observed and suggested to be involved in fluid secretion leading to cyst enlargement in polycystic kidney disease (PKD). The cyst expansion deteriorates the renal function and, therefore, therapies targeting cyst enlargement are of clinical interest. Of note, inhibition of vasopressin function using vasopressin 2 receptor (V2R) antagonist which decreased cAMP production along with AQP2 production and function can slow cyst growth in ADPKD. This finding supports the role of AQP2 in cyst enlargement. Steviol, a major metabolite of the sweetening compound stevioside, was reported to retard MDCK cyst growth and enlargement by inhibiting CFTR activity. Interestingly, its efficacy was found to be higher than that of CFTRinh-172. Since steviol was also found to produce diuresis in rodent, it is likely that steviol might have an additional effect in retarding cyst progression, such as inhibition of AQP2 expression and function. Here, we investigated the effect of steviol on AQP2 function and on cyst growth using an in vitro cyst model (MDCK and Pkd1−/− cells). We found that steviol could markedly inhibit cyst growth by reducing AQP2 expression in both Pkd1−/− and MDCK cells. Real-time PCR also revealed that steviol decreased AQP2 mRNA expression level as well. Moreover, a proteasome inhibitor, MG-132, and the lysosomotropic agent, hydroxychloroquine (HCQ) were found to abolish the inhibitory effect of steviol in Pkd1−/− cells. Increased lysosomal enzyme marker (LAMP2) expression following steviol treatment clearly confirmed the involvement of lysosomes in steviol action. In conclusion, our finding showed for the first time that steviol slowed cyst growth, in part, by reducing AQP2 transcription, promoted proteasome, and lysosome-mediated AQP2 degradation. Due to its multiple actions, steviol is a promising compound for further development in the treatment of PKD.

Tankyrase-mediated β-catenin activity regulates vasopressin-induced AQP2 expression in kidney collecting duct mpkCCDc14 cells.

Aquaporin-2 (AQP2) mediates arginine vasopressin (AVP)-induced water reabsorption in the kidney collecting duct. AVP regulates AQP2 expression primarily via Gsα/cAMP/PKA signaling. Tankyrase, a member of the poly(ADP-ribose) polymerase family, is known to mediate Wnt/β-catenin signaling-induced gene expression. We examined whether tankyrase plays a role in AVP-induced AQP2 regulation via ADP-ribosylation of G protein-α (Gα) and/or β-catenin-mediated transcription of AQP2. RT-PCR and immunoblotting analysis revealed the mRNA and protein expression of tankyrase in mouse kidney and mouse collecting duct mpkCCDc14 cells. dDAVP-induced AQP2 upregulation was attenuated in mpkCCDc14 cells under the tankyrase inhibition by XAV939 treatment or small interfering (si) RNA knockdown. Fluorescence resonance energy transfer image analysis, however, revealed that XAV939 treatment did not affect dDAVP- or forskolin-induced PKA activation. Inhibition of tankyrase decreased dDAVP-induced phosphorylation of β-catenin (S552) and nuclear translocation of phospho-β-catenin. siRNA-mediated knockdown of β-catenin decreased forskolin-induced AQP2 transcription and dDAVP-induced AQP2 expression. Moreover, inhibition of phosphoinositide 3-kinase/Akt, which was associated with decreased nuclear translocation of β-catenin, diminished dDAVP-induced AQP2 upregulation, further indicating that β-catenin mediates AQP2 expression. Taken together, tankyrase plays a role in AVP-induced AQP2 regulation, which is likely via β-catenin-mediated transcription of AQP2, but not ADP-ribosylation of Gα. The results provide novel insights into vasopressin-mediated urine concentration and homeostasis of body water metabolism.

Rosiglitazone increases aquaporin 2 transcription and apical expression in renal cells: possible implication for treating nephrogenic diabetes insipidus? (892.10)

Aquaporin 2 (AQP2) is the vasopressin‐regulated water channel palying a crucial role in urine concentration in the kidney. Rosiglitazone (RGZ), an agonist of the peroxisome proliferator‐activated receptor gamma (PPARγ), increases AQP2 expression in normal rats. Here we tested whether RGZ could also modulate AQP2 intracellular trafficking in renal cells.Wild type C57BL‐6 mice were treated for 3 days with 20mg/Kg RGZ to confirm the transcriptional effect on AQP2 in mice. In parallel, we used mouse MCD4 renal collecting duct cells to study the effect of RGZ on AQP2 trafficking to the apical membrane. Apical surface biotinylation and confocal analysis were used to semiquantify the effect. Single cell live microfluorimetry and FRET were used to measure changes in Ca2+ and cAMP intracellular concentrations, respectively.In mice, RGZ induced a twofold increase of AQP2 compared to control mice.In MCD4 cells, stimulation with 50µM RGZ for 30 min robustly increased AQP2 trafficking to the apical plasma membrane to an extent comparable to that elicited by forskolin stimulation. Interestingly, RGZ was able to increasely AQP2 phosphorylation at Ser256 concomitant with intracellular Ca++ increase and without apparent intracellular cAMP elevation.Taken together these results suggest that RGZ might be useful to increase AQP2 transcription and apical trafficking in renal cells bypassing the stimulation of vasopressin receptor which is defective in pathological conditions as in Nephrogenic Diabetes Insipidus.Grant Funding Source: Telethon GGP12040/ AIFA MRAR08P011

NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.

In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac

Urine concentration involves the hormone vasopressin (AVP), which stimulates cAMP production in renal principal cells, resulting in translocation and transcription of aquaporin-2 (AQP2) water channels, greatly increasing the water permeability, leading to a concentrated urine. As cAMP levels decrease shortly after AVP addition, whereas AQP2 levels still increase and are maintained for days, we investigated in the present study the mechanism responsible for the AQP2 increase after long-term 1-desamino-8-d-arginine vasopressin (dDAVP) application using mouse collecting duct (mpkCCD) cells. While 30 min of dDAVP incubation strongly increased cAMP, cAMP was lower with 1 day and was even further reduced with 4 days of dDAVP, although still significantly higher than in control cells. One day of dDAVP incubation increased AQP2 promoter-dependent transcription, which was blocked by the protein kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE-dependent transcription and CREB phosphorylation were not increased. Exchange factor directly activated by cAMP (Epac) 1 and 2 were found to be endogenously expressed in mpkCCD cells. Application of dDAVP increased the expression of Epac1, while Epac2 was reduced. Incubation with a specific Epac activator after dDAVP pretreatment increased both AQP2 abundance and transcription compared with cells left unstimulated the last day. In conclusion, the PKA-CRE pathway is involved in the initial rise in AQP2 levels after dDAVP stimulation but not in the long-term effect of dDAVP. Instead, long-term regulation of AQP2 may involve the activation of Epac.

Lithium reduces aquaporin-2 transcription independent of prostaglandins

Vasopressin (AVP)-stimulated translocation and transcription of aquaporin-2 (AQP2) water channels in renal principal cells is essential for urine concentration. Twenty percent of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder in which the kidney is unable to concentrate urine. In vivo and in mouse collecting duct (mpkCCD) cells, lithium treatment coincides with decreased AQP2 abundance and inactivation of glycogen synthase kinase (Gsk) 3β. This is paralleled in vivo by an increased renal cyclooxygenase 2 (COX-2) expression and urinary prostaglandin PGE(2) excretion. PGE(2) reduces AVP-stimulated water reabsorption, but its precise role in lithium-induced downregulation of AQP2 is unclear. Using mpkCCD cells, we here investigated whether prostaglandins contribute to lithium-induced downregulation of AQP2. In these cells, lithium application reduced AQP2 abundance, which coincided with Gsk3β inactivation and increased COX-2 expression. Inhibition of COX by indomethacin, leading to reduced PGE(2) and PGF(2α) levels, or dexamethasone-induced downregulation of COX-2 both increased AQP2 abundance, while PGE(2) addition reduced AQP2 abundance. However, lithium did not change the prostaglandin levels, and indomethacin and dexamethasone did not prevent lithium-induced AQP2 downregulation. Further analysis revealed that lithium decreased AQP2 protein abundance, mRNA levels and transcription, while PGE(2) reduced AQP2 abundance by increasing its lysosomal degradation, but not by reducing AQP2 gene transcription. In conclusion, our data reveal that in mpkCCD cells, prostaglandins decrease AQP2 protein stability by increasing its lysosomal degradation, indicating that in vivo paracrine-produced prostaglandins might have a role in lithium-induced NDI via this mechanism. However, lithium affects also AQP2 gene transcription, which is prostaglandin independent.

Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5μM) but not FK 506 (0.01-1μM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

Hypotonicity-induced Reduction of Aquaporin-2 Transcription in mpkCCD Cells Is Independent of the Tonicity Responsive Element, Vasopressin, and cAMP

The syndrome of inappropriate antidiuretic hormone secretion is characterized by excessive water uptake and hyponatremia. The extent of hyponatremia, however, is less than anticipated, which is ascribed to a defense mechanism, the vasopressin-escape, and is suggested to involve a tonicity-determined down-regulation of the water channel aquaporin-2 (AQP2). The underlying mechanism, however, is poorly understood. To study this, we used the mouse cortical collecting duct (mpkCCD) cell line. MpkCCD cells, transfected with an AQP2-promoter luciferase construct showed a reduced and increased AQP2 abundance and transcription following culture in hypotonic and hypertonic medium, respectively. This depended on tonicity rather than osmolality and occurred independently of the vasopressin analog dDAVP, cAMP levels, or protein kinase A activity. Although prostaglandins and nitric oxide reduced AQP2 abundance, inhibition of their synthesis did not influence tonicity-induced AQP2 transcription. Also, cells in which the cAMP or tonicity-responsive element (CRE/TonE) in the AQP2-promoter were mutated showed a similar response to hypotonicity. Instead, the tonicity-responsive elements were pin-pointed to nucleotides -283 to -252 and -157 to -126 bp. In conclusion, our data indicate that hypotonicity reduces AQP2 abundance and transcription, which occurs independently of vasopressin, cAMP, and the known TonE and CRE in the AQP2-promoter. Increased prostaglandin and nitric oxide, as found in vivo, may contribute to reduced AQP2 in vasopressin-escape, but do not mediate the effect of hypotonicity on AQP2 transcription. Our data suggest that two novel segments (-283 to -252 and -157 to -126 bp) in the AQP2-promoter mediate the hypotonicity-induced AQP2 down-regulation during vasopressin-escape.

Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase

Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.

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 kappaB (NF-kappaB) 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-kappaB by 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 IkappaB kinase beta 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 V(2) receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative kappaB elements. Mutation of either kappaB 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 kappaB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-kappaB subunits. We additionally found that hypertonicity activated NF-kappaB 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-kappaB is an important physiological regulator of AQP2 transcription.

Normalization of renal aquaporin-2 water channel expression by fosinopril, valsartan, and combination therapy in congestive heart failure: a new mechanism of action

This study investigated the effect of fosinopril (Fos), valsartan (Val), and combination of both drugs (Fos + Val) on the cardiac and renal expression of aquaporin-1 (AQP1) and aquaporin-2 (AQP2) in congestive heart failure (CHF). A rat model of CHF was created by ligating the left anterior descending coronary artery to induce acute myocardial infarction (AMI). Rats were treated by Fos, Val, or Fos + Val for 4 weeks. In renal medulla and cortex, AMI was associated with 2.2- and 1.8-fold increase in AQP2 mRNA expression when compared with Sham-operated rats (medulla: 23.6 ± 2.8 vs. 52.3 ± 8.7%; P < 0.001; cortex: 19.4 ± 3.9 vs. 35.5 ± 7.1%; P < 0.05). All the treatment regimens were able to normalize AQP2 transcription in the renal medulla (Fos, 19.9 ± 4.9%; Val, 22.8 ± 4.9%; Fos + Val, 20.1 ± 5.1%; P = NS vs. Sham) and in the cortex (Fos, 21.2 ± 6.7%; Val, 20.4 ± 6.0%; Fos + Val, 18.9 ± 7.5%; P = NS vs. Sham). Similarly, the AQP2 protein expression increased by 2.1-fold after CHF ( P < 0.05), and was normalized by the treatment regimens (Sham, 0.57 ± 0.19%; CHF, 1.22 ± 0.45%; Fos, 0.39 ± 0.36%; Val, 0.46 ± 0.34%; Fos + Val, 0.36 ± 0.15%; all P < 0.05 vs. CHF). These treatment regimens also prevented the increase in body weight as found in untreated CHF rats (analysis of variance P < 0.05). The renal and cardiac AQP1 gene and protein expressions were unaltered in CHF or by medical therapy. There was no observed cardiac AQP2 expression in all the study groups. Treatment with Fos, Val, or combination therapy was effective in preventing the upregulation of renal AQP2 gene and protein expressions in CHF rats caused by AMI.