Regulation Of Aquaporin-2 Research Articles

Bayesian mapping of protein kinases to vasopressin-regulated phosphorylation sites in renal collecting duct.

Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB).NEW & NOTEWORTHY Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2.

TNF inhibits AQP2 expression via a miR137-dependent pathway.

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.

Abstract 063: Soluble (Pro)renin Receptor Acts Via Direct Activation Of Angiotensin Type 1 Receptor To Enhance Renal Aquaporin-2 Expression And Urine Concentrating Capability

Abundant evidence has established an essential role of collecting duct (pro)renin receptor (PRR) in regulation of aquaporin-2 (AQP-2) expression and urine concentrating capability. Moreover, such AQP2-regulating and antidiuretic action of PRR is mediated by its cleavage product, the soluble PRR (sPRR). However, the mechanism of action of sPRR in this setting remains incompletely understood. In light of our recent report of direct interaction of sPRR and angiotensin type 1 receptor (AT1R) in the vascular endothelial cells, we examined the hypothesis that sPRR signals via AT1R in the kidney. In primary rat inner medullary collecting duct cells, exposure to a recombinant sPRR (termed as sPRR-His) at 10 nM for 24 h induced 4 fold increase in AQP2 protein abundance, which was blunted by `~50% by pretreatment with losartan. In C57/BL6 mice, administration of the V2R antagonist (OPC at 30 mg/kg/d via gavage for 3 days) resulted in symptoms of nephrogenic diabetes insipidus (NDI), including polydipsia, polyuria, and hypoosmotic urine, all of which were improved by subcutaneous infusion of sPRR-His at 30 μg/kg/d via minipump (urine volume [ml]: 5.3 ± 0.2 vs. 3.3 ± 0.3, n =5, p < 0.01; urine osmolality [Osm/kg. H2O]: 428.6 ± 29.2 vs. 704.6 ± 24.2, n =5, p < 0.01). Administration of losartan (5 mg/kg/d) almost completely reversed the therapeutic effect of sPRR-His (urine volume [ml]: 4.7 ± 0.2; urine osmolality [Osm/kg. H2O]: 486 ± 16.1). Immunoblotting demonstrated that sPRR-His treatment restored OPC-induced reduction of AQP2 protein abundance, which was reversed by losartan. Together, sPRR interacted with AT1R to control renal AQP2 expression and urine concentrating capability.

Alteration of N6-methyladenosine epitranscriptome profiles in bilateral ureteral obstruction-induced obstructive nephropathy in juvenile rats.

Urinary tract obstruction is a common cause of renal failure in children and infants, and the pathophysiological mechanisms of obstructive nephropathy are largely unclear. It has been reported that m6A modulation is involved in renal injury. However, whether m6A RNA modulation is associated with obstructive nephropathy has not yet been reported. The aim of this study was to investigate the m6A epitranscriptome profiles in the kidneys of bilateral ureteral obstruction (BUO) in young rats. The total level of m6A in the kidneys was measured by liquid chromatography-tandem mass spectrometry. The mRNAs of related genes were detected by real-time PCR. Methylated RNA immunoprecipitation sequencing was performed to map the epitranscriptome-wide m6A profile. Global m6A levels were increased after BUO, and the mRNA expression levels of m6A methyltransferases and demethylases were significantly decreased in BUO group rat kidneys; the expression levels of EGFR and Brcal were significantly upregulated, while the mRNA expression levels of Notch1 were downregulated (P < 0.05). A total of 154 genes associated with 163 m6A peaks were identified. The m6A epitranscriptome was significantly altered in BUO rat kidneys, which is potentially implicated in the pathophysiological processes of obstructive nephropathy. The m6A RNA modification was associated with the process of renal injury in ureteral obstructive nephropathy by participating in multiple dimensions. The dysregulation of m6A methyltransferases and demethylases may be related to the pathophysiological changes of BUO-induced obstructive nephropathy. The m6A RNA modulation of the genes EGFR, Brca1, and Notch1 that were related to the regulation of aquaporin2 might be the potential mechanism for the polyuresis after ureteral obstruction.

α-Actinin 4 Links Vasopressin Short-Term and Long-Term Regulation of Aquaporin-2 in Kidney Collecting Duct Cells.

Water permeability of the kidney collecting ducts is regulated by the peptide hormone vasopressin. Between minutes and hours (short-term), vasopressin induces trafficking of the water channel protein aquaporin-2 to the apical plasma membrane of the collecting duct principal cells to increase water permeability. Between hours and days (long-term), vasopressin induces aquaporin-2 gene expression. Here, we investigated the mechanisms that bridge the short-term and long-term vasopressin-mediated aquaporin-2 regulation by α-actinin 4, an F-actin crosslinking protein and a transcription co-activator of the glucocorticoid receptor. Vasopressin induced F-actin depolymerization and α-actinin 4 nuclear translocation in the mpkCCD collecting duct cell model. Co-immunoprecipitation followed by immunoblotting showed increased interaction between α-actinin 4 and glucocorticoid receptor in response to vasopressin. ChIP-PCR showed results consistent with α-actinin 4 and glucocorticoid receptor binding to the aquaporin-2 promoter. α-actinin 4 knockdown reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. α-actinin 4 knockdown did not affect vasopressin-induced glucocorticoid receptor nuclear translocation, suggesting independent mechanisms of vasopressin-induced nuclear translocation of α-actinin 4 and glucocorticoid receptor. Glucocorticoid receptor knockdown profoundly reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. In the absence of glucocorticoid analog dexamethasone, vasopressin-induced increases in glucocorticoid receptor nuclear translocation and aquaporin-2 mRNA were greatly reduced. α-actinin 4 knockdown further reduced vasopressin-induced increase in aquaporin-2 mRNA in the absence of dexamethasone. We conclude that glucocorticoid receptor plays a major role in vasopressin-induced aquaporin-2 gene expression that can be enhanced by α-actinin 4. In the absence of vasopressin, α-actinin 4 crosslinks F-actin underneath the apical plasma membrane, impeding aquaporin-2 membrane insertion. Vasopressin-induced F-actin depolymerization in one hand facilitates aquaporin-2 apical membrane insertion and in the other hand frees α-actinin 4 to enter the nucleus where it binds glucocorticoid receptor to enhance aquaporin-2 gene expression.

Aquaporin 2 regulation: implications for water balance and polycystic kidney diseases.

Targeting the collecting duct water channel aquaporin 2 (AQP2) to the plasma membrane is essential for the maintenance of mammalian water homeostasis. The vasopressin V2 receptor (V2R), which is a GS protein-coupled receptor that increases intracellular cAMP levels, has a major role in this targeting process. Although a rise in cAMP levels and activation of protein kinase A are involved in facilitating the actions of V2R, studies in knockout mice and cell models have suggested that cAMP signalling pathways are not an absolute requirement for V2R-mediated AQP2 trafficking to the plasma membrane. In addition, although AQP2 phosphorylation is a known prerequisite for V2R-mediated plasma membrane targeting, none of the known AQP2 phosphorylation events appears to be rate-limiting in this process, which suggests the involvement of other factors; cytoskeletal remodelling has also been implicated. Notably, several regulatory processes and signalling pathways involved in AQP2 trafficking also have a role in the pathophysiology of autosomal dominant polycystic kidney disease, although the role of AQP2 in cyst progression is unknown. Here, we highlight advances in the field of AQP2 regulation that might be exploited for the treatment of water balance disorders and provide a rationale for targeting these pathways in autosomal dominant polycystic kidney disease.

Olive Leaf Extract (OLE) impaired vasopressin-induced aquaporin-2 trafficking through the activation of the calcium-sensing receptor

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.

P0020THE NEPHRO-PROTECTIVE ROUTE OF CGMP DOES NOT COMPENSATE NPRHOGENIC DIABETES INSIPIDUS, BOTH CONDITIONS MEDIATED BY RENAL INTEGRIN LINKED KINASE (ILK)

Abstract Background and Aims Renal ILK transgenic deletion on mice (cKD-ILK) address basal polyuria compatible with nephrogenic diabetes insipidus (NDI), due to disrupted tubular water channel aquaporin 2 (AQP2) AQP2 expression and vesicular trafficking to the apical membrane [Cano-Peñalver JL et al. FASEB J. 2014; Mamuya FA et al. Am J Physiol Renal Physiol. 2016]. An early symptom of chronic renal diseases (CKD) is NDI. ILK depletion also have a nephron-protective effect in renal damage in mice due to the increase in cGMP [Cano-Peñalver JL et al. Mol Med. 2016; de Frutos S et al. Biochim Biophys Acta Mol Basis Dis. 2019], the latter an alternative anti-NDI mediator. Here we demonstrate that ILK-mediated regulation of AQP2, under basal or CKD contexts, does not depend on cGMP. Method cKD-ILK and controls (WT) were treated orally with a NO donor, as precursor of cGMP formation (IDN, 300mg / Kg / day) for 24 hours. Urine volume was determined as a measure of tubular functionality. Fresh kidneys from non-treated mice were cultured ex vivo with precursors of cGMP (NO donor SNP, 1 µM or cGMP analogue 8Br-cGMP, 0.2 mM) for 30 minutes. AQP2 in the apical membrane of the tubules was quantified by immunohistochemistry. More cKD-ILK and WT were subjected for 6 weeks to a diet supplemented with 0.2% adenine (A) as CKD inducer or baseline standard diet. Urine volume and total ranal AQP2 levels were determined. Results Compared to WT, cGMP axis activation in vivo did not compensate the basal NDI present in cKD-ILK and the activation ex vivo did not improve the apical presence of AQP2 in cKD-ILK. Both WT and cKD-ILK subjected to CKD showed exacerbated polyuria and decreased levels of renal AQP2, but no differences were observed between groups. Conclusion ILK regulates AQP2, both in a basal and pathological context (CKD) independent of cGMP which otherwise is nephron-protective. Our results probably explain the co-existence of cGMP increase together with AQP2 decrease observed in the cKD-ILK kidneys.

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A-Dependent Signaling in Kidney.

Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein aquaporin-2. Vasopressin signaling is initiated by binding to a G-protein-coupled receptor called V2R, which signals through heterotrimeric G-protein subunit Gs α, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteome-wide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2R-selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad-spectrum kinase inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide), and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic data sets have been made available online for modeling vasopressin signaling and signaling downstream from other G-protein-coupled receptors. SIGNIFICANCE STATEMENT: New developments in protein mass spectrometry are facilitating progress in identification of signaling networks. Using mass spectrometry, it is now possible to identify and quantify thousands of phosphorylation sites in a given cell type (phosphoproteomics). The authors describe the use of phosphoproteomics technology to identify signaling mechanisms downstream from a G-protein-coupled receptor, the vasopressin V2 subtype receptor, and its role of the regulation and dysregulation of water excretion in the kidney. Data from multiple phosphoproteomic data sets are provided as web-based resources.

CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Regulation of aquaporin-2 by RNA interference.

MicroRNAs (miRNAs) are short non-coding RNAs that interact with 3'-untranslated regions of mRNAs. miRNAs modulate gene expression by regulating mRNA translation and provide novel insights into the complex regulation of protein expression and function. In this chapter, we describe the general features of RNA interference, identification of miRNAs, and interaction of miRNAs with their target genes. In particular, we have shown that several miRNAs are responsive to arginine vasopressin or aldosterone stimulation in mouse cortical collecting duct mpkCCD cells. Moreover, we identified both miR-32 and miR-137 as AQP2-targeting miRNAs using in silico analysis and also identified several target genes of miR-32 and miR-137. As the target sequences of miR-32 and miR-137 are commonly found in mRNAs of vasopressin-regulated genes, further studies regarding the interaction of miRNAs with their target genes are required to obtain comprehensive understanding of miRNA-regulated AQP2 expression in kidney collecting duct cells.

Vasopressin-Independent Regulation of Aquaporin-2 by Tamoxifen in Kidney Collecting Ducts.

Arginine vasopressin (AVP) mediates water reabsorption in the kidney collecting ducts through regulation of aquaporin-2 (AQP2). Also, estrogen has been known to regulate AQP2. Consistently, we previously demonstrated that tamoxifen (TAM), a selective estrogen receptor modulator, attenuates the downregulation of AQP2 in lithium-induced nephrogenic diabetes insipidus (NDI). In this study, we investigated the AVP-independent regulation of AQP2 by TAM and the therapeutic effect of TAM on the dysregulation of AQP2 and impaired urinary concentration in a unilateral ureteral obstruction (UUO) model. Primary cultured inner medullary collecting duct (IMCD) cells from kidneys of male Sprague-Dawley rats were treated with TAM. Rats subjected to 7 days of UUO were treated with TAM by oral gavage. Changes of intracellular trafficking and expression of AQP2 were evaluated by quantitative PCR, Western blotting, and immunohistochemistry. TAM induced AQP2 protein expression and intracellular trafficking in primary cultured IMCD cells, which were independent of the vasopressin V2 receptor (V2R) and cAMP activation, the critical pathways involved in AVP-stimulated regulation of AQP2. TAM attenuated the downregulation of AQP2 in TGF-β treated IMCD cells and IMCD suspensions prepared from UUO rats. TAM administration in vivo attenuated the downregulation of AQP2, associated with an improvement of urinary concentration in UUO rats. In addition, TAM increased CaMKII expression, suggesting that calmodulin signaling pathway is likely to be involved in the TAM-mediated AQP2 regulation. In conclusion, TAM is involved in AQP2 regulation in a vasopressin-independent manner and improves urinary concentration by attenuating the downregulation of AQP2 and maintaining intracellular trafficking in UUO.

New insights into the transcriptional regulation of aquaporin-2 and the treatment of X-linked hereditary nephrogenic diabetes insipidus.

The kidney collecting duct (CD) is a tubular segment of the kidney where the osmolality and final flow rate of urine are established, enabling urine concentration and body water homeostasis. Water reabsorption in the CD depends on the action of arginine vasopressin (AVP) and a transepithelial osmotic gradient between the luminal fluid and surrounding interstitium. AVP induces transcellular water reabsorption across CD principal cells through associated signaling pathways after binding to arginine vasopressin receptor 2 (AVPR2). This signaling cascade regulates the water channel protein aquaporin-2 (AQP2). AQP2 is exclusively localized in kidney connecting tubules and CDs. Specifically, AVP stimulates the intracellular translocation of AQP2-containing vesicles to the apical plasma membrane, increasing the osmotic water permeability of CD cells. Moreover, AVP induces transcription of the Aqp2 gene, increasing AQP2 protein abundance. This review provides new insights into the transcriptional regulation of the Aqp2 gene in the kidney CD with an overview of AVP and AQP2. It summarizes current therapeutic approaches for X-linked nephrogenic diabetes insipidus caused by AVPR2 gene mutations.

A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging.

Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.

Altered expression of Aquaporin-2 in one-kidney, one-clip hypertension

AimsThe aim of the present study was to evaluate the regulation of Aquaporin-2 (AQP2) water channel in the kidney of one-kidney, one-clip rats (Goldblatt-1 model). In addition, some mechanisms that underlie the role of AQP2 in the Goldblatt-1 model were evaluated. Main methodsSprague-Dawley rats were divided in three groups: control two-kidney, no clip (C, 2 K-NC); nephrectomized one-kidney, no clip (N, 1 K-NC) and Goldblatt one-kidney, one-clip (G, 1 K-1C). AQP2 expression (by westernblot, real time PCR, immunohistochemistry and immunofluorescence), vasopressin V2 receptor expression (by real time PCR), cAMP concentration, NFkB and TonEBP (cytosol to nucleus ratio) were evaluated in the renal medulla. Key findingsAQP2 expression, V2 receptor expression and cAMP concentration were decreased in the renal medulla of 1 K-1C rats, NFkB translocation was favoured towards the nucleus suggesting its activation while TonEBP translocation was not altered in this model of hypertension. SignificanceIn this model of hypertension the decrease of AQP2 expression could be a mechanism that counteracts the high blood pressure promoting water excretion and this may be consequence of decreased vasopressin sensitivity and/or the increased activity of NFkB at renomedullary collecting duct level. Given that renovascular hypertension is among the most common causes of secondary hypertension, it is important to elucidate all the relevant mechanisms involved in the generation or in the compensation of the hypertensive state in order to improve the diagnoses and treatment of the patients.

Tamoxifen attenuates development of lithium-induced nephrogenic diabetes insipidus in rats.

Lithium is widely used in treatment of bipolar affective disorders but often causes nephrogenic diabetes insipidus (NDI), a disorder characterized by severe urinary-concentrating defects. Lithium-induced NDI is caused by lithium uptake by collecting duct principal cells and altered expression of aquaporin-2 (AQP2), which are essential for water reabsorption of tubular fluid in the collecting duct. Sex hormones have previously been shown to affect the regulation of AQP2, so we tested whether tamoxifen (TAM), a selective estrogen receptor modulator, would attenuate lithium-induced alterations on renal water homeostasis. Rats were treated for 14 days with lithium, and TAM treatment was initiated 1 wk after onset of lithium administration. Lithium treatment resulted in severe polyuria and reduced AQP2 expression, which were ameliorated by TAM. Consistent with this, TAM attenuated downregulation of AQP2 and increased phosphorylation of the cAMP-responsive element-binding protein, which induced AQP2 expression in freshly isolated inner-medullary collecting duct suspension prepared from lithium-treated rats. In conclusion, TAM attenuated polyuria dose dependently and impaired urine concentration and downregulation of AQP2 protein expression in rats with lithium-induced NDI. These findings suggest that TAM is likely to be a novel therapeutic option for lithium-induced NDI.

Dexamethasone enhances vasopressin-induced aquaporin-2 gene expression in the mpkCCD cells

The mouse cortical collecting duct cell (mpkCCD) has been an instrumental cell model for studying vasopressin-mediated aquaporin-2 regulation. This cell line was first developed by Vandewalle's group from a transgenic mouse carrying the transforming SV40 antigens driven by the pyruvate kinase promoter. To immortalize the cells, four hormone supplements (dexamethasone, epidermal growth factor, insulin, and triiodothyronine) were used to enhance SV40 antigen expression; however, these hormones appear to have various effects on aquaporin-2 gene expression in the cells. Here, we evaluated the effects of each hormone supplement and found that dexamethasone enhanced vasopressin-induced aquaporin-2 gene expression at both mRNA and protein levels in a dose- and time-dependent manner, without affecting mRNA or protein stability. The effects of dexamethasone were attributed largely to enhanced aquaporin-2 mRNA transcription in association with an enhanced aquaporin-2 promoter activity. Dexamethasone did not affect vasopressin-regulated aquaporin-2 phosphorylation and trafficking. In summary, we optimized the conditions to enhance vasopressin-induced endogenous aquaporin-2 gene expression in the mpkCCD cells. By increasing the amount of aquaporin-2 protein in the cells, our method will facilitate the study of aquaporin-2 cell physiology regulated by vasopressin.

Roflumilast and aquaporin-2 regulation in rat renal inner medullary collecting duct.

Roflumilast is a cyclic nucleotide phosphodiesterase inhibitor that is FDA‐approved for treatment of chronic obstructive pulmonary disease. With a view toward possible use for treatment of patients with X‐linked nephrogenic diabetes insipidus (NDI) due to hemizygous mutations in the V2 vasopressin receptor, this study sought to determine the effect of roflumilast on aquaporin‐2 (AQP2) phosphorylation, AQP2 trafficking, and water permeability in the rat inner medullary collecting duct (IMCD). In the presence of the vasopressin analog dDAVP (0.1 nmol/L), both roflumilast and its active metabolite roflumilast N‐oxide (RNO) significantly increased phosphorylation at S256, S264, and S269, and decreased phosphorylation at S261 (immunoblotting) in IMCD suspensions in a dose‐dependent manner (3–3000 nmol/L). Another commonly used phosphodiesterase inhibitor, IBMX, affected phosphorylation only at the highest concentration in this range. However, neither roflumilast nor RNO had an effect on AQP2 phosphorylation in the absence of vasopressin. Furthermore, roflumilast alone did not increase AQP2 trafficking to the plasma membrane (immunofluorescence) or increase water permeability in freshly microdissected perfused IMCD segments. We conclude that roflumilast can be used to enhance vasopressin's action on AQP2 activity in the renal collecting duct, but has no detectable effect in the absence of vasopressin. These findings suggest that roflumilast may not have a beneficial effect in X‐linked NDI, but could find useful application in acquired NDI.

Aquaporin-2 Ser-261 phosphorylation is regulated in combination with Ser-256 and Ser-269 phosphorylation

Aquaporin-2 (AQP2) is a water channel in collecting duct principal cells in the kidney. Vasopressin catalyzes AQP2 phosphorylation at several serine sites in its C-terminus: Ser-256, Ser-261, and Ser-269. Upon stimulation by vasopressin, Ser-269 phosphorylation increases and Ser-261 phosphorylation decreases. Ser-256 phosphorylation is relatively constant. However, whether these types of phospho-regulation occur independently in distinct AQP2 populations or sequentially in the same AQP2 population is unclear. Especially, the manner of vasopressin-mediated Ser-261 phospho-regulation has been in controversy. In this study, we established phospho-specific AQP2 immunoprecipitation assays and investigated how pS256-positive AQP2 and pS269-positive AQP2 are catalyzed by forskolin or vasopressin, focusing on their Ser-261 phosphorylation status in polarized Madin-Darby canine kidney (MDCK) cells and in mice. In forskolin-treated MDCK cells, Ser-269 phosphorylation preceded Ser-261 dephosphorylation and Ser-256 phosphorylation was constant. In both MDCK cells and mouse kidney, phospho-specific immunoprecipitation revealed that the regulated Ser-269 phosphorylation occurred in the pS256-positive AQP2 population. Importantly, basal-state Ser-261 phosphorylation and its regulated dephosphorylation occurred in the pS256- and pS269-positive AQP2 population. These results provide the direct evidence that the Ser-261 dephosphorylation is involved in the pS256- and pS269-related AQP2 regulation.

Serine/threonine phosphatases and aquaporin-2 regulation in renal collecting duct.

Phosphorylation of the aquaporin-2 (AQP2) water channel at four COOH-terminal serines plays a central role in the regulation of water permeability of the renal collecting duct. The level of phosphorylation at these sites is determined by a balance between phosphorylation by protein kinases and dephosphorylation by phosphatases. The phosphatases that dephosphorylate AQP2 have not been identified. Here, we use large-scale data integration techniques to identify serine-threonine phosphatases likely to interact with AQP2 in renal collecting duct principal cells. As a first step, we have created a comprehensive list of 38 S/T phosphatase catalytic subunits present in the mammalian genome. Then we used Bayes' theorem to integrate available information from large-scale data sets from proteomic and transcriptomic studies to rank the known S/T phosphatases with regard to the likelihood that they interact with AQP2 in renal collecting duct cells. To broaden the analysis, we have generated new proteomic data (LC-MS/MS) identifying 4538 distinct proteins including 22 S/T phosphatases in cytoplasmic fractions from native inner medullary collecting duct cells from rats. The official gene symbols corresponding to the top-ranked phosphatases (common names in parentheses) were: Ppp1cb (PP1-β), Ppm1g (PP2C), Ppp1ca (PP1-α), Ppp3ca (PP2-B or calcineurin), Ppp2ca (PP2A-α), Ppp1cc (PP1-γ), Ppp2cb (PP2A-β), Ppp6c (PP6C), and Ppp5c (PP5). This ranking correlates well with results of prior reductionist studies of ion and water channels in renal collecting duct cells.