AQP2 Trafficking Research Articles

#3314 DIURETIC AND RENOPROTECTIVE EFFECTS OF GOREISAN IN A RAT MODEL OF NEPHROTIC SYNDROME

Abstract Background and Aims Goreisan, a traditional Chinese medicine composed of five herbs, is commonly used for the purpose of control of edema and gastrointestinal symptoms. However, the detailed mechanisms have not been fully elucidated. In clinical settings, nephrotic syndrome often causes edema, mainly due to hypoalbuminemia and sodium retention. Thus, we investigated the effects of Goreisan on the kidney using a rat model of nephrotic syndrome. Method A model of nephrotic syndrome was created by a right nephrectomy and subsequent administration of adriamycin using Sprague Dawley rats. Nephrotic rats were randomly divided into three groups at 10 weeks of age as follows; vehicle (control), Goreisan 0.5 g/kg (GL), and Goreisan 1.0 g/kg (GH) groups. Goreisan was administered once a day for 4 weeks. At 14 weeks of age, the rats were sacrificed for the evaluation of blood, urine, mRNA expressions, and kidney histopathology. Results Urine volume was significantly increased in the GL and GH groups than in the control group (Δurine volume: control; −1.25±2.48 mL, GL group; 9.83±4.16 mL, GH group; 10.00±2.62 mL). The GH group showed significantly lower urine osmotic pressure compared to the control group. There were no significant differences in urinary sodium excretion and serum arginine-vasopressin levels among the three groups. Subsequently, we assessed the expression of aquaporin (AQP) in kidney tissue using real-time PCR and immunohistochemical staining. The mRNA expression of AQP1, AQP2, AQP3, and AQP4 did not differ among the three groups. However, immunohistochemical staining showed that the localization of AQP2 at the apical plasma membrane of the collecting ducts was decreased by treatment with Goreisan. Furthermore, deterioration of kidney function was prevented by treatment with Goreisan. Histological analysis revealed that glomerular and tubulointerstitial damages were ameliorated in the GL and GH groups compared to the control group (glomerular damage score: control; 2.19±0.05, GL group; 1.71±0.05, GH group; 1.10±0.05, tubulointerstitial damage score: control; 2.55±0.09, GL group; 2.28±0.10, GH group; 2.12±0.10). In addition, urinary 8-OHdG, which is an oxidative stress marker, was significantly lower in the GL and GH groups than in the control group. Conclusion Our results suggested that Goreisan may have diuretic and renoprotective effects by suppressing the trafficking of AQP2 to the apical plasma membrane of the collecting ducts and possibly reducing systemic oxidative stress.

#4082 TAZ-KNOCKDOWN AFFECTS THE VASOPRESSIN-INDUCED AQUAPORIN-2 (AQP2) TRAFFICKING AND PROTEIN ABUNDANCE IN KIDNEY COLLECTING DUCT CELLS

Abstract Background and Aims Transcriptional coactivator with PDZ-binding motif (TAZ), a downstream effector of the hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ KO mice were known to display multicystic kidneys with polyuria. We aimed to study the role of TAZ in vasopressin-induced AQP2 regulation. Method 1) siRNA-mediated knockdown of TAZ in mpkCCDc11 cells; 2) RT-qPCR, semiquantitative immunoblotting, immunocytochemistry of AQP2; 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, the mouse collecting duct cell line. Results Endogenous AQP2 expression was induced in mpkCCDc11 cells by dDAVP (10−9M) treatment. After starvation for 24 h, dDAVP (10−9M) stimulation for 15 and 30 min induced a significant AQP2 translocation to the cell membrane. In contrast, the dDAVP-induced AQP2 translocation was markedly attenuated in the cells with siRNA-mediated TAZ knockdown (TAZ-KD), despite no changes in cAMP production. Phalloidin staining demonstrated the excessive stress fiber formation in the TAZ-KD. dDAVP (10−9 M) treatment for 24 h induced AQP2 mRNA (12,608 ± 177% of the control) and AQP2 protein abundance (270 ± 18%). In contrast, dDAVP-induced increase of AQP2 mRNA (273 ± 14% of the control) and protein abundance (99 ± 17%) was significantly attenuated in TAZ-KD. Unchanged TonEBP protein abundance was observed in TAZ-KD. NGS identified several potential AQP2 transcription factors (TF), and Klf6, Irf3, Cebpb, and Nr4a1 were selected based on previous in silico database. Among them, Nr4a1 was chosen for further studies due to a significant decrease in the mRNA expression levels in TAZ-KD, as demonstrated by RT-qPCR. Conclusion TAZ is likely to affect dDAVP-induced AQP2 trafficking. This is not mediated by the changes in cAMP/PKA pathway, but other non-canonical pathways are involved. TAZ could regulate AQP2 abundance, possibly via an interaction with several TF.

Uncovering the functional role of GPR39 in the renal collecting duct

G protein-coupled receptors (GPCRs) are seven transmembrane domain receptors that comprise the largest class of proteins in the mammalian genome. GPR39 is part of the ghrelin subfamily of GPCRs, and is well-expressed in the kidney but has no reported function in renal physiology. To probe the renal role of GPR39, we first sought to localize it; in the absence of a validated immunofluorescence (IF) antibody, we used RNAscope. We found that Gpr39 localizes to nephron segments in the cortex and medulla which are positive for DBA (Dolichos Biflorus Agglutinin), a collecting duct marker. RNAScope expression of Gpr39 was highest in the inner medullary collecting duct, with lower levels in the outer medullary and cortical collecting ducts. Within the collecting duct, Gpr39 transcripts colocalized with Aqp2, indicating that Gpr39 is expressed specifically in principal cells. To address whether GPR39 is apical or basolateral, we cloned murine Gpr39 with a C-terminal EGFP tag and transiently transfected this construct into either polarized M-1 cells (murine collecting duct cell line) or polarized MDCK cells (Madin-Darby canine kidney cells); in both cases, cells were grown to confluence on permeable supports. In both cell lines, heterologous GPR39 trafficked to the basolateral membrane. Based on the localization of GPR39 to principal cells, we hypothesized that GPR39 activation may alter AQP2 expression or trafficking. To test this hypothesis in vitro, we first validated mpkCCD (mouse principal cell kidney cortical collecting duct) cells as a model. We cultured mpkCCD on transwells until polarized (TER >5kΩ·cm 2 ). In the absence of dDAVP, mpkCCD do not express AQP2 by western or IF. Basolateral treatment with 1nM dDAVP for 4 days induced AQP2 expression (western blot) and apical trafficking (confocal IF). We also confirmed that withdrawal of dDAVP (4h) resulted in a cytosolic accumulation of AQP2, while the re-introduction of dDAVP (1h) returned AQP2 to the apical membrane by IF. Moreover, we found that Gpr39 expression was unaffected by dDAVP by qPCR (n=3, ΔCt ± SEM: control = 7.9±0.3, dDAVP = 7.2±0.1) and western blot. In a preliminary study, we treated mpkCCD with 13nM of a GPR39 selective agonist, cpd1324, or with a vehicle control, and probed for changes in AQP2 by IF. Both groups were treated with 1nM dDAVP and with 10μM ZnCl2 (a GPR39 cofactor). We found that cpd1324 treatment decreased AQP2 by IF (mean integrated fluorescence density ± SEM (A.U.): cpd1324 = 233.5 ± 20.8, control = 373.8 ± 13.0). Additionally, cpd1324 treatment reduced the apical AQP2 abundance (mean integrated fluorescence density ± SEM (A.U.) of the most apical Z-slice: cpd1324 = 6.05e+05 ± 3.9e+04, control = 2.6e+06 ± 4.3e+05). These results suggest that GPR39 agonism may antagonize vasopressin-induced AQP2 trafficking and/or expression. NIH T32HL007534 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel signalling pathways in nephrogenic syndrome of inappropriate antidiuresis: functional implication of site-specific AQP2 phosphorylation.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X-linked disease caused by gain-of-function mutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp-In T-REx Madin-Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2-wt or non-phosphorylatable AQP2-S269A/AQP2-S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2-wt and significantly higher basal water permeability. In addition, V2R-R137L/C showed significantly higher activity of Rho-associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269-AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R-R137L/C mutants and AQP2-S269A showed a significant reduction in AQP2membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269-AQP2 phosphorylation in the gain-of-function phenotype. Conversely, V2R-R137L/C mutants retained the gain-of-function phenotype when AQP2-S256A was co-expressed. In contrast, cells expressing the F229V mutant and the non-phosphorylatable AQP2-S256A had a significant reduction in AQP2membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain-of-function V2R-R137L/C mutants causing NSIAD is protein kinaseA independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation. KEY POINTS: Nephrogenic syndrome of inappropriate antidiuresis is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R-F229V) and the other vaptan resistant (V2R-R137C/L). In renal cells, all three activating arginine vasopressin receptor 2 (V2R) variants display constitutive AQP2 plasma membrane expression and high basal water permeability. In cells expressing V2R-R137L/C mutants, disruption of the AQP2-S269 phosphorylation site caused the loss of the gain-of-function phenotype, which, in contrast, was retained in V2R-F229V-expressing cells. Cells expressing the V2R-F229V mutant were instead sensitive to disruption of the AQP2-S256 phosphorylation site. The serine/threonine kinase Rho-associated kinase (ROCK) was found to be involved in AQP2-S269 phosphorylation downstream of the V2R-R137L/C mutants. These findings might have clinical relevance for patients with nephrogenic syndrome of inappropriate antidiuresis.

β3 Adrenergic Receptor Agonist Mirabegron Increases AQP2 and NKCC2 Urinary Excretion in OAB Patients: A Pleiotropic Effect of Interest for Patients with X-Linked Nephrogenic Diabetes Insipidus.

We previously reported the novel finding that β3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of β3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the β3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

AQP2 trafficking in health and diseases: an updated overview.

Renal collecting duct principal cells play a key role in controlling body water balance. Principal cells express the water channels AQP2, AQP3, and AQP4 that mediate renal water reabsorption. AQP3 and AQP4 are expressed at the basolateral membrane constitutively. Conversely, AQP2 is localized in intracellular vesicles and translocates to the plasma membrane under vasopressin action. Stimulation with vasopressin activates the cAMP/PKA signal transduction pathway that induces the redistribution of AQP2 from an intracellular pool to the apical plasma membrane. AQP2 trafficking and function depend on multiple post-translational modifications. Moreover, several proteins control different steps activated by the vasopressin stimulation that triggers the redistribution of the AQP2 vesicles. A-kinase anchoring proteins (AKAPs) together with phosphodiesterases and adenylate cyclases play crucial roles in modulating local changes of cAMP. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE), cytoskeletal proteins, and the small GTPases of the Rho family regulate the fusion and the endocytotic retrieval of AQP2 vesicles. Abnormal vasopressin signaling and altered AQP2 expression or trafficking can lead to disorders characterized by deregulated mechanisms controlling water homeostasis. This review provides updated data on the molecular signals regulating vasopressin-induced AQP2 trafficking in health and disease.

Pdcd10-Stk24/25 complex controls kidney water reabsorption by regulating Aqp2 membrane targeting.

PDCD10, also known as CCM3, is a gene found to be associated with the human disease cerebral cavernous malformations (CCMs). PDCD10 forms a complex with GCKIII kinases including STK24, STK25, and MST4. Studies in C. elegans and Drosophila have shown a pivotal role of the PDCD10-GCKIII complex in maintaining epithelial integrity. Here, we found that mice deficient of Pdcd10 or Stk24/25 in the kidney tubules developed polyuria and displayed increased water consumption. Although the expression levels of aquaporin genes were not decreased, the levels of total and phosphorylated aquaporin 2 (Aqp2) protein in the apical membrane of tubular epithelial cells were decreased in Pdcd10- and Stk24/25-deficient mice. This loss of Aqp2 was associated with increased expression and membrane targeting of Ezrin and phosphorylated Ezrin, Radixin, Moesin (p-ERM) proteins and impaired intracellular vesicle trafficking. Treatment with Erlotinib, a tyrosine kinase inhibitor promoting exocytosis and inhibiting endocytosis, normalized the expression level and membrane abundance of Aqp2 protein, and partially rescued the water reabsorption defect observed in the Pdcd10-deficient mice. Our current study identified the PDCD10-STK-ERM signaling pathway as a potentially novel pathway required for water balance control by regulating vesicle trafficking and protein abundance of AQP2 in the kidneys.

Structural Insights into AQP2 Targeting to Multivesicular Bodies.

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.

Sorting nexin 27 regulates the lysosomal degradation of AQP2 protein in kidney collecting duct

We recently demonstrated that vacuolar protein sorting‐associated protein 35 (Vps35), a component of the retromer complex, interacts with the carboxyl terminus of aquaporin‐2, and depletion of Vps35 was associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2. Sorting nexin 27 (SNX27), a PDZ domain‐containing protein, is known to cooperate with the retromer complex in the recycling of early‐endosome to the plasma membrane. Since carboxyl terminus of aquaporin‐2 has the class I PDZ‐interacting motif (X‐Thr/Ser‐X‐Φ), we aimed to examine whether SNX27 could also play a role in the regulation of AQP2 trafficking and protein abundance in the kidney collecting duct cells. Immunohistochemistry revealed that SNX27 was diffusely labeled throughout the cytoplasm in the inner medullary collecting duct cells of rat kidney. Co‐immunoprecipitation assay of rat kidney inner medulla demonstrated that SNX27 interacted with AQP2. The role of SNX27 in the dDAVP‐responsive regulation of AQP2 trafficking and protein abundance was examined in mouse collecting duct mpkCCDc14 cells under siRNA‐mediated knockdown of SNX27. Cell surface biotinylation assay revealed that dDAVP‐induced translocation of AQP2 to the apical plasma membrane was decreased under SNX27 knockdown. Moreover, dDAVP‐induced AQP2 up‐regulation was significantly blunted in the cells with the siRNA‐mediated SNX27 knockdown. During the withdrawal period after dDAVP stimulation, the decrease of AQP2 protein abundance in the cells with SNX27 knockdown was attenuated by lysosomal inhibition with chloroquine co‐treatment. Taken together, SNX27 is an interacting protein with AQP2 and is likely to act as a component of the AQP2 sorting machinery after endocytosis, partly by regulating the lysosomal degradation of AQP2 protein abundance.Support or Funding InformationThis study was supported by the National Research Foundation of Korea Grants 2014R1A5A2009242 and 2016R1A2B4009365 funded by the Ministry of Science, ICT and Future Planning, KoreaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of urinary exosomal release of aquaporin-1 and -2 after renal ischemia-reperfusion in rats.

Acute kidney injury (AKI) is an important risk factor for the development of chronic kidney disease (CKD), and an alteration in renal water handling has been observed during the transition of AKI to CKD. Urinary exosomal release of aquaporin-1 (AQP1) and AQP2, important proteins for renal water handling, has recently been reported to predict their levels of renal expression. Therefore, we examined the patterns of urinary exosomal release of AQP1 and AQP2, and the exosomal marker proteins tumor susceptibility 101 protein (TSG101) and ALG-2 interacting protein X (Alix), in the acute and chronic phases following induction of AKI by renal bilateral ischemia/reperfusion (I/R) in rats. Blood tests and histological examinations indicated that AKI occurred before at 7 days after renal I/R ( day 7) and that renal fibrosis developed progressively thereafter. Immunoblotting demonstrated significant decreases in the urinary exosomal release of AQP1 and AQP2 during severe AKI. Urinary exosomal release of Alix and TSG101 was significantly increased on day 7. These data were also confirmed in rats with unilateral renal I/R causing more serious AKI. Urinary exosomal release of either the Ser-256- or Ser-269-phosphorylated form of AQP2, both of which are involved in apical trafficking of AQP2, was positively correlated with that of total AQP2. These results suggest that urinary exosomal release of AQP1 and AQP2 is reduced in I/R-induced AKI, whereas that of Alix and TSG101 is increased in the initial phase of renal fibrosis. Furthermore, apical trafficking of AQP2 appears to be related to urinary exosomal release of AQP2.

Sildenafil for the Treatment of Congenital Nephrogenic Diabetes Insipidus

Background: Congenital nephrogenic diabetes insipidus (NDI) is characterized by massive polyuria and polydipsia due to defects in the vasopressin-sensitive signaling system expression of the acuaporin-2 (AQP2) water channel of the kidney collecting duct principal cells. Current conventional treatment regimen including hydration, diuretics and non-steroidal anti-inflammatory drugs can only partially reduce polyuria. Recent experimental studies have suggested that treatment with sildenafil, a selective phosphodiesterase inhibitor, may enhance cyclic guanosine monophosphate (cGMP)-mediated apical trafficking of AQP2 and may be effective in increasing water reabsorption in patients with congenital NDI. Patient and Methods: A 4-year old boy with X-linked NDI resistant to conventional therapy was treated with sildenafil for 10 days after a 2-day washout period between the 2 treatment regimens. Aliquots of the 24-hour urine collections before and after treatment were analyzed for urine volume, osmolality, cGMP and AQP2 determinations. Blood samples were also obtained for sodium and osmolality measurements. The primary endpoint was 24-hour urine volume after 10 days of sildenafil and conventional treatments. Results: Compared to conventional therapy, treatment with sildenafil resulted in substantial reduction in 24-hour urine volume (1,764 vs. 950 ml) and serum sodium (148 vs. 139) mEq/l, and increased urine osmolality (104 vs. 215 mOsm/l), and AQP2 excretion (5 vs. 26 fmol/mg creatinine). The patient tolerated sildenafil well and experienced no adverse effects. Conclusions: Sildenafil citrate should be considered an alternative agent in the treatment of X-linked NDI resistant to conventional therapy.

Sympathetic Stimulation In The Kidney: A Possible Antidiuretic Effect Of BAR3?

Beta-adrenergic receptors BAR1 and BAR2 regulate important renal functions. However, the presence, localization and possible physiological role of BAR3 in the kidney is still debated. We immunolocalized BAR3 in wt mouse with markers of different nephron segments. Confocal analysis indicated that BAR3 is expressed at the basolateral membrane of the epithelial cells of the thin ascending limb (tAL), thick ascending limb (TAL), distal convolute tubule (DCT) and the cortical and outer medullary collecting duct (CD). The presence of BAR3 in the vasopressin-sensitive nephron segments prompted us to investigate the possible effect of BAR3 stimulation on the plasma membrane expression of the water channel AQP2. We incubated mouse kidney slices either with isoproterenol or with the BAR3 selective agonist BRL37344, in the presence or in the absence of the PKA inhibitor H89 or BAR3 antagonists. To uncover the possible physiological role of BAR3 stimulation in antidiuresis, we injected BRL37344 in mice lacking vasopressin V2 receptor (V2R). Interestingly, in kidney slices, isoproterenol or BRL37344 elicited a vasopressin-like effect on AQP2 trafficking that was counteracted by BAR3 antagonists or H89. Moreover, in V2R-defective mice, BRL37344 promoted a potent antidiuretic effect. Taken together, these findings are of great physiological importance as they uncover the antidiuretic effect of BAR3 stimulation in the kidney and suggest that BAR3 agonists might be useful to bypass V2R inactivating mutations.

Interaction of AQP2 and integrin b1 is important for the maintenance of epithelial structure and function

Kidney tubular epithelium contributes critically to the regulation and maintenance of electrolyte, acid and base, and water balance in the body. Aquaporin 2 is the water channel expressed in the principal cells of the collecting ducts (CDs) of the kidneys. It regulates water re-absorption in the CDs in response to the antidiuretic hormone, vasopressin (VP). Extensive studies from our group and others have revealed a complex regulatory network of AQP2 trafficking inside cells. More recently, study from our lab has shown that AQP2 is not only a water channel but also an integrin binding membrane protein. Through interacting with integrin b1, AQP2 modulates the internalization and trafficking of integrin b1, facilitates its turnover at the focal adhesions, and results in increased cell migration. Disrupting the interaction between AQP2 and integrin b1 by mutating the RGD motif led to reduced endocytosis, cell surface retention of integrin b1, defective cell migration and tubulogenesis. In addition, we show here that interaction of AQP2 and integrin b1 is also important for maintaining a polarized, subcellular distribution of integrin b1 in epithelium. Uncoupling of AQP2 and integrin interaction (through mutating the RGD-integrin binding motif of AQP2, or using AQP2 null animals) leads to redistribution of integrin b1 from the basal membrane domain to the lateral membrane domain in polarized cell culture and in AQP2 knock out animals. This redistribution of integrin b1 is associated with aberrant expression of the serine/threonine kinase Akt in cells. In summary, our data suggest that besides transporting water, the water channel AQP2 plays a novel role and contributes importantly to the structural and functional integrity of the renal tubular epithelium through interacting with integrin b1.

Combination of secretin and fluvastatin ameliorates the polyuria associated with X-linked nephrogenic diabetes insipidus in mice

X-linked nephrogenic diabetes insipidus (X-NDI) is a disease caused by inactivating mutations of the vasopressin (AVP) type 2 receptor (V2R) gene. Loss of V2R function prevents plasma membrane expression of the AQP2 water channel in the kidney collecting duct cells and impairs the kidney concentration ability. In an attempt to develop strategies to bypass V2R signaling in X-NDI, we evaluated the effects of secretin and fluvastatin, either alone or in combination, on kidney function in a mouse model of X-NDI. The secretin receptor was found to be functionally expressed in the kidney collecting duct cells. Based on this, X-NDI mice were infused with secretin for 14 days but urinary parameters were not altered by the infusion. Interestingly, secretin significantly increased AQP2 levels in the collecting duct but the protein primarily accumulated in the cytosol. Since we previously reported that fluvastatin treatment increased AQP2 plasma membrane expression in wild-type mice, secretin-infused X-NDI mice received a single injection of fluvastatin. Interestingly, urine production by X-NDI mice treated with secretin plus fluvastatin was reduced by nearly 90% and the urine osmolality was doubled. Immunostaining showed that secretin increased intracellular stores of AQP2 and the addition of fluvastatin promoted AQP2 trafficking to the plasma membrane. Taken together, these findings open new perspectives for the pharmacological treatment of X-NDI.

The effects of altered extracellular pH on dDAVP‐induced phosphorylation (serine 256) and intracellular trafficking of AQP2 (1137.1)

The effects of altered extracellular pH (pHe) on dDAVP‐induced phosphorylation (S256, p‐AQP2) and trafficking of AQP2 were examined in the rat kidney inner medullary collecting duct (IMCD) cells. Freshly prepared IMCD suspension was exposed to buffer with pH 6.4, 7.4, or 8.4 for 1 h. dDAVP (10‐10 M, 3 min)‐induced AQP2 phosphorylation was more prominent when tubule suspension was exposed to pH 7.4 or 8.4, compared to pH 6.4. When primary cultured IMCD cells were exposed to pHe 6.4, 7.4 or 8.4 for 1 h, intracellular pH was changed to 6.1, 7.2 and 8.1, respectively. IMCD cells cultured in transwell chamber were exposed to transepithelial pH gradient for 1 h (pH 6.4, 7.4 or 8.4 at the apical side vs. pH 7.4 at the basolateral side). Laser scanning confocal microscopy and cell surface biotinylation assay revealed that exposure to lower pH (pH 6.4, 1 h) significantly reduced dDAVP (10‐9M, 15 min, basolateral)‐induced AQP2 trafficking and surface expression of p‐AQP2. Importantly FRET analysis revealed that dDAVP (10‐10 M)‐induced increase of PKA activity was attenuated when cells were exposed to pHe 6.4, whereas forskolin (10‐7 M)‐induced PKA activation was not affected. Taken together, decreased pHe is likely to exert an inhibitory effect on dDAVP‐induced AQP2 phosphorylation and intracellular trafficking in IMCD cells, presumably via an inhibition of G‐protein‐cAMP‐PKA action.

Identifying transcriptional targets of vasopressin using combined ChIP‐seq and RNA‐seq analysis of mpkCCD cells (1181.6)

The peptide hormone vasopressin regulates transport in the renal collecting duct in part through regulation of gene expression. To obtain a global profile of genes regulated by vasopressin, we performed ChIP‐seq analysis of RNA polymerase II and acetylated histone H3K27 binding sites associated with transcriptionally active genes. Cultured mpkCCD cells were treated with either the vasopressin analog dDAVP (0.1 nM) or vehicle for 24 hours, and then processed for chromatin IP. DNA‐protein complexes were immunoprecipitated using either RNA pol II or H3K27ac antibodies. After reversal of crosslinks, DNA fragments were sequenced and mapped to a reference genome. ChIP‐seq data analysis allowed for precise mapping of genomic elements that were significantly enriched or de‐enriched with dDAVP treatment including exonic, intronic, intergenic, and promoter regions. We correlated these changes at the genomic level with corresponding changes in RNA using RNA‐seq analysis of ‘Poly(A)’‐enriched libraries from mpkCCD cells treated with or without dDAVP. Targets enriched in ChIP‐seq and RNA‐seq data sets in the presence of dDAVP included the water channel aquaporin‐2 (Aqp2), genes associated with AQP2 trafficking (Mal2) and AQP2 transcriptional regulation (Elf3), and many others. This study presents a wealth of new data needed for modeling the transcriptional response to vasopressin.Grant Funding Source: Supported by NHLBI

Visualization of vectorial trafficking of aquaporin‐2 tagged with photoconvertible fluorescence protein in mpkCCD cells (1181.15)

AQP2 tagged with green fluorescent protein (GFP‐AQP2) has been used to monitor AQP2 trafficking in kidney cell lines. However, vectorial trafficking of GFP‐AQP2 resembling to that in the principle cells of collecting duct has not been demonstrated in real time in vitro. mEos2 is a photoconvertible fluorescence protein. To visualize AQP2 trafficking, chimeras with mEos2 fused to either the amino‐terminus of AQP2 (Eos‐AQP2) or the carboxyl‐terminus (AQP2‐Eos) via a 17 amino‐acids linker were constructed and expressed in mpkCCD cells on glass‐bottom dishes. Forskolin‐induced apical targeting of AQP2 in real time was monitored using video‐rate confocal microscopy at four dimensions (XYZ‐T). AQP2‐Eos chimeras expressed in mpkCCD cells were localized constitutively to the apical plasma membrane. In contrast, the Eos‐AQP2 chimeras trafficked from intracellular vesicles to the apical membrane in response to forskolin stimulation, indicating similar vectorial trafficking of AQP2 as in collecting duct. Co‐transfection of these two chimeras in 1:1 ratio increased the rate of AQP2 apical accumulation triggered by forskolin (~2‐fold). However, the synergic effect was abolished when the linker was reduced to 8 amino‐acids. These observations suggested that proximity of mEos at the amino‐terminus and the carboxyl‐terminus within the AQP2 tetramer modulates the response to cAMP stimulation.

Statins increase AQP2 urinary excretion in hypercholesterolemic patients: a pleiotropic effect of statins of potential interest for patients with defects of AQP2 trafficking. (892.8)

We reported that statins treatment increases AQP2 membrane expression in both cultured renal cells and in the renal tubule of mouse, but the ultimate effect in human kidney is unknown. Here, we conducted a pilot clinical study on AQP2 membrane expression in hypercholesterolemic patients requiring statin therapy. In humans, urinary AQP2 (uAQP2) is a non‐invasive, reliable biomarker correlating with the amount of plasma membrane‐expressed AQP2.Nineteen naïve patients with sustained hypercholesterolemia were enrolled along with 15 patients already under statin treatment for at least 1 year. We quantified the 24 h uAQP by ELISA. First, uAQP2 levels were compared in the two groups of patients. Then, time‐dependent changes of uAQP2 levels were studied at baseline, week1 and week 2 in naïve patients starting statin treatment (simvastatin 10‐20 mg/day).uAQP2 was significantly higher in patients chronically treated with statins compared to naïve (3839±319 pmol/24 h vs. 2819±234 pmol/24 h).Following statins treatment in naïve patients, uAQP2 shifted from 2471±358 pmol/24 h at baseline to 4443±493 pmol/24 h, at the end of the week1, and to 4003±450 pmol/24 h at the end of week2 of treatment.We conclude that the pleiotropic effect of statins on AQP2 plasma membrane abundance is attainable in humans at pharmacological doses. This finding has considerable relevance for those pathological conditions in which AQP2 membrane trafficking is impaired as in nephrogenic diabetes insipidus.Grant Funding Source: Telethon GGP12040/ AIFA MRAR08P011

935 OVEREXPRESSION OF CREM ALPHA IN CD4+ T CELLS ACCELERATES ACUTE ConA-INDUCED HEPATITIS BUT DOES NOT INFLUENCE FIBROSIS

Introduction: Aquaporins (AQPs) are a family of channel proteins that facilitate the osmotic movement of water and small solutes across biological membranes. In the hepatobiliary tract, AQPs participate in formation and flow of bile. We have previously shown that Liver X Receptor b (LXRb), a nuclear receptor activated by oxysterols, controls the expression of AQP-1 in pancreas and kidney. Aim and Methods: In the present study we aimed to investigate the role of LXRb in controlling water channel homeostasis in the hepatobiliary tract, in-vivo. Both male and female WT and LXRb−/− mice were studied at the age of 12 months. Results: In WT mice, LXRb protein was strongly expressed in the nuclei of intrahepatic cholangiocytes while a weak expression was detected in hepatocytes. Male LXRb−/− mice demonstrated a significant increase of serum levels of both total bilirubin and alkaline phosphatase, indicating the presence of a mild cholestasis. In the whole liver, there was a significant reduction in the levels of both the mRNA of AQP-1 and AQP-4 in male LXRb−/− mice. Interestingly, AQP-4 protein expression was detected on the plasma membrane of male WT hepatocytes while in LXRb−/− mice, the immunoreactivity was identified in the cytosol, suggesting a possible defect in its transport to the membrane. No AQP-4 was detected in female mice either WT or LXRb−/−. In the kidney, AQP-2 trafficking is controlled by caveolin-1, a major component of caveolae, that is involved in transcytosis, endocytosis and signal transduction. In prostate cancer cells, caveolin-1 is upregulated by testosterone and acts as Androgen Receptor (AR) co-regulator being involved in non-genomic effects of AR. In LXRb−/− male mice, strong immunoreactivity of caveolin-1 was detected in the cytosol while in WT mice it was on the cell surface. Thus there is impaired translocation of caveolin-1 in the mutant mice. Interestingly, in LXRb−/− male mice, serum testosterone levels were in normal range. Conclusion: LXRb regulates the level of AQP-1 and AQP-4 mRNA as well as the cellular localization of AQP-4, via caveolin-1, in male mice.

Effect ofAtractylodes macrocephalaon Hypertonic Stress-Induced Water Channel Protein Expression in Renal Collecting Duct Cells

Edema is a symptom that results from the abnormal accumulation of fluid in the body. The cause of edema is related to the level of aquaporin (AQP)2 protein expression, which regulates the reabsorption of water in the kidney. Edema is caused by overexpression of the AQP2 protein when the concentration of Na+ in the blood increases. The rhizome of Atractylodes macrocephala has been used in traditional oriental medicine as a diuretic drug; however, the mechanism responsible for the diuretic effect of the aqueous extract from A. macrocephala rhizomes (AAMs) has not yet been identified. We examined the effect of the AAM on the regulation of water channels in the mouse inner medullary collecting duct (mIMCD)-3 cells under hypertonic stress. Pretreatment of AAM attenuates a hypertonicity-induced increase in AQP2 expression as well as the trafficking of AQP2 to the apical plasma membrane. Tonicity-responsive enhancer binding protein (TonEBP) is a transcription factor known to play a central role in cellular homeostasis by regulating the expression of some proteins, including AQP2. Western immunoblot analysis demonstrated that the protein and mRNA expression levels of TonEBP also decrease after AAM treatment. These results suggest that the AAM has a diuretic effect by suppressing water reabsorption via the downregulation of the TonEBP-AQP2 signaling pathway.