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The alteration of water balance and related disorders has emerged as being strictly linked to the state of activation of the vasopressin-aquaporin-2 (vasopressin-AQP2) pathway. The lack of responsiveness of the kidney to the vasopressin action impairs its ability to concentrate the urine, resulting in polyuria, polydipsia, and risk of severe dehydration for patients. Conversely, non-osmotic release of vasopressin is associated with an increase in water permeability in the renal collecting duct, producing water retention and increasing the circulatory blood volume. This review highlights some of the new insights and recent advances in therapeutic intervention targeting the dysfunctions in the vasopressin-AQP2 pathway causing diseases characterized by water balance disorders such as congenital nephrogenic diabetes insipidus, syndrome of inappropriate antidiuretic hormone secretion, nephrogenic syndrome of inappropriate antidiuresis, and autosomal dominant polycystic kidney disease. The recent clinical data suggest that targeting the vasopressin-AQP2 axis can provide therapeutic benefits in patients with water balance disorders., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca 2+ ] i . While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1 -/- and Epac2 -/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca 2+ ] i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1 -/- and Epac2 -/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na + and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2.

Background: The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms., Methods: We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout ( Tgr5 -/- ) mice., Results: Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5 mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated -/- mice, LCA treatment failed to prevent reduction of AQP2 expression.Tgr5 -/- mice, LCA treatment failed to prevent reduction of AQP2 expression., Conclusions: TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney., (Copyright © 2018 by the American Society of Nephrology.)

Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1 - Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1 - AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis . The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process.

The molecular mechanisms of melamine-induced renal toxicity have not been fully understood. The purpose of the study aimed to investigate whether melamine and cyanuric acid induced NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in the kidney, which may contribute to abnormal water and sodium handling in a rat model. Wistar rats received melamine (Mel; 200 mg·kg body wt -1 ·day -1 ), cyanuric acid (CA; 200 mg·kg body wt -1 ·day -1 ), or Mel plus CA (Mel + CA; 100 mg·kg body wt -1 ·day -1 , each) for 2 wk. Mel + CA caused damaged tubular epithelial structure and organelles, dilated tubular lumen, and inflammatory responses. Crystals were observed in urine and serum specimen, also in the lumen of dilated distal renal tubules. The combined ingestion of Mel and CA in rats caused a markedly impaired urinary concentration, which was associated with reduced protein expression of aquaporin (AQP)1, 2, and 3 in inner medulla and α-Na-K-ATPase and Na-K-2Cl transporters in cortex and outer medulla. Mel + CA treatment was associated with increased protein expression of CD3 and mRNA levels of CD68 and F4/80 as well as phosphorylation of NF-κB in the kidney. Mel + CA treatment increased protein and mRNA expression of NLRP3 inflammasome components apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, and IL-1β in the inner medulla of rats. NF-κB inhibitor Bay 11-7082 reduced IL-1β expression induced by Mel + CA and prevented downregulation of AQP2 in inner medullary collecting duct cell suspensions. In conclusion, Mel + CA treatment caused urinary-concentrating defects and reduced expression of renal AQPs and key sodium transporters, which is likely due to the inflammatory responses and activation of NLRP3 inflammasome induced by crystals formed in the kidney.

Congenital nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentrating ability. Symptomatic polyuria is present from birth, even with normal release of the antidiuretic hormone vasopressin by the pituitary. Over the last two decades, the aquaporin-2 (AQP2) gene has been cloned and the molecular mechanisms of urine concentration have been gradually elucidated. Vasopressin binds to the vasopressin type II receptor (V2R) in the renal collecting ducts and then activates AQP2 phosphorylation and trafficking to increase water reabsorption from urine. Most cases of congenital NDI are caused by loss-of-function mutations to V2R, resulting in unresponsiveness to vasopressin. In this article, we provide an overview of novel therapeutic molecules of congenital NDI that can activate AQP2 by bypassing defective V2R signaling with a particular focus on the activators of the calcium and cAMP signaling pathways.

Arsenicals were introduced as monotherapies for the treatment of human African trypanosomiasis, or sleeping sickness, over 100 years ago. Toxicity has always been an issue but these drugs have proven to be both effective and quite durable. Unfortunately, melarsoprol-resistant parasites emerged as early as the 1970s and were widespread by the late 1990s. Resistance was due to mutations affecting an aquaglyceroporin (AQP2), a parasite solute and drug transporter. This is the only example of widespread drug resistance in trypanosomiasis patients for which the genetic basis is known. This link between melarsoprol and AQP2 illustrates how a drug transporter can improve drug selectivity but, at the same time, highlights the risk of resistance when the drug uptake mechanism is dispensable for parasite viability and virulence., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Background: Inherited nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by impaired urinary concentrating ability. Little clinical data on long-term outcome exists., Method: This was a single-centre retrospective medical record review of patients with a diagnosis of NDI followed between 1985 and 2017. We collected available data on growth, weight, school performance, complications and comorbidities., Results: We identified 36 patients with available data and a clinical diagnosis of NDI, which was genetically confirmed in 33 of them. Patients presented at a median age of 0.6 years and median length of follow-up was 9.5 years. Chief symptoms at presentation were faltering growth, vomiting/feeding concerns, polyuria/polydipsia, febrile illness and hypernatraemic dehydration. Median weight standard deviation scores (SDS) improved from -2.1 at presentation to 0.2 at last follow-up. In contrast, height SDS remained essentially unchanged at -1.1 at presentation and -0.9 at last follow-up. Most patients were treated with prostaglandin synthesis inhibitors and thiazides, yet weaned off during school age without an obvious change in urine output. Median estimated glomerular filtration rate at last follow-up was 81 mL/min/1.73 m 2 . Urological complications were noted in 15 patients, constipation in 11 and learning difficulties in 5. Median age at resolution of nocturnal enuresis was 11 years. Estimated median daily fluid intake at median age of 13 years was 3800 mL/m 2 ., Conclusion: The overall prognosis in inherited NDI is favourable with regular treatment. As expected, most complications were related to polyuria. There is an apparent loss of efficacy of medications during school age. Our data inform the prognosis and management of patients with NDI.

An ideal inducible system should be cell specific and have absolutely no background recombination without induction (i.e., no leakiness), a high recombination rate after induction, and complete fidelity in cell specificity (i.e., restricted recombination exclusively in cells where the driver gene is expressed). However, such an ideal mouse model remains unavailable for collecting duct research. Here, we report a mouse model that meets these criteria. In this model, a cassette expressing ER T2 CreER T2 ( ECE) is inserted at the ATG of the endogenous Aqp2 locus to disrupt Aqp2 function and to express ECE under the control of the Aqp2 promoter. The resulting allele is named Aqp2 ECE . There was no indication of a significant impact of disruption of a copy of Aqp2 on renal function and blood pressure control in adult Aqp2 ECE/+ heterozygotes. Without tamoxifen, Aqp2 ECE did not activate a Cre-dependent red fluorescence protein (RFP) reporter in adult kidneys. A single injection of tamoxifen (2 mg) to adult mice enabled Aqp2 ECE to induce robust RFP expression in the whole kidney 24 h postinjection, with the highest recombination efficiency of 95% in the inner medulla. All RFP-labeled cells expressed principal cell markers (Aqp2 and Aqp3), but not intercalated cell markers (V-ATPase B1B2, and carbonic anhydrase II). Hence, Aqp2 ECE confers principal cell-specific tamoxifen-inducible recombination with absolutely no leakiness, high inducibility, and complete fidelity in cell specificity, which should be an important tool for temporospatial control of target genes in the principal cells and for Aqp2 + lineage tracing in adult mice.

The importance of the kidney distal convoluted tubule (DCT) and cortical collecting duct (CCD) is highlighted by various water and electrolyte disorders that arise when the unique transport properties of these segments are disturbed. Despite this critical role, little is known about which proteins have a regulatory role in these cells and how these cells can be regulated by individual physiologic stimuli. By combining proteomics, bioinformatics, and cell biology approaches, we found that the E3 ubiquitin ligase CHIP is highly expressed throughout the collecting duct; is modulated in abundance by vasopressin; interacts with aquaporin-2 (AQP2), Hsp70, and Hsc70; and can directly ubiquitylate the water channel AQP2 in vitro shRNA knockdown of CHIP in CCD cells increased AQP2 protein t and reduced AQP2 ubiquitylation, resulting in greater levels of AQP2 and phosphorylated AQP2. CHIP knockdown increased the plasma membrane abundance of AQP2 in these cells. Compared with wild-type controls, CHIP knockout mice or novel CRISPR/Cas9 mice without CHIP E3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. We did not observe significant changes in other water- or sodium-transporting proteins in the gene-modified mice. In summary, these results suggest that CHIP regulates AQP2 and subsequently, renal water handling.1/2 and reduced AQP2 ubiquitylation, resulting in greater levels of AQP2 and phosphorylated AQP2. CHIP knockdown increased the plasma membrane abundance of AQP2 in these cells. Compared with wild-type controls, CHIP knockout mice or novel CRISPR/Cas9 mice without CHIP E3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. We did not observe significant changes in other water- or sodium-transporting proteins in the gene-modified mice. In summary, these results suggest that CHIP regulates AQP2 and subsequently, renal water handling., (Copyright © 2018 by the American Society of Nephrology.)

Background: Tolvaptan slows progression of autosomal dominant polycystic kidney disease (ADPKD) by antagonizing the vasopressin-cAMP axis. Nitric oxide (NO) stimulates natriuresis and diuresis, but its role is unknown during tolvaptan treatment in ADPKD., Methods: Eighteen patients with ADPKD received tolvaptan 60 mg or placebo in a randomized, placebo-controlled, double blind, crossover study. L-NMMA (L-NG-monomethyl-arginine) was given as a bolus followed by continuous infusion during 60 min. We measured: GFR, urine output (UO), free water clearance (C H2O ), fractional excretion of sodium (FE Na ), urinary excretion of aquaporin-2 channels (u-AQP2) and epithelial sodium channels (u-ENaCγ), plasma concentrations of vasopressin (p-AVP), renin (PRC), angiotensinII (p-AngII), aldosterone (p-Aldo), and central blood pressure (cBP)., Results: During tolvaptan with NO-inhibition, a more pronounced decrease was measured in UO, C H2O (61% vs 43%) and FE Na (46% vs 41%) after placebo than after tolvaptan; GFR and u-AQP2 decreased to the same extent; p-AVP increased three fold, whereas u-ENaC γ , PRC, p-AngII, and p-Aldo remained unchanged. After NO-inhibition, GFR increased after placebo and remained unchanged after tolvaptan (5% vs -6%). Central diastolic BP (CDBP) increased to a higher level after placebo than tolvaptan. Body weight fell during tolvaptan treatment., Conclusions: During NO inhibition, tolvaptan antagonized both the antidiuretic and the antinatriuretic effect of L-NMMA, partly via an AVP-dependent mechanism. U-AQP2 was not changed by tolvaptan, presumeably due to a counteracting effect of elevated p-AVP. The reduced GFR during tolvaptan most likely is caused by the reduction in extracellular fluid volume and blood pressure., Trial Registration: Clinical Trial no: NCT02527863 . Registered 18 February 2015.

The abundance of integral membrane proteins in the plasma membrane is determined by a dynamic balance between exocytosis and endocytosis, which can often be regulated by physiological stimuli. Here, we describe a mechanism that accounts for the ability of the peptide hormone vasopressin to regulate water excretion via a phosphorylation-dependent modulation of the PDZ domain-ligand interaction involving the water channel protein aquaporin-2. We discovered that the PDZ domain-containing protein Sipa1l1 (signal-induced proliferation-associated 1 like 1) binds to the cytoplasmic PDZ-ligand motif of aquaporin-2 and accelerates its endocytosis in the absence of vasopressin. Vasopressin-induced aquaporin-2 phosphorylation within the type I PDZ-ligand motif disrupted the interaction, in association with reduced aquaporin-2 endocytosis and prolonged plasma membrane aquaporin-2 retention. This phosphorylation-dependent alteration in the PDZ domain-ligand interaction was explained by 3D structural models, which showed a hormone-regulated mechanism that controls osmotic water transport and systemic water balance in mammals., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background/aims: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation., Methods: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting., Results: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation., Conclusion: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aims to explore the therapeutic effect of bone marrow mesenchymal stem cells on adriamycin nephrosis, and the potential mechanism. The rat experimental nephropathy model was established by unilateral nephrectomy combined repeated injecting adriamycin (ADR). Thirty adriamycin nephrosis rats were randomly divided into three groups, including ADR (n=10), MSCs transplantation through peripheral veins groups (M-V, n=10), and MSCs transplantation through right renal artery groups (M-A, n=10), and there was another normal control group (N, n=10). This study lasted 8 weeks, 24 hours urine was collected through simple metabolic cage to measure urinary volume and urine protein quantitation in 24 hours. The levels of plasma albumin (ALB), sodium were measured by biochemical analysis. The expressions of AQP1-2 were measured by immuno-histochemistry assay. Kidney medulla ultramicroscopic structure was observed by TEM. The results indicated that the ALB and 24 h urinary volume have significant increased in M-V and M-A group compared to the ADR group (P<0.05). Furthermore, the serum sodium and urine protein quantitation in 24 hours were decreased in M-V and M-A group compared to ADR group (P<0.05). Protein expression of AQP1-2 had been remarkably decreased (P<0.05). It showed degenerative changes of kidney ultra microscopic structures of the ADR rats, while MSCs transplantation could significantly improve the damage. In conclusion, in adriamycin nephropathy rats, MSCs transplantation exerts its therapeutic effects by decrease urinary albumin excretion, increase ALB, decrease sodium and the expression of AQP1-2 in renal tubules.

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ethnopharmacological Relevance: Polyporus umbellatus (Pers.) Fries (Polyporaceae, Zhuling ) has been commonly used in medicine for a wide range of ailments related to the edema, scanty urine, vaginal discharge, urinary dysfunction, as well as jaundice and diarrhea., Aim of the Review: The present paper reviewed the traditional uses, propagation, phytochemistry, pharmacology, pharmacokinetics and quality control of Polyporus umbellatus., Materials and Methods: All the available information on Polyporus umbellatus was collected via a library and electronic search (using Web of Science, Pubmed, ScienceDirect, Splinker, Google Scholar, etc.)., Results: Phytochemical studies showed the presence of many valuable secondary metabolites such as steroids, polysaccharides, anthraquinones and nucleosides. Crude extracts and isolated compounds showed a wide spectrum of pharmacological activities including diuretic, nephroprotective, anti-cancer, immuno-enhancing, hepatoprotective, anti-inflammatory and antioxidative activities. The pharmacokinetic studies demonstrated that the ergosterol and ergone had a high distribution and absorption in the plasma and the two main components of Polyporus umbellatus were mainly excreted by faeces. The determination of multiple chemical components was successfully applied to the quality control of Polyporus umbellatus., Conclusions: Modern phytochemical, pharmacological and metabonomic investigations showed that the crude extracts and isolated compounds from Polyporus umbellatus possess many kinds of biological functions, especially in the diuretic activities and the treatment of kidney diseases as well as anti-cancer, immuno-enhancing and hepatoprotective activities. The pathways of the distribution, absorption, metabolism and excretion of main steroidal compounds were clarified by pharmacokinetic studies. Most of the pharmacological studies were conducted using crude and poorly characterized extracts of Polyporus umbellatus in animals especially in case of diuretic activities and the treatment of kidney diseases. Thus, more bioactive components especially diuretic compounds should be identified using bioactivity-guided isolation strategies and the possible mechanism of action as well as potential synergistic or antagonistic effects of multi-component mixtures derived from Polyporus umbellatus need to be evaluated integrating pharmacological, pharmacokinetic, bioavailability-centered and physiological approaches. In addition, more experiments including in vitro, in vivo and clinical studies should be encouraged to identify any side effects or toxicity. These achievements will further expand the existing therapeutic potential of Polyporus umbellatus and provide a beneficial support to its future further clinical use in modern medicine., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Remodeling of the actin cytoskeleton is required for vasopressin (VP)-induced aquaporin 2 (AQP2) trafficking. Here, we asked whether VP and forskolin (FK)-mediated F-actin depolymerization depends on AQP2 expression. Using various MDCK and LLC-PK1 cell lines with different AQP2 expression levels, we performed F-actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F-actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F-actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC-PK(1) cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F-actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane-associated F-actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F-actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells, and that this is not dependent on the polarity of AQP2 membrane insertion.

Background/aims: We have previously shown that aquaporin-2 (AQP2) is down-regulated in the renal medulla of rats made hypertensive by chronic inhibition of nitric oxide synthase. It has been shown that AQP2 expression is regulated by the calcineurin/nuclear factor of activated T cells (NFATc). Nitric oxide (NO) regulates the activity of NFATc via c-Jun-N-terminal kinase 2 (JNK2). Therefore, we hypothesized that increases in NO enhance NFATc-mediated up-regulation of AQP2 promoter activity., Methods: AQP2 mRNA and protein expression were detected in mouse renal papilla. AQP2 promoter luciferase reporter- and NFAT luciferase reporter-transfected MDCK cells were used to determine AQP2 promoter activity and NFATc activity, respectively. Cells were incubated with classic activators and inhibitors of NFATc and the NO pathway., Results: Our results demonstrate that both Ca(2+) and NO have a synergistic effect resulting in an increase in AQP2 mRNA and protein in mouse papilla and activation of the AQP2 promoter in kidney-derived cells. In addition, NO enhances Ca(2+)-induced NFATc activation. The underlying mechanism involves increased NFATc nuclear import and decreased export via protein kinase G-mediated inhibition of JNK1/2., Conclusions: This is the first study defining novel regulatory roles for NO and NFATc in the control of AQP2, which is an important renal protein.