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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.

Kidney function has two important elements: glomerular filtration and tubular function (secretion and reabsorption). A persistent decrease in glomerular filtration rate (GFR), with or without proteinuria, is diagnostic of chronic kidney disease (CKD). While glomerular injury or disease is a major cause of CKD and usually associated with proteinuria, predominant tubular injury, with or without tubulointerstitial disease, is typically non-proteinuric. CKD has been linked with cognitive impairment, but it is unclear how much this depends on a reduced GFR, altered tubular function or the presence of proteinuria. Since CKD is often accompanied by tubular and interstitial dysfunction, we explore here for the first time the potential role of the tubular and tubulointerstitial compartments in cognitive dysfunction. To help address this issue, we have selected a group of primary tubular diseases with preserved GFR, in which to review the evidence for any association with brain dysfunction.Cognition, mood, neurosensory, and motor disturbances are not well characterized in tubular diseases, possibly because they are subclinical and less prominent than other clinical manifestations. The available literature suggests that brain dysfunction in tubular and tubulointerstitial diseases is usually mild and is more often seen in disorders of water handling. Brain dysfunction may occur when severe electrolyte and water disorders in young children persist over a long period of time before the diagnosis is made. We have chosen as examples to highlight this topic, Bartter and Gitelman syndromes and nephrogenic diabetes insipidus. We discuss current published findings, some unanswered questions, and propose topics for future research.

Background: Chronic kidney diseases (CKD) are usually associated with dyslipidemia. Statin therapy has been primarily recommended for the prevention of cardiovascular risk in patients with CKD; however, the effects of statins on kidney disease progression remain controversial. This study aims to investigate the effects of statin treatment on renal handling of water in patients and in animals on a high-fat diet. Methods: Retrospective cohort patient data were reviewed and the protein expression levels of aquaporin-2 (AQP2) and NLRP3 inflammasome adaptor ASC were examined in kidney biopsy specimens. The effects of statins on AQP2 and NLRP3 inflammasome components were examined in nlrp3-/- mice, 5/6 nephroectomized (5/6Nx) rats with a high-fat diet (HFD), and in vitro. Results: In the retrospective cohort study, serum cholesterol was negatively correlated to eGFR and AQP2 protein expression in the kidney biopsy specimens. Statins exhibited no effect on eGFR but abolished the negative correlation between cholesterol and AQP2 expression. Whilst nlrp3+/+ mice showed an increased urine output and a decreased expression of AQP2 protein after a HFD, which was moderately attenuated in nlrp3 deletion mice with HFD. In 5/6Nx rats on a HFD, atorvastatin markedly decreased the urine output and upregulated the protein expression of AQP2. Cholesterol stimulated the protein expression of NLRP3 inflammasome components ASC, caspase-1 and IL-1β, and decreased AQP2 protein abundance in vitro, which was markedly prevented by statins, likely through the enhancement of ASC speck degradation via autophagy. Conclusion: Serum cholesterol level has a negative correlation with AQP2 protein expression in the kidney biopsy specimens of patients. Statins can ameliorate cholesterol-induced inflammation by promoting the degradation of ASC speck, and improve the expression of aquaporin in the kidneys of animals on a HFD.

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.

In response to the antidiuretic hormone arginine-vasopressin (AVP), inducing elevated levels of the second messenger 3’,5’-cyclic adenosine monophosphate (cAMP) and the activation of the cAMP-dependent protein kinase (protein kinase A, PKA), the water channel aquaporin-2 (AQP2) is redistributed from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. Its insertion into the plasma membrane induces the water reabsorption from primary urine and fine-tunes body water homeostasis. Dysregulation of this process causes or is associated with water balance disorders, such as diabetes insipidus (DI) (Olesen and Fenton, 2021; Christ-Crain and Gaisl, 2021). The mechanisms which control the AQP2 localization are not completely understood. The main part of this thesis describes the discovery of a hitherto unknown role of aurora kinase A (AURKA) in the control of the AQP2 localization. Two AURKA-selective inhibitors, aurora-A inhibitor I and alisertib, inhibited the cAMP-dependent AQP2 redistribution in two cell models by effects on the actin cytoskeleton. Novel derivatives of aurora-A inhibitor I provided insights into the structure–activity–relationship (SAR) of the inhibitor. Filamentous (F-) actin containing stress fibers can either serve as transport tracks for AQP2-bearing vesicles en route to the plasma membrane (Nedvetsky et al., 2007; Sasaki, Yui and Noda, 2014) or as a physical barrier by blocking the vesicles’ access to the plasma membrane (Klussmann et al., 2001; Tamma et al., 2001; Simon et al., 1993). Aurora-A inhibitor I induced the depolymerization of the actin transport tracks by activating the actin-depolymerizing protein cofilin-1 (CFL1). In contrast, alisertib promoted the formation of the actin barrier. Alisertib did not affect the CFL1 activity and must thus control the actin cytoskeleton and the AQP2 localization via different mechanisms. The second part builds on published work on the antimycotic drug fluconazole, which elicits antidiuretic effects by inducing the AQP2 redistribution to the plasma membrane of renal principal cells in the absence of AVP (Vukićević et al., 2019). The antimycotic drug tebuconazole was identified as another interesting candidate to modulate the AQP2 localization, which is of potential interest for treating certain forms of DI. In the third part, the molecular phenotype of a novel heterozygous mutation of the AQP2 gene, causing autosomal dominant nephrogenic DI (NDI), was characterized. The mutation led to an intracellular retention of AQP2 and an increase in the complex glycosylation of the protein. This thesis contributes to a better understanding of the molecular mechanisms underlying the AVP-induced AQP2 redistribution. Further, it opens new perspectives for the treatment of water balance disorders with modulators of the AQP2 localization., In Antwort auf das antidiuretische Hormon Arginin-Vasopressin (AVP), welches den Anstieg des Second Messengers 3’,5’-zyklischen Adenosinmonophosphat (cAMP) und die Aktivierung der cAMP-abhängigen Proteinkinase (Proteinkinase A, PKA) bewirkt, wird das Wasserkanalprotein Aquaporin-2 (AQP2) von intrazellulären Vesikeln in die Plasmamembran der Hauptzellen des renalen Sammelrohrs umverteilt. Seine Insertion in die Plasmamembran induziert die Wasserrückresorption aus dem Primärharn und dient der Feinregulation der Wasserhomöostase im Körper. Die Fehlregulation dieses Prozesses ist Ursache von oder assoziiert mit Störungen des Wasserhaushalts, wie z.B. dem Diabetes insipidus (DI) (Olesen and Fenton, 2021; Christ-Crain and Gaisl, 2021). Die Mechanismen, die der Kontrolle der AQP2-Lokalisation dienen, sind noch nicht vollständig aufgeklärt. Der Hauptteil dieser Arbeit beschreibt die Entdeckung einer bislang unbekannten Rolle der Aurorakinase A (AURKA) in der Kontrolle der AQP2-Lokalisation. Zwei AURKAselektive Inhibitoren, Aurora-A inhibitor I und Alisertib, inhibierten die cAMP-abhängige AQP2-Umverteilung in zwei Zellmodellen durch Effekte auf das Aktin-Zytoskelett. Neuartige Derivate von Aurora-A inhibitor I ermöglichten Einblicke in die Struktur–Wirkungs–Beziehung (SAR) des Inhibitors. Filamentöses (F-) Aktin-enthaltende Stressfasern können entweder als Transportschienen für AQP2-tragende Vesikel auf dem Weg zur Plasmamembran dienen (Nedvetsky et al., 2007; Sasaki, Yui and Noda, 2014) oder aber als physische Barriere den Zugang der Vesikel zu der Plasmamembran blockieren (Klussmann et al., 2001; Tamma et al., 2001; Simon et al., 1993). Aurora-A inhibitor I induzierte die Depolymerisierung der Aktin-Transportschienen, indem es das Aktin-depolymerisierende Protein Cofilin-1 (CFL1) aktivierte. Im Gegensatz dazu förderte Alisertib die Bildung der Aktin-Barriere. Alisertib beeinflusste nicht die CFL1-Aktivität, weshalb davon ausgegangen werden muss, dass es das Aktin-Zytoskelett und die AQP2-Lokalisation durch andere Mechanismen kontrolliert. Der zweite Teil baut auf publizierten Erkenntnissen über das Antimykotikum Flukonazol auf, welches antidiuretische Effekte aufweist, indem es die AQP2-Umverteilung in die Plasmamembran renaler Hauptzellen unabhängig von AVP induziert (Vukićević et al., 2019). Das Antimykotikum Tebukonazol wurde als weiterer interessanter Kandidat für die Modulation der AQP2-Lokalisation identifiziert. Dies ist von potentiellem Interesse für die Behandlung bestimmter Formen von DI. Im dritten Teil wurde der molekulare Phänotyp einer neuen heterozygoten AQP2-Genmutation charakterisiert, welche Ursache für einen autosomal dominanten nephrogenen Zusammenfassung XDI (NDI) ist. Die Mutation führte zu einer intrazellulären Retention von AQP2 und einer Erhöhung der komplexen Glykosylierung des Proteins. Diese Arbeit trägt zu einem besseren Verständnis der molekularen Mechanismen bei, denen die AVP-vermittelte AQP2-Umverteilung unterliegt. Darüber hinaus eröffnet sie neue Perspektiven für die Behandlung von Störungen des Wasserhaushalts mithilfe von Modulatoren der AQP2-Lokalisation.

Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.

Funding: This article is published as part of a supplement financially supported by the COST Action CA19127-Cognitive Decline in Nephro-Neurology: European Cooperative Target (CONNECT). Kidney function has two important elements: glomerular filtration and tubular function (secretion and reabsorption). A persistent decrease in glomerular filtration rate (GFR), with or without proteinuria, is diagnostic of chronic kidney disease (CKD). While glomerular injury or disease is a major cause of CKD and usually associated with proteinuria, predominant tubular injury, with or without tubulointerstitial disease, is typically non-proteinuric. CKD has been linked with cognitive impairment, but it is unclear how much this depends on a decreased GFR, altered tubular function or the presence of proteinuria. Since CKD is often accompanied by tubular and interstitial dysfunction, we explore here for the first time the potential role of the tubular and tubulointerstitial compartments in cognitive dysfunction. To help address this issue we selected a group of primary tubular diseases with preserved GFR in which to review the evidence for any association with brain dysfunction. Cognition, mood, neurosensory and motor disturbances are not well characterized in tubular diseases, possibly because they are subclinical and less prominent than other clinical manifestations. The available literature suggests that brain dysfunction in tubular and tubulointerstitial diseases is usually mild and is more often seen in disorders of water handling. Brain dysfunction may occur when severe electrolyte and water disorders in young children persist over a long period of time before the diagnosis is made. We have chosen Bartter and Gitelman syndromes and nephrogenic diabetes insipidus as examples to highlight this topic. We discuss current published findings, some unanswered questions and propose topics for future research. publishersversion published

International audience; Aquaporins (AQPs) are water channels widely distributed in living organisms and involved in many pathophysiologies as well as in cell volume regulations (CVR). In the present study, based on the structural homology existing between mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs), cholesterol consensus motif (CCM) and the extra-cellular vestibules of AQPs, we investigated the binding of corticosteroids on the AQP family through in silico molecular dynamics simulations of AQP2 interactions with cortisol. We propose, for the first time, a putative AQPs corticosteroid binding site (ACBS) and discussed its conservation through structural alignment. Corticosteroids can mediate non-genomic effects; nonetheless, the transduction pathways involved are still misunderstood. Moreover, a growing body of evidence is pointing toward the existence of a novel membrane receptor mediating part of these rapid corticosteroids' effects. Our results suggest that the naturally produced glucocorticoid cortisol inhibits channel water permeability. Based on these results, we propose a detailed description of a putative underlying molecular mechanism. In this process, we also bring new insights on the regulatory function of AQPs extra-cellular loops and on the role of ions in tuning the water permeability. Altogether, this work brings new insights into the non-genomic effects of corticosteroids through the proposition of AQPs as the membrane receptor of this family of regulatory molecules. This original result is the starting point for future investigations to define more in-depth and in vivo the validity of this functional model.

Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.

Background Congenital nephrogenic diabetes insipidus (NDI) is primarily caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R). Renal unresponsiveness to the antidiuretic hormone vasopressin impairs aquaporin-2 (AQP2) water channel activity and water reabsorption from urine, resulting in polyuria. Currently available symptomatic treatments inadequately reduce patients’ excessive amounts of urine excretion, threatening their quality of life. In the past 25 years, vasopressin/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) has been believed to be the most important signaling pathway for AQP2 activation. Although cAMP production without vasopressin is the reasonable therapeutic strategy for congenital NDI caused by V2R mutations, the efficacy of candidate drugs on AQP2 activation is far less than that of vasopressin. Results Intracellular distribution and activity of PKA are largely controlled by its scaffold proteins, A-kinase anchoring proteins (AKAPs). Dissociating the binding of AKAPs and PKA significantly increased PKA activity in the renal collecting ducts and activated AQP2 phosphorylation and trafficking. Remarkably, the AKAPs–PKA disruptor FMP-API-1 increased transcellular water permeability in isolated renal collecting ducts to the same extent as vasopressin. Moreover, derivatives of FMP-API-1 possessed much more high potency. FMP-API-1/27 is the first low-molecular-weight compound to be discovered that can phosphorylate AQP2 more effectively than preexisting drug candidates. Conclusion AKAP-PKA disruptors are a promising therapeutic target for congenital NDI. In this article, we shall discuss the pathophysiological roles of PKA and novel strategies to activate PKA in renal collecting ducts.

Congenital nephrogenic diabetes insipidus (NDI) is a rare genetic disorder characterized by renal inability to concentrate urine. We utilized a multicenter strategy to investigate the genotype and phenotype in a cohort of Chinese children clinically diagnosed with NDI from 2014 to 2019. Ten boys from nine families were identified with mutations in AVPR2 or AQP2 along with dehydration, polyuria–polydipsia, and severe hypernatremia. Genetic screening confirmed the diagnosis of seven additional relatives with partial or subclinical NDI. Protein structural analysis revealed a notable clustering of diagnostic mutations in the transmembrane region of AVPR2 and an enrichment of diagnostic mutations in the C-terminal region of AQP2. The pathogenic variants are significantly more likely to be located inside the domain compared with population variants. Through the structural analysis and in silico prediction, the eight mutations identified in this study were presumed to be disease-causing. The most common treatments were thiazide diuretics and non-steroidal anti-inflammatory drugs (NSAIDs). Emergency treatment for hypernatremia dehydration in neonates should not use isotonic saline as a rehydration fluid. Genetic analysis presumably confirmed the diagnosis of NDI in each patient in our study. We outlined methods for the early identification of NDI through phenotype and genotype, and outlined optimized treatment strategies.

Emerging evidence is showing that apelin plays an important role in regulating salt and water balance by counteracting the antidiuretic action of vasopressin (AVP). However, the underlying mechanism remains unknown. Here, we hypothesized that (pro) renin receptor (PRR)/soluble prorenin receptor (sPRR) might mediate the diuretic action of apelin in the distal nephron. During water deprivation (WD), the urine concentrating capability was impaired by an apelin peptide, apelin-13, accompanied by the suppression of the protein expression of aquaporin 2 (AQP2), NKCC2, PRR/sPRR, renin and nuclear β-catenin levels in the kidney. The upregulated expression of AQP2 or PRR/sPRR both induced by AVP and 8-Br-cAMP was blocked by apelin-13, PKA inhibitor (H89), or β-catenin inhibitor (ICG001). Interestingly, the blockage of apelin-13 on AVP-induced AQP2 protein expression was reversed by exogenous sPRR. Together, the present study has defined the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/sPRR pathway in the CD as the molecular target of the diuretic action of apelin.

Acquired forms of nephrogenic diabetes insipidus (NDI) include lithium (Li)‐induced and hypokalemia‐induced NDI. Both forms are associated with AQP2 downregulation and collecting duct (CD) cellular remodeling. Statins are cholesterol‐lowering drugs appearing to increase AQP2 membrane‐translocation and improve urine concentration in other NDI models. We have investigated if statins are able to prevent or rescue the Li‐induced changes in mice and in a mouse cortical CD cell line (mCCDc1l). Biotinylation assays showed that acute (1hr) atorvastatin, simvastatin, or fluvastatin increased AQP2 membrane accumulation in mCCDc1l cells showing that the cell line responds to acute statin treatment. To see whether chronic statin treatment abolish the Li effects, mCCDc1l cells were treated with 48 h Li, combined Li/atorvastatin or combined Li/simvastatin. Li reduced AQP2, but combined Li/atorvastatin or Li/simvastatin did not prevent AQP2 downregulation. In mice, chronic (21 days) Li increased urine output and reduced urine osmolality, but combined Li/atorvastatin did not prevent these effects. In inner medulla (IM), Li reduced total AQP2 and increased pS261‐AQP2. Combined Li/atorvastatin did not abolish these changes. Atorvastatin did not prevent a Li‐induced increase in intercalated cells and proliferation in IM. In mice with already established NDI, atorvastatin had no effect on the Li‐induced changes either. Mice subjected to 14 days of potassium‐deficient diet developed polyuria and AQP2 downregulation in IM. Co‐treatment with atorvastatin did not prevent this. In conclusion, atorvastatin does not appear to be able to prevent or rescue Li‐NDI or to prevent hypokalemic‐induced NDI., Patients with nephrogenic diabetes insipidus (NDI) suffer from severe polyuria and polydipsia. Statins have been suggested as potential treatment. However, we found that statins have no effect in two acquired forms of NDI.

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the G&alpha, 12/13&ndash, Rho&ndash, ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2.

Aldosterone indirectly regulates water reabsorption in the distal tubule by regulating sodium reabsorption. However, the direct effect of aldosterone on vasopressin-regulated water and urea permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether aldosterone regulates osmotic water permeability in isolated perfused rat IMCDs. Adding aldosterone (500 nM) to the bath significantly decreased osmotic water permeability in the presence of vasopressin (50 pM) in both male and female rat IMCDs. Aldosterone significantly decreased aquaporin-2 (AQP2) phosphorylation at S256 but did not change it at S261. Previous studies show that aldosterone can act both genomically and non-genomically. We tested the mechanism by which aldosterone attenuates osmotic water permeability. Blockade of gene transcription with actinomycin D did not reverse aldosterone-attenuated osmotic water permeability. In addition to AQP2, the urea transporter UT-A1 contributes to vasopressin-regulated urine concentrating ability. We tested aldosterone-regulated urea permeability in vasopressin-treated IMCDs. Blockade of gene transcription did not reverse aldosterone-attenuated urea permeability. In conclusion, aldosterone directly regulates water reabsorption through a non-genomic mechanism. Aldosterone-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. There may be a sex difference apparent in the inhibitory effect of aldosterone on water reabsorption in the inner medullary collecting duct. This study is the first to show a direct effect of aldosterone to inhibit vasopressin-stimulated osmotic water permeability and urea permeability in perfused rat IMCDs.

Hyperuricemia is characterized by abnormally high level of circulating uric acid in the blood and is associated with increased risk of kidney injury. The pathophysiological mechanisms leading to hyperuricemic nephropathy (HN) involve oxidative stress, endothelial dysfunction, inflammation, and fibrosis. Mangiferin is a bioactive C-glucoside xanthone, which has been exerting anti-inflammatory, anti-fibrotic, and antioxidative effects in many diseases. This study aimed to evaluate the effect of mangiferin treatment in HN. In a mouse model of HN, we observed lower circulating urate levels and ameliorated renal dysfunction with mangiferin treatment, which was associated with reduced renal inflammation and fibrosis. We next investigated the mechanism of urate lowering effect of mangiferin. Metabolic cage experiment showed that mangiferin-administrated mice excreted significantly more urinary uric acid due to elevated urine output, but no marked change in urine uric acid concentration. Expressions of water channels and urate transporters were further assessed by western blot. Renal AQP2 expression was decreased, yet urate transporters URAT1, GLUT9, and OAT1 expressions were not affected by mangiferin in HN mice. Moreover, mangiferin treatment also normalized xanthine oxidase and SOD activity in HN mice, which would decrease uric acid synthesis and improve oxidative stress, respectively. Therefore, our results reveal a novel mechanism whereby mangiferin can reduce serum uric acid levels by promoting AQP2-related urinary uric acid excretion. This study suggested that mangiferin could be a multi-target therapeutic candidate to prevent HN via mechanisms that involve increased excretion and decreased production of uric acid and modulation of inflammatory, fibrotic, and oxidative pathways.

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.

Water reabsorption along the collecting duct is dependent on the function of aquaporin 2 (AQP2). Currently, information on microRNA (miRNA)-mediated, post-transcriptional regulation of AQP2, which may influence water reabsorption, is limited. In mice, ablation of the Dicer enzyme (crucial for miRNA maturation) in AQP2-expressing cells induces nephrogenic diabetes insipidus (NDI) with dysregulation of the miRNA profile. A major finding is the identification of miRNAs associated with NDI through mediating epigenetic control of AQP2. This study offers novel targets for AQP2 regulation and potential treatment for governing renal water reabsorption.Background MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression.Methods Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice.Results The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409textendash3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre+ mice, resulting in decreased RNA Pol II association.Conclusions Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.

BackgroundPrimary nephrogenic diabetes insipidus (NDI) is a rare disorder and little is known about treatment practices and long-term outcome.MethodsPaediatric and adult nephrologists contacted through European professional organizations entered data in an online form.ResultsData were collected on 315 patients (22 countries, male 84%, adults 35%). Mutation testing had been performed in 270 (86%); pathogenic variants were identified in 258 (96%). The median (range) age at diagnosis was 0.6 (0.0–60) years and at last follow-up 14.0 (0.1–70) years. In adults, height was normal with a mean (standard deviation) score of −0.39 (±1.0), yet there was increased prevalence of obesity (body mass index >30 kg/m2; 41% versus 16% European average; P ConclusionThis large NDI cohort shows an overall favourable outcome with normal adult height and only mild to moderate CKD in most. Yet, while full-time employment was similar to the European average, educational achievement was lower, and more than half had urological and/or mental health problems.

SUMMARY: Aquaporins (AQPs) are members of the aquaporin water channel family that play an important role in reabsorption of water from the renal tubular fluid to concentrate urine. Using immunohistochemical staining on paraffin sections, We studied expression of AQP2, AQP3 and AQP4 in renal medulla of Bactrian camel (Camelus bactrianus). The renal medulla of cattle (Bos taurus) acted as the control. Compared with the control, strong expression of AQP2 was observed at the apical plasma membrane and intracellular vesicles, in both the outer medullary collecting duct (OMCD) and the inner medullary collecting duct (IMCD) of camel. Strong expression of AQP3 was observed at the basolateral plasma membrane of the IMCD of camel. Strong AQP4 expression, however, was observed at the basolateral plasma membrane in the OMCD of camel. Moreover, moderate AQP4 expression was detected in endothelium of capillary in medullary region of camels, whereas very weak/absent expression was detected in endothelium of capillary of cattle. We concluded that expression of AQP2, AQP3 and AQP4 in the camel kidney showed some differences from cattle in renal trans-epithelial water transport. It may enhance our better understanding of special water metabolism mechanisms that enable camels to survive in extreme environments.

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.

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.

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 m2. 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/m2. 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.

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

Diagnostic value of urinary retinol binding protein (RBP), albumin (ALB) and aquaporin-2 (AQP2) in neonatal hydronephrosis and their relationship with the expression of monocyte chemoattractant protein 1 (MCP-1) in the prenatal maternal peripheral blood was investigated. Forty-six child patients with hydronephrosis admitted to Hongqi Hospital Affiliated to Mudanjiang Medical College from December 2016 to November 2017 were selected as the observation group and the control included 46 normal newborn infants. The urinary RBP, ALB, AQP2 and the expression of MCP-1 in the prenatal maternal peripheral blood in the two groups were compared. The diagnostic value of the combination of urinary RBP, ALB and AQP2 for the neonatal hydronephrosis was accessed through the area under curve (AUC). The changes of urinary RBP, ALB and AQP2 of child patients were observed and the correlations between RBP, ALB, AQP2 and MCP-1 were analyzed. The concentrations of RBP and ALB in the observation group were obviously increased compared to those in the control group. The AQP2 concentration in the observation group was lower than that in the control group. In the observation group, the MCP-1 level in the prenatal maternal blood was significantly higher than that in the control group (P

Background Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder caused by mutations in the CYP21A2. Congenital nephrogenic diabetes insipidus (NDI) is a rare X-linked recessive or autosomal recessive disorder caused by mutations in either AVPR2 or AQP2. Genotype-phenotype discordance caused by genetic mosaicism in CAH patients has not been reported, nor the concomitant CAH and NDI. Case presentation We investigated a patient with concomitant CAH and NDI from a consanguineous family. She (S-1) presented with clitoromegaly at 3 month of age, and polydipsia and polyuria at 13 month of age. Her parents and two elder sisters (S-2 and S-3) were clinically normal, but elevated levels of serum 17-hydroxyprogesterone (17-OHP) were observed in the mother and S-2. The coding region of CYP21A2 and AQP2 were analyzed by PCR-sequencing analysis to identify genetic defects. Two homozygous CYP21A2 mutations (p.R357W and p.P454S) were identified in the proband and her mother and S-2. The apparent genotype-phenotype discordance was due to presence of small amount of wild-type CYP21A2 alleles in S-1, S-2, and their mother’s genome, thus protecting them from development of classic form of 21OHD (C21OHD). A homozygous AQP2 mutation (p.A147T) was also found in the patient. The patient was treated with hydrocortisone and hydrochlorothiazide. Her symptoms were improved with normal laboratory findings. The clitoromegaly is persisted. Conclusions Genetic mosaicism is a novel mechanism contributing to the genotype-phenotype discordance in 21OHD and small percentage of wild-type CYP21A2 alleles may be sufficient to prevent phenotype development. This is a first report of concurrent 21OHD and NDI caused by simultaneous homozygous CYP21A2 and AQP2 mutations.

SUMMARY: Aquaporins (AQPs) are members of the aquaporin water channel family that play an important role in reabsorption of water from the renal tubular fluid to concentrate urine. Using immunohistochemical staining on paraffin sections, We studied expression of AQP2, AQP3 and AQP4 in renal medulla of Bactrian camel (Camelus bactrianus). The renal medulla of cattle (Bos taurus) acted as the control. Compared with the control, strong expression of AQP2 was observed at the apical plasma membrane and intracellular vesicles, in both the outer medullary collecting duct (OMCD) and the inner medullary collecting duct (IMCD) of camel. Strong expression of AQP3 was observed at the basolateral plasma membrane of the IMCD of camel. Strong AQP4 expression, however, was observed at the basolateral plasma membrane in the OMCD of camel. Moreover, moderate AQP4 expression was detected in endothelium of capillary in medullary region of camels, whereas very weak/absent expression was detected in endothelium of capillary of cattle. We concluded that expression of AQP2, AQP3 and AQP4 in the camel kidney showed some differences from cattle in renal trans-epithelial water transport. It may enhance our better understanding of special water metabolism mechanisms that enable camels to survive in extreme environments. RESUMEN: Las acuaporinas (AQP) son miembros de las proteínas de transporte que desempeñan un papel importante en la reabsorción de agua del líquido tubular renal para concentrar la orina. Estudiamos la expresión de AQP2, AQP3 y AQP4 en la médula renal del camello bactriano (Camelus bactrianus) usando tinción inmunohistoquímica en secciones de parafina. La médula renal del bovino (Bos taurus) se usó como control. En comparación con el control, se observó una fuerte expresión de AQP2 en la membrana plasmática apical y vesículas intracelulares tanto en el conducto colector medular externo (CCME) como en el conducto colector medular interno (CCMI) del camello. Se observó una fuerte expresión de AQP3 en la membrana plasmática basolateral del CCMI del camello. También se observó una expresión fuerte de AQP4 en la membrana plasmática basolateral en el CCME de camello. Además, se detectó una expresión moderada de AQP4 en el endotelio de los capilares en la región medular de los camellos, mientras que en el endotelio de los capilares del bovino se detectó una expresión muy débil. Concluimos que la expresión de AQP2, AQP3 y AQP4 en el riñón de camello mostró algunas diferencias con el bovino en el transporte trans-epitelial de agua renal. El estudio podría mejorar nuestra comprensión de los mecanismos especiales del metabolismo del agua que permiten a los camellos sobrevivir en ambientes extremos.

FUNCTIONAL ANALYSIS OF MUTATIONS FOUND IN AQP2 GENE OF PATIENTS WITH DIABETES INSIPIDUS TUĞÇE KARADUMAN Master of Philosophy, Department of Biology Supervisor: Prof. Dr. Hatice MERGEN June 2018, 111 pages The water homeostasis in the body is provided by balancing water loss and intake. Arginine vasopressin (AVP, ADH, antidiuretic hormone) is a hormone synthesized in the hypothalamus region of the brain and stored in the posterior part of the pituitary gland to be secreted when stimulated. In Diabetes Insipidus (DI) patients, production and activity of this hormone is reduced. This hormone regulates a vital function by increasing the reabsorption of water in the kidney collecting channel. Once the AVP hormone is secreted, AVP bounds to the arginine vasopressin receptor 2 (AVPR2) which is a transmembrane protein localized on the basolateral membrane of polarized epithelial cells of kidney nephron. AVPR2 is a G protein bound receptor (GPCR). Thus, G protein becomes active and activates adenylate cyclase. As a result, produced cAMP stimulates protein kinase A. Protein kinase A phosphorylates the aquaporin (AQP2) proteins which are present in the cell to enable localization of these proteins in apical membrane in tetramers. Absorption of water from the urine to the kidney cells is ensured through these channels. When the water reabsorption stimulus is over, AQP2 is removed from the membrane by endocytosis. Any pathology that may occur within this control system causes impairment of water homeostasis of the body and generation of DI. AQP2 is a homotetrameric water channel responsible for the reabsorption of water in the kidney's main collecting duct cells. Mutations in AQP2 gene induce nephrogenic DI (NDI), a pathogenic condition in maintaining body water homeostasis, causing excess volume of urine to be produced. There are several hypotheses that have been proven by clinical and experimental observations in the literature regarding the role of these mutations in the pathogenesis of NDI. It has been proven that AQP2 gene mutations generally lead to misfolding of mutant protein or misleading of this protein; It has also been reported that nonfunctional water channels indicate correct membrane targeting. The purpose of this study is to perform functional analysis of A45T, R85X and A147T mutations identified in the AQP2 gene in NDI patients. It is aimed to contribute to the treatment approaches for the development of pharmacological agents which can be more effective in the treatment of the disease through the evaluation of the results obtained from the experimental studies in correlation with the clinical information of the patients. For the purposes of this study; expression vector carrying the wild-type coding AQP2 sequence was provided and appropriate expression vectors containing mutant AQP2 gene sequences were prepared using the site-directed mutagenesis method. Following this process, stable transfection of wild-type and mutant vectors into MDCK cells was performed. Deglycosylation experiments were conducted to investigate the intracellular maturation process of mutant proteins by expression of the vectors in MDCK cells. Cycloheximide analysis was also performed to determine the half-lives of the proteins and the results of both the deglycosylation experiments and the cyclohexamide experiments were evaluated by immunoblot analysis. Immunocytochemical analyses were performed to determine the intracellular traffic of mutant AQP2 proteins. Within the framework of functional analysis studies performed on the Xenopus laevis oocyte expression system of AQP2 gene mutations, oocyte expression vectors containing wild type and mutation were injected into oocyte cells and total membrane and plasma membranes were isolated in order to determine the expression levels of AQP2 protein in oocytes; wild type protein and mutant proteins membrane ratios were compared through immunoblot analysis. To determine the effects of mutant AQP2 proteins on water uptake mechanism, oocyte cells were tested for water permeability. As a result, within the framework of this thesis, all mutant AQP2 proteins showed alteration of function compared to the wild type AQP2. Keywords: Diabetes insipidus, AQP2, functional analysis İÇİNDEKİLER Sayfa ÖZET i ABSTRACT iv TEŞEKKÜR vii İÇİNDEKİLER ix ÇİZELGELER DİZİNİ xiii ŞEKİLLER DİZİNİ xiiii SİMGELER VE KISALTMALAR xvii 1. GİRİŞ 1 2. GENEL BİLGİ 3 2.1. Vücut Su Dengesi ve Böbrek Su Kanalları 3 2.1.1. Böbrek Su Kanalları 3 2.1.2. Vücuttaki Su Dengesinin Vazopressin ile Regülasyonu 6 2.1.3. Renal AQP2’nin Vazopressin ile Regülasyonu 6 2.1.4. AQP2 Regülasyonunun Hücresel ve Moleküler Mekanizması 8 2.2. Diabetes İnsipidus 11 2.2.1. Nefrojenik Diabetes İnsipidus 13 2.2.2. Konjenital NDI 14 2.2.2.1. X-Bağlantılı NDI ve AVPR2 mutasyonları 16 2.2.2.2. AQP2 Mutasyonları 18 2.2.2.3. Otozomal Çekinik NDI 20 2.2.2.4. Otozomal Baskın NDI 23 2.3. Nefrojenik Diabetes İnsipidus’da Genetik Test Değerlendirmesi 25 2.4. Nefrojenik Diabetes İnsipidus’da AQP2-Temelli Terapötik Yaklaşımlar 25 2.4.1. Fosfodiesteraz İnhibitörleri 25 2.4.1.1. Siklik GMP (cGMP) Yolak Aktivasyonu 25 2.4.1.2. Siklik AMP (cAMP) Yolak Aktivasyonu 26 2.4.2. Statinler 26 2.4.3. Prostaglandinler 27 2.4.4. Isı şoku proteini (Hsp90) 27 3. MATERYAL ve YÖNTEM 28 3.1. Fonksiyon Analizi Gerçekleştirilecek Olan Mutasyonların Belirlenmesi 29 3.2. Hücre Kültürü Çalışmalarında Kullanılan Mutant AQP2 Genini İçeren İfade Vektörlerinin Site-Directed Mutagenez Yöntemi ile Hazırlanması 31 3.2.1. AQP2 Geni Taşıyan İfade Vektörünün Temin Edilmesi 31 3.2.2. Yabanıl AQP2 cDNA Dizisinde Site-Directed Mutagenez Yöntemi ile Çalışılacak Olan Mutasyonların Oluşturulması, Oluşturulan Dizilerin İfade Vektörlerine Aktarılması ve DNA Dizi Analizi Yöntemi ile Doğrulanması 33 3.3. MDCK Hücre Hattı ile Gerçekleştirilen Hücre Kültürü Çalışmaları 43 3.3.1. MDCK Hücrelerinin Üretilmesi 44 3.3.2. Dondurulmuş Hücrelerin Açılması 44 3.3.3. Hücrelerin Pasajlanması 45 3.3.4. Hücrelerin Dondurulması 45 3.3.5. Yabanıl Tip ve Mutant AQP2 Dizilerini İçeren İfade Vektörlerinin MDCK Hücrelerine Stabil Transfeksiyon Protokolü 46 3.4. Forskolin Uygulaması 49 3.5. Deglikozilasyon Deneyleri 50 3.6. Siklohekzimid Analizi 52 3.7. İmmünblot Analizi 53 3.8. Yabanıl ve Mutant Tip Hücrelerde İmmünsitokimyasal Analiz 54 3.9. Xenopus laevis Oosit İfade Sistemi Çalışmaları 56 3.9.1. Xenopus laevis Oosit İfade Sistemi Çalışmalarında Kullanılacak Olan Yabanıl Tip ve Mutant pT7TS-AQP2 Vektörlerinin Hazırlanması 59 3.9.2. Xenopus laevis Oositlerine Enjeksiyonu Gerçekleştirilecek Yabanıl Tip ve Mutant cRNA’ların in vitro Eldesi için Yapılan Çalışmalar 60 3.9.2.1. Yabanıl ve Mutant Tip Oosit İfade Vektörlerinin Doğrusal Hale Getirilmesi (Linearizasyonu) 60 3.9.2.2. Yabanıl ve Mutant Tip Lineer Oosit İfade Vektörlerinin Pürifikasyonu 61 3.9.2.3. Yabanıl ve Mutant Tip cRNA’ların Eldesi/Ekstraksiyonu 62 3.9.3. Xenopus laevis Oositlerinin ve İlgili Besiyerlerinin Temini 64 3.9.4. Xenopus laevis Oositlerine Yabanıl Tip ve Mutant pT7TS-AQP2 Vektörlerinden in vitro Olarak Elde Edilen cRNA’ların Enjeksiyonun Yapılması 64 3.9.5. İmmünblot Analizleri/Xenopus laevis Oositlerinden Total ve Plazma Membranlarının İzole Edilerek, Yabanıl ve Mutant Protein Miktarlarının Karşılaştırılması 66 3.9.6. Su Geçirgenlik Testinin Uygulanması 70 4. SONUÇLAR VE TARTIŞMA 72 4.1. Hücre Kültürü Çalışmalarında Kullanılacak Mutant AQP2 İçeren İfade Vektörlerinin Site-Directed Mutagenez Yöntemi ile Hazırlanması 72 4.1.1. AQP2 Geni Taşıyan İfade Vektörü İzolasyonu 72 4.1.2. Yabanıl AQP2 cDNA Dizisinde Site-Directed Mutagenez ile Çalışılacak Olan Mutasyonların Oluşturulması, Oluşturulan Dizilerin İfade Vektörlerine Aktarılması ve Doğrulanması 73 4.2. MDCK Hücre Hattı ile Gerçekleştirilen Hücre Kültürü Çalışmalarına İlişkin Sonuçlar 76 4.2.1. Stabil Transfeksiyon Protokolü Uygulanarak Elde Edilen Hücre Hatlarına İlişkin Görüntüler 76 4.2.2. Yabanıl ve mutant AQP2 protein ifadelerinin, stabil MDCK hücre hattı ve Xenopus laevis ifade sisteminde immünblot analiz sonuçlarının değerlendirilmesinde ön bilgi 78 4.2.3. Forskolin Uygulaması Sonuçları 78 4.2.3.1. Forskolin Uygulama Sonuçlarının Değerlendirilmesi 79 4.2.4. Deglikozilasyon Deney Sonuçları 80 4.2.4.1. Deglikozilasyon Deney Sonuçlarının Değerlendirilmesi 80 4.2.5. Siklohekzimid Analizi Sonuçları 81 4.2.5.1. Siklohekzimid Analizi Sonuçlarının Değerlendirilmesi 83 4.2.6. Yabanıl ve Mutant Tip Hücrelerde İmmünsitokimyasal Analiz Sonuçları 83 4.2.6.1. Yabanıl ve Mutant Tip Hücrelerde İmmünsitokimyasal Analiz Sonuçlarının Değerlendirilmesi 84 4.3. Xenopus laevis Oosit İfade Sistemi Çalışmalarında Kullanılacak Olan Yabanıl Tip ve Mutant pT7TS-AQP2 Vektörlerinin Hazırlanmasına İlişkin Sonuçlar 85 4.4. Xenopus laevis Oositlerine İnjeksiyonu Gerçekleştirilecek Yabanıl Tip ve Mutant cRNA’ların in vitro Eldesi için Yapılan Çalışma Sonuçları 88 4.4.1. Yabanıl ve Mutant Tip Oosit İfade Vektörlerinin Linearizasyon Sonuçları 88 4.4.2. Yabanıl ve Mutant Tip cRNA’ların Eldesi/Ekstraksiyonu 88 4.4.3. İmmunblot Analiz Sonuçları/Xenopus laevis Oositlerinden Total ve Plazma Membranlarının İzole Edilerek, Yabanıl ve Mutant Protein Miktarlarının Karşılaştırılmasına İlişkin Veriler 89 4.4.3.1. Xenopus laevis Oosit İfade Sistemi İmmunblot Analiz Sonuçlarının Değerlendirilmesi 91 4.4.4. Su Geçirgenlik Testine İlişkin Sonuçlar 91 4.4.4.1. Su Geçirgenlik Testine İlişkin Sonuçların Değerlendirilmesi 94 KAYNAKLAR 100 ÖZGEÇMİŞ 112 ÖZET DİABETES İNSİPİDUS’LU HASTALARIN AQP2 GENİNDE TANIMLANAN MUTASYONLARIN FONKSİYONEL ANALİZLERİ Tuğçe KARADUMAN Doktora, Biyoloji Anabilim Dalı Tez Danışmanı: Prof. Dr. Hatice MERGEN Haziran 2018, 111 sayfa Vücutta su homeostazı, su alımı ve kaybının dengede olması ile sağlanır. Diabetes insipidus (DI) hastalarında yapımı ve etkisi azalmış olan arjinin vazopressin, (AVP, ADH, antidiüretik hormon) beynin hipotalamus bölgesinde sentezlenen ve hipofiz bezinin arka bölümünde depolanarak uyarılma durumunda salgılanan bir hormondur. Bu hormon, böbrek toplama kanalındaki suyun geri emilimini artırarak, hayati fonksiyona sahip olan bir düzenleme gerçekleştirmektedir. AVP hormonu salgılandıktan sonra, böbrek nefronlarının polarize epitel hücrelerinin bazolateral membranında lokalize bir transmembran protein olan arjinin vazopressin reseptör 2’ye (AVPR2) bağlanır. AVPR2, bir G protein bağlı reseptördür (GPCR). Bu şekilde G proteini aktive olur ve adenilat siklazı aktive eder. Sonuçta, oluşan cAMP, protein kinaz A’yı stimüle eder. Protein kinaz A, hücre içerisinde bulunan aquaporin su kanalı (AQP2) proteinlerini fosforilleyerek onların tetramerler halinde apikal membrana yerleşimlerini sağlar. Bu kanallar vasıtasıyla suyun idrardan böbrek hücrelerine geçişi sağlanır. Su geri emilimi uyarısı sona erdiğinde, AQP2 endositoz ile membrandan uzaklaştırılır. Bu kontrol sistemi içerisinde oluşabilecek herhangi bir patoloji vücudun su homeostazının bozulmasına ve DI gelişimine neden olur. AQP2, böbrek ana toplayıcı kanal hücrelerinde, suyun geri emiliminden sorumlu olan homotetramerik bir su kanalıdır. AQP2 genindeki mutasyonlar, fazla hacimde idrar üretilmesine neden olarak, vücut su homeostazının sağlanma sürecinde patojenik bir durum olan nefrojenik DI’yi (NDI) indüklemektedir. Bu mutasyonların NDI patogenezindeki rolü için literatürde yer alan klinik ve deneysel gözlemlerle kanıtlanmış çeşitli hipotezler vardır. AQP2 gen mutasyonlarının genelde mutant proteinin yanlış katlanmasına ya da bu proteinin yanlış hedeflenmesine yol açtığı tespit edilmiş olmakla birlikte; işlevsel olmayan su kanallarının, doğru membran hedeflemesi gösterdiği de bildirilmiştir. Bu çalışmanın amacı, NDI tanısı konmuş hastaların AQP2 geninde grubumuz tarafından tanımlanmış olan A45T, R85X ve A147T mutasyonlarının fonksiyon analizlerini gerçekleştirmektir. Bu amaç kapsamında yapılan deneysel çalışmalarda; yabanıl tip kodlayıcı AQP2 dizisi taşıyan ifade vektörü temin edilmiş, site-directed mutagenez yöntemi kullanılarak mutant AQP2 gen dizilerini içeren uygun ifade vektörleri hazırlanmıştır. Bu süreci takiben, yabanıl tip ve mutant vektörlerin MDCK hücrelerine stabil transfeksiyonu gerçekleştirilmiştir. Vektörlerin MDCK hücrelerinde ifadesi sağlanarak, mutant proteinlerin hücre içi olgunlaşma sürecinin karakterizasyonun araştırılması için deglikozilasyon deneyleri gerçekleştirilmiştir. Ayrıca proteinlerin yarı-ömürlerinin belirlenmesi amacıyla, siklohekzimid analizi yapılmış ve hem deglikozilasyon deneyleri hem de siklohekzimid deneylerine ait sonuçlar immünblot analizi ile değerlendirilmiştir. Mutant AQP2 proteinlerinin hücre içi trafiğinin belirlenmesi için immünsitokimyasal analizler gerçekleştirilmiştir. AQP2 gen mutasyonlarının Xenopus laevis oosit ifade sisteminde gerçekleştirilecek fonksiyon analiz çalışmaları kapsamında ise, yabanıl tip ve mutasyonu bulunduran oosit ifade vektörlerinden elde edilen cRNA’ların oosit hücrelerine injeksiyonu gerçekleştirilerek, AQP2 proteininin oositlerde ifade düzeylerini tespit edebilmek için total membran ve plazma membranları izole edilmiş, yabanıl tip protein ile mutant proteinlerin membranlardaki oranları immünblot analizi ile karşılaştırılmıştır. Mutant AQP2 proteinlerinin su alım mekanizması üzerine etkilerinin belirlenmesi için oosit hücrelerine su geçirgenliği testi uygulanmıştır. Sonuç olarak tez kapsamında, çalışılan tüm mutant proteinlerin MDCK hücreleri ve Xenopus laevis oosit ifade sisteminde yabanıl tipe göre farklı oranlarda fonksiyon değişikliği gösterdiği belirlenmiştir. Anahtar kelimeler: Diabetes insipidus, AQP2, Fonksiyonel analiz

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. 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.

During the evolutionary transition from land to water, cetaceans have undergone numerous critical challenges, with osmoregulation being the major one. Two subspecies of the narrow-ridged finless porpoise (Neophocaena asiaeorientalis), the freshwater Yangtze finless porpoise (N. a. asiaeorientalis, NAA) and the marine East Asian finless porpoise (N. a. sunameri, NAS), provide excellent subjects to understand the genetic basis of osmoregulatory divergence between freshwater and marine mammals. The kidney plays an important and well-established role in osmoregulation in marine mammals and thus, herein, we utilized RNA-seq to characterize the renal transcriptome and preliminarily analyze the divergence between the NAA and the NAS. Approximately 48.98 million clean reads from NAS and 49.40 million clean reads from NAA were obtained by RNA-Seq. And 73,449 (NAS) and 68,073 (NAA) unigenes were assembled. Among these annotations, 22,231 (NAS) and 21,849 (NAA) unigenes were annotated against the NCBI nr protein database. The ion channel complex GO term and four pathways were detected as relevant to osmoregulation by GO and KEGG pathway classification of these annotated unigenes. Although the endangered status of the study species prevented analysis of biological replicates, we identified nine differentially expressed genes (DEGs) that may be vital in the osmoregulation of the narrow-ridged finless porpoise and worthwhile for future studies. Of these DEGs, the differential expression and distribution of the aquaporin-2 (AQP2) in the collecting duct were verified using immunohistochemical experiments. Together, this work is the first report of renal transcriptome sequencing in cetaceans, and it will provide a valuable resource for future molecular genetics studies on cetacean osmoregulation.

Bardet-Biedl Syndrome (BBS) is a rare autosomal recessive disorder with renal and extra-renal involvement. The wide spectrum of clinical manifestations is associated to the high genetic heterogeneity. To date 21 genes have been identified in humans and the majority of them encode proteins located on the basal body of the primary cilium. For this reason the disease is has been included among the 'ciliopathies'. The renal involvement is extremely heterogeneous in BBS and is considered the main cause of morbidity and mortality. Recent evidences have suggested that mutations in BBS6, 10 and 12 are associated with a more severe renal dysfunction. The most common renal dysfunction is the urine concentrating defect, even though the underlying mechanism is not completely known. Recently we have demonstrated that hyposthenuria in BBS patients has a renal origin, and depends on desmopessin resistance. The majority of hyposthenuric BBS patients have a combined defect to both concentrate and dilute the urine. The combined defect is associated with a blunted increased urine Aquaproine-2 (u-AQP2) excretion in antidiuresis. A ccordingly, in vitro BBS10 silencing prevented AQP2 trafficking to the apical plasma membrane. However, after long term water restriction hyposthenuric BBS patients showed the same u-AQP2 excretion compared with controls, suggesting that other mechanisms are implicated into the pathogenesis of hyposhtenuria. The complete molecular mechanism underlying hyposhtenuria remains largely unknown in BBS. Whether this defect may represent a predictor factor for poor renal outcome remains to be elucidated.

Aquaporin 2 (AQP2) is a small protein located in the collecting tubules of kidneys; it plays an important role in the concentration and production of urine. The aim of this study was to determine the expression level of the AQP2 gene in the kidney of broiler chickens after the administration of renal­dose dopamine. Broiler chickens (25 days-old) were randomly divided into two groups (n=20 per group): intravenous administration of saline solution (control group) or renal-dose dopamine (dopamine group). The expression and localization of the AQP2 gene were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC), respectively. The protein level of AQP2 was analyzed by western blot analysis. The dopamine group presented no significant difference (p>0.05) in the biochemical criterion or mRNA expression of AQP2 compared with the control group. However, AQP2 protein level was significantly reduced (p

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 (CH2O), fractional excretion of sodium (FENa), 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, CH2O (61% vs 43%) and FENa (46% vs 41%) after placebo than after tolvaptan; GFR and u-AQP2 decreased to the same extent; p-AVP increased three fold, whereas u-ENaCγ, 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. 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 (CH2O), fractional excretion of sodium (FENa), 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, CH2O (61% vs 43%) and FENa (46% vs 41%) after placebo than after tolvaptan; GFR and u-AQP2 decreased to the same extent; p-AVP increased three fold, whereas u-ENaCγ, 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.

Prostaglandin E2 (PGE2), a metabolite of arachidonic acid, plays a role in water and sodium reabsorption in the collecting duct of the kidney. The collecting duct is responsible for the fine tuning of water and electrolytes. Only a small fraction of the filtered water and sodium is reabsorbed in the collecting duct, a fraction crucial to the regulation of water and electrolyte balance. This current study addresses the role of EP1, one of four PGE2 receptors, in the collecting duct. It is well documented that PGE2 inhibits sodium and water reabsorption in the collecting duct, however the exact mechanism is still debated. To determine whether the EP1 receptor mitigates AngII renal effects, an in vivo study was performed with EP1-/- mice. Global EP1-/- knockout mice were crossed with a renin overexpressing mouse line (herein denoted as “Ren”) and subjected to a high salt (HS) and low salt (LS) diet. Ren mice displayed an 11mmHg increase in systolic blood pressure (BP) on a HS diet and a decrease in BP of 14mmHg on a LS diet compared to the normal salt (NS) diet. Ren EP1-/- mice did not display a significant increase or decrease in BP on a HS or LS diet. On a LS diet, Ren EP1-/- displayed a drop in urine osmolarity (1641 mOsm/ kgH2O) vs. wild type (WT) mice (2107 mOsm/ kgH2O), consistent with increased sodium reabsorption. Narrowing in on the collecting duct, Ren EP1-/- mice had enhanced αENaC levels compared to Ren mice. In ex vivo microperfusion experiments, EP1-/- tubules show no response to PGE2 in the presence of AVP, whereas PGE2 inhibits AVP induced water reabsorption in WT mice. An increase in αENaC membrane accumulation due to EP1 gene ablation results in increased sodium reabsorption subsequently leading to a rise in BP. This contributes to the lack of salt sensitivity in EP1-/- mice. Overall, the EP1 receptor in the collecting duct represents a potential therapeutic target for the treatment of hypertension.

BACKGROUND:Tolvaptan is a selective vasopressin receptor antagonist. Nitric Oxide (NO) promotes renal water and sodium excretion, but the effect is unknown in the nephron's principal cells. In a dose-response study, we measured the effect of tolvaptan on renal handling of water and sodium and systemic hemodynamics, during baseline and NO-inhibition with L-NMMA (L-NG-monomethyl-arginine).METHODS:In a randomized, placebo-controlled, double blind, cross over study, 15 healthy subjects received tolvaptan 15, 30 and 45 mg or placebo. L-NMMA was given as a bolus followed by continuous infusion during 60 min. We measured urine output (UO), free water clearance (CH2O), fractional excretion of sodium (FENa), urinary aquaporin-2 channels (u-AQP2) and epithelial sodium channels (u-ENaCγ), plasma vasopressin (p-AVP) and central blood pressure (cBP).RESULTS:During baseline, FENa was unchanged. Tolvaptan decreased u-ENaCγ dose-dependently and increased p-AVP threefold, whereas u-AQP2 was unchanged. During tolvaptan with NO-inhibition, UO and CH2O decreased dose-dependently. FENa decreased dose-independently and u-ENaCγ remained unchanged. Central BP increased equally after all treatments.CONCLUSIONS:During baseline, fractional excretion of sodium was unchanged. During tolvaptan with NO-inhibition, renal water excretion was reduced dose dependently, and renal sodium excretion was reduced unrelated to the dose, partly via an AVP dependent mechanism. Thus, tolvaptan antagonized the reduction in renal water and sodium excretion during NO-inhibition. Most likely, the lack of decrease in AQP2 excretion by tolvaptan could be attributed to a counteracting effect of the high level of p-AVP. BACKGROUND: Tolvaptan is a selective vasopressin receptor antagonist. Nitric Oxide (NO) promotes renal water and sodium excretion, but the effect is unknown in the nephron's principal cells. In a dose-response study, we measured the effect of tolvaptan on renal handling of water and sodium and systemic hemodynamics, during baseline and NO-inhibition with L-NMMA (L-NG-monomethyl-arginine).METHODS: In a randomized, placebo-controlled, double blind, cross over study, 15 healthy subjects received tolvaptan 15, 30 and 45 mg or placebo. L-NMMA was given as a bolus followed by continuous infusion during 60 min. We measured urine output (UO), free water clearance (CH2O), fractional excretion of sodium (FENa), urinary aquaporin-2 channels (u-AQP2) and epithelial sodium channels (u-ENaCγ), plasma vasopressin (p-AVP) and central blood pressure (cBP).RESULTS: During baseline, FENa was unchanged. Tolvaptan decreased u-ENaCγ dose-dependently and increased p-AVP threefold, whereas u-AQP2 was unchanged. During tolvaptan with NO-inhibition, UO and CH2O decreased dose-dependently. FENa decreased dose-independently and u-ENaCγ remained unchanged. Central BP increased equally after all treatments.CONCLUSIONS: During baseline, fractional excretion of sodium was unchanged. During tolvaptan with NO-inhibition, renal water excretion was reduced dose dependently, and renal sodium excretion was reduced unrelated to the dose, partly via an AVP dependent mechanism. Thus, tolvaptan antagonized the reduction in renal water and sodium excretion during NO-inhibition. Most likely, the lack of decrease in AQP2 excretion by tolvaptan could be attributed to a counteracting effect of the high level of p-AVP.TRIAL REGISTRATION: Clinical Trial no: NCT02078973 . Registered 1 March 2014.

Protein kinase anchoring by scaffolding proteins is a cornerstone in the regulation of molecular pathways. One of the best studied examples is anchoring of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs). The present study investigates the involvement of PKA-containing signalosomes in two distinct contexts. The first part of this thesis relates to the role of AKAPs in the regulation of canonical Wnt signaling. Canonical Wnt signaling plays a role in a plethora of molecular mechanisms, including cell proliferation and fate determination, with important clinical implications in several pathologies, including cancer. Wnt signaling inhibition in basal state cells relies on the proteolysis of β-catenin. This depends on Glycogen Synthase Kinase 3β (GSK3β) phosphorylation of β-catenin, and is antagonized by PKA. The present study addresses the role of the cytosolic AKAP Glycogen Synthase Kinase Interaction Protein (GSKIP), and reveals how this scaffold scavenges both PKA and GSK3β away from β-catenin. The result is that GSKIP promotes and inhibits Wnt signaling, highlighting the importance of the GSKIP- mediated PKA-GSK3β crosstalk in the control of Wnt signaling. The second part of the thesis describes novel findings about the translocation of the water channel Aquaporin-2 (AQP2) into the plasma membrane of renal principal cells. AQP2 insertion increases water permeability of the collecting duct, fine- tuning water homeostasis. This process is not yet entirely understood. Deregulations of AQP2 translocation result in either excessive water excretion or retention, and are clinically relevant in both cases. Previous research established an automated immunofluorescence-based screening and identified the poorly characterized Cyclin Dependent Kinase 18 (CDK18) involvement in AQP2 regulation. In the second part of the thesis it is described how CDK18 is able to control AQP2 localization, abundance and phosphorylation. It was also discovered that the E3 ubiquitin ligase STIP1 Homology And U-Box Containing Protein 1 (STUB1) is interacting with CDK18 and mediates the CDK18-driven AQP2 ubiquitination. STUB1 is additionally responsible for the binding of PKA in proximity with CDK18 and AQP2, behaving in an AKAP-like way. The identification of a novel kinase involved in the control of AQP2 translocation could pave the way to a causal pharmacological window for AQP2-related pathologies., Die räumliche und zeitliche Kontrolle von Proteinkinasen durch Gerüstproteine ist ein Meilenstein der Regulation molekularer Signalwege. Eines der bisher am besten untersuchten Beispiele ist die intrazelluläre Lokalisation von Protein Kinase A (PKA) durch A-Kinase Ankerproteine (AKAP). Diese Arbeit untersucht die Rolle von PKA- basierten Signalkomplexen in zwei unterschiedlichen Modellen. Der erste Teil der Dissertation befasst sich mit der Rolle von AKAPs in der Regulation des kanonischen Wnt-Signalweges, der eine Vielzahl von molekularen Mechanismen reguliert. Unter anderem ist er für Zellproliferation und –differenzierung entscheidend und spielt daher eine wichtige Rolle bei der Entstehung von Tumorerkrankungen. Im Grundzustand der Zelle wird das Wnt- Signal durch Proteolyse des Effektorproteins β-catenin inhibiert. Der Abbau wird durch Glycogen Synthase Kinase 3β (GSK3β)-Phosphorylierung von β-catenin induziert; umgekehrt führt eine Phosphorylierung durch PKA zur Stabilisierung des Proteins. In dieser Arbeit wurde das AKAP Glycogen Synthase Kinase Interaction Protein (GSKIP) untersucht, das sowohl PKA als auch GSK3β binden kann, und gezeigt, wie es den Zugang beider Kinasen zu β-catenin regulieren kann. Somit kann GSKIP den Wnt- Signalweg sowohl fördern als auch inhibieren, was die Bedeutung von GSKIP für das PKA-GSK3β-Zusammenspiel im Wnt-Signalwegs hervorhebt. Im zweiten Teil der Dissertation werden neue Mechanismen beschrieben, die eine Umverteilung des Wasserkanals Aquaporin-2 (AQP2) in die Plasmamembran von renalen Hauptzellen regulieren. Der Einbau von AQP2 in die Plasmamembran erhöht die Wasserpermeabilität des renalen Sammelrohrs und trägt zur Feinregulation des Wasserhaushalts bei. Dieser Prozess unterliegt einem komplexen Zusammenspiel von Enzymen und Gerüstproteinen, das im Ganzen noch nicht verstanden ist. Fehlregulationen der AQP2-Umverteilung führen zu einem exzessiven Wasserverlust über den Primärharn bzw. zu einer abnormalen Wasserretention. In einer vorangegangenen Studie wurde ein automatisiertes Immunfluoreszenzmikroskopie-basiertes Verfahren entwickelt, das die bisher wenig charakterisierte Kinase CDK18 (Cyclin Dependent Kinase 18) als entscheidend für die AQP2 Umverteilung identifizierte. In dieser Arbeit wird beschrieben, wie CDK18 die Lokalisierung, Proteinmenge und den Phosphorylierungsstatus von AQP2 kontrolliert. Die E3 Ubiquitin-Ligase STUB1 (STIP1 Homology And U-Box Containing Protein 1) interagiert mit CDK18 und vermittelt über CDK18 die Ubiquitinierung von AQP2. STUB1 hält PKA in räumlicher Nähe zu CDK18 und AQP2 und weist damit eine AKAP-ähnliche Funktion auf. Die Identifizierung einer neuen, für die AQP2- 7 Umverteilung relevanten Kinase könnte neuartige pharmakologische Ansätze für die kausale Behandlung von AQP2-basierten Krankheiten liefern.

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.

Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the tetramer which contains just one or no phosphorylated monomers. This difference in diffusion rate may reflect behavior of AQP2 tetramers destined for either plasma membrane retention or endocytosis.

Renal ischemia/reperfusion (I/R) can lead to impaired urine concentration ability and increased fractional excretion of sodium (FeNa). Local ischemic pre conditioning improves renal water and sodium handling after I/R injury. Here, we investigate whether remote ischemic per conditioning (rIPeC) prevents dysregulation of renal water and salt handling in response to I/R injury and mechanisms that may be involved. Rats were subjected to right nephrectomy and randomized into a sham group or an I/R group. In the I/R group, rats were subjected to 37 min of renal ischemia and 3 days of reperfusion. rIPeC was applied to the abdominal aorta. Blood and urine were collected on day 3 postoperatively for clearance studies. The expression of aquaporins (AQPs) and the sodium transporter Na–K‐ATPase were analyzed using immunoblotting and immunohistochemistry. I/R injury resulted in polyuria, increased FeNa, and decreased urine osmolality compared to sham rats. rIPeC attenuated the increase in FeNa and the decrease in urine osmolality. Expression of AQP1, AQP2, phosphorylated AQP2 (pAQP2), and Na–K‐ATPase was downregulated in I/R rats. rIPeC attenuated the reductions in AQP2 and pAQP2 expression. Immunohistochemistry revealed decreased labeling of Na–K‐ATPase in the outer medulla in I/R kidneys compared to kidneys from sham and I/R + rIPeC rats. After renal ischemia, the expression of Na–K‐ATPase was substantially reduced in the outer medullary thick ascending limb. In conclusion, our data suggest that rIPeC might prevent dysregulation of renal water and salt handling via regulation of AQP2 expression and phosphorylation as well as via regulation of Na–K‐ATPase expression in I/R rat kidneys.

We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2/M phases of the cell cycle. This acceleration is due, at least in part; to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to augment cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na+/H+ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Two cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs) and AQP2-RCCD1 (transfected with AQP2). We here demonstrate, for the first time, that AQP2-expressing cells present both higher intracellular pH and higher NHE2 protein expression than WT-RCCD1 cells. In AQP2-expresing cells NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2/M phases. We also observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. Fil: Rivarola, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Di Giusto, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Christensen, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Ford, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Capurro, Claudia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

The renal phenotype in Bardet-Biedl syndrome (BBS) is highly variable. The present study describes renal findings in 41 BBS patients and analyzes the pathogenesis of hyposthenuria, the most common renal dysfunction. Five of 41 patients (12%) showed an estimated glomerular filtration rate 60 ml·min-1·1.73 m-2. Urine protein and urine albumin-to-creatinine ratio were over 200 and 30 mg/g in 9/24 and 7/23 patients, respectively. Four of 41 patients showed no renal anomalies on ultrasound. Twenty of 34 patients had hyposthenuria in the absence of renal insufficiency. In all 8 of the hyposthenuric patients studied, dDAVP failed to elevate urine osmolality (Uosm), suggesting a nephrogenic origin. Interestingly, water loading (WL) did not result in a significant reduction of Uosm, indicating combined concentrating and diluting defects. dDAVP infusion induced a significant increase of plasma Factor VIII and von Willebrand Factor levels, supporting normal function of the type 2 vasopressin receptor at least in endothelial cells. While urinary aquaporin 2 (u-AQP2) abundance was not different between patients and controls at baseline, the dDAVP-induced increased u-AQP2 and the WL-induced reduction of u-AQP2 were blunted in patients with a combined concentrating and diluting defect, suggesting a potential role of AQP2 in the defective regulation of water absorption. Urine Uromodulin excretion was reduced in all hyposthenuric patients, suggesting a thick ascending limb defect. Interestingly, renal Na, Cl, Ca, but not K handling was impaired after acute WL but not at basal. In summary, BBS patients show combined urinary concentration and dilution defects; a thick ascending limb and collecting duct tubulopathy may underlie impaired water handling. The renal phenotype in BardetBiedl syndrome (BBS) is highly variable. The present study describes renal findings in 41 BBS patients and analyzes the pathogenesis of hyposthenuria, the most common renal dysfunction. Five of 41 patients (12%) showed an estimated glomerular filtration rate < 60 ml.min(-1).1.73 m(-2). Urine protein and urine albumin-to-creatinine ratio were over 200 and 30 mg/g in 9/24 and 7/23 patients, respectively. Four of 41 patients showed no renal anomalies on ultrasound. Twenty of 34 patients had hyposthenuria in the absence of renal insufficiency. In all 8 of the hyposthenuric patients studied, dDAVP failed to elevate urine osmolality (Uosm), suggesting a nephrogenic origin. Interestingly, water loading (WL) did not result in a significant reduction of Uosm, indicating combined concentrating and diluting defects. dDAVP infusion induced a significant increase of plasma Factor VIII and von Willebrand Factor levels, supporting normal function of the type 2 vasopressin receptor at least in endothelial cells. While urinary aquaporin 2 (u-AQP2) abundance was not different between patients and controls at baseline, the dDAVP-induced increased u-AQP2 and the WL-induced reduction of u-AQP2 were blunted in patients with a combined concentrating and diluting defect, suggesting a potential role of AQP2 in the defective regulation of water absorption. Urine Uromodulin excretion was reduced in all hyposthenuric patients, suggesting a thick ascending limb defect. Interestingly, renal Na, Cl, Ca, but not K handling was impaired after acute WL but not at basal. In summary, BBS patients show combined urinary concentration and dilution defects; a thick ascending limb and collecting duct tubulopathy may underlie impaired water handling.

Arginine-vasopressin (AVP) stimulates the redistribution of water channels, aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. By this AVP directs 10 % of the water reabsorption from the 170 L of primary urine that the human kidneys produce each day. This review discusses molecular mechanisms underlying the AVP-induced redistribution of AQP2; in particular, it provides an overview over the proteins participating in the control of its localization. Defects preventing the insertion of AQP2 into the plasma membrane cause diabetes insipidus. The disease can be acquired or inherited, and is characterized by polyuria and polydipsia. Vice versa, up-regulation of the system causing a predominant localization of AQP2 in the plasma membrane leads to excessive water retention and hyponatremia as in the syndrome of inappropriate antidiuretic hormone secretion (SIADH), late stage heart failure or liver cirrhosis. This article briefly summarizes the currently available pharmacotherapies for the treatment of such water balance disorders, and discusses the value of newly identified mechanisms controlling AQP2 for developing novel pharmacological strategies. Innovative concepts for the therapy of water balance disorders are required as there is a medical need due to the lack of causal treatments.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β 1 - and β 2 -adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β 3 -adrenergic receptor (β 3 -AR) in mouse kidney. The β 3 -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 β 3 -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 β 3 -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 β 3 -AR stimulation in the kidney. Hence, β 3 -AR agonism might be useful to bypass AVPR2-inactivating mutations.

We have previously demonstrated that in renal cortical collecting duct cells (RCCD1) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT‐RCCD1 (not expressing aquaporins) and AQP2‐RCCD1 (transfected with AQP2). Our results showed that when most RCCD1 cells are in the G1‐phase (unsynchronized), the blockage of barium‐sensitive K+ channels implicated in rapid RVD inhibits cell proliferation only in AQP2‐RCCD1 cells. Though cells in the S‐phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down‐regulation in the rapid RVD response only in AQP2‐RCCD1 cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2—besides increasing water permeability—would play some other role. These observations together with evidence implying a cell‐sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G1, volume tends to increase but it may be efficiently regulated by an AQP2‐dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down‐regulated when volume needs to be increased in order to proceed into the S‐phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2. Fil: Di Giusto, Gisela. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Flamenco, María del Pilar. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina Fil: Rivarola, Valeria. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina Fil: Fernadez, Juan. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina Fil: Melamud, Luciana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina Fil: Ford, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina Fil: Capurro, Claudia Graciela. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Summary 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-PK1 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.

Previously we demonstrated that neonatally induced partial unilateral ureteral obstruction (PUUO) in rats is associated with changes in the abundance of renal acid-base transporters that were paralleled by reduction in renal functions dependent on the severity and duration of obstruction. The aim of the present study was to identify whether changes in renal aquaporin abundance are age-dependent. Semiquantitative immunoblotting and immunohistochemistry were used to examine the changes in abundance of AQP1, AQP2, p-S256AQP2 (AQP2 phosphorylated at consensus site Ser(256)) and AQP3 in the kidneys of rats with neonatally induced PUUO within the first 48 h of life, and then monitored for 7 or 14 weeks. Protein abundance of AQP2 and AQP3 increased in both obstructed and non-obstructed kidneys 7 weeks after induction of neonatal PUUO (PUUO-7W). In contrast, AQP1 and AQP2 protein abundance in the obstructed kidney were reduced after 14 weeks of PUUO (PUUO-14W). Importantly, pS256-AQP2 protein abundance was reduced in obstructed kidneys of both PUUO-7W and PUUO-14W. Immunohistochemistry confirmed the persistent pS256-AQP2 downregulation in both PUUO-7W and PUUO-14W rats. The study shows that the protein abundance of AQP1, AQP2, and AQP3 in the obstructed kidney is increased in PUUO-7W, which may be a compensatory phenomenon and reduced in PUUO-14W rats suggesting a time-/age-dependent dysregulation in response to PUUO. pS256-AQP2 protein abundance is reduced consistent with obstruction-induced direct effects in the apical part of the collecting duct principal cells in response to PUUO.

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE(2) production. Here we found that mRNA expression by the PGE(2)-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE(2) signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278-284. doi:10.1038/ki.2008.498.

Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood-brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Eine Hauptaufgabe der Niere ist die Regulation des Wasser-Salz-Haushaltes. Bei Wassermangel oder Hypernatriämie wird Vasopressin (AVP) ausgeschüttet. Es aktiviert V2-Rezeptoren und stimuliert dadurch die Konzentration des Urins in der Niere. Im Sammelrohr bewirkt die V2-Rezeptor-Signalkaskade eine erhöhte Expression und Translokation des Wasserkanals Aquaporin2 (AQP2) zur apikalen Plasmamembran. Über diesen Kanal wird Wasser aus dem Primärharn in das Interstitium rückresorbiert, um dann wieder den Blutgefäßen zugeführt zu werden. Störungen im AVP-System können sowohl Hypo- als auch Hypernatriämien verursachen. Brattleboro Ratten mit zentralem Diabetes insipidus werden verbreitet als Modellorganismus zur Untersuchung der V2-Rezeptor-Signalkaskade genutzt. Ihre Sammelrohrzellen besitzen zwar den zellulären Signaltransduktionsapparat für die AVP-vermittelte Translokation von AQP2, aufgrund ihres systemischen AVP-Mangels zeigen sie jedoch eine verminderte Expression von AQP2 an der apikalen Plasmamembran und 10-fach erhöhte Urinvolumina. Die hier vorliegende Arbeit belegt eine direkte und spezifische Rolle des kalziumabhängigen Phosholipidbindeproteins ANXA2 und seines Bindungspartners P11 bei der Translokation von AQP2. Dabei liefert sie eine umfassende Lokalisationsstudie von ANXA2 in der Niere. Nach Aktivierung der V2 -Rezeptor-Signalkaskade durch das AVP-Analogon dDAVP zeigt sie neben der Expressionssteigerung von AQP2 auch die erhöhte Expression von ANXA2 und liefert den ersten tierexperimentellen Nachweis der Ko-Translokation von AQP2, ANXA2 und P11. Sie bestätigt und verifiziert die Komplexierung von ANXA2 und AQP2, zeigt aber keine direkte Bindung zwischen den beiden Proteinen. Mittels Peptid SPOT Arrays belegt sie jedoch eine direkte Bindung zwischen P11 und dem C-Terminus von AQP2 und zeigte eine zusätzliche, direkte Bindung zwischen einem Tetramer, bestehend aus zwei ANXA2 und zwei P11 Molekülen, mit dem N-Terminus von AQP2. Diese Bindung ist abhängig vom Phosphostatus des ANXA2. Phosphomimetisches Aspartat anstelle von Serin 26 verhindert die Bindung, wobei phosphomimetisches Aspartat anstelle von Tyrosin 24 keinen Einfluss darauf hat. Die Ergebnisse dieser Studie liefern neue Bausteine zur Entschlüsselung der AVP-Signalkaskade, die im Zuge der Urinkonzentrierung aktiviert wird. Sie ist ein weiterer Schritt zur Entwicklung neuer Therapieansätze zur besseren Behandlung von AVP-induzierten Osmolaritätsstörungen., A major function of the kidney is the regulation of body water and electrolyte balance. In case of water deprivation or hypernatremia, vasopressin (AVP) is secreted to activate V2 receptors, and therefore stimulates the urinary concentrating mechanism in the kidney. The V2 receptor signaling cascade in the collecting duct leads to increased expression and translocation of the water channel AQP2 (aquaporin2) to the apical membrane. Water from inside the tubular system is reabsorbed by this channel to be delivered back to the vasculature. A disrupted AVP system leads to hyponatremia or hypernatremia. Brattelboro rats are broadly used as a model organism for the analysis of the V2 receptor signaling cascade. Though their collecting duct cells own the signaling transduction machine due to a mutation, they are not able to secrete AVP. Therefore, they show a reduced AQP2 expression at the apical membrane and tenfold increased urine volumes. This dissertation reveals a direct and specific role of the calcium and phospholipid binding protein ANXA2 and its binding partner P11 in the translocation process of AQP2. It provides a widespread localization study of ANXA2 in the kidney. During activation of the V2 receptor signaling cascade via AVP analogon dDAVP it shows not only increased expressions of AQP2 but also of ANXA2. For the first time it proves the co-translocation of AQP2 ANXA2 and P11 in an animal model, under these circumstances. It also confirms the complexation of ANXA2 and AQP2 but does not find direct binding properties. However, using Peptide SPOT Arrays it demonstrates direct linkage among P11 and the C-terminal end of AQP2 and also an additional linkage among a tetramer composed of two ANXA2 and two P11 molecules and the N-terminal end of AQP2. This linkage is dependent on the phosphorylation status of ANXA2. A phosphomimicking aspartate residue at the position of serine 26 prevents the binding, whereas a phosphomimicking aspartate residue at the position of tyrosine 24 does not interfere. These results extend our knowledge regarding the intracellular signaling cascade involved in AVP-mediated urine concentration and may facilitate the development of novel mechanism-based pharmacological tools for the treatment of AVP-related diseases.