Background: Renal water reabsorption is controlled by vasopressin (AVP) which activates phosphorylation of AQP2 at serine 256 (pS256) and translocation to the plasma membrane. Besides S256, AVP causes dephosphoryation of S261. Recent studies showed that cyclin-dependent kinases can phosphorylate S261 AQP2 peptides in vitro. In an attempt to investigate the possible role of cdks on AQP2 phosphorylation, we identified a PKA-independent pathway regulating AQP2 trafficking. Methods: MDCK cells or kidney slices were left untreated or forskolin stimulated with or without roscovitine (10μM), a specific cdks inhibitor. Results: In ex-vivo kidney slices and MDCK cells roscovitine increased pS256 and decreased pS261.The changes in AQP2 phosphorylation were paralleled by an increase in cell surface AQP2 expression and osmotic water permeability in the absence of forskolin stimulation. Of note, roscovitine did alter neither cAMP intracellular level nor PKA activity. Because phosphorylation results from the balance between kinase and phosphatase activity we evaluated the possible contribution of protein phosphatases PP1, PP2A and PP2B. Of these, roscovitine treatment specifically reduced PP2A activity in MDCK cells. Interestingly, in PKD1+/- mice displaying a syndrome of inappropriate antidiuresis with high level of pS256 despite unchanged AVP and cAMP (Alharabi et. al 2007) we found a reduced PP2A expression and activity. Indeed similarly to what previously found in PKD1+/- mice, roscovitine significantly decreased intracellular calcium in MDCK. Conclusions: Our data indicate that a reduced activity of PP2A, secondary to reduced intracellular Ca2+ levels, promotes AQP2 trafficking independently from the AVP-PKA axis. This pathway may be relevant for explaining pathological states characterized by inappropriate AVP secretion and positive water balance.

BACKGROUND Sodium-sensitive hypertension is caused by renal tubular dysfunction, leading to increased retention of sodium and water. Previous findings have suggested that single-nucleotide polymorphisms of the cytoskeletal protein, a-adducin, are associated with increased membrane expression of the Na/K pump and abnormal renal sodium transport in Milan hypertensive strain (MHS) rats and in humans. However, the possible contribution of renal aquaporins (AQPs) to water retention remains undefined in MHS rats. METHODS Kidneys from MHS rats were analyzed and compared with those from age-matched Milan normotensive strain (MNS) animals by quantitative-PCR, immunoblotting, and immunoperoxidase. Endocytosis assay was performed on renal cells stably expressing AQP4 and co-transfected either with wild-type normotensive (NT) or with mutated hypertensive (HT) a-adducin. RESULTS Semiquantitative immunoblotting revealed that AQP1 abundance was significantly decreased only in HT MHS whereas AQP2 was reduced in both young pre-HT and adult-HT animals. On the other hand, AQP4 was dramatically upregulated in MHS regardless of the age. These results were confirmed by immunoperoxidase microscopy. Endocytosis assays clearly showed that the expression of mutated adducin strongly reduced the rate of constitutive AQP4 endocytosis, thereby increasing its abundance at the plasma membrane. CONCLUSIONS We provide here the first evidence that AQP1, AQP2, and AQP4 are dysregulated in the kidneys of MHS animals. In particular, we provide evidence that alpha-adducin mutations may be responsible for AQP4 upregulation. The downregulation of AQP1 and AQP2 and the upregulation of AQP4 may be relevant for the onset and maintenance of salt-sensitive hypertension.

OBJECTIVE: The intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immunoglobulin A nephropathy (IgAN) pathogenesis, which is still largely undefined. Recently, vasopressin (AVP) has been advocated to contribute to the genesis and progression of chronic kidney diseases (CKD) directly, and indirectly, via RAS activation. Our aim is to explore the intrarenal activity of AVP, its relationship with RAS activity, as well as its modulation by therapies in IgAN. DESIGN: In this observational study, we measured plasma copeptin, a surrogate marker of AVP, the urine excretion of aquaporin 2 (AQP2), a protein reflecting renal AVP action, and angiotensinogen (AGT), a parameter of renal RAS activation, and their relationship with renal function in 44 IgAN patients at the time of renal biopsy, without any drug therapy, and after 6-month treatment with ACEi or steroid+ACEi. Twenty-one patients with other CKD and 40 healthy subjects were recruited as controls. METHODS: ELISAs were used to measure all variables of interest. RESULTS: At baseline, IgAN patients showed higher urinary levels of AQP2, compared with controls and patients with other CKD. Urinary AQP2 and AGT levels strongly correlated with the presence of arterial hypertension. Steroids+ACEi caused the decrease of all the variables examined. The fall of urinary AQP2 and AGT following drug treatments was associated with the decrease of daily proteinuria. CONCLUSION: Our findings would support the involvement of AVP-AQP2 axis, interacting with the RAS, in the progression of IgAN and candidate AQP2 as a possible novel marker of the disease.

We screened human kidney-derived multipotent CD133+/CD24+ ARPCs for the possible expression of all 13 aquaporin isoforms cloned in humans. Interestingly, we found that ARPCs expressed both AQP5 mRNA and mature protein. This novel finding prompted us to investigate the presence of AQP5 in situ in kidney. We report here the novel finding that AQP5 is expressed in human, rat and mouse kidney at the apical membrane of type-B intercalated cells. AQP5 is expressed in the renal cortex and completely absent from the medulla. Immunocytochemical analysis using segment- and cell type-specific markers unambiguously indicated that AQP5 is expressed throughout the collecting system at the apical membrane of type-B intercalated cells, where it co-localizes with pendrin. No basolateral AQPs were detected in type-B intercalated cells, suggesting that AQP5 is unlikely to be involved in the net trans-epithelial water reabsorption occurring in the distal tubule. An intriguing hypothesis is that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb. Future studies will unravel the physiological role of AQP5 in the kidney.

In an attempt to investigate the regenerative potential of adult multipotent renal progenitor/stem cells (ARPCs) isolated from human kidneys (Sallustio et al., 2009) we characterized them for the expression of aquaporins. ARPCs expressed measurable levels of the proximal tubule-specific AQP1, both at mRNA and protein levels. When ARPCs were differentiated in vitro into epithelial cells, the expression of the collecting duct-specific AQP2 was also induced. Surprisingly, ARPCs also expressed measurable levels of AQP5, an aquaporin known to be selectively expressed in lung, salivary and lachrymal glands in mammals. This evidence prompted us to investigate the presence and the localization of AQP5 in the mammalian kidney. Total RNA was isolated from adult human, rat and mouse kidneys and subjected to RT-PCR. Interestingly, AQP5 transcripts were found in all the species tested. Western blotting analysis, revealed an AQP5 band of 27 kDa as well as a glycosylated form. Consistent with that, neither the transcript nor the protein was found in AQP5 null mice. AQP5 abundance was higher in the renal cortex than in the medulla. Immunolocalization indicated that AQP5 was expressed at the apical membrane of the cortical collecting ducts (CCDs) epithelial cells with negligible staining in the inner medulla. Triple immunostaining indicated that, in rat CCDs, AQP5 did not colocalize either with AQP2 or with the intercalated cells marker V-ATPase, suggesting a cell specific expression of AQP5 in cells not expressing AQP2 but likely involved in water reabsorption. The ratio between AQP2- and AQP5-expressing cells was approximately 3:1. In conclusion, the expression of AQP5 in the ARPCs, might suggest a role in the differentiation/regeneration processes of the collecting duct epithelial cells. Moreover, its constitutive expression at the apical membrane in the CCD, renders AQP5 a possible target for improving water reabsorption in the collecting duct when AQP2 apical expression is unpaired as in nephrogenic diabetes insipidus.

Aims: Few investigations have explored the urinary aquaporin-2 (u-AQP2) excretion pattern after birth in preterm infants with conflicting results regarding the correlation between u-AQP2, urinary osmolality and vasopressin. The aims of this study were to evaluate u-AQP2 excretion during the first week of life in preterm infants, to correlate u-AQP2 with other markers of renal function and to investigate the relationship between u-AQP2, urinary tonicity and arginine-vasopressin in the immature kidney. Methods: In infants born less than 33 weeks daily diuresis, u-AQP2, urinary arginine-vasopressin, urine and plasma tonicity, creatinine and electrolytes were measured through the first 7 days of life. Results: Fifty-five infants were evaluated. u-AQP2 excretion showed the following profile: the highest u-AQP2 levels were found on day 2 and values remained significantly higher until day 5 with respect to day 1. On day 6, u-AQP2 levels significantly decreased to values closer to those found on day 1. u-AQP2 excretion was not associated with arginine-vasopressin while significant, but weak association was found with urinary tonicity (r = -0.20; -0.32 < r < -0.11; P < 0.05). u-AQP2 excretion and creatinine clearance were significantly associated during the study period (r = 0.19; 0.08 < r < 0.29; P < 0.05). There was a strong association between totally u-AQP2 excretion and diuresis over the week (r = 0.72; 0.66 < r < 0.76; P < 0.0001). Conclusion: Significant variations occur in AQP2 expression levels during the first week of life in preterm infants. AQP2 does not seem to contribute to the urinary concentration ability after birth. Further investigations are required to elucidate the mechanisms underlying the strong association between diuresis and u-AQP2 excretion in early postnatal life.

The localization of the water channel aquaporin-2 (AQP2) is subjected to regulation by vasopressin. Vasopressin adjusts the amount of AQP2 in the plasma membrane by regulating its redistribution from intracellular vesicles into the plasma membrane allowing water entry into the cells and water exit through AQP3 and AQP4. This permits water reabsorption and urine concentration. Following binding of vasopressin to its V2R receptor, the rise in cAMP activates protein kinase A, which in turn phosphorylates AQP2 and thereby triggers the redistribution of AQP2. Several proteins participating in the control of cAMP-dependent AQP2 trafficking have been identified including SNAREs, annexin-2, hsc70, AKAPs and small GTPases of the Rho family proteins. Moreover, AQP2 has been found to be regulated by posttranslational modifications (PTMs), such as ubiquitination and glutathionylation. Loss-of-function mutations of both V2R and AQP2 are associated with congenital nephrogenic diabetes insipidus characterized by a failure to concentrate urine. Conversely gain-of-function mutations of the V2R are associated with the nephrogenic syndrome of inappropriate antidiuresis characterized by positive water balance and hyponatremia. Vaptans, nonpeptide vasopressin receptor antagonists represent a new class of drugs developed for the treatment of euvolemic or hypervolemic hyponatremia. This chapter summarizes recent data elucidating molecular mechanisms underlying the trafficking of AQP2. The mechanism of action of vaptans and their current use in clinical practice is discussed.

High concentrations of urinary calcium counteract vasopressin action via the activation of the calcium-sensing receptor (CaSR) that is expressed in the luminal membrane of collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). Pendrin/NaCl cotransporter double-knockout (dKO) mice display significant calcium wasting and develop severe volume depletion, despite increased circulating vasopressin levels. We hypothesized that the CaSR-mediated impairment of AQP2 expression/trafficking underlies vasopressin resistance in dKO mice. Compared with wild-type mice, in renal inner medulla, dKO mice had reduced total AQP2 sensitive to proteasome inhibitors, higher levels of AQP2-pS261, ubiquitinated AQP2, and p38-MAPK, an enzyme that is activated by CaSR signaling and known to phosphorylate AQP2 at Ser261. CaSR inhibition with the calcilytic NPS2143 reversed these effects, which indicates that CaSR mediates the up-regulation of AQP2-pS261, ubiquitination, and degradation. Of note, dKO mice demonstrated significantly higher AQP2-targeting miRNA-137 that was reduced upon CaSR inhibition, supporting a critical role for CaSR in the down-regulation of AQP2 expression. Our data indicate that CaSR signaling reduces AQP2 abundance both via AQP2-targeting miRNA-137 and the p38-MAPK/AQP2-pS261/ubiquitination/proteasomal axis. These effects may contribute to the reduced renal concentrating ability that has been observed in dKO mice and underscore a physiologic mechanism of the CaSR-dependent regulation of AQP2 abundance via a novel microRNA pathway.-Ranieri, M., Zahedi, K., Tamma, G., Centrone, M., Di Mise, A., Soleimani, M., Valenti, G. CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

Background: The calcium-sensing receptor (CaSR) is a G protein coupled receptor, which plays an essential role in regulating Ca2+ homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Methods: Primary cells isolated from human urine sediment were infected with SV40T and hTERT vectors. Subconfluent cell layers were transferred to 33°C and selected by antibiotics for 15 days. Cells were subcloned and expanded to 70% confluence at 33°C. After maturation at 37°C for 10 days, the cloned cells were used. Results: The obtained ciPTEC cells expressed ZO-1 protein and aquaporin 1 thus confirming their epithelial and PT origin respectively. The expression of the endogenous CaSR in ciPTEC was confirmed by Western blotting revealing the immunodetection of both forms at 130 and ~200 kDa, corresponding to the monomeric and mature receptor. Of note, functional studies with Fura2-AM indicated that the physiological agonist, calcium (Ca2+), and the calcimimetic NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to low concentrations of its agonists. Cytosolic calcium levels were 46.2±2.22% (vs ATP 100%) after stimulation with 2.5μM Ca2+ and to the 37±1.76% (vs ATP 100%) after stimulation with 2.5μM NPS-R568. Calcium depletion from the ER using CPA (cyclopiazonic acid) abolished the increase in cytosolic calcium elicited by NPS-R568 confirming the origin of calcium exit from intracellular stores. Conclusions: We conclude that human proximal tubular ciPTEC cells express functional CaSR and respond to its activation with a release of calcium from the ER. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients. 

Kidney water reabsorption is regulated by the hormone vasopressin (AVP) which exerts its effect on the water channel AQP2. AVP binds to its V2 receptors and causes, via elevation of cAMP, activation of protein kinase A (PKA) and phosphorylation of AQP2 at serine 256 (pS256), AQP2 translocation to the plasma membrane. Besides S256, in vivo AVP action co-incides with de-phosphoryation of S261, but the kinases involved are unknown. Bio-informatical analysis suggests that cdk1/5 may phosphorylate S261 and their potential relevance in AQP2 regulation was investigated. Immunohistochemistry and immunoblot analysis indicated that cdk1 and cdk5 are both expressed in renal principal cells. In ex-vivo kidney slices and MDCK-AQP2 cells, the cdk1/5 inhibitor roscovitine, increased pS256 and decreased pS261. In MDCK-AQP2 cells, immunocytochemistry and cell surface biotinylation showed that roscovitine caused AQP2 translocation to the apical membrane in the absence of forskolin, resulting in increased osmotic water permeability. However roscovitine did not affect PKA activity and cAMP levels. Interestingly roscovitive modulated the phosphorylation state of DARPP-32, a potent inhibitor of PP1 a phosphatase possibly involved in AQP2 de-phosphorylation. Together, these data indicate that cdks are functionally involved in the regulation of AQP2 trafficking and suggest that inhibition of cyclin kinases may be an alternative approach for treatment of Nephrogenic diabetes insipidus.

In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Ca2+] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Ca2+, 300 mu M Gd3+ and with 10 mu M of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca2+](i) although displaying different features. Specifically, Ca2+ as well as stimulation with NPS-R 568 induced a rapid peak of [Ca2+](i) while stimulation with Gd3+ induced transient intracellular Ca2+ oscillations. PLC inhibition completely abolished any [Ca2+](i) increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca2+](i) oscillations induced by Gd3+, while the peak induced by high Ca2+ was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd3+. On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Ca2+ oscillations produced by Gd3+ are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca2+ in response to 5mM [Ca2+](o) is mainly due to PLC/IP3 pathway activation.

Background: We have recently shown that in MCD4 renal cells, cell surface AQP2 expression in cells exposed to CaSR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin. Those findings were in line with data obtained in hypercalciuric subjects displaying at baseline significantly higher AQP2 excretion and no significant increase in AQP2 excretion and urinary osmolarity after acute DDAVP administration compared to normocalciurics (Procino et al Plos One 2012). This indicates that CaSR-AQP2 interplay represents an internal renal defense to mitigate the effects of rising of calcium during antidiuresis on the risk of calcium precipitation. Methods: Human wild-type CaSR (hCaSR-wt) and its constitutively active variants (hCaSR-R990G; hCaSR-N124K) were functionally expressed in renal HEK cells stably expressing hAQP2. The N124K mutation is one of eight naturally occurring activating mutations in subjects with autosomal dominant hypocalcemia, whereas R990G is a gain-of- function of the CASR gene polymorphism. Western blotting analysis of a crude membrane fraction was performed using phospho-specific antibodies. Results: Compared to mock cells, pS256-AQP2 abundance was significantly increased in cells expressing either the wt-CaSR or its activating variants. Of note, we also found a significant increase in pS261-AQP2 in hCaSR-wt expressing cells compared to mock. Interestingly, the expression of pS261-AQP2 was significantly higher in cells expressing the constitutively active CaSR variants with respect to wt-CaSR expressing cells. No change in the pS269 was observed. Conclusions: Since previous data demonstrated that the amount of pS261 significantly decreases in response to short-term vasopressin exposure, it can be speculated that the increase in pS261 observed in cells expressing constitutively active CaSR variants might counteract the vasopressin response.

This book has been prepared under the auspice of the European Low Gravity Research Association (ELGRA). As a scientific organization the main task of ELGRA is to foster the scientific community in Europe and beyond in conducting gravity and space related research. This publication is dedicated to the science community, and especially to the next generation of scientists and engineers interested in space researches. ELGRA provides here a comprehensive description of space conditions and means that have been developed on Earth to perform space environmental and (micro-)gravity related research. We want to thank all our colleagues who contributed to the interesting and hopefully inspiring content of this book. It is the first in its kind to addressing a comprehensive overview of ground-based technologies and sciences related to (micro-)gravity, radiation and space environment simulation research.

The renal Na+-K+-2Cl- co-transporter (NKCC2) is expressed in kidney thick ascending limb (TAL) cells, where it mediates NaCl reabsorption regulating body salt levels and blood pressure. In this study we used a well-characterized NKCC2 construct (c-NKCC2) to identify NKCC2 interacting protein by an antibody shift assay coupled with Blue Native/SDS-PAGE (BN/SDS-PAGE) and Mass Spectrometry (MS). Among the interacting proteins we identified moesin, a protein belonging to ERM (Ezrin, Radixin, Moesin) family. Co-immunoprecipitation experiments confirmed that c-NKCC2 interacts with the N-terminal domain of moesin in LLC-PK1 cells. Moreover, c-NKCC2 accumulates in intracellular and sub-apical vesicles in cells transfected with a moesin dominant negative GFP-tagged construct. In addition, moesin knockdown by siRNA decreases by about 50% c-NKCC2 surface expression. Specifically, endocytosis and exocytosis assays showed that moesin knockdown does not affect NKCC2 internalization but strongly reduces exocytosis of the co-transporter. Our data clearly demonstrate that moesin plays a critical role in apical membrane insertion of NKCC2, suggesting a possible involvement of moesin in regulation of Na+ and Cl- absorption in the kidney.

Background: Cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates due to their ability to target cell cycle proteins. Of these, roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis because reduces the abnormal cell proliferation. The two roscovitine enantiomers (R and S) are both very promising therapeutic tools due to their ability to regulate cell proliferation, however they might exert distinct actions at tissue levels. Here we evaluated roscovitine effect on actin cytoskeleton and intracellular calcium signaling in MDCK cells. Methods: Intracellular calcium was evaluated by Fura-2AM microfluorimetry. Actin filaments were visualized by phalloidin-TRITC. Results: The two enantiomers had opposite effects on actin organization as R-roscovitine caused actin depolymerization whereas S-roscovitine stabilized actin filaments. Long term R-roscovitine treatment significantly reduced basal cytosolic calcium compared to control cells. In contrast, S-roscovitine treated cells showed a significant increase in basal intracellular calcium. Short term exposure to S-roscovitine induced a cytosolic calcium peak, which was abolished after store depletion with cyclopiazonic acid (CPA). Instead R-roscovitine caused cytosolic calcium oscillations followed by a small calcium plateau. Calcium oscillations were prevented after store depletion with CPA or treatment with the PLC inhibitor U73122. Bafilomycin, a selective vacuolar H+-ATPase inhibitor abolished the small calcium plateau. Conclusions: To our knowledge this is the first study revealing the differential effect of S- and R-roscovitine on cytoskeleton and intracellular calcium signaling in renal cells. Since calcium and CDKs are pleiotropic cellular regulators and both exert powerful effects on cell proliferation and regulation of membrane transporter trafficking through actin dynamics, the use of S- and R-roscovitine as therapeutic tools has to be carefully evaluated.

The water channel Aquaporin 2 (AQP2) is responsible for the vasopressin (VP)-dependent water reabsorption occurring in the kidney during antidiuresis. X-linked nephrogenic diabetes insipidus (XNDI), a severe rare disease characterized by impaired urine-concentrating ability of the kidney, is caused by inactivating mutations in the V2 type VP receptor (V2R) gene. Mutation prevents the VP-induced shuttling of AQP2 from intracellular storage vesicles to the apical plasma membrane of kidney collecting duct principal cells. This, in turn, dramatically reduces water reabsorption resulting in severe polyuria and constant risk of dehydration. Unfortunately, the current pharmacological approach for handling XNDI is unable to rescue AQP2 membrane expression. We have previously reported that the cholesterol-lowering drug lovastatin increases AQP2 membrane expression in renal cells in vitro. More recently we reported that, in mice, fluvastatin increases AQP2 membrane expression in the collecting duct in a VP-independent fashion and greatly increases the amount of water reabsorbed in the kidney. Additional experiments in vitro, performed on a cell culture model recapitulating AQP2 trafficking, indicate that this effect of fluvastatin is most likely caused by the statin-dependent inhibition of protein prenylation of key regulators of AQP2 trafficking in collecting duct cells. We identified members of the Rho and Rab families of proteins as possible key players whose reduced prenylation might result in the accumulation of AQP2 at the plasma membrane, by modulating the basal rate of exocytosis and/or endocytosis. Most importantly, preliminary results obtained using the conditional mouse model of human XNDI, characterized by severe polyuria and low urine osmolality, indicate that fluvastatin treatment significantly reduces diuresis and increases urine osmolality. Taken together, these results strongly suggest that statins may prove useful in the therapy of XNDI.

Background: Exposure to microgravity results in alterations of renal function, fluid redistribution and bone loss which contributes to the potential risk of renal stone formation. Hypercalciuria is recognized as a condition predisposing to calcium nephrolitiasis and long-term space flights cause bone loss coupled to a rise of urinary calcium excretion. Methods: AQP2 excretion was measured by ELISA in urines collected from healthy volunteers participating at the studies. Results: We recently demonstrated that high calcium delivery to the collecting duct reduces local Aquaporin 2 (AQP2) mediated water reabsorption under vasopressin action, thus limiting the maximal urinary concentration and reducing calcium saturation. To analyze alteration of renal water handling during microgravity, we evaluated two ground-based analog of spaceflight, thermoneutral water immersion and bed rest. AQP2 excretion and diuresis were measured in two separated studies mimicking acute adaptation (6 hours water immersion) or chronic adaptation (35 days bed rest) to microgravity. Water immersion resulted in a significant increase in urinary output apparently not related to AQP2 alteration and manly due to reduced vasopressin secretion. On the other hand 35 days bed rest resulted in an increase in urinary calcium, which coincided with a significant decrease in AQP2 excretion (645±7.4 fmol/ml to 569±10.3 fmol/ml), which is expected to result in urine dilution reducing the risk of calcium saturation. Conclusions: Our data indicate that calciuria and water balance have to be strictly controlled during microgravity and long-term bed rest as key elements for the risk of kidney stone formation.

Abstract: The renal-specific Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is the major salt transport pathway of the apical membrane of the mammalian thick ascending limb of Henle's loop. Here, we analyze the role of the tetraspan protein myelin and lymphocytes-associated protein (MAL)/VIP17 in the regulation of NKCC2. We demonstrated that 1) NKCC2 and MAL/VIP17 colocalize and coimmunoprecipitate in Lilly Laboratories cell porcine kidney cells (LLC-PK1) as well as in rat kidney medullae, 2) a 150-amino acid stretch of NKCC2 C-terminal tail is involved in the interaction with MAL/VIP17, 3) MAL/VIP17 increases the cell surface retention of NKCC2 by attenuating its internalization, and 4) this coincides with an increase in cotransporter phosphorylation. Interestingly, overexpression of MAL/VIP17 in the kidney of transgenic mice results in cysts formation in distal nephron structures consistent with the hypothesis that MAL/VIP17 plays an important role in apical sorting or in maintaining the stability of the apical membrane. The NKCC2 expressed in these mice was highly glycosylated and phosphorylated, suggesting that MAL/VIP17 also is involved in the stabilization of NKCC2 at the apical membrane in vivo. Thus, the involvement of MAL/VIP17 in the activation and surface expression of NKCC2 could play an important role in the regulated absorption of Na(+) and Cl(-) in the kidney.

Nephrogenic diabetes insipidus is treated with statins.

Background: The vasopressin receptor antagonist tolvaptan has emerged as tool in the management of hyponatremia. However, no direct evidence that the aquaretic effect of tolvaptan is based on impairment of vasopressin stimulated AQP2 phosphorylation and targeting to the plasma membrane has been provided. Methods: MDCK stably expressing hAQP2 or rat kidney slides were exposed to DDAVP or forskolin (FK) stimulation in the presence or in the absence of tolvaptan (10nM). The effect of these treatments on cAMP levels, AQP2 phosphorylation, intracellular calcium concentration and osmotic water permeability was analyzed. Results: In MDCK cells, DDAVP treatment significantly increased cAMP levels paralleled by an increase in p256AQP2. Pretreatment with tolvaptan significantly reduced both effects. Surprisingly, tolvaptan pretreatment strongly reduced the increase in p256AQP2 elicited by FK, a direct activator of adenylyl cyclase. Similar results were obtained in rat kidney slides. In line, tolvaptan prevented the increase in the osmotic water permeability promoted by either DDAVP or FK in MDCK. We therefore analyzed whether tolvaptan had, per se, a cellular effect. Calibration of cellular calcium in MCDK cells revealed that tolvaptan caused a significant increase in intracellular calcium (tolvaptan 64.0±2 nM; ctr 32±1.7 nM). Since p256AQP2 can be de-phosphorylated by PP2A, a calcium depended serine/threonine phosphatase, rat kidney slides were pretreated with tolvaptan and exposed to FK in the presence or absence of caliculyn (5pM) a specific inhibitor of PP2A. Under these conditions tolvaptan failed to prevent FK-induced increase in p256AQP2 suggesting that tolvaptan, activates PP2A. Conclusions: Tolvaptan prevents vasopressin induced increase in p256AQP2, AQP2 trafficking and increase in osmotic water permeability. Moreover tolvaptan increases basal intracellular calcium, which might have relevant consequences in modulating p256AQP2 levels and therefore the clinical response to the drug.

ObjectiveLoss-of-function calcium-sensing receptor (CaR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Though full CaR deletion is possible in mice, most human CaR mutations result from single amino acid substitutions which maintain partial function. However, here we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaRR392X), in effect a full "CaR-knockout".Case ReportThe infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6 mmol/l [2.15-2.65]) and elevated PTH concentrations (650-950 pmol/l [12-81]) together with skeletal demineralisation. NSHPT was confirmed by CaR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels.Design and MethodsThe R392X stop codon was inserted into human CaR and the resulting mutant (CaRR392X) expressed transiently in HEK-293 cells.ResultsCaRR392X expressed as a 54kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaRR392X-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity.ConclusionsIntriguingly, the patient remained normocalcaemic throughout childhood (2.5mM Ca2+, 11pg/ml PTH [10-71], age-8) but exhibited mild asymptomatic hypocalcaemia at age-10, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CaR-knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

Background: To evaluate some aspects of renal CaSR physiopathology, we analyzed the signaling underling two constitutively active variants of the CaSR. Methods: Constructs encoding human wild-type CaSR (hCaSR-wt) and its constitutively active (hCaSR-R990G; hCaSR-N124K) and inactive variants (hCaSR-del121) were transiently transfected in HEK cells. Results: Immunofluorescence studies revealed that both hCaSR-wt and its activating variants were expressed at the plasma membrane, whereas the inactive form localized intracellularly. The physiological agonist, calcium (Ca++ 5mM), and the calcimimetic NPS-R568 (5μM) induced a significant increase in cytosolic calcium in cells expressing hCaSR-wt and its active variants, compared to mock. We also found that the basal intracellular calcium was significantly lower in cells expressing hCaSR-wt and its activating variants compared to mock and hCaSR-del121 transfected cells. Low calcium levels are expected to make cells more sensitive to intracellular calcium changes in response to CaSR agonists. In line, FRET studies using D1ER probe, which detects [Ca++]ER directly demonstrated a significant higher calcium accumulation in cells expressing the activating CaSR variants. Since the storage of calcium in the ER is mainly regulated by SERCA, the activity and the expression of this pump were evaluated. Compared to hCaSR-wt expressing cells SERCA expression and activity were found significantly increased in cells expressing activating CaSR variants. An inverse correlation with PMCA was also found. Conclusions: Together, these findings indicate that for the efficiency of calcium signaling system, cells monitor cytosolic and ER calcium levels regulating the expression of the SERCA and PMCA. To our knowledge this is the first demonstration that a complex parallel adaptive feedback can explain the molecular basis of constitutively active variants of the CaSR. 

Abstract: The Milan hypertensive strain of rats (MHS) develops hypertension as a consequence of the increased tubular Na(+) reabsorption sustained by enhanced expression and activity of the renal tubular Na-K-ATPase. To verify whether the Na-K-2Cl cotransporter (NKCC2) is involved in the maintenance of hypertension in MHS rats, we have analysed the phosphorylation state and the activation of NKCC2 in Milan rats. Western blotting and immunofluorescence experiments were performed using specific antibodies against the regulatory phospho-threonines in the NKCC2 N terminus (R5 antibody). The phosphorylation levels of NKCC2 were significantly increased in the kidney of MHS rats. Moreover, the administration of furosemide in vivo decreased the blood pressure and increased the urine output and natriuresis in MHS rats demonstrating the actual involvement of NKCC2 activity in the pathogenesis of hypertension in this strain of rats. The up-regulation of NKCC2 activity is most probably mediated by a STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylation at serine-325 since it was significantly increased in MHS rats. Interestingly, aldosterone treatment caused an increase in NKCC2 phosphorylation in NKCC2-expressing MDCK cells. In conclusion, we demonstrated an increase in the activity of NKCC2 along the TAL that significantly contributes to the increase in systemic blood pressure in MHS rats. The elevated plasma levels of aldosterone, found in MHS rats, may influence Na(+) balance through a SPAK-dependent regulation of NKCC2 accounting for the maintenance of the hypertensive state in MHS rats.

We have previously shown that treatment with lovastatin accumulates the water channel AQP2 at the apical plasma membrane of cultured MCD4 renal cells by inhibiting its constitutive endocytosis. This observation might be of importance to rescue AQP2 apical expression in X-linked NDI. Here we propose a mechanism that might explain this effect. In addition to inhibiting cholesterol synthesis, statins also inhibit the synthesis of other sterol and non-sterol intermediate compounds produced by the mevalonate pathway including the isoprenoids, farnesyl- (FP) and geranylgeranyl-pyrophosphate (GGP). Proteins of the Rab GTPase family must be post-translationally prenylated by addition of two geranylgeranyl moieties in order to be properly targeted to membranes and to be active. Members of the Rab family, including Rab5, were found associated with immunoisolated AQP2 storage vesicles. Rab5 is expressed in early endosomes and in chlatrin-coated endocytic vesicles suggesting a role in regulating AQP2 endocytosis at the plasma membrane. In this study we found that, in MCD4 cells, mevalonate prevented apical accumulation of AQP2 induced by lovastatin incubation as demonstrated by confocal microscopy. Interestingly, similar results were obtained by provision of GGP, suggesting a role of prenylation in regulating AQP2 trafficking. Measurement of osmotic water permeability, obtained by a calcein-quenching-based assay on FlexStation, confirmed that the increase in water permeability induced by lovastatin treatment was completely abolished by mevalonate or GGP. In agreement with these results, analysis of Rab5 prenylation under lovastatin treatment, showed that its electrophoretic mobility on SDS-PAGE was slightly reduced, consistent with a reduction of protein isoprenylation. Conversely, addition of mevalonate or GGP prevented Rab5 electrophoretic shift. Taken together, these data suggest that statins might increase AQP2 apical expression by reducing Rab5 isoprenylation and its association to the apical plasma membrane where it regulates AQP2 endocytosis.

Na(+) is commonly designed as the culprit of salt-sensitive hypertension but several studies suggest that abnormal Cl(-) transport is in fact the triggering mechanism. This review focuses on the regulation of blood pressure (BP) by pendrin, an apical Cl(-)/HCO(3)(-) exchanger which mediates HCO(3)(-) secretion and transcellular Cl(-) transport in type B intercalated cells (B-ICs) of the distal nephron. Studies in mice showed that it is required not only for acid-base regulation but also for BP regulation as pendrin knock-out mice develop hypotension when submitted to NaCl restriction and are resistant to aldosterone-induced hypertension. Pendrin contributes to these processes by two mechanisms. First, pendrin-mediated Cl(-) transport is coupled with Na(+) reabsorption by the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger NDCBE to mediate NaCl reabsorption in B-ICs. Second, pendrin activity regulates Na(+) reabsorption by the adjacent principal cells, possibly by interaction with the ATP-mediated paracrine signalling recently identified between ICs and principal cells. Interestingly, the water channel AQP5 was recently found to be expressed at the apical side of B-ICs, in the absence of a basolateral water channel, and pendrin and AQP5 membrane expressions are both inhibited by K(+) depletion, suggesting that pendrin and AQP5 could cooperate to regulate cell volume, a potent stimulus of ATP release.

Obesity has increased dramatically during the past decade and is an established risk factor for the development of chronic kidney disease. In this study we focused our attention on the potential dysregulation of renal aquaporins (AQPs) in the kidney of a mouse model of obesity and type 2 diabetes mellitus. Mice were fed a high fat (HFD) or normal (ND) diet and analyzed at 20 weeks and at 34 weeks. At 20 weeks, western blotting of total lysates from HFD mice revealed higher immunoreactivity for AQP1 whereas no significant change in AQP2, AQP3 and AQP4 abundance was observed. However, AQP1, AQP2, AQP3 and AQP4 immunoreactivity decreased in a crude kidney membrane preparation suggesting a reduced cell surface expression of all these AQPs. Stopped flow light scattering studies showed a reduced osmotic water permeability (Pf) of the whole renal plasma membranes from 254.8±30.5 mm/s to 169.8±16.2 (P&lt;0.0001) in ND versus HFD mice, respectively, consistent with the observed decrease in AQPs cell surface expression. At 34 weeks, western blotting of total lysates or of a crude membrane preparation from HFD mice revealed significant downregulation of AQP1 and AQP2 whereas AQP3 and AQP4 immunoreactivity was unchanged in both preparations compared to ND mice. Altogether, these data reveal altered AQPs expression and osmotic water permeability of HFD mice kidney. These observations suggest a patho-physiological relevance for aquaporins in renal complications associated with metabolic syndrome.

In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.

Human pendrin (SCL26A4, PDS) is a 780 amino acid integral membrane protein with transport function. It acts as an electroneutral, sodium-independent anion exchanger for a wide range of anions, such as iodide, chloride, formate, bicarbonate, hydroxide and thiocyanate. Pendrin expression was originally described in the thyroid gland, kidney and inner ear. Accordingly, pendrin mutations with reduction or loss of transport function result in thyroid and inner ear abnormalities, manifested as syndromic (Pendred syndrome) and non-syndromic hearing loss with an enlarged vestibular aqueduct (ns-EVA). Pendred syndrome, the most common form of syndromic deafness, is an autosomal recessive disease characterized by sensorineural deafness due to inner ear malformations and a partial iodide organification defect that may lead to thyroid goiter. Later, it became evident that not only pendrin loss of function, but also up-regulation could participate in the pathogenesis of human diseases. Indeed, despite the absence of kidney dysfunction in Pendred syndrome patients, evidence exists that pendrin also plays a crucial role in this organ, with a potential involvement in the pathogenesis of hypertension. In addition, recent data underscore the role of pendrin in exacerbations of respiratory distresses including bronchial asthma and chronic obstructive pulmonary disease (COPD). Pendrin expression in other organs such as mammary gland, testis, placenta, endometrium and liver point to new, underscored pendrin functions that deserve to be further investigated.

La malattia policistica renale, nota come rene policistico, è una patologia genetica di cui esistono una forma autosomica dominante, più comune e tipica nell’adulto, e una autosomica recessiva, più rara e presente nell’infanzia. In particolare, la forma autosomica dominante (ADPKD) è la più comune malattia genetica letale ed è nel mondo la prima causa di insufficienza renale. Essa è causata da mutazioni nei geni PDK1 o PDK2 che codificano rispettivamente per la policistina 1 (PC1) e la policistina 2 (PC2). La PC1 interagisce con la PC2 per formare un complesso multifunzionale, ovvero un canale, che regola il passaggio del calcio intracellulare. Le mutazioni di PC1 o PC2 comportano alterata funzionalità del canale e disregolazione dei segnali cellulari controllati dal calcio. Tutto ciò si traduce nella formazione di cisti ripiene di fluido in entrambi i reni. La formazione delle cisti a lungo andare compromette severamente la normale funzione del rene portando alla insufficienza renale. Allo stato attuale non esiste una cura farmacologica per la malattia. L’unico farmaco riconosciuto e approvato per la sua efficacia nel rallentare l’ingrandimento delle cisti, e di conseguenza la progressione della malattia, è il tolvaptan, un antagonista di recettori V2 dell’ormone vasopressina. D’altra parte i calciomimetici, farmaci che agiscono attivando i recettori del calcio provocando incremento di calcio intracellulare, hanno mostrato una certa efficacia nel contrastare la malattia policistica in modelli animali. Scopo della presente invenzione è fornire un’associazione farmacologica dell’antagonista del recettore della vasopressina tolvaptan con un calciomimetico per il trattamento del rene policistico con l’obiettivo di ottenere una azione sinergica e contemporaneamente limitare gli effetti collaterali associati all’utilizzo dell’unico farmaco attualmente indicato per il trattamento di questa patologia. Gli inventori sono in possesso di convincenti dati in vitro e di preliminari dati in vivo sulla validità della strategia terapeutica oggetto della invenzione.

La presente invenzione si riferisce ad un nuovo metodo per trattare le conseguenze della malattia genetica rara diabete insipido (NDI) nefrogenico, in particolare il tipo di NDI legato all'X (X-NDI). Nelle persone che soffrono di X-NDI il gene per il recettore della vasopressina è mutato, quindi la proteina mutata non può trasdurre all'interno della cellula renale il segnale della presenza di vasopressina in circolo e indurre un aumento della quantità del canale per l’acqua acquaporina 2 (AQP2) sulla membrana plasmatica. Ciò impedisce il riassorbimento di acqua nel dotto collettore dei reni (antidiuresi) e determina una grave poliuria e disidratazione. Le statine possono aumentare la quantità di AQP2 alla membrana plasmatica in assenza di vasopressina circolante. È stato scoperto che le statine, ampiamente utilizzate nel trattamento dell'ipercolesterolemia, sono in grado di ridurre di conseguenza l'eccesso di produzione di urina nei pazienti affetti da NDI. Uno scopo della presente invenzione è di fornire un metodo per trattare l’ X-NDI, e in particolare poliuria correlata con l’ X-NDI, mediante un metodo di trattamento che superi gli inconvenienti della dei trattamenti attuali e non abbia effetti collaterali significativi. Lo scopo dell'invenzione è quindi raggiunto mediante un metodo per il trattamento dell’ NDI che consiste nella somministrazione di una o più statine al paziente.

Nephrogenic diabetes insipidus is treated with statins