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.

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.

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.

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.

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/-conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/-ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/-by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

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.

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.

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. 

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.

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.