X-linked nephrogenic diabetes insipidus (NDI) is a disease caused by inactivating mutations of the vasopressin (AVP) type 2 receptor (V2R) gene. V2R inactivation prevents plasma membrane expression of the water channel AQP2 in the kidney collecting duct (CD) cells and impairs the kidney concentration ability. In the present work we showed that secretin receptor (SCTR) is functionally expressed at the basolateral membrane of the kidney collecting duct cells. Based on this observation, we infused the mouse model of X-NDI with SCT via osmotic pumps for 14 days at a dose of 2.5 mmol/Kg/day. Urinary parameters were not altered in SCT-infused animals. Interestingly, however, SCT significantly increased AQP2 levels in the CD, although the protein was mostly accumulated in the intracellular storage vesicles and hardly detectable at the plasma membrane. As we previously reported that fluvastatin (Flu) treatment increases AQP2 plasma membrane expression in the CD cells of wt mice, SCT-infused X-NDI mice received a single injection of Flu (50 mg/Kg). Interestingly, during the following 6 hours of observation, the diuresis of mice treated with SCT+Flu was reduced by nearly 90% and urine osmolality doubled. Analysis of the kidneys confirmed that SCT increased intracellular stores of AQP2 and the addition of Flu promoted AQP2 accumulation at the plasma membrane. Taken together these evidence indicate that the association of SCT and Flu might represent a novel and effective pharmacological treatment for X-NDI.

La presente invenzione ha per oggetto l’impiego di agonisti selettivi dei recettori beta-adrenergici di tipo 3 (BAR3) nel trattamento del diabete insipido nefrogenico (NDI), in particolare del diabete insipido nefrogenico legato alla X (X-linked) (X-NDI).

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.

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.

X-linked nephrogenic diabetes insipidus (X-NDI) is a disease caused by inactivating mutations of the vasopressin (AVP) type 2 receptor (V2R) gene. Loss of V2R function prevents plasma membrane expression of the AQP2 water channel in the kidney collecting duct cells and impairs the kidney concentration ability. In an attempt to develop strategies to bypass V2R signaling in X-NDI, we evaluated the effects of secretin and fluvastatin, either alone or in combination, on kidney function in a mouse model of X-NDI. The secretin receptor was found to be functionally expressed in the kidney collecting duct cells. Based on this, X-NDI mice were infused with secretin for 14 days but urinary parameters were not altered by the infusion. Interestingly, secretin significantly increased AQP2 levels in the collecting duct but the protein primarily accumulated in the cytosol. Since we previously reported that fluvastatin treatment increased AQP2 plasma membrane expression in wild-type mice, secretin-infused X-NDI mice received a single injection of fluvastatin. Interestingly, urine production by X-NDI mice treated with secretin plus fluvastatin was reduced by nearly 90% and the urine osmolality was doubled. Immunostaining showed that secretin increased intracellular stores of AQP2 and the addition of fluvastatin promoted AQP2 trafficking to the plasma membrane. Taken together, these findings open new perspectives for the pharmacological treatment of X-NDI. © 2014 International Society of Nephrology.

Objective: Among the pleiotropic effects of statins, we have previously reported that fluvastatin increases the amount of plasma membrane-expressed AQP2 in renal collecting duct cells both in vitro and in vivo, independently of vasopressin. This effect may be of potential clinical significance for the treatment of patients affected by nephrogenic diabetes insipidus forms caused by inactivating mutations of the vasopressin type 2 receptor. Here we report the effect of fluvastatin on AQP2 plasma membrane abundance on an adult male XNDI patient treated with statins.

A novel mutation in the Lamin A/C gene (LMNA c.418_438dup) was detected in the index patient and in additional family members with diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) and history of sudden cardiac death. The functional characterization of this LMNA mutant was performed in cultured HL-1 cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs and subjected to confocal microscopy analysis and hypoxic stress conditions in 100% N2 for 8h. Mutated LMNA was clearly expressed in aggregates of different sizes and not uniformly distributed along the nuclear envelope as WT LMNA. Moreover, the mutated LMNA variant causes perturbation in nuclear shape and Nuclear Pore Complexes organization. Of note, we observed that under hypoxic conditions nuclear envelopes expressing mutated LMNA become leaky, leading a nuclear fluorescent marker to escape into the cytoplasm. This indicates that, under cell stressing conditions, the nucleo-cytoplasmic compartmentalization is affected in cardiomyocytes expressing this LMNA mutation, inducing, as final fatal consequence, cell apoptosis. In conclusion, we not only open new avenues to gain more insights in the pathogenesis of ARVC and to conceive novel therapeutic strategies but we also shed lights on the role of nuclear Lamin A in the physio-pathology of cardiomyocytes.

The antidiuretic hormone vasopressin regulates water reabsorption in the nephron by inducing apical plasma membrane exocytosis of the water channel aquaporin 2 in the kidney collecting duct principal cells. Disruption of this physiological mechanism by genetic alteration of either the vasopressin type 2 receptor gene or the aquaporin 2 gene, results in a rare genetic disorder known as nephrogenic diabetes insipidus, which main hallmark are polyuria and polydipsia. Over the last decades, analysis of patients affected by this disease helped genetists, clinicians, cell and molecular biologists and pharmacologists to better understand the physiology of water reabsorption in the kidney, the molecular basis of the disease and to propose protocols for rapid diagnosis and pharmacological handling of the disease. Much still remains to be done in terms of targeted therapy to make sure that these patients benefit from an improved quality of life. In this article we provide an overview on the most recent strategies under investigation for rescuing the mutated gene products activity or for bypassing defective vasopressin receptor signaling.

Background: The kidney-specific isoform of the Na-K-2Cl cotransporter NKCC2 is involved in the Na+ reabsorption in the TAL cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Indeed both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for hypertension treatment. Accordingly a high-throughput screening for NKCC inhibitors is extremely useful in the development and characterization of new anti-hypertensive drugs. So far the high-throughput screening of NKCC transporters activity has been done by 86Rb+ influx assays. Methods: We developed a Tallium (Tl+) based fluorescent influx assay that can accurately and rapidly measure NKCC transport activity in adherent epithelial cells in the high-throughput Flex station device (FLEXA). We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). Results: In the absence of Cl- in the assay buffer, Tl+ addition did not induce any increase in fluorescence. However a robust Tl+ influx was observed after Cl-addition in c-NKCC2 transfected cells but not in mock-transfected or in parental LLC-PK1 cells suggesting that the Tl+ influx is actually mediated by the c-NKCC2 cotransporter. The c-NKCC2-driven FLEXA signal displays a rapid linear increased phase within the first 20 s after Cl- addition followed by a slower increase and a plateau phase. The initial rate of Tl+-dependent Cl- influx observed in c-NKCC2 transfected cells is about 3-fold over the background signal in mock-transfected LLC-PK1 cells. The preincubation with furosemide prevented the Cl-dependent Tl+ influx confirming the specificity of the NKCC-mediated Tl+ influx. Conclusions: We demonstrated that this assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, as a continuous assay, it offers improved sensitivity over endpoint NKCC functional assay. 

Background: The kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na+ reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension. Results: Taking advantage of an existing Thallium (Tl+)-based kit, we set up a Tl+ influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays. Conclusions: So far the screening of NKCC transporters activity has been done by 86Rb+ influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs.

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 INFORMATION: 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. RESULTS: In this study we used a well-characterized NKCC2 construct (c-NKCC2) to identify NKCC2 interacting proteins 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 c-NKCC2 internalization but strongly reduces exocytosis of the co-transporter. CONCLUSIONS: 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.

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.

Objective: Bardet-Biedl syndrome (BBS) is a rare genetic disorder whose clinical features include renal abnormalities, which ranges from renal malformations to renal failure. Polyuria and iso-hyposthenuria are common renal dysfunctions in BBS patients even in the presence of normal GFR. The mechanism underlying this defect is unknown and no genotype-phenotype correlation has yet been reported. Here we report four BBS patients showing different renal phenotypes: one had polyuria with hyposthenuria associated with mutation of BBS10, while three patients with normal urine concentrating ability had mutations in BBS1.

Bardet-Biedl syndrome (BBS) is a autosomal-recessive ciliopathy characterized by defects in multiple organ systems. The clinical phenotype consists of major features that are considered the hallmarks of the disorder: retinal degeneration, obesity, hypogonadism, polydactyly, mental retardation, and renal dysfunction. In particular, polyuria and polydipsia, with impairment of renal concentration capacity, are the earliest signs of renal dysfunction. Multiple lines of evidence have indicated that the BBS phenotype is largely a consequence of ciliary dysfunction since most BBS proteins localize to the basal body and the ciliary axoneme. However, recent evidences revealed that BBS proteins might also be involved in non-ciliary-related microtubule-based transport. Among the 14 identified genes (BBS1-14), mutated in BBS patients, BBS10 alone contribute approximately 20% of all known mutations. In order to investigate whether the polyuria associated with BBS might be related to a defect in the shuttling of the water channel AQP2 in the kidney collecting duct, we studied the effect of selective BBS10 knockdown in AQP2-expressing renal cells. Interestingly, apical surface biotinylation indicated that BBS10 siRNA dramatically and specifically prevented the forskolin-induced exocytosis of AQP2 at the apical membrane. In the same experimental condition, immunofluorescence followed by confocal analysis showed that BBS10 silencing strongly affected the organization of the microtubules cytoskeleton within the cell. As a consequence, we observed that, upon FK, treatment AQP2 mostly redistributed to the basolateral membrane with negligible increase at the apical membrane. Taken together, these results suggest that lost of proper microtubule-based polarized transport in the collecting duct cells cause basolateral misrouting of AQP2 and might explain the polyuria associated with mutations of BBS10 causing BBS.

Background: Bardet-Biedl syndrome (BBS) is a autosomal-recessive ciliopathy characterized by defects in multiple organ systems causing retinal degeneration, obesity, hypogonadism, polydactyly, mental retardation, and renal dysfunction. In particular, polyuria and polydipsia, with impairment of renal concentration ability, are the earliest signs of renal dysfunction.

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.

OBJECTIVE:: The progression from prehypertensive to hypertensive state in spontaneous hypertensive rats (SHRs) is accompanied by a significant increase in membrane expression of Na-K-2Cl co-transporter isoform 2 (NKCC2), suggesting that the altered NKCC2 trafficking and activity are directly related with the development of hypertension in this strain. The aim of this work is to gain insights on the molecular mechanism that underlies this phenomenon. METHODS:: We performed a comparative analysis of NKCC2 multiprotein complexes (MPCs) in the kidney of SHRs versus Wistar Kyoto rats by Blue Native difference gel electrophoresis combined with mass spectrometry. RESULTS:: We found that the recruitment of the β-subunit isoform 1 of the Na+-K +-ATPase (β1NK) in NKCC2 MPCs was significantly increased in the kidneys of SHR compared with Wistar Kyoto rat control strain. Co-immunoprecipitation experiments showed that β1NK actually interacts with NKCC2 in the native tissue. The analysis of the physiological role of β1NK-NKCC2 interaction in human embryonic kidney cells showed that β1NK increased the steady-state membrane expression and activity of NKCC2 enhancing NKCC2 trafficking toward the plasma membrane. CONCLUSION:: We identify a new NKCC2-interacting partner involved in the modulation of NKCC2 intracellular trafficking and possibly involved in the regulation of blood pressure. © 2014 Wolters Kluwer Health Lippincott Williams & Wilkins.

The central role of Na+–K+–2Cl− cotransporter type 2 (NKCC2) in vectorial transepithelial salt reabsorption in thick ascending limb cells from Henle’s loop in the kidney is evidenced by the effects of loop diuretics, the pharmacological inhibitors of NKCC2, that are amongst the most powerful antihypertensive drugs available to date. Moreover, genetic mutations of the NKCC2 encoding gene resulting in impaired apical targeting and function of NKCC2 transporter give rise to a pathological phenotype known as type I Bartter syndrome, characterised by a severe volume depletion, hypokalaemia and metabolic alkalosis with high prenatal mortality. On the contrary, excessive NKCC2 activity has been linkedwith inherited hypertension in humans and in rodent models. Interestingly, in animal models of hypertension, NKCC2 upregulation is achieved by post-translational mechanisms underlining the need to analyse the molecular mechanisms involved in the regulation of NKCC2 trafficking and activity to gain insights in the pathogenesis of hypertension.

The mutation gain-of-function A843E of the Calcium Sensing Receptor (CASR) causes a Bartter syndrome type V. Patients carrying this CASR variant showed a remarkable reduced renal NaCl reabsorption in Thick Ascendent Limb (TAL) resulting in renal loss of NaCl in the absence of mutations in renal Na+ and Cl- ion transporters. The molecular mechanisms underlying this clinical phenotype are still unknown. Indeed, we investigated, in LLC-PK1 epithelial renal cells, the possible functional cross-talk of CASR-A843E mutant with NKCC2, the main transporter involved in the NaCl reabsorption in the TAL. The steady-state NKCC2 phosphorylation and activity were inhibited by about 60% in cells transfected with CASR-A843E mutant compared to CASR WT-transfected cells, used as control. Of note, the low-chloride-dependent NKCC2 activation was also strongly inhibited upon the expression of CASR-A843E mutant. Interestingly the maximal activation of CASR WT mimicked the effect of CASR-A843E on NKCC2 regulation. The expression of the CASR mutant did not alter the apical localization of NKCC2 in LLC-PK1 cells, suggesting that the CASR-A843E affected intracellular pathway/s modulating NKCC2 activity rather than NKCC2 intracellular trafficking. Our findings opened new avenues not only to gain more insight in the role of CASR in the physiopathology of the kidney, but also to conceive novel therapeutic strategies for the treatment of the Bartter syndrome type V.

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.

Nephrogenic Diabetes Insipidus (NDI) is a common complication of lithium (Li+) treatment. Li+ inhibits vasopressin-stimulated translocation of the water channel aquaporin 2 (AQP2) to the apical membrane of kidney collecting duct (CD) principal cells, thereby limiting water reabsorption in the CD (1). Long-term exposure to Li+ may also down-regulate AQP2 gene expression (2). This results in the excretion of a large volume of hypoosmotic urine. We previously showed that statins increase AQP2 abundance at the apical plasma membrane of CD principal cells in vivo in a mouse model of genetically-induced NDI (3). The preliminary data presented in the Letter by Elie et al., unambiguously showed that statins attenuate the drop in urine osmolality, observed also in Li+-induced NDI. They showed that none of the statins users observed in their study, had urine osmolality lower than 300mOsm/Kg during Li+ treatment. In line of principle, this is predictable on the base of the mechanism of action of statins on AQP2 trafficking. This interesting, preliminary evidence is of great therapeutic importance, for statins might help attenuating the side effects of lithium therapy in the kidney. Further studies in Li+-treated animals and measurements of AQP2 urine excretion in patients, are necessary to understand the mechanism by which statins counteract the drop of urine osmolality in Li-induced NDI.

Lamin A/C is a structural protein of the nuclear envelope (NE) and cardiac involvement in Lamin A/C mutations was one of the first phenotypes to be reported in humans, suggesting a crucial role of this protein in the cardiomyocytes function. Mutations in LMNA gene cause a class of pathologies generically named 'Lamanopathies' mainly involving heart and skeletal muscles. Moreover, the well-known disease called Hutchinson-Gilford Progeria Syndrome due to extensive mutations in LMNA gene, in addition to the systemic phenotype of premature aging, is characterised by the death of patients at around 13 typically for a heart attack or stroke, suggesting again the heart as the main site sensitive to Lamin A/C disfunction. Indeed, the identification of the roles of the Lamin A/C in cardiomyocytes function is a key area of exploration. One of the primary biological roles recently conferred to Lamin A/C is to affect contractile cells lineage determination and senescence. Then, in differentiated adult cardiomyocytes both the 'structural' and 'gene expression hypothesis' could explain the role of Lamin A in the function of cardiomyocytes. In fact, recent advances in the field propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can 'consequently' alter gene expression. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France.

Aquaporin 2 (AQP2) is the vasopressin-regulated water channel palying a crucial role in urine concentration in the kidney. Rosiglitazone (RGZ), an agonist of the peroxisome proliferator-activated receptor gamma (PPARγ), increases AQP2 expression in normal rats. Here we tested whether RGZ could also modulate AQP2 intracellular trafficking in renal cells. Wild type C57BL-6 mice were treated for 3 days with 20mg/Kg RGZ to confirm the transcriptional effect on AQP2 in mice. In parallel, we used mouse MCD4 renal collecting duct cells to study the effect of RGZ on AQP2 trafficking to the apical membrane. Apical surface biotinylation and confocal analysis were used to semiquantify the effect. Single cell live microfluorimetry and FRET were used to measure changes in Ca2+ and cAMP intracellular concentrations, respectively. In mice, RGZ induced a twofold increase of AQP2 compared to control mice. In MCD4 cells, stimulation with 50µM RGZ for 30 min robustly increased AQP2 trafficking to the apical plasma membrane to an extent comparable to that elicited by forskolin stimulation. Interestingly, RGZ was able to increasely AQP2 phosphorylation at Ser256 concomitant with intracellular Ca++ increase and without apparent intracellular cAMP elevation. Taken together these results suggest that RGZ might be useful to increase AQP2 transcription and apical trafficking in renal cells bypassing the stimulation of vasopressin receptor which is defective in pathological conditions as in Nephrogenic Diabetes Insipidus.

Preliminary results by our group showed that exposure to Rosiglitazone (RGZ) induces phosphorylation and apical translocation of AQP2 in mouse collecting duct clone 4 (MCD4) cells. Here we studied the effect of short-term exposure to 50 µM RGZ on cAMP- and Ca2+-mediated signaling pathways, both key players for AQP2-mediated water reabsorption in the collecting duct. Cytosolic Ca2+ and cAMP levels were imaged in real-time in MCD4 cells loaded with Fura-2 or transiently transfected with the EPAC-based fluorescent probe H90, respectively. Physiologically, AQP2 phosphorylation/translocation depends on cytosolic cAMP levels. Nonetheless, cAMP measurements showed that RGZ did not induce significant changes in cAMP levels. Conversely, 20 minutes RGZ stimulation of Fura-2 loaded MCD4 cells induced a large, transient cytosolic Ca2+ peak that was not the result of direct blockade of the SERCA pump since the rate of store empting elicited by CPA in the absence of external Ca2+ was not significantly different in the presence of RGZ. Importantly, removal of external Ca2+ and inhibition of Ca2+ channels with ruthenium red prevented the RGZ-induced increase in cytosolic Ca2+ indicating a prominent role for Ca2+ entry at the plasma membrane. In conclusion, RGZ-induced AQP2 phosphorylation/translocation process is likely initiated by a fast, large extracellular Ca2+ influx most likely via Ca2+-dependent transient receptor potential channels. Further studies to ascertain which cascade of kinases is involved in this scenario are in progress.

Il diabete insipido nefrogenico (NDI) è una patologia caratterizzata dalla incapacità del rene a concentrare le urine in risposta all'ormone antidiuretico, vasopressina. Di conseguenza, il paziente NDI produce un enorme volume di urine, è cronicamente assetato ed a rischio di disidratazione. La forma congenita di NDI più comune è dovuta alla mutazione del gene che produce un recettore della vasopressina (V2R) non correttamente funzionante. Il bersaglio della vasopressina nel rene è il canale per l'acqua AQP2 che permette il riassorbimento di acqua renale per cui quando il recettore V2R è alterato, l'AQP2 non è in grado di svolgere il suo ruolo fisiologico. Nessuna delle terapie correnti è risolutiva o mirata al recupero della funzionalità dell' AQP2. Il nostro gruppo ha chiare evidenze sperimentali che le statine, largamente usate nella cura della ipercolesterolemia, sono in grado di recuperare la funzionalità della AQP2 indipendentemente dalla vasopressina. Abbiamo inoltre evidenze sperimentali che la secretina, un ormone coinvolto nel processo digestivo, è in grado di stimolare il proprio recettore SCTR sulle cellule renali producendo un effetto sull’AQP2 simile a quello indotto dalla vasopressina. Di grande importanza è l’osservazione che la somministrazione combinata di fluvastatina e secretina migliora notevolmente i sintomi della malattia nei topi affetti da NDI. Queste osservazioni gettano le basi per l’elaborazione di nuovi interventi terapeutici per il trattamento dell’NDI nell’uomo.

Abstract of the project and progress report X-linked nephrogenic diabetes insipidus (X-NDI) is a rare disease characterized by resistance of the kidney to the action of the antidiuretic hormone arginine-vasopressin (AVP) and is accompanied by severe polyuria and risk of dehydration. It is caused by inactivating mutations in the AVP type 2 receptor (V2R) gene that prevent the AVP-induced trafficking of the water channel AQP2 to the luminal plasma membrane of the kidney collecting duct (CD), and, consequently, reduces kidney concentration ability. In addition, as AVP also regulates AQP2 gene transcription, AQP2 protein is very low in NDI. This represents one of the principal limitations toward the establishment of an AQP2-targeted therapy that could bypass AVP signaling in NDI patients. Even if expressed at the plasma membrane, the residual amount of AQP2 expressed by an NDI patient might be insufficient to guarantee proper water reabsorption. These considerations prompted us to explore alternative signaling pathways that could restore physiological levels of AQP2 in the CD. One of the best candidates that could fulfill this purpose is the secretin receptor (SCTR), a G-proteins-coupled receptor (GPCR) that, like V2R, elevates intracellular cAMP levels upon binding its ligand secretin (SCT), a hormone physiologically released by the intestinal mucosa. We report here that SCTR is functionally expressed at the basolateral membrane of kidney collecting duct principal cells. To investigate a possible beneficial effect of SCT on NDI phenotype, X-NDI mice, the only viable mouse model of X-linked NDI, were subcutaneously infused with SCT (1nmol/Kg/day, via osmotic minipumps) for 14 days. Urinary parameters were not altered in SCT-infused animals. Interestingly, however, SCT significantly increased AQP2 levels in the CD, although the protein was mostly accumulated in the intracellular storage vesicles and hardly detectable at the plasma membrane. We previously reported that fluvastatin (Flu) treatment, mimicking the action of AVP, transiently increases AQP2 abundance at the luminal membrane of the kidney CD in vivo in wild type mice in the absence of AVP stimulation. SCT-infused X-NDI mice were injected intraperitoneally with Flu (50 mg/Kg). Diuresis and urine osmolality were monitored for 6 hours after Flu injection. Interestingly, Flu was able to potentiate the effect of SCT: cumulative diuresis was reduced by nearly 90% in mice treated with SCT+Flu and, accordingly, urine osmolality almost doubled in the same interval of time. Immunofluorescence confirmed that SCT increased intracellular stores of AQP2 and the addition of Flu promoted AQP2 accumulation at the plasma membrane. Taken together these data strongly indicate that the combination of SCT and Flu might represent a new and effective pharmacological treatment for X-NDI.

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

Mutations in the Lamin A/C gene (LMNA), which encodes A-type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co-segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease-causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP- (or mCherry)-tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK-CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry-R321X also induced impaired ER Ca(2+) handling, reduced capacitative Ca(2+) entry at the plasma membrane and abnormal nuclear Ca(2+) dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.

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.

Scopo della presente invenzione è l'uso di agonisti selettivi dei recettori beta-adrenergici di tipo 3 (BAR3) nel trattamento della malattia genetica rara diabete insipido nefrogenico (NDI), in particolare del diabete insipido nefrogenico X-legato (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 aumentare la quantità del canale per l’acqua acquaporina 2 (AQP2) sulla membrana plasmatica. Ciò impedisce il riassorbimento di acqua nel dotto collettore dei reni (antidiuresi) che porta a una grave poliuria e disidratazione. Pertanto, scopo della presente invenzione è fornire una molecola alternativa che siano utilizzate nel trattamento del diabete insipido nefrogenico (NDI), in particolare del diabete insipido nefrogenico X-legato (X-NDI), che consenta il ripristino di livelli normali di acquaporina 2 ( AQP2) sulla membrana luminale delle cellule principali del dotto collettore renale. L'uso di agonisti selettivi di BAR3, tra cui il composto BRL37344 adatto all'uso su roditori e il composto YM-178 già testato nell'uomo, porta alla cura di tale condizione. Secondo la presente invenzione è stato sorprendentemente osservato che, nel tubulo renale murino, le cellule del tratto spesso ascendente ascendente (TAL) dell’ansa e del dotto collettore corticale (CCD) esprimono i recettori beta-adrenergici di tipo 3 (BAR3). Questi recettori agiscono con lo stesso meccanismo di trasduzione del segnale attivato dal recettore per la vasopressina AVPR2. Si è visto che, stimolando i recettori BAR3 anche in soggetti con la condizione sopra descritta di diabete insipido nefrogenico X-linked (X-NDI), è possibile aggirare l'inattivazione del recettore AVPR2 e, totalmente o in parte, ripristinare un fenotipo normale.

Object of the present invention is the use of selective agonists of beta-adrenergic type 3 receptors (BAR3) in the treatment of nephrogenic diabetes insipidus (NDI), in particular of X-linked nephrogenic diabetes insipidus (X-NDI).

Nephrogenic diabetes insipidus is treated with statins