Na(+) -K(+) -2Cl(-) cotransporter type 2 trafficking and activity: The role of interacting proteins.

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

Novel anticancer drug 5h-pyro[3,2-a] phenoxazin-5-one (PPH) regulates lncRNA HOTAIR and HOXC genes in human MCF-7 cells

Breast cancer in women is the second most commonly cancer, after skin cancer. The percentage of mortality risk for breast cancer is 1 in 37 women (2.7%), which makes breast cancer represent the second cause of cancer death in women. Recently, new research based on previously published work in systemic chemotherapy and endocrine therapy field, have improved the incidence rates. The quinonic nucleus is common to many natural and synthetic products associated with anticancer and antibacterial activities, these compounds are typically DNA-intercalating agents. The Class I Homeobox genes (HOX in human and hox in mouse) control embryonic development and specific determination of positional identity anteroposterior axis of the human body. The HOX genes, are 39 transcription factors related to morphological, physiological disease. It has been demonstrated that any deregulation into the network is able to induce neoplastic transformation. Particularly, HOXC locus collaborating with lncRNA HOTAIR play a key role in breast cancer. In this study, our group evaluated the chemical and metabolic stability of new anticancer molecule 5H-pyro[3,2-a] phenoxazin-5-one (PPH). In a recent paper, we have already demonstrated that a new and potent anticancer synthetic iminoquinone, the 5H-pyrido[3,2-a]phenoxazin-5-one (PPH), is able to inhibit a large number of lymphoblastoid and solid-tumor-derived cells at submicromolar concentrations. Based on our previous research, we decided to analyze the cytotoxic effect and capability of PPH to control the lncRNA HOTAIR and HOXC locus gene expression in human breast cancer cells MCF-7, in order to demonstrate its role like potential new breast cancer antitumor drug

A novel formulation of glucose‐sparing peritoneal dialysis solutions with l‐carnitine improves biocompatibility on human mesothelial cells

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end‐stage renal disease (ESRD) lies in the fact that the currently used glucose‐based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and L‐carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro‐inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro‐inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility

Novel Anticancer Drug 5H-pyro[3,2-a] Phenoxazin-5-one (PPH) Regulates lncRNA HOTAIR and HOXC genes in Human MCF-7 Cells

Breast cancer in women is the second most commonly cancer, after skin cancer. The percentage of mortalityrisk for breast cancer is 1 in 37 women (2.7%), which makes breast cancer represent the second cause of cancerdeath in women. Recently, new research based on previously published work in systemic chemotherapy andendocrine therapy field, have improved the incidence rates. The quinonic nucleus is common to many naturaland synthetic products associated with anticancer and antibacterial activities, these compounds are typicallyDNA-intercalating agents. The Class I Homeobox genes (HOX in human and hox in mouse) control embryonicdevelopment and specific determination of positional identity anteroposterior axis of the human body. The HOXgenes, are 39 transcription factors related to morphological, physiological disease. It has been demonstratedthat any deregulation into the network is able to induce neoplastic transformation. Particularly, HOXC locuscollaborating with lncRNA HOTAIR play a key role in breast cancer. In this study, our group evaluated the chemical and metabolic stability of new anticancer molecule 5H-pyro[3,2-a] phenoxazin-5-one (PPH). In a recent paper, we have already demonstrated that a new and potent anticancersynthetic iminoquinone, the 5H-pyrido[3,2-a]phenoxazin-5-one (PPH), is able to inhibit a large number oflymphoblastoid and solid-tumor-derived cells at submicromolar concentrations. Based on our previous research, we decided to analyze the cytotoxic effect and capability of PPH to control thelncRNA HOTAIR and HOXC locus gene expression in human breast cancer cells MCF-7, in order to demonstrateits role like potential new breast cancer antitumor drug

NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells.

The gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop 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 incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na(+):K(+):2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL. RESULTS: The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl(-)-dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 ω-hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites. CONCLUSIONS: The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5

Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome

The K+ voltage-gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K+ current. The aim of this study was to characterize the biophysical properties of a C-terminus-truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12-lead electrocardiogram, transthoracic echocardiography and 24-hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co-expressed with KCNH2, (b) G1006fs/49 exerts a dominant-negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage-sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic