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Hypertension is one of the major health problems leading to the development of cardiovascular diseases. Despite a rapid expansion in global hypertension prevalence, molecular mechanisms leading to hypertension are not fully understood largely due to the complexity of pathogenesis involving several factors. Salt intake is recognized as a leading determinant of blood pressure, since reduced dietary salt intake is related to lower morbidity and mortality, and hypertension in relation to cardiovascular events. Compared with salt-resistant populations, salt-sensitive individuals exhibit high sensitivity in blood pressure responses according to changes in salt intake. In this setting, the kidney plays a major role in the maintenance of blood pressure under the hormonal control of the renin–angiotensin–aldosterone system. In the present review, we summarize the current overview on the molecular mechanisms for modulation of blood pressure associated with renal ion channels/transporters including sodium–hydrogen exchanger isoform 3 (NHE3), Na+-K+-2Cl– cotransporter (NKCC2), sodium–chloride cotransporter (NCC), epithelial sodium channel (ENaC) and pendrin expressed in different nephron segments. In particular, recent studies on experimental animal models with deletion of renal ion channels led to the identification of several crucial physiological mechanisms and molecules involved in hypertension. These findings could further provide a potential for novel therapeutic approaches applicable on human patients with hypertension.

BACKGROUND: Mutations in SLC37A4, which encodes the intracellular glucose transporter G6PT, cause the rare glycogen storage disease type 1b (GSD1b). A long-term consequence of GSD1b is kidney failure, which requires KRT. The main protein markers of proximal tubule function, including NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, are downregulated as part of the disease phenotype.METHODS: We utilized an inducible mouse model of GSD1b, TM-G6PT-/-, to show that glycogen accumulation plays a crucial role in altering proximal tubule morphology and function. To limit glucose entry into proximal tubule cells and thus to prevent glycogen accumulation, we administered an SGLT2-inhibitor, dapagliflozin, to TM-G6PT-/- mice.RESULTS: In proximal tubule cells, G6PT suppression stimulates the upregulation and activity of hexokinase-I, which increases availability of the reabsorbed glucose for intracellular metabolism. Dapagliflozin prevented glycogen accumulation and improved kidney morphology by promoting a metabolic switch from glycogen synthesis toward lysis and by restoring expression levels of the main proximal tubule functional markers.CONCLUSION: We provide proof of concept for the efficacy of dapagliflozin in preserving kidney function in GSD1b mice. Our findings could represent the basis for repurposing this drug to treat patients with GSD1b.

EAST/SeSAME syndrome is a rare disease affecting the Central Nervous System (CNS), inner ear, and kidney. The syndrome is due to loss-of-function mutations in the KCNJ10 gene encoding the inward-rectifying potassium channel Kir4.1. EAST/SeSAME syndrome is mainly diagnosed during childhood with a tonic-clonic seizure being the usual first symptom. Due to a limited number of patients and recent identification of the disease, few data are available on the clinical progress of this disease in adulthood. In particular, neurologic and nephrological outcomes have not been reported. We present a case series of 4 adult patients harbouring homozygous missense mutation p.Ala167Val and homozygous frameshift mutations p.Asn232Glnfs*14 and p.Gly275Valfs*7. Effects of these mutations were predicted by in silico modelling and bioinformatic tools. Patients with truncating mutations were associated with more severe outcomes, both in tubulopathy severity and neurological symptomatology. Conversely, either missense or truncating mutations were correlated with similar severity of epilepsy, with a long free-of-event period up to 20 years old. No eGFR decline was documented. Modelling predicted that truncating mutations lead to complete Kir4.1 dysfunction. Finally, all patients had a mild increase in urinary protein excretion. Our study indicates that the prognosis of patients suffering from EAST/SeSAME syndrome is related to the severity of the mutation causing the disease. As predicted by in silico modelling, truncating mutations of KCNJ10 are associated with more severe disease, with recurrence of symptomatic hypokalemia and more severe neurological phenotype. The type of mutation should be considered for the therapy tailored to patients' phenotype.

Background and Aims Kidney plays a central role on the maintenance of water homeostasis, acid-base and electrolytes balances through the activity of different types of ion channels/transporters expressed along the nephron segments. Mutations in genes encoding these transporters could subsequently lead to aberrant transporter activities, resulting in abnormal renal handling of electrolytes, thus represent monogenic form of rare kidney diseases. Accumulating number of mutations identified in genes responsible for such monogenic disorders demonstrated that eventual disease phenotypes may vary according to the type and localization of the mutation within the gene. Thus, careful evaluation of gene variation would be crucial prior to designing the strategy for the therapy in each case. Here we present various mutations from our patients, identified in genes including kcnj10, SLC12A1, SLC26A4 and clcn7 which are associated with rare tubulopathies EAST/SeSAME syndrome, Bartter’s syndrome, Pendred syndrome and Fanconi syndrome, respectively. In order to explore molecular mechanisms underlying the observed disease conditions in our patients, we have performed computational modeling analyses of these mutations in comparison with wild-type models. Method Three-dimensional homology models of Kir4.1, NKCC2, Pendrin and CLC-7 proteins were generated by Swiss-Model protein structure homology-modelling server (http://swissmodel. expasy.org) and I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). Disease-related mutations including novel mutations identified from our patients were mapped onto the three-dimensional models and compared with wild-type models in terms of atomic interactions as well as secondary, tertiary and quaternary structures. Furthermore, we assessed possible effects of missense mutations on the function of ion channels/transporters using online bioinformatic prediction tools PolyPhen-2, Mutation taster, PROVEAN and SIFT. Results The three-dimensional model comparison between wild-type Kir4.1 and Ala167Val variant, which is related to EAST/SeSAME syndrome, revealed that Ala167Val located at the junction between transmembrane domain 2 (TM2) and C-terminus is predicted not to interrupt the sequence of the hydrogen bonds, thus not altering the TM2 alpha-helix structure. In addition, while PolyPhen-2 and Mutation taster evaluated Ala167Val as ‘probably damaging’, PROVEAN and SIFT predicted Ala167Val as ‘neutral’ and ‘tolerated’. These observations are in line with the clinical data demonstrating the milder phenotype in patients with Ala167Val mutation compared with the ones harboring frameshift mutations leading to truncated Kir4.1 channel (Figure. 1). Furthermore, computational modeling of wild-type NKCC2 and frameshift mutation Arg302Glyfs*3 variant clearly demonstrated that Arg302Glyfs*3 results in a loss of large part of the protein, indicating that NKCC2-Arg302Glyfs*3 is practically nonfunctional (Figure. 2). Conclusion Computational modeling of disease-related mutations in various ion channels/transporters represents a novel, powerful approach for comprehensive interpretation of the disease phenotypes observed in patients with rare tubulopathies. In addition, combination of in silico modeling and clinical data could provide us with further insight into molecular mechanisms underlying the renal transporter activities. Furthermore, this in silico computational modeling approach can be applicable and suggestive for novel pharmacological intervention as well as the visual disease severity assessment.

Nephrogenic diabetes insipidus (NDI) is a rare tubulopathy characterized by urinary concentration defect due to renal resistance to vasopressin. Loss-of-function mutations of vasopressin V2 receptor (V2R) gene (AVPR2) is the most common cause of the disease. We have identified five novel mutations L86P, R113Q, C192S, M272R, and W323_I324insR from NDI-affected patients. Functional characterization of these mutants revealed that R113Q and C192S were normally localized at the basolateral membrane of polarized Madin-Darby Canine Kidney (MDCK) cells and presented proper glycosylation maturation. On the other side, L86P, M272R, and W323_I324insR mutants were retained in endoplasmic reticulum and exhibited immature glycosylation and considerably reduced stability. All five mutants were resistant to administration of vasopressin analogues as evaluated by defective response in cAMP release. In order to rescue the function of the mutated V2R, we tested VX-809, sildenafil citrate, ibuprofen and tolvaptan in MDCK cells. Among these, tolvaptan was effective in rescuing the function of M272R mutation, by both allowing proper glycosylation maturation, membrane sorting and response to dDAVP. These results show an important proof of concept for the use of tolvaptan in patients affected by M272R mutation of V2R causing NDI.

Mitogen-activated protein kinases (MAPKs) are intracellular molecules regulating a wide range of cellular functions, including proliferation, differentiation, apoptosis, cytoskeleton remodeling and cytokine production. MAPK activity has been shown in normal kidney, and its over-activation has been demonstrated in several renal diseases. The extracellular signal-regulated protein kinases (ERK 1,2) signalling pathway is the first described MAPK signaling. Intensive investigations have demonstrated that it participates in the regulation of ureteric bud branching, a fundamental process in establishing final nephron number; in addition, it is also involved in the differentiation of the nephrogenic mesenchyme, indicating a key role in mammalian kidney embryonic development. In the present manuscript, we show that ERK1,2 signalling mediates several cellular functions also in mature kidney, describing its role along the nephron and demonstrating whether it contributes to the regulation of ion channels and transporters implicated in acid-base and electrolytes homeostasis.

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 &times, 105 M-1 s-1 against paraoxon hydrolysis at 70&deg, C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 &Aring, We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

We developed a liquid phase ZnO nanoparticles (NPs) fabrication method which employed the cage-shaped protein apoferritin as a biotemplate. The suppression of the chemical reaction outside apoferritin by stabilizing Zn2+ with ammonia allowed the selective NP synthesis inside the cavity at room temperature. The composition of synthesized NPs was confirmed to be that of ZnO, and their size was determined to be 5.1 ± 0.8 nm in diameter through energy-dispersive X-ray spectroscopy and high-resolution transmission electron microscopy. In-depth studies about the effect of anion species on the NP formation proved that the anion introduction through the channels was competitive and also that the decrease in hydroxide ion (OH–) concentration ratio in the reaction solution led to less ZnO NPs formation.

We developed an environmentally friendly method for the synthesis of cerium (Ce) oxide nanoparticles (NPs) and their two- and three-dimensional array formation using the cage shaped protein apoferr...