Salt Therapy as a Complementary Method for the Treatment of Respiratory Tract Diseases, With a Focus on Mold-Related Illness.

Salt therapy has been used for millennia, but modern salt therapy can be traced to the salt mines and caves in Europe and Russia from the early 19th century. Today, breathing in the microclimate of caves with their stable air temperature and moderate to high humidity in the presence of sodium, potassium, magnesium and calcium and the absence of airborne pollutants and pollen is called speleotherapy. The inhalation of natural pure sodium chloride (NaCl) in a controlled environment (air temperature 18° to 24°C and relative humidity 40% to 60%) is called halotherapy. The main active ingredient in halo- and speleotherapy is NaCl aerosol particles, which penetrate all layers of the respiratory tract. In addition to their antibacterial and anti-inflammatory properties, salt particles also facilitate mucociliary transport and reduce immunogloblin E (IgE) levels. Clinical trials have confirmed that salt therapy is an effective option for relieving symptoms and improving functional parameters in sinusitis, bronchiectasis, chronic bronchitis, mild and moderate asthma and chronic obstructive pulmonary disease (COPD). Rinsing with hypertonic saline has been found to be beneficial in reducing airway inflammation in patients with bronchiolitis. In addition to avoidance, salt therapy should be recommended as a complementary therapy in patients with prolonged exposure to indoor air dampness microbiota, which may cause damage to the respiratory mucosa. Salt therapy is safe and well tolerated.

Cold Saline Perfusion before Ischemia-Reperfusion Is Harmful to the Kidney and Is Associated with the Loss of Ezrin, a Cytoskeletal Protein, in Rats.

BACKGROUND: Organ protection for transplantation is perfusion with ice-cold preservation solutions, although saline is also used in animal experiments and living donor transplantations. However, ice-cold perfusion can contribute to initial graft injury. Our aim was to test if cytoskeletal damage of parenchymal cells is caused by saline itself or by the ice-cold solution. METHODS: F344 rat kidneys were flushed with cold (4 °C) saline, ischemic and sham kidneys were not perfused. In a separate set, F344 kidneys were flushed with saline or preservation solution at 4 or 15 °C. Ischemia time was 30 min. RESULTS: Renal injury was significantly more severe following cold ischemia (CI) than after ischemia-reperfusion without flushing (ischemia/reperfusion (I/R)). Functional and morphologic damage was accompanied by severe loss of ezrin from glomerular and tubular epithelial cells after CI. Moreover, saline caused serious injury independently from its temperature, while the perfusion solution was more beneficial, especially at 4 °C. CONCLUSIONS: Flushing the kidney with ice-cold saline can cause more severe injury than ischemia-reperfusion at body temperature even during a short (30 min) ischemia. Saline perfusion can prolong recovery from ischemia in kidney transplantation, which can be prevented by using preservation solutions.

Septins in kidney: A territory little explored.

Septins are a conserved family of GTP-binding proteins that assemble into cytoskeletal filaments to function in a highly sophisticated and physiologically regulated manner. Originally septins were discovered in the budding yeast as membrane-associated filaments that affect cell polarity and cytokinesis. In the last decades, much progress has been made in understanding the biochemical properties and cell biological functions of septins. In line with this, mammalian septins have been shown to be involved in various cellular processes, including regulation of cell polarity, cytoskeletal organization, vesicle trafficking, ciliogenesis, and cell-pathogen interactions. A growing number of studies have shown that septins play important roles in tissue and organ development and physiology; yet, little is known about their role in the kidney. In the following review, we discuss the structure and functions of septins in general and summarize the evidence for their presence and roles in the kidney.

Glucose Transporters in Diabetic Kidney Disease-Friends or Foes?

Diabetic kidney disease (DKD) is a major microvascular complication of diabetes and a common cause of end-stage renal disease worldwide. DKD manifests as an increased urinary protein excretion (albuminuria). Multiple studies have shown that insulin resistance correlates with the development of albuminuria in non-diabetic and diabetic patients. There is also accumulating evidence that glomerular epithelial cells or podocytes are insulin sensitive and that insulin signaling in podocytes is essential for maintaining normal kidney function. At the cellular level, the mechanisms leading to the development of insulin resistance include mutations in the insulin receptor gene, impairments in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, or perturbations in the trafficking of glucose transporters (GLUTs), which mediate the uptake of glucose into cells. Podocytes express several GLUTs, including GLUT1, GLUT2, GLUT3, GLUT4, and GLUT8. Of these, the most studied ones are GLUT1 and GLUT4, both shown to be insulin responsive in podocytes. In the basal state, GLUT4 is preferentially located in perinuclear and cytosolic vesicular structures and to a lesser extent at the plasma membrane. After insulin stimulation, GLUT4 is sorted into GLUT4-containing vesicles (GCVs) that translocate to the plasma membrane. GCV trafficking consists of several steps, including approaching of the GCVs to the plasma membrane, tethering, and docking, after which the lipid bilayers of the GCVs and the plasma membrane fuse, delivering GLUT4 to the cell surface for glucose uptake into the cell. Studies have revealed novel molecular regulators of the GLUT trafficking in podocytes and unraveled unexpected roles for GLUT1 and GLUT4 in the development of DKD, summarized in this review. These findings pave the way for better understanding of the mechanistic pathways associated with the development and progression of DKD and aid in the development of new treatments for this devastating disease.

Functional proteomics of cellular mechanosensing mechanisms.

Mechanosensing enables cells to coordinate their phenotype with the mechanical properties of their tissue microenvironment. In this process, cells probe their surroundings by applying contractile forces, which produces different amounts of mechanical strain within the cells as a function of the stiffness of their extracellular substrates. Tension within cells can then affect the structure and composition of most cellular organelles, including cell adhesions, the cytoskeleton, the plasma membrane and the nucleus. On a molecular level, the conformations, modifications, interactions, and subcellular localizations of proteins have been shown to be altered by biomechanical forces. Functional proteomics aims at the analysis of these effects in a proteome wide, unbiased and high throughput manner. Emerging methods, such as crosslinking mass spectrometry and advanced protein correlation profiling, will enable future analysis of mechanosensing on the level of protein interactions in situ, and subcellular protein localization, which can now be determined with very high accuracy from whole cell analysis for thousands of proteins at once. Combined use of these mass spectrometry toolsets with the analysis of posttranslational modifications will ultimately move the field to a comprehensive list of molecular alterations in cellular mechanosensing. We will give an overview on current developments in functional proteomics and the latest applications on questions related to mechanobiology.

Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion.

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane.

Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation.

Integrin-based adhesions play critical roles in cell migration. Talin activates integrins and flexibly connects integrins to the actomyosin cytoskeleton, thereby serving as a 'molecular clutch' that transmits forces to the extracellular matrix to drive cell migration. Here we identify the evolutionarily conserved Kank protein family as novel components of focal adhesions (FAs). Kank proteins accumulate at the lateral border of FAs, which we term the FA belt, and in central sliding adhesions, where they directly bind the talin rod domain through the Kank amino-terminal (KN) motif and induce talin and integrin activation. In addition, Kank proteins diminish the talin-actomyosin linkage, which curbs force transmission across integrins, leading to reduced integrin-ligand bond strength, slippage between integrin and ligand, central adhesion formation and sliding, and reduced cell migration speed. Our data identify Kank proteins as talin activators that decrease the grip between the integrin-talin complex and actomyosin to regulate cell migration velocity.

Ezrin is down-regulated in diabetic kidney glomeruli and regulates actin reorganization and glucose uptake via GLUT1 in cultured podocytes.

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes.

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking.

Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.