The role of urea in neuronal degeneration and sensitization: an in vitro model of uremic neuropathy

Background: Uremic neuropathy commonly affects patients with chronic kidney disease (CKD), with painful sensations in the feet, followed by numbness and weakness in the legs and hands. The symptoms usually resolve following kidney transplantation, but the mechanisms of uremic neuropathy and associated pain symptoms remain unknown. As blood urea levels are elevated inpatients with CKD, we examined the morphological and functional effects of clinically observed levels of urea on sensory neurons. Methods: Rat DRG neurons were treated with 10or50 mMol/L urea for 48 hours, fixed and immunostained for PGP9.5 and βIII tubulin immunofluorescence, ,. Neurons were also immunostained for TRPV1, TRPM8 and Gap43 expression, and the capsaic insensitivity of urea or vehicle treated neurons was determined.Results: Urea treated neurons had degenerating neurites with diminished PGP9.5 immunofluorescence,and swollen, retracted growth cones. βIII tubulin appeared clumped after urea treatment. Neurite lengths were significantly reduced to 60 ± 2.6%(10 mMol/L, **P<0.01), and to 56.2± 3.3 %, (50 mMol/L, **P<0.01),urea treatmentfor 48 hours, compared with control neurons. Fewer neurons survived urea treatment,with 70.08 ± 13.3% remaining after 10 mMol/L (*P<0.05), and 61.49 ± 7.4 % after 50 mMol/L ureatreatment (**P<0.01), compared with controls. The proportion of neurons expressing TRPV1 wasreduced after urea treatment, but not TRPM8 expressing neurons. In functional studies, treatment with urea resulted in dose-dependent neuronal sensitization.Capsaicinresponses were significantly increased to 115.29 ± 3.4%(10 mMol/L, **P<0.01) and 125.3 ± 4.2%(50 mMol/L,**P<0.01), compared with controls. Sensitization due to urea was eliminated in the presence of the TRPV1 inhibitor SB705498, the MEKinhibitor PD98059,the PI3 kinase inhibitor LY294002, and the TRPM8 inhibitor AMTB. ConclusionNeurite degenerationandsensitization are consistent with uremic neuropathy,, and provide a disease-relevant model to test new treatments

On-demand delivery of single DNA molecules using nanopipettes

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM

Nanoscale, Voltage-Driven Application of Bioactive Substances onto Cells with Organized Topography

With Scanning Ion Conductance Microscopy (SICM), a non-contact scanning probe technique, it is possible to obtain information about both the surface topography of live cells and to apply molecules onto specific nanoscale structures. The technique is therefore widely used to apply chemical compounds and to study the properties of molecules on the surface of various cell types. The heart muscle cells, the cardiomyocytes, possess a highly elaborate, unique surface topography including T-tubule openings leading into a cell internal system which exclusively har-bors many proteins necessary for the cells physiological function. Here we applied Isoproterenol into these surface openings by changing the applied voltage over the SICM nanopipette. To determine the grade of precision of our application we used finite element simulations to inves-tigate how the concentration profile varies over the cell surface. We first obtained topography scans of the cardiomyocytes using SICM and then determined the electrophoretic mobility of Isoproterenol in a high ion solution to be -7×10-9 m2/Vs. The simulations showed that the delivery to the T-tubule opening is highly confined to the underlying Z-groove and especially to the first T-tubule opening, where the concen-tration is approximately 6.5 times higher compared to on a flat surface under the same delivery settings. Delivery to the crest, instead of the T-tubule opening, resulted in a much lower concentration, emphasizing the importance of topography on agonist delivery. In conclusion SICM, unlike other techniques, can reliably deliver precise quantities of compounds to the T-tubules of cardiomyocyte

On-Demand Delivery of Single DNA Molecules Using Nanopipets

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM

Nanoscale Imaging of Primary Cilia with Scanning Ion Conductance Microscopy

Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM

Short-term angiotensin II treatment regulates cardiac nanomechanics: Via microtubule modifications

Mechanical properties of single myocytes contribute to the whole heart performance, but the measurement of mechanics in living cells at high resolution with minimal force interaction remains challenging. Angiotensin II (AngII) is a peptide hormone that regulates a number of physiological functions, including heart performance. It has also been shown to contribute to cell mechanics by inducing cell stiffening. Using non-contact high-resolution Scanning Ion Conductance Microscopy (SICM), we determine simultaneously cell topography and membrane transverse Young's modulus (YM) by a constant pressure application through a nanopipette. While applying pressure, the vertical position is recorded and a deformation map is generated from which YM can be calculated and corrected for the uneven geometry. High resolution of this method also allows studying specific membrane subdomains, such as Z-grooves and crests. We found that short-term AngII treatment reduces the transversal YM in isolated adult rat cardiomyocytes acting via an AT1 receptor. Blocking either a TGF-β1 receptor or Rho kinase abolishes this effect. Analysis of the cytoskeleton showed that AngII depletes microtubules by decreasing long-lived detyrosinated and acetylated microtubule populations. Interestingly, in the failing cardiomyocytes, which are stiffer than controls, the short-term AngII treatment also reduces the YM, thus normalizing the mechanical state of cells. This suggests that the short-term softening effect of AngII on cardiac cells is opposite to the well-characterized long-term hypertrophic effect. In conclusion, we generate a precise nanoscale indication map of location-specific transverse cortical YM within the cell and this can substantially advance our understanding of cellular mechanics in a physiological environment, for example in isolated cardiac myocytes

Short-term angiotensin II treatment regulates cardiac nanomechanics via microtubule modifications.

Mechanical properties of single myocytes contribute to the whole heart performance, but the measurement of mechanics in living cells at high resolution with minimal force interaction remains challenging. Angiotensin II (AngII) is a peptide hormone that regulates a number of physiological functions, including heart performance. It has also been shown to contribute to cell mechanics by inducing cell stiffening. Using non-contact high-resolution Scanning Ion Conductance Microscopy (SICM), we determine simultaneously cell topography and membrane transverse Young's modulus (YM) by a constant pressure application through a nanopipette. While applying pressure, the vertical position is recorded and a deformation map is generated from which YM can be calculated and corrected for the uneven geometry. High resolution of this method also allows studying specific membrane subdomains, such as Z-grooves and crests. We found that short-term AngII treatment reduces the transversal YM in isolated adult rat cardiomyocytes acting via an AT1 receptor. Blocking either a TGF-β1 receptor or Rho kinase abolishes this effect. Analysis of the cytoskeleton showed that AngII depletes microtubules by decreasing long-lived detyrosinated and acetylated microtubule populations. Interestingly, in the failing cardiomyocytes, which are stiffer than controls, the short-term AngII treatment also reduces the YM, thus normalizing the mechanical state of cells. This suggests that the short-term softening effect of AngII on cardiac cells is opposite to the well-characterized long-term hypertrophic effect. In conclusion, we generate a precise nanoscale indication map of location-specific transverse cortical YM within the cell and this can substantially advance our understanding of cellular mechanics in a physiological environment, for example in isolated cardiac myocytes

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis.

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements

Ionic conduction, rectification, and selectivity in single conical nanopores

Modern track-etching methods allow the preparation of membranes containing a single charged conical nanopore that shows high ionic permselectivity due to the electrical interactions of the surface pore charges with the mobile ions in the aqueous solution. The nanopore has potential applications in electrically assisted single-particle detection, analysis, and separation of biomolecules. We present a detailed theoretical and experimental account of the effects of pore radii and electrolyte concentration on the current-voltage and current-concentration curves. The physical model used is based on the Nernst-Planck and Poisson equations. Since the validity of continuum models for the description of ion transport under different voltages and concentrations is recognized as one of the main issues in the modeling of future applications, special attention is paid to the fundamental understanding of the electrical interactions between the nanopore fixed charges and the mobile charges confined in the reduced volume of the inside [email protected]

Porous silicon nanoneedles modulate endocytosis to deliver biological payloads

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies