cytochalasin-d and Nephrotic-Syndrome

Scanning and transmission electron microscopy were used to evaluate free surface microprojections on the kidney glomerular epithelium in response to various experimental situations. Normally glomerular epithelial podocytes exhibit a rather sparse population of finger-like or bleb-like projections. Some investigations, however, have suggested that microvillous projections may increase in number during the differentiation of podocytes and in the aging kidney. With the onset of puromycin aminonucleoside induced nephrotic syndrome, podocyte microprojections become very knob-like and irregular in their shapes. In response to the in vitro environment, the number of podocyte microprojections increases dramatically. Neuraminidase removal of the sialic acid component of the glomerular glycocalyx and exposure to either cytochalasin B (25 microgram/ml) or cytochalasin D (2 microgram/ml), prevent the in vitro formation of podocyte microprojections. In vitro incubation in compounds which depolymerize cytoplasmic microtubules (e.g. vinblastine sulfate 10(-5); colchicine 10(-5) M), however, result in the formation of unusually long glomerular epithelial microprojections. Investigations utilizing the cationic ligand polycationized ferritin (PCF) have shown that podocyte microprojections are the most anionic sites on the free surfaces of these cells. During prolonged incubation of PCF treated glomeruli, the tips of podocyte microprojections are the last sites to shed the anionically bound PCF. It is suggested that the highly anionic sialic acid components of the glomerular free surface glycocalyx may play principal roles in the formation, maintenance and shapes of glomerular podocyte microprojections.

Topics: Animals; Cell Membrane; Cytochalasin B; Cytochalasin D; Cytochalasins; Epithelium; Ferritins; Kidney Glomerulus; Microscopy, Electron, Scanning; Microtubules; Microvilli; Nephrotic Syndrome; Neuraminidase; Polymers; Rats

Gain-of-function mutations in the transient receptor potential (TRP) cation channel subfamily C member 6 (TRPC6) gene and mutations in the NPHS2 gene encoding podocin result in nephrotic syndromes. The purpose of this study was to determine the functional significance of biochemical interactions between these proteins. We observed that gating of TRPC6 channels in podocytes is markedly mechanosensitive and can be activated by hyposmotic stretch or indentation of the plasma membrane. Stretch activation of cationic currents was blocked by small interfering RNA knockdown of TRPC6, as well as by SKF-96365 or micromolar La(3+). Stretch activation of podocyte TRPC6 persisted in the presence of inhibitors of phospholipase C (U-73122) and phospholipase A2 (ONO-RS-082). Robust stretch responses also persisted when recording electrodes contained guanosine 5'-O-(2-thiodiphosphate) at concentrations that completely suppressed responses to ANG II. Stretch responses were enhanced by cytochalasin D but were abolished by the peptide GsMTx4, suggesting that forces are transmitted to the channels through the plasma membrane. Podocin and TRPC6 interact at their respective COOH termini. Knockdown of podocin markedly increased stretch-evoked activation of TRPC6 but nearly abolished TRPC6 activation evoked by a diacylglycerol analog. These data suggest that podocin acts as a switch to determine the preferred mode of TRPC6 activation. They also suggest that podocin deficiencies will result in Ca(2+) overload in foot processes, as with gain-of-function mutations in the TRPC6 gene. Finally, they suggest that mechanical activation of TRP family channels and the preferred mode of TRP channel activation may depend on whether members of the stomatin/prohibitin family of hairpin loop proteins are present.

Topics: Animals; Calcium Channel Blockers; Cell Line; Cell Membrane; Chlorobenzoates; Cinnamates; Cytochalasin D; Diglycerides; Estrenes; Guanosine Diphosphate; HEK293 Cells; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Ion Channel Gating; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; ortho-Aminobenzoates; Peptides; Phosphodiesterase Inhibitors; Phospholipase A2 Inhibitors; Podocytes; Pyrrolidinones; Rats; RNA Interference; RNA, Small Interfering; Spider Venoms; Thionucleotides; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases