GLOMERULAR MESANGIAL CELL ALTERED CONTRACTILITY IN HIGH GLUCOSE IS CA2+ INDEPENDENT

In diabetes, loss of renal arteriolar smooth muscle cell contractility leads to intraglomerular hypertension. In glomeruli isolated from streptozotocin (STZ)-induced diabetic rats, the mesangial cells (smooth muscle-like) display loss of contractile responsiveness to angiotensin II. This study examines the mechanistic relationship between altered mesangial cell contractility and vasopressor hormone-stimulated Ca2+ signaling in high glucose. Glomeruli were isolated from normal or STZ-induced diabetic rats to observe ex vivo mesangial cell contractile function. Also, rat mesangial cells were cultured (10-20 passages) in normal (5.6 mmol/l) or high (10-25.6 mmol/l) glucose for 1-5 days. Reduction of glomerular volume and decreased planar surface area of cultured mesangial cells in response to vasoconstrictor stimulation over 60 min were measured by videomicroscopy and personal computer-based morphometry. Contraction of glomenruli isolated from STZ-administered rats in response to endothelin (ET)-1 (0.1 mu mol/l) or the Ca2+ ionophore A23187 (5 mu mol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AW) (1.0 mu mol/l) or ET-1 (0.1 mu mol/l), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6-25.6 mmol/l) within 24 h of high-glucose exposure, which was sustained for 5 days. Mesangial cells in high glucose were consistently smaller in size compared with those in normal glucose. Mesangial cells were preloaded with myo-[2-H-3]inositol and intracellular [H-3] inositol phosphate release in response to AVP (1.0 mu mol/l) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/l) versus high (25.6 mmol/l) glucose, we observed no significant difference in stimulated inositol phosphate levels from 10 to 60 s. The Ca2+-signaling response of cultured mesangial cells preloaded with Fura 2 or Indo 1 was measured by spectrofluorometry. After culture in 25.6 mmol/l glucose, 0.1 mu mol/l AVP or 0.1 mu mol/l ET-1 stimulated a cytosolic Ca2+ signal, with first and second phases, identical to that exhibited by mesangial cells in normal glucose. Fluorescence imaging of cultured mesangial cell cytoskeletal filamentous actin (F-actin) stained with rhodamine-phalloidin demonstrated reduced F-actin staining in the basal state and loss of the normal F-actin disassembly response to ET-1 in high glucose. Glomerular mesangial cells display glucose-induced altered contraction and F-actin disassembly to vasoactive stimuli, which occur in the presence of normal Ca2+ signaling.