Glomerular Mesangial Cell Altered Contractility in High Glucose Is Ca²⁺ Independent Free

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/1) or high (10–25.6 mmol/1) 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 glomeruli isolated from STZ-administered rats in response to endothelin (ET)-1 (0.1 μmol/1) or the Ca²⁺ ionophore A23187 (5 μmol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AVP) (1.0 μmol/1) or ET-1 (0.1 μmol/1), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6–25.6 mmol/1) 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-[2-³H]inositol and intracellular [³H] inositol phosphate release in response to AVP (1.0 μmol/1) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/1) versus high (25.6 mmol/1) 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/1 glucose, 0.1 μmol/l AVP or 0.1 μmol/1 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.