Satoshi Ochi

Endothelin-1 receptors in rat renal glomeruli

Summary Renal glomeruli form a dynamic structure capable of regulating the glomerular filtration rate (GFR). To explore possible regulating effects of endothelin-1 (ET-1) upon glomerular functions, the presence of specific ET-1 receptors and the biological actions of ET-1 were studied in isolated rat renal glomeruli. The specific binding of [125I]ET-1 to glomeruli was time and temperature dependent. Scatchard analysis of the binding data indicated the presence of a single class of high-affinity binding sites with the apparent dissociation constant of 8.3 nM and the maximal binding capacity of 1.5 pmol/mg protein. ET-1 stimulated prostaglandin (PG) E2 production in glomeruli with the time course and the concentration dependency similar to those found in [125I]ET-1 binding to glomeruli. These results indicate the presence of high-affinity binding sites for ET-1 linked to PGE2 production in glomeruli, suggesting a possible role of ET-1 in the regulation of glomerular function.

Calcium-activated, phospholipid-dependent protein kinase in cultured rat mesangial cells.

The analysis of the 100000 × g supernatant fraction of cultured rat glomerular mesangial cells with DEAE‐cellulose ion‐exchange chromatography revealed a large peak showing the activity of a protein kinase (protein kinase C) which depended on phospholipid and diolein as well as Ca2+ Furthermore, it was shown that angiotensin II (AII) (10−6 M) induced rapid hydrolysis of phosphatidylinositol 4,5‐bisphosphate, leading to production of diacylglycerol rich in arachidonic acid, in the cultured rat mesangial cells. These results suggest that activation of protein kinase C resulting from enhancement of phosphoinositide metabolism may be important as an intracellular regulatory mechanism of AII upon cultured mesangial cells.

Signal transduction mechanism of interleukin 6 in cultured rat mesangial cells

Interleukin 6 (IL‐6) is one of the potent autocrine growth factors for mesangial cells. We investigated the signal transduction mechanism or IL‐6 in cultured rat mesangial cells. IL‐6 induced a transient increase of inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3) followed by a transient and sustained increase of intracellular calcium concentration, suggesting that IL‐6 stimulates phosphoinositide turnover. IL‐6 also stimulated prostaglandin E2 (PGE2) production. The IL‐6‐concentration dependency in PGE2 production was similar to that in Ins 1,4,5‐P3 production. We concluded that the action of IL‐6 on mesangial cells is exerted at least partially through the enhancement of phosphoinositide turnover and PGE2 production.

Stimulatory effect of thrombin on endothelin-1 production in isolated glomeruli and cultured mesangial cells of rats.

To determine whether endothelin-1 (ET-1) is released in close proximity to its binding sites on glomerular mesangial cells, and also to elucidate the regulatory factors responsible for its release, we measured immunoreactive ET-1 (ir-ET-1) in the incubation media of isolated glomeruli and cultured mesangial cells of rats using enzyme immunoassay under both basal and thrombin-stimulated conditions. ir-ET-1 was released time-dependently, from isolated glomeruli and cultured mesangial cells. Thrombin (2 U/ml) stimulated the release from both preparations. The release of immunoreactive big endothelin-1 (ir-big ET-1), which was assayed by using enzyme immunoassay, was also time-dependent, and the release was increased by thrombin (2 U/ml; at 4 h, 8 h, and 24 h) in cultured mesangial cells. The releases of big ET-1 and its converted product ET-1 from mesangial cells, an established target for ET-1, suggests that ET-1 acts as an autocrine factor for the cells.

Phosphoinositide turnover enhanced by angiotensin II in isolated rat glomeruli

To clarify the signal transduction mechanism of angiotensin II in renal glomeruli, we studied the effect of the hormone on phospholipid metabolism using isolated rat glomeruli. Stimulation of the glomeruli pulse-chase labeled with [3H]glycerol by angiotensin II caused a rapid (within 15 s) breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) with a concurrent production of 1,2-diacylglycerol. This effect of angiotensin II was in a dose-dependent manner within the range from 10(-12) M to 10(-6) M, and was inhibited by saralasin. Angiotensin II also decreased the 3H radioactivity of PIP slightly only at 15 s and increased that of phosphatidic acid after 15 s, with no significant effect upon the labelings of phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) within 1 min. The change in phospholipid metabolism by angiotensin II was similar when the glomeruli were labeled with [32P]orthophosphate: the decrease in the labeling of PIP2 and the increase in the labeling of phosphatidic acid after 15 s. In addition, 32P labeling of PI increased after 2 min. These results suggest that angiotensin II, after binding to glomerular receptors, induces initial PIP2 hydrolysis to diacylglycerol and subsequent resynthesis of PIP2 through phosphoinositide turnover.

Evaluation of Hemodialysis Efficiency by an Edema Fluid Model

The concentrations of urea, creatinine, and uric acid were measured in edema fluid and plasma during hemodialysis and 18 h after hemodialysis. The concentrations of these solutes in plasma were 15-17% lower than in the edema fluid after hemodialysis. Eighteen hours after hemodialysis, however, the concentrations in plasma were almost the same as those in edema fluid. These data suggest that the removal of these solutes from the extracellular space is delayed during hemodialysis. The plasma concentrations obtained at 18 h after hemodialysis are better indicators of hemodialysis efficiency.