Fig 2 - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrating the presence of EP2 and EP4 receptor mRNA in mouse glomeruli. The expression of EP2 and EP4 receptor mRNA was determined by RT-PCR as detailed in the Methods and in the text.
Source publication
Recently we immunohistochemically demonstrated that prostaglandin E2 (PGE2) promoted the clearance of aggregated bovine serum albumin (a-BSA) deposited in glomeruli. Herein, we investigated the role of PGE2 and its signal transduction in the disposal of macromolecules in glomeruli. EP2 and EP4 receptor mRNA was detected in glomeruli by RT-PCR analy...
Context in source publication
Context 1
... and EP4 receptor mRNA in glomeruli (Fig. 2) The yield of total RNA was 14 mg. The cDNAs for EP2 and EP4 receptors of mice were subjected to amplification by PCR. When PCR products were analyzed on 2% agarose gels, each single ethidium bromide staining band was observed. A 401-bp EP2 cDNA and a 423-bp EP4 cDNA were generated, ...
Similar publications
Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer...
The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK wa...
Citations
... Pharmacologically, there are four kinds of prostaglandin E 2 -receptor subtypes, namely, EP 1 , EP 2 , EP 3 , and EP 4 ; and the expression of mRNA of all four receptor subtypes has been demonstrated in the kidney (5,6). We have previously demonstrated the expression of mRNA of EP 2 and EP 4 in the glomeruli by reverse transcription polymerase chain reaction (RT-PCR) analysis (7). EP receptors have a 7-transmembrane structure, and EP 2 and EP 4 are coupled to the Gs protein that stimulates adenylate cyclase. ...
... We previously reported that prostaglandin E 1 delayed the development of experimental nephritis in rats by injection of antiglomerular basement membrane antibody (4,8). The glomeruli produce prostaglandin E 2 in the basal condition and generate cyclic AMP in response to prostaglandin E 2 (7,9). Prostaglandin E 2 , cyclic AMP analogues, and cyclic AMP-elevating agents exert various effects, including reducing excessive fibronectin production and increasing disposal of aggregated protein, on the glomeruli and mesangial cells (10,11). ...
... Preparation of anti-mouse glomerular antiserum (12) The glomeruli were isolated from mice by the procedure of Assmann et al. with slight modifications (7,13). Briefly, mouse kidneys were perfused with 1 mg/ ml of iron oxide in phosphate-buffered saline (PBS) and removed. ...
Prostaglandin E2-receptor subtypes, EP1, EP2, EP3, and EP4, are present in the kidney. The aim of this study was to elucidate the anti-nephritic effect of an EP4-receptor agonist on an experimental nephritic model. Mice were injected i.v. with anti-glomerulus antiserum to induce nephritis. Nephritic glomeruli generated more prostaglandin E2 (2.6 and 0.7 ng) and less cyclic AMP than normal glomeruli (11 and 26 pmol). The production of cyclic AMP in nephritic glomeruli increased 67% in response to AE1-329, an EP4 agonist, at 10−5 M. Nephritic glomeruli expressed a lesser amount of mRNA of prostaglandin E2-receptor subtypes as compared with normal glomeruli. AE1-329 was administered s.c. at 100 μg/kg per day for 3 weeks. AE1-329 suppressed the increase in creatinine and cholesterol compared to those in the control nephritic mice. AE1-329-treated nephritic mice had less crescentic glomeruli and less deposition of rabbit IgG (anti-glomerular basement membrane antibody) in glomeruli than the control mice. AE1-329 prevented the development of glomerulonephritis. These findings suggest that EP4-receptor agonists are a promising drug to prevent the development of glomerulonephritis.
... Pharmacologically, there are four kinds of PGE 2 receptor subtypes, namely EP 1 , EP 2 , EP 3 and EP 4 , and the expression of mRNA for all the four receptor subtypes has been demonstrated in the kidney (Sugimoto et al., 1994; Nemoto et al., 1997). We have previously demonstrated the expression of mRNA for EP 2 and EP 4 in glomeruli by reverse transcription – polymerase chain reaction (RT – PCR) analysis (Nagamatsu et al., 2001). EP receptors have a seven-transmembrane structure and EP 2 and EP 4 are coupled to the Gs protein that stimulates adenylate cyclase. ...
... At 10 À6 M forskolin decreased the levels of fibronectin by 42% compared with the vehicle-treated control nephritic glomeruli (Figure 3a). As the kidney produces large amounts of PGE 2 , not PGE 1 and the glomeruli express prostaglandin E receptor subtypes (EP 4 ) (Nagamatsu et al., 2001), we investigated the effects of PGE 2 on fibronectin production in the nephritic glomeruli. The accumulation of fibronectin decreased dose-dependently in the nephritic glomeruli incubated with PGE 2 compared with the vehicle-treated nephritic controls (Figure 3b). ...
Excessive production of extracellular matrix is thought to be involved in the progression of glomerulonephritis and glomerulosclerosis. In chronic glomerulonephritis, fibronectin has been shown to accumulate in the glomeruli, accompanied by cell proliferation.
Glomerulonephritis was induced in rats by the injection of anti‐glomerular basement membrane antibody. The rats showed proteinuria and histological alterations in the glomeruli. An increase in fibronectin levels in the culture medium of isolated nephritic glomeruli was confirmed, and was associated with the development of nephritis. Immunohistochemical staining demonstrated a marked accumulation of fibronectin in the glomeruli of nephritic rats.
Attenuated generation of cyclic AMP was also observed in the nephritic glomeruli treated with forskolin, prostaglandin E1 or adenosine.
Forskolin, prostaglandin E2 and 8‐bromo‐cyclic AMP markedly reduced the production of fibronectin by the nephritic glomeruli compared with controls in a dose‐dependent manner. 8‐Bromo‐cyclic AMP suppressed the production of fibronectin by cultured mesangial cells.
These findings suggest that the attenuated generation of cyclic AMP in response to ligands is connected to the augmented accumulation of fibronectin in nephritic glomeruli, and may facilitate the development of methods for treating glomerulonephritis and glomerulosclerosis.
British Journal of Pharmacology (2003) 140 , 1245–1251. doi: 10.1038/sj.bjp.0705564
... These findings raise the question of why production of prostaglandin E2 was increased in glomeruli by deposition of aggregated protein. We reported previously that prostaglandin E 2 accelerates the process of disposal of aggregated protein in glomeruli and in cultured mesangial cells (22,23). As shown in the present study, an increase in cyclooxygenase-2 expression is, therefore, required to produce prostaglandin E2 and accelerate removal of aggregated protein from glomeruli. ...
Cyclooxygenase has two isozymes, a constitutive type (cyclooxygenase-1) and an inducible type (cyclooxygenase-2). The aim of the present study was to determine whether cyclooxygenase -2 is associated with the increased production in prostaglandin E-2 in glomeruli by aggregated protein. Mice were injected with aggregated bovine serum albumin. Glomeruli were isolated using sieves and a magnet: Production of prostaglandin E-2 was increased in glomeruli after injection of aggregated bovine serum albumin. RT-PCR analysis indicated enhanced expression of cyclooxygenase-2 mRNA in aggregated bovine serum albumin-loaded glomeruli. Western blotting analysis indicated an increase in cyclooxygenase-2 protein in glomeruli by aggregated bovine serum albumin. Glomeruli were incubated with indomethacin, NS-398 or niflumic acid in the presence of arachidonic acid. Indomethacin resulted in remarkable reduction of prostaglandin E-2 levels in aggregated bovine serum albumin-loaded glomeruli. Niflumic acid also inhibited prostaglandin E-2 production, and its inhibitory rate was more than that of NS-398. In conclusion, aggregated protein induces cyclooxygenase-2 in glomeruli, suggesting that cyclooxygenase-2 is involved in the process of disposal of aggregated protein in glomeuli.
We previously demonstrated the cAMP-PKA pathway to be associated with the reduction in aggregated proteins such as immune complex in glomeruli. The aim of this study was to clarify whether PKC is involved in the reduction of aggregated protein and phosphorylation of CREB in aggregated protein-loaded glomeruli. Mice were injected with aggregated bovine serum albumin (a-BSA), and glomeruli were isolated. The a-BSA-injected mice produced more cyclic AMP and had more phosphorylated serine and phosphorylated CREB in their glomeruli than the controls. The expression of phospho-CREB increased with the accumulation of a-BSA. KT5720 and H7 suppressed the increase in phosphorylated CREB in a-BSA-loaded glomeruli and the decrease in accumulated a-BSA in the glomeruli. These findings suggest that PKC is associated with the reduction of aggregated protein and phosphorylation of CREB in aggregated protein-loaded glomeruli.
The aim of this study was to clarify the influence of hyperglycemia on the deposition of aggregated protein in the glomeruli of diabetic mice. KK-A(y) mice injected with aggregated bovine serum albumin accumulated more of it in the glomeruli than did ICR mice. There were no histological alterations in the glomeruli of KK-A(y) mice. KK-A(y) mice given voglibose in mouse-chow for 2 weeks had significantly reduced blood glucose, glycated albumin, and hemoglobin A(1C) levels compared with control mice. The voglibose-treated KK-A(y) mice were injected with aggregated bovine serum albumin and accumulated significantly less albumin in the glomeruli than did the control mice. Pioglitazone decreased blood glucose levels compared with the control, and reduced the glomerular deposition of aggregated albumin. Glomerular aggregated bovine serum albumin levels and blood glucose levels were reduced significantly by the injection of insulin. Six times more advanced glycation endproducts were produced from aggregated bovine albumin than from non-aggregated bovine albumin on incubation with glucose and L-lysine in vitro. Glucose-loaded ICR mice generated more advanced glycation endproducts from aggregated albumin, and had more aggregated bovine albumin in the glomeruli. It was suggested that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli even early on in diabetes.
Cyclooxygenase has two isozymes, a constitutive type (cyclooxygenase-1) and an inducible type (cyclooxygenase-2). The aim of the present study was to determine whether cyclooxygenase-2 is associated with the increased production in prostaglandin E2 in glomeruli by aggregated protein. Mice were injected with aggregated bovine serum albumin. Glomeruli were isolated using sieves and a magnet. Production of prostaglandin E2 was increased in glomeruli after injection of aggregated bovine serum albumin. RT-PCR analysis indicated enhanced expression of cyclooxygenase-2 mRNA in aggregated bovine serum albumin-loaded glomeruli. Western blotting analysis indicated an increase in cyclooxygenase-2 protein in glomeruli by aggregated bovine serum albumin. Glomeruli were incubated with indomethacin, NS-398 or niflumic acid in the presence of arachidonic acid. Indomethacin resulted in remarkable reduction of prostaglandin E2 levels in aggregated bovine serum albumin-loaded glomeruli. Niflumic acid also inhibited prostaglandin E2 production, and its inhibitory rate was more than that of NS-398. In conclusion, aggregated protein induces cyclooxygenase-2 in glomeruli, suggesting that cyclooxygenase-2 is involved in the process of disposal of aggregated protein in glomeuli.
The aim of this study was to investigate the disposal of aggregated protein in the glomeruli of spontaneously diabetic mice. Diabetic mice, KK-A(y) and db/db, and age-matched ICR mice were injected intravenously with aggregated bovine serum albumin (a-BSA) at 0.6 mg/g, and the glomeruli and the blood were obtained. Diabetic mice had larger amounts of a-BSA in their glomeruli than the ICR mice, threefold in KK-A(y) and twofold in db/db, at 3 h after the a-BSA injection. Additionally, the disappearance of a-BSA was retarded in the diabetic glomeruli. KK-A(y) displayed a-BSA in the glomeruli 24 h after the a-BSA injection and db/db did after 12 h, while the ICR did by 8 h. In spite of increases of insulin to similar degrees in both strains of diabetic mice after the a-BSA injection, blood glucose levels markedly decreased in KK-A(y) compared with db/db. There were no histopathological alterations in the glomeruli of the diabetic mice. Depositions of a-BSA were confirmed to be higher in the diabetic glomeruli by the immunofluorescence technique, and KK-A(y) displayed higher depositions of a-BSA than did db/db. The present study suggests that hyperglycemia is involved in the increased deposition of aggregated protein in the glomeruli and that the degradation of aggregated protein is retarded in diabetic glomeruli.
To elucidate the involvement of cyclooxygenase (COX) in degradation of aggregated protein in diabetic glomeruli, we used streptozotocin (STZ)-induced diabetic mice and aggregated bovine serum albumin (a-BSA) as a model protein. There was a higher deposition of a-BSA in diabetic glomeruli compared to normal glomeruli 18 h after a-BSA injection at 4 and 8 weeks after STZ. Degradation of a-BSA was confirmed using isolated glomeruli. Diabetic glomeruli produced prostaglandin E(2) (PGE(2)) more than normal glomeruli in the basal level at 8 weeks. a-BSA caused further increase of PGE(2) production in normal glomeruli, but not in diabetic glomeruli. Niflimic acid, a selective COX-2 inhibitor, reduced PGE(2) production of normal glomeruli in the a-BSA loading group, but not that in the control group. In diabetic glomeruli, niflimic acid reduced PGE(2) production in both the control group and a-BSA loading group. In normal glomeruli, a-BSA increased expressions of both COX-2 mRNA and protein. However, in diabetic glomeruli, a-BSA increased COX-2 mRNA expression but not COX-2 protein expression. These results suggest that retarded degradation of aggregated protein in diabetic glomeruli is associated with lack of further expression of COX-2 protein and further production of PGE(2) in response to aggregated protein.
Prostaglandin E(2)-receptor subtypes, EP(1), EP(2), EP(3), and EP(4), are present in the kidney. The aim of this study was to elucidate the anti-nephritic effect of an EP(4)-receptor agonist on an experimental nephritic model. Mice were injected i.v. with anti-glomerulus antiserum to induce nephritis. Nephritic glomeruli generated more prostaglandin E(2) (2.6 and 0.7 ng) and less cyclic AMP than normal glomeruli (11 and 26 pmol). The production of cyclic AMP in nephritic glomeruli increased 67% in response to AE1-329, an EP(4) agonist, at 10(-5) M. Nephritic glomeruli expressed a lesser amount of mRNA of prostaglandin E(2)-receptor subtypes as compared with normal glomeruli. AE1-329 was administered s.c. at 100 microg/kg per day for 3 weeks. AE1-329 suppressed the increase in creatinine and cholesterol compared to those in the control nephritic mice. AE1-329-treated nephritic mice had less crescentic glomeruli and less deposition of rabbit IgG (anti-glomerular basement membrane antibody) in glomeruli than the control mice. AE1-329 prevented the development of glomerulonephritis. These findings suggest that EP(4)-receptor agonists are a promising drug to prevent the development of glomerulonephritis.
