The kallikrein-kinin system (KKS) is associated with the development of diabetic complications, diabetic retinopathy (DR), and diabetic nephropathy (DN). DR is a major complication of both type 1 and type 2 diabetes mellitus and is a leading cause of visual impairment and blindness. Retinal pigment ...
The kallikrein-kinin system (KKS) is associated with the development of diabetic complications, diabetic retinopathy (DR), and diabetic nephropathy (DN). DR is a major complication of both type 1 and type 2 diabetes mellitus and is a leading cause of visual impairment and blindness. Retinal pigment epithelial cells support the function of retina photoreceptors and are implicated in the onset of DR. In addition, DN is also a leading cause of end-stage renal disease (ESRD), and accounts for significant morbidity and mortality. Mesangial cells and podocytes are major cells in the development of DN.
Bradykinin (BK), a major effector of KKS, has a pathophysiological function in the various organs and its effects are mediated by two subtypes of G-protein coupled receptor (GPCR), BK B1 receptor (B1R) and BK B2 receptor (B2R). Thus, regulation of B1R and B2R is also a predictor of retinal and renal dysfunction and an important feature of diabetic complications. However, the pathophysiological mechanism of B1R and B2R in DR and DN, especially retina pigment epithelial cells, mesangial cells, and podocytes, has not been elucidated. Therefore, this study was focused on the effects of B1R and B2R, during hyperglycemia condition on structural and functional changes in retina pigment epithelial cell, mesangial cells, and podocytes, which are important cells in the onset of DR and DN. That is, this study investigated 1) the effect of 25 mM glucose (HG)-induced B1R and B2R activation on glutamate uptake and its related signal cascades in human ARPE-19 cells, 2) the effect of HG-induced fatty acid amide hydrolase (FAAH) 1, BK receptors, and cannabinoid 1 receptor (CB1R)expression on apoptosis and its related signal cascades in human ARPE-19 cells, 3) the effect of HG-induced B1R and B2R activation on apoptosis and its related signal cascades in rat podocytes, 4) the effect of HG-induced B1R and B2R activation on fibrosis and its related signal cascades in rat mesangial cells, and 5)the expression of B1R and B2R in type 1 and type 2 diabetic models.
Results were as followings:
1) HG increased the level of D-[2,3-3H]-aspartate uptake in time- and dose-dependent manners and the expression of excitatory amino acid transporter-4 (EAAT-4)mRNA, and these were blocked by treatment with small interfering RNA(siRNA)for B1R and B2R, but not scrambled siRNA, supporting an involvement of B1R and B2R in this process. HG-increased D-[2,3-3H]-aspartate uptake was also blocked by [des-Arg10]-HOE140 (a B1R antagonist)and HOE140 (a B2R antagonist). HG increased B1R and B2R mRNA and protein expression in a time-dependent manner, increased B1R and B2R translocation from the membrane to the nucleus, and stimulated KKS components such as, kininogen, kallikrein, and kininase I mRNA expression. I examined whether BK receptors were involved in HG-induced signaling pathways. HG stimulated[3H]-arachidonic acid (AA) release, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2)proteins; these events were blocked by treatment with B1R and B2R siRNAs, but not by scrambled siRNA. HG-induced stimulation of D-[2,3-3H]-aspartate uptake was blocked by the cPLA2 and COX-2 inhibitors arachidonyl trifluoromethyl ketone (AACOCF3), mepacrine, 5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl-]-thiophene (Dup697), and N-[2-cyclohexy-loxy-4-nitrophenyl]methane-sulfonamide (NS398). Inaddition, HG stimulated nuclear factor-κB (NF-κB) and inhibitor-κB (I-κB)activation; these events were blocked by treatment with B1R and B2R siRNAs, but not by scrambled siRNA. HG-induced stimulation of D-[2,3-3H]-aspartate uptake was blocked by by the NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and SN50.
2) HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, HG upregulated the expression of CB1R mRNA and protein. HG-induced internalization of CB1R was blocked by the overexpression of FAAH 1 and CB1R-specific antagonist (AM251) in HEK 293 cells or ARPE-19 cells. HG-induced generation of reactive oxygen species (ROS) and lipid peroxide formation (LPO) were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced cytochrome c relases in cytosolic fraction. I also investigated whether the overexpression of FAAH 1 protect HG-induced apoptosis. HG increased the Bax/Bcl-2 ratio, cleaved Poly(ADP-ribose) polymerase(PARP), cleaved caspase-3 and cleaved caspase-9 levels and decreased the cell viability. HG-induced apoptotic effects were decreased with the overexpression of FAAH 1, AM251 and CB1R siRNA transfection. In addition, I confirmed crosstalk between BK receptors and CB1R on HG-induced apoptosis. HG-induced expression of B1R and B2R were decreased by the overexpression of FAAH 1 and AM251 treatment. HG-induced apoptotic effects were decreased by both [des-Arg10]-HOE140 and HOE140 treatment, as well as B1R and B2R siRNA transfection.
3) HG decreased cell viability and increased DNA fragmentation in rat podocytes. HG-induced DNA fragmentation and cleaved PARP and cleaved caspase-3 were blocked by both [des-Arg10]-HOE140 and HOE140. HG also increased phosphorylation of Akt, NF-κB/I-κB, and endoplasmic reticulum (ER) stress-related proteins such as PERK and eukaryotic initiation factor 2α(eIF2α)in rat podocytes, which was blocked by both [des-Arg10]-HOE140 and HOE140. In addition, B1R and B2R siRNA transfection prevented HG-induced phosphorylation of Akt, NF-κB, PERK, and eIF2α in rat podocytes. Moreover, HG upregulated the expression of CB1R mRNA and protein. AM251 treatment and CB1R siRNA transfection blocked the HG-induced BK receptors expression, signaling pathway.
4) HG increased the fibrosis related protein, fibronectin in rat mesangial cells. HG also increased tissue inhibitors of metalloproteinase-1 (TIMP-1) and decreased matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activity. In addition, both [des-Arg10]-HOE140 and HOE140 completely blocked the HG-induced increases in mRNA and protein expression of fibronectin. HG-induced TIMP-1 and MMP-2 and MMP-9 activity, which were blocked by both [des-Arg10]-HOE140 and HOE140. HG also increased the hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) activation and Smad2/3 translocation from the cytosol to the nucleus. HG-induced signaling molecules were blocked by both [des-Arg10]-HOE140 and HOE140. This study confirmed these results, observing that HG-induced activation of signaling molecules were blocked B1R and B2R siRNA, but not scrambled siRNA.In addition, HG-induced TGF-β1 activation and Smad2/3 translocation were not blocked by HIF-1α siRNA, suggesting HG-induced HIF-1α and TGF-β1/Smad2/3 signaling pathway independently control fibrosis in rat mesangial cells.
5) Increased B1R and B2R mRNA and protein expression shown in kidney of streptozotocin (STZ)-rats and ZDF rats using immunohistochemical stain, RT-PCR, and western blotting. In addition, pro-apoptotic proteins (caspase-3, CHOP, and Bax) and anti-apoptotic protein (Bcl-2) were increased in kidney of STZ- and Zucker rats better than normal rats.
In summary, 1) HG increases the EAAT-4 gluramate transporter activity and D-[2,3-3H]-aspartate uptake through B1R and B2R activation in ARPE-19 cells, and its action is associated with the cPLA2, COX-2 and NF‐kB signaling pathways, 2) HG increased oxidative stress and induced apoptosis via B1R, B2R, and CB1R activation and through FAAH 1 downregulation in ARPE-19 cells, 3)HG stimulates cell death such as apoptosis that is mediated by B1R and B2R throught CB1R activation as well as the Akt, NF‐κB/I-κB, and ER stress related proteins, PERK and eIF2α, signal pathways in podocytes, 4) HG stimulates fibronectin accumulation through HIF-1α and TFG-β1/Smad 2/3 througt B1R and B2R activation in mesangial cells, finally 5) B1R and B2R expression as well as apoptotic molecules were increased in kidney section of type 1 and type 2 diabetic models. These results shown a similar pattern to results of in vitro study. Therefore, the present results suggest that blockade of bradykinin receptors, B1R and B2R, may be a potential anti-diabetic therapy for the treatment of DR and DN.
The kallikrein-kinin system (KKS) is associated with the development of diabetic complications, diabetic retinopathy (DR), and diabetic nephropathy (DN). DR is a major complication of both type 1 and type 2 diabetes mellitus and is a leading cause of visual impairment and blindness. Retinal pigment epithelial cells support the function of retina photoreceptors and are implicated in the onset of DR. In addition, DN is also a leading cause of end-stage renal disease (ESRD), and accounts for significant morbidity and mortality. Mesangial cells and podocytes are major cells in the development of DN.
Bradykinin (BK), a major effector of KKS, has a pathophysiological function in the various organs and its effects are mediated by two subtypes of G-protein coupled receptor (GPCR), BK B1 receptor (B1R) and BK B2 receptor (B2R). Thus, regulation of B1R and B2R is also a predictor of retinal and renal dysfunction and an important feature of diabetic complications. However, the pathophysiological mechanism of B1R and B2R in DR and DN, especially retina pigment epithelial cells, mesangial cells, and podocytes, has not been elucidated. Therefore, this study was focused on the effects of B1R and B2R, during hyperglycemia condition on structural and functional changes in retina pigment epithelial cell, mesangial cells, and podocytes, which are important cells in the onset of DR and DN. That is, this study investigated 1) the effect of 25 mM glucose (HG)-induced B1R and B2R activation on glutamate uptake and its related signal cascades in human ARPE-19 cells, 2) the effect of HG-induced fatty acid amide hydrolase (FAAH) 1, BK receptors, and cannabinoid 1 receptor (CB1R)expression on apoptosis and its related signal cascades in human ARPE-19 cells, 3) the effect of HG-induced B1R and B2R activation on apoptosis and its related signal cascades in rat podocytes, 4) the effect of HG-induced B1R and B2R activation on fibrosis and its related signal cascades in rat mesangial cells, and 5)the expression of B1R and B2R in type 1 and type 2 diabetic models.
Results were as followings:
1) HG increased the level of D-[2,3-3H]-aspartate uptake in time- and dose-dependent manners and the expression of excitatory amino acid transporter-4 (EAAT-4)mRNA, and these were blocked by treatment with small interfering RNA(siRNA)for B1R and B2R, but not scrambled siRNA, supporting an involvement of B1R and B2R in this process. HG-increased D-[2,3-3H]-aspartate uptake was also blocked by [des-Arg10]-HOE140 (a B1R antagonist)and HOE140 (a B2R antagonist). HG increased B1R and B2R mRNA and protein expression in a time-dependent manner, increased B1R and B2R translocation from the membrane to the nucleus, and stimulated KKS components such as, kininogen, kallikrein, and kininase I mRNA expression. I examined whether BK receptors were involved in HG-induced signaling pathways. HG stimulated[3H]-arachidonic acid (AA) release, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2)proteins; these events were blocked by treatment with B1R and B2R siRNAs, but not by scrambled siRNA. HG-induced stimulation of D-[2,3-3H]-aspartate uptake was blocked by the cPLA2 and COX-2 inhibitors arachidonyl trifluoromethyl ketone (AACOCF3), mepacrine, 5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl-]-thiophene (Dup697), and N-[2-cyclohexy-loxy-4-nitrophenyl]methane-sulfonamide (NS398). Inaddition, HG stimulated nuclear factor-κB (NF-κB) and inhibitor-κB (I-κB)activation; these events were blocked by treatment with B1R and B2R siRNAs, but not by scrambled siRNA. HG-induced stimulation of D-[2,3-3H]-aspartate uptake was blocked by by the NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and SN50.
2) HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, HG upregulated the expression of CB1R mRNA and protein. HG-induced internalization of CB1R was blocked by the overexpression of FAAH 1 and CB1R-specific antagonist (AM251) in HEK 293 cells or ARPE-19 cells. HG-induced generation of reactive oxygen species (ROS) and lipid peroxide formation (LPO) were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced cytochrome c relases in cytosolic fraction. I also investigated whether the overexpression of FAAH 1 protect HG-induced apoptosis. HG increased the Bax/Bcl-2 ratio, cleaved Poly(ADP-ribose) polymerase(PARP), cleaved caspase-3 and cleaved caspase-9 levels and decreased the cell viability. HG-induced apoptotic effects were decreased with the overexpression of FAAH 1, AM251 and CB1R siRNA transfection. In addition, I confirmed crosstalk between BK receptors and CB1R on HG-induced apoptosis. HG-induced expression of B1R and B2R were decreased by the overexpression of FAAH 1 and AM251 treatment. HG-induced apoptotic effects were decreased by both [des-Arg10]-HOE140 and HOE140 treatment, as well as B1R and B2R siRNA transfection.
3) HG decreased cell viability and increased DNA fragmentation in rat podocytes. HG-induced DNA fragmentation and cleaved PARP and cleaved caspase-3 were blocked by both [des-Arg10]-HOE140 and HOE140. HG also increased phosphorylation of Akt, NF-κB/I-κB, and endoplasmic reticulum (ER) stress-related proteins such as PERK and eukaryotic initiation factor 2α(eIF2α)in rat podocytes, which was blocked by both [des-Arg10]-HOE140 and HOE140. In addition, B1R and B2R siRNA transfection prevented HG-induced phosphorylation of Akt, NF-κB, PERK, and eIF2α in rat podocytes. Moreover, HG upregulated the expression of CB1R mRNA and protein. AM251 treatment and CB1R siRNA transfection blocked the HG-induced BK receptors expression, signaling pathway.
4) HG increased the fibrosis related protein, fibronectin in rat mesangial cells. HG also increased tissue inhibitors of metalloproteinase-1 (TIMP-1) and decreased matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activity. In addition, both [des-Arg10]-HOE140 and HOE140 completely blocked the HG-induced increases in mRNA and protein expression of fibronectin. HG-induced TIMP-1 and MMP-2 and MMP-9 activity, which were blocked by both [des-Arg10]-HOE140 and HOE140. HG also increased the hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) activation and Smad2/3 translocation from the cytosol to the nucleus. HG-induced signaling molecules were blocked by both [des-Arg10]-HOE140 and HOE140. This study confirmed these results, observing that HG-induced activation of signaling molecules were blocked B1R and B2R siRNA, but not scrambled siRNA.In addition, HG-induced TGF-β1 activation and Smad2/3 translocation were not blocked by HIF-1α siRNA, suggesting HG-induced HIF-1α and TGF-β1/Smad2/3 signaling pathway independently control fibrosis in rat mesangial cells.
5) Increased B1R and B2R mRNA and protein expression shown in kidney of streptozotocin (STZ)-rats and ZDF rats using immunohistochemical stain, RT-PCR, and western blotting. In addition, pro-apoptotic proteins (caspase-3, CHOP, and Bax) and anti-apoptotic protein (Bcl-2) were increased in kidney of STZ- and Zucker rats better than normal rats.
In summary, 1) HG increases the EAAT-4 gluramate transporter activity and D-[2,3-3H]-aspartate uptake through B1R and B2R activation in ARPE-19 cells, and its action is associated with the cPLA2, COX-2 and NF‐kB signaling pathways, 2) HG increased oxidative stress and induced apoptosis via B1R, B2R, and CB1R activation and through FAAH 1 downregulation in ARPE-19 cells, 3)HG stimulates cell death such as apoptosis that is mediated by B1R and B2R throught CB1R activation as well as the Akt, NF‐κB/I-κB, and ER stress related proteins, PERK and eIF2α, signal pathways in podocytes, 4) HG stimulates fibronectin accumulation through HIF-1α and TFG-β1/Smad 2/3 througt B1R and B2R activation in mesangial cells, finally 5) B1R and B2R expression as well as apoptotic molecules were increased in kidney section of type 1 and type 2 diabetic models. These results shown a similar pattern to results of in vitro study. Therefore, the present results suggest that blockade of bradykinin receptors, B1R and B2R, may be a potential anti-diabetic therapy for the treatment of DR and DN.
Keyword
#Kallikrein-kinin system
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