The Potential Role Of Angiopoietin-1/Tie-2 Signaling On Peritoneal Vascular Permeability In Uremic Peritoneal Dialysis Rats

Background.Protection of the peritoneal vascular endothelium is an essential aspect of preventing increased peritoneal solute transport rate during long-term peritoneal dialysis(PD).Angiopoietin-1(Ang-1)is an important endothelial-specific protective factor,and the aim of the present study was to explore the potential role of Ang-1 on peritoneal vascular permeability in uremic PD ratsMethod.Male SD rats(n=36)were randomly grouped into sham group(n=12),uremia group(n=12)and uremic PD group(n=12).5/6 nephrectomy was used to construct uremia model,and uremic PD model was subjected to 5/6 nephrectomy plus intraperitoneal infusion of 4.25%glucose PD solution for 4 weeks..Functional and structural changes of the peritoneum were examined.Expression of Ang/Tie-2 and inter-endothelial junction protein(Occludin and VE-cadherin)in the peritoneum were determined by western blot.Pericyte coverage was quantified by double immunofluorescence staining.Peritoneal vascular permeability was investigated by measuring the leakiness of FITC-dextran(4 kDa)and FITC-BSA(69 kDa).Uremic PD rats were transfected by Ad-FLAG(n=10)and Ad-COMP-Ang-1(n=10),and the effects of COMP-Ang-1 on the peritoneal morphology and vascular permeability were investigatedResults:1.Compared to sham rats,morphological changes of peritoneal membrane were noted in uremia rats,including obvious mesothelial cells loss,thickened submesothelial extracellular matrix(177.49±42.48 vs 14.01±6.12?m,P=0.027)and increased peritoneal vessel density(6.43±1.13 vs 2.00±0.71/HP,P<0.001).After PD solution infusion for 4 weeks,these changes were more intensive,including more mesothelial cells loss,submesothelial extracellular matrix further thickened(728.68± 103.47 vs 177.49±42.48?m,P=0.002),and number of peritoneal vessel further increased(18.60±1.14 vs 6.43±1.13/HP,P<0.001).Compared to sham rats,uremic rats were also characterized by decreased pericyte coverage(0.475±0.078 vs 0.890±0.059,P=0.0001)and downregulated VE-cadherin expression(0.77±0.18 vs 1.28±0.27,P<0.05).There was no obvious change of Occludin expression in uremia rats compared to sham rats(0.69±10.25 vs 0.92±0.14,P>0.05).These trends were more remarkable in uremia PD rats,including pericyte coverage further decreased(0.121 ±0.056 vs 0.475±0.078,P<0.0001),VE-cadherin(0.40±0.16 vs 0.77±0.18,P=0.04)and Occludin(0.08±0.02 vs 0.69±0.25,P=0.015)expression further reduced significantly.Permeability of peritoneal microcirculation to FITC-BSA(128.88± 16.51 vs 2.56±0.42,P<0.0001)and FITC-dextran(165.73±52.28 vs 9.43±0.98,P=0.004)in uremia rats were higher than that in sham rats.After 4-week's PD solution infusion,the permeability of peritoneal microcirculation to FITC-BSA(2151.28± 151.34 vs 128.88± 16.51,P<0.001)and FITC-dextran(5695.14±1266.02 vs 165.73±52.28,P<0.001)were further increased.Uremic rats had higher D/Pcr(0.65±0.13 vs 0.50±0.09,P<0.05)and less net ultrafiltration(16.10±7.25 vs 22.91 ±4.04,P<0.05)than that in the sham group.D/Pcr further increased(0.92±0.06 vs 0.65±0.13,P<0.05)and net ultrafiltration further decreased(10.43± 1.08 vs 16.10±7.25,P<0.05)in uremia PD rats compared to uremia rats.2.Peritoneal expression of Ang-1 protein(0.91 ±0.14 vs 1.50±0.90,P=0.031)and Tie-2 protein phosphorylation level(0.22±0.04 vs 0.33±0.09,P<0.05)in uremic rats were both decreased compared to sham rats,and further down-regulated after 4-week's PD solution infusion(0.59±0.02 vs 0.91 ±0.14,P=0.032;0.16±0.03 vs 0.33±0.09,P<0.05 respectively).Compared to sham rats,peritoneal expression of Ang-2 protein was significantly upregulated in uremia PD rats,but not in uremia rats(0.52±0.05 vs 0.40±0.12,P>0.05).3.Compared to Ad-control group and uremia PD group,Ad-COMP-Ang-1 group had upregulated Ang-1 protein expression and higher Tie-2 protein phosphorylation level.Pericyte coverage increased(0.656±0.078 vs 0.121 ±0.056,P<0.0001;0.656±0.078 vs 0.170±0.067,P<0.0001),VE-cadherin(1.13±0.03 vs 0.72±0.16,P<0.05;1.13±0.03 vs 0.97±0.21,P<0.05)and Occludin(0.55±0.06 vs 0.16±0.06,P<0.001;0.55±0.06 vs 0.25±0.05,P<0.001)protein expression upregulated in Ad-COMP-Ang-1 group compared with Ad-control group and uremia PD group.Peritoneal microcirculation permeability to both FITC-BSA(227.371± 16.703 vs 2151.283±151.338,227.371 ±16.703 vs 2151.283± 151.338,P<0.001)and FITC-dextran(695.419± 107.166 vs 5695.13 6± 1266.021,695.419±107.166 vs 5746.325± 165.676,P<0.001)was significantly decreased in Ad-COMP-Ang-1 group than that in Ad-control group and uremia PD group.Ad-COMP-Ang-1 group had lower D/Pcr than that in Ad-control group(0.58±0.10 vs 0.93±0.06,P<0.05)and uremia PD group(0.58±0.10 vs 0.92±0.06,P<0.05).4.Compared to sham rats and uremia rats,both PI3K and AKT protein phosphorylation levels were significantly decreased in uremia PD group(0.460±0.038 vs 0.605±0.081,P=0.04;0.419±0.106 vs 0.752±0.037;P=0.01)Compared to Ad-control group and uremia PD group,COMP-Ang-1 upregulated both PI3K and AKT protein phosphorylation levels in Ad-COMP-Ang-1 group(1.219±0.063 vs 0.419±0.106,P<0.0001;2.155±0.103 vs 0.419±0.106,P=0.0001)Conclusion:1.Uremia and PD solution could reduce pericyte coverage,downregulate inter-endothelial junction proteins expression,increase permeability of peritoneal microcirculation,and induce higher peritoneal solute transport rate and less ultrafiltration.2.Uremia and PD solution down-regulated Ang-1 protein expression and Tie-2 protein phosphorylation level in peritoneal membrane.3.Enhance Ang-1/Tie-2 pathway improved pericyte coverage and inter-endothelial junction proteins expression,inhibited the high permeability of peritoneal microcirculation and lower peritoneal solute transport rate induced by uremia and PD solution.4.The protective mechanism of Ang-1/Tie-2 pathway on peritoneal vascular endothelium might be achieved through PI3K/Akt-dependent signaling pathway...