The Mechanism Of Angiogenesis Caused By Peritoneal Dialysis Ultrafiltration Failure And Its Intervention Studies

Backgroud and ObjectiveEnd-stage renal disease (ESRD) has become one of the major public health problems which mankind must face up in the 21st century. Peritoneal dialysis (PD) is an effective way to treat end-stage renal disease. Compared with hemodialysis, PD has its own advantages, such as protecting residual renal function and lower cost. However, some complications often occur during peritoneal dialysis procedure, for example infections, malnutrition, ultrafiltration failure and so on. In particular, the rate of ultrafiltration failure was as high as 36% in these patients who have maitained peritoneal dialysis for 4-5 years. Ultrafiltration failure has become the main reason for peritoneal dialysis patients to withdraw from peritoneal dialysis. So it is greatly important to explore the reason of ultrafiltration failure and furthermore to intervent the occurrence of ultrafiltration failure of peritoneal dialysis.The pathogenesis of ultrafiltration failure was currently considered to relate with three main factors. The first was the increased peritoneal vascular surface area:about 50% to 75% ultrafiltration failure was due to peritoneal angiogenesis that lead to the increased peritoneal vascular surface area, which would make the transport of small solutes speeded up and then induced abdominal osmotic pressure gradient rapidly disappearing, then the peritoneal ultrafiltration capacity lost.The second was the loss of function of aquqporins(AQPs) and the last was the increasing of peritoneal interstitial/lymph fluid reabsorbing rate.Peritoneal neoangiogenesis was the main mechanism that dued to ultrafiltration failure. Regulating angiogenesis inducers included vascular endothelial growth factor (VEGF), fibroblast growth factor (aFGF and bFGF) and other soluble factors. Inhibitors included angiogenesis inhibin, Endostatin (ES) and so on. At present angiogenesis was regulated by inducing agents and inhibitors. Under normal circumstances they were in equilibrium, once the inducer was dominant, it would activate the blood vessels grow and shape the new blood vessels. Otherwise angiosclerosis would occur. A number of angiogenesis inducers increased in the peritoneal dialysis effluents of a long time peritoneal dialysis patients, such as VEGF, bFGF etc. Currently VEGF was recognized as the key factor in the pathogenesis of ultrafiltration failure and the promotion of angiogenesis. VEGF was in the dominant position during neoangiogenesis. The positive expression of VEGF was found in the peritoneal microvascular endothelial cells and peritoneal mesothelial cells (PMc), which was a hot topic in the field of international medical research. But the specific mechanism of angiogenesis factors was still not clear. In conclusion, it was necessary to study the role and the mechanism of peritoneal angiogenesis in peritoneal dialysis patients and to explore the ways to delay the occurrence of peritoneal dialysis ultrafiltration failure and provide a theoretical basis and experimental evidence. So it would provide an effective treatment for multiple control the occurrence of peritoneal dialysis ultrafiltration failure.Therefore, the study was further divided into three parts.Part I:Build 5/6 nephrectomy uremic rat model and then build uremic peritoneal dialysis rats. Detect the gene and protein expression of VEGF, bFGF and the ES, as well as detecting the microvessel desity of peritoneal tissue. Explore the mechanism of peritoneal angiogenesis. Set up the theoretical foundation for the intervention study.Part II:Select peritoneal biopsies which were obtained from normal subjects, uremic predialysis patients and PD patients. And then the gene and protein expressions of VEGF,bFGF and ES were detected by human's peritoneum. Microvessel Density (MVD) was counted in each group. Set up a theoretical foundation for the further intervention study.Partâ…¢:Use recombinant human endostatin (Endostar) to inhibit peritoneal neoangiogenesis of urimic peritoneal dialysis rats. The aim is to provide clinical experimental basis for treating the ultrafiltration failure of peritoneal dialysis.Partâ… Animal experimentent Study of Neoangiogensis in Peritoneal Dialysis Ultrafiltration failureMaterials and MethodsThe study was done in male SD rats of clean grade weighing 180-200g.We randomly selected 8 rats as normal group and established the uremia rats model of 5/6 nephrectomy and then from these uremia rats, we randomly established the peritoneal dialysis (PD) rats model. At last, there were 4 groups in this study:normal rats (n=8), uremia rats (n=8), dialysized rats exposed to PD 1.5% solution (n=8) and 4.25%(n=8) respectively. The catheters implanted in dialysis rats were tunneled subcutaneously to the neck and solutions 20ml per rat were kept in the abdominal cavity for 2 hours per day without anesthesia. After regularly peritoneal dialysis for 28 days, tissue immunohistochemistry methods and reverse transcript polymerase chain reaction (RT-PCR) were applied to detect the mRNA and protein expressions of VEGF,bFGF and ES in rats'peritoneal tissues of each group. Statistical analysis was performed to compare expressions and co relationship in each group. Significance was defined as a=0.05.Results1. VEGF,bFGF and ES were positively expressed in peritoneal tissues of normal rat.2. Compared with normal group, the protein expression of VEGF,bFGF and ES were up-regulated in uremia rats and dialysized rats. 3. Compared with normal group, the mRNA expression of VEGF and bFGF were up-regulated in uremia rats and dialysized rats (p<0.05), but there was no significant difference (p>0.05).4. In uremia rats (r=0.990, P=0.000),1.5% dialysized rats (r=0.925, P=0.000), 4.25% dialysized rats (r=0.968, P=0.000), there was a positive correlation between VEGF and bFGF in mRNA level.5. MVD was up-regulated in uremia rats and dialysized rats compared with normal group (P<0.05).Partâ…¡Clinical research of the Mechanism of Neoangiogensis inPeritoneal dialysis Ultrafiltration failureMaterials and MethodsPeritoneal biopsies were obtained from normal controls (from non-uremic patients with abdominal surgery, excluding any abdominal membrane disease), uremic predialysis patients at catheter insertion and PD patients at the time of catheter remove, reinsertion or renal transplantation. RT-PCR techniques and Immunohistochemical staining were used to investigate VEGF, bFGF and ES expression in peritoneal tissue. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody.Results1. The mRNA expression of VEGF and bFGF were found in all peritoneal samples in each group. Base on normal group, the mRNA expression of VEGF,bFGF in uremic predialysis and PD group were significantly up-regulated (P<0.05) Compared with uremic predialysis group, the mRNA expression of VEGF, bFGF in PD group were significantly up-regulated (P<0.05). The mRNA expression of ES was no significantly difference in each group.2. The protein expression of VEGF and bFGF were found in all peritoneal samples in each group. Compared with normal group, the protein expression of VEGF, bFGF and ES in uremic predialysis and PD group were significantly up-regulated (P<0.05). Compared with uremic predialysis group, the protein expression of VEGF, bFGF and ES in PD group were significantly up-regulated (P<0.05)3. The new micro vascular vessels in normal group shows little or none. MVD was up-regulated in uremic predialysis and PD group compared with normal group (P<0.05).Partâ…¢Intervention Study of Preventing the occurrence of Peritoneal Dialysis Ultrafiltration FailureMaterials and Methods43 male SD rats of clean grade weighing 180-200g were used in the study. The uremia rats model was established by 5/6 nephrectomy and then from these uremia rats, we randomly established the peritoneal dialysis (PD) rats model. At last, there were 5 groups in this study:A:normal group (control group:n=8), B:uremia group (5/6 nephrectomy group:n=8), C:uremia peritoneal dialysis group (4.25% dialysized group:n=8), D1:the first treatment group with Endostar (10mg/kg treatment group: n=8), D2:the second treatment group with Endostar (40mg/kg treatment group:n=8). The rats from C, D1, D2 groups were given regular peritoneal dialysis. The catheters implanted in dialysis rats were tunneled subcutaneously to the neck and exchanged PD solutions per rat were kept in the abdominal cavity for 2 hours per day without anesthesia. The rats from D1, D2 groups accepted subcutaneous injection of Endostar during peritoneal dialysis period every other days, total administration 14 times. After regularly peritoneal dialysis for 28 days, tissue immunohistochemistry methods and reverse transcript polymerase chain reaction (RT-PCR) were applied to detect the mRNA and protein expressions of VEGF and in rats'peritoneal tissues of each group. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody. Statistical analysis was performed to compare with their expressions and co relationship in each group. Significance was defined as a=0.05.Results1. Base on normal group, the mRNA and protein expression of VEGF were significantly up-regulated in uremia group and uremia peritoneal dialysis group (P<0.05). Compared with uremia peritoneal dialysis group, the mRNA and protein expression of VEGF were significantly down-regulated in the first and second treatment groups with Endostar (P<0.05). Compared with the first treatment group with Endostar, the mRNA and protein expression of VEGF were significantly down-regulated in the second treatment group with Endostar (P<0.05)2. Base on the normal group, MVD was up-regulated in uremia group and uremia peritoneal dialysis group (P<0.05). Compared with uremia peritoneal dialysis group, MVD was significantly down-regulated in the first and second treatment groups with Endostar (P<0.05). Compared with the first treatment group with Endostar, MVD was significantly down-regulated in the second treatment group with Endostar (P<0.05)3. There was a positive correlation between VEGF and MVD in protein level (r=0.987, P=0.000)Conclusions1. The expression of VEGF,bFGF and ES was positive in peritoneal tissues both normal rats and human; the expression of VEGF,bFGF and ES was up-regulated by uremia circumstance and non-physiological compatibility peritoneal dialysis liquid.2. The upregulation of VEGF and bFGF in peritoneal dialysis rat and patient peritoneum might participate in the increasement of the peritoneum neoangiogensis and then cause the incidence of ultrafiltration.3. The protein level expression of ES was up-regulated by uremia circumstance and non-physiological compatibility peritoneal dialysis liquid. ES might play an important role in inhibitting peritoneum neoangiogensis4. Recombinant human endostatin (Endostar) can effectively inhibit rat peritoneum neoangiogensis inducing by peritoneal dialysis, and the role of inhibit strength is related with the dose of the drug.5. Recombinant human endostatin (Endostar) can inhibit rat peritoneum neoangiogensis by down-regulating the expression of VEGF in peritoneum.