| ObjectiveArisolochic Acid Nephropathy (AAN) is a renal disease reported after the introduction of Chinese herbs contained aristolochic acid. The chronic Aristolo-chic Acid nephropathy (CAAN) is the commonest in different AANs. Rapidly progressive tubulointerstitial fibrosis and early, severe anaemia and urothelial malignancy of the upper urinary is the characteristics of AAN. Recently, attention has focused on the mechanism of fibrosis and anaemia in AAN.Many Chinese herbs contain aristolochic acid, which were extensively used to treat disgestive and urogenital system diseases. Although the government has adopted lots of measures, there are many drug abused people in China. The task is very difficult to obviate and treat AAN in future.After about ten - year research, the doctors of department of nephrology had many theories about the mechanism of AAN. But the role of microvessel abnormality in the kidney and bone marrow injury was not paid close attention. Our investigation is to explore the role of aristolochic acid to the microvessel injury of kidney and bone marrow in rats. We used EGB to interfere in the CAAN rats and observed the role of protection. Our investigation included three parts: 1 The peritubular capillary injury on the progressive chonic Aristolochic Acid Nephropathy rats. 2 The microvascular injury of bone marrow in chronic Aristolochic Acid Nephropathy. 3 Effects of ginkgo biloba extract on microvascular injury of kidney and bone marrow in the rat model of chronic Aristolochic Acid Nephropathy.Materials and MethodsPart 1;All animal procedures were conducted after approval of protocol by the Chinese Medical University Animal Care Committee. Female Wistar rats (180 to 220 g, age, 7 to 8 weeks) were kept under standard conditions with unrestricted access to food and water in a 12;12 - h light - dark cycle. The CAAN Group ( n = 30) were established by administering with caulis aristolochiae man-shuriensis ( CAM) decoction by gavage at a dose of 10ml kg'1 d"1 (twice a day) for 8 weeks. Animals were sacrificed at 8, 12 and 16 weeks after CAM decoction administration. The Control Group (n = 24) were administered with drinking water by gavage at a dose of 10ml ? kg"1 ? d"1 (twice a day) for 8 weeks, and animals were sacrificed at 8, 12 and 16 weeks. Urine was collected for 24h in metabolic cages before death. At death, sample of serum was collected. The kidneys were rapidly removed and transferred to a metal plate cooled on ice for sectioning. One kidney was divided into two parts and snap frozen in liquid nitrogen and stored at -70^. The other kidney was sectioned coronally and immersed in 4% neutral — buffered formalin for 24h, dehydrated in graded alcohols, and embedded in paraffin. 24h urinary protein excretion rates were measured using the sulfosalicylic acid method, and blood urea nitrogen (BUN) and serum creatinine ( Scr) levels were determined with a Keysys analysator. CD34, HIF - la, VEGF and TNF - a were detected in the CAAN rat model.Part 2: The CAAN Group (n = 30) were established by administering with CAM decoction by gavage at a dose of 10ml ? kg"1 ? d~' (twice a day) for 8 weeks. Animals were sacrificed at 8, 12 and 16 weeks after CAM decoction administration. The Control Group (n = 24) were administered with drinking water by gavage at a dose of 10ml ? kg ~ d ~ (twice a day) for 8 weeks, and animals were sacrificed at the 8' , 12' and 16' week. Urine was collected for 24h in metabolic cages before death. At death, sample of serum was collected. The thigh bones were immersed in 4% neutral - buffered formalin for 24h, dehydrated in graded alcohols, and embedded in paraffin. 24h urinary protein excretion rates were measured using the sulfosalicylic acid method, and BUN and Scr lev-els were determined with a Keysys analysator. The hemoglobin (Hb) , reticulo-cyte ( Ret) and mean corpuscular volume ( MCV) were determined with a Sys-mex - 2100 analysator. The electron microscope was used to evaluate the mi-crovessel injury of bone marrow. CD34, HIF -la, VEGF and TNF - a were detected in the CAAN rat model.Part 3: 20 Female Wistar rats were treated with CAM decoction for 8 weeks, and then they were divided into two groups stochasticly: Group A were then treated with EGB for 8 weeks;Group B were treated with drinking water for 8 weeks. In addition, 8 rats were set up for the control groups (Group C). All of rats were scarificed at the end of 16th week. Specimens of blood, urine and kidney and bone marrow were taken before the animals were killed. Pathological examination, immunohistochemical analysis, western - blotting and RT - PCR were made to the renal specimens.Statistical Comparisons: Values are reported as mean ± SD. We used SPSS11.5 software to conduct statistical analyses. The relationships between variables were assessed by Pearson correlation analysis. All comparisons were two - tailed. Significance was defined as P <0. 05.ResultsPart 1: A significant increase in BUN and Scr levels was observed for the CAAN rats from the 8th week, compared with the Control Group, P <0. 05 at 8' week and P <0. 01 at the 12th and 16th week. Pathologic proteinuria ( defined as >25 mg/24h) was present in the CAAN rats after 8 weeks and then exhibited a continous increase up to week 16. In the Control Group, no significant abnormality was observed in the renal tissue samples obtained on week 8, 12 and 16. On the contrary, at week 8, in the CAAN rats, there was moderate interstitial e-dema and partial tubular necrosis, but no significant interstitial fibrosis. At week 12, partial foci of tubular atrophy and mononuclear cells infiltration, surrounded by moderate interstitial fibrosis, were found in the deep cortex, the outer medulla, and the medullary rays from the CAAN rats. In the Control Group, PTC was normal. But in the CAAN rats, partial PTC basement membrane thick-ening was clearly confirmed at week 8. At week 12, PTC basement membrane was fuzzy. At week 16, PTC endothelial cell was destructive and PTC basement membrane was fuzzy and dissolved. The protein and mRNA expression of CD34 and VEGF decreased, but that of HIF -1 a increased gradually, and the expression of TNF - a increased firstly and decreased later. At week 16, the PTC density of the Control Group was 42. 80 ±4.49/0. 13 mm compared to that of the AAN rats 8.10 ±2.28/0.13 mm2, P <0.01;The HIF - la - IOD was 0.49 ±0. 06 x 103 compared to that of the CAAN rats 7. 11 ± 1. 20 x 103, P < 0. 01;The VEGF - IOD was 26.94 ±9.34 x 103 compared to that of the CAAN rats 2. 78 ±0.78 xlO3, P<0.01;TheTNF-a-IOD was 4. 52 ±0.35 x 103 compared to that of the CAAN rats 18.96 ±3. 63 x 103. At the 8th week, the area of renal interstitium in the Control Group decreased greatly than that in the CAAN rats, but it increased at the 12' and increased more significantly at the 16th week.Part 2: The variation of the renal function and urine protein was same as the Part 1. the CAAN rats were anaemic at the 12th week, and anemia became worse at the 16* week. The level of Hb of the CAAN rats compared with the Control Group, P <0.01. The TNF - a in myeloid tissue of the CAAN rats increased significantly, and CD34 expression and MVD decreased following the time. Compared with the Control Group, P <0. 01. The level of EPO of each group was normal,and the CAAN rats compared with the Control Group, P <0.01. In the Control Group, the microvessel of bone marrow was normal. But in the CAAN rats, the microvessel was dilated and congested. At week 12, the cell membrane of microvessel was fuzzy and destructive. At week 16, the microvessel was entirely dissolved. The protein and mRNA expression of CD34 and VEGF decreased, but that of HIF -1 a increased gradually, and the expression of TNF - a increased firstly and decreased later. At week 16, the MVD of the Control Group was 27.13 ±2. 80/0.13 mm2 compared to that of the CAAN rats 4. 80 ± 1.48/0.13 mm2, P <0.01;The HIF - la -IOD was 3. 65 ±0.76 x 103 compared to that of the CAAN rats 15.26 ±3. 89 x 103, P <0.01;The VEGF -IOD was 33.05 ±4.61 x 103 compared to that of the CAAN rats 3.94 ±0. 62 x 103, P <0.01;The TNF - a - IOD was 1. 99 ±0.43 x 103 compared to that ofthe CAAN rats 6.26 ± 1.13 x 103.Part 3: It was found that there was significant tubulointerstitial injury and interstitial fibrosis, with more markedly decreased PTC density in the Group A. Group A compared with Group B and Group C, PTC density, real function and the area of renal tubule decreased more (all of P values were less than 0.01) , but the area of renal interstitial is significantly increased (P value was less than 0. 01). The PTC density is positive correlation with the level of Scr and the area of renal tubule, but negative with the area of renal interstitial ( all of P values were less than 0. 01). The protein and mRNA expression of HIF - la and TNF - a decreased significantly in Group B compared that of Group A. The protein and mRNA expression of VEGF increased significantly in Group B compared that of Group A.ConclusionThese data suggested that:1 Following significant reductions in PTC densities and VEGF expression, HIF - 1 a expression increased markedly in these experiments, which contributed to the progressive tubulointerstitial fibrosis. PTC loss, with consequent tissue hypoxia and ischemia, may play an important role in progressive CAAN. The decreased VEGF expression and the increased TNF - a expression may be the causes of micro vascular injury of kidney in the CAAN rats.2 The microvessel of bone marrow was markedly injured and the MVD decreased following the time, but the expression of HIF - 1 a increased. The mi-crovascular injury of bone marrow is correlative with the early, severe anemia. The decreased VEGF expression and the increased TNF - a expression may be the causes of microvasculair injury of bone marrow in the CAAN rats.3 The intervention by EGB can significantly increase the protein and mRNA expression of VEGF and decrease the protein and mRNA expression of TNF - a, which ameliorated PTC injury and anemia, and protected the renal function. It expanded a new therapeutic way to CAAN. |
PDF Full Text Request