The Theory Of The "Turbid Toxin Causing Blood Stasis" In Renal Interstitial Fibrosis And Protective Effect Of Chinese Herbs

Objective: Chronic kidney disease(CKD) was seriously harmful to the health of human quality in our country, which had occupied in the spectrum of disease for a long time. Renal interstitial fibrosis can be seen in the progression of chronic kidney disease. At present, a large number of clinical and experimental results show that the degree of renal interstitial fibrosis is closely related to the renal function and prognosis of patients. And we need to take effective methods to alleviate the inflammatory cell infiltration, reduce inflammatory response, prevent the occurrence and development of interstitial fibrosis.In the formation of renal interstitial fibrosis, renal interstitial inflammatory cells infiltration, inflammatory reaction, renal tubular cell damage, phenotype transformation, the excessive deposition of extracellular matrix(ECM), renal interstitial fibroblasts activation and proliferation can lead to the formation of scar hardening. And the cell proliferation induced by inflammatory injury plays an important role in the progression of renal interstitial fibrosis.In the early study of adrenomedullin(ADM) inhibited cell phenotype conversion process by the regulation of Huayu Ditan Tongluo prescription, we found that it is difficult to slow down disease progression by inhibition of cell proliferation. And cell proliferation, which can be promoted by inflammatory lesions, plays a key role in renal interstitial fibrosis.At the end-stage renal disease of spleen and kidney deficiency, wet turbidity toxin connotation can be found and long illness come into collaterals. Based on inflammatory injury and cell proliferation in renal interstitial fibrosis, we proposed hypothesis of blood stasis caused by "Zhuodu".In this study, animal models of renal interstitial fibrosis were established by subjecting wild-type(WT) and ADM knockout mice to unilateral ureteral obstruction(UUO). Overexpression of inflammatory mediators was induced by activation of aldosterone, which then activated nuclear factor kappa B(NF-κB) by protein kinase-mediated phosphorylation. Consequently, cell proliferation was induced, and the overproduction of the ECM in turn increased the progression of renal interstitial fibrosis. In our study, we have realized signal transduction pathway in fibrosis induced by inflammatory mediators with cell proliferation and the protective effects of adrenomedullin(ADM). At the same time, treatment with eplerenone and Yiqi Huayu Jiedu decoction, we realized the mechanism and signal pathway of inflammatory mediators( "Zhuodu") induced cell proliferation and fibrosis(blood stasis) formation. And the effect of ADM in the proliferation of cells and the regulation target of traditional Chinese medicine were also observed.Part one The theory of Turbid Toxin Induced Blood Stasis in renal interstitial fibrosis and the protective effect mechanism of traditional Chinese medicine for Yiqi Huayu JieduMethods: According to renal interstitial fibrosis at the end stage of chronic kidney disease, symptom matched corresponding TCM disease category, referring to the ancient Chinese medical literatures, we found the commonness. Combined with the theory of traditional Chinese medicine, modern Chinese medicine science research and long-term clinical application, we could realize the main function of traditional Chinese medicine in experiment and clinical application on RIF’s treatment.Sixty-four refractory nephrotic syndrome patients were divided into treatment group and control group, with 32 in each group. The control group was treated by standard prednisone. The treatment group was taken Chinese medicine based on benefiting qi, removing blood stasis and removing toxicity over half a year in addition to treatment in the control group. The 24 h urinary protein(URPO), serum albumin(ALB), total cholesterol(CHOL), triglyceride(TG) fibrinogen(FIB) and partial thromboplastin time(APTT) changes were detected in both groups.Results: Summarized and proposed the theory of blood stasis caused by Zhuodu in renal interstitial fibrosis, to solve the pathogenesis, we adopted the therapy of "Yiqi Huayu and Jiedu". In the experimental study and clinical application, we had realized the protective effects of Yiqi Huayu Jiedu traditional Chinese medicine on renal interstitial fibrosis.The levels of URPO, CHOL, TG and FIB in the treatment group were lower than before treatment(P < 0.05), and the levels of ALB and APTT were significantly increased(P < 0.05). The levels of URPO, CHOL and FIB in the control group were lower than before treatment(P < 0.05), and the levels of ALB and APTT were significantly increased(P < 0.05). At the end of treatment, the levels of URPO, CHOL, TG and FIB in the treatment group were significantly decreased compared with the control group(P < 0.05), and the levels of ALB and APTT were significantly increased(P < 0.05).Part two Reproduction and identification of ADM gene knockout miceMethods: Female C57BL/6J mice weighing 25-30 g were obtained from Beijing HFK Bioscience Co., Ltd. and male ADM knockout(AMKO) mice(+/-) were kindly provided by Tatsuo Shimosawa from The University of Tokyo. The mice had free access to tap water and a wet mash non-sodium-depleted diet. They fed by standards of SPF. After adaptive feeding, 1 male and 2 female mice were caged. After hybridization, three month old male ADM gene knockout mice(+/-) and female WT mice were caged toghter, female mice get pregnant immediately recorded the paternally derived. DNA was amplified by PCR from tail tissues of mice, and then 2% agarose gel electrophoresis was carried out to identify ADM gene knockout mice.Results: Genotype fragment of mice tail tissue by agarose gel electrophoresis displayed ADM gene knockout mice heterozygous(+/-) double channels and we can identify adrenomedullin(ADM) gene knockout mice by such gene band.Part three The effect of Yiqi Huayu Jiedu decoction on morphology, aldosterone and cell proliferation of wild-type(WT) and ADM gene knockout(AMKO) mice with UUOMethods: Wild type(WT) and ADM gene knockout(AMKO) mice were divided into eight groups, including the WT-Sham, WT-UUO, WT-UUOE, WT-UUOZ, AMKO-Sham, AMKO-UUO, AMKO-UUOE and AMKOUUOZ groups. The left ureter was ligated in all mice except those in the sham operation groups, which underwent the same procedures except that the left ureter was not ligated. WT-UUOE and AMKO-UUOE groups received eplerenone mixed in the feed at the dose of 100 mg/kg/day. WT-UUOZ and AMKOUUOZ were treated with Yiqi Huayu Jiedu Decoction(Huangqi 20 g, Danshen 20 g, Cubiejia, Jiangcan, Wushaoshe, Dilong, Chishao, Huangqin, Jinyinhua, Pugongying and Dahuang each 10 g, these drugs according to the adult dose of 50 kg body weight daily for a translation) 6.5g/kg·d oral irrigation. All mice in the sham operation groups and UUO groups were treated with normal saline and normal food. The drug was administered for 10 days, during which time the general appearance and health of mice in each group were observed. Blood and kidneys were harvested at the end of the experiment. Serum creatinine(Scr) and blood urea nitrogen(BUN) levels were determined by the Jaffé and diacetyl monoxime methods, respectively. Aldosterone was measured with a radioimmunoassay. Part of each left kidney was immediately snap frozen on liquid nitrogen(stored at-74℃) for Western blotting, while the remaining tissue was fixed in 4% paraformaldehyde for histopathology. Tissues were dehydrated by bathing in increasing concentrations of methanol or isopropanol. After embedding in paraffin, 3μm sections of the kidney were cut and then prepared by hematoxylin and eosin(H&E) staining and Masson’s trichrome staining, to observe histopathological changes. And PCNA was detected by using immunohistochemistry. All data are presented as means ± S.D. Statistical analysis was performed using one-way analysis of variance(ANOVA) and Chi-square test by SPSS 17.0. A P value < 0.05 and 0.01 was considered statistically significant.Result:1 Experimental results of the general observation of mice During the experiment, two sham operated mice were found to have no significant abnormality in the drinking water and the other groups, and the incision healed well. At the end of the experiment, the removal of mice in each group of the left kidney, observation showed that two sham operated mice kidney shape and color are consistent with that of the normal mice, the model group showed kidney volume was significantly increased water, color from the normal bright red to yellow, very dark brown: two in accordance with eplerenone group and Chinese medicine group kidney volume is also increasing, but compared with the model group, the relatively small, less the amount of water, the color rendering dark red. 2 Pathological changes in kidneys of mice H&E staining showed no obvious abnormalities in the sham operation groups of WT and ADM knockout mice. In UUO mice, obstructed kidneys showed edema, necrosis and shedding in the renal tubular epithelial cells and even extreme atrophy. A large number of inflammatory cells infiltrated the interstitial areas, especially in ADM knockout mice. Compared with the UUO mice, eplerenone and traditional Chinese medicine treated mice showed similar signs of injury, such as cell necrosis and distended renal tubules and ureters, but inflammatory cell infiltration was inhibited. Masson’s trichrome staining showed no obvious abnormality of the renal structure in the sham operation group, and the renal tubular basement membrane was intact. A small amount of collagen was observed in the renal interstitium in WT and AMKO mice. In UUO mice, the collagen increased markedly in the renal interstitum in AMKO mice. The collagen deposition was decreased in the eplerenone and traditional Chinese medicine treated group, but the injury in AMKO mice was more severe than that in WT mice. 3 Tests of renal function BUN levels were statistically significantly different between the WT and AMKO groups, as well as among the sham, UUO, UUOE and UUOZ groups(P<0.05), and WTUUOZ group was different from AMKOUUOZ group(P<0.05). As shown in Fig. 2-6;Table 1-2, Scr levels were significantly different(P<0.05) between the model groups and the corresponding sham groups(WT and AMKO mice). Levels of Scr were significantly decreased in the UUOE and UUOZ groups compared with the corresponding UUO groups in the same type(P<0.05).And WTUUOZ group was different from AMKOUUOZ group(P<0.05). 4 Tests of aldosterone Measurements of the biological activity of aldersterone also showed significant differences among the sham, UUO, UUOE and UUOZ groups(P<0.05), and the two model groups were statistically significant in the other groups(P<0.05). WTUUOE and WTUUOZ groups were different from AMKOUUOE and AMKOUUOZ groups(P<0.05). 5 The effect of cell proliferation As demonstrated in Fig.2-4,2-8;Table.1-4,PCNA was detected. Few PCNA positive cells were observed in renal tubular epithelial cells in mice of the sham group, while their numbers were increased significantly in the dilated distal tubule in UUO mice. Compared with UUO mice, PCNA positive cells were reduced in the eplerenone and traditional Chinese medicine treated groups. Compared with WT mice, the number of positive cells increased clearly in the UUO, eplerenone and traditional Chinese medicine treated groups of AMKO mice(P<0.05).Part four The effect of Yiqi Huayu Jiedu decoction on SGK-1, NF-кB(P65), ERK,P-ERK and ADM of wild-type(WT) and ADM gene knockout(AMKO) mice with UUOMethods: Animal groups, model replication, drug delivery methods were the same as the second part, a total of 10 days. At the end of the experiment, Part of each left kidney was immediately snap frozen on liquid nitrogen(stored at-74°C) for Western blotting, and SGK-1, NF-кB(p65), ERK and P-ERK expression wre detected by using immunohistochemistry. The statistical method was the same as the second part.Result:1 Detection of SGK-1, NF-кB(p65), ERK and P-ERK by Western Blot 1.1 Test of SGK-1 by Western Blot Semi-quantitative results show that there was a small amount of expression in WT-Sham and AMKO-Sham groups, WT-UUO and AMKO-UUO groups were significantly higher than sham group of the same type(P<0.05), and AMKO-UUO group was higher than WT-UUO group(P<0.05). The treatment groups were significantly lower than UUO groups of the same type(P<0.05). There was no significant difference between the traditional Chinese medicine group and the eplerenone group in the same treatment group of WT and AMKO mice(P>0.05). 1.2 Test of NF-кB by Western Blot Western blot analysis was used for the detection of NF-кB protein expression, semi-quantitative results show that there was a small amount of expression in WT-Sham and AMKO-Sham groups, WT-UUO and AMKO-UUO groups were significantly higher than sham group of the same type(P<0.05), and AMKO-UUO group was higher than WT-UUO group(P<0.05). The treatment groups were significantly lower than UUO groups of the same type(P<0.05). There was no significant difference between the traditional Chinese medicine group and the eplerenone group in the same treatment group of WT and AMKO mice(P>0.05). Compared with WT mice, there was significant difference in the traditional Chinese medicine group and the eplerenone group of AMKO mice(P<0.05). 1.3 Test of P-ERK by Western Blot Semi-quantitative results show that there was a small amount of expression in WT-Sham and AMKO-Sham groups, WT-UUO and AMKO-UUO groups were significantly higher than sham group of the same type(P<0.05), and AMKO-UUO group was higher than WT-UUO group(P<0.05). The treatment groups were significantly lower than UUO groups of the same type(P<0.05). There was no significant difference between the traditional Chinese medicine group and the eplerenone group in the same treatment group of WT and AMKO mice(P>0.05). Compared with WT mice, there was significant difference in the traditional Chinese medicine group and the eplerenone group of AMKO mice(P<0.05). 1.4 Test of P-ERK/ERK by Western Blot By test of Western blot, the results showed that little expression of P-ERK/ERK protein in WT-Sham and AMKO-Sham groups, WT-UUO and AMKO-UUO groups were significantly higher than sham group of the same type(P<0.05), and AMKO-UUO group was higher than WT-UUO group(P<0.05). The treatment groups were significantly lower than UUO groups of the same type(P<0.05). There was no significant difference between the traditional Chinese medicine group and the eplerenone group in the same treatment group of WT and AMKO mice(P>0.05). 1.5 Test of ADM by Western Blot Western blot analysis was used for the detection of ADM expression, semi-quantitative results show that there was a small amount of expression in WT-Sham and AMKO-Sham groups, WT-UUO and AMKO-UUO groups were significantly lower than sham group of the same type(P<0.05), and AMKO-UUO group was lower than WT-UUO group(P<0.05). The treatment groups were significantly higher than UUO groups of the same type(P<0.05). There was significant difference between the traditional Chinese medicine group and the eplerenone group in the same treatment group of WT and AMKO mice(P>0.05). Compared with WT mice, there was significant difference in the traditional Chinese medicine group and the eplerenone group of AMKO mice(P<0.05). 2Immunohistochemical analysis of SGK-1,NF-кB and P-ERK 2.1 Immunohistochemical analysis showed expression of SGK-1 in the cytoplasm of collecting duct epithelial cells, under the microscope, WT-Sham and AMKO-Sham groups were no positive expression in glomerular and renal tubule. SGK-1 increased significantly in the cytoplasm of the epithelial cells of the collecting duct, some parts of tubular epithelial cells and surrounding stromal area was significant, especially AMKO-UUO group. Compared with the model groups, the expression of SGK-1 in the two types of treated groups were significantly lower than the model groups.2.2 Immunohistochemical analysis showed expression of NF-кB in the cytoplasm of cortical proximal tubules, WT-Sham and AMKO-Sham groups were was no positive expression in glomerular and renal tubule, but NF-кB increased significantly in the cytoplasm of renal cortical proximal tubule, especially AMKO-UUO group. Compared with the model groups, the expression of NF-кB in the two types of treated groups were significantly lower than the model groups. 2.3 Immunohistochemical analysis showed a little of expression of P-ERK in the glomerular and renal tubule. The expression of P-ERK in WT-UUO and AMKO-UUO groups can be seen in renal tubular epithelial cells, especially AMKO-UUO group. Compared with the model groups, the expression of P-ERK in the two types of treated groups were significantly lower than the model groups. And the AMKO mice were more serious than the WT mice.Conclusion: 1 "Turbid Toxin Causing Blood Stasis" is the pathogenesis of chronic kidney disease. 2 Obstructive nephropathy can activate aldosterone, induce inflammatory damage, stimulate cell proliferation, and involve in renal interstitial fibrosis. 3 There is a closely relationship between ADM and renal interstitial fibrosis. Chinese herb can inhibit renal interstitial fibrosis by the way of regulating ADM.