| Objective: Tubulointerstitial fibrosis (TIF) is a common pathological feature of most end-stage kidney disease. Numbers of clinical pathology observations had shown that tubulointerstitial injury played more important role than glomerular damage on renal failure. So looking for those none or lower toxicity drugs and reject tubulointerstitial fibrosis in different channel and stage are significantly on renal disease treating. The disease of tubuloninterstitial was associated with cell proliferation and it played an important role in the progress of renal fibrosis. The model of unilateral ureteral obstruction induced the serious injury of tubuloninterstitial. It showed tubuloninterstitial cell proliferation, unicellular infiltrated and comply with the tubular epithelial cell hyperplasia and myofibroblast. Cell proliferation was related to Angiotensin II and transforming growth factor-β1. It could be inhibited by the Angiotensin II receptor blocker. Adrenomedullin is a kind of vasoactive peptide that be found in recently. It existed in many important organs and tissues. It mainly physiological function was to make the vessel dilated, lowered blood pressure, discharged Na+ and diuresis, and also played a key role in regulating the function of heart and kidney.Recently investigation showed that adrenomedullin could reject the tubulointerstitial fibrosis through to inhibit transforming growth factor-β1which stimulated collagen synthesis and secretion. It becomes a hot spot in kidney disease study. The traditional Chinese herbs plays important role in treating kidney disease. Animal experiment showed that Chinese herbs which invigorate the blood circulation could inhibit Angiotensin II and transforming growth factor-β1 expression and reduce the deposit of extracellular matrx (ECM), alleviate the degree of tubulointerstitial fibrosis. But we can not know if this effect is related to any other vasoactive peptide. In order to prove the possible operating mechanism, we used the adrenomedullin knockout(AMKO) mice induced the tubulointerstitial fibrosis model to investigate the change of cell proliferation in the condition of gene absence, the difference between wild mice and adrenomedullin knockout(AMKO) mice and Angiotensin II receptor blocker was used to treat. Then, Biejiajian pill was gave to tubulointerstitial fibrosis rat with UUO by oral ,observed its effect on cell proliferation and the expression of adrenomedullin in kidney, inquired into the possible mechanism of Biejiajian pill on inhibiting of the tubulointerstitial fibrosis.Part I The expression of cell proliferation in wide mice with UUOMethods:SPE57BL/6J KWL female wild-type mice, weighting 16-22 g, at 8 weeks of age were used in this study. Both of them were divided into three groups respectively, Sham group, UUO group, and UUO plus Valsartan (UUOV) group (n=10). Under ethyl ether anesthesia, left ureters of mice were ligated. The mice of Valsartan group got the medicine 10 mg.Kg-1.day-1 by oral at the day before obstruction for six days, sham and UUO group mice got the same volume of saline. At the 6 day after surgery, obstructed kidneys were harvested. Kidney tissues for light microscopy were fixed with 4% paraformaldehyde and embedded in paraffin, stained with HE and Masson. PCNA, Col III and TGF-β1 protein expression were examined by immunohistochemical staining and PCNA, TGF-β1 and Col III mRNA level were measured in a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). In situ hybridization were used for expression of TGF-β1 mRNA .Results1 Morphological observation resultsHE staining shown that renal sections from the control kidney of Sham group mice was normal, sections from the obstructed kidneys of mice with UUO appeared that the cortical and medullary interstitial spaces increased due to edema, the convoluted and straight segments of the proximal tubule were dilated, epithelial cells were absent,necrosis. In Valsartan treated group the damage still showed seriously but it better than that of UUO group. Masson staining showed there were no abnormal in Sham group kidneys, but in UUO group, tubular dilation, tubular basement thinning and tubular atrophy have been shown , collagen type III increased in interstitium, in Valsartan treated group collagen type III still showed seriously but lighted than that of in UUO group.2 The expression of PCNA,Col III,TGF-β1 in the level of protein detected by Immunohistochemical stainingPCNA: There are a few PCNA positive cells in Sham-operation groups. But the amount of PCNA positive cells increased obviously in UUO group (p<0.001). And Valsartan inhibited the expression of PCNA positive cells (p<0.001). Collagen type III immunohistochemical staining showed that there were no expression of collagen type III in sham groups , but it was expressed strongly in UUO group(p<0.05). It was observed in the tubulointerstitium and inhibited after Valsartan treated (p<0.05). TGF-β1 immunohistochemical staining showed that the expression of it had been seen weakly in Sham group mice kidney, after surgeried, TGF-β1 was strongly expressed in kidney (p<0.001), it was inhibited by Valsartan (p<0.001).3 The expression of PCNA,Col III and TGF-β1 mRNA detected by RT-PCR and in situ hybridization3.1 The expression of PCNA,Col III and TGF-β1 mRNA detected by RT-PCR : The expression of PCNA was weakly in Sham group,It was up-regulated strongly by UUO (p<0.001),Valsartan can down-regulate it(p<0.05). Gene expression of collagen type III was weakly in Sham group, but up-regulated strongly in UUO group (p<0.05), Valsartan can down-regulate it (p<0.05). TGF-β1 gene level in Sham group was weakly, in UUO group they were up-regulated obviously (p<0.05) and also was inhibited by Valsartan(p<0.05).3.2 The expression of TGF-β1 mRNA detected by in situ hybridization TGF-β1 mRNA detected by in suit hybridization showed that the expression of it was weakly in Sham group mice kidney , in UUO group TGF-β1 was expressed apparently (p<0.05) , it was inhibited by Valsartan (p<0.05).Part II The effect of adrenomedullin on cell proliferation in renal interstitial fibrosis induced by unilateral ureteral obstructionMethods:We used adrenomedullin knockout (AMKO) mice, Female, weighting 16-22 g, at 8 weeks of age. All of them were located gene and were knocked out the adrenomedullin gene. It was divided into three groups respectively, AMKO-Sham group, AMKO-UUO group, and UUO plus Valsartan group (AMKO-UUOV) (n=5). Under ethyl ether anesthesia, left ureters of mice were ligated. The group of Valsartan got the medicine 10 mg.Kg -1.day-1 by oral at the day before obstruction for six days, Sham and UUO groups got the same volume of saline. At the 6 day after surgery, obstructed kidneys were harvested. Kidney tissues for light microscopy were fixed with 4% paraformaldehyde and embedded in paraffin, stained with HE and Masson. PCNA, Col III and TGF-β1 protein expression were examined by immunohistochemical staining; PCNA, TGF-β1 and Col III mRNA level were measured in a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). In situ hybridization were used for expression of TGF-β1 mRNA .Results1 Morphological changeHE staining showed that there were no abnormal in AMKO-Sham group mices kidney. But AMKO-UUO group showed that the proximal tubule epithelial cells dropsy and distal tubule dilated , epithelial cells were absent,necrosis. In Valsartan treated group the damage still showed seriously but better than that of in AMKO-UUO group. Masson staining showed that there were no abnormal in AMKO-Sham group, but in AMKO-UUO group, tubular dilation, tubular basement thinning and tubular atrophy had been shown. Collagen type III increased in interstitium, in AMKO-UUOV group collagen type III still showed seriously but better than AMKO-UUO group. Compared with wild mice collagen type III increased in AMKO-UUO group.2 The expression of PCNA,Col III,TGF-β1 detected by immunohistochemical stainingPCNA: There were a few of PCNA positive cells in AMKO-Sham group mice kidney. While the number of PCNA positive cells were increased obviously in AMKO-UUO group (p<0.001). And Valsartan inhibited the expression of PCNA positive cells (p<0.001),it was more than that of wild mice. Collagen type III immunohistochemical staining showed that there were no expression of collagen type III in AMKO-Sham group , but it was expressed obviously in AMKO-UUO group(p<0.01). It was observed in the tubulointerstitium and improved after Valsartan treated (p<0.05). All these expression were more remarkably than those of wild mice. TGF-β1 immunohistochemical staining showed that the expression of it was weakly in AMKO-Sham group mice kidney, and in AMKO-UUO mice kidney it was more obveriously than that of in AMKO-Sham group (p<0.001) , it was inhibited by Valsartan (p<0.001). All these expression were more remarkablly than those of wild mice.3 The expression of PCNA,Col III and TGF-β1 mRNA detected by RT-PCR and in situ hybridization3.1 The expression of PCNA,Col III and TGF-β1 mRNA detected by RT-PCR : The expression of PCNA was weakly in AMKO-Sham group mice kidney,It was up-regulated strongly by UUO (p<0.001),Valsartan can down-regulate it(p<0.05). Gene expression of collagen type III was weakly in AMKO-Sham group, it was up-regulated strongly in AMKO- UUO group (p<0.001), Valsartan can down-regulate it (p<0.05). Expression of TGF-β1 mRNA was weak in AMKO-Sham group, in AMKO-UUO group they were up-regulated obviously (p<0.001) and also was inhibited by Valsartan (p<0.05). All these expression were more remarkably than those of wild mice.3.2 The expression of TGF-β1 mRNA with in situ hybridization TGF-β1 mRNA detected by in suit hybridization showed that the expression of it was weakly in AMKO-Sham group mice kidney , after surgery , in AMKO-UUO group TGF-β1 was up-regulated strongly in kidney(p<0.05) , it was inhibited by Valsartan(p<0.05). All these expression were more remarkably than those of wild mice.Part III The effect of Biejiajian pill on the expression of cell proliferation Methods: SD female wild-type mice, weighting 180±10g, at 8 weeks of age were used in this study and divided into four groups at randomly, Sham group, model (UUO) group, and UUO plus Valsartan group(UUOV),UUO plus Biejiajian pill group (UUOB)(n=10). Excepted Sham group, left ureters of rat were ligated. The group of Biejiajian pill and Valsartan were given 1.6g.Kg-1.day-1 and 10 mg.Kg -1.day-1 by oral respectively until before killed it. Sham and UUO groups got the same volume of saline. After surgery 14 day, obstructed kidneys were harvested. The blood was got and kept in cold storage for serum creatinine and BUN. Left kidney was harvested to weigh. Part of kidney tissues was used to detect the dose of adrenomedullin by radioimmunoassay method. Part of kidney tissues were fixed with 4% paraformaldehyde and embedded in paraffin, stained with HE and Picrosirius-polarization. Part of kidney tissues fixed with 70% ethyl alcohol to detect PCNA positive cells proliferation, PCNA apoptosis and it cell cycle by flow cytometry. The expression of PCNA tested with immunohistochemical staining and RT-PCR methods.Results1 Kidney weight/body weight and serum creatinine, blood urea nitrogenThe body weight of rats in UUO group increased slowly. At the end of test, the left kidney expanded. The index of kidney weight/body weight was higher than that of Sham group remarkably(p<0.05﹚; In UUOB and UUOV group, it was lower than that of UUO group obviously(p<0.05, p<0.05﹚;The index of UUOV group was higher than that of UUOB group, but there were no difference between them(p﹥0.05). In UUO group ,the index of serum creatinine and blood urea nitrogen were higher than those of Sham group remarkably(p<0.001, p<0.01﹚, Biejiajian pill and Valsartan group were lower than that of UUO group significantly(p<0.001,p<0.001; p<0.01,p<0.01﹚;The serum creatinine and blood urea nitrogen of UUOV group were higher than that of UUOB group, but there were no difference between them.(p﹥0.05, p﹥0.05).2 The morphological change Morpathological: The renal tissues were stained with HE and picrosirius-polarization. There was no change in Sham group. But in UUO group, interstitial macrophage and monocyte infiltrated, renal tubular atrophy, emphraxis or expand were shown. The number of glomerular reduced markedly, and the area of glomerular decreased. The renal capsule adhered or expanded. Fibrous tissues proliferated around the renal capsule. While the lesions in treatment groups were slighter than that in UUO group. Pathologic image analysis shows: In UUO group, the area of fibrosis expanded significantly; the area of fibrosis in UUOB,UUOV groups were decreased remarkably compared with the UUO group(P<0.001, P<0.001). It means that Biejiajian pill and Valsartan could inhibit fibrosis in obstructive nephropathy. Among them, the inhibition of UUOB group was higher than that of UUOV group, but there were no difference between them(p﹥0.05). 3 The expression of PCNA detected by immunohistochemical staining.There were a few PCNA positive cells in Sham group. While the amount of PCNA positive cells increased obviously in UUO group than in Sham group(p<0.001).In UUOB and UUOV groups, it decreased remarkably than those of in UUO group(p<0.001, p<0.001).Among of them , the number of PCNA in UUOB was fewer than that of UUOV group, but there were no difference between them (p﹥0.05).4 The expression of PCNA mRNA detected by RT-PCRThe expression of PCNA mRNA showed that it was weak expression in Sham group. It increased remarkably in UUO group than that of Sham group(p<0.001). In UUOB and UUOV groups ,it was decreased significantly than those of in UUO group (p<0.01,p<0.01). The expression of PCNA mRNA in UUOB group was lower than that of UUOV group, but there were no difference between them. ( p﹥0.05)5 The effect of Biejiajian pill on PCNA positive cell, cell apoptosis and cell cycle detected by flow cytometry5.1 The changing of the PCNA positive cell protein in groupsResult showed that PCNA positive cell protein expression of UUO group higher than that of Sham group(p<0.001).And it decreased remarkably in UUOB and UUOV groups than that of UUO group(p<0.05,p<0.05).Though the expression of it lower in UUOB group than that of UUOV group, there were no statistics meaning(p﹥0.05).All above results mean that both Biejiajian pill and Valsartan could inhibit cell proliferation.5.2 The changing of apoptosis rate and apoptosis / proliferation ratio of PCNA in groups5.2.1 The apoptosis of PCNA of UUO group was higher than that of Sham group(p<0.001). These index were decreased significantly in Biejiajian pill and Valsartan groups than that of UUO group(p<0.05,p<0.05), and UUOV group was higher than that of UUOB group, but there were no difference between them(p﹥0.05).5.2.2 The apoptosis /proliferation ratio It decreased remarkably in UUO group than in Sham group(p<0.01),The index was increased significantly in Biejiajian pill and Valsartan groups than that of UUO group(p<0.05,p<0.05), and UUOB group was higher than UUOV group, but there were no difference between them(p﹥0.05).These results showed that both Biejiajian pill and Valsartan could induce PCNA cell apoptosis and inhibit tubulointerstitial fibrosis.5.3 The changing of cell cycle in every group:5.3.1 The cell proportion of PCNA in S stageResult showed that the cell proportion of PCNA in S stage was the lowest in Sham group, others increased than that of Sham group(p<0.001, p<0.001, p<0.001),among of them it was the highest in UUO group, UUOB and UUOV groups decreased remarkably than that of UUO group(p<0.05,p<0.05 ), UUOV group was lower than that of UUOB group, but there were no statistics meaning(p﹥0.05).5.3.2 The cell proportion of PCNA in G0/G1 stageResult showed that PCNA cell proportion of G0/G1 stage was the highest in Sham group, others decreased than that of Sham group(p<0.001, p<0.001, p<0.001),Among of them, it was the lowest in UUO group, UUOB and UUOV groups increased remarkably than that of UUO group(p<0.01,p<0.01 ), Valsartan group was lower than Biejiajian pill group, but there were no difference between them(p﹥0.05).5.3.3 The cell proportion of PCNA in G2/M stageResult showed that there was no obviously change of PCNA cell proportion of G2/M stage. there were no statistics meaning in every group (p﹥0.05)All above results mean that both Biejiajian pill and Valsartan could induce PCNA cell apoptosis, inhibit cell mitosis, keep the PCNA cell stayed in G0/G1 stage, they could inhibit cell from G1 stage get into S stage and inhibit the cell proliferation.PartⅣThe effect of Biejiajian pill on the expression of TGF-β1,Col III Methods: The methods and animals were the same as the part III. Immunohistochemical staining and RT-PCR methods were used to detect the expression of TGF-β1 and Col III. The effect of Biejiajian pill also Investigated in this study.Results1 The expression of Col III,TGF-β1 detected by immunohistochemical staining1.1 The expression, distribution and semi-quantitative analysis of ColⅢ: The expression of ColⅢwas weakly in Sham group, while in other groups the expression of ColⅢincreased . The positive area and density of ColⅢin UUO group were higher than that of Sham group(P<0.001, P<0.001);Compared with the UUO group, the positive area and density of ColⅢin UUOB,UUOV groups decreased obviously(P<0.001, P<0.001; P<0.001, P<0.001). It means that Biejiajian pill and Valsartan could inhibit the expression of ColⅢin obstructive kidney. The inhibition of UUOB group was stronger than that of UUOV group, but there were no difference between them (p﹥0.05, p﹥0.05).1.2 The expression , distribution and semi-quantitative analysis of TGFβ1 :The expression of TGF-β1 was weakly in Sham group, while in other groups the expression of TGF-β1 was up-regulated. They can be seen in renal tubular epithelial cell, interstitial cell and glomeruli. The positive area and density of TGF-β1 in UUO group was higher than that of Sham group(P<0.001, P<0.001);Compared with the UUO group, the positive area and density of TGFβ1 in UUOB,UUOV groups decreased obviously ( P<0.001, P<0.001;P<0.001, P<0.001).It means that Biejiajian pill and Valsartan can inhibit the expression of TGF-β1 in obstructive kidney. Among of them, the inhibition of UUOB group was stronger than that of UUOV group, But there were no bifference between(p﹥0.05, p﹥0.05).2 The expression of Col III and TGF-β1 mRNA detected by RT-PCR2.1 The expression of Col III mRNA showed that it was weak expression in Sham group. It was up-regulated remarkably in UUO group than that of Sham group(p<0.001). In UUOB and UUOV groups, it was down-regulated significantly than that of UUO group (p<0.01,p<0.01). The expression of Col III mRNA in UUOB group was lower than that of UUOV group, but there were no difference between them ( p﹥0.05).2.2 The expression of TGF-β1mRNA showed that it was weak expression in Sham group. It was up-regulated remarkably in UUO group than that of Sham group(p<0.001). In UUOB and UUOV groups , it was down-regulated significantly than that of UUO group (p<0.01,p<0.01). The expression of TGF-β1 mRNA in UUOB group was lower than that of UUOV group, but there were no difference between them ( p﹥0.05) .Above all results showed that Biejiajian pill and Valsartan could down-regulate the expression of PCNA,Col III,TGF-β1 mRNA, It played an protective effect in the progress of tubulointerstitial fibrosis.PartⅤThe effect of Biejiajian pill on the expression of ADMMethods: The methods and animals were the same as the part III. Parts of kidney tissues were used to detect the dose of adrenomedullin by radioimmunoassay method. Immunohistochemical staining and RT-PCR methods were used to detect the expression of ADM in the level of protein and gene. In situ hybridization method used to detect the expression of ADM mRNA . We observed the effect of Biejiajian pill on the expression of ADM.Results1 The dose of adrenomedullin protein in kidney tissueThe dose of adrenomedullin protein in UUO group rat kidney tissue was lower than that of Sham group remarkably(p<0.001); Compared with that of UUO group, it was up-regulated in UUOB and UUOV groups apparently(p<0.001,p<0.05﹚; The dose of adrenomedullin protein in UUOB group was higher than that of UUOV group, but there were no difference between them(p﹥0.05).2 The expression of ADM detected by immunohistochemical stainingThe expression, distribution and semi-quantitative analysis of ADM: It was the strongest expression of ADM in Sham group, the expression of model group obviously weakly than Sham group(P<0.001, P<0.001) . In UUOB and UUOV group the positive area and density of ADM up-regulated obviously than those of UUO group(P<0.001, P<0.001 ;P<0.001, P<0.001). Among them, the positive area and density of ADM expression of ADM in protein level in UUOB group were higher than that of UUOV group; but there were no difference between them(p﹥0.05, p﹥0.05).It means that both Biejiajian pill and Valsartan could up-regulate the expression of ADM in the level of protein.3 The expression of ADM,PCNA,Col III and TGF-β1 mRNA detected by RT-PCR and in situ hybridization3.1 The expression, distribution and semi-quantitative analysis of ADM mRNA with in situ hybridization:The result showed that ADM mRNA mainly expression in renal tubular epithelial cell cytoplasmic and expressed. It expressed a little stronger on distal convoluted tubular. The positive area and density of it in UUO group down-regulated remarkably than that of in Sham group(P<0.001, P<0.001)and it only to be seen in proximal convoluted tubular epithelial cell . The expression of ADM mRNA increased significantly in UUOB and UUOV group compared with that of in UUO group(p<0.001,p<0.001; P<0.001, P<0.001), It was weaken on distal convoluted tubular epithelial cell because unilateral ureteral obstruction induced cell dropped, but it became stronger on proximal convoluted tubular. ADM mRNA expression in UUOB group was a little higher than that of in UUOV group; but there were no difference between them(p﹥0.05, p﹥0.05).3.2 The expression of mRNA detected by RT-PCRThe expression of ADM mRNA showed that it was the strongest in Sham group. In UUO group, it weakened remarkably than that of Sham group(p<0.001),but its expression up-regulated significantly in UUOB and UUOV groups than that of in UUO group(p<0.05 ,p<0.05 ).Above all results showed that Biejiajian pill and Valsartan could up-regulate the expression of ADM mRNA in rat with UUO.ConclusionsWe succeed in copy tubulointerstitial fibrosis model with wild mice, Adrenomedullin Knockout (AMKO) mice and SD rat, treated with Biejiajian pills and the Valsartan. Aggregate analysis the results, the conclusions were got as follow:1 Proliferating Cell Nuclear Antigen (PCNA) can reflect the degree of cell proliferation in the process of interstitial fibrosis. In unilateral ureteral obstruction (UUO) model, the expression of PCNA was up-regulated apparently. The expression of all TGF-β1 protein and mRNA up-regulated and were closely related to cell proliferation. The expression of collagen III in UUO model increased and participated in the process of interstitial fibrosis.2 AT1RA Valsartan inhibited the excessive expression of TGF-β1, alleviated the pathological injury of kidney tissue, improved the component of ECM accumulation in kidney. It also inhibited the renal interstitial fibrosis.3 Use adrenomedullin knockout (AMKO) mice to copy the model of tubulointerstitial, observed the changing of cell proliferation in the condition of adrenomedullin absent, results showed that adrenomedullin played an important role in the process of cell proliferation, it could inhibit the expression of PCNA.4 The degree of tubulointerstitial fibrosis was more heavyer in adrenomedullin knockout (AMKO) mice than in wild mice, and it could be inhibited by AT1RA Valsartan, it showed that adrenomedullin and Valsartan had the intersectic function, this function perhaps fulfilled through to inhibit the excessive expression of TGF-β1.5 Adrenomedullin could inhibit ECM accumulation through inhibited to induce TGF-β1. At mean while, it possible inhibited cell proliferation and displayed protective effect on kidney through inhibit TGF-β1.6 Biejiajian pill could improve the dose of adrenomedullin in kidney tissue, up-regulated the expression of adrenomedullin in the level of protein and gene, through inhibited the expression of TGF-β1 to reduce cell proliferation,lessened the collagen III composed, played the preventative function on tubulointerstitial fibrosis, it was one of the possible operating mechanism that traditional herb Chinese for treatment of kidney disease.7 Flow cytometry result showed that Biejiajian pill and Valsartan could induce PCNA cell apoptosis, inhibit PCNA cell proliferation, keep the PCNA cell stay in G0/G1 stage, and inhibit cell from G0/G1 stage to get into S stage and inhibit cell proliferation, delay the procession of tubulointerstitial fibrosis.
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