The Effect Of Chinese Herbs On Tubular Epithelial Myofibroblast Transdifferentiation In Adrenomedullin Knockout Mice With UUO

Chronic renal failure (CRF) is one of the most serious diseases in China andthe incidence of CRF has been increasing in recent years. A large number ofclinical and animal experimental results showed that the renal interstitialfibrosis (RIF) plays an important role in chronic kidney diseases and CRF canbe slow down by preventing RIF. RIF is a common pathological process ofchronic kidney diseases and it can be induced by a variety of factors, in whichcell proliferation and myofibroblast transdifferentiation are involved mainly.Tubular epithelial-myofibroblast transdifferentiation (TEMT) was put forwardby Strutz in1994and was proved to be a core pathological change of RIF andthe α-smooth muscle actin (α-SMA) was considered to be the marker ofTEMT. TEMT also has a complex mechanism related to various factors andit is hard to be prevented effectively by single pathway. However, it isnecessary to searched for the pharmaceuticals with more physiological effectsand more action pathways. Discovered in1993, adrenomedullin (ADM) is akind of biologically active polypeptide and distribute in many vital organs andtissues of the body with expressing more in kidney. ADM has variousphysiological effects including inhibiting the expression of transforminggrowth factor beta1(TGF-β1) and angiotensin II (AngⅡ), anti-oxidation andanti-inflammatory. The TGF-β1is the key cytokine in the process of TEMTand it is therefore believed that ADM can inhibit TEMT by down-regulatingthe expression of TGF-β1and then can delay the process of RIF. However, itsspecific mechanism remains to be explored. Meanwhile, it has been proved inour former experiments that the Chinese herbs can antagonize TEMT byinhibiting the expression of TGF-β1but it’s still not clear whether Chineseherbs are in the way of regulating ADM in the process. To figure out the mechanism of Chinese herbs on antagonizing TEMT and the correlation toAMD, this study was designed to observe Chinese herbs’ effects on theexpression of α-SMA, TGF-β1, Col Ⅲand ADM in adrenomedullinknockout(AMKO) mice with unilateral ureteral obstruction(UUO), whileeplerenone, the aldosterone receptor blocker, was used as the comparator.PartⅠ: Effect of Chinese herbs on renal function and pathomorphologyin TEMT In adrenomedullin knockout mice with UUOMethods: Both of wild and adrenomedullin knockout (AMKO) mice weredivided into four groups respectively. They are WT-Sham group, WT-UUOgroup, WT-UUOE group, WT-UUOS group, AMKO-Sham group,AMKO-UUO group, AMKO-UUOE group and AMKO-UUOS group. Exceptthe mice in two Sham groups, the others in the rest groups were ligated theirleft ureters under ethyl ether anesthesia. The mice in two UUOS groups weregiven Chinese herbs27.6g/(kg.d) by oral gavage and two UUOE groups weregiven eplerenone100mg/(kg.d) in their forage. The others got the samevolume of saline. At the10day after surgery, all mice were sacrificed andspecimens of blood were got to detect the contents of Blood Urea Nitrogen(BUN) and Serum Creatinine (SCr). And the left kidneys were cut to bemeasured the index of kidney weight/body weight (KW/BW) and parts of leftkidneys of mice were prepared respectively for HE and Masson staining.The data were expressed as mean±SD. SPSS16.0software was adoptedand one way ANOVA was applying to comparison among many groups. Allresults are considered significant at P<0.05and P<0.01.Results:1Index of kidney weight/body weightThe Index of KW/BW of the mice in UUO，UUOE and UUOS groups wassignificantly higher than that of two Sham groups respectively (P<0.01) butthere was no obvious difference among them (P<0.05).2The result of pathomorphologyHE staining showed that no abnormalities in two Sham groups. In thekidneys of UUO，UUOE and UUOS groups, the necrosis and detachment of the renal tubules epithelial cells were observed with a large of inflammatorycells infiltrated in renal interstitial space. Mssson staining showed that therewas no abnormality in two Sham groups. There was many collagen depositedin renal interstitium just like strip-shaped and spotty-shaped in the two UUOgroups and the AMKO-UUO group was more serious than that of WT-UUOone.3The result of renal functionThere was no significant difference of Scr in all the8groups. Neither wasBUN.(P>0.05)Part Ⅱ: Effect of Chinese herbs on the express of α-SMA in TEMT inadrenomedullin knockout mice with UUOThe methods of animal grouping，model establishment, medication andstatistics were the same to PartⅠ,10days. The expressions of α-SMA inkidneys were detected by RT-PCR, Western Blot and immunohistochemistryrespectively.Results:1The results of RT-PCR Compared with corresponding Sham groups, theexpression of α-SMA mRNA was enhanced obviously in UUO groups(P<0.01; P<0.01), and the AMKO-UUO was higher than that of WT-UUO onesignificantly(P<0.05). The high expression of α-SMA mRNA wasdown-regulated respectively in both UUOE and UUOS groups (P<0.01;P<0.01).2The Results of Western Blot A little of expression of α-SMA protein inkidney tissues was observed in WT-Sham and AMKO-Sham groups，while theexpression of α-SMA in WT-UUO and AMKO-UUO groups was enhancedremarkably (P<0.01; P<0.01), and the AMKO-UUO was higher than that ofWT-UUO one significantly (P<0.05). Compared with corresponding UUOgroups, the high expression of α-SMA was significantly decreased in UUOEand UUOS groups (P<0.01; P<0.01) and there was no significant differencebetween the UUOE and UUOS groups (P>0.05; P>0.05).3The Results of Immunohistochemistry showed that there was a little expression of α-SMA only in vascular smooth muscle cells in two Shamgroups and it was remarkably increased in WT-UUO and AMKO-UUO groupswith the expanding scope of tubulointerstitium in cortical andcorticomedullary area. And the AMKO-UUO was more serious than that ofWT-UUO one. The expression of α-SMA in UUOE and UUOS groups wasreduced significantly.Part Ⅲ: Effect of Chinese herbs on the express of TGF-β1and Col Ⅲ inTEMT in adrenomedullin knockout mice with UUOThe methods of animal grouping，model establishment, medication andstatistics were the same to Part Ⅰ,10days. The expressions of TGF-β1and ColⅢ in kidneys were detected by RT-PCR, Western Blot andimmunohistochemistry respectively. In addition, the expression of Collagentissues was detected by sirius red staining.Results:1The expression of TGF-β1mRNA and Col III mRNA detected by RT-PCR1.1The result of TGF-β1mRNA detected by RT-PCR showed that a littleexpression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO one (P<0.05). Compared with correspondingUUO groups, the expression was reduced in both UUOE and UUOS groupssignificantly (P<0.05; P<0.05).1.2The result of Col III mRNA detected by RT-PCR showed that littleexpression of Col III in WT-Sham and AMKO-Sham groups was observed andit was increased obviously in UUO groups (P<0.01; P<0.01)with expressingmore in AMKO-UUO one (P<0.01). Compared with corresponding UUOgroups, the expression was reduced in both UUOE groups and UUOS groupssignificantly (P<0.01; P<0.01). And there was more expression inAMKO-UUOE and AMKO-UUOS groups than corresponding groups of wildtype mice (P<0.05; P<0.05).2The expression of TGF-β1and Col III protein detected by Western Blot2.1The result of TGF-β1protein detected by Western Blot showed that a little expression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO(P<0.01). Compared with correspondingUUO groups, the expression was reduced in both UUOE and UUOS groupssignificantly (P<0.01; P<0.01).2.2The result of Col III protein detected by Western Blot showed that a littleexpression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO(P<0.01). Compared with correspondingUUO groups, the expression was reduced in both UUOE groups and UUOSgroups significantly (P<0.05; P<0.01). And there was more expression inAMKO-UUOE and AMKO-UUOS groups than corresponding groups of wildtype mice.(P<0.05; P<0.05)3The expression of TGF-β1and Col III detected by immunohistochemistry3.1The result of TGF-β1detected by immunohistochemistry demonstrated thatthere was weak expression in glomerular and tubular epithelial cells in twoSham groups, and the expression was remarkably increased in WT-UUO andAMKO-UUO groups. The AMKO-UUO group expressed more than that ofWT-UUO ones. Compared with corresponding UUO groups the expression ofTGF-β1in UUOE and UUOS groups was reduced with expressing more inAMKO-UUOE and AMKO-UUOS groups.3.2The result of Col III detected by immunohistochemistry demonstrated thatthere was modest expression in glomerular and interstitial in two Sham groupsand the expression was remarkably increased in WT-UUO and AMKO-UUOgroups, especially in tubulointerstitium. The mice in the AMKO-UUO groupexpressed more than WT-UUO one. Compared with corresponding UUOgroups the expression of Col III in UUOE and UUOS groups was reducedwith expressing more in AMKO-UUOE and AMKO-UUOS groups.4The result of collagen tissues detected by sirius red staining demonstratedthat there was modest expression in glomerular, tubular and interstitial in twoSham groups and there was no obviously difference between them. The expression was remarkably increased in WT-UUO and AMKO-UUO groupmainly in renal tubulointerstitium with expression more in AMKO-UUOgroup. Compared with corresponding UUO groups the expression of collagentissues in UUOE and UUOS groups was reduced with expressing more inAMKO-UUOE and AMKO-UUOS ones.Part Ⅳ: Effect of Chinese herbs on the express of ADMin renalinterstitial fibrosis mice with UUOThe methods of animal grouping，model establishment, medication andstatistics were the same to PartⅠ,10days.The expressions of ADM inkidneys were detected by RT-PCR, Western Blot and immunohistochemistryrespectively.Results:1The Results of RT-PCR The expression of ADM in AMKO-Sham groupwas weaker than that of WT-Sham group significantly(P<0.01). Comparedwith corresponding Sham groups, the expression of ADM mRNA was reducedobviously in UUO groups (P<0.01; P<0.01) and the AMKO-UUO was weakerthan that of WT-UUO one significantly (P<0.01). The low expression of ADMmRNA was up-regulated respectively in both UUOE and UUOS groups(P<0.05).2The Results of Western Blot There was some expression of ADM inkidney tissues was observed in WT-Sham and AMKO-Sham groups，while theexpression of ADM in AMKO-Sham group is weaker than that of WT-Shamgroup(P<0.01). The expression in WT-UUO and AMKO-UUO groups wasreduced remarkably (P<0.01; P<0.01) and the AMKO-UUO was lower thanthat of WT-UUO one significantly (P<0.01). Compared with correspondingUUO groups the low expression of ADM was significantly up-regulated in theUUOE and UUOS groups (P<0.01; P<0.01).3The Results of Immunohistochemistry In WT-Sham and AMKO-Shamgroups, there was some expression in cortical and corticomedullary area,especially in medullary. And the expression in AMKO-Sham group is weakerthan that of WT-Sham one. The expression was remarkably reduced in WT-UUO and AMKO-UUO groups and AMKO-UUO expressed less thanWT-UUO group. There was increasing expression of ADM in UUOE andUUOS groups than that of corresponding UUO groups respectively withexpressing more in wild mice.Conclusions:1The animal models of renal interstitial fibrosis were established byunilateral ureteral obstruction. The expression of collagen tissues in the kidneywere increased remarkably in UUO mice, especially in adrenomedullinknockout mice. Chinese herbs and eplerenone can inhibit collagen depositionin renal interstitial and the effect was more significant in wild-type mice withUUO.2The expression of α-SMA, which is the marker of TEMT, was strongerobviously in UUO mice, especially in adrenomedullin knockout mice. Chineseherbs and eplerenone can down-regulate the expression level of α-SMAmRNA and protein.3Chinese herbs have the function of de-gradating collagen tissues andreducing the deposition of ECM by down-regulating the expression of TGF-β1and Col Ⅲ mRNA and protein.4Compared with Sham groups, The expression of ADM was significantlyreduced in UUO groups of wild type and adrenomedullin Knockout mice andChinese herbs can effectively up-regulated its expression level, especially inwide mice.5Chinese herbs can inhabit TEMT and antagonize the progress of RIF bydown-regulating the expression of TGF-β1and Col Ⅲ while the expressionof ADM was up-regulated. It may be the one of the targets of the mechanismin Chinese herbs on RIF prevention.