The Experimental Studies On The Preventive Effect Of KXI On Alcoholic Kidney Injury And Its Influence On PDGF-BB

IntroductionEthanol can induce multiorgan lesion among which the injury of liver is especially significant. There have been numerous reports on alcoholic liver disease (ALD) ,but the renal lesion hasnt got extensive attention.We succeeded in establishing the rat model of ALD by direct intragastric infusion of ethanol in 2002 and found that kidney presented associated pathological changes during the different stages of ALD among which the change of tubulo -interstitial fibrosis(TIF)is significant. Extracellular matrix(ECM)of glomeru-lar mesangium increased and collagen fibers deposited in electron microscope and the expression of PDGF - BB which indicates the proliferation of fibers increased, compared with control group.In 2003 Brzoska reported that blurred trabecular structure, vacuolar degeneration and increased density of nuclear chromatin with very compact nuclear structure was found in hepatocytes of zones 2 and 3 in rats exposed to 10% ( w/ v) ethanol (EtOH) for 12 weeks. Moreover,in the kidney tubules, degeneration and hypertrophy of epithelial cells and dilation in the glomeruli were also observed.At present few studies have been performed to investigate alcoholic tubulo -interstitial fibrosis( ATIF) and there is no report on the prevention of ATIF. This study made use of the traditional Chinese medicine(TCM) ,i. e,KangXian-FuFang I ( KXI) mainly composed of many blood - activating and tasis - eliminating drugs and observed the protect effect of KXI on ATIF and its influence on the expression of PDGF - BB.Material and Method1. Experimental materials and equipments1.1 Experimental animals51 male Wistar rats (170 ±20g) , provided by Laboratory Animal Center of China Medical University.1.2 Experimental regentsRabbit - anti - rat antibody: PDGF - BB (1:50 - 100) , Strept - Avidin -Biotin (SABC) test kit and diaminobenzidine ( DAB ) show color reagent. KXI ( mainly composed of safflower, angelica, astragalus membranaceus, saliva milt-iorrhiza, tetrandrine and so on).1. 3 Experimental equipmentsParaffin microtome, light microscope, photography apparatus, JEM - 1200 EM transmission electron microscope, Micro ( fluorescence) - picture analyze system2. Method of establishing rat model2.1 The establishment of alcoholic kidney injury animal model (method of direct intragastric infusion of ethanol).Referrence to the method established by our experimental department (reference 6 ).2.2 The establishment of KXI preventing experimental animal model. Referrence to the method established by our experimental department ( reference 26 ).3. Procedure of experiment 3.1 Pathological examinationAnesthetized the rat peritoneally, perfuse fixing through heart, then collected the kidney tissue. Fixed the specimen with formalin and covered with paraffin. The kidney tissue was cut into 4jxm - thick continuous section and stainedwith hematoxylin and eosin. Pathologic changes were observed under the light microscope.3. 2Electroscopic techniquesThe specimen were fixed with glutaraldehyde,then covered with epoxy resin. The renal tissue were cut into 60um of ultrathin sections with a diamond knife. The ultrathin sections were first dyed by uranyl acetate and then by citrate. Observed the changes by JEM - 1200EX transmission electron microscope.3. 3 Detection PDGF - BB by ABC immunohistochemistryThe paraffin sections were dewaxed to water,inactivate the endogenetic enzyme , repair the antigen, add normal goat serum, add the first antibody PDGF -BB( 1:50), 4X. to stay overnight and add the second antibody 20 - 371 for twenty minutes. ABC compound was added for twenty minutes under 20 — 37t. All steps were completely washed by 0. 01M PBS . DABshowed color. The sections were dyed again slightly by hematoxylin and observed under the light microscope. The cells containing light brown particulate at cell plasm were positive cells. Utilize the micro( fluorescence ) picture analyze system to analyze the integrated intensity of immunohistochemistry PDGF - BB posotive signals in selective field of vision.4. Statistic analysisThe measurement data was analyzed with t — test and chi square test.Results1. Renal pathologic changes in light microscopeAt the end of 4 weeks of ethanol group, the renal tissue shew slight proliferation of glomerular mesangial cells, focal infiltration of inflammatory cells in tu-bulo - interstitium and vacuolation of renal tubular epithelial cell. At the end of 8 weeks of ethanol group, these pathologic changes became more obvious. At the end of 12 weeks of ethanol group,renal tubules were atrophy,epithelium got thin and flat,and tubular lumen were dilated.KXI group showed focal infiltration of inflammatory cells and vacuolation of renal tubular epithelial cell at the end of 8 weeks. At the end of 12 weeks major-ity of the tissue are almost normal.2. Changes of transmission electron microscopeAt the end of 8 weeks of ethanol group, lysosome increased in renal tubular epithelial cell with numerous vacuoles. At the end of 12 weeks of ethanol group , collagen fibers deposit can be observed in interstitium and foot processes of glo-merulus vesceral layer epithelial cells were partly confluent. At the end of 12 weeks of KXI group collagen fibers deposit decreased significantly and there were no confluence of foot processes.3. Expression of PDGF - BB in immunohistochemisty assayThere were no differences between two groups at the end of 4 weeks (P >0. 05). At the end of 8 and 12 weeks,the expression of PDGF - BB in KXI group decreased markedly compared with ethanol group and the difference is significant (P<0.05).DiscussionsAbout 5-10 percent of ethanol is oxidized and excreted through kidney. When the amount of ethanol is beyond the capacity of hepatic metabolism, the excretion of kidney increases significantly. Due to plentiful blood supply, ethanol and acetaldehyde in circulation accumulates and concentrates in renal cells and interstitial tissue, resulting in the injury of kidney. At past and present experimental studies we observed the kidney shew corresponding pathologic changes in the different stages of ALD and the changes of tubulo - interstitium were especially significant while glomerular mesangium shew slight proliferation. In TEM collagen fibers increased and deposited in glomerular mesangium which may be the pathological basis of chronic renal failure.The mechanism of alcoholic kidney injury is not clear at present and may be associated with the consumption of oxygen during the metabolim of ethanol, the production of cytokines and lipid peroxidation. The role of PDGF - BB in renal fibrosis got extensive attentions. In the past studies we noted the dynamic changes of PDGF - BB in the whole course of rat model and found that as time proceeded and dose of ethanol increased the expression of PDGF - BB increasedgradually. This was similar to the outcome of renal damages caused by other etio-logical factors. This study confirmed the results again.The mechanism of renal fibrosis hasnt been explained exactly, so there is no satisfactory therapy. In the studies of alcoholic hepatic fibrosis it was tesified that KXI could inhibit the proliferation of HSC stimulated by acetaldehyde, the expression of matrix metallproteinase( MMP) -2, -9,the excretion of TGF - (^ and PDGF, and interfered with the production of collagen. By this mechanism it prevented and treat ALD.In our study we applied KXI to the prevention of ATIF and found that KXI not only alleviated the histological lesion but also decreaced the expression of PDGF - BB in alcoholic kidney injury. We thought the mechanism may be that KXI acceleratd the metabolism of alcohol ascribed to its protective effect on alcoholic liver , thus decreased the harmful effect of ethanol and of its metabolites on kidney; the effect of TCM on PDGF - BB was more complicated and may be versatile. It was possible that harmful factors decreased or the production of PDGF - BB decreased resulting from the inhibition of infiltration of inflammatory cells such as lymphocytes, monocytes, and therefore inhibit the proliferation and activation of fibroblast and transdifferentiation of renal tubular epithelial cell into MFB. TIF is the outcome of interaciton of glumerular intrinsic cells ( mesangial cells ^endothelial cells ^ foot process cells )^ renal tubular epithelial cells ^ fibro-blasts >, inflammatory cells % chemokines^ growth factors and inflammtory mediators. The interaction of different mechanisms need further investigation.Conclusions1. The kidney shew different pathologic injuries as time of stimulation went on and the dose of ethanol increased, such as the slight proliferation of glomeru-lar mesangium , vacuolation of renal tubular epithelial cells, focal infiltration of inflammatory cells, swelling of renal tubular epithelial cells and the dilatation of tubular lumen among which tubulo - intersitial lesion was more significant. KXI could prevent this histological damage.2. The expression of PDGF - BB in alcoholic renal damages increased, indi-...