The Effect Of Hydroxyethyl Starch On The Histopathology And Renal Function Of Autotransplant Kidney

ObjectiveTo observe the tissue deposits of hydroxyethyl starch in the autotransplantkidney I/R model and it's effect on renal function, study the evidence of whetherhydroxyethyl starch harmful to the transplanted kidney, and look for the feasibility ofthe use of hydroxyethyl starch in the fluid treatment during kidney transplantationoperation.Materials and methods18 adult New Zealand white rabbits, female or male, body weight 1.8～2.6kilogram, were divided randomly into three group, group voluven ( group V, n=6),group HES 200/0.5 (group Y, n=6) and group normal saline for control (group S, n=6).After anesthesia administered by intramuscular injection with 2mg-kg-1 of ketamine,the right marginal ear vein was catheterized for fluid administration, and 3mg·kg-1·h-1 of ketamine was pumped in for the maintenance of anesthesia, 8ml'kg-1·h-1 ofRinger's solution was pumped in for the physiological need and the fluid loss duringoperation. The carotid artery was catheterized with a 22G heparinized Catheter tomonitor the blood pressure.To establish the autotransplant kidney ischemia/reperfusion injury model: AMedian abdominal incision was made, separate the left kidney carefully from thepeplos, and then separate the renal artery and the renal vein. Separate the right kidney and 2 pieces of thread preparing for nephrectomy. Occlusion the left renal artery by aatraumatic artery clip, then occlusion the left renal vien by another atraumatic arteryclip. A 24G Catheter was catheterized into the left renal artery immediately, andanother 22G Catheter was catheterized into the left renal vein meanwhile. Then theleft kidney was perfused with low-temperature (0℃to 4℃) HCA solution10～15ml (pressure 60-80mmHg, the hot ischemia time during this procession isabout 3min), massage the kidney during the perfution until it tumed pale and theoutflow from the left kidney vein became clear. The perfused kidney was preservedwith the low-temperature preservation system (0℃to 4℃) for 1 hours, the catheterwas removed, and the kidney artery and vein were repaired with 7-0 atraumatic suture.After 60min of low-temperature preservation, the left kidney was reperfused and theright kidney was removed. The autotransplant kidney ischemia/reperfusion injurymodel was established and the abdominal incision was sewed.Factors: After the reperfusion of the left kidney, the treatments below wereadministered: In group V, 10 ml·kg-1 of Voluven(?) was pumped in by the rightmarginal ear vien with the speed of 20 ml·h-1; 10 ml·kg-1 of 6% HES 200/0.5 wasadministered with the speed of 20 ml·h-1 in group Y; and in group S, 10 ml·kg-1 ofnormal saline was pumped in with the same speed. After this procession, the pump ofketamine was stoped. 2 hours after the reperfusion, the artery catheter was removedand the carotid artery was then ligated to prevent bleeding. The incision of the neckwas sewed, and the rabit was put back to the cage, free of water and food.Data and specimen collection, test and treatment: The systolic blood pressure,the diastolic blood pressure and the pulse were recorded when the operation wasstarted (T1), the renal artery was occluded (T2), 10rain after the renal artery wasoccluded (T3), the left kidney was reperfused (T4) and 2 hous after the left kidneywas reperfused (T5). The rabbit's blood samples were collected when the operationwas started (Ta) 2 hours (Tb) and 24 hours (Tc) after the left kidney was reperfused.24 hours after the left kidney was reperfused, 20ml of air was injected into therabbit's marginal ear vein to kill it quickly. The kidney was harvested and the sample of kidney was put into 4% buffered paraformaldehyde for 24 hours, then the samplewas changed into 70%, 80%, 90%, 95%, 100%, 100% alchol respectively todehydration. After transparent in the dimethyl benzene, the sample then wasembedded in paraffin. The 3～6μm paraffin sections were stained withHaematoxylin-eosin (HE) and unstained paraffin sections for immunohistochemistry.Slides were reviewed blindly and scored with a semiquantitative scale evaluatingchanges according to Paller. Specifically, for each kidney 10 different areas werescored, and care was taken to avoid repeated scoring of different convolutions of thesame tubule, with points given for the presence and extent of tubular epithelial cellflattening (I point), brush border loss (I point), cell membrane bleb formation (I or 2points), interstitial edema (I point), cytoplasmic vacuolization (I point), cell necrosis(I or 2 points), and tubular lumen obstruction (I or 2 points). Higher scoresrepresented more severe damage (maximum score per area was 10).Immunohistochemistry: The unstained paraffin sections were immersed in thedimethyl benzene for I0min twice to dewax, then was changed into 100%, 100%,95%, 90%, 80%, 70%, alchol respectively to hydration, after blocking endogenousperoxidases, paraffin sections were incubated with a well defined polyclonal rabbitantibody against HES, followed by swine antirabbit immunoglobulin taged withhorseradish peroxidase, then stained with DAB. Sections were counterstained withhaematoxylin. Controls included dilution series, omission of primary antibody,replacement of the primary antibody by PBS. The picture was taken withCoolSNAP-Pro_cf system. 5 pictures were selected randomly from each slice, thepictures were analyzed with Image-Pro Plus 5.0 software and calculated the totalareae of all of the DAB stained particles.Statistical analysis. All data are reported as the mean+standard deviation((?)±s).Test of homogeneity of variances was analyzed firstly, then analysis ofone-way-anova was used to evaluate the basic status data such as body weight, bloodpressure, pulse, BUN, Cr. The Paller's scores were also analyzed with one-way-anova.Analysis of variance of repeated measure data was used to analyze the repeatedmeasures data. t-test was used to analyze the areae of the DAB stained particles. SPSS 13.0 were used to analyze the data. P values of＜0.05 were accepted assignificant.ResultThe baseline data and it's assessment were shown in table 1 (P＞0.05). There isno significant difference between the baseline data. There is significant defferencewith the blood pressure and pulse in the test of within-subjects effect (P＜0.05), andbetween-subjects effect, no difference (P＞0.05). There is significant defference withthe BUN result in the test of within-subjects effect and between-subjects effect(P＜0.05), and there is interaction between the BUN and the group (P=0.003).Pairwise comparisons showed significant difference between each time point(P＜0.05). The BUN result in group V was significantly different from that of group Sand group Y (P＜0.05). There is significance between the groups of the Paller's score(P＜0.05). The result of the Paller's score in group Y was was significantly differentfrom that of group S and group V (P＜0.05). There was positive staining of DAB inthe immunochemistry test in group Y and group V. There is significance of the totalareae of all of the DAB stained particles between group Y and group V(t=2.538,P=0.015), the means of that areae in group Y (508.51μm²) was higher than that ofgroup V (226.87μm²).Conclusion1. There was IRI in the rabbit's kidney during autotransplant of the kidney. Theinjury of 6% hydroxyethyl starch 200/0.5 to the autotransplant kidney was moresignificant. The new preparation of 6% hydroxyethyl starch 130/0.4 might do mild orno harm to the autotransplant kidney.2. There was tissue deposit in the autotransplant kidney IRI model when theHES was used to maintain the blood volume. There was more tissue deposits in theautotransplant kidney of 6% hydroxyethyl starch 200/0.5 than that of 6%hydroxyethyl starch 130/0.4.3. The renal deleterious effects of 6% hydroxyethyl starch 200/0.5 on the renalfunction of the autotransplant kidney were much severe while that of 6%hydroxyethyl starch 130/0.4 were mild.