A Study Of The Expression Of UCP2 Gene And Caspase-9 Protein In Hypothyroid Rats Kidney

Objective: Iodine is a sort of vital microelement for human body. Thyroid has the function of gathering iodine to synthesize thyroid hormone.Thyroid hormone plays very important function in various organs including the metabolism, growth, development and maturity. Because the body lacking of thyroid hormone when hypothyroidism could be resulted in hyperlipemia, atherosclerosis , the increase of Osteopontin, monocyte chemotactic factor-1,transforming growth fator-βetc, impact sodium-kalium ATP enzyme, sodium-hydrogen exchange body, sodium-calcium transport body etc,and other manifold ion pump, thus result in the changes of kidney structure AND function. Recently the study of Gnanalingham etc, proclaim that TH has the positive regulating of uncoupling protein-2(UCP2),So lanes etc,also, confirm that the mechanism of TH regulating UCPs mostly in genetictranscription AND molecular level. UCP2 as a anion transport protein which could regulate proton's transportation spaning membrane on the mitochondrial inner membrane could reduced the product of reactive oxygen species(ROS). Furthermore,as a result of the recognition and research area ,deepen and widen, recent years,it is found that UCP2 not only participated in the energy metabolism, lipid metabolism, regulating insulin secretion, atherosclerosis, immune response mediated macrophage AND inflammation AND macrophage differentiation, mature. Whereas caspase-9 as the principal initiator of Caspase family plays a vital function in mitochondrin apoptosis pathway.The present study aims to explore in the state of hypothyroidism whether it is apoptosis in rats kidney and its likelihood mechanism from the expression of UCP2 gene and caspase-9 protein.Method: 1. The experimental rats grouping and/or hypothyroid model reproducing : select four - five weeks(seventy-centigramme) femail wistar rats, random divided into Iodine deficiency group(IDG)AND normal control group(CL), each group 10 rats,adapted feeding three month;Low iodine feedstuff came from sever iodine deficiency mountainous area of xinlong in hebei province. Actual measurement iodine content of feedstuff was 50μg/kg, Tianjin tap water was 8μg/L,KI adding in dringking water per litre : IDG:0μg,CL:381.7μg, acording food-intake 20g per day, water-intake 30ml per day to estimate total iodine intaking per day(IDG:1.24μg/d;CL:10μg/d),took blood before executing and detected the serums FT3,FT4. 2. The measurem- -ent of serums TH:Before executing animals respectively took each group animal's anticoagulation blood 4ml,centrifuged, extracted serums about 2ml,adopted chemiluminescent method to measur serums FT3 and FT4 level. 3.Kidney's morphologic alter of hypothyroid rats:Taking model as foundation,to observe morphologic changes of the kidney by HE sraining below the optical microscope. 4.The analysis of immunohisto- -chemistry:The protein expression of caspase-9 and UCP2 were compared between the two groups of rat's kidney by immunohistochemistry method. adopted image-Proplus 5.0, speciality image analysis system,to count each sample positive regional average optical density below differ optical microscope,using SPSS 13.0 to analysis the result. 5.The gene expression of UCP2 in hypothyroid rats kidney:Total RNA from kidney parenchyma using Trizol one-step-method were reversed transcribed to be cDNA. The cDNA and the differert primes were mixed in PCR reaction system to amplify target gene(UCP2) ,Utilizingβ-actin as inside reference. After 1.2% agarose gel electrophoresis , apply BIO -Rad 2000 gathering image,QuantityOne,image analysis system,to measure gradation value. Take target gene and inner reference ratio to proceed semiquantitative analysis,using spss13.0 to proceed relevance Statistics analysis.Results: 1.Serums hormone level:IDG group serums hormone level was considerably decreased comparing to CL. IDG group:FT4=11.38±1.74,FT3=2.28±0.97;CL group:FT4 =29.27±0.95,FT3=2.99±0.44;comparison between groups p<0.01,has statistics meaning. 2.The pathology changes in rat kidney:HE staining below optical microscope, compared with control group,IDG group cortex AND medullae outside thined,mesentric cell and matrices increased,foot cells number decreased,remote terminal renal tubule and outer medullae renal tubule nucleoli dying sharpened, nucleole evident,chromatin side gathered. 3.The expression of caspase-9 protein in rat kidney:It prove that comparing to CL group the caspase-9 protein expressiong of cortical glomerular, remote terminal renal tubule and outer medullae renal tubule increased in IDG group rats and changed more evidently in remote terminal renal tubule and outer medullae renal tubule. IDG group:IOD/area(glomerule)= 0.25±0.03;IOD/area(outer medullae renal tubule)=0.25±0.03 CL group:IOD/area(glomerule)=0.18±0.03;IOD/area(outer medullae renal tubule)=0.16±0.01;comparison between groups p<0.01,had statistics meaning. 4.The expression of UCP2 protein in rat's kidney:It prove that comparing to CL group the UCP2 protein expressiong of cortical glomerular, remote terminal renal tubule and outer medullae renal tubule decreased in IDG group rats and changed more evidently in outer medullae renal tubule. IDG group:IOD/area(glomerule)=0.17±0.02;IOD/area(outer medullae renal tubule)=0.19±0.02 CL group:IOD/area(glomerule)=0.24±0.04;IOD/area(outer medullae renal tubule)=0.25±0.01,comparison between groups p<0.01,had statistics meaning. 5.The expression of UCP2 gene in rat's kidney:Outcomes displayed that comparing to CL group IDG group rats kidney UCP2mRNA expression evdently decreased:IDG group:UCP2/β-actin=0.70±0.19;CL group:UCP2/β-actin=1.30±0.09,comparison between groups p<0.01, had statistics meaning.Conclution:1 Iodine insufficiency can result in wistar rat thyroid function reducing, Hypothyroidism animal model reproduces successfully.2 Hypothyroidism can cause the increase of apoptosis of cortical glomerular, remote terminal renal tubule and outer medullae renal tubule , medullae renal tubule Aggravates glomeruli.3 Hypothyroidism can cause the increase of the expression of capase-9 protein in rat's kidney including cortical glomerular, remote terminal renal tubule and outer medullae renal tubule,and medullae renal tubule more severly.4 Hypothyroidism can cause the decrease of the expression of UCP2 mRNA and it's protein in rat's kidney.