Expression Changes Of Adrenomedullin In Kidney Of Streptozotocin-induced Diabetic Rats

Objective:Diabetic nephropathy(diabetic nephropathy,DN) is a common microvascular disease in diabetes mellitus(diabetes mellitus,DM)patients,which is the most dangerous complication in diabetic microvascular disease with diabetic retinopathy and neuropathy,but the exact mechanism of the disease have not yet been fully clarified.We employed STZ-induced male adult Sprague-Dawle diabetic rats to study the alteration of the ADM expressions in the kidney by immunohistochemistry. Changes in kidney ultrastructure under optical microscope were determined by hematoxylin eosin staining(HE staining) and transmission electron microscopy.To research the expression of kidney adrenomedullin in STZ-induced diabetic rats,and explore the mechanism of diabetic nephropathy,This can provide theory basis and curative ways in clinical practice.Materials and Methods:1.Animal groups 20 SD rats that weighing 150-180g(2 months old) were divided into two groups at random.They are the normal control group and the diabetes model group,each group has 10 rats.2.Animal model The normal control group were fasted for ten hours.Then injected Juyuan sodium salt solution with 3ml/kg.The diabetes model group were aslo fasted for ten hours.Rats were injected streptozotocin intraperitoneally with 3ml/kg in diabetes model group.48 hours after injection,cut off the rats' tail to test the blood sugar by Monitor blood sugar and test urine sugar levels by test paper.We found that their blood sugar was over 16.7mmol/L and urine sugar was over ++ demostrating the animal acute diabetes model was succeeded.All animals are free to consume water and food.We checked blood sugar and urine sugar every week.3.Immunohistochemistry(1)MaterialAfter the successful model,we keep rats feading 4 weeks,and then fetch seven rats from each group with injecting sodium pentobarbital anesthesia;we intubated from the left ventricle of rats and perfusion with PBS solution infusion about 100 ml;then anesthetized with 4 percent solution of POM Reperfusion about 300 ml,kidney was removed in the same kind of stationary liquid immersion in 30 min,and washed with PBS,all operations are conducted under 4℃condition.(2)Slice preparation(3)Hybridication,washing and staining (4)Patch and Fengpian4.The control group used an PBS instead of Rabbit anti-rat ADM polyclonal antibody,and the remaining steps are same.5.HE staining6.Obsrevation kidney ultrastructure by TEM7.Image analysis and statistical analysis We analysed all sections through the same magnification(×400) and the same light intensity,and choose 10 sections which have the same position of DAB staining from each group,each section was measured five perspective at random,the measurement results from the average;use UTHSCSA Image Tools 3.0(U.S.University of Texas Medical School Development) image analysis system for analysis,statistics the number and light density of positive cell within the unit area(mm~2);data using SPSS 10.0 for statistical analysis,with results that the use of t-test,P＜0.05 for the significant difference.Result:1.Weigh,blood sugar and urine sugar:Before modeling diabetic rats,the normal control group weights 164.2±6.3 g,glucose was 3.7±0.5 mmol / L,urine sugar(-);the diabetes model group weights 167.2±7.9 g,glucose was 3.6±0.4 mmol/L,urine sugar was(-).Between the two groups no significant difference(P＞0.05).after four weeks:the control group weights 223.8±12.1 g,glucose was 3.9±0.5 mmol/L,urine sugar was(-);the diabetes model group weights 159.1±18.1 g,glucose was 30.4±2.8 mmol/L,urine sugar was(+ +) to(+++).Differences between the two groups was significant(P＜0.01).2.The expression of ADM in kidney:The yellow particles of ADM immunoreactive can be found in the cytoplasm of tubular epithelial cells both the normal control group and the diabetic model group.However,compared with the control group,ADM in the diabetic model group were enhanced.In particular,ADM positive immune cells and optical density values in the control rats renal unit area were 59.1±14.6/mm2 and 84.2±8.4,and ADM group was immune cells and optical density values in the diabetic model group were 197.3±47.4 / mm~2 and 117.8±12.9(P＜0.01).3 Observation HE staining in kidney through the light microscope:Under the light microscope,we can discover that the capillary of glomerulus was thicking,the cavity of renal capsule was fractured,the basement membrane was thicking,the membrane was hypertrophic,and tubular epithelial cells showed vacuoles and the degeneration of granular through the light microscope,and Renal tubular was mildly expansion,the mesenchym of renal tubules was widened.4 Observation the changes of myocardial ultrastructure through electron microscope:electron microscope:the basement membrane of glomerulus was segmental thickening,the podocytic process of the epithelial cells was integrative and widened,mitochondria was obviously swelling,the number of crest was decreased or fractured,Cilia was damaged,the part membrane of renal cell and the chromatin in the nuclear was enriched and gathered.Conclusion1.Constructed diabetes rat model:In order to study on the mechanism of diabetes,to construct an ideal diabetes animal model was very important.In the practice alloxan and streptozotocin were injected intraperitoneally to make diabetes animal model and using streptozotocin was commonly.STZ could add alkyl on special bases of DNA then ruin ADP ribosome synthase so the B cell was destroyed directly.The standard of diabetes rat model was that its blood sugar was up 16.65mmol/L(300mg/dl) through scholar studying.In this experiment we choosed SD rats and fasted them for 10 hours then injected streptozotocin via tail vein with 3ml/kg.48 hours after injection rat's blood sugar was≥16.7 mmol/L and had polydipsia,polyuria and polyphagia symptom. This is accorded to standard of diabetes model.2.The change of Diabetic Nephropathy pathologyDN typically pathological change in the structure is increasing the volume of kidney and glomerular,and is gradually emerging the thickening of GBM and the gathering of the glomerular extracellular matrix(extracellular matrix,ECM),finally, tubulointerstitial extracellular matrix(ECM) is aslo accumulation.And this study observed that the capillary of glomerulus was thicking,the cavity of renal capsule was fractured,the basement membrane was thicking,the membrane was hypertrophic,and tubular epithelial cells showed vacuoles and the degeneration of granular through the light microscope.Renal tubular was mildly expansion,the mesenchym of renal tubules was widened,the basement membrane of glomerulus was segmental thickening,the podocytic process of the epithelial cells was integrative,widened,and accorded with the reported.3.The relationship between adrenomedullin and diabetic nephropathyThe first successful separation and purification of ADM was from human pheochromocytoma in 1993 by Kitamura.In recent years,the function of ADM in the kidneys gradually is attached great importance,and we aslo know that high blood sugar can increase the expression of ADM.This may be due to that high blood sugar can activate the activity of PKC,and increase the expression of ADM,however,if we add PKC inhibitor into the medium,this function of high blood sugar increasing the expression of ADM will be blocked.High blood sugar and high concentration of FFA will increase the synthesis of DAG,and activate PKC.And high oxidative stress under the condition of high blood sugar can also enhance the activity of PKC.Another study shows that the levels of ADM shows the trend of first increase and then decease in rats'plasma which gets DM disease,and the plasma levels of ADM is considered to reflect the seriousness of the DM disease.And our study confirms the first time that in STZ-induced diabetic rats' kidney the expression of ADM is increasing,this demonstrate that ADM play an important role in the process incidence of diabetic nephropathy.Because high blood sugar can increase the expression of ADM through stimulating PKC,so,the expression of ADM in local kidney is likely a compensatory mechanism of body for combating all kinds of injury signals in diabetes which are caused by metabolism disorder under the conditions of high blood sugar,such as the activation of Ang II system,the increase of oxidative stress,the excessive activation of PKC and other adverse effects on the kidneys.Occurrence of the decompensation under the circumstances of chronic high blood sugar may be the important reason why the trend of ADM increase first and then decrease in rats'plasma with getting DM disease.This study first reveals that ADM in the pathogenesis of diabetic nephropathy has an important position,and it also provides a laboratory basis for the target of treating diabetic nephropathy through ADM and PKC signal pathway in clinical.