| Hypertension,a common and high incident disease,is seriously endangering human health.It is estimated that over 25%of world adult population have been affected by the disorder and more than 100 million patients in china experience hypertension.Moreover,the incidence of hypertension is gradually increasing.A large number of epidemiological and clinical trials have shown that hypertension is one of the main causes of cardiovascular diseases,renal diseases and peripheral vascular diseases.Hypertension has become a major global public health problem. Kidney plays an important role in the regulation of blood pressure while it is also one of the main damage organ caused by hypertension.Damage to renal function will contribute to the development of hypertension.During the past few years,many studies have founded that activation of renal renin-angiotensin-aldosterone system (RAS)play important roles in hypertensive renal injury.AngiotensinⅡ(AngⅡ)is the main active substances of the RAS.It can stimulate the production of TGF-β(transforming growth factor protein)and osteopontin that may cause local inflammation,stimulate vascular and mesangial cell proliferation and hypertrophy. Losartan is a widely used clinical AngⅡreceptor antagonist.The effect of anti-hypertension and nephroprotection of losartan have been proved by many clinical trials,however the exact mechanism underling which are still not well known. The human genome project has been completed.However,it is not enough for learning the mechanisms how disease happened by gene analysis,the majority of diseases is associated with the expression of protein.The study of proteins,product of the gene expression,is thought to be the final stage of revealing the mechanisms of life and disease.Proteomics is a new subject from genome.Proteome means proteins expressed by a genome while proteomics is a subject to study the proteome by various kinds of technological means.Although Proteomics by name is now over 10 years old,it is widely used in biomedical fields.Understanding the protein expression profiles of kidney in hypertension may provide further information how hypertension caused renal injury.Twelve male SHR and six age-matched WKY rats were obtained at 6 weeks of age.SHR were divided into two groups at random,untreated group and losartan group.Losartan was dissolved in distilled water and given by gastric gavage at the dose of 20 mg/kg/day from 7 to 14 weeks of age.Drug concentrations were adjusted every week according to the body weight of rats.At 14 weeks of age,all rats were sacrificed by decapitation.The proteins were extracted by TCA method.The protein concentrations were determined by a Bradford assay.200μg of protein was loaded on 24cm,nonlinear IPG strips of pH 3-10 for first dimension electrophoresis, followed by vertical 12.5%SDS-PAGE for second dimension electrophoresis.The gels were visualized by silver staining and analysed with ImageMaster 2D Elite software.The protein spots with significant changes were selected for further identification.After digested by trypsin,the peptides were mixed with matrix solution and analyzed by MS.The proteins were identified by searching the NCBInr protein database using the MASCOT search engine.The identified proteins were verified by western blot.Results:(1)Systolic blood pressure(SBP)levels were monitored in all rats every week.Compared to WKY rats,the SBP of SHR rats was significantly elevated at 6 weeks(150.72±5.07 mmHg vs 110.11±3.74,p＜0.01).The differences in SBP between SHR and WKY rats were also seen at 8,10,12 and 14 weeks.At 8 weeks of age,the SBP of Los group was significantly lower than that of SHR rats(142.22±13.40 vs 162.33±8.86,p＜0.05).The differences between these two groups were also significant at 10,12 and 14 weeks(p＜0.05).(2)After the rats were sacrificed, the kidneys were removed for histopathologic examination.Compared with WKY rats,marked damage was evident in the kidney sections from SHR.Renal arteriole wall was thickening.Vascular lumen was narrowing.(3)The protein profiles of kidney of WKY,SHR and SHR treated with losartan were established by two-dimensional gel electrophoresis.1144±35,1215±78 and 1219±44 spots were isolated in WKY,SHR and SHR treated with losartan respectively.Compared with the WKY rats,25 spots had changed significantly in SHR.These spots were excised for identification by MALDI-TOF MS based on PMF.11 spots were identified.Of these spots,4 spots(spot 1,4,8 and 19)were newly appeared,5 spots(spot 2,5,6, 12 and 22)were up-regulated and 2 spots(spot 18 and 24)were down-regulated.The expression levels of 8 spots(spot 1,4,6,8,18,19,22 and 24)were reversible by treatment with anti-hypertensive drug losartan while 3 spots(spot 2,5 and 12)were not affected.(4)Spot 2,8 and 22 were identified as the same protein.(5)The successful rate of silver staining is only 44%.(6)Spot 4(Enol protein)and spot 18 (NADP~+-ICDH)were confirmed by western blot.Eno1 protein was up-regulated in SHR and can be reversible by treatment with anti-hypertensive drug losartan. NADP~+-ICDH was down-regulated in SHR and up-regulated after treatment.Conclusion:(1)Systolic blood pressure of SHR was significantly elevated at 6 weeks.The SBP increased with increasing age.Marked damage was evident in the kidney sections from SHR at 14 weeks.SHR can be used for the study of hypertensive renal damage.(2)After treated for 2 weeks,the SBP of Los group was significantly lower than that of SHR.(3)The study established a protein profile for kidney of SHR.25 spots had changed significantly and 11 proteins were identified by PMF.The expression levels of some proteins were reversible by treatment with anti-hypertensive drug losartan.The identified spots were mainly involved in energy metabolism,lipid transferring between membranes and cell proliferation.Several identified proteins may be as candidates for further investigation of pathophysiological mechanism of renal injury in hypertension.(4)One protein (lamininγ2 chain)was expressed as multiple spots on the reference map,suggesting that they were either charge isoforms or fragments of the same protein.(5)The successful rate of silver staining is low,suggesting that more effective method of staining may be needed in the study of proteomics. |
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