Effect Of Fluvastatin On Renal Cell Cycle Negative Regulation Protein P27 In Diabetic Nephropathy Rats

Background and ObjectiveThe pathological changes of diabetic nephropathy(DN) include hypertrophy of renal inherent cells in the early stage, glomerular basement membrane (GBM) thickening and extracellular matrix (ECM) progressing accumulation mainly distributing in glomerular mesangial region.Those proliferation, hypertrophy and apoptosis of renal inherent cells and ECM thickening are the important components of the proceeding and development in DN. The regulation of cell grownth phase of DN and repression of cell proliferation and hypertrophy produce a marked effect to postpone DN proceeding. The cell grownth phase is regulated by cell cycle regulatory protein, including Cyclins, Cyclin-dependent kinases(CDKs) and Cyclin–dependent kinase inhibitors(CKIs). The CKI p27 is one of the important cell cycle negative regulation proteins.The objective was to study the effect of Flu on p27 in the Early Stage of DN rats, approached its effect and effective mechanisms at cell cycle level and provided clues for provention and cure for DN.MethodOur experiment included two parts:Part I1.Modeling and Administration WayAdult male Wistar rats weighing 180g to 220g were studied and randomly divided into four groups: 1. normal control group(Group C);2. Flu-treated control group(Group CF);3. DN model group (Group DN);4.Flu-treated DN group (GroupDNF).Group DN and Group DNF were induced by STZ,55mg/Kg, intraperitoneal injection one time. Group C and Group CF were by the equal dose of citric acid-natrium citricum intraperitoneal injection. Blood glucose from caudal vein were measured after 48 hours.Rats in experimental groups whose random blood glucose was at least 16.7mmol/L were regard as sucessful models. Once the models was sucessfully established, Fluvastatin was given to Group CF and DNF, 2mg/Kg/d,by intragastric administration, distilled water to Group C and DN.2.Sample collectionDuring the experiment, body weight and blood glucose from caudal vein were measured weekly.Urinary production was collected exactly for quantity of 24 hour urinary protein before sacrificed. All the rats were sacrificed in the experimental period of the 8th week. blood was gotten from heart,then serum was stored below -70℃,then biochemical indicators were examined,and then kidney hypertrophy Index(KHI) and Ccr were calculated.After sacrificed, left kidneys were taken out and weighed.Renal tissues were imbedded by paraffin,cut sections of 2μm,then stained with PAS and PAM.The p27,TGF-β1 and FN expressions were assessed by immunohistochemistry. Cortex of right kidneys were stored below-70℃for Western blot to detect p27 expression.PartⅡMesangial cell culture: cells were devided into two groups: (1)Normal control group:(NG,5.6mmol/L D-glucose); (2)High glucose group (HG,30 mmol/L,D-glucose).At the 24th ,48th,72th ,120th hour, cells were taken out for Western blot analysis to detect p27 expression.Statistical analysisData was presented as Mean±SD, statistical analysis was performed using one-way ANOVA, P value <0.05 or P value <0.01 was considered significantly.ResultPart IAfter 8 weeks,BG level of Group DN and DNF was evidently increased and maintained at a very high level compared with Group C and CF(P<0.01). The BW grew rapidly in Group C and CF,however slowly in Group DN and DNF(P<0.01).The level of TC and TG of Group DN and DNF was higher than Group C and CF(P<0.01),but the difference between Group DN and DNF was not statistically significant(P>0.05).The levels of 24h UP and KHI were all significantly higher than Group C and CF(P<0.01),and Group DNF was lowerer than Group DN. Ccr of Group DN was higher than Group C and CF(P<0.01), Ccr of Group DNF was higher than Group C and CF(P<0.05),and Group DNF was decreased compared with Group DN.The levels of Creatinine of Group DN and DNF were higher than those of non-diabetic rats, but not reach statistical significance.Renal pathological sections showed that renal glomeruli size increased,Mesangial cells emerged gently proliferation, mesangial regions diffusely widened,partly blood capillary was Compressed and collapsed,mesangial matrix diffusely expanded,partly capillary basement membrane(GBM) thickened,renal tubular epithelial cells emerged hypertrophy and vacuolar degeneration.Above-mentioned changes of Group DNF were lighter than Group DN, mesangial matrix lightly expanded,and mesangial region was lightly widening.Semiquantitative analysis with immunohistochemistry technique showed that p27,TGF-β1 and FN expressions in Group DN and DNF were much higher than Group C and CF(P<0.0l),but p27,TGF-β1 and FN expressions in Group DNF were decreased compared with Group DN(P<0.05).Western blot showed that p27 expression in Group DN and DNF was much higher than Group C and CF(P<0.0l),Fluvastatin could inhibit p27 expression increase,and the relative p27 expression of Group DNF was markly lower than Group DN( P<0.05).Part IIWestern blot showed that p27 expression of Mesangial cells began to increase after 24 h in HG environment and maintain 120 h.DiscussionAnimal model of DN rats induced by STZ injection is one of good Models to study diabetes.The 8th week after models were successfully established,DN rats emerged kidney hypertrophy, accompanied with proteinuria and Ccr increasing, Renal pathological sections showed that renal glomerulus increased, mesangial matrix diffusely expanded,capillary basement membrane(GBM) thickened,which signed model rats had progressed into the early stage of DN.In the experiment once the models were sucessfully established, Fluvastatin was given to Group DNF, 2mg/Kg/d,8 weeks later,24h UP and KHI of Group DNF decreased compared with Group DN,which showed that Fluvastatin had effect on kidney protection and postponed DN progression.The result was in accordance with the animal experiments before which showed HCRI could relieve kidney damage of DN rats.Recently,many researches have showed that kidney protection of HCRI notonly depend on decreasing blood lipid,but also depend on nondecreasing blood lipid,such as resisting cell proliferation, anti-inflammatory, immunological regulation etc.One of kidney protection mechanisms of HCRI is regulating the biological course,which includes hypertrophy, proliferation and apoptosis of renal cells through cell cycle regulation.Western blot of part II showed that p27 expression of mesangial cells began to increase after 24 h in HG environment and maintained 120h,Which illustrated p27 expression of Mesangial cells in HG culture markly increased.During cell proliferation, p27 had great role in initiating the G1-S-phase transit. High-level p27 can block the G1-S-phase to transit and led to cell cycle arrest in G1-phase.In the experiment,the effects of the fluvastatin on p27 expression of renal inherent cells of the early DN contain two aspects:firstly,HCRI(HCRI) can repress the degradation of the p27;secondly, through kidney protection function of decreasing blood-lipid or nondecreasing blood-lipid,HCRI decreased genetic transcription and protein expression of TGF-β1.Then the decrease of TGF-β1 can induced p27 expression decreasing.Thus, HCRI repesses not only the expression of p27,but also the degradation of the p27.After being treated by the Fluvastatin, expression of p27 in the kidneys of DN rats stepped up compared to the normal control group,and stepped down compared to the diabetes control group.So we could suppose that the fluvastatin decreased the genic transcription and the protein expression of the TGF-β1. Speaking in a lively way that because the"source head"was cut off,the effects of"strangling"were very little,even though the degradation of p27 was repressed. So in the Fluvastatin-treated group,the increased degree of the expression of p27 was lowerer than the diabetes control group,which relieved the cell hypertrophic degree,furthermore decreased secretion of ECM,blocked kidney damage,thuseduced kidney protection.Conclusion1. The p27 protein expression of mesangial cells increased markly under the stimulation of HG environment.2. Fluvastatin had little influence on blood glucose,but decreased the excretion of early DN protein and KHI,then protect the kidney function.3. The protection of Fluvastatin to the kidney, through the effect of independent decreasing blood lipid,one of effect mechanisms is adjusting the CKI p27, repessing the abnormal proliferation and hypertrophy of renal cells and decreasing ECM synthesis.4. It is effective to influence kidney cells growth through intervening the cell cycle regulation mechanism in the early stage, then relieve the damage in the early stage of DN and delay its advancement.