| Background&ObjectiveDiabetic nephropathy (DN), one of serious microvascular complications ofdiabetes (DM), is the leading reason of the death caused by diabetes, which can oftenlead to end-stage renal disease (ESRD). Podocyte early injury in DN morphologicallyshows foot process fusion and disappear, podocytes get smaller, then negative chargebecomes less, and finally podocytes detach from GBM and are excreted in the urine.Nowadays, with the establishment and development of podocyte cell line, more andmore attention was paid on the research of podocyte, the importance of podocyteinjury in the occurrence of proteinuria and glomerular sclerosis in DM has beengradually acknowledged.Forkhead transcription O1(FoxO1), a member of Forkhead protein family, itsactivity and the expression of its target gene can be decreased when it isphosphorylated. Our previous studies have shown that the activity of FoxO1isreduced in renal contex tissues of DN rats. Then our research group found that in thegroup of DN rats with treatment FoxO1phosphorylation is decreased, the activity ofFoxO1is increased and foot process fusion is alleviated when compared with that in the group of DN rats without treatment, the fact shows that the podocyte injury ofdiabetic rats is probably related to the bioactivity of FoxO1. But the relationshipremains unclear at home or abroad. It is also reported that PI3K/Akt signal pathwaymay participate in the development and progression of DN. As a downstream factorof this signal pathway, we believe that the reduction of FoxO1activity probably takepart in the podocyte injury.In our study, we will detect the effects of upregulation of FoxO1on the podocyteinjury of DM rats.Materials&Methods120Male Sprague-Dawley rats (100±20g) were raised in IVC system。Whenthe rats reached8weeks old, about (220±20) g, from which chose90rats to establishdiabetic rats model, then divided these diabetic rats randomly into three groups:diabetic rats (group d), rats transfected with empty lentiviral vectors (DM plusLV-pSC-GFP group, served as group a) and rats which were transfected withLV-CA-FoxO1lentiviral vectors (DM plus LV-CA-FoxO1group, served as group b).The rats which received an injection of diluent buffer served as normal control (groupc). These diabetes rats were induced by a single intraperitoneal injection of1%STZ ina dose of60mg/kg after fasting for12hours. Blood glucose was measured threetimes of continuous72hours after the injection. Rats in the diabetic group wereexcluded if plasma glucose was less than16.7mM. Rats took food and water freelyand were not given any hypoglycemic agent in the experimental period. When the ratsmodel of diabetic were established successfully and blood glucose had been stableabout5days, inject100ul the recombinant lentivirus vector（LV-pSC-GFP andLV-CA-FoxO1) at different sites of renal cortex in group a and group b.(Thetransfection titer was measured as7×108TU/mL, take the transfection efficiency andcytotoxicity of lentiviral vectors into consideration, the proper amount of transfectionis100ul). Rats in group c and d were given the same dose of saline solution. At theend of2、4、8weeks,24-hour urine from every rat was collected for24hours urinary protein (24hUPro), urine albumin (UAlb) measurement, and podocalyxin (PCX) inthe urinary sediment, then the rats were intraperitoneally anesthetized and blood wascollected from angular vein for Blood glucose (BG), serum creatinine (Scr) and bloodurea nitrogen (BUN) examination. The right kidney weight/body weight ratio (KI) ofrats were detection and calculated. All the kidneys were cut in a coronal plane. A halfof the dorsal part of right kidney from each rat in group a and group b was drawn forobservation under inverted fluorescence microscope and light microscope. A half ofthe dorsal part of right renal cortex from each rat in group c and group d was fixed in4%paraformaldehyde for HE stain. Another quarter of the dorsal part of right renalcortex in each group was cut into small pieces (1mm3) for electron microscopeobservation and the rest dorsal part of cortical tissues were removed and stored at-80°C for further study. The expression of FoxO1, PCX, nephrin, COL4A3, COL4A5and desmin were measured by Real-time PCR and Western blotting.Results1. Under inverted fluorescence microscopy, we can observe expression of greenfluorescent protein (GFP) in the renal cortex of group a and group b, which means thelentiviral vectors was transfected into rats successfully.2. At the end of2weeks, there were no significant differences in UPro/24h,UAlb, Scr and BUN levels among the four groups (p>0.05); compared with group c,in group a and group d, BG and KI levels were significantly increased and bodyweight (BW) was decreased（all P<0.05）; compared with group a and group d, ingroup b, there were no significant differences in BG and BW levels (p>0.05), KI wassignificantly decreased（P<0.05）. At the end of4,8weeks, compared with group c, ingroup a and group d, BG, KI, UPro/24h, UAlb, Scr and BUN levels were decreasedsignificantly (all P<0.05); compared with group a and group d, in group b, KI,UPro/24h, UAlb, Scr and BUN levels were reduced (all P<0.05), there were nosignificant differences in BG and BW levels (p>0.05). There were no significantdifferences in all indicators between group a and group d (all P<0.05). 3. At the end of2,4,8weeks, compared with group c, in group a and group d,the content of PCX in urinary sediment was markly elevated (P<0.05); compared withgroup a and group d, in group b, the content of PCX in urinary sediment wassignificantly decreased (P<0.05); There were no significant differences betweengroup a and group d (P<0.05).4. At the end of2,4,8weeks, compared with group c, in group a and group d,the mRNA expression of FoxO1, nephrin, and PCX were markly decreased (allP<0.05); compared with group a and group d, in group b, the mRNA expression ofFoxO1, nephrin, and PCX were significantly increased (all P<0.05); smilarly, themRNA expression of FoxO1in group b was significantly increased when comparedwith group c (P<0.05); compared with group c, in group a and group d, the mRNAexpression of COL4A3, COL4A5and desmin were markly incressed (all P<0.05);compared with group a and group d, in group b, the mRNA expression of COL4A3,COL4A5and desmin were significantly reduced (all P<0.05); There were nosignificant differences between group a and group d (P<0.05).5. At the end of2,4,8weeks, compared with group c, in group a and group d,there was no significant difference in the protein expression of FoxO1, but theexpression of nephrin, PCX and the ratio of FoxO1/p-FoxO1were significantlydecreased (all P<0.05); compared with group a and group d, in group b, the proteinexpression of FoxO1, nephrin, and PCX were markly elevated (all P<0.05); theprotein expression of FoxO1and the ratio of FoxO1/p-FoxO1in group b wassignificantly increased when compared with group c (P<0.05); compared with groupc, in group a and group d, the protein expression of COL4A3, COL4A5and desminwere significantly incressed (all P<0.05); compared with group a and group d, ingroup b, the protein expression of COL4A3, COL4A5and desmin were marklyreduced (all P<0.05); There were no significant differences in all indicators betweengroup a and group d (P<0.05). ConclusionsUpregulation of FoxO1can improve the podocyte injury in diabetic rats, whichis probably related to regulating the expression of PCX, nephrin and desmin byFoxO1. |
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