| Background&ObjectiveThe excessive proliferation of mesangial cells plays a pivotal role in thedevelopment of diabetic nephropathy (DN), which is a major chronic complication ofdiabetes. Forkhead transcription factor O1, a member of FoxO subfamily, is regulatedby the PI3K/AKT signaling pathway thus gathering multiple downstream signalingmolecules to regulate the intracellular and extracellular stress signals, which play akey role in the aspects of anti-oxidative stress, glucose regulation and lipidmetabolism and cell cycle. At present, many researches have focused on theanti-tumor function of FoxO1in regulating cell cycles; however, few studies areconcentrating on whether FoxO1can inhibit the proliferation of mesangial cells indiabetes. Our previous research found that FoxO1mRNA levels reduced in the renalcortex of diabetic rats which accompanied by proliferation of mesanginal cells andaccumulation of extracellular matrix, indicating a close relationship between FoxO1and diabetic nephropathy.As a cell cycle-dependent kinase inhibitor, p27Kip1is an important target geneof FoxO1, which plays a pivotal role in aspects of regulating cell proliferation,differentiation and apoptosis. Although previous studies have uncovered the function of p27Kip1in the occurrence of a variety of tumors, it is still unclear whether FoxO1can inhibit the proliferation of mesangial cells by regulating the expression ofp27Kip1. To investigate the effect and mechanism of FoxO1in diabetic,constitutively active FoxO1lentiviral vectors were injected into the renal cortex ofdiabetic rats, followed by assessing the proliferation of mesangial cells and theaccumulation of extracellular matrix in diabetic rats.Materials&MethodsThe empty lentiviral vector (LV-NC-GFP) and the constitutively active FoxO1lentiviral vector (LV-CA-FoxO1) were constructed.8-week-old SPF male SD rats(220±20g) were fed one week at room temperature. After12h fasting,100rats wererandomly selected to induce diabetes by a single intraperitoneal injection of60mg/kgstreptozocin (STZ) solution which was dissolved in fresh0.05M citrate buffer(PH=4.5), while30rats were injected with considerable volume of citrate bufferwhich served as normal group (NG group). Blood glucose was measured to validatethe induction of diabetes (>16.7mmol/L). Diabetic rats were randomly divided intothree groups: diabetic group (DM group), diabetes with LV-NC-GFP group (NCgroup), diabetes with LV-CA-FoxO1group (CA group). As surgery entrance, anincision was made at costalspinal angle in right back to expose the right kidney of rats,followed by injecting100μl LV-CA-FoxO1or LV-pSC-GFP into renal cortex atseveral sites, whereas rats of DM group were injected with an equal volume of saline.Then muscle and skin were sutured. At the end of two weeks, four weeks and eightweeks after lentiviral vectors injection,24-hour urine was collected for thedetermination of24-hour urinary protein (UPro/24h) and urinary albuminquantification (UAIb). After the rats were anesthetized with chloral hydrate, bloodwas collected from orbital vein to test serum creatinine (Scr) and blood urea nitrogen(BUN). The renal cortex of the injection sites were obtained and kept in liquidnitrogen precooled for2minutes, followed by preparing the frozen sections whichwere observed under inverted fluorescence microscope to determine whetherlentivirus was successful transfected. Tissue of the injection site was cut into small pieces (1mm3) and fixed in4%pre-cooling glutaraldehyde for electron microscopicexamination. To observe the pathological changes of kidney, the renal cortex fromeach rat was fixed in4%paraformaldehyde for HE stain and immunohistochemistry.The remaining kidney cortex was preserved in liquid nitrogen rapidly for subsequentexperiments. The mRNA levels of FoxO1, p27Kip1, plasminogen activator inhibitor(PAI-1), fibronectin (FN), glomerular collagen Ⅳ (Col Ⅳ) were detected byreal-time PCR. The protein expression of FoxO1, p-FoxO1, p27Kip1and PAI-1weretested by western bloting. Expressions of collagen Ⅳ and fibronectin in glomeruliwere assessed by immunohistochemical staining.Results1. Massive GFP were observed around injection sites in renal cortex at differenttimes, indicating that the sequences in lentiviral vectors had successfully transfectedinto rat renal cortex and stablely expressed.2. Rats in DM group and NC group were apathetic, along with the increase ofwater intakes and urine outputs, compared to NG group. Although the psychosissymptoms of rats in CA group were close to normal rats, water intakes and urineoutputs were still higher. Compared with NG group, BW decreased significantly atdifferent time periods, and BG, KI were elevated in DM group (P﹤0.05); althoughthere were no difference in UPro/24h, UAlb, Ser and BUN levels (P>0.05) in twoweek, these indesxes were significantly increased at the end of four week and eightweek (P <0.05).We found that every indexes of CA group showed no difference withDM group at end of two week (P>0.05), but KI, UPro/24h, UAlb, Ser and BUN weresignificantly lower at the end of four week and eight week (P <0.05). All indexes hadno difference between NC group and DM group (P>0.05).3. HE staining showed that the structures of glomeruli were clear in rats of NGgroup, whereas the mesangial cells proliferated significantly and mesangial matrixexpanded in rats of DM group and NC group; On the contrary, the renal pathologicalchanges were obviously ameliorated in CA group. Under electron microscopy, theglomerular basement membrane was uniform thickness with evenly distributed foot process and no mesangial cells proliferation and mesangial matrix accumulationswere observed in NG group; Along with the time progress, the basement membranebecame thickening or not smooth and foot process fusion gradually increased, whilemesangial cells and matrix became nodular hyperplasia in rats of DM group; Footprocess fusion was improved in CA group, accompanied with mild hyperplasia ofmesangial cells and matrix.4. Compared with NG group, the mRNA expressions of FoxO1and p27Kip1significantly decreased (P <0.05) wherase the levels of PAI-1，FN and Col Ⅳmarkedly increased(P <0.05) in DM group at each time period. Compared with DMgroup, the mRNA levels of FoxO1and p27Kip1significantly increased (P <0.05),and PAI-1, FN, Col Ⅳ mRNA expressions decreased (P <0.05) in CA group.5. In each time period, compared with NG group, the protein expression ofFoxO1did not change (P>0.05), the level of p-FoxO1and ratio of p-FoxO1/FoxO1significantly increased as time progressed in DM group (P <0.05). The proteinexpression of FoxO1drastically increased in renal cortex in CA group (P <0.05),whereas the protein level of p-FoxO1had no statistical difference compared with DMgroup (P>0.05). Moreover, the ratio of p-FoxO1/FoxO1decreased after transfection(P <0.05), which indicated the relative increased bioactivity of FoxO1. Comparedwith NG group, protein expression of p27Kip1significantly decreased (P <0.05), andthe levels of PAI-1increased markedly (P <0.05) in DM group. The proteinexpression of p27Kip1elevated (P <0.05) and the level of PAI-1significantly reduced(P <0.05) in CA group. Every indicator had no significant difference between the NCgroup and DM group (P>0.05).6. Immunohistochemistry showed that the expression of FN and Col Ⅳ in DMgroup increased gradually (P <0.05), which was significantly decreased in CA group(P <0.05). There was no significant difference between NC group and DM group ineach index (P>0.05).Conclusions1. In the renal cortex of diabetic rats, the mRNA level of FoxO1reduced wherase the protein level of p-FoxO1increased, accompanied with proliferation ofmesangial cell and accumulation of mesangial matrix.2. The elevation of FoxO1expression alleviated the pathological changes of DN,possibly through the mechanisms of which inhibited the proliferation of mesangialcells by upregulating the expression of its target gene p27Kip1, as well as attenuatedextracellular matrix accumulation by downregulating PAI-1. |
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