| Background Diabetic nephropathy(DN) is one of microvascular complication with largest harm to survival rate. It is also the chief and independent pathogen which caused final stage kidney disease. Podocyte injury is regarded as the key to cause DN proteinuria and glomerular sclerosis in DN morbidity. Proteinuria is mainly caused by damage of glomerular filtration barrier, which increases increasing of protein permeability and decreasing of permselective function. Podocyte cell is well differentiated cell which attached to GBM. It stretches a lot of neuritis from soma, and form first grade and secondary foot process. The diameter between adjacent foot processes is about 30-40 nm. The clearance is filled by infiltrative slit diaphragm. As the last layer construction of glomerular filtration barrier, the integrity of podocyte cell and slit diaphragm has important function to maintain glomerular filtration function. Forkhead transcription factor O1 is one of Forkhead transcription factor O subfamily. Its function involves multiple physiological pathological processes like anti-oxidative stress, cell cycle stationary, apoptosis, proliferation, inflammatory response, glucolipid metabolism, and autophagy, and it has close relationship with the appearing and developing of diabetic nephropathy. It is found by research that, in DN mouse model renal cortex induced by STZ, the activity level of Fox O1 decreases. Mitochondrion is the major place where eukaryocyte does metabolic activity, the major position which generates ROS, and plays key function in cell oxidative damage. Under normal physiological status, cells constantly clear away damaged or aging mitochondria through mitochondria autophagy to keep stable cell internal environment. PTEN induced putative kinase 1(PINK1) is a kind of survival factor induced under oxidative stress status of cells. It is one of important functional proteins which attend mitochondria autophagy. Under high glucose cultivation, the expression quantity of podocyte cells decreases. It is showed by research that, PINK1 is a downstream gene of Fox O1 in cardiomyocyte. A hypothesis is put forward: Fox O1 might adjust mitochondria autophagy through activation of PINK1, decrease excessive releasing of ROS, and improve diabetic podocyte cell damage.Objective To study the roles and mechanisms of Fox O1 on mitophagy and mitochondrial dysfunction in mouse podocyte cells under high glucose condition and in glomeruli of DN mice.Methods Constitutively active Fox O1 lentiviral vectors(LV-CAFox O1), Fox O1 short hairpin RNA(LV-sh Fox O1), PINK1 short hairpin RNA(LV-sh PINK1) or empty lentiviral vectors(LV-NC) were constructed.27 mice were randomly selected for diabetes models from 36 SPF male KM mice, the other 9 mice as normal control group(NG group). Diabetic mice were randomly divided into three groups: diabetic group(DM group), diabetes with LV-CA-Fox O1 group(CA group), diabetes with LV-NC group(NC group). CA-Fox O1 or empty lentiviral vectors(LV-NC)(10?l)was intravitreally injected in right kidney glomeruli of diabetic mice before they are were anesthetized using a dissecting microscope. The mice were anesthetized with chloral hydrate at the end of twelve weeks, and the kidneys were immediately removed and fixed in 4% paraformaldehyde for HE staining, and in 4% pre-cooling glutaraldehyde for electron microscopic examination. The glomeruli were preserved in liquid nitrogen rapidly for detecting the m RNA level of Fox O1 by q RT-PCR and relative protein expression of Fox O1?p-Fox O1?PINK1?parkin?MFN2?p62 by Western blotting analysis.Conditionally immortalized mouse podocyte cell line(CIMPs) were transfected with constitutively active Fox O1 lentiviral vectors(LV-CAFox O1), Fox O1 short hairpin RNA(LV-sh Fox O1), PINK1 short hairpin RNA(LV-sh PINK1) or empty lentiviral vectors(LV-NC) when cultured in normal glucose(5.6mmol/L) medium. CIMPs were then divided into 6 groups: normal CIMPs cultured in normal glucose(5.6mmol/L)medium or high glucose( 25mmol/L) medium were served as normal control group( group NG,(1)) or high glucose group(group HG,(2)), while the CIMPs transfected with LV-CA-Fox O1 or LV-NC and treated in high glucose medium( 25mmol/L) were served as group LV-CA((3)) or group LV-NC((5)), the CIMPs transfected with both LV-CA-Fox O1 and LV-sh PINK1 and treated in high glucose medium( 25mmol/L) was served as group(4), and the CIMPs transfected with LV-sh Fox O1 and treated in normal glucose medium( 5.6mmol/L) were served as group(6). After cultured in corresponding conditions for 72 h, the m RNA level of Fox O1 of CIMPs in each group were measured by real-time quantitative PCR. The expression levels of protein were assessed by Western blotting, including PINK1, parkin, MFN2, and p62. The expressions and distributions of Fox O1 were detected by immunofluorescence. The bonding of Fox O1 to the promoter sequence of PINK1 was detected by chromatin immunoprecipitation. The mitochondrial ultrastructure is observed by transmission electron microscope. The level of mitophagy was detected by the combination of both adenovirus vector GFP-LC3 and Mito-Tracker Red. The intracellular production of ROS was measured using DCFH-DA. The mitochondrial membrane potential were quantified using the JC-1 Assay KitResults 1. The expression of Fox O1 were increased in the kidney glomeruli of diabetic mice with transfection of CA-Fox O1. In group CA, the protein levels of Fox O1 and p-Fox O1 were both increased(all P<0.05), but the ratio of p-Fox O1/Fox O1 was decreased.2. Up-regulation bioactivity of Fox O1 slowed down progression of DN in mice. Western blotting showed an increase of protein expression of PINK1, parkin and MFN2 in group CA compared with group DM(P<0.05) while p62 level showed a decrease(P<0.05). HE staining and scanning electron microscope showed that the structures of glomeruli were clear in group NC, whereas foot process fusion increased in group DM.3. LV-CA-Fox O1 transfection promoted the expression of Fox O1 in CIMPs. In group LV-CA, the protein levels of Fox O1 m RNA was increased(P<0.05), while group NG+sh Fox O1 showed a decrease in m RNA level of Fox O1(P>0.05).4. The results of chromatin immunoprecipitation showed Fox O1 is capable of bonding to the promoter sequence of PINK1, and this could be inhibited by high glucose(all P<0.05).5. Overexpression of Fox O1 led to the increase of mitophagy related proteinCompared with group NG, the expression levels of PINK1, parkin and MFN2 were significantly decreased(all P<0.05), the expression levels of p62 was significantly increased(P<0.05), while these indexes in group LV-CA were increased(all P<0.05). And LV-sh PINK1 could reverse those changes(all P<0.05). Variation tendency of PINK1 m RNA kept the same with protein(all P<0.05).6. The results of immunofluorescence suggested that the staining of extranuclear Fox O1 was increasd in HG group compared with NG(all P<0.05). LV-CA could inhibited this effect of high glucose(all P<0.05), but sh PINK1 would block it up(all P<0.05).7. The results of DCFH-DA suggested that the content of ROS was increasd in HG group compared with NG(all P<0.05). LV-CA could inhibited this effect of high glucose(all P<0.05), but sh PINK1 would block it up(all P<0.05).8. The colocalization of adenovirus vector GFP-LC3 and Mito-Tracker Red suggested that the number of bicolor-labeled dots was increasd in HG group compared with NG(all P<0.05). LV-CA could inhibited this effect of high glucose(all P<0.05), but sh PINK1 would block it up(all P<0.05).9. The images of transmission electron microscope showed a series of mitochondrial injury in HG group compared with NG. LV-CA could inhibited this effect of high glucose, but sh PINK1 would block it up.10. The images of JC-1 showed a decrease in mitochondrial membrane potential in HG group compared with NG(all P<0.05). LV-CA could inhibited this effect of high glucose(all P<0.05), but sh PINK1 would block it up(all P<0.05).Conclusions1. Injection of CA-Fox O1 in DN mice deferred disease progress, and activated PINK1/parkin pathway.2.Overexpression of constitutively active Fox O1 can obviously improve the level of CIMPs mitophagy, thus alleviating mitochondrial dysfunction, possibly through the mechanisms of bounding to the promoter of PINK1 in CIMPs. |
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