The Role Of PI3K/Akt Pathway In The Podocyte Injury Of Diabetic Nephropathy

Objectives: Diabetic nephropathy (DN) is one of the main microvascularcomplications of diabetes mellitus, with increasing morbidity and proportionin chronic dislysis nephropathy in china, as well as being the capital leadingcause of end stage renal disease in Western countries. To explore thepathogenesis of diabetic nephropathy and search available therapeutic measureare very important for patients with DN. The pathogenesis of diabeticnephropathy is very complicated, and many factors are involved such ashyperglycemia, lipid metabolism disorder, abnormal hemodynamics,inflammatory cytokines, oxidative stress, cell apoptosis and so on. Alongwith the construction of podocyte cell strain and the rising development ofbiochemistry technology, podocyte is gradually known and studied in recentyears. Podocyte injury is identified to play an important role in thepathogenesis of DN and considered as a pivotal factor to induce proteinuriaand glomerular sclerosis. This provides a novel means for preventing andtreating DN.Podocytes are also called glomerular visceral epithelial cell and made upglomerular filtration barrier together with a fenestrated endothelial layer, theglomerular basement membrane (GBM). Early podocyte injurymorphologically shows foot process fusion or effacement, followed bypodocyte becoming smaller, negative charge becoming less, detaching fromthe basement membrane and excreting in the urine at last. Podocytes arehighly differentiated cells and have little proliferative ability. When thenumber of exfoliative cells exceeds that of proliferative cells, glomerularfiltration barrier is destroyed on the one hand, which lead to massiveproteinuria, podocytes persistently excret collogen Ⅳon the other hand,which lead to glomerular sclerosis in the end. Hyperglycemia, angiotensin Ⅱ, lipid metabolism disorder, oxidative stress, and inflammatory cytokines canstimulate podocytes to be disfunctional and impaired. Recent study revealsthat adnormal podocyte function is related to the gene mutation or delation ofsome specific structural protein locating in podocytes. Slitdiaphragm-associated proteins such as nephrin, CD2-associated protein, ZO-1,podocin and so on that maintain the functional integrity of glomerular slitdiaphragm, are widely researched. In the last years, the role of podocalyxin inpodocyte injury of many glomerular diseases has been emphasized.Podocalyxin, as a maturation marker, is expressed in an epiphragm of footprocess and rich in negative charge that responsible for glomerular chargebarrier. Both down-regulation in renal tissue and up-regulation in urine ofpodocalyxin expression indicate podocyte injury. Desmin is a cytoskeletonand the same muscle-derived cell marker as α-smooth muscle actin (α-SMA).In normal conditions, desmin may slightly express in mesangial cells, but doesnot emerge in podocytes. While podocyte is damaged and phenotypicalterations happen, desmin will overtly express in podocytes.Phosphoinositide3kinese/protein kinase B (PI3K/Akt) signaling pathwaycan mediate the differentiation, proliferation, apoptosis, and migration of cells,however, the excessive activation will result in cell functional disorder.LY294002, a classic inhibitor of PI3K/Akt pathway, can prevent activating ofPI3K by inhibiting subunit p110, so as to inhibit phosphorylation of PDK andAkt. Phosphase and tensin homology deleted on chromosome ten (PTEN) hasthe activity of phosphoesterase and antagonize the activity of PI3K bydephosphorylating PIP3to PIP2, therefore it is the negative regulator ofPI3K/Akt pathway. It has been suggested that PI3K/Akt might mediateepithelial-mesenchymal transition in tubular epithelial cells and mesangial cellhypertrophy of DN. Moreover, our previous study also has identified thatPI3K/Akt pathway participates in high glucose-induced lipogenesis andextracellular matrix in renal tubular cells. However, there are few reportsabout podocyte injury of DN, especially in patients with DN. In the presentstudy, many methods have been used to examine the activation of PI3K/Akt pathway and the alterations of podocyte marker proteins in patients with DNand in high glucose-induced mouse podocytes. Besides, LY294002or PTENgene transfection has been used to explore the effect of PI3K/Akt pathway onphenotypic alterations of podocytes and further to investigate the role ofPI3K/Akt pathway in podocyte injury, so as to provide a novel means forpreventing and treating DN.Methods:1Selection of patients and detection of podocalyxin, Col Ⅳ,desmin, p-Aktand PTENThirty patients diagnosed as diabetic nephropathy by case history, clinicaldate and renal biopsy from October2009to October2011at the SecondHospital of Hebei Medical University were included in this study. The patientsincluded16men and14women (M:F=8:7), whose age were51.5±15.7yearsold. The secondary cases such as HBV associated glomerulonephris, lupusnephris, Henoch-Schonlein purpura nephris, amyloidosis and myelomenephropathy were excluded. Those cases complicated with idiopathicnephropathy such as Membranous nephropathy, FSGS, IgA nephropathy werealso excluded. All patients were divided into three groups according to2010pathologic classification of diabetic nephropathy proposed by the AmericanSociety of Nephrology: class Ⅰg roup (n=9), classⅡ group (n=10),class Ⅲgroup (n=11). Clinical data of every patient were well recorded beforebiospy. Urinary samples of all patients were collected on the day of biospy,followed by centrifugating urine and retaining clear supernatant liquid. Theconcentration of podocalyxin in supernatant liquid were evaluated by ELISA.10healthy volunteer with normal level of blood sugar, renal function andurine analysis were collected for control group. The expression of ColⅣ,desmin, p-Akt and PTEN in glomeruli were detected byimmunocytochemistry.10case renal tissues without evident renal diseasewere obtained from distant portion of renal tumors as control group.2Podocytes culture and detection of p-Akt, Akt, PTEN, podocalyxin,nephrin, desmin and α-SMA in podocytes treated by high glucose or LY294002Mouse podocytes were cultured at33°C in humidified atmosphere of5%CO2in DMEM-F12media containing10%fetal bovine serum (FBS),penicillin/streptomycin (100U/ml and100μg/ml, respectively) and2-3dropsMouse Interferon γ (10u/ml) to promote propagation. After being passaged,cells had been transfered to37°C, DMEM-F12media containing10%FBSand penicillin/streptomycin (without Interferon γ) to promote differentiation.About10to14days, cells were incubated in serum-free DMEM-F12for24hours to synchronize the cell growth, then treated by high glucose (HG,30mmol/L glucose) for0,3,6,12,24,48h, with normal glucose (NG,5mmol/Lglucose) as control group. Again, to study LY294002intervention, cells weresynchronized, then were divided into five groups: I: NG group, II: HG group,III~Ⅴ: HG+LY294002intervention (the concentration of LY294002:10,20,40μmol/L respectively). The intervented time was determined by the peak ofp-Akt expression. The cells were harvested to extract total and nuclear proteinand total RNA. The expression of p-Akt, Akt, PTEN, podocalyxin anddesmin were detected by immunocytochemistry. Western blot was also usedto detect the expression of p-Akt, Akt, PTEN, podocalyxin, nephrin, desminand α-SMA protein. The mRNA levels of podocalyxin and desmin wereexamined by real time-PCR.3Transfection of PTEN into mouse podocytes and detection of p-Akt, Akt,PTEN, podocalyxin, nephrin, desmin and α-SMA in podocytespcDNA3.1-PTEN-EGFP expression vector was constructed and transfectedinto mouse podocytes with lipofectamine2000in vitro. The cells are dividedinto four groups: NG group, HG group, HG control vector group (a vectorwithout specific PTEN gene), HG PTEN vector group (a vector with specificPTEN gene). At6h after transfection, the medium was replaced with normalDMEM-F12medium with10%FBS for24h. Then cells were cultured for48h under HG medium. Fluorescence microscopy was used to examine EGFPexpression. The cells were harvested to extract total and nuclear protein andtotal RNA.Western blot was used to detect the expression of p-Akt, Akt, PTEN, podocalyxin, nephrin, desmin and α-SMA protein. The mRNA levelsof PTEN, podocalyxin and desmin were examined by real time-PCR.Results:1Clinical and pathological manifestation of patients with DN, theconcentration of podocalyxin and the expression of Col Ⅳ, desmin, p-Akt andPTEN①G lomerular morphological changes: there was no abnormal change incontrol group by light microscopy. The class Ⅰpatients showed that glomerulibecame bigger accompanied with thick GBM, without specific changes inmesangial area. Mesangial expansion and matrix accumulation appeared in theclass Ⅱ patients in the absence of nodular sclerosis (Kimmelstiel–Wilsonlesions). There were nodular sclerosis in the class Ⅲpatients but thepercentage of global glomerular sclerosis was less than50%. It was showedthat GBM had become thicker uniformly, accompanied with foot processfusion or effacement by electron microscopy.②Immunocytochemical stainingshowed that the expression of Col Ⅳand desmin in golmeruli of DN weremore obvious than those in control group. The severer the lesions were, themore remarkable the expression were. ColⅣ was expressed in mesangial areaand GBM, while desmin was expressed only in mesangial area. Theexpression of p-Akt and PTEN in golmeruli of DN were also more obviousthan that in control group. The expression of p-Akt was the most remarkablein class Ⅱ changes, and the expression of PTEN was the most remarkable inclass Ⅰ changes.PTEN was expressed in cell cytoplasm, while p-Akt wasexpressed in cell nucleus and cytoplasm.③T he concentration of podocalyxinin urine of patients with DN was higher than that in control group (t=27.149,P=0.000). The severer the renal lesions of patients were, the higher theconcentration of podocalyxin was(P＜0.05).④The concentration ofpodocalyxin had a positive correlation with the levels of urine proteinexcretion, Scr, HbA1C and the expression of Col Ⅳ,desmin in glomeruli,whereas a negative correlation with the levels of ALB, eGFR and theexpression of PTEN in glomeruli. 2The effects of high glucose and LY294002on the activation of PI3K/Aktpathway and the expression of the marker protein in mouse podocytes①Immunocytochemical staining showed that p-Akt was expressed in cellnucleus and cytoplasm,while Akt and PTEN were expressed in cell cytoplasm.It was displayed by western blot that in response to high glucose, theexpression of p-Akt in podocytes was increased in a time-dependentmanner(P＜0.01)and the expression of Akt had no change. However, theexpression of PTEN was incipiently increased with the peak on3h anddecreased from24h with the lowest on48h (P＜0.01). LY294002decreasedthe expression of p-Akt in podocytes in a dose-dependent manner (P＜0.01),whereas had no effect on the expression of Akt.②Immunocytochemicalstaining showed that podocalyxin was expressed in cell membrane andcytoplasm, then desmin was expressed mainly in cell cytoplasm. The results ofwestern blot and real time-PCR showed that in high glucose conditions, theexpression of podocalyxin was gradually decreased and the expression ofdesmin was gradually increased (P<0.05), which suggested that podocytes hadunderwent phenotypic alterations. Furthermore, LY294002prevented thephenotypic alterations in a dose-dependent manner, as demonstrated by thesuppression of high glucose-induced podocalyxin down-regulation anddesmin up-regulation (P<0.05).③H ighglucose-induced podocytes haddisplayed decreased nephrin and increased α-SMA by western blot (P<0.01).LY294002also prevented the above alterations in a dose-dependent manner(P<0.05).3The effects of PTEN over-expression on the activation of PI3K/Aktpathway and the phenotypic alterations of high glucose-induced podocytes①The expression of PTEN protein and mRNA in HG PTEN vector groupwere significant higher than those in HG control vector group, HG group orNG group (P<0.05), which suggested a successful gene transfection andhigher expression at protein level. The expression of PTEN protein andmRNA in HG control vector group were significant lower than those in NGgroup (P<0.05), but there was not significantly different between HG control vector group and HG group.②Western blot analysis indicated that theexpression of p-Akt in HG PTEN vector group was significant lower than thatin HG control vector group or HG group (P<0.01), and that there was notsignificantly different for the expression of Akt among all groups. Theexpression of podocalyxin and nephrin in HG PTEN vector group weresignificantly higher than that in HG control vector group or HG group butlower than that in NG group, whereas the expression of desmin and α-SMAwere significant lower than that in HG control vector group or HG group buthigher than that in NG group (P<0.05).③Real time-PCR analysis showed thatthe expression of podocalyxin mRNA and desmin mRNA were the same astheir protein respectively.Conclusions:1There are podocyte injury and the the activation of PI3K/Akt pathway inpatients with diabetic nephropathy. The concentration of urinary podocalyxinmay forecast the severity of patient's condition and correlate with theexpression of p-Akt and PTEN, which suggests that PI3K/Akt pathway maybemediate podocyte injury of diabetic nephropathy.2High glucose could induce the expression of p-Akt, the down-regulationof epithelial markers (podocalyxin, nephrin) and up-regulation ofmuscle-derived cell markers (desmin, α-SMA) in podocytes in atime-dependent manner. LY294002, a specific inhibitor of PI3K/Akt pathway,may depress the expression of p-Akt and ameliorate the phenotypic alterationsof podocytes in a dose-dependent manner, which suggests that high glucosemight take part in podocyte injury by mediating phenotypic alterations ofpodocytes.3PTEN over-expression may depress the activation of Akt and amelioratethe phenotypic alterations, suggesting that PTEN might relieve podocyteinjury via negatively mediating PI3K/Akt pathway, which provides a novelinsight for preventing and treating diabetic nephropathy.