Effcts Of Knockout Receptor Activator Of NF-κB(Rank) On Proteinuria In Podocytes In Mice

BACKGROUNDAs economy develops, people’s living standards have been improved well. The incidence of hypertension and diabetes is consistently rising.All of these diseases have become high-risk factors of Chronic Kidney Disease.A survey shows that the prevalence of CKD reaches to 10.8% among adults.Therefore, CKD has become a threat to human public health.In recent years, the number of patients entered end-stage renal disease is still rising.How to delay its progress has become a world-level problem.Numerous researches demonstrate that proteinuria is an independent risk factor of CKD.The level of proteinuria will influence CKD prognosis.Therefore,how to reduce the level of proteinuria is of great value of medical research.In the past studies, proteinuria is related to the function and structure of Glomerular Filtration Barrier.GBM consists of at least 5 layers:endothelial cell surface membrane structure, endothelial cell, endothelial cell fenestration, GBM, podocyte gap and podocyte.The membrane between podocytes is the last filtration barrier. More and more researches demonstrate that podocyte plays a vital role in proteinuria.Podocyte consists of cell body, main projection and foot process.Related foot process is interdigitated to each other.Only small moleculer objections can go through the SD.Protein can not go through it. Podocalyxin in the upper is the negative charge barrier of podocyte. Special structure of podocyte relays on actin and some specific moleculer protein.The injury of podocyte will damage GBM and leads to proteinuria.In some human kidney diseases, such as membranous nephropathy, minimal change nephropathy and focal segmental glomerular sclerosis are related to podocyte injury. Immune complexes, complements and antibodies will also injure podocyte. Morphology and non-morphology changes acure in injured podocytes,such as foot process effacement,cell hypertrophy and apoptosis.For example,LPS can change dynamic system of foot process through influencing rearrangement of actin fibrosis.It perfprmance as increased activity in vitro and foot process effacement in vivo.In human glomerular disease and LPS-induced mice proteinuria model, we could observe the increasing expression of uPAR.uPAR,a glycosylphosphatidylinositol(GPI)-anchored protein, has important roles in metastasis and inflammation.However, podocytes are in the mitosis anaphase.The ability of proliferation and division growth is limited.Once the damage, they will hardly regenerate.Once the damage,the acroteric podocytes will be hypertrophied to cover the bare GBM and its vulnerability will also increase.Glomerular sclerosis will be the endpoint event.TNF receptor superfamily consists of RANK and RANKL.RANKL is the vital factor in osteoclast differention and maturation.In the osteoclast,RANKL conbines with RANK,and collect with TNF receptor correlative factors to activate NF-κB and MAPKs.OPG is the decoy recptor.It conbines with RANK to block this acess.In the past studies,this signal pathway is focused on bone tumors and ostroporosis.But in some recent studies,it also play a part in Renal Osteodystrophy,prostatic cancer,mammary cancer and autoimmune diseases.What’s more,it also works in podocytes.The expression of RANK is low on normal podocytes.But in some podocytes’diseases,the expression is high.RANK can be induced by PAN in rat,and what’s more, the aptosis of podocytes will be observed.In the 5/6 nephrectomy animal model,RANK is also highly expressed.All of these may be the example that RANK is harmful to podocytes.In order to discuss the reason of podocyte injury, our research obtain podocyte-specific KO mice with Cre-LoxP system, by then to make LPS-induced mice proteinuria model.We all know that LPS can induce AKI in mice.The basic mechanism is to cause the micro thrombus, microcirculatory disturbance and metabolic disorders through inflammatory reaction and some specific cell factors.It has nothing to do with T and B cells, but through recombination of actin to disorder the dynamic system of podocyte.In vitro,it represents as enhanced migrated and invaded ability.In vivo,it represents as foot process effacement,leading to proteinuria.We observe the difference among KO group mice,W group and normal C57 group,to dicuss the mechanism of podocyte damage,specific cell factors and to find a brand new way to cure glomerular disease.Objectives1. The role of RANK expression in podocyte injury.2. The signal pathway of RANK in podocyte injury.MethodsAnimals The animal study has been approved by Research Ethics Committee,Guangdong General Hospital,Guangdong Academy of Medical Sciences(NO.GDREC 2015220A).We purchased B6.Cg-Tg(NPHS2-cre)295Lbh/J mice from American Jackson Laboratory and RANK-loxP mice from University of Western Australia.All the mice are feeding and breeding in Nanjing University-Nanjing Biological Medicine Research Institute.We purchased the ordinary C57 mice from Laboratory Animal Center,Sun-Yat-sen University,China.Animal models and treatment with LPS The LPS-induced mice proteinuria model was performanced in three groups of mice(C57 group,W group and KO group,initial age 6-8 weeks) by performancing LPS injection. PCR analysis was carried out to identify the genotypes of podocyte-specific RANK knockout mice. Six (6-8 weeks old)female RANK-/- mice were chosen as KO group. Six (6-8 weeks old) littermates and six(6-8 weeks old) C57/B6j mice were separately chosen as W group and C57 group. Each group was injected with LPS to create podocyte-injured proteinuria model.24h urine was collected before and after injection. Urinary albumin, urine creatinine and UACR were detected by automatic biochemical analyzer. The mice were sacrificed after 48h.Immunohistochemistry Three group of tissues were made into paffrin section,dyeing with PAS.WT1 as the specific marker to sign podocyte.Antibody WT1(SC-192) was perchased from American Santa Cruz.All the operation steps were according to the reagent kit.Each section was selected randomly 10 glomerulus and count the numer of them.Transmission Electron Microscopy (TEM) In order to observe under transmission electron microscopy, ultrasections were (60-100 nm) cut from cortical kidney tissue samples embedded with epon resin, using an ultramicrotome (Leica),collected on cppor grids,and stained with an uranyl acetate and lead citrate.Ultrathin sections were stained with uranyl acetate for 10 min. Consequently, in Reynolds lead citrate for 2 min.Ultrastructral analysis was performanced by transmission electron microscopy.Immunofluorescence Murine kidney tissue were harvested and snap-frozen according to standard protocols and fixed after sectioning in ice-cold acetone for 10 min.For immunohischemistry labeling,tissues were washed once with PBS,permeabilized with 0.5% Triton-X 100 in PBS and incubated with blocking solution(5% BSA) for 20 min at room temperature before futher incubation with one of the primary antibodies(synaptopodin),integrin-β1(RANK,integrin-β1 and uPAR) for 2 hours in room temperature.For double labeling,sections were washed 3 times with PBS for 10min,and one of the secondary antibodies(Thermo 10088 Donkey anti-goat IgG-TRITC; Introvegen 555 anti-Rabbit IgG-FITC)was applied for 2 hours.Pictures were captured with confocal microscopy.All images were analysed by 2 investigators blinded to the identity of the samples.Westernblot Protein expression with integrin-β1, integrin-β3 and uPAR were determined by westernblot analysis.Briefly,kidney cortex isolated from murine was homogenized in lml of tissue lysis buffer. Samples were centrifuged at 3000g for 15min, and the supernants were assayed.After being mixed with SDS-PAGE sample buffer and boiled for 5min,samples were electrophoresed on 10% SDS polyacrylamide gels and transferred to PVDF membranes for 2 hours at 120V.Membranes were blocked for 30min with tris-buffered asline that contained 5% BSA(5%BSA/TBS) and incubated with diluted primary antibody including anti-integrin-β1,anti- integrin-β3 and anti-uPAR overnight at 4℃.The membranes were washed and developed using the enhanced chemiluminmescence system.Satistical analysis All the experiments were repeated 3 times. We assessed statistical significance by one-way ANOVA analysis of variance for comparison between three groups in SPSS19.0. P<0.05 was considered significant.All values are expressed as x±s.ResultsComparing with C57 group and W group, there is no difference in the weight and kidney morphology.We selected randomly 6 female mice in each group at the age of 6-8 weeks.The weight of the mice are C57 group(25.47±6.03)vs W group(22.45±2.38)vs KO group(23.72±5.41).There has no statistically significant difference.The weight of kidney respectively are C57 group(1.27±0.05)vs W group(1.12±0.11)vs KO group(1.15±0.10).And there has no statistically significant difference,too.After LPS injection, comparing with the other two groups,the increase of ACR was lesser than them.Before LPS injection,the ACR of three groups are respectively:C57 group(6.34±0.68)vs W group(7.39±1.84)vs KO group(7.40±1.60).The difference between three groups has no statistically significant.After LPS injection,the ACR started to rise soon,which reminds us the model was successful.The ACR are seperatedly C57 group(132.13±14.26)vs W group(107.56±22.32) vs KO group(23.70±9.90),and KO group was lesser than the other two groups.The results were statistically significant. (P<0.05).After LPS injection, the pathology has no difference, but the number of podocytes decreased was lesser in KO group.Before and after LPS injection, the pathology of glomeruli has no significant change.But after LPS injection, the number of podocytes decreased was lesser in KO group. The difference was statistically significant.In the TEM, there was lesser podocyte foot process effacement in KO group after LPS injection.Before LPS injection,the kidneys were normal in three groups.After LPS injection, we could observe foot process effacement in three groups’podocytes.However,the effacement was lesser in the KO group.In the LPS-induced mice proteinuria model,the effect of expression of integrin-β1、integrin-β3 and uPAR.1. ImmunofluorescenceSynaptopodin located as podocyte.After LPS injection,the expression of integrin-β1 were decreased in all the groups.But the decrease in KO group was more than other two groups.The expression of integrin-β3 were all increased but the KO group was lesser than others.2. WesternblotWesternblot results of integrin-β1 and integrin-β3 confirm the immunofluorescence.The expression of uPAR were all increased in three groups.But it was increased lesser in the KO group.Conclusions1. Comparing with C57 group and W group, there is no difference in the weight and kidney morphology.2. After LPS injection, comparing with the other two groups,the increase of ACR was lesser than them.It shows that there is an important significance of RANK in the podocyte injury.3. After LPS injection, the pathology has no difference, but the number of podocytes decreased was lesser in KO group.4. In the LPS-induced proteinuria model,the expression of integrin-β1 were decreased in all the groups.But the decrease in KO group was more than other two groups.The expression of integrin-β3 were all increased but the KO group was lesser than others. The expression of uPAR were all increased in three groups.But it was increased lesser in the KO group.The results reminds us that RANK may interfere with integrin-β1, integrin-β3 and uPAR to affect proteinuria.