| Objective: Diabetic nephropathy (diabetic nephropathy, DN) is one ofthe most common microvascular complications of diabetes, and also the firstcause of end-stage renal disease (ESRD) in the world. It is becaming the hugeburden of health care in developing and developped countries. The patientswith DN mainly show edema, proteinuria, and progressive renal functiondecline. Renal pathology is mainly characterized by excessive amassing ofextracelluar matrix (ECM) with thickening of glomerular and tubularbasement membranes and increased amount of mesangial matrix, whichultimately progress to glomerulosclersis and tubulo-interstitial fibrosis. Recentresearches have shown that the disturbance of the glomerular filtration barrier,especially the podocyte injury (which is an important component ofglomerular filtration barrier), play crucial role in the progression of diabeticnephropathy.Podocytes are terminally differentiated visceral epithelial cell. Podocytesand their foot processes are unique in that they comprise the outer layer of thekidney ultrafiltration filter and form the glomerula slit diaphragm, which is acomplex cellular structure that prevents the development of the proteinuria inan actin cytoskeleton-dependent manner. In order to highlight the importanceof podocyte in diabetic nephropathy, DN is also called diabetic podocytopathy.In the onset and development in this setting, the pathological changes ofpodocyte mainly include the number decrease, apoptosis, foot effacement,detached from the GBM and GBM bare. However, the detailed mechanism ofpodocyte injury is remaining unclear. The developmental gene gremlinre-emergence in diabetic nephropathy is of great interesting.Gremlin, an antagonist of bone morphogenetic protein (BMP), is a23-28KD highly conservative secreted glycoprotein, which is present as both soluble and cell-associated forms and is found within the Golgi apparatus andendoplasmic reticulum. It consists of184amino acid residues and regulatesembryonic development, growth and differentiation. There are potentialnuclear localization sites near its carboxyl-terminus, N-linked glycosylationsites, and a number of phosphorylation sites, which mediate the interactionsbetween proteins. During embryonic development, gremlin regulates thedevelopment of limb bud、lung and kidney branching morphogenesis viaSHH/FGF-4(sonic hedgehog/fibroblast growth factor4) feedback loopbetween stromal polarization area (ZPA: zone of polarizing activity) and theapical ectodermal ridge (AER). This process is achieved through the control ofapoptosis. In adult, normal kidney tissues has hardly expression of gremlin,wheras in conditions of diabetic nephropathy the expression of gremlinre-emerges and co-localizes with TGF-beta1. Gremlin results in epithelialmesenchymal transition of proximal tubular epithelial cell and mesangial cellscultured in high ambient glucose, drive them to secrete large amounts ofcollagen and fibronectin and promotes renal fibrosis. Whether gremlin causespodocyte injury, there are limited reports.The podocyte injury results in proteinuria in the early stage of diabeticnephropathy. The marker proteins’ expression and apoptosis of podocyte aremajor indicators of podocyte injury. The present study aims to investigate therole and mechanisms of gremlin in hyperglycemia-induced podocyte injuryand to explore the probably involved signaling pathway to provide theoreticalbasis for the prevention and management of DN.Methods:1The expression and distribution of gremlin in podocyte treated with highglucose.The conditionally immortalized mouse podocytes were cultured at33℃with γ–IFN (the permissive conditions) to induce proliferation, and thencultured at37℃withoutγ–IFN (the unpermissive conditions) for10-14daysto induce podocytes differentiation and mature. F-actin and synaptopodin weredetected by immunofluorescence to validate the morphology and differentiation of podocytes. Qualified podocytes were used in experiments.Differentiated podocytes were randomly divided into3groups: normal control(NC) group, high glucose group (HG) and normal glucose+mannitol group(MG) group. NC group was treated with D-glucose5.5mmol/L, HG groupwas treated with D-glucose30mmol/L and MG group was treated with5.5mmol/L D-glucose+24.5mol/L mannitol in order to control the influence ofhigh osmotic pressure to the experimental results. After synchronous culturedfor48hours three group cells were harvested to abstract the total protein andmRNA. The expression of gremlin was detected by western blot and real-timePCR. The distribution and localizing characteristics of gremlin in podocyteswere obseved by the Laser confocal microscope.2The effect of recombinant mice gremlin on the markers and apoptosis ofpodocytes in high glucose ambient.Recombinant mice gremlin and high glucose co-stimulated the podocytesto mimic the conditions of diabetic nephropathy in vivo.①Firstly, to explorethe time and dose relationship between gremlin and high glucose podocytes,the differentiated podocytes were cultivated with high glucose and gremlin (0,0.1,0.25,0.5,0.75,1μg/ml) for48hours respectively. Using western blotand real-time PCR method found out the maximal negative regulatoryconcentration of gremlin on nephrin and synaptopodin, then this optimalconcentration was used in the experiments below. Secondly, podocytes werecultivated with the optimal concentration of gremlin contaminating highglucose for0,6,12,24,48and72hours to find out the maximal negativeregulatory time of gremlin on nephrin and synaptopodin.②Normal control(NC) group, high glucose control (HG) group and high glucose+gremlin (HG+grem1) group were set. According to the results of experiment①, thepodocytes were incubated with0.75ug/ml gremlin for48h, then theexpression and distribution of nephrin and synaptopodin were visualized bylaser confocal microscope to analyze the podocyte injury.③Setting controlsas above, the apoptosis podocytes were detected by TUNEL and TUNELpositive cells were counted.④Bcl-2, Bax and Cleaved Casepase–3, which compensate for the false-positive of TUNEL, were putative indicators ofapoptosis. They were detected by western blot and real-time PCR method tofurther assess the podocyte injury.3The effect of gremlin-siRNA on the markers and apoptosis of podocyte inhigh glucose ambient.Differentiated podocytes were divided into three groups: high glucosegroup (HG), high glucose+gremlin-siRNA group (HG+grem.siRNA) andhigh glucose+control siRNA group (HG+con.siRNA).①Optimizing thetransfection conditions: The optimal dose of gremlin–siRNA was confirmedby abviously knockdown the expression of gremlin gene.②The effect ofgremlin-siRNA on the location and distribution of nephrin and synaptopodinin high glucose ambient podocyte was observed by laser confocal microscope.③The effect of gremlin-siRNA on the expression of nephrin andsynaptopodin in high glucose ambient podocyte was assessed by western blotand real time PCR.④The apoptosis effect of gremlin-siRNA on podocyteswere detected by TUNEL. TUNEL positive cells were counted.⑤Bcl-2,Bax and Cleaved Casepase-3were detected by western blot and real-timePCR to further confirm the apoptosis effect of gremlin-siRNA on podocyte.4The probably signal mechanism involved in gremlin-mediated podocyteinjury in high glucose ambient.①Due to TGF–β1colocalization with gremlin in renal tissue of DN,there maybe exists a certain relationship between TGF–β1and gremlin inhigh glucose environment. Differentiated mature podocytes were divided intotwo groups: one group was treated with high glucose+gremlin (0.75μg/ml)for48h, the protein and mRNA expression of TGF-β1was detected. Theother group was treated with high glucose+TGF-β1(5ng/ml) for48h, theprotein and mRNA expression of gremlin was detected. Additionally，normalcontrol (NC) and high glucose control (HG) were set.②The cannoniacl andnoncannonical TGF–β1signaling molecules detection in high glucosepodocyte exposed to gremlin: Podocytes were incubated with high glucoseand recombinant mice gremlin (0.75μg/ml) for0,15,30,60,120and240 minutes, the cannonical (smad2/p-smad2, smad3/p-smad3) and noncannonical(p38/p-p38, JNK1/2/p-jnk1/2) signling molecules were detected to find outthe signal pathway mediating the role of gremlin on hyperglycemic podocyte.③The above experimental results implicated that the TGF–β/smad2/3signaling pathways involved in the pathogenesis of gremlin. SB431542, aTGF–β receptor blocker, blocked the suprression of gremlin on the expressionof nephrin and synaptopodin protein in hyperglycemic podocytes.④Podocytes treated with high glucose and gremlin was transfected withsmad2/3–siRNA and harvested after48hours to verified the knocking downeffect. Smad2/3knocking down inhibited the supression of gremlin on theexpression of nephrin and synaptopodin protein.Results:1The expression and distribution of gremlin1in high glucose ambientpodocytes.①F-actin immunofluorescence assay showed that there were twodifferent phenotypes in cultured podocytes. At33℃with γ–IFN (thepermissive conditions), cells proliferated and maintained cobblestone-likemorphology. When cultured at37℃without γ–IFN (nonpermissivecondition), cells stretched out differently shaped long processes from cellbodies，arised many spindle-like projections from their primary processes andstopped proliferation. Synaptopodin is one marker indicating maturedifferentiated podocytes. Immunofluorescence staining showed that there wassparsely expression of synaptopodin in undifferentiated podocytes, while itsexpression in differentiated podocytes was enhanced obviously.②The resultsof Western blot showed that in NC group and MG group, there was hardlyexpression of gremlin protein, while in HG group its expression was obviouslyenhanced (p<0.01).③The results of Real-time PCR displayed that comparedwith NC group and MG group, the expression level of gremlin mRNA in HGgroup was increased significantly when the podocyte was cultured for48hours (p<0.01).④Confocal staining demonstrated that the distribution ofgremlin1in podocytes was located predominantly in the cytoplasm with a punctate pattern., and sparsely on the cell membrane with a thin linear or fineparticles pattern.2The effect of recombinant mice gremlin on the markers and apoptosis ofpodocytes in high glucose environment①The dose-effect relationship experiment showed that the protein andmRNA expression of nephrin and synaptopodin decreased significantly at agremlin concentration of0.75μg/ml (p <0.01).②The time-effect relationshipexperiment disclosed that the protein and mRNA expression of nephrin andsynaptopodin decreased significantly when cells were treated with HG+0.75μg/ml gremlin for48h (p <0.01).③The laser confocal image showedthat gremlin aggravated the hyperglycemia-induced podocyte injury. Thedistribution of nephrin differed markedly between podocytes cultured in thepresence of gremlin and control cells. Control cells expressed nephrin in cellprojectons or exhibited a filamentous nephrin appearance. By contrast, cellscultured with gremlin displayed a cytoplastic and non-filamentous distribution.In gremlin-cultured podocytes, synaptopodin distributed mainly at peri-orintra-nuclear regions. In normal control cells, synaptopodin localized mainlyin the foot process or cytoplasm in a punctuated pattern along actin filaments.High-glucose induced synaptopodin shifting to the peri-nucleus anddepolymerization of actin mirofilaments. Gremlin aggravated such injury andinduced a marked decreasement in podocyte size, and rearrangement ofsynaptopodin from a punctuated pattern to a granular mass pattern.④Theresults of TUNEL showed that compared with the HG group, the cellapoptosis rate of HG+gremlin group was increased significantly (p <0.05).⑤The experimental results of apoptosis related proteins displayed thatcompared with the HG group, gremlin decreased the levels of Bcl-2proteinand mRNA further, while increased those of Bax and Cleaved Caspase-3.Collectively, these results disclosed that gremlin aggravatedhyperglycemia-induced podocytes apoptosis.3The effect of gremlin1-siRNA on the markers and apoptosis of podocyte inhigh glucose environment ①The verification experiment results of gremlin1-siRNA showed that at80pmol/well gremlin-siRNA knocked down the protein and mRNAexpression of gremlin significantly in podocytes cultured in6well plate (p <0.01).②The confocal images displayed that gremlin-siRNA rescued theexpression and distribution of nephrin and synaptopodin in hyperglycemicpodocyte. Gremlin-siRNA attenuated the distribution of nephrin from thecytoplasm and non-filamental to the cell membrane and filament pattern, andthat of synaptopodin from massive granular to punctate pattern.③Westernimmunoblot and real-time PCR analysis indicated that gremlin1siRNArescued the expression of nephrin and synaptopodin at both the mRNA andprotein levels.④Gremlin1-siRNA decreased the percentage ofTUNEL-positive cells. There were7.021%apoptosis cells in HG group and4.986%apoptosis cells in HG+grem.siRNA group (p <0.05).⑤GremlinsiRNA increased the expression of Bcl-2and decreased that of Bax andCleaved Caspase-3in HG ambient (p <0.01). Gremlin1-siRNA attenuated HGinduced apoptosis effectively.4The signal mechanism mediating the pathogenesis of gremlin inhyperglycemic podocytes①Podocytes that were cultured in gremlin1(0.75μg/ml) and highglucose (30mmol/L) expressed TGF-β1more abundantly than high glucoseand normal controls at both the protein (p<0.05) and mRNA (p<0.05) levels.Conversely, podocytes that were cultured in TGF-β1(5ng/ml) concomitantlywith high glucose (30mmol/L) expressed gremlin1more abundantly than highglucose and normal controls at both the protein (p<0.05) and mRNA (p<0.05)levels.②The protein of TGF-β signaling molecules (smad2/p-smad2,smad3/p-smad3, p38/p-p38and JNK1/2/p-JNK1/2) was analyzed by westernblot. In gremlin-activated podocytes, smad2and smad3were phosphorylatedin a time-dependent manner. By contrast, neither JNK1/2nor p38MAPK wereactivated. Gremlin activated canonical, but not non-canonical signalingpathway in podocytes (p<0.05, versus0min controls).③TGF-β receptorinhibitors (SB431542) blocked the ability of gremlin1to suppress nephrin and synaptopodin expression. Inhibition of the TGF-β receptor with SB431542(5μM/L) blocked the effect of gremlin1on high glucose cultured podocytes andrescued the expression of nephrin and synaptopodin (p<0.05). Gremlin injuredpodocytes required endogenous TGF-β1.④Smad2/3-siRNA treatmentinhibited the ability of gremlin to down-regulate the expression of nephrin andsynaptopodin in high-glucose ambient podocytes. Podocytes were transfectedwith smad2/3siRNA (60pmol/well) for6hour before treatment with gremlinand high glucose. Control cells were transfected with scrambled siRNAsequences. Smad2/3siRNA knockdown rescued the expression of bothnephrin and synaptopodin and attenuated injury of podocytes cultured in highglucose ambient (p <0.01).Conclusions:1Gremlin is clearly elevated in high glucose cultured mouse podocytes.The distribution of gremlin1in podocytes locates predominantly in thecytoplasm in a punctate pattern and sparsely on the cell membrane in a thinlinear or fine particle pattern.2Gremlin aggravates hyperglycemia induced podocyte injury, supressesthe expression of nephrin and synaptopodin and deteriorates the apoptosis ofpodocytes.3Treatment high glucose ambient podocyte with gremlin-siRNArescues the expression of nephrin and synaptopodin and declines apoptosisrate of podocyte.4Gremlin and TGF-β1induce each other in high glucose conditions.Canonical smad2/3pathway, but not non-canonical pathway of TGF-βsignaling is activated by gremlin, which exerts its effect on the podocytethrough endogenous TGF-β signaling.In summary, gremlin plays a crucial role in the pathogenesis of podocyteinjury in DN, perhaps by disrupting the homeostatic balance between TGF-βand BMP. There also appears to be a feedback loop between gremlin andTGF-β1whereby they are capable of inducing each other and accelerating thepathogenesis of the disease process. Gremlin activates a canonical smad2/3 pathway, but does not activate a non-canonical pathway of TGF-β signaling.New therapies targeting gremlin may protect the kidney from the developmentand progression of DN. |
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