Expression Of ANGPTL3 In Kidneys Of Children With Primary Nephritic Syndrome And The Mechanism Responsible For Podocyte Damage

Primary nephrotic syndrome is a common glomerular disease in childhood, the majority of which are due to minimal change disease. It is characterized as heavy proteinuria, hypoalbuminaemia, hyperlipidemia and edema that caused by the disorder of glomerular filtration barrier. Podocytes and GBM cooperatively construct the glomerular filtration barrier, which prevents protein leakage into urine filtrates. In addition to the profound involvement of podocytes in the pathogenesis of glomerular diseases the sophisticated function of podocytes and the molecules associated with podocytes are highly attractive in the research of the mechanisms of proteinuria. Among the proteins expressed by podocytes some are inherent proteins of podocytes such as the slit diaphragm protein or the cytoskeletal components, some are the podocyte-derived cytokines such as angiopoietin protein with influence on the function of endothelium or heparan sulfate proteoglycans (HSPGs) which contribute to the glomerular charge barrier. These interactive molecules take part in the development of proteinuria via a complicated podocyte molecular network. More and more new molecules in the network are going to be revealed with the knowing of the pathogenesis of proteinuria.In our previous study, it was found by Affymetrix GeneChip technology the mRNA expression of angiopoietin-like 3 protein (ANGPTL3) in kidneys of children with minimal change nephrotic syndrome increased significantly compared with the normal control. And the expression of ANGPTL3 mRNA was found increased in the glomerulus of adriamycin rats along with the development of proteinuria by laser microdissection. ANGPTL3, a member of angiopoietin family, is a 70kD secretory protein. It was reported to be involved in angiogenesis and in the regulation of lipid metabolism.While few is known about the role of ANGPTL3 in kidney. In this study the expression of ANGPTL3 is to be examined in kidneys of children with primary nephrotic syndrome to find the relation to the changing of the glomerular filtration barrier. The expression of ANGPTL3 or HSPGs of podocytes and the detachment of podocytes are to be evaluated in puromycin induced pdocyte damage and inpodocytes with gene transfection of ANGPTL3 to explore the possible mechanism of pdocyte injury and proteinuria.Part I Expression of ANGPTL3 in kidneys of children with primary nephrotic syndromeObjective To examine the expression of ANGPTL3 in kidneys among children with primary nephrotic syndrome.Methods A total of 69 renal biopsies were enrolled including MCD (n=31), MN(n=6), FSGS (n=6), TBMN (n=10), IgA nephropathy with mesangial proliferation (n=16), and 2 cases with nephrectomy for tumor as normal control. According to the period from episode to renal biopsy, cases were divided into four groups ("<1 month" "1-6 months" "6-12 months" and ">12 months"). Immunohistochemistry and image analysis system were performed to analyze the expression of ANGPTL3 or perlecan by use of positive index (PI). Immunofluorescence for ANGPTL3 co-labelling with WT1 and perlecan was to show the distribution of ANGPTL3. It was measured the foot process width (FWP) in 10 cases of MCD to analysize the fusion of podocytes. Correlation analysis was performed for the expression of ANGPTL3 with FWP, urine protein or the expression of perlecan.Results (1) The PI level for ANGPTL3 in glomerulus of patients with MCD (7.49±1.96) and MN (6.27±0.98) was significantly higher than that of TBMN (0.02±0.001), FSGS (3.14±0.49) or normal control (0.02±0.001) respectively (P <0.05). The PI level for ANGPTL3 in glomerulus of patients with IgA nephrosis accompanied with proteinuria was significantly higher than that of patients with IgA nephrosis accompanied with hematuria (1.90±0.81 vs 0.03±0.01, P<0.05). (2) Co-labeling with WT1, a marker of podocyte, showed ANGPTL3 was expressed in the cytosol of WT1 positive cells under immunofluorescent microscope. Co-labeling with perlecan, a molecule secreted by podocyte and expressed in GBM, showed ANGPTL3 with a strong positivity selectively at periphery of glomerular capillary loops. (3) The PI level for ANGPTL3 in glomerular was correlated with the ratio of urinary protein and creatinine positively, with FPW positively ( r= 0.86, 0.84; P<0.05), and with the PI level for perlecan in glomerular negatively( r=-0.83; P<0.05). (4) No difference was found for the expression of ANGPTL3 or perlecan among the four groups withdifferent course of disease.Conclusions In human kidneys, ANGPTL3 is selectively expressed in podocytes. The expression of ANGPTL3 in glomerular increases with the urinary protein leakage and with the fusion of foot processes. The expression of ANGPTL3 was correlated with the expression of perlecan negatively.Part II Expression of ANGPTL3, perlecan and agrin in puromycin-induced pdocyte damageObjective To investigate the possible role of ANGPTL3, perlecan and agrin in puromycin-induced pdocyte damage.Methods MPC5, a conditionally immortalized mouse podocyte cell line in vitro, were induced propagate and passage by y-IFN at 33°C which were subsequently cultured at 37°C for differentiation for 14 days. Western blot and real time PCR were processed to evaluate the expression of ANGPTL3 in puromycin induced podocyte damage with different doses (10mg/ml, 20mg/ml, 50mg/ml) at distinct time intervals (6,8, 10, 12, 24,48 hours).Results ANGPTL3 was only weakly expressed in normal podocytes. The induction of puromycin upregulated both protein expression and mRNA expression of ANGPTL3 on podocytes. In response to puromycin, a time-dependent up-regulation of ANGPTL3 could be observed starting as early as 12 h after incubation with 10ug/ml puromycin (P<0.05) and reaching the peak after 24 h. And a time-dependent down-regulation of perlecan or agrin started as early as 8 hs ( by 46.6%, 44.4%, P <0.05) and reached the minimum after 24 h ( by 55.9%, 74.8%, P <0.05). The mRNA expression of ANGPTL3 was correlated with that of perlecan or agrin negatively (Pearson coefficient -0.407, -0.573 , P=0.03, 0.02 ).Conclusions In puromycin induced podocyte damage the expression of perlecan or agrin decreased prior to the up regulation of ANGPTL3. The expression of ANGPTL3 is up-regulated in time dependent manner accompany with the expression of perlecan or agrin down-regulated within 24hous in puromycin induced podocyte damage.Part III The changes of perlecan and agrin expression on podocyte by ANGPTL3 gene transfectionObjective To study the influence of ANGPTL3 gene transfection on the expression of perlecan and agrin and its possible role in puromycin-induced pdocyte damage.Methods (1)Immunofluorescence staining and real time PCR were adopted to detect the expression of ANGPTL3, perlecan and agrin on normal and transfected MPC5 compared with puromycin induced podocyte damage. (2) Detachment assay was performed on normal and transfected podocyte with or without puromycin stimulation through cell number counting and attachment ratio evaluation.Results (1) The staining of perlecan and agrin was strengthened and co-localized with ANGPTL3 on podocyte transfected by gene ANGPTL3. (2) Compared with normal control, the mRNA expression of perlecan or agrin increased significantly on podocytes transfected by gene ANGPTL3 ( by 3.54, 1.22, P<0.05). (3) Comaring in normal, untransfected podocyte and podocyte transfected by pcDNA3.1-ANGPTL3, there were significant difference in cell adhesion after puromycin treatment (P<0.05). 24h or 48h after puromycin treatment, the attachment ratio was shown 95.7%±3.3%, 80.6%±2.7% on podocytes transfected by gene ANGPTL3 compared with 38.6% ±4.7%, 30.8%±7.5% on normal podocytes, 27.4%±3.5%, 20.6%±5.5% on podocytes untransfected with puromycin induced detachment. While there was no difference of the expression of perlecan or agrin among the three groups after puromycin inducement (P>0.05).Conclusions ANGPTL3 gene transfection could up-regulate the mRNA expression of perlecan and agrin, and moreover, can ameliorate puromycin induced podocyte detachment without changing the puromycin induced down-regulation of HSPGs.