Expression Of ANGPTL3 In Kidney, Serum And Urine Of The Adriamycin-induced Nephrotic Rat

BackgroundPrimary nephrotic syndrome (PNS) is a common disease during childhood. About 80 percentage of PNS in children is minimal change nephrotic syndrome (MCNS). Although most of the patients were sensitive to steroid, some patients were prone to frequent-relapse or to be resistant to steroid treatment, even developing into renal insufficiency. Clinical manifestations of PNS is characterised as heavy proteinuria, hyperlipidemia, hypoproteinemia and edema. The pathogenesis of MCNS is not well known yet in spite of the many studies on the pathogenesis of MCNS, in particular proteinuria. Recently lots of studies focus on glomerular podocyte because the unique pathological character in kidney of MCNS is extensive fusion foot processes and loss of the nomal architectecture of slit diaphragms. Many kinds of protein expressed in podocyte, such as nephrin, podocin and so on, were found to play a role in proteinuria. These interactive molecules take part in the development of proteinuria via a complicated podocyte molecular network. However, the other important molecules in the network and their roles in the pathogenesis of MCNS will be recognized gradually, when with more new studies come up.In our previous study, by Affymetrix GeneChip technology we have found that the mRNA expression of angiopoietin-like 3 protein (ANGPTL3) in kidney of children with primary minimal change nephrotic syndrome increased significantly when compared with the normal control. ANGPTL3 is a 70kD secretory protein and a member of angiopoietin family. The C-terminal fibrinogen-like domain of ANGPTL3 was reported to be involved in angiogenesis and reparation of vascular endothelial cells, while coiled-coil domain of ANGPTL3 is involved in the regulation of lipid metabolism by inhibiting LPL activity. Thus, ANGPTL3 seems to be a multifunctional factor. Up to now, ANGPTL3 was reported to be expressed predominantly in liver. There is no report about the expression and role of ANGPTL3 in kidney. Therefore, we used animal model and successively observed the ANGPTL3 expression and changes in kidney, serum and urine of normal and nephrotic rats at protein and gene level respectively, analyzed the assosiation of ANGPTL3 with the development ofproteinuria and hyperlipidemia, being able to disclose the possible role of ANGPTL3 in the pathogenesis of MCNS. A eukaryotic expression plasmid of human ANGPTL3 will be constructed, which will provide a sound basis for further study on the role of ANGPTL3 in MCNS.Part I Expression of ANGPTL3 Protein in Kidney of the Adriamycin-induced Nephrotic RatObjective To successively observe the ANGPTL3 protein expression and change in kidney of normal and adriamycin-induced nephrotic rats and analyze the assosiation of ANGPTL3 with the development of proteinuria and hyperlipidemia, and in order to disclose the role of ANGPTL3 in the pathogenesis of MCNS.Methods Adriamycin(ADR)-induced nephrotic rat models were established by a single injection of 7mg/kg adriamycin via the tail vein. 64 Sprague-Dawley rats were divided randomly into two groups: ADR group injected with adriamycin and control group injected with normal saline. 8 ADR rats and 8 control rats were sacrificed at 7days, 14days, 21 days and 28days after adriamycin injection respectively. 24hr urinary protein, serum cholesterol, serum triglyceride and renal function of rats were measured by routine biochemical method. The expression and distribution of ANGPTL3 in kidney were studied by immunohistochemical staining (IHC) and Western-blot, and were quantitatively analyzed with IMS color image analysis system.Results (1) In ADR rats, urinary protein and the level of triglyceride and cholesterol in serum increased significantly at 14th day (P<0.01, 0.05, 0.01, respectively), at same time, albumin in serum decreased significantly (P<0.01). Changes of these indices reached the peak level at 28th day. Under electron microscope, ADR rats were found loss of the normal architecture of foot processes, although not complete at 14 days after adriamycin injection. At 28 days after adriamycin injection, extensive fusion of foot processes was observed in ADR rats. (2) The protein of ANGPTL3 in kidney of ADR rat increased significantly at 21st day and 28th day compared with control by Western blot (both P<0.05). (3) The IHC index of ANGPTL3 in ADR rats' glomeruli increased significantly by 42% and 148% of control level at 14th day (PO.05) and 21st day (PO.01) respectively. In corticaltubuli ,as compared with control, ADR rats increased significantly in the IHC index of ANGPTL3 at 21st day (PO.01). The peak level of the expression of ANGPTL3 in glomeruli and tubuli were both observed at 28th day (both PO.01). The major site of ANGPTL3 expression in kidney was located in cytoplasm of podocyte as well as that of tubular epithelial cell. (4) The expression of ANGPTL3 in glomeruli correlated significantly to the proteinuria level (r=0.81,P<0.01). There was also significant correlativity between the expression of ANGPTL3 in renal tubuli and hyperlipidemia (rchoiesteroi=0.70, PO.01; rtrigiyceride=0.65, PO.01, respectively).Conclusions The expression of ANGPT13 protein is presented in cytoplasm of glomerular podocytes and tubular epithelial cells of rat's kidney tissue. During the period of the present study , the expression of ANGPTL3 in kidney increases gradually, along with the aggravation of proteinuria and hyperlipidemia. Both of them are correlated with the expression of ANGPTL3 in kidney.Part II Analyze the Expression of ANGPTL3mRNA in Kidney of the Adriamycin-induced Nephrotic Rat by LaserMicrodissectionObjective To determine the expression of ANGPTL3mRNA in glomeruli and tubuli of adriamycin-induced nephrotic rats and the secretion of ANGPTL3 in kidney, to analyze the assosiation of ANGPTL3 change resulted from kidney in ADR rats with the development of proteinuria, and to further disclose the role of ANGPTL3 inMCNS.Methods Establishing of animal model and animal group were same as part I. Twenty glomeruli and forty cortical tubuli were dissected from every piece of frozen section of kidney by laser microdissection system. Real time quantitative RT-PCR was used to determine the expression of ANGPTL3mRNA respectively in renal cortex, glomeruli and tubuli of ADR rats at 7 days, 14 days, 21 days and 28 days successively after adriamycin injection.Results (l)Twenty glomeruli and forty cortical tubuli of rats from every section were dissected successfully by laser microdissection and RNA obtained from themwas enough. (2)The mRNA of ANGPTL3 in total cortex of kidney in ADR rat increased significantly at 21st day(P<0.01 ) and at 28th day (P<0.05) compared to control.(3) The tendency of ANGPTL3mRNA expression in glomeruli of ADR rats was same as that in total cortex. ADR rats had a higher expression level of ANGPTL3mRNA in glomeruli at 21st and 28th day compared to control (both PO.05). (4) There was no significantly change of ANGPTL3mRNA expression in cortical tubuli after adriamycin injection.Conclusions ANGPTL3mRNA can be expressed in kidney of normal and ARD rats and ANGPTL3 can be secreted in kidney. The expression of ANGPTL3mRNA increases in glomeruli of ADR rats during the development of proteinuria. It is suggested that ANGPTL3 has an effect on the function of glomerulus and play an important role in the development of proteinuria.Part m Expression of ANGPTL3 in Serum and Urine of the Adriamycin-induced Nephrotic RatObjective To successively detect the ANGPTL3 level in serum and urine of adriamycin-induced nephrotic rats, and to discuss kidney filtration and reabsorption of ANGPTL3 in nephrotic syndrome.Methods Establishing of animal model and animal group were same as part I. The level of ANGPTL3 in serum and urine of every group rats were detected by Dot-ELISA.Results (1) In ADR rats ,the level of ANGPTL3 in serum decreased significantly at 7th day compared to control (P<0.01). No significant difference of ANGPTL3 in serum was found between ADR rat and control at 14th day and 21st day, while at 28th day ADR rats had a significantly higher ANGPTL3 level in serum than control (P<0.05). (2) There was no significant difference of ANGPTL3 in urine of ADR rats after adriamycin injection compared with control.Conclusions The level of ANGPTL3 in ADR rat's serum gradually increases after adriamycin injection, but is lower than or equal to the control in the initialpart of study.The level of ANGPTL3 in urine of ADR rats have not change. It is indicated that ANGPTL3 can be filtrated through the glomerular filtration barrier and be reabsorbed by renal tubuli.Part IV Construction of Eukaryotic Expression Plasmid ofHuman ANGPTL3Objective To construct a eukaryotic expression plasmid pcDNA3-ANGPTL3, and to provide a basis for further study on the role of ANGPTL3 in MCNS.Methods The full-length coding region of human ANGPTL3 was cloned from human liver cDNA library by PCR and digested with restriction endonucleases EcoR I and Xho I . Vector pcDNA3 was treated in same way. Then ANGPTL3 gene was inserted into eukaryotic expression vector pcDNA3 and recombinant plasmid pcDNA3-ANGPTL3 was identified by restriction analysis and sequencing.Results Recombinant plasmid DNA successfully produced a band which had the same size as ANGPTL3. The inserted sequence was identical with that published on GeneBank.Conclusions A eukaryotic expression plasmid of human ANGPTL3 was constructed successfully.CONCLUSIONS1. ANGPTL3 can be secreted by kidney of rat and presents in the cytoplasm of glomerular podocyte and tubular epithelial cell.2. ANGPTL3 is secreted increasingly in glomeruli of adriamycin-induced nephrotic rats and participates in the development of proteinuria possibly the role of reparation on glomerular filtration barrier. The increase of ANGPTL3 in renal tubuli of adriamycin-induced nephrotic rats resulted from renal tubuli reabsorption reflects the change of ANGPTL3 in serum, and is correlated with hyperlipidemia. Therefore, ANGPTL3 plays an important role in the development of proteinuria and hyperlipidemia in MCNS.