Developing a system for regulating expression of human hepatocyte growth factor using tetracycline in NRK52E cells.

INTRODUCTION: Hepatocyte growth factor (HGF) is a target of gene therapy for renal fibrosis. The aim of this study was to establish a human HGF gene expression system that is regulated by tetracycline (Tet) in normal rat kidney tubular epithelial cells (NRK52E cells). MATERIALS AND METHODS: The plasmids pTet-on, pBI-L-HGF and pTK-Hyg were transfected sequentially into NRK52E cells using Lipofectamine 2000. The expression of HGF gene was measured, and the activity of expressed HGF was detected. RESULTS: A clone of pBI-L-HGF/NRK52E cells showing strong reaction to doxycycline (Dox) was selected using a luciferase reporter assay system. The expression of both HGF mRNA and protein was significantly higher (both p < 0.01) in the Dox group than that in the control group. Furthermore, the bioactivity of expressed HGF was confirmed in the assay. CONCLUSIONS: A Tet-regulated human HGF gene expression system in NRK52E cells has been established. This cell line may prove useful for gene therapy against renal fibrosis.

[Podocyte injury and expression of nephrin and VEGF in rat nephrosis model induced by adriamycin].

OBJECTIVE: To investigate podocyte injury and the expression of nephrin and VEGF in rat nephrosis model induced by adriamycin. METHODS: The rat adriamycin induced nephrosis model was established, while the biochemical indicators in blood and urine were measured and the pathological changes of the renal tissue were evaluated by light microscope and electron microscope. The podocyte number was counted, and the expression levels of nephrin, VEGF were examined at different time by means of immunohistochemistry. RESULTS: After second injected with adriamycin,the model group nephrin presented a weak signal in the end of the first week (P < 0.05), and the expression of VEGF started to increase at the end of the eighth week (P < 0.05). The podocyte number decreased at the end of the eighth week (P < 0.05). The expression of nephrin and the number of podocyte were negatively correlated with the 24-hour urine protein, blood urea nitrogen and serum creatinine; while the expression of VEGF was positively correlated with the 24-hour urine protein, blood urea nitrogen and serum creatinine. CONCLUSION: The decrease of nephrin expression and the change of its distribution might be the significant factors resulting in considerable proteinuria. VEGF participated in the process of proteinuria and glomerular sclerosis in the development of rat adriamycin nephrosis.

[Construction and identification of tetracycline-inducible human hepatocyte growth factor eukaryotic expression vector].

OBJECTIVE: To construct a tetracycline-inducible eukaryotic expression vector containing human hepatocyte growth factor (HGF) cDNA. METHODS: Human HGF cDNA fragment was obtained by PCR from pUC-SRalpha/HGF plasmid and inserted into the restriction site between Mlu I and Sal I of the tetracycline-inducible eukaryotic expression vector pBI-L. pBI-L-HGF was constructed by DNA recombination in vitro, and was identified by restriction endonucleases digestion and sequencing. RESULTS: The fragment of pBI-L-HGF digested with restriction endonucleases well corresponded to expectation, and the sequence of inserted HGF cDNA was correct according to the GenBank. CONCLUSION: The tetracycline-inducible eukaryotic expression vector of human HGF pBI-L-HGF has been constructed successfully, which allows further study of HGF gene therapy with much safety and easy manipulation.

[Construction and identification of tetracycline-inducible rat Smad7 eukaryotic expression vector].

OBJECTIVE: To construct a tetracycline-inducible eukaryotic expression vector of rat Smad7. METHODS: The total RNA was extracted from normal rat kidney with Trizol agent. Rat Smad7 cDNA fragment was cloned by RT-PCR, and was inserted into the restriction site between Nhe I and Hind III of the inducible eukaryotic expression vector pBI-L by tetracycline. pBI-L-Smad7 was constructed by digestion and ligation, and detected by restriction endonuclease digestion and sequencing. RESULTS: The recombinant eukaryotic expression vector pBI-L-Smad7 was constructed correctly as confirmed by restriction endonuclease digestion and sequencing. The fragment of pBI-L-Smad7 digested with restriction endonucleases and the sequence of inserted Smad7 cDNA were consistent with the results of theoretical analysis. CONCLUSION: The tetracycline- inducible eukaryotic expression vector of rat Smad7, pBI-L-Smad7, is constructed successfully, which may facilitate further clinical study of Smad7 gene therapy for tissue and organ fibrosis.

[The protective effects of three components isolated from Astragalus membranaceus on shock wave lithotripsy induced kidney injury in rabbit model].

OBJECTIVE: Extracorporeal shock wave lithotripsy (ESWL) is has been shown to reduce renal parenchymal injury subject to application of shock wave lithotripsy in our pervious study. To investigate the protective action of three main components from Astragalus membranaceus, including total saponins of astragalus (TSA), total flavonoids of astragalus (TFA) total polysaccharide of astragalus (TPA) in alleviating shock wave induced kidney damage. METHODS: Sixty four male rabbits were randomly assigned to a control group or to 3 groups that were premedicated with TSA TFA and TPA respectively prior to application of ESWL. Each group of animals underwent shock wave lithotripsy (18 kV) to the right kidneys and received a total of 1500 shocks. Peripheral blood samples were collected to evaluate the levels of plasma endothelin-1 (ET-1), plasma nitric oxide (NO) and serum malondialdehyde (MDA) before and after shock wave treatment. The concentrations of these markers in the treated kidney tissues were also detected 3 days, 7 days and 14 days after application of ESWL. The changes of histopathology and cells ultrastructure were observed through light microscope and electron microscope. Untreated contralateral kidneys were evaluated as controls. RESULTS: In control serials the levels of ET-1 and MDA were elevated significantly while the level of NO was significantly decreased after application of shock wave lithotripsy (P < 0.05). The comparison between the controls and premedicated groups demonstrated that all these three components especially TSA and TFA significantly inhibited shock wave induced increasing of ET-1 and MDA (P < 0.05). TSA also significantly suppressed the decrease of NO and made the recovery time earlier compare to the results of controls (P < 0.05). However, TFA and TPA had almost no effects on the change of NO. (P > 0.05). The results in histopathology showed noticeably damage of glomerular and tubular epithelial cells in the treated kidneys in the controls. The histological alterations in the TPA group were similar to those of the controls. These alterations were significantly milder in the TSA and TFA particular the TSA group. CONCLUSION: TFA and TSA, especially TSA seemed to play the key role in alleviating ESWL induced kidney damage.

[Effects of components isolated from Astragalus mongholicus on expression of p-selectin in shock wave induced kidney injury in rabbit model].

OBJECTIVE: To investigate the effective components of Astragalus mongholicus and their mechanisms in alleviating extracorporeal shock wave lithotripsy (ESWL) induced kidney injury. METHOD: 69 male rabbits were randomly assigned into control group, sham treatment group, Total Saponins of Astragalus (TSA) group, Total Flavonoids of Astragalus (TFA) group, and Total Polysaccharide of Astragalus group (TPA). The shock wave treated kidneys were observed for the expression of p-selectin and the change of cells ultrastructure pre and post ESWL. The concentration of Maleic Dialdehyde (MDA) as well as activity of superoxide dismutase (SOD) in the kidney tissues was also analyzed. RESULT: After application of shock wave treatment, p-selectin was expressed extensively in renal glomerulus, renal tubules and renal interstitium of the treated kidneys. It showed a significant increase of MDA levels and decrease of SOD activity in control group (P < 0.05) after ESWL. The comparison between controls and TSA group demonstrated that TSA could significantly reduce the positive rate of p-selectin P < 0.05) and alleviate the injuries of cells ultrastructure. The results also showed a significant decrease of MDA levels and increase of SOD activity in TSA group compared to controls (P < 0.05). The protective effects of TFA and TPA in alleviating kidney injury induced by shock wave were lower than those of TSA; they had no effects of the expression of P-selectin. CONCLUSION: TSA is the main components of A. mongholicus in alleviating shock wave induced kidney injury not only by scavenging oxygen free radicals but also inhibiting the expression of p-selectin.