Hepatocyte Growth Factor Protects Podocyte From Injury

Objective:The important components of glomerular filtration barrier--podocyte plays an important role on the pathogenesis of proteinuria and glomerular sclerosis. Hepatocyte growth factor (HGF) is a damage-mediated tissue repair factor which can lead to cell growth, proliferation and differentiation. But whether HGF could prevent kidney from podocyte injury and proteinuria or not is unclear. thus,using mouse model of puromycin nephropathy, we are going to observe the effects of HGF on injury of podocyte and proteinuria induced by puromycin aminonucleoside(PAN)。Methods:Male, 18 ~ 22g, BALB / c mice were enrolled in the study. the mice were divided randomly into normal control group (NC, n = 6), PAN nephropathy model group (PAN, n = 6), HGF plus PAN group (H + P, n = 6). PAN group: the puromycin nephropathy model of mice was established by single tail vein injection of 144mg/kg body weight puromycin aminonucleoside. H + P group: rapidly tail vein injected the normal saline containing hepatocyte growth factor plasmid (pCMV-HGF) (20ug/1.6ml) to the mice, after 24 hours took 144mg/kg body weight of puromycin aminonucleoside tail vein injection, and the normal control group and PAN group were injected the same amount of normal saline containing 20ug pCDNA3. In modeling before and after 3 days, 24-hour urine samples were collectted after puromycin aminonucleoside injection in metabolic cages, then ,the mice were killed and collecting specimens of renal tissue. Using ELISA method to detect a 24-hour urinary protein excretion, and. light and electron microscopy were used to study the tissues lesion and the ultrastructure changes of podocyte foot process. investigate the expression and distribution of WT1 by Western blot and immunohistochemical staining .meanwhile, the expression changes of the synaptopodin was detected by indirect immunofluorescence and immune histochemical . In vitro study with immortalized mouse podocyte, using RT-PCR and Western blot method to detect expression changes of podocyte WT1 gene and protein . the amount of albumin leakage of Boyden chamber was detected by BCA protein quantitative.Results:(1) Compared with NC group , 24h urinary protein excretion was increasd in PAN group (*P <0.05), HGF may decrease urine protein excretion (*P <0.05).(2) The results of light microscope showed the structures of glomerular were no obvious difference between NC and PAN group.Transmission electron microscopy revealed a large number of podocyte foot process fusion, even shedding in PAN group, yet in the injection of human hepatocyte growth factor (hHGF) plasmid mice, found that the extent and scope podocyte foot process fusion were lower than that in PAN group.(3) The results of western blot and immunohistochemistry staining showed that the expression of WT1 in PAN group was lower than the NC group, yet compared with the PAN group, the expression of WT1 increased in H+P group.(4) In the NC group,the distribution of synaptopodin appeared to be continuous linear , yet staining of the linear were interrupted even effacement in PAN group. The expression and distribution of synaptopodin almost recovered in H+P group.(5) In vitro, HGF may alleviate expression reduction of WT1 mRNA and protein induced by PAN .meanwhile ,the results of Boyden chamber showed that amount of leakage of BSA increased in PAN group,yet HGF reduced the leakage of albumin caused by podocyte injury.Conclusions:Granting exogenous HGF can decrease proteinuria ,reduce the puromycin aminonucleoside-mediated podocyte injury, and improve the foot process fusion. That suggestes that protective effect of HGF to podocyte may be accomplished by upregulating the expression of WT1.