| BACKGROUDWhen the’lipid nephrotoxicity hypothesis’was proposed in 1982, it brought together several disparate experimental findings in hyperlipidemia and renal disease to suggest that concomitant hyperlipidemia and proteinuria would cause self-perpetuating renal disease once the initial glomerular insult was no longer present. This process would be analogous to atherosclerosis. It has been accepted by lots of scientists, and many clinical and animal model studies have supported the lipid nephrotoxicity hypothesis. the lipid profile of patients with CKD is typified by high circulating levels of very-low-density lipoprotein triglycerides, intermediate-density lipoprotein cholesterol and chylomicron remnants, and by low plasma levels of high-density lipoprotein cholesterol. Reduced clearance and increased plasma levels of small dense LDL particles facilitates their entrance into arterial walls, where accelerated oxidation of small dense LDL causes renal and vascular damage owing to reduced antioxidant activity of these particles. The LDL level is not usually increased in patients with CKD and might even be reduced. Researches showed that Low density lipoprotein(LDL) is one of the most representative lipoproteins in the process of chronic kidney disease(CKD), which could be converted to modified or oxidized low density lipoprotein(Ox-LDL) under pathological conditions, such as oxidative stress and inflammation. Ox-LDL could deposit in kidney, and attracts inflammatory cells such as macrophage infiltration, which recognized and uptaked this toxic ox-LDL and developed into foam cells. These physical changes made renal cells damage and cellular matrix accumulation, therefore, leading to glumerulosclerosis.Podocyte injury is the central part of glumerulo disease, and has play an important part in the genesis and development of glumerulosclerosis. Previous studies about lipid induced kidney injury were more focus on renal mesangial cells and endothelial cells. However, as the technologies of podocyte culture has been mature and the diagnostic criteria of focal segmental glomerulosclerosis(FSGS) has been developed, reseach on podocyte lipid injury has became a hot area. For example, Joles et al reported that oxLDL could cause damage in renal mesangial cells, endothelial cells and podocytes, and podocytes might be the major victim of ox-LDL insult. Podocytes are a kind of highly differentiated renal cells, whose foot processes wrap the glomerular basement membrane (GBM) to form slit diaphragm(SD) complex, and it has play an important role in the glomerular filtration barrier (GFB). Researchers have found that ox-LDL could induce nephrin---a crucial molecule of SD expression deficiency and resulted in cells damage. However, how ox-LDL induces the damage of podocytes remains to be elucidated.Scavenger receptors (SRs), first described by Brown and Goldstein, were a kind of glycoprotein widely expressed on the cell surface of macrophages, vascular smooth muscle cells and endothelial cells. SRs can bind acetylated LDL (Ac-LDL) and Ox-LDL, getting involved in the process of atherosclerosis, host defense, cell adhesion, cell proliferation and cell apoptosis10. The scavenger receptor family can be classified into nine classes (A-I) according to their structures. Among all these SRs, class A, class B(CD36), class E(LOX-1) and class G(CXCL-16) scavenger receptors were found expressed on lipid cells, thus can uptake ox-LDL and play a role in lipid-induced renal injury. Unlike other lipoprotein receptors, SR-A is not regulated via negative feedback by cytoplasmic cholesterol, and hence plays a key role in the formation of foam cells. However the reason why SR-A is not regulated via negative feedback by cytoplasmic cholesterol is unclear.Phosphatase and tensin homologue deleted on chromosome ten (PTEN) was originally identified as a tumor suppressor gene frequently lost from a region of chromosome 10q23 in a variety of human tumors, including those of the brain, breast, and prostate. PTEN is a dual-specificity protein and lipid phosphatase, and its primary cellular substrate is the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which it hydrolyzes to phosphatidylinositol (4,5)-bisphosphate (PIP2). PTEN blocks phosphatidylinositol 3 kinase (PI3K) signaling by inhibiting PIP3-dependent processes such as the membrane recruitment and activation of AKT, thereby inhibiting cell survival, growth, and proliferation. Research showed that increased stability of PTEN could inhibite the expression of SR-A and shows protection on the development of atherosclerosis. In kidney area, mesangial cell hypertrophy by high glucose is mediated by downregulation of the tumor suppressor PTEN, which indicated that PTEN may play a role in the process of glumerulosclerosis. As glomerulosclerosis and progressive glomerular and tubulointerstitial diseases are part of a spectrum of inter-related clinical disorders that include atherosclerosis, dyslipidemia, and oxidative and inflammatory stress, we guess that PTEN may have involved in lipid induced kidney disease.This study supposed to establish Ox-LDL induced podocyte injury model in vitro, and observe the change of PTEN, SR-A, nephrin expression when podocytes were exposed to Ox-LDL. To further confirm the relationship between PTEN and SR-A, PTEN inhibitor bpv(hOpic) (dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate), PTEN siRNA and PTEN adenovirus(ad PTEN) were used to dual-directional regulate PTEN expression, so as to observe the change of SR-A, nephrin, cell cholesterol accumulation and lipid uptake and explore the possible role of PTEN in the pathogenesis of lipd induced injury.METHODSPart one Culturing, identification and dividing subgroups of podocytes1. Cultivation of mouse podocytesTo induce proliferation, The frozen conditionally immortalized mouse podocyte cell lines(MPC) were thawed rapidly and cultured in RPMI 1640 containing 10% FBS and 20-100U/ml recombinant mouse IFN-y under 5% CO2,33℃ environment. And then, podocytes were transferred to plates coated with type I rat-tail collagen under 5% CO2,37℃ environment with 5% FBS but IFN-y. Cultured for 8-12 days, podocytes were differentiated enough to used in the research.2. Observation and identification of mouse podocytesFirst of all, Podocytes, cultured in 33 ℃ with IFN-y and 37℃ without IFN-y for 8-12 days, were observed and compared the morphological differences under the inverted phase contrast microscope. And then, We stained the podocytes that cultured in 37℃ without IFN-y for 8-12 days with podocyte-specific cytoskeletal protein synaptopodin, which would distrubute along cytoskeleton when podocytes have differentiated mature. In this research, all studies involving podocytes were carried out with the differentiated and matured podocytes.3. Intervention and treatment of differentiated Podocytes as follows(1) The influence of Ox-LDL on lipid accumulation and expression of nephrin, PTEN and SR-A.(2) The influence of PTEN on lipid accumulation and expression of nephrin, and SR-A when exposed to Ox-LDL.(3)The influence of PTEN on the uptake of lipidPart three DetectionsDetection of the content of cholesterol using enzymic method; the mRNA level of PTEN, nephrin, SR-A by RT-PCR; the protein level of PTEN, nephrin, SR-A by Western Blot; uptake of lipid by immunofluorescence.Part four Statistical analysesAll values were expressed as x±SD. Statistical analyses was performed with SPSS 19.0. one-way ANOVA was used for multiple comparisons among groups and for the heterogeneity of variance. LSD-t test was used when the data meet the homogeneity of variance. Otherwise, Dunnett’s T3-test would be used. If there was statistical differences in groups, the comparision between two groups would be uesd. Significance was difined as P<0.05.RESULTSPart one The influence of Ox-LDL on lipid accumulation and expression of nephrin, PTEN and SR-A1. Ox-LDL induces podocyte lipid accumulation.Differentiated podocytes were divided into control group, Ox-LDL group(podocytes were cultured with 10μg/ml,20μg/ml,40μg/ml,80μg/ml Ox-LDL for 24h Or 20μg/ml Ox-LDL for 12h,24h,48h). Cellular cholesterol content was examined by enzymic method. The result shows that Ox-LDL could increase cellular cholesterol content, and its increase extent depends on the dose and exposure time of Ox-LDL, which means as the dose of Ox-LDL increased or the exposure time of Ox-LDL extends, podocyte cellular cholesterol content increased. There was significant between co-BSA group and control group.2. PTEN and SR-A are expressed on mouse podocytes.Kidney tissues were harvesting from normal C57 mice and then develop into frozen slide for immunofluorescence stain. The result found that PTEN and SR-A are bothe expressed on mouse kidney. However, PTEN was mainly localized in mesangium and podocytes, while the expression of SR-A on normal glomerulus are few, also localizing in podocytes and mesangium.3. Ox-LDL inhibit the expression of PTEN and nephrin, and stimulate the expression of SR-A.Differentiated podocytes were divided into groups as above, the mRNA level of PTEN, nephrin, SR-A by RT-PCR; the protein level of PTEN, nephrin, SR-A by Western Blot. Our results shows that PTEN and SR-A protein were expressed on differentiated podocytes, and Ox-LDL exposure could increase the level of SR-A and its increase depends on the dose of Ox-LDL and exposure time of Ox-LDL; On contrast, Ox-LDL leads to decrease the level of nephrin and PTEN. As the dose of Ox-LDL increased or the exposure time of Ox-LDL extends, the expression of nephrin and PTEN decreased. There was significant between co-BSA group and control group.Part two The influence of PTEN on lipid accumulation and expression of nephrin, and SR-A when exposed to Ox-LDL.1. The influence of PTEN on lipid accumulation.Differentiated podocytes were transfected with 100MOI ad PTEN or 50nmol/L PTEN siRNA for 48h or pre-treated with specific PTEN inhibitor bpv(hOpic) for 1 h and treated with 20μg/ml Ox-LDL for 24h, and cellular cholesterol content was examined by enzymic method. Our result find that there was a obvious increase in Ox-LDL group compared with control group. However, compared with Ox-LDL group, si PTEN+Ox-LDL group and bpv(hOpic)+Ox-LDL group shows much more cellular cholesterol content, and there was significant. Consistently, cellular cholesterol content of ad PTEN+Ox-LDL group was lower than Ox-LDL group and there was significant between Ox-LDL and ad PTEN+Ox-LDL group. These results together shows that inhibition of PTEN could increased Ox-LDL induced cellular cholesterol further, while up-regulation of PTEN will inhibit Ox-LDL induced cellular cholesterol.2. The influence of PTEN on the expression of nephrin and SR-A when exposed to Ox-LDL.Differentiated podocytes were transfected with 100MOI ad PTEN or 50nmol/L PTEN siRNA for 48h or pre-treated with specific PTEN inhibitor bpv(hOpic) for 1h and treated with 20μg/ml Ox-LDL for 24h, and the expression of nephrin, PTEN were examined by western blot. Our Results show that modulat PTEN has an effect on the expression of nephrin and SR-A. there was a obvious increase in Ox-LDL group compared with control group. However, compared with Ox-LDL group, si PTEN+Ox-LDL group and bpv(hOpic)+Ox-LDL group shows an increase in the expression of SR-A, while a decrease in the expression of nephrin, and there was significant. Consistently, the expression SR-A in ad PTEN+Ox-LDL group was lower than Ox-LDL group and the expression of nephrin in ad PTEN+Ox-LDL group was higer than Ox-LDL group, and also there was significant between Ox-LDL and ad PTEN+Ox-LDL group. These results together shows that inhibition of PTEN could lead Ox-LDL induced SR-A up-regulation and nephrin decrease futher, while up-regulation of PTEN shows an opposite effect.Part three The influence of PTEN on the uptake of lipidDifferentiated podocytes were transfected with 100MOI ad PTEN or 50nmol/L PTEN siRNA for 48h or pre-treated with specific PTEN inhibitor bpv(hOpic) for 1h and treated with 10mg/L Dil-Ox-LDL for 6h, the uptake of lipid was detected by immunofluorescence. The result shows that podocyte could uptake Dil labeled Ox-LDL. Compared with DiI-Ox-LDL group, siPTEN+DiI-Ox-LDL group and bpv (hOpic)+DiI-Ox-LDL group uptake much more DiI labeled Ox-LDL, while ad PTEN+DiI-Ox-LDL group uptake less DiI labeled Ox-LDL. These means that inhibit PTEN could promote podocyte uptake lipid while up-regulation of PTEN inhibit podocyte uptake lipid.CONCLUSIONMouse renal podocyte could express PTEN and SR-A protein. Ox-LDL up-regulates SR-A through decreasing the expression of PTEN, and contributes to podocyte injury. |
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