Protective effect ofACTH4-10 on adriamycin-induced podocyte injury

Objective To observe the influence of adrenocorticotropic hormone (ACTH4-10) in the changes of podocyte proliferation, apoptosis and expression of nephrin and podocin on adriamycin(ADR) -induced podocyte injury and investigate the protective effect ofACTH4-10. Methods All podocytes were randomly divided into following groups: normal control, ADR-induced group andACTH4-10 intervention group (low, middle and high concentration). Normal control group was not treated, ADR-induced group was induced to set the model of podocyte injury by ADR (1 μmol/L) for 24 hours andACTH4-10 intervention groups were intervened by 1 μg/L, 10 μg/L and 100 μg/LACTH4-10 for 1 hours respectively, prior to setting the model of podocyte injury. Cell counting kit (CCK-8) was used to detect the multiplication of podocytes and TUNEL apoptosis detection kit was used to detect podocyte apoptosis. Real-time PCR and Western blotting were used to examine the expression of nephrin and podocin. Results Compared with control group, podocyte proliferation and expression of nephrin and podocin was decreased significantly in ADR-induced group (P＜0.05), meanwhile podocyte apoptosis was increased obviously (38.14% vs 5.12%). Compared with ADR-induced group, podocyte proliferation and expression of nephrin and podocin was increased generally with concentration ofACTH4-10. Although podocyte apoptosis rates (20.45%, 17.39%, 11.02%) were increased inACTH4-10 intervention group (low, middle and high concentration) while comparing with normal control group, podocyte apoptosis decreased obviously while comparing with ADR-induced group. ConclusionsACTH4-10 can stabilize the expression of nephrin and podocin on slid diaphragm, and has the protective effect on podocyte injury induced by ADR, while the effect depends on the concentration ofACTH4-10.     

Fluvastatin ameliorates podocyte injury in proteinuric rats via modulation of excessive Rho signaling

Statins have been reported to confer renoprotection in several experimental models of renal disease through pleiotropic actions. The roles of statins in glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of fluvastatin on podocyte and tubulointerstitial injury in puromycin aminonucleoside (PAN)-induced nephrosis. PAN induced massive proteinuria and serum creatinine elevation on day 7, which were significantly suppressed by fluvastatin. Immunofluorescence studies of podocyte-associated proteins nephrin and podocin revealed diminished and discontinuous staining patterns in rats with PAN nephrosis, indicating severe podocyte injury. Fluvastatin treatment dramatically mitigated the abnormal staining profiles. Reduction of nephrin expression by PAN and its reversal by fluvastatin were confirmed by quantitative analyses. By electron microscopy, effacement of foot processes was ameliorated in fluvastatin-treated rats. Fluvastatin also mitigated tubulointerstitial damage in PAN nephrosis, with the repression of PAN-induced NF-kappaB and activator protein-1 activation in the kidneys. In addition, expression of activated membrane-bound small GTPase RhoA was markedly increased in the glomeruli of PAN nephrosis, which was inhibited by fluvastatin treatment. In cultured podocytes, fluvastatin suppressed PAN-evoked activation of RhoA and actin cytoskeletal reorganization. Furthermore, fasudil, a specific Rho-kinase inhibitor, successfully ameliorated PAN-induced podocyte damage and proteinuria. In summary, fluvastatin alleviated podocyte and tubulointerstitial injury in PAN nephrosis. The beneficial effects of fluvastatin on podocytes can be attributable to direct modulation of excessive RhoA activity. Our data suggest a therapeutic role for statins in clinical conditions that are relevant to podocyte injury.     

The relationship among nephrin, podocin, CD2AP, and alpha-actinin might not be a true 'interaction' in podocyte

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1, NPHS2, CD2AP, and ACTN4, can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or alpha-actinin-4 mRNA by using reconstructed RNA interference vector--psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of alpha-actinin-4, whereas alpha-actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.     

Cloning of rat homologue of podocin: expression in proteinuric states and in developing glomeruli

Podocin is identified as a product of the gene mutated in a patient with autosomal recessive steroid-resistant nephrotic syndrome. Although podocin is reported to be located at the slit diaphragm area, the precise role of podocin for maintaining the barrier function of the slit diaphragm has not been clearly elucidated. A rat homologue of podocin was cloned, and the expression of podocin was investigated and then compared with the nephrin and the ZO-1 expressions in rat experimental proteinuric models and in developing glomeruli. Amino acid sequences of rat and human podocin are highly homologous (84.3% identity). The domain structure of podocin is also highly conserved between rat and human. The mRNA expression for podocin was detected in glomeruli and the nerve tissues. The localization of podocin has close proximity to that of nephrin in normal adult rat glomeruli. Podocin staining was restricted to the basal side of the podocyte of the early developing stage, whereas nephrin staining was detected on the basolateral surface of podocyte. The redistribution of podocin was observed in the anti-nephrin antibody (ANA)-induced nephropathy and puromycin aminonucleoside (PAN) nephropathy. The redistribution of podocin paralleled with nephrin in ANA nephropathy but not in PAN nephropathy. Podocin is observed at the site of tight junction newly formed in proteinuric state in PAN nephropathy. It is postulated that podocin is one of the critical components of a slit diaphragm for maintaining the barrier function of the glomerular capillary wall.     

雷公藤内酯醇干预高糖影响小鼠肾脏足细胞Nephrin蛋白表达的研究

目的：通过观察雷公藤内酯醇（triptolide,TP）及对照药物缬沙坦（valsartan,Val）干预高糖环境培养的足细胞肾小球足细胞裂孔隔膜（GPSD）核心蛋白nephrin的表达,来深入探讨雷公藤治疗糖尿病肾病（DN）蛋白尿的分子生物学机制。方法：（1）将培养成熟的足细胞分为对照组1、对照组2、模型组、雷公藤内酯醇干预组及缬沙坦干预组（干预组均设低、中、高3个剂量）,培养24h后以RT-PCR法检测各组足细胞nephrin mRNA的表达。（2）将培养成熟的足细胞设为对照组、模型组、雷公藤内酯醇干预组及缬沙坦干预组,培养24h后行间接免疫荧光法检测nephrin蛋白的表达。结果：（1）与模型组相比,不同剂量TP和Val组均能提高nephrin mRNA表达（P〈0.05）,并且中剂量TP组和高剂量Val组效果最好（P〈0.05）。（2）与对照组相比,间接免疫荧光法可见模型组nephrin表达下降。经TP和Val干预24h后,TP和Val组足细胞nephrin蛋白的表达均有一定程度提高。结论：雷公藤内酯醇和缬沙坦均能上调高糖诱导的足细胞nephrin的表达下降,这可能是雷公藤及缬沙坦在临床上有效治疗DN蛋白尿的机制之一。     

三种不同药物作用下足细胞分子的变化

目的 从足细胞分子的角度探讨抗蛋白尿药物作用的细胞分子机制。方法 建立阿霉素肾病大鼠模型。注射阿霉素后次日分别给予利生普利、泼尼松以及全反式维甲酸(ATRA)干预蛋白尿。注射阿霉素后第3、7、14、28 天每组处死6 只大鼠，留取肾脏标本。应用间接免疫荧光染色、实时PCR、Western印迹分别检测各个时间点nephrin、podocin、CD2相关蛋白(AP)、α辅肌动蛋白(actinin)-4 的分布、mRNA 和蛋白表达量的变化。应用免疫沉淀检测nephrin 与podocin、nephrin与CD2AP 分子间作用以及nephrin 磷酸化水平。结果 与对照组相比，第14天时肾病组尿蛋白显著增加(P < 0.01)。与肾病组相比，利生普利、泼尼松和ATRA干预后均显著降低了蛋白尿( P < 0.05)， 减轻了足突融合。通过分析不同时间点足细胞分子的表达，显示3种干预药物均引起了nephrin、podocin、CD2AP 表达的变化，维持了正常的nephrin 磷酸化水平，而且利生普利和泼尼松首先抑制了podocin 分子，而ATRA首先抑制了CD2AP 分子的异常变化。与此同时，nephrin、podocin、CD2AP 和α-actinin-4 分子的分布在干预后也趋于正常。此外，无论在肾病组还是干预组大鼠，nephrin 与podocin、nephrin 与CD2AP 分子间一直保持着共沉淀关系。 结论 利生普利、泼尼松和ATRA 都通过稳定重要的足细胞分子nephrin、podocin、CD2AP 来发挥它们的抗蛋白尿作用。     

1,25(OH)2D3 ameliorates high glucose-induced podocyte injury via PI3K/p-Akt signalling pathway

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced podocyte injury and its signal transduction mechanism. Methods Differentiated mouse podocytes were exposed to normal glucose, high glucose, and different concentrations of 1,25(OH)2D3 or LY294002 (a selective PI3K inhibitor) for 24 h. PCR and immunofluorescent staining were used to detect nephrin, podocin, and desmin. Western blotting was used to detect protein expression of nephrin, podocin, desmin, PI3K, Akt and p-Akt. Results Compared with high glucose group, 1,25(OH)2D3 (100 nmol/L and 1000 nmol/L) significantly up-regulated the expression of podocin and nephrin in podocytes induced by high glucose (P＜0.05). Meanwhile, 1,25(OH)2D3 (100 nmol/L) significantly reduced the expression of desmin (P＜0.05). PI3K and p-Akt were obviously reduced in high glucose group. In the presence of 1,25(OH)2D3, the trends were reversed. However the above effects of 1,25(OH)2D3 were abolished when p-Akt was blocked by the PI3K inhibitor LY294002. Conclusions 1,25 (OH)2D3 can inhibit high glucose-induced podocyte injury through PI3K/p-Akt signaling pathway.     

Attenuation of high glucose induced podocyte injury by ursolic acid up-regulating autophagy level

Objective To investigate the effects of ursolic acid (UA) on autophagy and podocyte injury induced by high glucose. Methods Conditionally immortalized murine podocyte were cultured in high glucose, the effect of PI3K inhibitor LY294002 and ursolic acid treatment were observed. The miR-21 expression was detected using RT-qPCR. The activation of PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related protein and podocyte marker protein were determined by Western blot. Immunofluorescence staining showed the expression of podocyte marker protein and endogenous accumulation of LC3. Autophagosomes were observed using electron microscopy. Results Compared with normal control group,the cells exposed to high glucose condition showed down-regulated synaptopodin, podocin and nephrin expression (P＜0.01), up-regulated miR-21 expression (P＜0.01), down-regulated PTEN expression (P＜0.01), up-regulated p85-P13K, phospho(p)-Akt, p-mTOR,p62/SQSTMI, expression and down-regulated LC3II and Beclin1 expression (all P＜0.01). Ursolic acid and LY294002 promoted synaptopodin, podocin and nephrin expression (all P＜0.01), up-regulated LC3II, Beclin1 expression and down-regulated p62/SQSTM1 expression (all P＜0.01), down-regulated p85-PI3K, p-Akt, p-mTOR expression (all P＜0.01). However, LY294002 did not affect the expression of miR-21 and PTEN. Ursolic acid inhibited miR-21 expression and upregulated PTEN level. Conclusions The podocyte injury is associated with defective autophagy level under high glucose condition. Ursolic acid could reduce podocyte injury by increasing autophagy level via inhibition of miR-21 expression and PTEN/Akt/mTOR pathway.     

尿毒清颗粒对糖尿病大鼠足细胞损伤的保护作用研究

目的：探讨尿毒清颗粒对糖尿病大鼠足细胞损伤的保护作用。方法：将SD大鼠制备成STZ糖尿病模型,实验分3组：正常对照组、糖尿病未干预组、尿毒清颗粒治疗组（2.6g·kg-1·d-1灌胃）,于实验第4周、8周末检测血糖、血肌酐、尿肌酐及尿白蛋白排泄率,光镜、电镜下观察肾组织病理改变并计数足突宽度,免疫组化观察足细胞相关蛋白分子nepherin、podocin的表达,Real time-PCR检测肾皮质nepherin、podocin mRNA表达。结果：尿毒清颗粒可以改善糖尿病大鼠肌酐清除率,降低尿白蛋白排泄,改善肾脏病理,减轻足突融合,维持足细胞相关蛋白分子的分布与表达。结论：初步证实尿毒清颗粒能通过上调足细胞相关蛋白分子水平减轻足细胞损伤,对糖尿病大鼠足细胞损伤具有保护作用。     

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.     

The effect of acteoside on puromycin nephropathy and podocyte injury model

Objective To explore the effect of acteoside (one of the ingredients of Total Glycosides Extracted from Rehmannia capsules) on treatment of proteinuria and protection of podocytes. Methods In this study, puromycin nephropathy rat model was successfully established. After detecting the degree of proteinuria, the expression of podocyte injury markers and the degree of podocyte foot process fusion were investigated by electron microscope. In addition, puromycin treated podocyte injury model was also successfully established in vitro. Podocyte viability, migration, cytoskeleton and injury marker were detected. Results In vivo study showed that acteoside could effectively reduce proteinuria (P＜0.05), restore the expression of podocyte injury markers such as nephrin and synaptopodin (all P＜0.05), and alleviate the degree of podocyte foot process fusion. In vitro study showed that acteoside could effectively restored podocyte viability (P＜0.05), reduce abnormal migration ability (P＜0.05), protecte cytoskeleton and restore the expression of podocyte injury marker nephrin (P＜0.05). Conclusions This study confirms that acteoside can reduce the degree of proteinuria in puromycin nephropathy rat model in vivo and alleviate the degree of podocyte injury in vitro as well as enrich the molecular mechanism of Total Glycosides Extracted from Rehmannia capsules in treatment of proteinuria.     

ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo

BACKGROUND: Podocytes are terminally differentiated and highly specialized epithelial cells. The factors governing podocyte differentiation are poorly understood. We tested the hypothesis that all-trans retinoic acid (ATRA), a vitamin A derivative, induces podocyte differentiation in vitro and in vivo. METHODS: We tested the effects of ATRA on podocytes. Primary rat, primary mouse, and immortalized mouse podocytes were exposed to ATRA (1, 5, 10, 20, 40, 50, 80, 160, and 200 micromol/L) or control (ethanol) for 72 hours. Cell morphology was examined by electron microscopy, the expression of podocyte specific proteins was measured by immunoflourescence and Western blot analysis, cell number and apoptosis were measured by 3-[4,5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst staining, respectively. To determine if ATRA alters podocyte differentiation in vivo, experimental injury was induced in C57BL6 mice using the antiglomerular antibody. Animals were given either daily intraperitoneal ATRA (16 mg/kg) or vehicle (corn oil). For end points, we measured proteinuria, podocyte-specific protein immunostaining, and proliferation [proliferating cell nuclear antigen (PCNA)] at days 5 and 14 (N= 5/group/time point). RESULTS: ATRA induced podocyte process formation in vitro, and significantly increased the expression of nephrin and podocin. This coincided with a reduction in proliferation. ATRA also significantly prevented the decrease in staining for synaptopodin, nephrin, and podocin in experimental animals (P < 0.05 vs. control). This was accompanied by reduced proteinuria and decreased podocyte proliferation (P < 0.05 vs. control). CONCLUSION: ATRA induces podocyte differentiation in vitro and in vivo and alters the expression of certain podocyte-specific proteins. Further studies are ongoing to delineate the mechanism of this effect.     

Overexpression of interleukin-13 induces minimal-change-like nephropathy in rats

IL-13 has been implicated in the pathogenesis of minimal-change nephrotic syndrome. This study aimed to investigate the role of IL-13 on the development of proteinuria and expression of podocyte-related genes that are associated with nephrotic syndrome. IL-13 was overexpressed in Wistar rats through transfection of a mammalian expression vector cloned with the rat IL-13 gene, into the quadriceps by in vivo electroporation. Serum IL-13, albumin, cholesterol, and creatinine and urine albumin were measured serially. Kidneys were harvested after day 70 for histology and electron microscopy. Glomerular gene expression of nephrin, podocin, dystroglycan, B7-1, and IL-13 receptor subunits were examined using real-time PCR with hybridization probes and expressed as an index against beta-actin. Protein expression of these molecules was determined by immunofluorescence staining. The IL-13-transfected rats (n = 41) showed significant albuminuria, hypoalbuminemia, and hypercholesterolemia when compared with control rats (n = 17). No significant histologic changes were seen in glomeruli of IL-13-transfected rats. However, electron microscopy showed up to 80% of podocyte foot process fusion. Glomerular gene expression was significantly upregulated for B7-1, IL-4Ralpha, and IL-13Ralpha2 but downregulated for nephrin, podocin, and dystroglycan. Immunofluorescence staining intensity was reduced for nephrin, podocin, and dystroglycan but increased for B7-1 and IL-4Ralpha in IL-13-transfected rats compared with controls. In conclusion, these results suggest that IL-13 overexpression in the rat could lead to podocyte injury with downregulation of nephrin, podocin, and dystroglycan and a concurrent upregulation of B7-1 in the glomeruli, inducing a minimal change-like nephropathy that is characterized by increased proteinuria, hypoalbuminemia, hypercholesterolemia, and fusion of podocyte foot processes.     

方格星虫提取物对膜性肾病模型的治疗作用及机制初探

目的:初步探讨方格星虫粗提物(SNP)对膜性肾病(MN)的治疗作用及其可能机制。方法:50只SD大鼠随机分为模型组和对照组,模型组大鼠腹腔一次注入抗FxlA血清建立被动性Heymann肾炎模型,模型建立1周后依24 h尿蛋白排泄量(24 h UPE)随机将Heymann肾炎大鼠分为SNP治疗组和未治疗组,治疗组用SNP灌胃治疗。考马斯亮蓝法每周测定24 h UPE。治疗4周结束后处死大鼠取双肾,光镜、电镜观察肾脏病理改变,RT-PCR法检测肾组织nephrin mRNA和podocin mRNA的表达量。结果:SNP治疗4周后大鼠24 h UPE较未治疗组降低(P<0.05);模型组大鼠肾小球基底膜增厚,上皮下电子致密物沉积,足细胞足突融合等病理改变,SNP治疗组大鼠上述病变较未治疗组减轻,经测定,足细胞足突融合率较未治疗组显著降低(P<0.01);SNP治疗组nephrin mRNA和podocin mRNA表达量较未治疗组升高(P<0.05)。结论:SNP可通过上调膜性肾病大鼠足细胞nephrin和podocin基因表达水平,一定程度上减轻足细胞足突融合,进而减少尿蛋白滤过,可能对人类膜性肾病具有辅助治疗作用。     

中药合剂对早期糖尿病肾病大鼠足细胞nephrin和podocin表达的影响

也页目的：观察中药合剂（ TCM mixture）对糖尿病肾病（ DKD）大鼠足细胞裂孔隔膜蛋白分子（ nephrin和podocin）表达的影响,探讨其对DKD的防治作用。方法：高脂高糖喂养8周联合小剂量STZ（30 mg/kg）建立T2DM大鼠模型,将DM模型大鼠随机分为中药合剂治疗组（B）及T2DM模型对照组（A）;另设正常对照组（NC）。各组于治疗前及干预后4、8、12周末检测大鼠血糖浓度及尿微量白蛋白;第12周末称大鼠体重（ BW）、肾重（ KW）,计算肾肥大指数（ KW/BW）,观察血清尿素氮（ BUN）、肌酐（ Scr）、总胆固醇（ TC）、三酰甘油（ TG）变化;光镜观察肾脏病理改变;应用免疫组化技术观察足细胞相关蛋白Nephrin、Podocin肾组织分布与表达;采用Western blot技术测定肾皮质Nephrin、Podocin 蛋白的表达。结果：应用中药合剂干预后与T2DM模型对照组相比,大鼠的尿微量白蛋白总量、肾肥大指数、血糖、尿素氮、血肌酐较T2DM模型组明显降低（ P〈0.05）,体重增加（P〈0.05）,病理改变减轻,nephrin和podocin蛋白表达增加（P〈0.05）。结论：中药合剂能提高nephrin和podocin的表达,促进足细胞损伤修复,减少尿微量白蛋白的排泄,减缓糖尿病肾病的进展。     

Nephrotic plasma alters slit diaphragm-dependent signaling and translocates nephrin, Podocin, and CD2 associated protein in cultured human podocytes

Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm (SD) proteins nephrin, podocin, and CD2-associated protein (CD2AP) to function optimally. The effects of normal human plasma and nephrotic plasma on podocytes were tested, focusing particularly on the SD complex. With the use of a conditionally immortalized human podocyte cell line, it first was shown that exposure to normal and non-nephrotic human plasma leads to a concentration of nephrin, podocin, CD2AP, and actin at the cell surface. Next, the effects of plasma from patients with nephrotic conditions to non-nephrotic conditions were compared. When exposed to all nephrotic plasma samples (and a non-human serum control), nephrin podocin and CD2AP assumed a cytoplasmic distribution; nephrin and synaptopodin were selectively downregulated, and the relocation of nephrin induced by nephrotic plasma could be rescued back to the plasma membrane by co-incubation with non-nephrotic plasma. Furthermore, intracellular calcium signaling was altered by nephrotic plasma, which was mediated by tyrosine kinase phosphorylation. With the use of nephrin mutant human cell lines, it was shown that this signaling and translocation response to normal plasma is nephrin dependent. This work demonstrates that nephrotic plasma seems to be deficient in factors that act via the podocyte SD complex, which are essential in maintaining its physiologic function.     

Reduced podocin expression in minimal change disease and focal segmental glomerulosclerosis is related to the level of proteinuria

Background

Glomerular podocyte molecules are involved in the pathogenesis of congenital nephrotic syndrome. However, their role in primary nephrotic syndrome is not clear. This study investigated the expression of nephrin, podocin and synaptopodin in primary nephrotic syndrome.

Methods

Eighty-seven patients with primary nephrotic syndrome including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and membranoproliferative glomerulonephritis Type I (MPGN) were included in the study. Glomerular expression of nephrin, podocin and synaptopodin was studied in renal biopsies by immunofluorescence and immunohistochemistry. Correlation of expression with clinical and biochemical parameters was performed.

Results

The pattern of expression for all podocyte proteins in controls was uniform fine granular along the capillary walls towards the visceral epithelial cell aspect. Glomerular expression of nephrin was present in all renal biopsies and was similar to that in controls. Glomerular synaptopodin expression was seen in all MN and MPGN patients, while it was seen in 74 % (17/23) MCD and 93.5 % (29/31) FSGS. Reduced synaptopodin expression showed no correlation with clinical and biochemical factors. Podocin expression was present in 5/23 MCD (22 %), 3/31 FSGS (9.6 %), 13/17 MN (76.4 %) and 13/16 MPGN (81 %) patients. The reduced expression of podocin significantly correlated with the degree of proteinuria (p = 0.032). No correlation with age, gender and serum creatinine level was observed.

Conclusion

Reduction of glomerular podocin expression found in MCD and FSGS is related to the amount of proteinuria. Our findings suggest that alteration in podocyte phenotype may not be a primary event and may reflect the degree of podocyte injury in primary nephrotic syndrome.     

EP1 receptor mediates Adriamycin-induced podocyte injury through activating p38 MAPK

Objective To investigate the effect of prostaglandin E2 receptor 1 (EP1) on Adriamycin (ADR)-induced glomerular podocytes injury and its possible mechanism. Methods (1) In vivo experiments: 6-8 weeks old male Balb/c mice were randomly divided into four groups: Control group; ADR group; EP1 agonist 17-phenyl PGE2+ADR group; EP1 antagonist SC-19220+ADR group. The mouse model of nephrotic syndrome was induced by injection of ADR (10 mg/kg) into tail vein, and then EP1 agonist (1 μg/g) and antagonist (25 μg/g) were administered respectively. Six weeks later, all mice were sacrificed and urine, blood and kidney tissues were collected. Detecting urine protein, blood chemistry, changes of renal pathology and podocyte-related proteins, electron microscopy changes of podocytes. (2) In vitro experiments: Podocytes were cultured in vitro and divided into different groups: Control group; ADR group (0.2 μmol/L); EP1 agonist (0.1, 1, 10 μmol/L)+ADR (0.2 μmol/L) group; antagonist (0.1, 0.5, 1 μmol/L)+ADR (0.2 μmol/L) group. The proliferation of podocytes was measured by CCK-8. Expression of PGE2 in podocytes was detected by ELISA. Indirect immunofluorescence was used to determine the localization of podocyte-related proteins nephrin, podocin and CD2AP. Expression of nephrin, podocin, CD2AP, COX2 in podocytes was detected by Western blotting and Real-time quantitative PCR. p38 MAPK or phospho-p38 MAPK was measured by Western blotting as well. Flow cytometry was used to detect cell apoptosis. Results (1) In vivo experiments: Compared with control group, obvious proteinuria, blood biochemical changes and renal pathological changes were observed in ADR group, proteinuria, blood biochemical and renal pathological changes were more serious in mice dealt with agonist, while antagonist could reduce ADR-induced injury (all P＜0.05). Results of immunohistochemistry showed that the expression of podocyte-related proteins nephrin, podocin and CD2AP in ADR group were significantly lower than those in control group, and EP1 agonist could further inhibit expression of these proteins, while antagonist could reverse this inhibitory effects (P＜0.05). Electron microscopic results showed that mice in ADR group appeared foot enlargement and fusion, and the agonist group further aggravated the injury, while antagonist intervention could inhibit the injury of podocytes. (2) In vitro experiments: Compared with control group, expression of PGE2 and COX2 were increased; mRNA and protein expression of nephrin, podocin, CD2AP were decreased, p38 MAPK activity and podocytes apoptosis were increased in ADR group (P＜0.05); Agonist could aggravate podocytes damage (P＜0.05), while Antagonist could down-regulate the expression of PGE2 and COX2, promote the expression of nephrin, podocin and CD2AP, and inhibit the activity of p38 MAPK and podocytes apoptosis (P＜0.05). The addition of p38 MAPK inhibitor(10 μmol/L) could reduce the inhibitory effect of EP1 agonist on the expression of podocyte-related proteins nephrin, podocin and CD2AP (P＜0.05). Conclusions EP1 receptor may activate the p38 MAPK signaling pathway to inhibit podocytes-related proteins nephrin, podocin and CD2AP, as well as mediate the ADR induced podocyte injury. Inhibition of EP1 receptor however have a protective effect.     

红景天对AOPPs诱导小鼠足细胞转分化的影响

目的:探讨红景天(salidroside)对晚期氧化蛋白产物(AOPPs)诱导小鼠足细胞转分化的影响。方法:以小鼠永生性足细胞系为研究对象。给予200μg/ml的AOPP刺激小鼠足细胞24h,同时加入红景天(浓度分别为0.1μmol、1μmol、10μmol、100μmol、200μmol)进行干预,采用Westernblot检测内质网应激标志性蛋白Grp78、CHOP以及平滑肌激动蛋白(α-SMA)、nephrin、podocin的表达水平。结果:200μg/mlAOPP条件下小鼠足细胞表达高水平的Grp78、CHOP及α-SMA,表达低水平的nephrin和podocin;予不同浓度红景天作用后,足细胞中Grp78、CHOP及α-SMA的表达水平降低,neph-rin和podocin的表达水平升高。结果表明红景天可部分逆转AOPPs的作用,且存在一定的剂量依赖性。结论:红景天对AOPPs诱导的足细胞Grp78、CHOP过度表达有很好的抑制作用,这可能是它减轻肾脏纤维化的机制之一。