帕立骨化醇抑制血管紧张素活性减轻足细胞损伤

目的：观察维生素D受体激动药帕立骨化醇对尿毒症大鼠肾脏的保护作用。方法：雄性SD大鼠切除右肾并结扎左肾动脉前支诱导尿毒症模型。将20只尿毒症鼠随机分为尿毒症组（UC组）、UC＋帕立骨化醇治疗组（UA组）。另设正常SD大鼠为对照组（NC组）。第10周检测大鼠血清Ca、P、1,25（OH）2D3、成纤维细胞生长因子23（FGF23）、甲状旁腺素（PTH）、血管紧张素Ⅱ（AngⅡ）、血肌酐（Scr）、白蛋白（Alb）、24h尿蛋白（24hUP）。RT—PCR检测肾小球血管紧张素原（Ang）、血管紧张素转化酶（ACE）、nephrin、synaptopodin的表达。胞核WT-1阳性细胞计数测定每个肾小球足细胞数量。结果：UC组P、FGF23、PTH、AngⅡ、Scr、Alb、24hUP较NC组显著升高（P〈0．01）,Ca、1,25（OH）2D3、Alb降低（P〈0．05或0．01）。UC组Ang、ACE mRNA的表达较NC组显著增加（P〈0．01）;nephrin、synaptopodin mRNA的表达较NC组显著降低（P〈0．01）;肾小球足细胞数目减少。帕立骨化醇改善尿毒症鼠的矿物质代谢,下调AngⅡ水平,减少尿蛋白的排泄;抑制肾小球Ang、ACE mRNA的表达,恢复nephrin、synaptopodin的表达,并维持肾小球足细胞数量。结论：帕立骨化醇可抑制尿毒症鼠肾小球血管紧张素活性,减轻足细胞损伤和脱落。     

骨形态发生蛋白-7对足细胞的保护作用

目的探讨血管紧张素Ⅱ(AngⅡ)、转化生长因子-β1(TGF-β1)对体外足细胞(PC)凋亡的影响,及骨形成发生蛋白-7(BMP-7)对AngⅡ或TGF-β1介导PC凋亡及活力是否有保护作用。方法制备PC,进行如下实验:1.PC与10-8mol.L-1、10-7mol.L-1、10-6mol.L-1AngⅡ或TGF-β1培养24 h;2.PC分别与10-7mol.L-1的AngⅡ或TGF-β1培养12 h、24 h、36 h;3.BMP-710-7mol.L-1预处理PC,再与10-7mol.L-1AngⅡ或TGF-β1一起培养24 h。末端脱氧核糖核苷酸转移酶介导的dUTP切口末端标记技术(TUNEL)法检测PC的凋亡,四甲基偶氮唑蓝(MTT)测定PC活力,ELISA测定PC和AngⅡ一起培养时上清液中TGF-β1水平。结果随AngⅡ作用时间延长及浓度增加,PC凋亡愈明显,PC活力降低,上清液中TGF-β1水平亦升高。TGF-β1亦以剂量时间依赖性增加PC凋亡,降低PC活力。BMP-7减少PC凋亡,增加PC活力,并降低PC和AngⅡ一起培养的上清液中TGF-β1水平。结论 BMP-7可下调TGF-β1水平,减轻AngⅡ诱导PC凋亡,增加PC活力,对PC损伤有保护作用。     

高糖环境及雷帕霉素干预对肾小球足细胞Ⅳ型胶原和MMP-9表达的影响

目的探讨高糖培养环境及雷帕霉素干预对肾小球足细胞Ⅳ型胶原和基质金属蛋白酶9（MMP-9）表达的影响。方法体外培养的小鼠肾小球足细胞分为空白对照组、高渗对照组、高糖组和高糖＋雷帕霉素干预组。Real-time PCR和Western blotting检测各组肾小球足细胞Ⅳ型胶原α3链（COL4A3）、α5链（COL4A5）mRNA及MMP-9mRNA和蛋白的表达;ELISA法检测细胞培养上清液中Ⅳ型胶原的含量。结果与对照组比较,高糖组足细胞COL4A3、COL4A5mRNA表达及培养上清液中Ⅳ型胶原含量均升高（P〈0.05）,而足细胞MMP-9mRNA和蛋白表达均降低（P〈0.05）。与高糖组比较,雷帕霉素干预组足细胞COL4A3、COL4A5mRNA表达及培养上清液中Ⅳ型胶原含量均降低（P〈0.05）,足细胞MMP-9mRNA和蛋白表达均升高（P〈0.05）。结论雷帕霉素对高糖环境中足细胞Ⅳ型胶原和MMP-9表达异常具有逆转作用。     

雷公藤免煎剂对IgA肾病患者尿蛋白及尿足细胞的影响

目的:观察雷公藤免煎剂对IgA肾病患者蛋白尿及尿足细胞的影响。方法:64例IgA肾病患者随机分为治疗组和对照组,每组32例。两组患者均给予西医基础治疗,治疗组另给予雷公藤颗粒免煎剂20 g/d冲服,对照组予等量颗粒模拟剂20 g/d冲服。观察并比较两组患者的尿足细胞阳性率、尿足细胞计数、24 h尿蛋白定量及尿nephrin的变化,评价两组降尿蛋白的疗效。结果:降尿蛋白疗效,治疗组总有效率为78.1%,对照组总有效率为43.8%,两组比较差异具有统计学意义(P<0.01)。治疗后8周、16周、24周,治疗组患者的尿足细胞计数和尿蛋白定量较治疗前均显著降低(P<0.05),且治疗组尿足细胞计数和尿蛋白定量水平较对照组同期亦显著下降(P<0.05);治疗后16周、24周,治疗组患者的尿足细胞阳性率较治疗前显著降低(P<0.05),且在治疗后24周,治疗组患者的尿足细胞阳性率亦显著低于对照组患者(P<0.05);而对照组患者在治疗后24周其尿足细胞计数和尿蛋白定量较治疗前才出现显著降低(P<0.05)。治疗后,两组患者的尿nephrin及尿nephrin/尿肌酐值较治疗前均显著降低(P<0.01),且治疗组较对照组降低更加明显(P<0.01)。结论:雷公藤免煎剂能明显减少IgA肾病患者的尿蛋白量,其机制可能与改善足细胞损伤、减少尿足细胞排泄有关。     

针对CTGF的siRNA对高糖诱导小鼠足细胞nephrin、podocin减少的影响

目的 探讨结缔组织生长因子(CTGF)在高糖诱导小鼠足细胞nephrin、podocin表达减少中的作用,及针对CTGF基因的siRNA对其影响.方法 将体外培养的小鼠足细胞分为6组:(1)正常对照组,1640培养基含D-葡萄糖1 g/L;(2)等渗对照组,1640培养基合D-葡萄糖lg/L,甘露醇3.5 g,L;(3)高糖组,1640培养基含D.葡萄糖4.5 g,L;(4)高糖+空白对照组:细胞转染空白质粒后于舍D-葡萄糖4.5 g/L中的1640培养基中培养;(5)高糖+阴性对照组:细胞转染含无关序列的重组质粒后于含D-葡萄糖4.5 g/L中的1640培养基中培养;(6)高糖+干扰组:细胞转染针对CTGF的siRNA表达质粒后于含D-葡萄糖4.5g/L中的1640培养基中培养.应用Reahime PCR检测nephrin、podocin、CTGFmRNA水平,Western blot检测CTGF,nephrin、podocin蛋白表达水平.结果 高糖可诱导CTGF mRNA及蛋白表达增加,下调足细胞的nephrin、podocin mRNA及蛋白水平,而通过特异性siRNA抑制CTGFmRNA表达后,CTGF表达减少,同时伴有细胞nephrin、podocin表达升高.结论 CTGF是高糖诱导小鼠足细胞损伤的重要介质,高糖可通过CTGF诱导nephrin,podocin表达减少.针对CTGF的siRNA能明显改善这种损伤,为DN发病机制的研究提供新的实验依据.     

Lessons from studies on focal segmental glomerulosclerosis: an important role for parietal epithelial cells?

Glomerular diseases are caused by multiple mechanisms. Progressive glomerular injury is characterized by the development of segmental or global glomerulosclerosis independent of the nature of the underlying renal disease. Most studies on glomerular disease focus on the constituents of the filtration barrier (podocytes, glomerular basement membrane (GBM), endothelial cells) or the mesangial cells. Little attention is given to the epithelial cells lining Bowman's capsule, the so called parietal epithelial cells (PECs). This 'lack of attention' is partly explained by the presumed 'passive' function of PECs, which are large, flattened cells that cover Bowman's capsule in a single cell layer and form a barrier between the ultrafiltrate and the periglomerular interstitium, in normal glomerular physiology. A more important reason has been the lack of an established primary role for the parietal epithelium in glomerular diseases. However, in recent years, several studies have demonstrated that PECs are involved in extracapillary proliferation. In addition, PECs can become highly active, proliferating cells, expressing many growth factors, chemokines, cytokines, and their receptors. It was recently demonstrated that PECs also play a part in the development of focal segmental glomerulosclerosis (FSGS). This review summarises current knowledge of the PEC, with emphasis on the role of PECs in the development of FSGS.     

尿足细胞阳性IgA肾病患者临床病理特点

目的探讨尿足细胞阳性原发性IgA肾病(IgAN)患者临床病理特点。方法50例肾活检明确诊断的IgAN患者和10例健康志愿者,利用足细胞的标记蛋白Podocalyxin(PCX)标记尿液和肾组织足细胞,采集IgAN患者肾活检时临床资料、肾活检光镜结果,其中光镜参照Hass分级,各项病理指标参照Fofi半定量积分法进行评分,荧光显微镜观察肾组织足细胞PCX荧光表达程度,电镜观察足细胞足突形态学改变。结果①尿足细胞阳性的IgAN患者尿蛋白水平、血肌酐水平、平均动脉压较尿足细胞阴性患者增高,血浆白蛋白、肾小球滤过率(GFR)较尿足细胞阴性患者降低(P<0.05);②光镜示尿足细胞阳性的IgAN患者肾小球硬化程度、新月体发生率较尿足细胞阴性患者增高(P<0.05),而两组比较,系膜增生、基质增多、肾小管萎缩、间质纤维化、间质炎细胞浸润、间质血管硬化程度的差异无统计学意义(P>0.05);③肾组织足细胞PCX抗体荧光表达显示:肾小球节段硬化和球性硬化处足细胞PCX抗体荧光表达缺失;④电镜观察提示,无论是否伴足细胞尿,其足细胞足突均可出现一系列形态学改变。结论足细胞尿是反映肾脏疾病轻重的一个指标,与肾脏病理类型有一定关系。     

Effect of TRPC6 knockdown on puromycin aminonucleoside-induced podocyte injury

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.     

通络方剂减轻糖尿病大鼠肾小球足细胞损伤

目的：采用糖尿病大鼠模型研究通络方剂对肾小球足细胞的保护作用及其可能的机制。 方法：SD大鼠分为正常对照组、糖尿病对照组、通络方剂组和α硫辛酸组。干预组大鼠分别予以通络方剂（0.4g/kg/d）、α硫辛酸（100mg/kg/d）灌胃,干预12周后处死大鼠,留取血清和尿标本,测定肾重体重比,24h尿蛋白定量、BUN、Cr、Alb/Cr等肾功能指标,留取肾组织标本,体视学方法计数足细胞数量,计算足细胞相对密度,western blot方法检测肾皮质Nephrin的表达,同时测定肾皮质MDA含量和SOD活力。 结果：通络方剂组和α硫辛酸组大鼠24小时尿蛋白定量低于DM对照组,足细胞计数和Nephrin表达量高于DM对照组,差异有统计学意义（p<0.05）;与DM组相比,通络方剂组和α硫辛酸组大鼠肾皮质MDA含量较少,SOD活力较高（p<0.05）。 结论：通络方剂对糖尿病大鼠肾小球足细胞损伤具有保护作用,其机制可能与其抗氧化作用有关。     

Inhibition of integrin-linked kinase by angiotensin II receptor antagonist, irbesartan attenuates podocyte injury in diabetic rats

Background  Integrin-linked kinase (ILK) dysregulation is involved in the progression of diabetic nephropathy (DN). The aim of this study was to investigate the effects of angiotensin II receptor blocker (ARB), irbesartan, on ILK expression and podocyte injury in DN.

Methods  DN was induced by the combined feeding of high-sucrose, high-fat diet and intra-peritoneal injection of low dose of streptozotocin (35 mg/kg) in spontaneously hypertensive rats. Diabetic rats were treated with irbesartan (50 mg×kg⁻¹×d⁻¹) by gavage for 8 weeks. The renal morphologic changes and podocyte injury were investigated by light and electron microscopy, and the ILK expression was evaluated by real-time RT-PCR and Western blotting analysis.

Results  Diabetic rats exhibited with the similar clinical feature of type 2 DN. Morphologically, they were characterized by expansion of mesangial matrix, loss of podocyte and podocyte injury. Impressively, compared to controls, the ILK expression in diabetic rats were upregulated, which were positively correlated with both podocyte injury and albuminuria. Irbesartan significantly prevented ILK overexpression, along with the amelioration of podocyte injury and albuminuria.

Conclusions  ILK plays an important role in mediating podocyte injury in DN; irbesartan inhibits ILK upregulation and attenuates podocyte injury, which might offer a new insight into the role of ARB in preventing DN progression.