Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1

Aims/hypothesis  Peroxisome proliferator-activated receptor (PPAR) γ agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARγ agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARγ agonist rosiglitazone on human podocytes. Methods  Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. Results  Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. Conclusions/interpretation  In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARγ agonists may improve podocyte function in diabetic nephropathy.     

Role of MKK3–p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice

Aims/hypothesis  Obesity and diabetes are associated with increased intracellular p38 mitogen-activated protein kinase (MAPK) signalling, which may promote tissue inflammation and injury. Activation of p38 MAPK can be induced by either of the immediate upstream kinases, MAP kinase kinase (MKK)3 or MKK6, and recent evidence suggests that MKK3 has non-redundant roles in the pathology attributed to p38 MAPK activation. Therefore, this study examined whether MKK3 signalling influences the development of obesity, type 2 diabetes and diabetic nephropathy. Methods  Wild-type and Mkk3 (also known as Map2k3) gene-deficient db/db mice were assessed for the development of obesity, type 2 diabetes and renal injury from 8 to 32 weeks of age. Results   Mkk3 ^+/+ db/db and Mkk3 ^−/− db/db mice developed comparable obesity and were similar in terms of incidence and severity of type 2 diabetes. At 32 weeks, diabetic Mkk3 ^+/+ db/db mice had increased kidney levels of phospho-p38 and MKK3 protein. In comparison, kidney levels of phospho-p38 in diabetic Mkk3 ^−/− db/db mice remained normal, despite a fourfold compensatory increase in MKK6 protein levels. The reduced levels of p38 MAPK signalling in the diabetic kidneys of Mkk3 ^−/− db/db mice was associated with protection against the following: declining renal function, increasing albuminuria, renal hypertrophy, podocyte loss, mesangial cell activation and glomerular fibrosis. Diabetic Mkk3 ^−/− db/db mice were also significantly protected from tubular injury and interstitial fibrosis, which was associated with reduced Ccl2 mRNA expression and interstitial macrophage accumulation. Conclusions/interpretation  MKK3–p38 MAPK signalling is not required for the development of obesity or type 2 diabetes, but plays a distinct pathogenic role in the progression of diabetic nephropathy in db/db mice.     

环氧合酶-2基因敲低对足细胞相关蛋白表达的影响

目的:观察足细胞环氧合酶-2(COX-2)基因敲低对足细胞相关蛋白表达的影响。方法:以条件永生性小鼠足细胞株为研究对象,分为足细胞正常对照组、足细胞COX-2基因敲低组和足细胞阴性转染组,用流式细胞仪检测足细胞凋亡水平,用Western bolt检测COX-2、Nephrin、Podocin、CD2相关蛋白(CD2AP)的表达水平。结果:与足细胞正常对照组及阴性转染组相比,足细胞COX-2基因敲低组COX-2蛋白表达明显降低、细胞凋亡率明显增高,而Nephrin、Podocin、CD2AP蛋白表达则显著降低。结论:足细胞COX-2基因敲低可以导致足细胞凋亡,且足细胞Nephrin、Podocin、CD2AP蛋白的表达下调。     

足细胞损伤与膜性肾病的研究进展

膜性肾病是常见的原发性肾小球疾病,是以肾小球脏层上皮细胞下免疫复合物弥漫性沉积、基底膜增厚伴钉突形成为其病理学特征。其发病机制复杂,近年来,随着对足细胞的一些标志分子的深入研究,证实足细胞的异常与蛋白尿的产生密切相关。在此对足细胞的异常与膜性肾病的发生发展等相关进展作简单综述。     

高血压大鼠足细胞的超微病变和podocalyxin的表达及其作用研究

目的观察高血压大鼠足细胞的超微结构病变,探讨足细胞蛋白podocalyxin(PCX)在高血压大鼠肾组织中的表达及作用。方法随机将30只雄性SD大鼠以改进的"两肾一夹"方法建立高血压大鼠模型,分为高血压组和对照组。分别于造模前和造模后1、5、10周检测两组大鼠的尿β2-微球蛋白(β2-MG)、血尿素氮(BUN)及血肌酐(Scr)水平;免疫荧光方法观察PCX在肾组织的表达,并用Image-pro plus 6.0软件,以面积密度值进行分析;光镜、电镜观察肾小球及足细胞超微结构改变。结果 (1)术后2周起高血压组收缩压(SBP)较对照组显著升高(137.2±9.4mmHg vs 92.3±10.3mmHg,P<0.01),并且逐渐升高至术后4周趋于平稳。(2)术后5周起高血压组尿β2-MG显著高于对照组[(110.28±11.25)ng/L vs(56.68±9.51)ng/L,P<0.01],并且有继续增高的趋势。两组BUN、Scr在整个实验过程中没有显著性差异。(3)光镜、电镜观察均可见高血压组肾脏发生病理改变;足细胞超微结构发生病变。(4)免疫荧光可见高血压组PCX表达明显减少,其面积密度值显著低于对照组(0.204±0.042 vs 0.296±0.039,P<0.01),且PCX表达与尿β2-MG呈显著负相关(r=-0.927,P<0.01)。结论 PCX蛋白表达降低可能是参与高血压肾损害的机制之一,其可能是导致肾小球电荷屏障受损、尿蛋白排泄增加、肾功能受损、肾脏病理及足细胞超微结构发生病变的基础之一。     

Urinary podocyte-associated mRNA profile in Egyptian patients with diabetic nephropathy

IntroductionPodocyte injury and subsequent excretion in urine play a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA expression in urinary sediment by real-time PCR is emerging as a noninvasive method of screening DN-associated biomarkers. We aimed to study the expression of podocyte-associated genes in urinary sediment and their relation to disease severity in type 2 diabetic Egyptian patients with diabetic nephropathy.Methodology: Sixty patients with type 2 diabetes mellitus were recruited in addition to twenty non diabetic healthy volunteers. Relative mRNA abundance of nephrin, podocalyxin, and podocin were quantified, and correlations between target mRNAs and clinical parameters were examined.ResultsThe urinary mRNA levels of all genes studied were significantly higher in diabetics compared with controls (p < 0.001), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both UAE and HbA_1c. The expression of nephrin, podocalyxin, and podocin mRNA correlated with serum creatinine {(r = 0.397, p value = 0.002), (r = 0.431, p value = 0.001), (r = 0.433, p value = 0.001) respectively}.ConclusionThe urinary mRNA profiles of nephrin, podocalyxin, and podocin were found to increase with the progression of DN, which suggested that quantification of podocyte-associated molecules will be useful biomarkers of DN.     

a-硫辛酸治疗43例Ⅲ期糖尿病肾病患者疗效分析

目的 观察α-硫辛酸治疗糖尿病肾病的疗效。方法78例2型糖尿病肾病Ⅲ期患者,随机分为硫辛酸组43例和对照组35例,硫辛酸组予以a-硫辛酸注射液600mg静脉滴注,一日一次,半月后改为a-硫辛酸胶囊300mg,2次／d及氯沙坦100mg／d,对照组给予氯沙坦100mg／d,总疗程12周。治疗前后分别进行一般项目监测和24h尿微量白蛋白（24hA,b）、层黏连蛋白（LN）、IV型胶原（C-Ⅳ）、超敏C反应蛋白（hsCRP）的定量测定及尿中足细胞计数。结果治疗前2组患者的一般项目和监测指标相比均无统计学意义;硫辛酸组和对照组HbA。24hA,b、LN、C—Ⅳ、hsCRP、足细胞计数治疗后较治疗前均有显著下降,差异有统计学意义（均P〈0．05）,高密度脂蛋白胆固醇（HDL—C）治疗后较治疗前有显著上升,差异有统计学意义（均P〈0．05）;硫辛酸组治疗后较对照组治疗后24hA,b有明显下降（P〈0．05）;硫辛酸组HbA小24hA。b、C—IV、足细胞计数下降幅度较对照组有显著降低,差异有统计学意义（均P〈0．05）。用药过程中2组患者均无严重不良反应发生。结论a-硫辛酸可以减少糖尿病肾病尿微量白蛋白及足细胞的凋亡,氧化应激在DN的发生发展中起重要的作用。     

足细胞异常与IgA肾病

IgA肾病是最常见的原发性肾小球疾病,以肾小球系膜区IgA沉积为病理特征。以往研究亦多侧重于系膜细胞在肾小球硬化中的作用,然而随着足细胞相关分子的深入研究,证实足细胞损伤与IgA肾病蛋白尿产生、新月体形成、肾小球硬化及间质纤维化密切相关。针对足细胞的治疗也有了新的认识。现就足细胞异常与IgA肾病临床病理指标的关系及IgA肾病治疗等相关进展进行综述。     

Perilipin2 inhibits diabetic nephropathy-induced podocyte apoptosis by activating the PPARγ signaling pathway

Podocyte apoptosis plays a pivotal role in the pathogenesis of diabetic nephropathy (DN). The main purpose of this study was to investigate the effects of perilipin2 on high glucose (HG)-induced podocyte apoptosis and associated mechanisms. Differentially expressed genes (DEGs) in BTBR ob/ob mice vs. nondiabetic mice kidneys were obtained from GSE106841 dataset and picked out using the ‘limma’ package. The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) and was visualized by Cytoscape. Perilipin2 was a hub gene using the cytoHubba plug-in from Cytoscape. Gene ontology (GO) analysis revealed that the 126 overlapping DEGs were mainly enriched in ‘oxidation reduction’ [biological process, (BP)], metal ion binding’ [molecular function, (MF)] and ‘extracellular region’ [cellular component, (CC)]. KEGG pathway analysis revealed that perilipin2 was mainly involved in ‘PPAR signaling pathway’. DN inhibited perilipin2 expression and PPARγ expression, as by both in vitro and in vivo studies. In vitro experiments demonstrated that perilipin2 inhibition could not only reduced PPARγ expression in podocytes, it could also promote the apoptosis, and inhibit the viability in HG treated podocytes using western blot, CCK8 and flow cytometry assays. Perilipin2 overexpression reversed the effects of HG on inhibiting podocalyxin, nephrin, precursor (pro)-caspase-3/-9 and PPARγ protein expression and increasing cleaved caspase-3/-9 protein expression. Furthermore, the functions of perilipin2 overexpression reversing HG-induced podocyte apoptosis were inhibited by PPARγ inhibitor. In conclusion, the functions of DN-induced podocyte apoptosis were inhibited by activation of the PPARγ signaling pathway caused by perilipin2 overexpression.     

Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.