The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy

Micro-RNAs (miRNAs) are short (average 22 nucleotides) noncoding regulatory RNAs that inhibit gene expression by targeting complementary 3'-untranslated regions of protein-encoding mRNAs for translational repression or degradation. miRNAs play key roles in both the function and differentiation of many cell types. Drosha and Dicer, two RNAase III enzymes, function in a stepwise manner to generate a mature miRNA. Previous studies have shown that podocyte-specific deletion of Dicer during development results in proteinuric renal disease and collapsing glomerulopathy (CG); however, Dicer has functions other than the generation of miRNAs. Here we found that the podocyte-specific deletion of Drosha results in a similar phenotype to Dicer mutants, confirming that the Dicer mutant phenotype is due to the loss of miRNAs. Moreover, the inducible deletion of Drosha in 2- to 3-month-old mice (Tet-On system) resulted in CG. Thus, continuous generation of miRNAs are required for the normal function of mature podocytes and their loss leads to CG. Identifying these miRNAs may provide new insight into disease pathogenesis and novel therapeutic targets in various podocytopathies.     

The next level of complexity: post-transcriptional regulation by microRNAs

MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs that regulate expression of about half of human genes. Canonical miRNA biosynthesis involves two RNase III-type proteins, Drosha and Dicer. A podocyte-specific knockout of Dicer resulted in foot process effacement. In this issue, Zhdanova et al. report on targeting Drosha in a podocyte-specific fashion. Their study demonstrates an essential role of canonical miRNAs in maintaining the fully differentiated podocyte phenotype.     

Expression Profile of MicroRNA Biogenesis Components in Renal Transplant Patients

Background

MicroRNAs (miRNAs) and the miRNA biogenesis components are potential biomarkers of some prevalent diseases, such as cancer and diabetes. In light of this information, we aimed to investigate the expression profiles of miRNA biogenesis components in renal transplant patients before and after transplantation and how these profiles are related to immunosuppressive treatment and clinical outcomes of these patients.

人肝癌细胞株与正常肝细胞株MicroRNA差异表达谱研究

目的 建立SMMC-7721人肝癌细胞株与CCC-HEL-1人正常肝细胞株MicroRNA(miRNA)差异的表达谱,确定差异的miRNA,为进一步研究miRNA在肝细胞癌变机制中的作用和肝癌的治疗提供新的线索.方法 体外培养SMMC-7721人肝癌细胞株和CCC-HEL-1人正常肝细胞株.用TRIzol法提取细胞的总...     

Review: The role of microRNAs in kidney disease

MicroRNAs (miRNAs) are short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression via blockade of protein translation or by inducing mRNA degradation. These miRNAs potentially regulate the expression of thousands of proteins. As a result, miRNAs have emerged rapidly as a major new area of biomedical research with relevance to kidney disease. MiRNA expression has been shown to differ between the kidney and other organs as well as between different kidney regions. Furthermore, miRNAs have been found to be functionally important in models of podocyte development, diabetic nephropathy and polycystic kidney disease. Of particular interest, podocyte-specific deletion of Dicer, a key enzyme in the biogenesis of miRNA, results in proteinuria and severe renal impairment in mice. One miRNA (miR-192) can also act as an effector of transforming growth factor-β activity in the high-glucose environment of diabetic nephropathy. Differential expression of miRNAs has been reported in kidney allograft rejection. It is anticipated that future studies involving miRNAs will generate new insights into the complex pathophysiology underlying various kidney diseases, generate diagnostic biomarkers and might be of value as therapeutic targets for progressive kidney diseases. The purpose of this review is to highlight key miRNA developments in kidney diseases and how this might influence the diagnosis and management of patients with kidney disease in the future.     

Increased expression of vascular endothelial growth factor in kidney leads to progressive impairment of glomerular functions

Vascular endothelial growth factor (VEGF) is an important mediator in maintaining normal kidney functions. In addition, several lines of evidence suggest that upregulation of VEGF in glomeruli may be associated with or cause renal dysfunction such as diabetic nephropathy. For elucidation of the pathologic consequences of high levels of VEGF in glomeruli, transgenic (Tg) rabbits that express human VEGF(165) isoform in both kidney and liver under the control of the human alpha-1-antitrypsin promoter were generated and characterized. With the use of heterozygous Tg rabbits and their littermates aged 8 to 55 wk, renal functions and structures were investigated. Compared with control rabbits, Tg rabbits exhibited progressive proteinuria with increased GFR at the early stage and decreased GFR at the later stage. Histologic examinations revealed that Tg rabbit kidneys were characterized by considerable glomerular hypertrophy as a result of increased proliferation of both glomerular capillaries and mesangial cells accompanied by prominent podocyte hypertrophy. With increasing age starting from 20 wk, Tg rabbit kidneys showed prominent formation of microaneurysms and capillary proliferation at the vascular pole area. At a later stage (55 wk), many glomeruli showed sclerosis and tuft collapse with the formation of glomerular cysts on a background of tubular atrophy and interstitial fibrosis. This study provides the first evidence that increased expression of VEGF in glomeruli directly causes the glomerular hypertrophy that is associated with proteinuria, suggesting that VEGF exerts multiple effects on the glomerular pathophysiologic processes.     

Differential expression of nephrin according to glomerular size in early diabetic kidney disease

Diabetic nephropathy (DN) is clinically characterized by proteinuria. Many studies tried to demonstrate a relationship between proteinuria and changes in nephrin in various forms of glomerular diseases including DN, but the results are not consistent. Glomerular hypertrophy occurs in DN, yet hypertrophy does not develop in all glomeruli concurrently. For investigation of the differences in nephrin expression according to glomerular size, glomeruli were isolated from 10 control and 10 streptozotocin-induced diabetic rats at 6 wk after the induction of diabetes by a sieving technique using sieves with pore sizes of 250, 150, 125, and 75 microm. Glomeruli then were classified into large glomeruli (LG; on the 125-microm sieve) and small glomeruli (SG; on the 75-microm sieve) groups. Glomerular volumes were determined using an image analyzer, and mRNA and protein expression was determined by real-time PCR and Western blot, respectively. The mean volumes of diabetic LG (1.51 +/- 0.06 x 10(6) microm(3)) and control LG (1.37 +/- 0.05 x 10(6) microm(3)) were significantly higher than those of diabetic SG (0.94 +/- 0.03 x 10(6) microm(3)) and control SG (0.87 +/- 0.03 x 10(6) microm(3); P < 0.01). Nephrin mRNA expression was significantly reduced in the diabetic LG group compared with the diabetic SG and control glomeruli groups (P < 0.05). In contrast, nephrin mRNA expression was significantly higher in the diabetic SG group compared with the diabetic LG and control glomeruli groups (P < 0.05). Even after correction for 18s rRNA and Wilms' tumor-1 mRNA expression, the differences in nephrin mRNA expression remained significant. The expression of nephrin protein showed a similar pattern to the mRNA expression. In conclusion, these data suggest that the nephrin gene is differentially expressed according to glomerular size. Furthermore, more hypertrophied glomeruli with lesser nephrin expression may be responsible for albuminuria in the early stage of DN.     

Glomerular injury and tubular loss in adriamycin nephrosis.

Glomerular injury manifested by sustained proteinuria usually leads to tubule injury and reduction of the GFR. The current study explored the link between these processes in rats with adriamycin nephrosis. One group of nephrotic rats received a vasopressin V2 receptor blocker (V2X) from 4 to 16 wk after injection of adriamycin, whereas a second group received no treatment (NoRx). V2 receptor blockade increased urine volume without affecting protein excretion. At 16 wk, both groups of nephrotic rats exhibited a marked reduction in GFR in comparison with normal controls (V2X, 0.22 +/- 0.19 ml/min; NoRx, 0.20 +/- 0.11 ml/min; control, 1.23 +/- 0.11 ml/min). Morphologic studies revealed that the majority of glomeruli in nephrotic rats were no longer connected to normal tubule segments (V2X, 81 +/- 21%; NoRx, 85 +/- 18%; control, 1 +/- 2%). Glomeruli without tubules were not, however, globally sclerosed. Disruption of the glomerular tubular junction was associated with the presence of amorphous material separating damaged tubule cells from the basement membrane. Serial sections revealed that this material spread from extensive areas of adhesion between the glomerular tuft and capsule to invest the tubular neck. Reduction of the GFR was strongly correlated with the fraction of glomeruli not connected to normal tubules (r(2) = 0.82; P < 0.0001). V2 receptor blockade did not preserve renal function or structure. These findings suggest that local extension of glomerular injury to destroy the tubule neck is an important cause of loss of renal function in adriamycin nephrosis.     

Late consequences of acute ischemic injury to a solitary kidney

The sequelae of acute ischemic injury to a solitary kidney were assessed in rats subjected to right nephrectomy and transient occlusion of the left renal artery; control rats underwent right nephrectomy alone. Incomplete recovery from ischemic injury at 2 wk (serum creatinine levels of 1.1 +/- 0.2 versus 0.5 +/- 0.1 mg/dl, P < 0.05 for ischemia versus control) was followed by deterioration of renal function at 20 wk (serum creatinine levels of 1.7 +/- 0.4 versus 0.7 +/- 0.1 mg/dl, P < 0.05 for ischemia versus control). Morphologic studies showed that impairment of function after ischemic injury was associated with widespread tubulointerstitial disease. Some tubule segments were atrophic and others exhibited cystic dilation, so that the tubular cell volume fraction was reduced (37 +/- 4 versus 53 +/- 2%, P < 0.05), while the tubular lumen and interstitial volume fractions were increased (31 +/- 4 versus 23 +/- 2% and 29 +/- 2 versus 20 +/- 1%, respectively, both P < 0.05). Many glomeruli retained open capillary loops but were no longer connected to normal tubule segments (63 +/- 8 versus 15 +/- 7% of glomeruli, P < 0.05). There was a strong inverse correlation between the prevalence of such glomeruli and the GFR at 20 wk after ischemia (r2 = 0.79, P < 0.001). Tubulointerstitial disease at that time was accompanied by proteinuria and widespread segmental glomerular tuft injury. The occurrence of similar processes in human patients could contribute to the loss of graft kidneys that suffer ischemic injury during transplantation.     

The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells.

Bone marrow stem cells (BMC) develop into hematopoietic and mesenchymal lineages but have not been known to differentiate into glomerular cells. To investigate whether such differentiation is possible, a search was made for donor glomerular cells in lethally irradiated C57BL/6j (B6) mice given transplants of BMC from syngeneic mice transgenic for green fluorescence protein (GFP) ([GFP-->B6] mice). After the recipients of donor BMC manifested GFP-positive cells in their glomeruli, the numbers of such cells increased markedly, in a time-dependent manner, from 2 wk to 24 wk after bone marrow transplantation. Immunohistochemical analyses revealed that most GFP-positive cells in the glomeruli were neither macrophages nor T cells. With the use of a laser-scanning confocal microscope, GFP-positive cells were observed within the mesangium of [GFP-->B6] mice. Furthermore, indirect immunofluorescence assays demonstrated that desmin-positive cells in the glomeruli of these chimeric mice were also positive for GFP. Among glomerular cells isolated from [GFP-->B6] mice 24 wk after bone marrow transplantation and then cultured, the majority of cells (approximately 84%) stained for desmin and approximately 60% of the desmin-positive cells expressed GFP. In addition, these GFP-positive cells in the cultures contracted in response to angiotensin II stimulation. These results suggest that bone marrow-derived cells may have the potential to differentiate into glomerular mesangial cells.     

自发性2型糖尿病KKAy小鼠肾小球microRNA表达谱和氯沙坦治疗效应

目的 分析糖尿病肾病(DN)发病过程中肾小球miRNA表达谱的变化,观察血管紧张素受体拮抗剂(ARB)氯沙坦对DN肾小球miRNA表达谱的影响,确认在DN发病过程中发挥关键作用的miRNA.方法 8周龄KKAy小鼠随机分为氧沙坦治疗组(10 mg·kg-1·d-1)和非治疗组,C57BL/6小鼠作为正常对照组.于20周龄检测体质量、随机血糖、尿微量白蛋白、尿肌酐,观察肾脏形态改变.应用磁珠灌注法分离肾小球,提取总RNA,应用Affymetrix GeneChip miRNA芯片,分析KKAv小鼠肾小球microRNA表达谱的变化,以及氯沙坦对microRNA表达谱的影响.结果 KKAy小鼠的体质量和血糖较正常对照C57BL/6组小鼠显著升高(均P＜ 0.05),氯沙坦治疗显著改善2型糖尿病KKAy小鼠的尿白蛋白/肌酐比值[( 539.71±100.23) mg/g比(728.00±177.19) mg/g,P＜0.05]和肾脏病理损害,而对血糖无影响.miRNA芯片分析结果发现,与正常对照C57BL/6小鼠相比,20周龄KKAy小鼠肾小球内10个miRNA的表达上调;12个miRNA的表达下调.与KKAy非治疗组小鼠相比,20周龄氯沙坦治疗组KKAy小鼠肾小球内共有4个miRNA表达下调,其中miR-503和miR-181d在KKAy非治疗组小鼠肾小球内的表达显著上调,氯沙坦治疗可抑制其过表达.结论 miR-503和miR-181d在糖尿病KKAy小鼠肾小球内的表达显著上调,氯沙坦治疗可抑制其在糖尿病状态下的异常表达,可能为糖尿病肾病新的治疗靶点.     

创伤性脊髓损伤后环状非编码RNA-微小RNA潜在调控网络信息挖掘

目的通过高通量测序获取创伤性脊髓损伤(traumatic spinal cord injury,TSCI)后环状非编码RNA(circular RNA,circRNA)与微小RNA(microRNA,miRNA)表达谱,预测潜在circRNA-miRNA调控网络。方法取48只雄性C57BL/6小鼠(体质量18~22 g)随机均分为两组(n=24),TSCI组采用Allen’s打击器械制备TSCI模型,假手术组(Sham组)仅切开椎板不损伤脊髓。术后3 d,两组取材行HE染色,观察脊髓组织结构;提取组织总RNA建库,高通量测序鉴定circRNA和miRNA差异表达谱,基因本体分析(gene ontology,GO)注释差异表达的circRNA宿主基因功能,筛选显著差异表达的miRNA,通过TargetScan和miRanda预测circRNAmiRNA靶向结合,筛选关键circRNA,构建潜在调控网络。结果HE染色示Sham组小鼠脊髓结构完整无破裂,TSCI组脊髓结构有明显损伤破裂。测序共鉴定出17440个circRNA、1228个miRNA。差异表达的circRNA宿主基因主要富集在细胞质,生物过程中差异基因主要富集在转录的正调控和蛋白磷酸化过程。差异表达最显著的miRNA为mmu-miR-21-5p,筛选出可与其靶向结合的circRNA6730,以circRNA6730为核心构建潜在circRNAmiRNA调控网络。结论通过表达谱分析和功能注释分析,显著差异表达的circRNA和miRNA有潜在的临床标志物价值,以circRNA6730为核心包含mmu-miR-21-5p的靶向互作网络可能在TSCI的发生发展过程中起重要调控作用,有助于阐明TSCI的病理生理进程机制,为临床诊疗提供新思路。     

Glomerular localization and expression of Angiotensin-converting enzyme 2 and Angiotensin-converting enzyme: implications for albuminuria in diabetes

Angiotensin-converting enzyme 2 (ACE2) expression has been shown to be altered in renal tubules from diabetic mice. This study examined the localization of ACE and ACE2 within the glomerulus of kidneys from control (db/m) and diabetic (db/db) mice and the effect of chronic pharmacologic ACE2 inhibition. ACE2 co-localized with glomerular epithelial cell (podocyte) markers, and its localization within the podocyte was confirmed by immunogold labeling. ACE, by contrast, was seen only in glomerular endothelial cells. By immunohistochemistry, in glomeruli from db/db mice, strong ACE staining was found more frequently than in control mice (db/db 64.6 +/- 6.3 versus db/m 17.8 +/- 3.4%; P < 0.005). By contrast, strong ACE2 staining in glomeruli from diabetic mice was less frequently seen than in controls (db/db 4.3 +/- 2.4 versus db/m 30.6 +/- 13.6%; P < 0.05). For investigation of the significance of reduced glomerular ACE2 expression, db/db mice were treated for 16 wk with a specific ACE2 inhibitor (MLN-4760) alone or combined with telmisartan, a specific angiotensin II type 1 receptor blocker. At the end of the study, glomerular staining for fibronectin, an extracellular matrix protein, was increased in both db/db and db/m mice that were treated with MLN-4760. Urinary albumin excretion (UAE) increased significantly in MLN-4760-treated as compared with vehicle-treated db/db mice (743 +/- 200 versus 247 +/- 53.9 microg albumin/mg creatinine, respectively; P < 0.05), and the concomitant administration of telmisartan completely prevented the increase in UAE associated with the ACE2 inhibitor (161 +/- 56; P < 0.05). It is concluded that ACE2 is localized in the podocyte and that in db/db mice glomerular expression of ACE2 is reduced whereas glomerular ACE expression is increased. The finding that chronic ACE2 inhibition increases UAE suggests that ACE2, likely by modulating the levels of glomerular angiotensin II via its degradation, may be a target for therapeutic interventions that aim to reduce albuminuria and glomerular injury.     

Differential expression of microRNA (miRNA) in chordoma reveals a role for miRNA‐1 in Met expression

Emerging evidence suggests that microRNA (miRNA) expression signatures in cancer may have important diagnostic, prognostic, and therapeutic value, but there is no data on miRNA expression in chordoma. The purpose of this study was to identify the role of miRNAs in human chordoma. We analyzed miRNA expression in chordoma‐derived cell lines and chordoma tissue by using miRNA microarray technology with unsupervised hierarchical clustering analysis. The relative expression levels of these miRNAs were confirmed by real‐time quantitative RT‐PCR and Northern blot analysis. To characterize the potential role of miRNA‐1, miRNA‐1 was stably transfected into a chordoma cell line, UCH1. The expression of miRNA‐1 targeted gene Met in chordoma tissues was also studied. We observe that human chordoma tissues and cell lines can be distinguished from normal muscle tissue by comparing miRNA expression profiles. Several miRNAs were differentially expressed in chordoma cell lines compared to controls, and similar expression patterns were found in primary chordoma tissues. Importantly, we were able to show for the first time, to our knowledge, that expression of miRNA‐1 and miRNA‐206, two miRNAs implicated in a number of other cancer types, were markedly decreased in both chordoma tissues and cell lines. When chordoma cell lines were transfected with miRNA‐1, downregulation of known miRNA‐1 targets was observed. These targets included Met and HDAC4—two genes that were observed to be overexpressed in chordoma. Our results demonstrate that some miRNAs are differentially expressed in chordoma and, in particular, miRNA‐1 may have a functional effect on chordoma tumor pathogenesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:746–752, 2010