Article

Discovery of new glomerular disease-relevant genes by translational profiling of podocytes in vivo

Kidney International (Impact Factor: 8.52). 06/2014; 86(6). DOI: 10.1038/ki.2014.204
Source: PubMed

ABSTRACT Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. We expressed enhanced green fluorescent protein-tagged to ribosomal protein L10a in podocytes under the control of the collagen-1α1 promoter, enabling one-step podocyte-specific mRNA isolation over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed Col1α1-eGFP-L10a mice with the Actn4(-/-) and Actn4(+/K256E) models of FSGS and analyzed podocyte transcriptional profiles at 2, 6, and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression data sets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and defines novel biomarkers and therapeutic targets for human glomerular disease.Kidney International advance online publication, 18 June 2014; doi:10.1038/ki.2014.204.

1 Follower
 · 
53 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP-2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP-2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non-CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both CHR and non-CHR patients. Longitudinal analysis indicated that increase in urine MMP-2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP-2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP-2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.
    American Journal of Transplantation 11/2010; 10(11):2463-71. DOI:10.1111/j.1600-6143.2010.03290.x · 6.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Focal segmental glomerulosclerosis (FSGS) is a common form of idiopathic nephrotic syndrome defined by the characteristic lesions of focal glomerular sclerosis and foot process effacement; however, its etiology and pathogenesis are unknown. We used mRNA isolated from laser-captured glomeruli from archived formalin-fixed, paraffin-embedded renal biopsies, until recently considered an unsuitable source of mRNA for microarray analysis, to investigate the glomerular gene expression profiles of patients with primary classic FSGS, collapsing FSGS (COLL), minimal change disease (MCD), and normal controls (Normal). Amplified mRNA was hybridized to an Affymetrix Human X3P array. Unsupervised (unbiased) hierarchical clustering revealed two distinct clusters delineating FSGS and COLL from Normal and MCD. Class comparison analysis of FSGS + COLL combined versus Normal + MCD revealed 316 significantly differentially regulated genes (134 up-regulated, 182 down-regulated). Among the differentially regulated genes were those known to be part of the slit diaphragm junctional complex and those previously described in the dysregulated podocyte phenotype. Analysis based on Gene Ontology categories revealed overrepresented biological processes of development, differentiation and morphogenesis, cell motility and migration, cytoskeleton organization, and signal transduction. Transcription factors associated with developmental processes were heavily overrepresented, indicating the importance of reactivation of developmental programs in the pathogenesis of FSGS. Our findings reveal novel insights into the molecular pathogenesis of glomerular injury and structural degeneration in FSGS.
    American Journal Of Pathology 10/2010; 177(4):1674-86. DOI:10.2353/ajpath.2010.090746 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Drebrins are actin-binding proteins (ABP) initially identified in and thought to be specific for neuronal cells, where they appear to contribute to the formation of cell processes. Recent studies have also detected the isoform drebrin E2 in a wide range of non-neuronal cell types, notably in and near actin-rich lamellipodia and filopodia. The present study demonstrates drebrin enrichment in renal glomeruli. Immunohistochemistry and double-label confocal laser scanning microscopy have shown intense drebrin reactions in the mesangial cells of diverse mammalian species. In adult human and bovine kidneys, drebrin is, in addition, markedly enriched in the foot processes of podocytes, as also demonstrable by immunoelectron microscopy. By contrast, the podocytes of rodent glomeruli appear to contain significant drebrin concentrations only during early developmental stages. In differentiated murine podocytes cultured in vitro, however, drebrin is concentrated in the cell processes, where it partially codistributes with actin and other ABP. In biochemical analyses using protein extracts from renal cortices, large (approximately 20S) complexes ("drebrosomes") were found containing drebrin and actin. These findings confirm and extend our hypothesis that drebrin is involved in the regulation of actin dynamics also outside the nervous system. Clearly, drebrin has to be added to the ensemble of ABP regulating the actomyosin system and the dynamics of mesangial cells and foot processes in podocytes.
    Journal of the American Society of Nephrology 07/2003; 14(6):1452-63. DOI:10.1097/01.ASN.0000069222.63700.DE · 9.47 Impact Factor

Preview

Download
0 Downloads
Available from