A molecular profile of focal segmental glomerulosclerosis from formalin-fixed, paraffin-embedded tissue

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.

Figure 1

Isolation of a glomerulus by laser capture microdissection from an eosin-stained section cut 6-μm thick on a membrane-covered slide. A glomerulus is easily identified (center of A) and separated from surrounding tubulointerstitium using a laser to cut both the tissue and the clear membrane. B: A laser pulse (dot in C) catapults the liberated glomerulus and portion of membrane into a digestion buffer for RNA extraction.

Figure 2

RNA quality and gene expression profiles of FFPE versus frozen FRZN tissue. A: We analyzed RNA quality using 25 to 75 ng RNA isolated from renal cortex with the Agilent 2100 Bioanalyzer. The x-axis is in seconds, the y-axis is fluorescence units. RIN denotes RNA integrity number. B: Scatter plots demonstrate high correlation (R) of FFPE versus FRZN glomerular gene expression profiles. The x- and y-axis are log-transformed array signals. Each point represents one gene.

Figure 3

Unsupervised hierarchical clustering analysis demonstrates two main clusters of similar glomerular gene expression signatures. In cluster I, with the exception of three cases, Normal and MCD cluster together. In cluster II, FSGS and COLL cluster together.

Figure 4

Validation of FSGS + COLL versus Normal + MCD gene expression profiling using TaqMan quantitative real-time RT-PCR with 10 genes, normalized to GAPDH expression. *P < 0.05; **P < 0.1; ***P = 0.15, after Bonferroni correction for multiple comparisons.

Figure 5

Glomerular SOX9 protein expression. Immunohistochemistry for SOX9 reveals strong nuclear staining in podocytes (arrows) and parietal cells (arrowheads). Most of the nuclei within the center of the glomerular tuft (mesangial and endothelial cells) are negative or weakly positive, although some demonstrate strong staining. The nuclei of surrounding tubular cells show variable staining. Glomerular laser-capture does not collect the parietal cells or surrounding tubular cells.

Figure 6

Correlation of gene expression with segmental sclerosis. Among genes differentially regulated in the FSGS + COLL group, the expression of three were found to significantly correlate (P < 0.05) with the percentage of collapsing and classic FSGS lesions (AIF1) or the percentage of classic FSGS lesions alone (DSP and CCDC91).