Gene Expression Profiles of Podocyte-Associated Molecules as Diagnostic Markers in Acquired Proteinuric Diseases

Abstract

Abstract. For identifying potential diagnostic markers of proteinuric glomerulopathies, glomerular mRNA levels of molecules relevant for podocyte function (␣-actinin-4, glomerular epithelial protein 1, Wilms tumor antigen 1, synaptopodin, dystroglycan, nephrin, podoplanin, and podocin) were determined by quantitative real-time RT-PCR from microdissected glomeruli. Biopsies from 83 patients with acquired proteinuric diseases were analyzed (minimal change disease [MCD; n ϭ 13], benign nephrosclerosis [n ϭ 16], membranous glomerulopathy [n ϭ 31], focal and segmental glomerulosclerosis [FSGS; n ϭ 9], and controls [n ϭ 14]). Gene expression levels normalized to two different housekeeping transcripts (glyceraldehyde-3-phosphate-dehydrogenase and 18 S rRNA) did not allow a separation between proteinuric disease categories. However, a significant positive correlation between ␣-actinin-4, glomerular epithelial protein 1, synaptopodin, dystroglycan, Wilms tumor antigen 1, and nephrin was found in all analyzed glomeruli, whereas podocin mRNA expression did not correlate. Because varying amounts of housekeeper cDNA per glomerulus can confound expression ratios relevant for a subpopulation of cells, an "in silico" microdissection was performed using a podocyte-specific cDNA as a reference gene. Expression ratio of podocin to synaptopodin, the two genes with the most disparate expression, allowed a robust separation of FSGS from MCD and nephrosclerosis. Segregation of FSGS from MCD via this ratio was confirmed in an independent population of formaldehyde-fixed archival biopsies (MCD, n ϭ 5; FSGS, n ϭ 4) after glomerular laser capture microdissection. In addition, the expression marker was able to predict steroid responsiveness in diagnostically challenging cases of MCD versus FSGS (n ϭ 6). As the above approach can be performed as an add-on diagnostic tool, these molecular diagnostic parameters could give novel information for the management of proteinuric diseases.Proteinuric glomerular diseases still pose a formidable challenge to nephrology. Recent studies have highlighted the importance of proteinuria both as a clinical prognostic marker and as a factor predicting progressive loss of renal function (1-3). Alteration of the glomerular filtration barrier leads via damage of the glomerular podocyte to leakage of proteins into the ultrafiltrate (4). The filtration barrier consists of a specialized fenestrated endothelium, the fibrillar, hydrated meshwork of the glomerular basement membrane (GBM), and interdigitating podocyte foot processes with intervening filtration slits as the final filtration barrier (5). Identification of mutations in podocyte-associated molecules in various hereditary nephrotic disorders has highlighted the key role of the podocyte for the pathophysiology of proteinuria. The gene product of NPHS1, nephrin, mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the podocyte slit diaphragm (6). Mutations in a gene termed NPHS2, encoding the novel protein podocin, are associate...

Focal Segmental Glomerulosclerosis

“…Biomarkers for responsiveness to glucocorticoid therapy would be valuable to reduce or avoid the need to expose patients to drugs to which they will not respond. Preliminary studies that await validation in larger cohorts include the ratio of podocin mRNA to synaptopodin mRNA, particularly when the diagnostic uncertainty with regard to MCD versus FSGS exists (134). Immunohistochemical detection of reduced expression of synaptopodin and receptor-type tyrosineprotein phosphatase O (PTPRO or GLEPP1) may be useful in identifying steroid-resistant patients (135).…”

Induction of TRPC6 Channel in Acquired Forms of Proteinuric Kidney Disease

“…Control samples were obtained from the healthy kidney poles of individuals who underwent tumor nephrectomies. By real-time quantitative PCR, we determined the ratio of TRPC6 mRNA to 18S ribosomal RNA (17). We found significantly increased levels of TRPC6 mRNA in patients with MCD (n ϭ 13, P ϭ 0.006) and MGN (n ϭ 28, P ϭ 0.024) as compared with control patients (n ϭ 9).…”

Dystroglycan in the Diagnosis of FSGS

“…The presence of glomerular hypertrophy in patients with initial diagnosis of apparent MCD predicts an increased risk of subsequently developing overt FSGS (18). More recent molecular studies also support that MCD and FSGS differ from the onset (26). Thus, the definition of patterns of marker expression may aid in the distinction of MCD from FSGS.…”