Comparative proteomic analysis of children FSGS FFPE tissues

Abstract

Background: In children, focal segmental glomerulosclerosis (FSGS) is the main cause of steroid resistant nephrotic syndrome (SRNS). To identify specific candidates and the mechanism of steroid resistance, we examined the formalin-fixed paraffin embedded (FFPE) renal tissue protein profiles via liquid chromatography tandem mass spectrometry (LC-MS/MS).

Methods: Renal biopsies from seven steroid-sensitive (SS) and eleven steroid-resistant (SR) children FSGS patients were obtained. We examined the formalin-fixed paraffin embedded (FFPE) renal tissue protein profiles via liquid chromatography tandem mass spectrometry (LC-MS/MS). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) analysis, as well as the construction of protein-protein interaction (PPI) network were performed. Two proteins were further valiadated by immunohistochemistry staining in FSGS patients and mice models.

Results: In total, we quantified more than 4000 proteins, of which 325 were found to be differentially expressed proteins (DEPs) between the SS and SR group (foldchange ≥2, P<0.05). The results of GO revealed that the most significant up-regulated proteins were primarily related to protein transportation, regulation of the complement activation process and cytolysis. Moreover, clustering analysis showed differences in the pathways (lysosome, terminal pathway of complement) between the two groups. Among these potential candidates, validation analyses for LAMP1 and ACSL4 were conducted. LAMP1 was observed to have a higher expression in glomerulus, while ACSL4 was expressed more in tubular epithelial cells.

Conclusions: In this study, the potential mechanism and candidates related to steroid resistance in children FSGS patients were identified. It could be helpful in identifying potential therapeutic targets and predicting outcomes with these proteomic changes for children FSGS patients.

Keywords: Focal segmental glomerulosclerosis; Proteomics; Steroid resistance.

Fig. 1

Overview of the study design. The human FFPE kidney tissue samples were subjected to proteomics analysis, and proteins were identified. Subsequently, DAVID online software was used to analyse the identified differentially expressed proteins. Finally, different laboratory methods were used to confirm the identified proteins

Fig. 2

Differential expression (DE) analysis of quantitative proteomic profiling data. A The identified peptides (grey) and the quantified proteins (blue) in each sample were visualized as bar charts. B Volcano plot of DE proteins, a total of 302 proteins were up-regulated, a total of 23 proteins were down-regulated. Red dots stood for upregulated proteins, blue dots stood for downregulated proteins and grey indicated no significant difference. C Proteins differentially expressed between FSGS-SR and FSGS-SS samples, depicted as a heatmap and hierarchical clustering pattern. A1-A11 indicated FSGS-SR patients. B1-B7 indicated FSGS-SS patients

Fig. 3

Enrichment GO function analysis of the upregulated and downregulated proteins with significant differences between the groups. Only the leading terms of each group are presented. A GOBP terms analysis of upregulated proteins. B GO analysis of downregulated proteins. C KEGG pathway analysis. GOCC, gene ontology term for cellular compoents; GOBP, gene ontology term for biological process; GOMF, gene ontology term for molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes

Fig. 4

Validation analysis of ACSL4 and LAMP1. A Immunohistochemical stain for ACSL4 and LAMP1 of mouse kidney tissue after injury induced by LPS. B Immunohistochemical stain for ACSL4 and LAMP1 of the FSGS patients and controls. Original magnification, × 400