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Proteomic analysis of the formalin-fixed paraffin embedded (FFPE) renal tissue between steroid-sensitive and steroid-resistant (SR) children FSGS patients

ABSTRACT: In children, focal segmental glomerulosclerosis (FSGS) is the main cause of steroid resistant nephroticsyndrome (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). 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. In total, we quantified 4794 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

DISEASE(S): Nephrotic Syndrome

SUBMITTER: Jiajia Ni

LAB HEAD: Hong Xu

PROVIDER: PXD036164 | Pride | 2022-11-19

REPOSITORIES: Pride

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Project description:Steroid-refractory acute rejection is a risk factor for inferior renal allograft outcome. We aimed to gain insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid-refractory acute rejection. Eighty-three kidney transplant recipients (1995-2005), who were treated with methylprednisolone during a first acute rejection episode, were included in this study. Gene expression patterns were investigated in a discovery cohort of 36 acute rejection biopsies, and verified in a validation cohort of 47 acute rejection biopsies. In the discovery set, expression of metallothioneins (MT) was significantly (P<0.000001) associated with decreased response to steroid treatment. Multivariate analysis resulted in a predictive model containing MT-1 as an independent covariate (AUC=0.88, P<0.0000001). In the validation set, MT-1 expression was also significantly associated with steroid resistance (P=0.029). Metallothionein expression was detected in macrophages and tubular epithelial cells. Parallel to the findings in patients, in vitro experiments of peripheral blood mononuclear cells from 11 donors showed that non-response to methylprednisolone treatment is related to highly elevated MT levels. High expression of metallothioneins in renal allografts is associated with resistance to steroid treatment. Metallothioneins regulate intracellular concentrations of zinc, through which they may diminish the zinc-requiring anti-inflammatory effect of the glucocorticoid receptor. Transcriptional profiles of 40 renal allograft biopsy samples were analyzed using Illumina HumanRef-8 v3.0 BeadChips (Illumina, San Diego, CA). Expression profiles were investigated in total RNA isolated from biopsy samples of 18 patients with steroid responsive acute rejection and 18 patients with steroid resistant acute rejection. Four biopsies, taken during acute decrease of graft function but with no histomorphologic indication of rejection, were included as controls.

Dataset Information

Proteomic analysis of the formalin-fixed paraffin embedded (FFPE) renal tissue between steroid-sensitive and steroid-resistant (SR) children FSGS patients

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