FcER1: A Novel Molecule Implicated in the Progression of Human Diabetic Kidney Disease - PubMed (original) (raw)
FcER1: A Novel Molecule Implicated in the Progression of Human Diabetic Kidney Disease
Swastika Sur et al. Front Immunol. 2021.
Abstract
Diabetic kidney disease (DKD) is a key microvascular complication of diabetes, with few therapies for targeting renal disease pathogenesis and progression. We performed transcriptional and protein studies on 103 unique blood and kidney tissue samples from patients with and without diabetes to understand the pathophysiology of DKD injury and its progression. The study was based on the use of 3 unique patient cohorts: peripheral blood mononuclear cell (PBMC) transcriptional studies were conducted on 30 patients with DKD with advancing kidney injury; Gene Expression Omnibus (GEO) data was downloaded, containing transcriptional measures from 51 microdissected glomerulous from patients with DKD. Additionally, 12 independent kidney tissue sections from patients with or without DKD were used for validation of target genes in diabetic kidney injury by kidney tissue immunohistochemistry and immunofluorescence. PBMC DKD transcriptional analysis, identified 853 genes (p < 0.05) with increasing expression with progression of albuminuria and kidney injury in patients with diabetes. GEO data was downloaded, normalized, and analyzed for significantly changed genes. Of the 325 significantly up regulated genes in DKD glomerulous (p < 0.05), 28 overlapped in PBMC and diabetic kidney, with perturbed FcER1 signaling as a significantly enriched canonical pathway. FcER1 was validated to be significantly increased in advanced DKD, where it was also seen to be specifically co-expressed in the kidney biopsy with tissue mast cells. In conclusion, we demonstrate how leveraging public and private human transcriptional datasets can discover and validate innate immunity and inflammation as key mechanistic pathways in DKD progression, and uncover FcER1 as a putative new DKD target for rational drug design.
Keywords: DKD; FcER1; chronic kidney disease; diabetes; mast cells.
Copyright © 2021 Sur, Nguyen, Boada, Sigdel, Sollinger and Sarwal.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
Figure 1
Overall study design and workflow. Transcriptome profiling with gene microarray performed on PBMCs from demographically matched sets of patients at different stages of DKDs (Cohort1) integrated with the publicly available microarray experiments on four microdissected glomeruli of patients with DKD (Cohort 2). Customized bioinformatics allowed for the selection of genes that correlated with DKD severity in both data sets by hypergeometric enrichment. Network and pathway analysis was performed with significant overlapping genes. One target with the most significant expression with DKD severity and the associated cell type was validated in an independent set of DKD samples (Cohort 3). IHC and IF for protein product performed on human kidney tissue. T2D, Type 2 diabetes; HC, healthy control; D1, normoalbuminuria; D2, microalbuminuria; D3, macroalbuminuria; IHC, immunohistochemistry; IF, immunofluorescence.
Figure 2
(A) Principal component analysis plot with all samples and unique genes from the microarray analysis performed on peripheral blood mononuclear cells (PBMCs) from patients at different stages of DKDs (Cohort 1). Outliers are marked with black arrows. (B) Dendrogram of unsupervised clustering for all samples of diabetes (D1, D2, D3), ESRD, and healthy controls without the outliers is based on the significant genes determined by ANOVA (p < 0.05). (C) The dendrogram of unsupervised clustering for samples with diabetes only without the outliers is based on the top 100 significant genes, whose expression increased with disease severity as determined by the JT test. (D) Facet grid plot of representative genes from cohort 1 with a positive correlation between its expression and degree of albuminuria shown as log fold change compared to control. Candidate genes were selected by taking the top ten statistically significant genes. The analysis demonstrates the higher expression of FcER1 in more advanced stages of diabetes. (E) Dot-plot showing the top pathways in cohort 1. HC, Healthy control; D1, diabetes with normoalbuminuria; D2, diabetes with microalbuminuria; D3, diabetes with macroalbuminuria; ESRD, end stage renal disease.
Figure 3
(A) Different glomerular microarray platforms in GEO were converted to Entrez Gene identifiers and analyzed by eGWAS (Cohort 2). Using a one-tailed T-test P values between DKD and controls were calculated, which were converted to Z-scores and meta-analysis was performed based on the weighted Z-method. Gene expression leading to a Z-score above 5 or below -5 was considered significantly up or downregulated. (B) Dot-plot showing the top pathways in cohort 2.
Figure 4
Pathway analysis of the overlapped gene expression that is common in cohorts 1 and 2. (A) Table with the top significant canonical pathways in the 28 genes were found to be significant for DKD and overlapping between both datasets. (B) FcER1 signaling was identified as the center node of the significantly enriched canonical pathway connecting most of the identified significant genes including Tec Kinase, NF-B, IL-10, and T Helper cell differentiation pathways. Nodes outside the gene-set with known interactions are displayed in pink. (C) It is the known signaling pathway of FcER1, where the proteins including FcER1, TNF, SLP, and Ras are also present in the common 28 genes list.
Figure 5
Representative Immunohistochemistry staining for FcER1 in human kidney tissue. (A) Normal kidney at 20x. (B) Diabetic kidney at 20x. (C) Normal kidney at 40x. (D) Diabetic kidney at 40x. (E) The intensity of FcER1 staining in human kidney tissue. (F) Gene expression of FcER1 from PBMCs at different stages of diabetic kidney disease. (G) Gene expression of FcER1 in control and diabetic kidney disease; *p < 0.05, **p < 0.01, ***p < 0.001; DKD, diabetic kidney disease; D1, diabetes with normoalbuminuria; D2, diabetes with microalbuminuria; D3, diabetes with macroalbuminuria.
Figure 6
Representative immunofluorescence staining for coexpression of FcER1 and inflammatory cell markers in human kidney tissue. Coexpression of FcER1 and mast cell marker. MCD, Minimal changed disease; DN, diabetic nephropathy.
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