Transcriptome analysis of human diabetic kidney disease

Abstract

Objective: Diabetic kidney disease (DKD) is the single leading cause of kidney failure in the U.S., for which a cure has not yet been found. The aim of our study was to provide an unbiased catalog of gene-expression changes in human diabetic kidney biopsy samples.

Research design and methods: Affymetrix expression arrays were used to identify differentially regulated transcripts in 44 microdissected human kidney samples. DKD samples were significant for their racial diversity and decreased glomerular filtration rate (~25-35 mL/min). Stringent statistical analysis, using the Benjamini-Hochberg corrected two-tailed t test, was used to identify differentially expressed transcripts in control and diseased glomeruli and tubuli. Two different web-based algorithms were used to define differentially regulated pathways.

Results: We identified 1,700 differentially expressed probesets in DKD glomeruli and 1,831 in diabetic tubuli, and 330 probesets were commonly differentially expressed in both compartments. Pathway analysis highlighted the regulation of Ras homolog gene family member A, Cdc42, integrin, integrin-linked kinase, and vascular endothelial growth factor signaling in DKD glomeruli. The tubulointerstitial compartment showed strong enrichment for inflammation-related pathways. The canonical complement signaling pathway was determined to be statistically differentially regulated in both DKD glomeruli and tubuli and was associated with increased glomerulosclerosis even in a different set of DKD samples.

Conclusions: Our studies have cataloged gene-expression regulation and identified multiple novel genes and pathways that may play a role in the pathogenesis of DKD or could serve as biomarkers.

FIG. 1.

Differentially expressed transcripts in healthy glomeruli compared with the tubulointerstitium. A: Statistical significance was determined using a fold-change cutoff of 1.5 and a Benjamini-Hochberg multiple-testing correction P value < 0.05. The graph represents the number of increased (red) or decreased (blue) probesets in the glomerular compartment with the indicated fold change (1.5- to 8.0-fold). B: Hierarchal cluster (Manhattan distance and complete linkage) of the 100 transcripts with highest fold change showing increased expression in glomeruli (i.e., glomerular specific). One row represents one gene and one column represents one sample. Blue color signifies downregulation and red color signifies upregulation.

FIG. 2.

Differentially expressed transcripts in DKD glomeruli. A: Statistical significance was determined using a fold-change cutoff of 1.5 and a Benjamini-Hochberg multiple-testing correction P value < 0.05. The graph represents the number of increased (red) or decreased (blue) probesets in the glomerular compartment with the indicated fold change (1.5- to 4.0-fold). B: Hierarchal cluster (Manhattan distance and complete linkage) of the 100 genes with highest fold change differentially expressed in control and DKD glomeruli.

FIG. 3.

Differentially expressed transcripts in DKD tubulointerstitium compared with control samples. A: Statistical significance was determined using a fold-change cutoff of 1.5 and a Benjamini-Hochberg multiple-testing correction P value < 0.05. The graph represents the number of increased (red) or decreased (blue) probesets in the glomerular compartment with the indicated fold change (1.5- to 3.8-fold). B: Hierarchal cluster (Manhattan distance and complete linkage) of the 100 transcripts with highest fold change differentially expressed in control and DKD tubulointerstitium.

FIG. 4.

Increased expression of complement in diabetic glomeruli. A: Relative mRNA level of C3 in individual glomerular samples control (blue bars) and from DKD samples (red bars). B: Representative images of periodic acid-Schiff staining and C3 immunostaining of individual kidney-tissue samples. (A high-quality digital representation of this figure is available in the online issue.)