The renal transcriptome of db/db mice identifies putative urinary biomarker proteins in patients with type 2 diabetes: a pilot study

Abstract

We sought to identify novel urinary biomarkers of kidney function in type 2 diabetes. We screened the renal transcriptome of db/db and db/m mice for differentially expressed mRNA transcripts that encode secreted proteins with human orthologs. Whether elevated urine levels of the orthologous proteins correlated with diminished glomerular filtration rate was tested in a cross-sectional study of n = 56 patients with type 2 diabetes. We identified 36 putative biomarker genes in db/db kidneys: 31 upregulated and 5 downregulated. Urinary protein levels of six selected candidates (endothelin-1, lipocalin-2, transforming growth factor-β, growth and differentiation factor-15, interleukin-6, and macrophage chemoattractant protein-1) were elevated in type 2 diabetic patients with subnormal glomerular filtration rate (i.e., <90 ml·min(-1)·1.73 m(-2)), independent of microalbuminuria, age, sex, race, and use of angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists. In contrast, urinary levels of fibroblast growth factor were not increased. A composite variable of urine albumin and any of the six candidate markers was associated with subnormal estimated glomerular filtration rate more closely than albumin alone. In addition, urinary endothelin-1, growth and differentiation factor-15, and interleukin-6 were associated with a marker of proximal tubule damage, N-acetyl-β-d-glucosaminidase activity. These results suggest that gene expression profiling in diabetic mouse kidney can complement existing proteomic-based approaches for renal biomarker discovery in humans.

Fig. 1.

Putative biomarker genes identified from db/db kidneys sorted by hierarchical clustering. All mRNA expression values are normalized to 8-wk db/m kidneys (first column, db/m8). Subsequent columns are mRNA expression values from 16-wk db/m (db/m16, 1–3), 8-wk db/db (db/db8, 1–3), and 16-wk db/db (db/db16, 1–3) mice. The intensity of red and green are proportional to the magnitude of mRNA induction or repression, respectively.

Fig. 2.

Quantitative PCR (qPCR) measurements of candidate biomarker mRNAs in db/db and db/m kidneys. We selected 17 of the 36 putative biomarkers from Fig. 1 for verification studies by qPCR in an independent set of db/db and db/m mouse kidneys (n = 4 each). The changes in renal mRNA by qPCR were concordant with the microarray measurements shown in Fig. 1. Fibroblast growth factor mRNA, a negative control, was not differentially expressed in db/db vs. db/m kidneys. edn1, Endothelin 1; edn3, endothelin 3; bmp6, bone morphogenetic protein 6; gdf15, growth differentiation factor 15; gdf5, growth differentiation factor 5; tgfb1, transforming growth factor-β₁; erd1, erythroid differentiation regulator 1; cxcl2, chemokine (C-X-C motif) ligand 2; mcp1, macrophage chemoattractant protein-1; ccl9, chemokine (C-C motif) ligand 9; il6, interleukin-6; ctf1, CCAAT transcription factor 1; scgbla1, secretoglobin family 1A member 1; lcn2, lipocalin 2; tnfsf11, tumor necrosis factor ligand superfamily member 11; fgf, fibroblast growth factor. Values are mean ± SD of fold expression normalized to mRNA levels in 8-wk db/m control kidneys. *P < 0.05 and **P < 0.01.

Fig. 3.

Annotations for biological function associated with the putative diabetic kidney disease biomarkers. A: gene ontology annotations for biological function associated with 753 differentially expressed genes in the db/db gene set. Dots represent the false discovery rate-corrected P value for the annotation category (all P < 0.05). The number of genes in the db/db gene set associated with each annotation category is indicated under the dots. B: biological annotations for individual biomarker candidates, assigned in the db/db gene set in A, two-way clustered by biomarker and annotation category. A dot indicates that the biomarker belongs to the annotation category for biological function. The absence of a dot indicates that the biomarker is not associated with the biological function within the context of the db/db gene set.

Fig. 4.

Distribution of candidate biomarkers in urine from controls and participants with type 2 diabetes. Spot urine collections were obtained from 12 volunteers (open boxes) with apparently normal kidney function and 56 participants (shaded boxes) with type 2 diabetes. Urine levels of the autocrine/paracrine first messengers were measured by ELISA and corrected for the concentration of urine creatinine. Boxes represent the 75th to 25th percentile (i.e., interquartile range), and lines indicate the 1.5 and 3 interquartile ranges. Circles above each box represent potential outliers. Two-sided P values, calculated by independent sample t-tests, are reported. ET-1, endothelin-1; Cr, creatinine; TGF, transforming growth factor; NGAL, neutrophil gelatinase-associated lipocalin; Alb, albumin.

Fig. 5.

The selected urinary biomarker candidates were inversely associated with estimated glomerular filtration rate (eGFR) in a pilot study of participants with type 2 diabetes. A: albumin; B: ET-1; C: TGF-β; D: LCN2; E: GDF15; F: MCP-1; G: IL-6; H: FGF. Spot urine collections were obtained from n = 56 participants with type 2 diabetes and a range of eGFRs. GFR was estimated (Modification of Diet in Renal Disease equation) using clinical data obtained at the study visit. Urine protein levels of the biomarker candidates were measured by ELISA and normalized for creatinine. Urinary albumin normalized to creatinine was measured by ELISA in the same spot collection. The inset plots eGFR vs. urine albumin/creatinine in the range 0–600 μg/mg. The regression line (solid) and 95% confidence lines (dashed) are indicated. The Pearson's correlation coefficient r is reported.

Fig. 6.

Urinary ET-1 (A), GDF-15 (B), and IL-6 (C) correlated with a urine marker of proximal tubule damage, N-acetyl-β-d-glucosaminidase (NAGase) activity. Enzyme activity of NAGase was assessed in spot urine collections (n = 56) and correlated with ET-1, GDF-15, and IL-6 measured by ELISA in the same specimen. Biomarker values and NAGase activity are normalized for urine creatinine. The regression line (solid) and 95% confidence lines (dashed) are indicated. Pearson's correlation coefficient r is reported.