TRAIL deficiency contributes to diabetic nephropathy in fat-fed ApoE-/- mice

Abstract

Background: We recently demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) is protective of diet-induced diabetes in mice. While TRAIL has been implicated in chronic kidney disease, its role in vivo in diabetic nephropathy is not clear. The present study investigated the role of TRAIL in the pathogenesis of diabetic nephropathy using TRAIL(-/-)ApoE(-/-) mice.

Methods: TRAIL(-/-)ApoE(-/-) and ApoE(-/-) mice were fed a high fat diet for 20 w. Plasma glucose and insulin levels were assessed over 0, 5, 8 and 20 w. At 20 w, markers of kidney function including creatinine, phosphate, calcium and cystatin C were measured. Changes in mRNA expression of MMPs, TIMP-1, IL-1β and IL-18 were assessed in the kidney. Functional and histological changes in kidneys were examined. Glucose and insulin tolerance tests were performed.

Results: TRAIL(-/-)ApoE(-/-) mice had significantly increased urine protein, urine protein:creatinine ratio, plasma phosphorous, and plasma cystatin C, with accelerated nephropathy. Histologically, increased extracellular matrix, mesangial expansion and mesangial cell proliferation in the glomeruli were observed. Moreover, TRAIL(-/-)ApoE(-/-) kidneys displayed loss of the brush border and disorganisation of tubular epithelium, with increased fibrosis. TRAIL-deficient kidneys also had increased expression of MMPs, TIMP-1, PAI-1, IL-1β and IL-18, markers of renal injury and inflammation. Compared with ApoE(-/-) mice, TRAIL-/-ApoE-/- mice displayed insulin resistance and type-2 diabetic features with reduced renal insulin-receptor expression.

Conclusions: Here, we show that TRAIL-deficiency in ApoE(-/-) mice exacerbates nephropathy and insulin resistance. Understanding TRAIL signalling in kidney disease and diabetes, may therefore lead to novel strategies for the treatment of diabetic nephropathy.

Figure 1. TRAIL^-/-ApoE^-/- mice have increased renal injury.

Representative sections (40X magnification) of mouse kidney after 20 w of HFD stained with (a) Periodic acid Schiff showing relative increased mesangial matrix (yellow arrows) and increased cellularity in mesangial regions with local areas of tubular degeneration and loss of brush boarder (red asterix), (b) vimentin demonstrating increased matrix, (c) collagen IV (total kidney) showing increased collagen staining throughout the interstitium and (d) glomeruli. Stains were quantified as described in the Methods. Measurements are from n = 5 mice/genotype, 20–25 glomeruli per mouse (vimentin and collagen IV), or 11–15 images each of inner and outer cortex for collagen IV. Results are expressed as mean ± SEM, ****p<0.0001 by Mann-Whitney U test.

Figure 2. Kidneys from TRAIL^-/-ApoE^-/- mice have increased expression of genes indicative of fibrosis.

mRNA expression for (a) fibronectin, (b) PAI-1, (c) TIMP-1, (d) MMP-2 and (e) MMP-9 from kidneys. All levels were normalized to β-actin; n = 6/ genotype. Results are expressed as mean ± SEM, *p<0.05 and **p<0.01 by Mann-Whitney U test.

Figure 3. TRAIL^-/-ApoE^-/- mice have increased macrophage infiltration and genes of inflammation.

Representative sections (40X magnification) of mouse kidney after 20 w HFD stained for the pan macrophage marker (a) F4/80. Kidney mRNA expression for (b) IL-1β, (c) IL-18, (d) osteopontin (e) PPAR-γ and (f) TNF-α. All levels were normalized to β-actin; n = 5-6/genotype. Results expressed as mean ± SEM, *p<0.05, **p<0.01 and ****p<0.0001 by Mann-Whitney U test.

Figure 4. TRAIL^-/-ApoE^-/- mice show impaired glucose and insulin tolerance and kidneys have altered insulin signalling.

(a) Glucose and (b) insulin tolerance tests of ApoE^-/- (open circles) and TRAIL^-/-ApoE^-/- (▪) mice (n = 6–11/genotype). mRNA levels for (c) GLUT2, (d) GLUT4 and (e) insulin receptor were measured in the kidney. All mRNA levels were normalized to β-actin; n = 5-6/genotype. Results expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by Mann-Whitney U test and ANOVA.