The death ligand TRAIL in diabetic nephropathy

Abstract

Apoptotic cell death contributes to diabetic nephropathy (DN), but its role is not well understood. The tubulointerstitium from DN biopsy specimens was microdissected, and expression profiles of genes related to apoptosis were analyzed. A total of 112 (25%) of 455 cell death-related genes were found to be significantly differentially regulated. Among those that showed the greatest changes in regulation were two death receptors, OPG (the gene encoding osteoprotegerin) and Fas, and the death ligand TRAIL. Glomerular and proximal tubular TRAIL expression, assessed by immunohistochemistry, was higher in DN kidneys than controls and was associated with clinical and histologic severity of disease. In vitro, proinflammatory cytokines but not glucose alone regulated TRAIL expression in the human proximal tubular cell line HK-2. TRAIL induced tubular cell apoptosis in a dosage-dependant manner, an effect that was more marked in the presence of high levels of glucose and proinflammatory cytokines. TRAIL also activated NF-kappaB, and inhibition of NF-kappaB sensitized cells to TRAIL-induced apoptosis. It is proposed that TRAIL-induced cell death could play an important role in the progression of human DN.

Figure 1.

Changes in cell death gene expression in DN. Unsupervised hierarchical cluster analysis of biopsy samples from control subjects (Con) and patients with DN. Cluster analysis sorts genes and patient samples according to their similarities in expression levels. The clustering algorithm computes the distance between all of genes in the data set, and the two closest genes are joined by a branch and removed from the list. The distances between this branch and all other genes are calculated once again, and the process repeated until all genes have been clustered. After processing the data, the tree structure of the resulting dendrogram reflects the relationship among the samples. Each row represents a gene, and each column represents a biopsy; green shows mRNA repression, and red shows induction relative to the mean expression value. The clustering process is unbiased, and the operator is blinded with respect to the histologic categorization of the samples.

Figure 2.

TRAIL and OPG array expression correlate with severity of human DN. The panels show the correlation between TRAIL and OPG array expression and diverse parameters of severity of DN. Top and bottom left panels show the correlation between TRAIL and OPG gene expression in arrays and real-time qRT-PCR. TRAIL and OPG array mRNA data are expressed as Log2 signal intensity values. Linear regression was used to perform the best fit lines.

Figure 3.

Differential TRAIL protein expression in DN. Immunohistochemistry for TRAIL in control (A and B) and DN kidneys (C and D). In control kidneys, TRAIL staining is negative at the glomerular level (A) and shows a faint labeling in a limited number of tubular cross-sections (B). (C) Glomerular TRAIL positivity in DN. (D) Intense and diffuse tubular TRAIL positivity in DN. (E) Patients with DN and higher proximal tubular TRAIL expression (Table 2: immunohistochemistry score 2) show more severe tubular atrophy, interstitial fibrosis, and interstitial infiltrate than patients who have DN with a score of 1 or control subjects. Data are means ± SEM. *P < 0.01 versus the other two groups; **P < 0.05 versus the other two groups. Magnifications: ×100 in A; ×200 in B through D.

Figure 4.

Tubular epithelial TRAIL expression is regulated by proinflammatory cytokines. (A) Expression of TRAIL and OPG in HK-2 cells assessed by Western blot. (B) Semiquantitative RT-PCR. Expression plasmids for TRAIL-R1, -R2, and -R3 were used as controls. RT-PCR for TRAIL-R2 yielded two bands corresponding to its two transcript variants. (C and D) TRAIL protein expression in HK-2 cells. Cells were cultured in 5.5 or 25 mM glucose (C) or in 11 mM glucose and treated with a combination of cytokines (TNF-α and IFN-γ; D). Data are means ± SEM of three independent experiments. *P < 0.05. ADU, arbitrary densitometry units.

Figure 5.

High glucose sensitizes cells to TRAIL-induced cell death. (A) Apoptosis induction. Dose-response at 24 h. ▪, Cells cultured in 5.5 mM glucose; □, cells cultured in 25 mM glucose. *P < 0.05 versus absence of TRAIL 5.5 mM glucose; **P < 0.05 versus absence of TRAIL 25 mM glucose. Note the difference in the scale from B. (B) Apoptosis of HK2-cells treated with TRAIL 10 ng/ml and cytokines (TNF-α and IFN-γ) for 24 h. ▪, Cells cultured in 5.5 mM glucose; □, cells cultured in 25 mM glucose. Quantification of apoptosis by flow cytometry of DNA content. Experiments were performed five times, and each experiment consisted of triplicates. *P < 0.05. (C) Representative flow cytometry diagrams of cells cultured in 25 mM glucose. The line encompasses hypodiploid cells.

Figure 6.

TRAIL induces activation of NF-κB in tubular epithelial cells. (A) NF-κB DNA binding is detected by electrophoretic mobility shift assay after TRAIL stimulation (10 ng/ml). (B) Western blot. Parthenolide (P) induces transient accumulation of IκBα and prevents TRAIL-induced IκBα degradation. The fragment corresponding to activated caspase-3 can be detected in cells treated with parthenolide and TRAIL. B, basal. (C) Parthenolide (P) enhances TRAIL-induced apoptosis. Apoptotic cells quantified by flow cytometry of cell DNA content. The pictures show apoptotic nuclei (white arrows) present in permeabilized, propidium iodide–stained cells treated with parthenolide and TRAIL for 24 h. Data are means ± SEM of five independent experiments. *P < 0.05 versus other groups. Magnification, ×40.

Figure 7.

OPG interferes with TRAIL-induced NF-κB activation and TRAIL-induced loss of tubular cell survival in vitro. Recombinant OPG (rOPG) acts as a soluble ligand for TRAIL, blocking its binding to the target cells, and anti-OPG blocking antibodies (αOPG) inhibit OPG blockade of TRAIL, thus enhancing its activity on target cells. (A) Quantitative analysis of NF-κB activation by luciferase assay. RLU, relative light units. *P < 0.05 versus basal unstimulated; #P < 0.01. Experiments were conducted three or more times. Samples were prepared at least in triplicate. (B) Cell survival was assessed using MTS. P, parthenolide. *P < 0.05 versus all other groups; **P < 0.05 versus basal control. Experiments were conducted six times. Samples were prepared at least in triplicate. Data are means ± SEM. (C through F). Proposed interaction between TRAIL and OPG. (C) TRAIL induces a low level of apoptosis in cultured tubular epithelial cells. The concurrent activation of NF-κB by TRAIL contributes to the low level of apoptosis, because the NF-κB inhibitor parthenolide sensitizes to TRAIL-induced apoptosis (D). (E) OPG is a soluble decoy receptor for TRAIL and is able to block both apoptosis and NF-κB activation in response to TRAIL. (F) OPG limits the increased apoptosis induced by TRAIL when NF-κB is inhibited. We propose that in vivo the relative local concentrations of TRAIL versus OPG in the cell microenvironment determines the outcome of the interaction.

Figure 8.

The diabetic milieu renders cells more sensitive to TRAIL-induced apoptosis. Diagram representing the hypothetical role of TRAIL in human DN. In the normal kidney, tubular cells express low levels of TRAIL that may regulate inflammation and tumor transformation. During diabetes, TRAIL expression is increased, probably as a consequence of a proinflammatory milieu, sensitizing cells to TRAIL-induced apoptosis.