Donor Toll-like receptor 4 contributes to ischemia and reperfusion injury following human kidney transplantation

Abstract

While studies in animal models have linked Toll-like receptor (TLR) 4 signaling to kidney injury induced by ischemia and reperfusion, the relevance of TLR4 activation to allograft injury in human kidney transplants is unknown. Here we show that TLR4 is constitutively expressed within all donor kidneys but is significantly higher in deceased-, compared with living-donor organs. Tubules from deceased- but not living-donor kidneys also stained positively for high-mobility group box-1 (HMGB1), a known endogenous TLR4 ligand. In vitro stimulation of human tubular cells with HMGB1, in a TLR4-dependent system, confirmed that HMGB1 can stimulate proinflammatory responses through TLR4. To assess the functional significance of TLR4 in human kidney transplantation, we determined whether TLR4 mutations that confer diminished affinity for HMGB1 influence intragraft gene-expression profiles and immediate graft function. Compared with kidneys expressing WT alleles, kidneys with a TLR4 loss-of-function allele contained less TNFalpha, MCP-1, and more heme oxygenase 1 (HO-1), and exhibited a higher rate of immediate graft function. These results represent previously undetected evidence that donor TLR4 contributes to graft inflammation and sterile injury following cold preservation and transplantation in humans. Targeting TLR4 signaling may have value in preventing or treating postischemic acute kidney injury after transplantation.

Fig. 1.

TLR4 expression in implantation biopsies. (A) Preanastomosis biopsy sections (×200) from living- (n = 15) and deceased-donor kidneys (n = 9) were analyzed by immunohistochemistry for TLR4. TLR4 was expressed in proximal and distal tubuli, with higher expression in deceased-donor kidneys compared with living donors (P = 0.016). The amount of TLR4 protein quantified correlated with their respective mRNA expression levels (P = 0.0014). (B) TLR4 mRNA expression levels in preanastomosis biopsies was significantly higher in samples from deceased donors (n = 28) compared to samples from living donors (n = 18). Box and whisker blots show the medians, and the percentile values (10, 25, 75, 90) for normalized mRNA.

Fig. 2.

HMGB1 expression in implantation biopsies. HMGB1 was localized in distal and proximal tubules from deceased-donor kidneys (DD) obtained prior anastomosis. However, no HMGB1 staining was observed in living-donor kidneys (LD). Glomeruli (*indicates border) were negative for HMGB1 for both types of donors (n = 5 each group; ×200).

Fig. 3.

HMGB1-induced TLR4-mediated inflammation. (A) qRT-PCR analysis of cytokine genes of proximal tubular cell line (HK-2). Cells were cultured without (Unstim) or with the TLR4-specific ligand LPS (1 μg/ml) or with rHMGB1 (5 μg/ml) (n = 3–7 per group; *P < 0.05 compared with unstimulated controls). (B) Western blot shows TLR4 expression of HK-2 cells transfected with control siRNA (lane 1: 62.5 nM; lane 2: 125 nM), or TLR4 siRNA (lane 3: 62.5 nM; lane 4: 125 nM). GAPDH and tubulin (not shown) staining was used as a loading control. HK-2 cells were transfected transiently with control siRNA or siRNA (125 nM each) against TLR4 and then stimulated with rHMGB1 as described in (A) (n = 3 each; *P < 0.05). (C) HK-2 cells were cultured for 30 min with monoclonal anti-TLR4 antibody or isotype (IgG) control (20 μg/ml each) and then stimulated with rHMGB1 (5 μg/ml) as described in (A) (n = 4 each; *P < 0.05).

Fig. 4.

Messenger RNA levels in kidney tissue in relation to TLR4 mutation and kidney function. (A) Gene expression in relation to the TLR4-donor genotype (WT n = 38; TLR4 mutated n = 10). (B) Expression levels of tubular injury marker IL-18, TNFα, MCP-1, T-cell marker (CD3ε), and HO-1 were analyzed in kidneys with IGF (n = 22) or DGF (n = 26). Box and whisker blots show the medians, and the percentile values (10, 25, 75, 90) for normalized mRNA.