Toll-like receptor 4 signalling mediates inflammation in skeletal muscle of patients with chronic kidney disease

Abstract

Background: Inflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia but why uremia up-regulates pro-inflammatory cytokines is unknown. Toll-like receptors (TLRs) regulate locally the innate immune responses, but it is unknown whether in chronic kidney disease (CKD) TLR4 muscle signalling is altered. The aim of the study is to investigate whether in CKD muscle, TLRs had abnormal function and may be involved in transcription of pro-inflammatory cytokine.

Methods: TLR4, phospho-p65, phospho-ikBα, tumour necrosis factor (TNF)-α, phospho p38, Murf 1, and atrogin were studied in skeletal muscle from nondiabetic CKD stage 5 patients (n = 29) and controls (n = 14) by immunohistochemistry, western blot, and RT-PCR. Muscle cell cultures (C2C12) exposed to uremic serum were employed to study TLR4 expression (western blot and RT-PCR) and TLR-driven signalling. TLR4 signalling was abrogated by a small molecule chemical inhibitor or TLR4 siRNA. Phospho AKT and phospho p38 were evaluated by western blot.

Results: CKD subjects had elevated TLR4 gene and protein expression. Also expression of NFkB, p38 MAPK and the NFkB-regulated gene TNF-α was increased. At multivariate analysis, TLR4 protein content was predicted by eGFR and Subjective Global Assessment, suggesting that the progressive decline in renal function and wasting mediate TLR4 activation. In C2C12, uremic serum increased TLR4 as well as TNF-α and down-regulated pAkt. These effects were prevented by blockade of TLR4.

Conclusions: CKD promotes muscle inflammation through an up-regulation of TLR4, which may activate downward inflammatory signals such as TNF-α and NFkB-regulated genes.

Keywords: Chronic kidney disease; Inflammation; Muscle wasting; Toll-like receptors; Tumour necrosis factor-α.

Figure 1

Expression of TLR2 (a) and TLR3 (b) mRNAs and proteins in skeletal muscle of controls (n = 14) and patients with chronic kidney disease (CKD) (n = 29). TLR2 and TLR3 mRNA expression was determined by real‐time PCR and their protein expression by immunohistochemistry and image analysis. Values are expressed as fold increase ± SEM to the control muscle. TLR2 and TLR3 mRNAs and proteins were unchanged with respect to control subjects (P = NS). CKD = chronic kidney disease. (Magnification: ×400–1000).

Figure 2

Expression of TLR4 mRNA (a) and protein (b–d) in normal skeletal muscle (n = 14) and in patients chronic kidney disease (CKD) (n = 29). TLR4 mRNA was evaluated by real‐time PCR, and its protein by immunohistochemistry followed by image analysis (b, c) and western blot (d) of muscle lysates. Values are expressed as fold increase ± SEM to the control muscle. TLR4 mRNA was approximately two‐folds increased vs. controls. TLR4 protein was absent or very faintly expressed in the normal muscle, while was overexpressed (by 1.5–3‐folds) in CKD muscle (panel C). Western blots show up‐regulated TLR4 in CKD with respect to controls (panel d). Blots were stripped and reprobed with anti β‐actin antibody. The gel is representative of 12 CKD and 5 controls. CKD = chronic kidney disease. (Magnification: ×400–1000). The arrows indicate positive cells. *P < 0.05, **P < 0.001 vs. controls.

Figure 3

(Panel a). Phospho‐p65 (P‐p65) expression in the skeletal muscle of CKD patients and controls. Control (n = 11) muscle showed p‐p65 positive nuclei in a very small percentage. This percentage increased significantly in patients with CKD (n = 20). The degree of positive nuclei was estimated by counting the number of p‐p65 positive cells for 100 cells examined in average of five high‐power fields. (Panel b) Phospho‐IkB‐α (p‐IkB‐α) expression in the skeletal muscle of CKD patients and controls. P‐IkB‐α was highly up‐regulated in muscle of patients with CKD.(Panel c) Expression of TNF‐α mRNA and protein in CKD (n = 25) and control (n = 11) muscle. TNF‐α m‐RNA was two‐folds overexpressed in CKD samples with respect to the control tissue. The protein expression of TNF‐α was minimally detectable in control samples, while it was markedly increased (by approximately five‐folds) in muscle of CKD patients. (Panel d) Immunohistochemistry analysis for p‐p38 in normal and CKD subjects. Staining was weakly diffused in normal tissue, but intensely expressed in uremia. (Panel e) Expression of Murf 1 and atrogin mRNA. Murf 1 and atrogin mRNAs expression level was determined by real time PCR. Both genes were over expressed in CKD muscle (n = 12) with respect to controls (n = 10). C = controls; CKD = chronic kidney disease. (Magnification: ×400–1000). The arrows indicate positive areas. Data are expressed as fold increase ± SEM to normal muscle. *P < 0.05 vs. C; **P < 0.01 vs. C.

Figure 4

(Panel a). The effect of normal serum (NS) and uremic serum (US) on TLR4 mRNA and protein in C2C12 myotubes. Cells were incubated with 10% serum for 6 h. TLR4 mRNA expression was determined by real time PCR at different times and protein by western blot after six hours. (Panel b) The effect of uremic serum (US) on TNF‐α gene expression in C2C12 myotubes. TNF‐α mRNA expression was determined by real time PCR after 5 h treatment. (Panel c) Down‐regulation of pAkt in uremic serum (US)‐treated cells. pAkt was evaluated by western blot analysis after 24 h exposition to normal serum (NS) or US. Blots were stripped and reprobed with anti‐pAkt antibody.(Panel d) Uremic serum (US) induces p‐p38 during time course (0–240 min) in C2C12 myotubes. Blots were stripped and reprobed with anti β actin antibody. All results represent means ± SEM obtained from five independent experiments.*P < 0.05 vs. T0; **P < 0.01 vs. T0; §P < 0.001 vs. T0 and NS; +P < 0.01 vs. T0 and NS. NS = normal serum; US = uremic serum; pAkt = phospho‐Akt; p‐p38 = phospho‐p38.

Figure 5

Effects of p38 inhibitor (10 μM SB203580), and pKC inhibitors (0.2–4 μM staurosporine or 10 μM chelerythrine) on uremic serum (US)‐induced TLR4 mRNA. To further investigate the mechanism of uremic serum‐induced TLR4 expression, we examined the role of p38 and its related signal pathway including protein kinase C (PKC). Pretreatment of C2C12 myotubes with the p38 inhibitor SB203580 (10 μM) and the PKC inhibitors chelerythrine (5 μM) and staurosporine (0.2–0.4 μM) 1 h before serum exposure, resulted in a marked decrease in the serum‐induced TLR4 mRNA overexpression. All results represent means ± SEM obtained from three independent experiments.*P < 0.05 vs. T0;.*P < 0.05 vs. US; **P < 0.01 vs. US. US = uremic serum; che = chelerythrine; stau = staurosporine.

Figure 6

Effect of uremic serum (US) on TNF‐α gene expression and pAkt in C2C12 myotubes treated with TLR4 inhibitors or silenced for TLR4. (Panel a) Preincubation of myotubes with VIPER, a specific TLR4 inhibitor, prevented the ability of US to up‐regulate TNF‐α. (Panel b) As a next step, we employed gene silencing as an independent method to examine the role of US on TLR4 regulation in muscle. C2C12 were transfected with 60 nM siRNA NC and TLR4‐specific siRNA and the respective mRNA and protein were evaluated after 24 h. TLR4 siRNA decreased TLR4 mRNA and protein in C2C12 myotubes. (Panel c) Effect of US on TNF‐α mRNA in C2C12 silenced for TLR4. TLR4 gene silencing blunted US‐regulated TNF‐α. (Panel d) Effect of US on Akt phosphorylation. C2C12 with no knockdown and transfected with TLR4 siRNA were exposed for 24 hours to NS or US. pAkt was evaluated by western blot and membrane was stripped and reprobed with anti Akt antibody. TLR4 knockdown restored pAkt signalling. All results represent means ± SEM obtained from three independent experiments. #P < 0.01 vs. NC; §P < 0.01 vs. NC + US; °P < 0.05 vs. US; *P < 0.001 vs. US and CP + US. NS = normal serum; US = uremic serum; pAkt = phospho‐Akt; NC = non‐specific negative control siRNA; VIPER = viral inhibitory peptide of TLR4; CP7 = inert control peptide.