Contribution of the uremic milieu to an increased pro-inflammatory monocytic phenotype in chronic kidney disease

Abstract

Intermediate (CD14⁺⁺CD16⁺) monocytes have important pro-inflammatory and atherogenic features and are increased in patients with chronic kidney disease (CKD). The present study aims to elucidate the role of the uremic milieu and of platelet activation in monocyte differentiation. Monocyte subtypes were analyzed in CKD patients (n = 193) and healthy controls (n = 27). Blood from healthy controls (Ctrl; n = 8) and hemodialysis patients (HD; n = 8) was centrifuged, and plasma (pl) was exchanged between Ctrl and HD (Ctrlcells/HDpl and HDcells/Ctrlpl) or reconstituted as original (Ctrlsham and HDsham) and incubated for 24 h (T24). Monocyte differentiation and platelet aggregation to monocytes (MPA) was assessed by flow cytometry. Especially, a higher proportion of CD14⁺⁺CD16⁺ monocytes was found in hemodialysis (HD) patients (p < 0.01). In plasma exchange experiments, Ctrl cells/HD pl T24 showed an increased percentage of CD14⁺⁺CD16⁺ monocytes versus Ctrl sham (33.7% ± 15 vs. 15.7% ± 9.6; P < 0.005), comparable to the level of CD14⁺⁺CD16⁺ monocytes in the HD sham condition. The percentage of CD14⁺⁺CD16⁺ monocytes was lowered by suspending HD cells in Ctrl pl (18.4% ± 7.8 vs. 36.7% ± 15 in HD sham; P < 0.005) reaching the level of the Ctrl sham condition (15.7% ± 9.6). A mixture of uremic sulfates increased CD14⁺⁺CD16⁺ monocytes compared to control (19.8 ± 9.6% vs. 15.8 ± 10.9%; P < 0.05), paralleled by a rise MPA. Blocking MPA by abciximab, a potential therapeutic strategy, or anti-CD62P did not inhibit differentiation towards the CD14⁺⁺CD16⁺ monocytes. In conclusion, in the present cohort, CD14⁺⁺CD16⁺ monocytes are especially increased in HD patients and this can at least in part be attributed to the presence of the uremic milieu, with uremic sulfates inducing a reversible shift towards pro-inflammatory CD14⁺⁺CD16⁺ monocytes.

Figure 1

Monocyte subpopulations in CKD patients versus healthy control (Ctrl). CKD: patients with chronic kidney disease stages 1 to 5; HD: hemodialysis patients; PD: peritoneal dialysis patients. Results are presented a mean ± SD. One significance symbol: p < 0.05, two symbols: p < 0.01; * vs. Ctrl; ° vs. CKD; § vs. HD. CD14⁺⁺CD16⁻: classical monocytes; CD14⁺⁺CD16⁺: intermediate monocytes; CD14⁺CD16⁺⁺: non classical monocytes.

Figure 2

Monocyte subpopulations (A) and monocyte subpopulation-platelet aggregates (B) in healthy controls (Ctrl; white bars) and HD patients (grey bars) immediately after collection. Sodium citrate blood from healthy control (Ctrl; n = 8) and hemodialysis patients (HD; n = 8) Results are presented a mean ± SD. *p < 0.05, **p < 0.005 vs. Ctrl. CD14⁺⁺CD16⁻: classical monocytes; CD14⁺⁺CD16⁺: intermediate monocytes; CD14⁺CD16⁺⁺: non classical monocytes; CD14⁺CD16⁻: negatives; MPA: monocyte-platelet aggregates.

Figure 3

Effect of the uremic milieu (HD plasma (pl)) on healthy monocyte subpopulations compared to the effect of the healthy milieu (Ctrl plasma (pl)) on HD monocyte subpopulations. Sodium citrate blood from healthy controls (Ctrl) (n = 8) and hemodialysis patients (HD; n = 8) was centrifuged and the autologous plasma was completely removed and re-added (sham) or exchanged for blood type matched HD plasma and vice versa and incubated for 24 h. Data express monocytes subpopulations (%); upper right: zooms in on intermediate monocytes (CD14⁺⁺CD16⁺). Results are presented a mean ± SD. One significance symbol: p < 0.05, two symbols: p < 0.005 and three symbols: p < 0.001. *vs. Ctrl sham T0; ^§§vs. Ctrl sham T24; °vs. Ctrl cell + HD pl; ªvs. HD sham T0; ^#vs. HD sham T24.

Figure 4

Effect of uremic toxins on monocyte differentiation. Sodium citrate blood from healthy donors (n = 8) was incubated for 24 h at 37 °C in the presence of the salt control solution (Ctrl), sulfate mixture [indoxyl sulfate (IxS), p-cresyl sulfate (pCS), phenyl sulfate (PhS); sulfates], glucuronide mixture [indoxyl glucuronide (IG), p-cresyl glucuronide (pCG), phenyl glucuronide (PhG); glucuronides] or thrombine receptor activator peptide (TRAP). Data express the percentage of monocyte subtypes. Results are presented a mean ± SD. *p < 0.05; **p < 0.0005 vs. Ctrl.

Figure 5

Effect of uremic toxins on monocyte subpopulation-platelet aggregation (%). Sodium citrate blood from healthy donors (n = 8) was pre-incubated in the absence and presence of abciximab or anti-CD62P, and incubated for 24 h in the presence of salt control solution (Ctrl), (A) sulfate mixture or (B) thrombine receptor activator peptide (TRAP). Results are presented a mean ± SD. *p < 0.05 vs. Ctrl; ^#/°p < 0.05 vs. sulfates/TRAP; ^##/°°p < 0.005 vs. sulfates/TRAP; °°°p < 0.0005 vs. TRAP. MPA: monocyte-platelet aggregates. CD14⁺⁺CD16^-: classical monocyte-platelet aggregates; CD14⁺⁺CD16⁺: intermediate monocyte-platelet aggregates; CD14⁺CD16⁺⁺: non classical monocyte-platelet aggregates; CD14⁺CD16⁻: CD14⁺CD16^–platelet aggregates.

Figure 6

Representative plot illustrating identification of monocyte subpopulations. (A) Gating of monocytes based on CD86 positivity and (B) gating of monocyte subpopulations based on CD14 and CD16 positivity.