ApoL1 renal risk variants induce aberrant THP-1 monocyte differentiation and increase eicosanoid production via enhanced expression of cyclooxygenase-2

Abstract

Apolipoprotein L1 ( ApoL1) genetic variants are strongly associated with kidney diseases. We investigated the role of ApoL1 variants in monocyte differentiation and eicosanoid production in macrophages, as activated tissue macrophages in kidney might contribute to kidney injury. In human monocyte THP-1 cells, transient overexpression of ApoL1 (G0, G1, G2) by transfection resulted in a 5- to 11-fold increase in CD14 and CD68 gene expression, similar to that seen with phorbol-12-myristate acetate treatment. All ApoL1 variants caused monocytes to differentiate into atypical M1 macrophages with marked increase in M1 markers CD80, TNF, IL1B, and IL6 and modest increase in the M2 marker CD163 compared with control cells. ApoL1-G1 transfection induced additional CD206 and TGFB1 expression, and ApoL1-G2 transfection induced additional CD204 and TGFB1 expression. Gene expression of prostaglandin E₂ (PGE₂) synthase and thromboxane synthase and both gene and protein expression of cyclooxygenase-2 (COX-2) were increased by ApoL1-G1 and -G2 variants compared with -G0 transfection. Higher levels of PGE₂ and thromboxane B₂, a stable metabolite of thromboxane A₂, and transforming growth factor (TGF)-β1 were released into the supernatant of cultured THP-1 cells transfected with ApoL1-G1 and -G2, but not -G0. The increase in PGE₂, thromboxane B₂, and TGF-β1 was inhibited by COX-2-specific inhibitor CAY10404 but not by COX-1-specific inhibitor SC-560. These results demonstrate a novel role of ApoL1 variants in the regulation of monocyte differentiation and eicosanoid metabolism, which could modify the immune response and promote inflammatory signaling within the local targeted organs and tissues including the kidney.

Keywords: TGF-β1; apolipoprotein L1; cyclooxygenase-2; macrophage polarization.

Fig. 1.

Apolipoprotein L1 (ApoL1) overexpression in transiently transfected THP-1 cells. A: ApoL1 protein expression was observed in THP-1 cells transiently transfected with ApoL1-G0, ApoL1-G1, and ApoL1-G2 and compared with empty vector (EV) or mock transfection, as indicated (see methods for definition of constructs). Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-ApoL1 antibody (top) or anti-actin antibody (bottom) for loading control. Immunoblotting shown is a representative of 2 independent experiments. The density of ApoL1-G0, ApoL1-G1, and ApoL1-G2 over β-actin was quantified by densitometry and normalized to EV; n = 6. Expression levels of each of the ApoL1 variants were similar; ns, not significanct (P > 0.05). B: THP-1 cells (20,000/well) transfected with EV for 12 h and with ApoL1-G0, -G1, and -G2 for the indicated time were incubated for 12 h in complete culture medium containing 0.4 mg/ml MTT in 5% CO₂ at 37°C. Formazan production was measured spectrophotometrically at 570 nm in a microplate reader. Cell viability was normalized with EV transfection after 12 h; n = 3. P < 0.05 vs. EV.

Fig. 2.

Characterization of macrophage subtypes differentiated from THP-1 cells. THP-1 cells were treated with phorbal myristol acetate (PMA; A), IFNγ and LPS (B), and IL-4 and IL-13 (C). mRNA expression was quantified by qRT-PCR. Values of relative mRNA levels of each marker were expressed as fold increase compared with vehicle treatment. PMA was used to generate the M0 state, whose macrophage markers are CD14 and CD68; IFNγ and LPS were used to induce M1 state, the putative markers are CD80, TNF, IL1B, and IL6; and IL-4 and IL-13 were used to induce the M2 state, whose macrophage markers are CD163, CD204, CD206, and TGFB1. Veh, vehicle; n = 8–9. †P < 0.05 vs. vehicle, §P < 0.05 vs. M0.

Fig. 3.

Profile of cell surface markers induced by ApoL1 overexpression. Cultured THP-1 cells were transfected with EV or ApoL1-G0 (A), ApoL1-G1 (B), or ApoL1-G2 (C) variants as indicated. The average value of empty vector was used to normalize the relative mRNA expression for each marker. For all ApoL1 variants, mRNA expression of M1 markers was markedly increased and there was modest increase in gene expression of some M2 markers, indicating atypical M1 macrophage differentiation. As shown, M0 macrophage markers are CD14 and CD68, M1 macrophage markers are CD80, TNF, IL1B, and IL6, and M2 macrophage markers are CD163, CD204, CD206, and TGFB1. Values of relative mRNA level of each marker are expressed as fold increase compared with empty vector; n = 8–10. *P < 0.05 vs. EV, #P < 0.05 vs. ApoL1-G0.

Fig. 4.

ApoL1 risk variants induce cyclooxygenase (COX)-2 expression in THP-1 cells. Cultured THP-1 cells were transfected with EV or ApoL1-G0, ApoL1-G1, or ApoL1-G2 variants as indicated. A: relative COX-1 and COX-2 mRNA expression in ApoL1 transiently transfected THP-1 cells. Total mRNA was isolated and mRNA expression quantified by qRT-PCR. The average value of empty vector was used to normalize the relative mRNA expression for each cyclooxygenase. Values of relative mRNA level were expressed as fold increase compared with empty vector; n = 11–12. *P < 0.05 vs. EV; #P < 0.05 vs. ApoL1-G0. B: COX-1 and COX-2 protein expression in ApoL1 transiently transfected THP-1 cells. Cell lysates were subjected to SDS-PAGE before immunoblotting with indicated antibodies. Density of COX-1/2 normalized to β-actin was quantified by densitometry and further normalized to EV. n = 6; ns, not significant (P > 0.05).

Fig. 5.

ApoL1 risk variants increase prostanoid mRNA expression in THP-1 cells. Cultured THP-1 cells were transfected with EV or ApoL1-G0 (A), ApoL1-G1 (B), or ApoL1-G2 (C) variants as indicated. mRNA expression of prostanoid synthases was determined by qRT-PCR. PGIS, PGI2 synthase; PGDS, PGD2 synthase; PGES, PGE2 synthase; TBXAS1, thromboxane A₂ synthase. The average value of empty vector was used to normalize the relative mRNA expression for each prostanoid synthase. Values of relative mRNA level are expressed as fold increase compared with empty vector; n = 9–10. *P < 0.05 vs. EV; #P < 0.05 vs. ApoL1-G0.

Fig. 6.

ApoL1 risk variants increase prostanoid secretion in THP-1 cells. PGE₂ (A) and TBX₂ (B) concentrations in supernatant of ApoL1-transfected THP-1 cells were determined using respective ELISA kits. Data were summarized from 2 independent experiments; n = 9. *P < 0.05 vs. EV; #P < 0.05 vs. ApoL1-G0.

Fig. 7.

ApoL1 risk variant-mediated prostanoid secretion is inhibited by a COX-2 inhibitor in THP-1 cells. PGE₂ (A) and TBX₂ (B) concentrations in supernatant of ApoL1-transfected THP-1 cells were determined using respective ELISA kits. SC, SC-560, a COX-1 specific inhibitor; CAY, CAY10404, a COX-2 specific inhibitor. CAY10404 but not SC-560 inhibited ApoL1-G1- and ApoL1-G2-induced increase in PGE₂ and TBX₂ production, suggesting a central role for COX-2 in prostanoid production in these cells in response to overexpression of ApoL1 variants. Data are summarized from 2 independent experiments; n = 8–9. †P < 0.05 vs. vehicle; *P < 0.05 vs. EV.

Fig. 8.

ApoL1 risk variants induce TGF-β1 secretion from THP-1 cells. TGF-β1 concentration in cell lysates or the supernatant of ApoL1-transfected THP-1 cells were determined by ELISA. A: the majority of TGF-β1 was observed in the supernatant rather than within cells in THP-1 cells with EV or with ApoL1 overexpression; n = 9. *P < 0.05 vs. EV; #P < 0.05 vs. ApoL1-G0. B: TGF-β1 levels in the supernatant of ApoL1-transfected THP-1 cells were reduced by a COX-2 inhibitor. Data are combined from 3 independent experiments. # P < 0.05 vs. ApoL1-G0; †P < 0.05 vs. vehicle.