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Multicenter Study
. 2020 Apr 1;318(4):F861-F869.
doi: 10.1152/ajprenal.00433.2019. Epub 2020 Jan 31.

Soluble Fas affects erythropoiesis in vitro and acts as a potential predictor of erythropoiesis-stimulating agent therapy in patients with chronic kidney disease

Daniela Mendes Chiloff  1 , Danilo Candido de Almeida  1 , Maria A Dalboni  1 , Maria Eugênia Canziani  1 , Sunil K George  2 , Alshaimaa Mahmoud Morsi  2 , Nadia El-Akabawy  2   3 , Christopher D Porada  2 , Marcelino Souza Durao  1 , Abolfazl Zarjou  4 , Graca Almeida-Porada  2 , Miguel Angelo Goes  1   2
Affiliations
Multicenter Study

Soluble Fas affects erythropoiesis in vitro and acts as a potential predictor of erythropoiesis-stimulating agent therapy in patients with chronic kidney disease

Daniela Mendes Chiloff et al. Am J Physiol Renal Physiol. .

Abstract

Serum soluble Fas (sFas) levels are associated with erythropoietin (Epo) hyporesponsiveness in patients with chronic kidney disease (CKD). Whether sFas could predict the need for erythropoiesis-stimulating agent (ESA) usage and its influence in erythropoiesis remain unclear. We evaluated the relation between sFas and ESA therapy in patients with CKD with anemia and its effect on erythropoiesis in vitro. First, we performed a retrospective cohort study with 77 anemic patients with nondialysis CKD. We performed in vitro experiments to investigate whether sFas could interfere with the behavior of hematopoietic stem cells (HSCs). HSCs were isolated from umbilical cord blood and incubated with recombinant sFas protein in a dose-dependent manner. Serum sFas positively correlated with Epo levels (r = 0.30, P = 0.001) but negatively with hemoglobin (r = -0.55, P < 0.001) and glomerular filtration rate (r = -0.58, P < 0.001) in patients with CKD at baseline. Elevated sFas serum levels (4,316 ± 897 vs. 2,776 ± 749, P < 0.001) with lower estimated glomerular filtration rate (26.2 ± 10.1 vs. 33.5 ± 14.3, P = 0.01) and reduced hemoglobin concentration (11.1 ± 0.9 vs. 12.5 ± 1.2, P < 0.001) were identified in patients who required ESA therapy compared with patients with non-ESA. Afterward, we detected that the sFas level was slight correlated with a necessity of ESA therapy in patients with nondialysis CKD and anemia. In vitro assays demonstrated that the erythroid progenitor cell frequency negatively correlated with sFas concentration (r = -0.72, P < 0.001). There was decreased erythroid colony formation in vitro when CD34+ HSCs were incubated with a higher concentration of sFas protein (1.56 ± 0.29, 4.33 ± 0.53, P < 0.001). Our findings suggest that sFas is a potential predictor for ESA therapy in patients with nondialysis CKD and that elevated sFas could affect erythropoiesis in vitro.

Keywords: anemia and hematopoietic stem cells; chronic kidney disease; erythropoiesis-stimulating agents; soluble Fas.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the author(s).

Figures

Fig. 1.
Fig. 1.
Workflow diagram of the patient selection. A total of 151 patients with nondialysis chronic kidney disease (ND-CKD) with anemia were selected from Nephrology Service at the Federal University of São Paulo and from other previously published studies. After two exclusion criteria, the remaining patients (n = 77) were subdivided into two subgroups: 1) patients with ND-CKD with erythropoiesis-stimulating agent (ESA) therapy (n = 35) and 2) patients with ND-CKD without ESA therapy (n = 42). MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin (Hgb).
Fig. 2.
Fig. 2.
Global correlations between the variables analyzed. Correlations were performed using our data collection comprising 77 patients with nondialysis chronic kidney disease patients with anemia and not submitted to erythropoiesis-stimulating agent (ESA) therapy at baseline. We observed a positive correlation between serum erythropoietin (EPO) versus soluble Fas (sFas), hemoglobin (Hgb) versus estimated glomerular filtration rate (eGFR) and Hgb versus transferrin. In contrast, we detected a negative correlation among Hgb versus sFas, eGFR versus sFas, and EPO versus transferrin. P < 0.05.
Fig. 3.
Fig. 3.
Direct comparison of soluble Fas (sFas), hemoglobin (Hgb), and estimated glomerular filtration rate (eGFR) levels in patients with erythropoiesis-stimulating agent (ESA) and non-ESA therapy. sFas, Hgb, and eGFR were compared between patients that needed ESA therapy and those who did not need ESA therapy at their baseline. The violin-plot chart shows clearly that patients who further needed ESA therapy in our followup presented higher levels of sFas and a lower index of Hgb and eGFR at baseline than patients who did not submitted to ESA therapy. After 6 yr of followup, we verified that eGFR and Hgb index remained decreased in patients who needed ESA treatment. P < 0.05.
Fig. 4.
Fig. 4.
Flow cytometry characterization of enriched CD34+ human umbilical cord blood mononuclear cells (UCB-MNCs). First, we verified the general profile of unfractionated UCB-MNCs regarding the expression of CD34+, CD38+, CD133+, CD71+, glycophorin A (gly A), and CD36+. In addition, unfractionated cells presented higher levels of CD38+ (38.6%) compatible with a more differentiated phenotype. Afterward, we performed magnetic selection and enriched the UCB-MNC population with CD34+ cells (22.9%). This enriched population presented a typical hematopoietic progenitor stem cell phenotype, which was not detected in unfractionated cells (CD34+CD38−CD133+). The expression of CD133+, CD71+, glycophorin A, and CD36+ between both populations (CD34+ and unfractionated) was not statistically affected. SSC, side scatter.
Fig. 5.
Fig. 5.
Analyze of the human recombinant soluble Fas (sFas) influence on in vitro erythropoiesis. The concentration of sFas protein negatively correlated with the number of erythroid progenitor colonies (BFU-e/CFU-e). Also, it was observed that higher concentrations of recombinant human sFas protein presented smaller number of global erythroid colonies. BFU-e, burst forming unit-erythroid; CFU-e, colony forming unit-erythroid; Lc-sFas group, low concentrations of recombinant human sFas protein (0, 0.5, and 1 ng/mL); Hc-sFas group, high concentrations of recombinant human sFas protein (2, 4, and 8 ng/mL). P < 0.05.
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