Epoetin delta reduces oxidative stress in primary human renal tubular cells

Abstract

Erythropoietin (EPO) exerts (renal) tissue protective effects. Since it is unclear whether this is a direct effect of EPO on the kidney or not, we investigated whether EPO is able to protect human renal tubular epithelial cells (hTECs) from oxidative stress and if so which pathways are involved. EPO (epoetin delta) could protect hTECs against oxidative stress by a dose-dependent inhibition of reactive oxygen species formation. This protective effect is possibly related to the membranous expression of the EPO receptor (EPOR) since our data point to the membranous EPOR expression as a prerequisite for this protective effect. Oxidative stress reduction went along with the upregulation of renoprotective genes. Whilst three of these, heme oxygenase-1 (HO-1), aquaporin-1 (AQP-1), and B-cell CLL/lymphoma 2 (Bcl-2) have already been associated with EPO-induced renoprotection, this study for the first time suggests carboxypeptidase M (CPM), dipeptidyl peptidase IV (DPPIV), and cytoglobin (Cygb) to play a role in this process.

Figure 1

Induction of oxidative stress in confluent hTECs by incubation with GO (0 to 100 IU/mL) during 0 to 240 minutes. By measuring DCF fluorescence, a concentration-dependent accumulation of ROS was observed during the time.

Figure 2

GO-induced oxidative stress (240 minutes) in hTECs either pre/coincubated with epoetin delta or not. Epoetin delta attenuated oxidative stress in a dose-dependent way by reducing ROS formation. The data are expressed as the mean ± SD of 4 monolayers per condition from a single experiment representative of 6 separate experiments. *P < .05, °P < .01 compared to the control.

Figure 3

Comparison of epoetin delta- and epoetin alfa-induced effects on GO-induced oxidative stress (240 minutes) in cell cultures originating from two different kidney specimens, one showing EPO-induced anti-oxidative effects (a) and one where neither epoetin delta nor epoetin alfa had any effect on oxidative stress (b). Both ESA's showed a similar effect on GO-induced oxidative stress: either they were both able to reduce the amount of ROS formed or not. The data are expressed as the mean ± SD of 4 monolayers per condition from a single experiment representative of 2 separate experiments. *P < .05 compared to the control.

Figure 4

Immunofluorescent staining of EPOR in two cultures of hTECs that show an epoetin delta-induced effect on oxidative stress (a) or not (b). EPOR signal is visualized using the M-20 anti-EPOR antibody and an FITC-labeled (green fluorescence) secondary antibody. Punctate intracellular staining is seen in both cultures while membranous staining is only seen in cultures that showed epoetin delta-induced effects on oxidative stress.

Figure 5

Quantitative real-time RT-PCR analysis of HO-1 (a), AQP-1 (b), and Bcl-2 (c) expressions in mixed hTECs under basal conditions and after GO-induced oxidative stress (1 IU/mL) either or not in the presence of epoetin delta (100 IU/mL). GAPDH was used as endogenous control housekeeping gene. Preconditioning the cells with EPO resulted in a significant upregulation of HO-1 and AQP-1 mRNA. GO-induced oxidative stress further increased HO-1, AQP-1, and Bcl-2 mRNA expressions with maximum levels 60 minutes after induction of oxidative stress. Data are presented as the mean ± SD of triplicate determinations of 2 runs (i.e., 6 values each). *P < .05 versus no GO, °P < .05 versus epoetin delta 0 IU/mL, ^#P < .05 versus 30 minutes GO incubation.

Figure 6

Comparison of the relative (normalized to GAPDH and to calibrator sample) expressions of CPM (a), DPPIV (b), and Cygb (c) under basal conditions and after GO-induced oxidative stress (1 IU/mL) in cell cultures originating from two different kidney specimens, one showing EPO-induced anti-oxidative effects and one without any effect of EPO on oxidative stress. Preconditioning the cells with EPO resulted in a significant upregulation of CPM and DPPIV, mRNA. GO-induced oxidative stress further increased CPM, DPPIV and Cygb mRNA expressions with maximum levels 60 minutes after induction of oxidative stress. Remarkably, the cell culture in which epoetin delta was not able to induce a protective effect against oxidative stress was also not able to induce upregulation of those mRNA's. Data are presented as the mean ± SD of triplicate determinations representative of 2 separate runs (i.e., 6 values each). *P < .05 versus no GO, °P < .05 versus epoetin delta 0 IU/mL.