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.
Oxidative stress occurs when there is an imbalance between the generation of reactive oxygen species (ROS) and a biological system's ability to detoxify the reactive intermediates or repair the resulting damage [
Although it is known that EPO is able to attenuate (acute and chronic) kidney failure [
As a next step, we investigated (i) whether the EPO receptor (EPOR) is involved herein, (ii) the contribution of genes previously linked to EPO-induced protective mechanisms, such as heme oxygenase-1 (HO-1), aquaporin-1 (AQP-1), and B-cell CLL/lymphoma-2 (Bcl-2), and (iii) a possible role for carboxypeptidase M (CPM) dipeptidyl peptidase IV (DPPIV) and cytoglobin (Cygb) genes not yet linked to EPO-induced renoprotective mechanisms but to oxidative stress in general.
Primary hTECs were isolated as previously described [
Confluent hTECs were incubated with different concentrations of epoetin delta (Dynepo, Shire Pharmaceuticals Ltd.; 0–5–100 IU/mL) or epoetin alfa (Eprex, Janssen-Cilag; 100 IU/mL) for 24 hours before or concomitantly with the induction of oxidative stress (at least 4 wells per condition).
Oxidative stress was induced by exposure of confluent hTECs to different concentrations of GO (Sigma; 0–0.1–1–5–10–50–100 IU/mL) for different time periods (20 minutes, 30 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, and 4 hours) [
The GO-induced oxidative stress, assessed as the amount of generated cellular radicals, was measured by the
The mRNA expression of HO-1, AQP-1, Bcl-2, CPM, DPPIV, and Cygb in hTECs was analyzed by means of the quantitative real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) using the fluorescent TaqMan methodology and the ABI Prism 7000 Sequence Detection System (Applied Biosystems). According to the manufacturer’s instructions, cDNA was synthesized from total RNA extracted with the High Pure RNA Isolation Kit (Roche) using the High-Capacity cDNA Archive kit (Applied Biosystems). Ready to use, predesigned, primer and probe sets (Applied Biosystems) for human genes of interest (Hs00157965_m1 for HO-1, Hs00166067_m1 for AQP-1, Hs00153350_m1 for Bcl-2, Hs00266395_m1 for CPM, Hs00175210_m1 for DPPIV, Hs00370478_m1 for Cygb) and the housekeeping gene GAPDH (Hs99999905_m1) were used according to the manufacturer's guidelines. The mRNA quantity of the investigated genes was analyzed in triplicate, normalized against GAPDH, and expressed in relation to a calibrator sample using the comparative Ct method. Control cell cultures, that is, cultures receiving neither GO nor epoetin delta served as the calibrator sample, which was given a gene of interest/GAPDH mRNA expression ratio of 1.
Confluent hTECs were fixed in 4% formaldehyde for 10 minutes. Cells were incubated overnight with the M-20 anti-EPOR antibody (Santa Cruz Biotechnology) and subsequently with FITC-labeled goat anti-rabbit IgG antibody during 2 hours. Sections in which the primary antibody was omitted served as negative control.
Because of the interindividual variation in DCF fluorescence and mRNA expression levels of some investigated genes in cell monolayers derived from individual kidney samples, some data are represented as single representative experiments. Data are presented as mean ± SD. Statistics were performed with SPSS 16.0. Comparisons between the study groups for each time point and/or GO dose were assessed using a Kruskal-Wallis test, followed by a Mann-Witney
Quantification of oxidative stress resulting from the addition of GO to hTECs at different concentrations (0 to 100 IU/mL) and time points (10 to 240 minutes) shows that incubation of hTECs with GO resulted in a concentration-dependent accumulation of ROS during time (Figure
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.
Preincubation of hTECs with epoetin delta (24 hours before addition of GO; 0–0.1–1–10 IU/mL) significantly protects the cells against oxidative stress as indicated by a dose-dependent, attenuated ROS production. Furthermore, it was found that epoetin delta added to the culture medium concomitantly with the induction of oxidative stress attenuated the formation of ROS as well, be it in a less pronounced way. The epoetin delta-induced effects on ROS formation were only observed in 6 out of 9 experiments (hTECs cultures of 9 different kidney specimens) (Figure
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. *
Since epoetin delta did not reduce ROS formation in all hTECs cultures, it was investigated whether this was due to either cell culture and/or EPO characteristics. This was done by comparing the effects of epoetin delta to these of epoetin alfa. It became clear that the outcome of these two erythropoiesis-stimulating agents (ESA) was similar as both of them either or not protected the same cell cultures from oxidative stress (Figure
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. *
Protection
No protection
As epoetin delta and alfa induced similar effects on oxidative stress in the same culture, the presence or absence of protective mechanisms must be due to culture characteristics. Because EPO exerts its actions via the EPOR, its expression was investigated in two different cultures: one of them showing epoetin delta-induced protection whereas the other did not. In protective as well as nonprotective cultures, EPOR expression was seen in intracellular vesicles (Figure
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.
In the present study, the mRNA expression of HO-1, AQP-1, and Bcl-2 was assessed in cultures that were either preincubated with epoetin delta before the induction of oxidative stress or not. Under basal conditions (no GO incubation), preconditioning of hTECs with epoetin delta (100 IU/mL) resulted in a significant upregulation of HO-1 (1.00 ± 0.31 versus 1.56 ± 0.37,
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). *
HO-1/GAPDH expression
AQP-1/GAPDH expression
Bcl-2/GAPDH expression
Figure
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). *
In the present study, oxidative stress was induced using glucose oxidase (GO), an enzyme that, in the presence of glucose, produces H2O2, the most important source of cellular ROS. We showed that GO indeed induced a concentration-dependent accumulation of ROS in primary hTECs during time. Pretreatment of the primary hTECs with epoetin delta (5 or 100 IU/mL) resulted in a statistically significant reduction of GO-induced ROS production. As this effect was less pronounced when epoetin delta was administered together with GO, one may assume that EPO (epoetin delta) exerts a direct effect on renal tubular cells by (pre)conditioning these cells towards protection. Interestingly, epoetin delta pretreatment did not protect against oxidative stress in all cultures investigated. In further experiments, we found that epoetin delta and epoetin alfa (a widely used ESA, known to have renoprotective effects both in vivo [
In an attempt to explain this intriguing finding, the EPOR expression was investigated in these cell cultures using the M-20 antibody, recently identified as a specific anti-EPOR antibody [
In order to try to identify some specific pathways through which EPO could exert these observed anti-oxidative effects, the EPO-induced effects on HO-1, AQP-1, and Bcl-2 mRNA were investigated. These three genes have already intensively been studied in in vivo experimental setups and in line with previous studies performed in our laboratory [
Induction of HO-1 (or HSP32), known as a protein with antiapoptotic, anti-inflammatory, and cytoprotective properties [
The cytoprotective effects of EPO preconditioning have also been linked to the prevention of renal IRI-induced downregulation of AQP-1 [
In agreement with the observation that EPO exerts antiapoptotic effects and that Bcl-2 has been reported to be involved in several models of kidney injury [
Interestingly, the epoetin delta-induced upregulation of HO-1, AQP-1, and Bcl-2 expression could only be observed in cell cultures in which the compound induced an attenuation of ROS production again pointing to a direct protective effect on the tubular cells.
As we also aimed to get further insight into the mechanisms underlying EPO-induced antioxidant cytoprotection, expressions of three other genes potentially involved in the protective process were investigated: CPM, DPPIV, and Cygb. Epoetin delta treatment resulted in an upregulation of these genes under basal conditions, which was further increased in cell cultures that showed an epoetin delta-induced protection against GO-induced oxidative stress (and not in cultures that did not show this protection). Although data of the present study do not allow drawing clear-cut conclusions about a role for CPM, DPPIV, and Cygb in the EPO’s anti-oxidative properties, the observed EPO-induced alterations in their mRNA expression are worth being considered in view of the already known functions mentioned below.
CPM is a membrane glycoprotein which specifically removes C-terminal basic residues from peptides and proteins [
DPPIV is the most intensively studied member of the proline-specific dipeptidyl peptidase (DPP) family. Widely expressed among organs and body fluids and having different substrates in different organs [
Both being cell surface peptidases, CPM and DPPIV have a lot in common and are frequently studied together [
Cygb, sharing a common ancestry with myoglobin, is a recently discovered member of the vertebrate globin family [
In the present work, evidence was found for a direct cytoprotective effect of epoetin delta on renal tubular cells. This EPO-induced effect on renal tubular cells may be an important contribution to the EPO-induced renal protective effect seen in vivo. Furthermore, comparison of the localization of the EPOR in EPO-responding versus nonresponding cultures allows to suggest that the cytoprotective effect of epoetin delta in primary hTECs is mediated by the EPOR. Interestingly, the expression of six genes under study was significantly upregulated upon EPO administration in and only in those cell cultures in which an EPO-induced cytoprotective effect was seen, allowing (i) to confirm a role of HO-1, AQP-1, and Bcl-2 in the EPO-induced preconditioning of cells and (ii) to speculate about a role of CPM, DPPIV, and Cygb in this process. Further research with regard to the effects of EPO on the protein expression and function of these proteins is warranted.
This work would not have been possible without the generous cooperation of Dr. Braeckman (University Hospital Brussels), Dr. Verkoelen, and Dr. Asselman (Erasmus Medical Center Rotterdam), Dr. Dekuyper (AZ Maria Middelares, Ghent), and Professor Oosterlinck (University Hospital, Ghent). This work was funded by BOF (Bijzonder Onderzoeksfonds) University of Antwerp and by a research Grant from Shire Pharmaceuticals. Anja Verhulst is a (post) doctoral fellow of the Fund for Scientific Research Flanders (FWO). Xiang-hua Hou is a student of the Shandong University in China and was a fellow of a coculture program between China and Belgium that has been financed by both the China scholarship council and the department of Pathophysiology, University of Antwerp, Belgium. Professor De Meester and Dr. Deiteren (Laboratory of Medical Biochemistry, University of Antwerp) are thanked for their advice concerning the literature dealing with DPPIV and CPM. Annelies De Beuf and Xiang-hua Hou equally contributed to this manuscript