Abstract
The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin β pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.
Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules.
These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.
Introduction
Anemia is one of the most common diseases in humans [1]. Severe anemia and hypoxia stimulate the production of erythropoietin (Epo) by the kidney [2-8]. The increase in Epo production is measured by the increases in serum and urine Epo concentrations and in Epo mRNA expression in the kidney [4-11]. Serum or urine Epo concentrations have been measured by radio immunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) using antibodies against Epo [4, 9-12]. However, Epo protein expression in the kidney or liver has not been measured accurately, since Western blotting of Epo has not been possible. Serum/urine Epo concentrations, and Epo mRNA and HIF1α/2α expressions in the kidney and liver have been used as a substitute for Epo protein expression in the kidney and liver [4-13]. However, the increase of kidney-produced Epo has not been shown to increase to the same degree. This suggest the possibility that Epo production by the liver may have some role for the increase of Epo production in response to severe hypoxia [2, 3, 14].
The discovery of Epo led to the invention of erythropoiesis stimulating agents (ESAs) to treat anemic patients with chronic kidney disease (CKD) [15-17]. ESAs have also been illegally used by athletes to improve physical activity, leading to tests for doping [18]. The World Anti-Doping Agency (WADA) Technical Documents for Epo (TD2014EPO in TD2019INDEX) recommended the use of isoelectrical focusing (IEF) and/or SAR-PAGE after enrichment for Epo through ultrafiltration, selective protein precipitation or immunopurification to detect Epo in the urine or serum/plasma [19]. ELISA or Liquid Chromatography/Mass Spectrometry (LC/MS) after the pre-purification of urine are also useful. These recommendations clearly show that the detection of Epo by Western blotting is difficult.
We have reported a new method of Western blot analysis succeeding in the detection of kidney-produced Epo [20]. We have reported that Epo is produced by the cortical nephrons in control condition using in situ hybridization, immunohistochemistry and real time PCR with microdissected nephron segments. We also showed that Epo production by the intercalated cells of the collecting ducts is regulated by renin-angiotensin-aldosterone system [20]. We modified our method to detect plasma and urinary Epo. We report the new Western blot method for the detection of Epo protein in the plasma or urine. Using our new method, we investigated the role of kidney and liver for Epo production in response to severe hypoxia.
Methods
Materials and animals
Male Sprague Dawley rats (Japan SLC, Hamamatsu, Japan) were used in our study. In the severe hypoxia experiments, rats were exposed to 7% O2 and 93% N2 for 1-4 hr, which is known to stimulate rapid Epo production and is closer to the conditions at the summit of Mount Everest [9, 21]. For the detection of ESAs in plasma and urine, large doses of ESAs were administered to some rats through the vena cava, and plasma and urine were collected after 30 min from the aorta and bladder, respectively. Animal experiments were conducted in accordance with the Kitasato University Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (Approval No. 2018-030, 25-2). Blood and urine were collected from patients with CKD who received ESAs and from patients with severe anaemia. Urine was concentrated using a Vivaspin (GE Healthcare Bio-Science AB, Sweden). Our protocols were checked and approved by the above committee and the Ethics Committee at Kitasato University Medical Center (25-2, 2018032, 2019029). Informed consent was obtained from all patients.
Real-time PCR in control and hypoxic rats
The renal cortex and liver were collected from control and hypoxic rats. RNA was extracted using the RNeasy Mini Kit (Qiagen, 74106) and Qiacube. cDNA was synthesized using a Takara PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara, 6210). Real-time PCR was performed using probes from Applied Biosystems and Premix Ex Taq (Takara, RR39LR). Probes were obtained from Applied Biosystems (Epo, Rn01481376_m1; HIF2α, Rn00576515_m1; HIF1α, Rn01472831_m1; PHD2, Rn00710295_m1, Thermo Fisher Scientific, USA). β-actin (Rn00667869_m1) was used as an internal standard.
Western blot analysis
Western blot analysis was performed as described previously [20, 22]. Protein was collected from the renal cortex and liver using CelLytic MT (Sigma-Aldrich, C-3228) plus protease inhibitor (Roche, 05892970001). Urine samples were obtained from rats injected large doses of ESAs 30 min before the collection and from anemic patients. Plasma was obtained from rats injected large amount of ESAs and from patients with iron deficiency anaemia or CKD. An anemic patient was treated by iron supplementation and blood was collected at severe and mild anemia and after complete recovery. Blood was also collected form CKD patients who were treated by the injection of epoetin β pegol and control subject. Informed consent was obtained from all patients. Urine samples were concentrated by Vivaspin (GE Healthcare Bio-Science AB) and used for western blot. Plasma samples were used directly or after deglycosylation as described below. After SDS-PAGE, proteins were transferred to a PVDF membrane (Immobilon-P, Merck Millipore, IPVH00010) with 160 mA for 90 min. The membrane was blocked with 5% skim milk (Morinaga, Japan) for 60 min and incubated with the antibody against Epo (Santa Cruz, sc-5290, 1:500-2,000) for 60 min at room temperature. After washing, the membrane was incubated with a secondary antibody (the goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, 115-035-166, 1:5,000) for 60 min. Bands were visualized by the ECL Select Western Blotting Detection System (GE Healthcare Bio-Science AB, RPN2235) and LAS 4000 (Fujifilm). The band intensity was normalized against that of β-actin (MBL, M177-3), which was measured after stripping and reprobing the membrane (stripping solution, Wako, RR39LR). In some experiments, another antibody against Epo (clone AE7A5, MAB2871, R & D Systems) was used to compare the specificity of the antibody.
Deglycosylation study
Since the Epo protein is a glycosylated protein, deglycosylation was performed. N-glycosidase F (PNGase, Takara, 4450) was used as previously reported [22]. In brief, a mixture of 7.5 μl of plasma, 2.5 μl of water, and 1μl of 10% SDS was boiled for 3 min. Then, 11 μl of 2x stabilizing buffer was added, and 2 μl of PBS or PNGase was added. The samples were incubated in a water bath at 37°C for 15-20 hr. After incubation, the samples were spun down, and the supernatant was collected. For urine analysis, 7.5 μl – 30 ml of urine was used either directly or after concentration by Vivaspin. To 10 μl of concentrated urine, 1 μl of 10% SDS was added and boiled for 3min. The subsequent steps were the same as those performed for plasma. In the kidney and liver samples, 10 μl samples were treated in the same manner as urine. The 2x stabilizing buffer contained 62.5 mM Tris-HCl (pH 8.6), 24 mM EDTA, 2% NP-40 and 4% 2-mercaptoethanol.
Plasma Epo concentration measurements
Plasma and urine were collected from control and hypoxic rats. Plasma, serum and urine were also collected from patients with renal anaemia treated with ESAs or from patients with iron-deficient anaemia. Plasma, serum and urine Epo concentrations were measured by CLEIA (SRL, Tokyo, Japan, using Access Epo by Beckman Coulter, Brea, USA).
Immunohistochemistry of Epo production sites
Immunohistochemistry (IHC) of Epo expression was performed in control and severe hypoxic rats as previously reported [20, 23, 24]. A polyclonal antibody against the same sequences as sc-5290 was used, namely, sc-7956. Images were obtained using an optical microscope (Axio Imager. M2, Carl Zeiss, Oberkochen, Germany) with a digital camera (AxioCam 506, Carl Zeiss). Captured images were analysed using an image analysis system (ZEN 2, Carl Zeiss).
LC/MS analysis of band from western blot
The 22 kDa band of the western blot was excised and subjected to LC/MS as previously reported [25]. Negative staining was used to detect deglycosylated recombinant Epo. The negatively stained protein bands were excised from the SDS-PAGE gel, and in-gel tryptic digestion was carried out using ProteaseMAX reagent (Promega, WI, USA) according to the manufacturer’s protocol. The peptides were separated by L-column2 ODS (3 μm, 0.1 x 150 mm, CERI, Tokyo, Japan) at a flow rate of 500 nl/min using a linear gradient of acetonitrile (5% to 45%). Nano-LC-MS/MS analyses were performed with an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, MA, USA) as previously described [25].
Statistical analyses
Statistical analyses were performed using Excel Statics (BellCurve, Tokyo, Japan). Statistical significance was analysed using ANOVA and multiple comparison with Dunnett test, or non-parametric analysis by the Kruskal-Wallis test and multiple comparisons by the Shirley-Williams test. P<0.05 was considered statistically significant.
Results
Detection of Epo protein
We have reported that our western blot recognized hypoxic rat kidney Epo protein and the deglycosylated protein at 34-43 and 22 kDa, respectively. The specificity of sc-5290 was better than that of AE7A5 (Fig. 1A, B). ESAs were also detected by Western blot, and deglycosylation caused a shift of the bands to 22 kDa, except for that of epoetin β pegol (Fig. 2A1, A2). The deglycosylated band at 22 kDa showed a 10-100 times lower limit of detection than the non-deglycosylated band at 34-43 kDa (Fig. 2B1, 2B2).
Detection of Epo protein and ESAs in urine
The direct analysis (green line) and incubation with deglycosylation buffer (blue line) of anemic patient’s urine both volume-dependently showed an Epo protein band at 36-40 kDa. Deglycosylation (red line) shifted the bands to 22 kDa (Fig. 2C). Epoetin α (lane 1) and darbepoetin (lane 2) were detected by the direct application of rat urine after bolus injection. Epoetin β pegol (lane 3) was not detected, probably due to its limited excretion into the urine (Fig. 2D).
Detection of Epo protein and ESAs in plasma
The direct analysis of plasma from control and hypoxic rats by Western blotting showed no band (Fig. 3A, green line). Incubation of the plasma with deglycosylation buffer showed bands at 34-43 kDa in 4-hr hypoxic rats but not in control rats (Fig. 3A, blue line). Deglycosylation shifted the broad band at 34-43 kDa to 22 kDa (Fig. 3A, red line). Next, direct analysis of plasma from anemic patient also showed no band (Fig. 3B, green line). Incubation of the plasma with deglycosylation buffer showed a broad band at 36-40 kDa only in the case of severe anemia (Fig. 3B, lane 1, blue line). The partial recovery of anemia caused a faint band at 36-40 kDa, and complete recovery revealed no broad band at approximately 36-40 kDa. Deglycosylation caused an intense band at 22 kDa in anemia, and partial recovery of anemia caused a very faint band at 22 kDa (Fig. 3B, red line). No band was observed at 22 kDa after complete recovery.
The detection of ESAs in plasma was tested in rats after the intravenous injection of large doses of ESAs. The plasma Epo concentration was more than 100 times higher than under severe hypoxia. In this condition, epoetin α and epoetin β pegol were detected by the direct analysis of plasma (Fig. 3C, green line). The band of darbepoetin overlapped with the non-specific band, which was removed by the incubation of plasma with deglycosylation buffer (Fig. 3C, blue line). The bands of epoetin α and darbepoetin were shifted to 22 kDa by deglycosylation (Fig. 3C, red line). The band of epoetin β pegol shifted from 95-120 to 80-95 kDa. In contrast, no band representing epoetin β pegol was detected by the direct analysis of plasma from anemic CKD patients (Fig. 3D, green line). The incubation of plasma with deglycosylation buffer induced the appearance of a band at 95-120 kDa (Fig. 3D, lane 1 in blue line), which was shifted to 80-95 kDa by deglycosylation (Fig. 3D, lane 1 in red line).
Detection of Epo protein by LC/MS
To confirm that the band at 22 kDa is Epo protein, the 22 kDa band of recombinant human Epo and anemic patient’s urine were excised and analysed by LC/MS (Fig. 4A, B). Seven, and three peptide sequences of human Epo protein (sequence coverage 20% and 12%) were identified in the sample of recombinant human Epo and anemic patient, respectively (Table. 1). Recombinant rat Epo was also identified by LC/MS (Table. 1).
Epo protein expression in hypoxia
Epo mRNA and protein expression in the kidney and liver in hypoxia were examined in rats. HIF1α, HIF2α and Epo mRNA expression in the kidney reached a maximum at 2 hr after hypoxia, and PHD2 mRNA expression in the kidney reached its maximum at 4 hr (Fig. 5A-D). Epo mRNA showed a 200-fold increase in the kidney with no changes in the liver. (Fig. 5A). The plasma Epo concentration showed a 600-fold increase at 4 hr compared with zero time (Fig. 5E). Epo protein expression in the kidney reached its maximum at 4 hr, while the changes in Epo protein expression in the liver were small (Fig. 6A, B). Ususal Western blot showed an approximately 10-fold increase in Epo protein expressions in the kidney, respectively (Fig. 6A, B). Incubation of the kidney samples with deglycosylation buffer without PNGase made the bands clear and the increase of Epo protein expression reached 20-fold increase (Fig. 6C, D). In contrast, deglycosylated Epo protein expression showed an approximately 400-fold increase (Fig. 6C, F), which is very close to the changes in plasma Epo concentration. A very faint band of deglycosylated Epo was observed in the hypoxic liver (Fig. 6E, F).
Immunohistochemical Epo protein expression
Immunohistochemistry showed that renal proximal and distal tubules in the cortex were weakly stained under basal conditions (proximal tubules < thick ascending limbs, distal convoluted tubules) (Fig. 7A, C). Severe hypoxia caused increased Epo staining of the interstitial cells around proximal tubules in the deep cortical area but decreased staining in tubular cells, as in our previous report using in situ hybridization (Fig. 7B, D).
Discussion
We detected Epo protein and ESAs by the combination of usual Western blotting and LS/MS for the first time. Using a new method of Western blotting, we succeeded in the detection of urinary Epo and ESAs. However, intrinsic Epo and ESAs in plasma could not be detected even by our Western blot. The incubation of plasma in deglycosylation buffer resulted in the appearance of bands at 34-43 kDa, and deglycosylation caused a shift of those bands to 22 kDa, except for that of epoetin β pegol (CERA). LC/MS analysis of the 22 kDa band from anemic patient’s urine revealed human Epo. The sensitivity of our Western blotting is higher than that of LC/MS.
One of the findings of our new method is that detection limit of Epo protein is increased by deglycosylation. Detection limit of glycosylated and deglycosylated recombinant human Epo was 370 and 37 pg, respectively (Fig 2B1). The detection limit of deglycosylaed recombinant rat Epo was 3.7 pg (Fig 2B2). Therefore, the deglycosylation increased the detection limit of Eo by 10-100 times. Therefore, accurate quantitative estimates of Epo can be obtained by measuring deglycosylated Epo. Although Epo is detected directly in the urine, the estimation of deglycosylated Epo in the urine would be more accurate.
Our new method will change the tests for Epo doping. Currently, Epo doping is detected by IEF and/or SAR-PAGE or LC/MS after pre-purification of the samples [18, 19]. Our method does not require any pre-purification of the samples. Concentrated urine can be used directly for Western blotting. Blood samples should be deglycosylated to reduce non-specific bands. Intrinsic Epo and ESAs are distinguished simply by band size. To completely confirm the presence of ESAs, cut gels should be checked by LC/MS. More than 1-2 ng of Epo was required to detect Epo by LC/MS, while the detection limit of Epo by our Western blotting is 3.7-37 pg. Since plasma or serum contains a lot of proteins, concentrated plasma becomes very high osmolality and is difficult to use for Western blotting. In contrast, urine has usually no protein except patients with CKD, concentrated urine can be used for Western blotting.
Our new method allowed conclusions regarding unsolved questions about the sites of Epo production in response to severe hypoxia/anemia. Since the increase in Epo production in the kidney was not high enough compared to the changes in plasma Epo concentration and gene expression in the kidney, liver participation has been suggested [2-5, 14]. The difficulty of Epo protein detection by Western blot was the main reason. We showed that deglycylation increased the sensitivity of Epo detection by 10-100 times. Deglycosylated Epo expression showed a 400-fold increase, which is very close to the change of Epo concentration in plasma. Deglycosylated Epo expression in the hypoxic liver was very low. The increases of HIF1α and HIF2α mRNA expression as well as Epo mRNA were observed in the hypoxic kidney but not in the hypoxic liver. The increase of PHD2 mRNA expression and a large decrease of Epo mRNA expression were observed in the kidney 4 hr after hypoxia. HIF2α has a key role for Epo production and PHD2 has a key role for the degradation of Epo [26-28]. These data clearly show that the kidney is the main and sole site of Epo production in response to severe hypoxia. Although plasma Epo is very low in normal rats and humans, control rat kidneys showed deglycosylated Epo production, and immunohistochemistry showed Epo production in the cortical nephrons. Mujais and colleagues reported Epo mRNA expression in renal tubules using microdissected nephron segments in cobalt chloride-injected rats [29]. We have previously shown that fludrocortisone stimulated Epo production by the intercalated cells of the collecting ducts [20]. Our immunohistochemistry also showed that kidney interstitial cells respond to severe hypoxia by producing Epo. Yamamoto and colleagues showed that the site of Epo production by severe anemia is the interstitial cells using EPO promoter-driven GFP expression [8, 13]. Since 27 kDa GFP goes into nucleus, they may have overestimated the role of Epo production by interstitial cells in severe anemia. Since the cytoplasm of interstitial cells is very pale, Epo production by interstitial cells under hypoxia may not be as strong as expected. These data show that kidney nephrons produce Epo under control conditions and that kidney interstitial cells produce Epo in response to severe hypoxia or anaemia.
In conclusion, our data showed that Epo protein can be detected in urine and tissue samples by direct Western blot analysis and in blood after deglycosylation. Our data also showed that the kidneys have dual Epo production systems, low production by the nephron under normal conditions and hypoxia or anemia-induced high production by the interstitial fibroblast-like cells, and that the kidney is the main and sole site of Epo production in response to hypoxia or anaemia. Our method will fundamentally change Epo doping and detection.
Funding
This study was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan (24591244, 26461259, 26893202, 16K19493, 16K08505, 17K16578 and 19K09226) and by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
Author contributions
YY, YI, KK and HN designed the research; YY, YN, HI, YoS, YN, and HN performed the animal research; YI, TF, KY, TU, and HN performed western blot analysis; YY, TO, YuS, and KK performed IHC, TF, TaY, NK and HN performed RNA extraction and PCR; YI and HN performed the statistical analyses; and TeY performed LC/MS. MM and YuS advised on the experimental design and data interpretation.
Competing interests
The authors have no financial conflicts to declare.
Additional information
Correspondence and requests for materials should be addressed to E-mail: nono{at}insti.kitasato-u.ac.jp.
Acknowledgements
Our manuscript was edited for proper English language by NPG Language Editing Service (4221-D9DA-8D07-E1B1-3D9P).