Specific pathogen-free (SPF) adult male Lewis rats (n=64) and Brown Norway rats (n=64), 12–16 weeks old, weighing 250–350 g were purchased from Charles River Laboratories (Beijing, China). All animal experiments were performed in accordance with the Experimental Animal Regulations established by The Ministry of Science and Technology of the People's Republic of China, and the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA). The study received ethical approval from the Ethics Committee of Sun Yat-sen University. Prior to performing the experiments, all the animals were subjected to an overnight fast with unlimited access to water.

The SPF rats were randomly allocated to three groups (control, EPO and EPO-siRNA; n=8). The Lewis rats were used as the recipients, and the Brown Norway rats were used as donors in the renal transplants. Kidney transplantation was performed as previously described (28). In brief, the rats were anesthetized with inhalation of 10% chloral hydrate 0.3 ml/100 g body weight. The left kidney was prepared by freeing the ureter from the attachments. The donor kidney and ureter were removed en bloc. Donor kidney grafts were washed with 4°C saline solution and stored in 4°C HCA solution. The right kidney was removed from the recipient, and the donor kidney was transplanted. An anastomosis was created between the donor and recipient renal artery, renal vein and ureters with end-to-end anastomosis, to establish the unilateral orthotopic renal transplantation rat model. Rat rectal temperature was maintained at 35–37°C during surgery. After surgery, the rats were placed on a heat pad until they recovered.

After establishment of the unilateral orthotopic renal transplantation rat model, continuous administration of saline solution, recombinant human EPO (500 U/kg), or lentivirus-mediated EPO siRNA (1×10⁷ infectious units) (sc-37220-V; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed, once a day for 7 days. After the last administration, the rats were sacrificed and the kidneys were removed and processed for immunohistochemistry (IHC) and immunofluorescence (IFC) assessment of EPO levels, hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), PAS-methenamine (PASM) and Masson's trichrome staining, for detection of the levels of creatinine (Cr), EPO, IL-4, IL-10 and IFN-γ, and for western blot analysis of thymic stromal lymphopoietin (TSLP), STAT6, NF-κB, JNK, p38, and caspase-1 and -3 in renal tissues.

To further investigate the role of the STAT6/MAPK/NF-κB pathway in EPO-mediated renoprotection. The other 40 rats were divided into 5 groups (control, and 4 groups of the different inhibitors; n=8). The animals received the STAT6 inhibitor AS1517499 (10 mg/kg), the JNK inhibitor SP600125 (30 mg/kg), the p38 MAPK inhibitor SB203580 (15 mg/kg), or the NF-κB inhibitor lactacystin (10 mg/kg) in the presence of EPO and using the same route of administration of EPO following renal transplantation. The rats were then sacrificed and the kidneys were removed and processed for H&E, PAS, PASM, and Masson's trichrome staining, for detection of the levels of Cr, EPO, IL-4, IL-10 and IFN-γ, and for western blot analysis of TSLP, STAT6, NF-κB, JNK, p38, and caspase-1 and -3 in renal tissues.

The kidneys were processed for IHC and IFC according to standard protocols. For IHC, 3-μm paraffin sections using rabbit anti-mouse EPO monoclonal antibody (1:100; sc-5290) and goat anti-rabbit IgG poly-HRP (1:100; sc-2004) (both from Santa Cruz Biotechnology, Inc.). For IFC, the sections were fixed in 4% paraformaldehyde and incubated with rabbit anti-human EPO monoclonal antibody (1:50; sc-80995) and FITC-labeled goat anti-rabbit secondary antibody (1:150; sc-2004) (both from Santa Cruz Biotechnology, Inc.). The fluorescence intensity of EPO was calculated by using ImageJ software (National Institutes of Health). For nuclear staining, DAPI was used and confocal images were acquired as previously described (29,30) using the LSM 510 microscope (Carl Zeiss AG, Oberkochen, Germany). Formalin-fixed paraffin-embedded tissues were also stained with H&E and PAS, PASM and Masson's trichrome staining.

Total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA, USA) from homogenated whole kidneys. RNA was reverse transcribed to cDNA using SuperScript II Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific). RT-qPCR was performed on an ABI Prism 5700 Sequence Detection system (Applied Biosystems, Foster City, CA, USA) with SYBR-Green PCR Core reagents. The primers are listed in Table I. GAPDH served as internal control. The relative mRNA expression levels of each target gene were normalized to those of the controls using the 2^−ΔΔCq method.

Serum Cr, EPO, IL-4, IL-10 and IFN-γ levels were detected in a 96-well microtiter plate by using a commercial ELISA kit (Biosource, Camarillo, CA, USA) according to the manufacturer's instructions. All samples were tested in duplicate. The plate was read at 450 nm on a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The levels were calculated from a standard curve and expressed in pg/ml.

The protein lysates were prepared with RIPA and subjected to three freeze/thaw cycles using liquid nitrogen. The lysates were then electrophoresed in 10% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA), transferred to nitrocellulose membrane, blocked with 0.5% skimmed milk and incubated with TSLP, STAT6, NF-κB, JNK, p38 and caspase-1 and -3 primary antibodies (all from Santa Cruz Biotechnology, Inc.) for 1 h. Polyclonal anti-GAPDH was used as an internal control. After three washes, the membrane was incubated with horseradish peroxidase-conjugated IgG and developed under chemiluminescence condition (Amersham Pharmacia Biotech, Piscataway, NJ, USA) using ChemiDoc XRS (Bio-Rad Laboratories, Inc.). The quantification and analysis for the blots were performed with ImageJ software.

All data are presented as means ± standard deviation. Data between groups were compared using two-tailed unpaired t-test or analysis of variance. P<0.05 was considered to indicate statistically significant differences. All analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).

Following continuous administration of saline solution, recombinant human EPO (500 U/kg), or lentivirus-mediated EPO-siRNA for 7 days, histological analysis of kidneys was performed (Fig. 1). The images of H&E, PAS, PASM and Masson's staining revealed that IRI occurred after renal transplantation. Infiltration of inflammatory cells, with a large number of lymphocytes around the blood vesselst was observed. Furthermore, EPO protected the kidney from IRI, through decreasing the extent of tissue congestion and inflammatory cell infiltration; however, EPO-siRNA did not exert the same protective effect.

The level of EPO was detected in the kidneys (Fig. 2). Both IHC (Fig. 2A) and IFC images (Fig. 2B), and data qualification (Fig. 2C) revealed that the EPO levels in EPO-treated rats were higher compared with those in the saline solution and EPO-siRNA groups. Thus, the EPO level appears to be inversely correlated to the renal IRI.

The expression of EPO, IFN-γ, IL-4 and IL-10 in tissues and serum were detected by RT-qPCR and ELISA, respectively (Fig. 3A–G). As expected, EPO tissue and serum levels were significantly upregulated in rats with EPO administration, and was significantly downregulated by EPO-siRNA (Fig. 3A and E). EPO treatment reduced the expression of IFN-γ (Fig. 3B and F) and IL-4 (Fig. 3C and G), as well as the serum levels, whereas it increased the expression of IL-10 at the mRNA level and in the serum (Fig. 3D and H). All these effects induced by EPO were reversed by EPO-siRNA.

The Cr level in the serum was also investigated (Fig. 3I). The results revealed that, compared with controls, EPO administration decreased the serum level of Cr, and EPO-siRNA increased the serum level of Cr.

To analyze the underlying molecular mechanisms for EPO-mediated renoprotection, the changes in TSLP and STAT6/MAPK/NF-κB pathway were detected by western blot analysis (Fig. 3J and K). EPO administration upregulated the expression of TSLP, STAT6, p-JNK, p-p38, but downregulated the expression of caspase-3. By contrast, EPO-siRNA decreased the expression of EPO, TSLP, STAT6, p-JNK, p-p38 and caspase-1, but increased the expression of caspase-3, indicating that EPO protects the kidney from IRI via the STAT6/MAPK/NF-κB signaling pathway.

We further investigated whether the STAT6 inhibitor AS1517499, the JNK inhibitor SP600125, the p38 MAPK inhibitor SB203580, or the NF-κB inhibitor lactacystin suppressed the effects of EPO in inhibiting renal IRI (Figs. 4 and 5). The histological analysis revealed that EPO-mediated renoprotection was obviously inhibited by AS1517499, SP600125, SB203580 and lactacystin (Fig. 4).

The chemokine levels were also measured (Fig. 5A–G). IFN-γ (Fig. 5A and C) and IL-4 (Fig. 5B and D) at the mRNA level and in the serum levels were significantly increased by these inhibitors in the presence of EPO, while IL-10 levels (Fig. 5C and F) were significantly decreased in the presence of EPO. The serum level of Cr in the presence of EPO was also increased by each of these inhibitors (Fig. 5G).

The STAT6 inhibitor AS1517499 inhibited the expression of STAT6, p-JNK, p-p38 and p65, while it increased the expression of caspase-1 and -3 in the presence of EPO (Fig. 5H and I), suggesting that STAT6 acted upstream of MAPK (p-JNK and p-p38) and NF-κB. The JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 inhibited the expression of p-JNK and p65, while they increased the expression of caspase-1 and -3 in the presence of EPO (Fig. 5J and K), suggesting that MAPK (p-JNK and p-p38) acted upstream of NF-κB. The NF-κB inhibitor lactacystin upregulated the expression of caspase-1 and -3 in the presence of EPO (Fig. 5L).

Therefore, all the inhibitors of STAT6, JNK, p38 MAPK and NF-κB reversed the changes in the chemokine levels, Cr levels, and caspase-1 and -3 expression. These results suggest that the STAT6/MAPK/NF-κB signaling pathway was involved in EPO-mediated renoprotection from IRI.