Volume 84, Issue 3, Pages 482-490 (September 2013) Erythropoietin attenuates acute kidney dysfunction in murine experimental sepsis by activation of the β-common receptor  Sina M. Coldewey, Areeg I. Khan, Amar Kapoor, Massimo Collino, Mara Rogazzo, Michael Brines, Anthony Cerami, Peter Hall, Michael Sheaff, Julius E. Kieswich, Muhammed M. Yaqoob, Nimesh S.A. Patel, Christoph Thiemermann  Kidney International  Volume 84, Issue 3, Pages 482-490 (September 2013) DOI: 10.1038/ki.2013.118 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Effect of erythropoietin (EPO) on renal dysfunction in endotoxemic wild-type (WT) and β-common receptor (βcR) knockout (KO) mice. (a, b) Serum urea and (c, d) serum creatinine were measured 18h subsequent to sham operation or lipopolysaccharide (LPS) administration. Mice received either LPS (9mg/kg intraperitoneally (i.p.)) or vehicle (5ml/kg 0.9% saline i.p.). At 1h after induction of endotoxemia, mice were treated with either EPO (1000IU/kg EPO subcutaneously (s.c.)) or vehicle (10ml/kg 0.9% saline s.c.). (a, c; WT mice): sham+saline (n=3), sham+EPO (n=3), LPS+saline (n=9), and LPS+EPO (n=10). (b, d; βcR KO mice): sham+saline (n=3), sham+EPO (n=3), LPS+saline (n=9), and LPS+EPO (n=20). Data are expressed as means±s.e.m. for n number of observations. *P<0.05 vs. LPS+saline of WT or KO animals, respectively. Kidney International 2013 84, 482-490DOI: (10.1038/ki.2013.118) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Effects of endotoxemia and/or erythropoietin (EPO) on renal morphology (hematoxylin and eosin (HE) staining) and immunohistochemical staining for cleaved caspase-3 (CC-3) and kidney injury molecule-1 (Kim-1). (a–f) Morphological evaluation (HE staining) and immunohistochemistry (IHC)-based scoring of (g) CC-3 and (h) Kim-1 were performed in kidneys obtained from mice 18h after sham operation or lipopolysaccharide (LPS) administration. Mice received either LPS (9mg/kg intraperitoneally (i.p.)) or vehicle (5ml/kg 0.9% saline i.p.). At 1h after induction of endotoxemia, mice were treated with either EPO (1000IU/kg EPO subcutaneously (s.c.)) or vehicle (10ml/kg 0.9% saline s.c.). (a–f) Evaluation of HE-stained specimens revealed that there was no overt morphological evidence of proximal tubular epithelial cell injury, interstitial edema, interstitial inflammation, vasculopathy, or glomerular abnormality in any of the study groups and no treatment-related changes could be observed. Wild-type (WT) mice: (a) sham+saline (n=4), (b) LPS+saline (n=3), and (c) LPS+EPO (n=3). β-Common receptor (βcR) knockout (KO) mice: (d) sham+saline (n=4), (e) LPS+saline (n=4), and (f) LPS+EPO (n=4). (g) Analysis of CC-3 sections revealed no staining in sham-operated animals and a small increase in staining in all animals subjected to endotoxemia with no significant treatment-related changes in the CC-3 score (P>0.05). (h) The results of the Kim-1 IHC pathology evaluation showed slightly increased staining in the endotoxemic WT mice, which appeared reduced after treatment with EPO. There was no treatment-related change in the βcR KO mice. Overall, none of the observed changes in Kim-1 IHC scores were statistically significant (P>0.05). Kidney International 2013 84, 482-490DOI: (10.1038/ki.2013.118) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Effect of erythropoietin (EPO) on the phosphorylation of Akt, glycogen synthase kinase-3β (GSK-3β), and endothelial nitric oxide synthase (eNOS), and on the nuclear translocation of the p65 nuclear factor (NF-κB) subunit in the kidneys of wild-type (WT) and β-common receptor (βcR) knockout (KO) mice with endotoxemia. Mice received either lipopolysaccharide (LPS; 9mg/kg intraperitoneally (i.p.)) or vehicle (5ml/kg 0.9% saline i.p.). At 1h after induction of endotoxemia, mice were treated with either EPO (1000IU/kg EPO subcutaneously (s.c.)) or vehicle (10ml/kg 0.9% saline s.c.). Each band is from a single western blot experiment that is representative of three separate experiments. Data are expressed as means±s.e.m. for n number of observations. *P<0.05 versus LPS+saline of WT or KO animals, respectively (white bars). •P<0.05 vs. LPS+saline of WT or KO animals respectively (black bars). (a–c) Densitometric analysis of the bands (semiquantitative western blot analysis) is expressed for all groups studied as relative optical density (OD) of (a) phosphorylated Akt (pSer473) and the corresponding total Akt content (Σ Akt); (b) phosphorylated GSK-3β (pSer9) and the corresponding total GSK-3β content (Σ GSK-3β); and (c) phosphorylated eNOS (pSer1177) and the corresponding total eNOS content (Σ eNOS). All values were corrected for the corresponding β-actin and normalized using the related sham-operated band. (d) Densitometric analysis of the bands is expressed for all groups studied as relative OD for NF-κB p65 subunit levels in both cytosolic and nuclear fractions normalized using the related sham-operated band. Kidney International 2013 84, 482-490DOI: (10.1038/ki.2013.118) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 The development of renal dysfunction in aging mice following cecal ligation and puncture (CLP). Serum creatinine levels were measured 24h subsequent to sham operation or CLP surgery in wild-type (WT) mice at different ages (2, 5, and 8 months). Data are expressed as means±s.e.m. for n number of observations. *P<0.05 versus sham. Sham (2 months, n=3); CLP (2 months, n=3); CLP (5 months, n=5); and CLP (8 months, n=10). Kidney International 2013 84, 482-490DOI: (10.1038/ki.2013.118) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Effect of erythropoietin (EPO) on renal dysfunction in septic wild-type (WT) and β-common receptor (βcR) knockout (KO) mice. Serum creatinine levels were measured 24h subsequent to sham operation (no cecal ligation and puncture (CLP)) or CLP surgery in (a) WT and (b) βcR KO mice. At 1h after CLP surgery, mice were treated subcutaneously (s.c.) with either EPO (1000IU/kg EPO s.c.) or vehicle (10ml/kg 0.9% saline s.c.). (a; WT mice): sham+saline (n=3), sham+EPO (n=3), CLP+saline (n=10), and CLP+EPO (n=12). (b; βcR KO mice): sham+saline (n=3), CLP+saline (n=6), and CLP+EPO (n=20). Data are expressed as means±s.e.m. for n number of observations. *P<0.05 versus CLP+saline of WT or βcR KO animals, respectively. Kidney International 2013 84, 482-490DOI: (10.1038/ki.2013.118) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Detection of Csf2rb gene expression in kidney medulla and cortex. The Csf2rb gene encodes the mouse colony stimulating factor 2 receptor β (βcR). Messenger RNA was extracted from medulla or cortex of wild-type (WT) sham mice (n=3) and subjected to reverse transcription. PCR amplification of a Csf2rb gene fragment and a fragment of the murine glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene (mRNA extraction and gel loading control) was performed using the cDNA from medulla or cortex of the kidneys as template. When separated in a 1% agarose gel, amplicons with a calculated molecular size of 326bp for the Csf2rb fragment could be detected for both kidney cortex (lanes 1–3) and medulla (lanes 3–6). Intensity of the 175bp Gapdh band in lanes 1 to 6 confirmed comparative loading of the gel. Kidney International 2013 84, 482-490DOI: (10.1038/ki.2013.118) Copyright © 2013 International Society of Nephrology Terms and Conditions