Volume 79, Issue 1, Pages 77-88 (January 2011) Amelioration of cisplatin nephrotoxicity by genetic or pharmacologic blockade of prostaglandin synthesis  Zhanjun Jia, Ningning Wang, Toshinori Aoyagi, Haiping Wang, Haiying Liu, Tianxin Yang  Kidney International  Volume 79, Issue 1, Pages 77-88 (January 2011) DOI: 10.1038/ki.2010.331 Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 1 Assessment of renal function after cisplatin treatment. Blood urine nitrogen (BUN) (a) and plasma creatinine (Cr) (b) in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice at 72h following treatment with vehicle or cisplatin. Wild-type (WT)/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=16; KO/cisplatin: N=16. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 2 Morphological analysis of cisplatin-induced renal injury in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice. (a) Gross kidney appearance. (b) Hematoxylin and eosin staining (magnification: × 200 shown) of renal cortex. (c) Renal injury score in cisplatin-treated mPGES-1 +/+ and -/- mice. Wild-type (WT)/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=9; KO/cisplatin: N=10. N=9 per group. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 3 Effects of cisplatin-treatment on hematocrit (Hct) and body weight. Changes in hematocrit (Hct) (a) and body weight (b) in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice following treatment with vehicle or cisplatin. Hct was determined at 72h following cisplatin treatment and body weight was monitored daily. Wild-type (WT)/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=9; KO/cisplatin: N=10. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 4 The levels of proinflammatory cytokines in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice following treatment with vehicle or cisplatin. (a) Immunoblotting of tumor necrosis factor-α (TNF-α) and α-tubulin in the kidney. The densitometric value of TNF-α protein was normalized by α-tubulin. The mean values are shown below the immunoblot. *P<0.05 vs control. (b) Quantitative reverse transcriptase (qRT)-PCR analysis of renal TNF-α. (c) Enzyme-linked immunosorbent assay analysis of circulating TNF-α. (d), qRT-PCR analysis of renal interleukin (IL)-1β. Wild-type (WT)/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=9; KO/cisplatin: N=10. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 5 Evaluation of oxidative stress after cisplatin treatment. Measurements of kidney thiobarbituric acid-reactive substances (TBARS) (a) and quantitative reverse transcriptase (qRT)-PCR analysis of renal expression of p47phox (b) and gp91phox (c), NOX1 (d), NOX3 (e), superoxide dismutase (SOD1) (f), SOD2 (g), and SOD3 (h) in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice following treatment with vehicle or cisplatin. Wild-type (WT)/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=9; KO/cisplatin: N=10. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 6 Assessment of apoptotic pathway after cisplatin treatment. Quantitative reverse transcriptase (qRT)-PCR analysis of renal Bak (a), Bax (b), and Bcl-2 (c) in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice following treatment with vehicle or cisplatin. Wild-type (WT)/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=9; KO/cisplatin: N=10. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 7 Stimulation of renal membrane-associated prostaglandin E synthase-1 (mPGES-1) expression by cisplatin. (a) Immunohistochemistry of mPGES-1 in the renal cortex of wild-type (WT) mice treated with vehicle or cisplatin. Specificity of the antibody was validated by the using the competing mPGES-1 peptide. Arrow denotes the collecting duct (CD). Shown are representative photomicrographs (hematoxylin and eosin staining; magnification: × 200) from three independent experiments. (b) Immunoblotting analysis of renal mPGES-1 protein expression in WT mice treated with vehicle or cisplatin. Immunoblotting of α-tubulin serves as a loading control. The densitometric value of mPGES-1 protein was normalized by α-tubulin. The mean values are shown below the immunoblot. *P<0.05 vs control. (c) Quantitative reverse transcriptase (qRT)-PCR analysis of mPGES-1 in the kidneys of vehicle or cisplatin-treated mPGES-1 +/+ and -/- mice. (d) qRT-PCR analysis of renal mPGES-2. (e) qRT-PCR analysis of renal cytosolic PGES (cPGES). (f) Kidney prostaglandin E2 (PGE2) content in the two genotypes treated with vehicle or cisplatin. PGE2 was determined by enzyme immunoassay and normalized by protein content. (b) N=4–5 per group; (c–f) WT/vehicle: N=6; knockout (KO)/vehicle: N=6; WT/cisplatin: N=9; KO/cisplatin: N=10. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 8 Renal functional and structural damage in control mice and in mice treated with cisplatin alone or in combination with celecoxib. Celecoxib was started at 72h before cisplatin treatment. Blood urine nitrogen (BUN) (a) and plasma creatinine (Cr) (b), and renal histology (c: hematoxylin and eosin staining; d: renal injury score) were evaluated at 72h after cisplatin. In c, the magnification is × 200. Control: N=6; cisplatin: N=12; cisplatin+celecoxib: N=8 per group. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 9 The levels of circulating or renal proinflammatory cytokines and oxidative stress in control mice and in mice treated with cisplatin alone or in combination with celecoxib. (a) Enzyme-linked immunosorbent assay analysis of circulating tumor necrosis factor-α (TNF-α). (b) Quantitative reverse transcriptase (qRT)-PCR analysis of renal TNF-α. (c) qRT-PCR analysis of renal interleukin (IL)-1β. (d) Measurements of renal thiobarbituric acid-reactive substances (TBARS). (e) qRT-PCR analysis of renal gp91phox. (f) qRT-PCR analysis of renal p47phox. The gene expression was normalized by GAPDH. Control: N=6; cisplatin: N=12; cisplatin+celecoxib: N=8 per group. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 10 Stimulation of renal cyclooxygenase 2 (COX-2) expression by cisplatin. (a) COX-2 immunoreactivity in the kidney of wild-type (WT) mice treated with vehicle or cisplatin. Shown are representative photomicrographs (hematoxylin and eosin staining; magnification: × 200 shown) from three independent experiments. (b) Quantitative reverse transcriptase (qRT)-PCR analysis of renal COX-2 in vehicle or cisplatin-treated mice. (c) Enzyme-linked immunosorbent assay analysis of kidney prostaglandin E2 (PGE2) content (normalized by protein content). Control: N=6; cisplatin: N=12; cisplatin+celecoxib: N=8 per group. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 11 Role of membrane-associated prostaglandin E synthase-1 (mPGES-1) in renal ischemia–reperfusion (I/R) injury in mice. mPGES-1 +/+ and -/- mice were subjected to bilateral renal ischemia for 30min by clamping renal artery, followed by reperfusion. Blood urine nitrogen (BUN) (a) and plasma creatinine (Cr) (b) were determined at 24h after I/R injury. In a separate experiment, mPGES-1 +/+ mice were sham operated or subjected to the I/R procedure. Renal mRNA expression of mPGES-1 (c), mPGES-2 (d), cytosolic PGES (cPGES) (e), and cyclooxygenase 2 (COX-2) (f) was determined at 24h after I/R injury. N=5–7 per group. *P<0.05 vs sham. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 12 Analysis of renal function and plasma TNF-α after lipopolysaccharide (LPS) treatment. Blood urine nitrogen (BUN) (a), plasma creatinine (Cr) (b), and plasma tumor necrosis factor-α (TNF-α) (c) in membrane-associated prostaglandin E synthase-1 (mPGES-1) +/+ and -/- mice at 24h following an intraperitoneal injection of vehicle or lipopolysaccharide (LPS) (10mg/kg). N=8 per group. Data are mean±s.e. Kidney International 2011 79, 77-88DOI: (10.1038/ki.2010.331) Copyright © 2011 International Society of Nephrology Terms and Conditions