1. Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity 
Jinu Kim, Kelly E. Long, Kang Tang, Babu J. Padanilam 
Kidney International 
Volume 82, Issue 2, Pages (July 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
Poly(ADP-ribose) polymerase 1 (PARP1) expression and activation are increased by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) 129S1/SvImJ male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20mg/kg body weight) injection (n=6 in each group). (a) PARP1 expression and activation were examined by western blot analysis using anti-PARP1 and anti-PAR antibody, respectively. Anti-β-actin antibody was used as a loading control. (b–d) The intensities of protein bands (full length of PARP1, 116kDa; cleaved PARP1, 89kDa; PAR, 116kDa; all approximate) were quantified using the Lab Works analysis software. *P<0.05 vs. 0 days. ND, not detected.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

3. Figure 2
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency reduces kidney dysfunction, histological damage, and ATP depletion induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20mg/kg body weight) injection. (a, b) Plasma creatinine and blood urea nitrogen (BUN) concentrations after cisplatin injection (n=6 in each group). (c, d) Histological damage in the cortex or outer medulla of hematoxylin and eosin (H&E)–stained kidney sections was scored by counting the percentage of tubules that displayed tubular necrosis, cast formation, tubular dilation as follows: 0=normal; 1=<10%; 2=10–25%; 3=26–50%; 4=51–75%; and 5=>75%. Ten fields ( × 200 magnification) per kidney were used for counting (n=6 in each group). (e) Adenosine triphosphate (ATP) concentration after cisplatin injection (n=4 in each group). *P<0.05 vs. 0 days, #P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

4. Figure 3
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency does not affect apoptosis induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20mg/kg body weight) injection. (a) Cleaved caspase–3 expression was examined by western blot analysis. Anti-β-actin antibody was used as a loading control. The expression of cleaved caspase–3 was not significantly changed in Parp1-KO mouse kidneys compared with WT mouse kidneys at each time point. (b) The intensities of protein bands (∼17kDa) were quantified using the Lab Works analysis software. (c) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)–positive cells were counted in 10 fields ( × 200 magnification) per kidney (n=4 in each group). The number of TUNEL-positive cells was not significantly changed in Parp1-KO mouse kidneys compared with WT mouse kidneys at each time point. *P<0.05 vs. 0 days.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency reduces necrosis, not apoptosis, induced by cisplatin treatment in primary culture of proximal tubule epithelial cells. The proximal tubule epithelial cells were isolated from Parp1-knockout (KO) and -wild-type (WT) mouse kidneys, respectively. After 18h of starvation, the cells were treated with 400μmol/l cisplatin for 8h to induce necrosis (a, b, c) and 25μmol/l cisplatin for 24h to induce apoptosis (d, e, f), respectively. Dimethyl sulfoxide (DMSO) was used as vehicle. (a) The percentage of propidium iodide (PI)–positive cells was counted in five fields ( × 400 magnification) per well on 12-well plates (n=4 in each group). (b) Intracellular adenosine triphosphate (ATP) concentration after cisplatin or vehicle treatment in 100-mm dishes (n=4 in each group). (c) Lactate dehydrogenase (LDH) release was measured for 0, 2, 4, and 8h of cisplatin treatment in 24-well plates (n=6 in each group). (d) The percentage of transferase dUTP nick end labeling (TUNEL)-positive cells was counted in five fields ( × 400 magnification) per well (n=4 in each group). The percentage of TUNEL-positive cells was not significantly changed in Parp1-KO cells compared with WT cells. (e) Cleaved caspase–3 expression was examined by western blot analysis. Anti-β-actin antibody was used as a loading control. (f) The intensities of protein bands (∼17kDa) were quantified using the Lab Works analysis software. The expression of cleaved caspase–3 was not significantly changed in Parp1-KO cells compared with WT cells at each time point. *P<0.05 vs. vehicle or 0h, #P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

6. Figure 5
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency inhibits inflammation induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20mg/kg body weight) or saline (control) injection. (a) Polymorphonuclear neutrophil (PMN)–positive cells were counted in 10 fields ( × 200 magnification) per kidney (n=4 in each group). (b) The expression of intercellular adhesion molecule–1 (ICAM–1), tumor necrosis factor-α (TNF-α), and Toll-like receptor 4 (TLR4) in kidneys was examined by western blot analysis. Anti-β-actin antibody was used as a loading control. (c–e) The intensities of protein bands (ICAM–1, 110kDa; TNF-α, 26kDa; TLR4, 90kDa; all approximate) were quantified using the Lab Works analysis software (n=6 in each group). *P<0.05 vs. 0 days, #P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

7. Figure 6
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency suppresses JNK and p38 MAPK activation induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin injection. (a) Phosphorylated c-Jun N-terminal kinase (JNK) at Thy183/Tyr185 (p-JNK), JNK, phosphorylated p38 mitogen-activated protein kinase (MAPK) at Thr180/Tyr182 (p-p38), p38, phosphorylated MAPK kinase 3/6 at Ser189/207 (p-MKK3/6), and phosphorylated MKK4 at Thr261 (p-MKK4) were examined by western blot analysis. Anti-β-actin antibody was used as a loading control. (b–e) The intensities of protein bands (p-MKK3/6, 40kDa; p-JNK and JNK, 46 and 55kDa; p-MKK4, 46kDa; p-p38 and p38, 38kDa; all approximate) were quantified using the Lab Works analysis software (n=6 in each group). *P<0.05 vs. 0 days; #P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

8. Figure 7
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency suppresses NF-κB phosphorylation induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20mg/kg body weight) injection. (a) Phosphorylated nuclear factor-κB (NF-κB) RelA at Ser276 (p-RelA), RelA, and IκBα were examined by western blot analysis. Anti-β-actin antibody was used as a loading control. (b, c) The intensities of protein bands (p-RelA and RelA, 65kDa; IκBα, 39kDa; all approximate) were quantified using the Lab Works analysis software (n=6 in each group). *P<0.05 vs. 0 days; #P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

9. Figure 8
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency blocks TLR4/MAPK/NF-κB/TNF-α signaling pathway during necrotic cell death induced by cisplatin treatment in primary culture of proximal tubule epithelial cells. The proximal tubule epithelial cells were isolated from Parp1-knockout (KO) and -wild-type (WT) mouse kidneys. After 18h of starvation, the cells were treated with 400μmol/l cisplatin for 4h. Dimethyl sulfoxide (DMSO) was used as vehicle. To downregulate Toll-like receptor 4 (TLR4) expression, 500pmol of small interference RNA (siRNA) was transfected on a 100-mm culture dish for 24h before cisplatin or vehicle treatment. The same concentration of scramble siRNA was used as control. Pharmacological inhibitors (100μmol/l of SB against p38 activation, 10μmol/l of SP against c-Jun N-terminal kinase (JNK) activation, 100μmol/l of ammonium pyrrolidinedithiocarbamate against nuclear factor-κB (NF-κB) activation, or 100μmol/l of thalidomide against tumor necrosis factor-α (TNF-α) synthesis) or the same volume of DMSO (control) were added 2h before cisplatin or vehicle treatment. Results are representative of experiments repeated three times.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

10. Figure 9
Poly(ADP-ribose) polymerase 1 (Parp1) deficiency reduces oxidative stress induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20mg/kg body weight) injection. (a, b) Lipid hydroperoxide concentration and ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were measured using kits (see Materials and Methods; n=4 in each group). *P<0.05 vs. 0 days; #P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

11. Figure 10
Poly(ADP-ribose) polymerase 1 (PARP1) inhibitor reduces kidney dysfunction and histological damage induced by cisplatin injection in kidneys. The kidneys in 129S1/SvImJ male mice were harvested 0, 3, or 5 days after cisplatin injection. The mice were administered either PJ34 (10mg/kg body weight) or saline (control) twice daily intraperitoneally from 24h before cisplatin injection up to the time that they were killed. (a, b) Plasma creatinine and blood urea nitrogen (BUN) concentrations after cisplatin injection (n=4 in each group). (c, d) Histological damage in the cortex or outer medulla of H&E-stained kidney sections was scored by counting the percentage of tubules that displayed tubular necrosis, cast formation, and tubular dilation as follows: 0=normal; 1=<10%; 2=10–25%; 3=26–50%; 4=51–75%; and 5=>75%. Ten fields ( × 200 magnification) per kidney were used for counting (n=4 in each group). *P<0.05 vs. 0 days, #P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

12. Figure 11
Poly(ADP-ribose) polymerase 1 (PARP1) inhibitor reduces inflammation induced by cisplatin injection in kidneys. The kidneys in 129S1/SvImJ male mice were harvested 0, 3, or 5 days after cisplatin injection. The mice were administered either PJ34 (10mg/kg body weight) or saline (control, Con) twice daily intraperitoneally from 24h before cisplatin injection up to the time that they were killed. (a) Polymorphonuclear neutrophil (PMN)-positive cells were counted in 10 fields ( × 200 magnification) per kidney (n=4 in each group). (b) The expression of Poly(ADP-ribose) (PAR), intercellular adhesion molecule–1 (ICAM–1), tumor necrosis factor-α (TNF-α), and Toll-like receptor 4 (TLR4) in kidneys was examined by western blot analysis. Anti-β-actin antibody was used as a loading control. (c–f) The intensities of protein bands (PAR, 116kDa; ICAM–1, 110kDa; TNF-α, 26kDa; TLR4, 90kDa; all approximate) were quantified using the Lab Works analysis software (n=4 in each group). *P<0.05 vs. 0 days; #P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions