Poly(ADP-ribose) polymerase-1 is a key mediator of cisplatin-induced kidney inflammation and injury

Abstract

Cisplatin is a commonly used chemotherapeutic drug, the clinical use of which is limited by the development of dose-dependent nephrotoxicity. Enhanced inflammatory response, oxidative stress, and cell death have been implicated in the development of cisplatin-induced nephropathy; however, the precise mechanisms are elusive. Overactivation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) by oxidative DNA damage under various pathological conditions promotes cell death and up-regulation of key proinflammatory pathways. In this study, using a well-established model of nephropathy, we have explored the role of PARP-1 in cisplatin-induced kidney injury. Genetic deletion or pharmacological inhibition of PARP-1 markedly attenuated the cisplatin-induced histopathological damage, impaired renal function (elevated serum BUN and creatinine levels), and enhanced inflammatory response (leukocyte infiltration; TNF-α, IL-1β, F4/80, adhesion molecules ICAM-1/VCAM-1 expression) and consequent oxidative/nitrative stress (4-HNE, 8-OHdG, and nitrotyrosine content; NOX2/NOX4 expression). PARP inhibition also facilitated the cisplatin-induced death of cancer cells. Thus, PARP activation plays an important role in cisplatin-induced kidney injury, and its pharmacological inhibition may represent a promising approach to preventing the cisplatin-induced nephropathy. This is particularly exciting because several PARP inhibitors alone or in combination with DNA-damaging anticancer agents show considerable promise in clinical trials for treatment of various malignancies (e.g., triple-negative breast cancer).

Figure 1. Time-dependent changes in cisplatin-induced renal oxidative/nitrative stress, inflammation, apoptosis, PARP activation and dysfunction

Cisplatin time-dependently increased markers of oxidative/nitrative stress, inflammation and cell death (Panels A–D) in the kidneys following its administration to mice, which paralleled with the development of renal dysfunction (Panel E). Results are mean±S.E.M. of 6–7/group. *P<0.05 vs. 0 time point.

Figure 2. Pharmacologic inhibition of PARP attenuates cisplatin-induced kidney damage

Pretreatment with PARP inhibitors (Panels A and C) ameliorates cisplatin-induced profound kidney damage as evidenced by the attenuation of the increase in blood urea nitrogen (BUN) and creatinine values (Panel A) and histological tubular damage (Panel C, PAS Staining) in the kidney 72h after cisplatin administration to mice. PARP inhibitors were also effective in decreasing cisplatin-induced renal dysfunction (B) when administered 12 or 24 hours following the drug injection. Results are mean±S.E.M. of 6–8 experiments/group for panel A, 4–8 for panel B. Panel C (400x magnification) depicts representative sections from 4 kidneys of 4 animals/group, and quantifications from 8 representative fields with 400x magnification/group. *P<0.05 v.s. Vehicle, #P<0.05 Vehicle+Cisplatin vs. Cisplatin+AIQ/PJ34.

Figure 3. Genetic deletion of PARP-1 attenuates cisplatin-induced kidney damage

Genetic deletion of PARP-1 ameliorates the cisplatin-induced profound kidney damage as evidenced by the attenuation of the increase in blood urea nitrogen (BUN) and creatinine values (Panel A) and histological tubular damage (Panel B, PAS Staining) in the kidney 72h after cisplatin administration to mice. Results are mean±S.E.M. of 6–8 experiments/group. *P<0.05 vs. Vehicle, #P<0.05 Cisplatin in PARP^+/+ vs. Cisplatin in PARP^−/−mice.

Figure 4. Pharmacologic inhibition of PARP attenuates cisplatin-induced adhesion molecules expression and leukocyte and macrophage infiltration

Cisplatin significantly increased mRNA expression of adhesion molecules ICAM-1 and VCAM-1 mRNA (panels A and B), F4/80 (a marker of macrophages, panel C) and renal myeloperoxidase (MPO) activity/staining (panel D, E; an indicator of leukocyte infiltration), indicating enhanced inflammatory response. These were attenuated by treatment with AIQ or PJ34 (panels A–E; n=6–8/group). Panel E shows representative MPO staining (brown) of 4-4 kidneys/group (1000x magnification) from mice treated with cisplatin or cisplatin in combination with PARP inhibitors, and quantifications from 8 representative fields with 400x magnification/group. Results are mean±S.E.M. *P<0.05 vs. Vehicle, #P<0.05 Vehicle+Cisplatin vs. Cisplatin+AIQ/PJ34.

Figure 5. Genetic deletion of PARP-1 attenuates cisplatin-induced adhesion molecules expression, leukocyte and macrophage infiltration

Cisplatin significantly increased mRNA expression of adhesion molecules ICAM-1 and VCAM-1 (panels A and B), F4/80 (marker of macrophages, panel E), and renal myeloperoxidase staining (panel C) or activity (panel D) (an indicator of leukocyte infiltration), in PARP-1^+/+ mice (n=8). Panel D: Quantitative MPO assay indicating enhanced inflammatory cell infiltration in the kidneys of cisplatin-treated PARP-1^+/+ mice (n=8). These changes were attenuated in PARP-1^−/− mice (n=8). Panel C shows representative MPO staining (brown) of 4-4 kidneys/group (1000x magnification) from PARP-1^+/+ and PARP-1 mice treated with vehicle (n=6) or cisplatin (n=8), and quantifications from 8 representative fields with 400x magnification/group. Results are mean±S.E.M. *P<0.05 vs. Vehicle in PARP^+/+, #P<0.05 +Cisplatin in PARP^+/+ vs. Cisplatin+PARP^−/−

Figure 6. Pharmacologic inhibition or genetic deletion of PARP-1 attenuates cisplatin-induced increased expression of mRNA of TNF-α and IL-1β

Cisplatin significantly increased renal mRNA expression of TNF-α and IL1β mRNA (panels AD), indicating enhanced inflammatory response 72 h following its administration to mice. These were attenuated by treatment with AIQ or PJ34 (left side: panels A and C), and also in PARP-1^−/− mice compared to PARP-1^+/+ mice (right side: panels B and D). Results are mean±S.E.M. of 6–8/group *P<0.05 vs. Vehicle in C57Bl/6J/PARP^+/+, #P<0.05 Cisplatin in C57Bl/6J/PARP^+/+ vs. Cisplatin+AIQ/PJ34/PARP^−/−

Figure 7. Pharmacologic inhibition or genetic deletion of PARP-1 attenuates cisplatin-induced increased oxidative and nitrative stress

Cisplatin significantly increased renal HNE protein adduct, 8-OHdG, and 3-NT levels in the kidneys (panels A–F), indicating enhanced oxidative/nitrative stress 72 h following its administration to mice. These were attenuated by treatment with AIQ or PJ34 (left side of panels A, C and E), and also in PARP-1^−/− mice compared to PARP-1^+/+ mice (right side of panels B, D and F). Results are mean±S.E.M. of 6–8/group *P<0.05 vs. Vehicle vs. Cisplatin, #P<0.05 C57Bl/6J/PARP^+/++Cisplatin vs. Cisplatin+AIQ/PJ34/PARP^−/−

Figure 8. Pharmacologic inhibition or genetic deletion of PARP-1 attenuates cisplatin-induced increased expression of ROS generating NADPH oxidase isoforms (NOX4 (RENOX), NOX2 (gp91phox))

Cisplatin significantly increased renal mRNA expression of NOX4 and NOX2 (Panels A–D) 72 h following its administration to mice. These were attenuated by treatment with AIQ or PJ34 (left side of panels A and C), and also in PARP-1^−/− mice compared to PARP-1^+/+ littermates (right side of panels B and D). Results are mean±S.E.M. of 6–8/group *P<0.05 Vehicle/PARP^+/+ vs. Cisplatin, #P<0.05 C57Bl/6J/PARP^+/++Cisplatin vs. Cisplatin+AIQ/PJ34/PARP^−/−

Figure 9. Pharmacologic inhibition or genetic deletion of PARP-1 attenuates cisplatin-induced apoptosis

Cisplatin significantly increased markers of renal apoptosis (caspase 3/7 activity and TUNEL staining; Panels A–C) 72 h following its administration to mice. These were attenuated by treatment with AIQ or PJ34 (Panels A and C), and also in PARP-1^−/− mice compared to PARP-1^+/+ littermates (panels B and C) (n= 6–8/group for Panels A and B). Panel C shows representative TUNEL staining of 4-4 kidneys/group (400x magnification) from mice treated with cisplatin or cisplatin in combination with PARP inhibitors, and quantifications from 12 representative fields/group with 400x magnification. The images are overlays of nuclear staining (blue) and TUNEL (green) and the TUNEL positive cells are shown in light blue/green. Results are mean±S.E.M. *P<0.05 Vehicle vs. Cisplatin, #P<0.05 C57Bl/6J/PARP^+/++Cisplatin vs. Cisplatin+AIQ/PJ34/PARP^−/−.

Figure 10. Pharmacologic inhibition of PARP promotes cisplatin-induced cell demise in cancer cells

Cisplatin (panel A) and PARP inhibitors AIQ and PJ34 (panel B) induced concentration-dependent cell death in T24 cancer cells measured by XTT assay (left panels) and flow cytometry (right panels). Cisplatin combined with PARP inhibitor even at concentrations which did not induce cell death by themselves, decreased cell viability of cancer cells. Results are mean±S.E.M. of 4–8/group *P<0.05 vs. corresponding Vehicle, #P<0.05 vs. Vehicle+Cisplatin..,.,