Poly (ADP-Ribose) Polymerase Inhibitor Treatment as a Novel Therapy Attenuating Renal Ischemia-Reperfusion Injury

Abstract

Intrarenal robust inflammatory response following ischemia-reperfusion injury (IRI) is a major factor in the pathogenesis of renal injury in ischemic acute kidney injury (AKI). Although numerous studies have investigated various agents of immune modulation or suppression for ischemic AKI, few showed reproducible effects. We hypothesized that poly (ADP-ribose) polymerase (PARP) inhibitor may favorably change post-ischemic intrarenal immunologic micromilieu by reducing damage-associated molecular pattern (DAMP) signals and improve renal outcome in ischemic AKI. The effects of JPI-289 (a PARP inhibitor) on early renal injury in a murine IRI model and hypoxic HK-2 cell model were investigated. Bilateral IRI surgery was performed in three groups of 9-week-old male C57BL/6 mice (control, JPI-289 50 mg/kg, and JPI-289 100 mg/kg; n = 9-10 in each group). Saline or JPI-289 was intraperitoneally injected. Renal function deterioration was significantly attenuated in the JPI-289 treatment groups in a dose-dependent manner. Inflammatory cell infiltration and proinflammatory cytokine/chemokine expressions in the post-ischemic kidneys were also attenuated by JPI-289 treatment. JPI-289 treatment at 0.5 and 0.75 μg/ml facilitated the proliferation of hypoxic HK-2 cells. PARP inhibition with JPI-289 treatment showed favorable effects in ischemic AKI by attenuating intrarenal inflammatory cascade in a murine model and facilitating proliferation of hypoxic HK-2 cells.

Keywords: Ischemia-reperfusion injury; acute kidney injury; inflammation; parthanatos; poly(ADP-ribose) polymerase (PARP) inhibitor; translational immunology.

Figure 1

Functional and structural renal injury following IRI. (A, B) Blood urea nitrogen (BUN) and plasma creatinine levels were significantly lower in the JPI-289-treated groups compared with the IRI control group in a dose-dependent manner. (C) Hematoxylin and eosin staining of the cortex of postischemic kidneys. Arrows indicate damaged or necrotic tubules (×200). (D) On day 3 after IRI, the JPI-289-treated groups showed less damaged or necrotic tubules compared to the IRI control group. A total of 10 fields magnified 200× were scored for each mouse by a pathologist blinded to the groups. Data are from three independent experiments. *P < 0.05, compared with the IRI control group (n = 6–10 in each IRI group, n = 5 in the sham control group). Statistical analyses were performed with two-way ANOVA test followed by Newman-Keuls test (A, B) or the Mann-Whitney U test (D).

Figure 2

Leukocyte trafficking into the postischemic kidneys. (A) There were more pronounced infiltrations of leukocytes into the postischemic kidneys of IRI control mice compared with JPI-289-treated groups. Arrows indicate CD45-positive leukocytes (×200). (B) Semiquantitative analysis of CD45-positive leukocytes in the postischemic kidney using automated imaging analysis system (TissueFAXS). The whole fields of slides including both cortex and medulla were evaluated. (C) The percentages of total leukocytes expressing CD45 among total nucleated cells were lower in the postischemic kidneys of JPI-289-treated mice compared with those of IRI control mice. Data are from three independent experiments. *P < 0.05, compared with the IRI control group (n = 6–10 in each IRI group, n = 5 in the sham control group). Statistical analysis was performed using the Mann-Whitney U test.

Figure 3

Flow cytometry analyses of KMNCs isolated from postischemic kidneys on day 3 after IRI. (A, B) The infiltration of NK T cells were comparable among the 3 groups. The infiltration of neutrophils tended to be lower in the JPI-289 treated group. JPI-289 reduced the infiltration of intrarenal NK cells and macrophages. Data are from two independent experiments. *P < 0.05, compared with the control group (n = 6–8 in each group). Statistical analysis was performed with the Kruskal-Wallis test followed by Dunn’s test. Detailed gating strategies for each population were as follows; Lymphocytes, monocytes, and granulocytes were first identified on the basis of their FSC and SSC. CD45⁺ cells were gated to identify lymphocytes within the FSC and SSC based lymphocytes population. NK T cells were identified by CD3⁺ and NK1.1⁺ gate within lymphocytes. NK cells were identified by CD3^- and NK1.1⁺ gate within lymphocytes. Macrophages were identified by CD45⁺ and F4/80⁺ gate within the FSC and SSC based monocyte population. Neutrophils were identified by CD45⁺ and GR1⁺ gate within FSC and SSC based granulocyte population (more details are provided in Supplemental Figure 3 ).

Figure 4

The expression of intrarenal cytokines and chemokines in the postischemic kidneys on day 3 after IRI. JPI-289 treatment significantly decreased the expression of IFN-γ, CCL2, and IL-2 and increased the intrarenal expression of VEGF. *P < 0.05, compared with the IRI control group (n = 6–10 in each IRI group, n = 3 in the sham control group). Kidney protein extracts were obtained from three independent experiments. Statistical analysis was performed with the Kruskal-Wallis test followed by Dunn’s test.

Figure 5

Western blotting of protein samples extracted from postischemic kidneys and densitometry analyses. (A) The expression of NFκB was significantly decreased by JPI-289 treatment. The expression of TLR4 tended to be lower with JPI-289 treatment. (B) Bax/Bcl-2 ratios were lower in the JPI-289 treatment groups. *P < 0.05, compared with the control group. Kidney protein extracts (n = 6–10 in each group) were obtained from two independent experiments. Statistical analysis was performed with the Mann-Whitney U-test.

Figure 6

Proliferation of hypoxic HK-2 cells according to the dose of JPI-289. (A) JPI-289 treatment at 0.5 or 0.75 µg/ml facilitated proliferation of hypoxic HK-2 cells compared with the hypoxia control group. Day 0 corresponds to the day when HK-2 cells were taken out from the multi-gas incubator after 48 h of hypoxia. Data are from eight independent experiments. *P < 0.05, compared with the hypoxia control group. Statistical analysis was performed with the Mann-Whitney U test. (B) The proliferation of normoxic HK-2 cells treated with JPI-289 was comparable with that of the normoxia control group. Data are from eight independent experiments.

Figure 7

Analyses of cellular proinflammatory signaling pathways and apoptosis of hypoxic HK-2 cells. (A) Western blotting of TLR4 and NFκB showed that JPI-289 treatment reduced the expressions of TLR4 and NFκB compared with the hypoxia control group. Data are from six independent experiments. *P < 0.05, compared with the hypoxia control group. Statistical analysis was performed with the Mann-Whitney U-test. CD0, normoxia control group on day 0; CD1, normoxia control group on day 1; CD2, normoxia control group on day 2; HD0, the hypoxia group on day 0 in normoxia; HD1, the hypoxia group on day 1 in normoxia; HD2, the hypoxia group on day 2 in normoxia. The concentration of JPI-289 (ng/ml) is expressed in the parentheses. (B) Enzyme-linked immunosorbent assay of Bax and Bcl-2 showed that JPI-289 treatment reduced Bax/Bcl-2 ratios. Data are from six independent experiments. *P < 0.05, compared with the hypoxia control group. Statistical analysis was performed with the Mann-Whitney U test.

Figure 8

PARP-1 activities in the postischemic kidney protein extracts on day 3 after IRI. (A) JPI-289 effectively suppressed PARP-1 activity in a dose dependent fashion. Data are from two independent experiments. *P < 0.05, compared with the JPI-289 0.25 µg/ml treated group. Statistical analysis was performed with the Mann-Whitney U test. (B) Intrarenal PARP-1 activities in postischemic kidney protein extracts were lower in the JPI-289-treated mice. Data are from two independent experiments. *P < 0.05, compared with the control group. Statistical analysis was performed with the Mann-Whitney U test.