The intratracheal instillation of LPS induced metabolic acidosis and hypotension, decreased arterial oxygenation, and increased the lung wet/dry ratio and mRNA expressions of TNF-α, IL-1β and IL-6 in the lung and kidney. In the animals additionally given the PARP inhibitor 3-AB, the PARP inhibitory effect improved arterial oxygenation and pulmonary edema and prevented LPS from inducing metabolic acidosis and hypotension or increasing the mRNA expressions of TNF-α, IL-1β and IL-6 in the lung and kidney. Histopathological analysis further revealed perivascular edema of the lung and kidney in the LPS group, but not in the control group or LPS + 3-AB group. The PARP inhibitor 3-AB thus seems to confer protective effects in reducing pulmonary edema and kidney edema. Taken together, these findings demonstrate the important role of PARP activation in the development of lung and kidney inflammation following the administration of LPS. The current study is also the first to demonstrate that LPS-induced lung-kidney crosstalk is related to proinflammatory cytokines, and that PARP inhibition attenuates the lung-kidney crosstalk partly via the modulation of NF-kB dependent proinflammatory cytokines.
The intratracheal administration of LPS initiates a lung inflammatory response [12, 15–17] useful for preparing an animal model of acute lung injury and ARDS [18, 19]. LPS has the potential to induce symptoms of sepsis, even when administered intratracheally . DNA strand breakage activates PARP in states of endotoxemia and inflammatory response, and excessive activation induces cell dysfunction and cell death by depleting the cellular stores of adenosine triphosphate [4, 5]. Pharmacological inhibition of PARP has been investigated in various experimental conditions of acute lung injury and LPS-induced organ injury [10, 11, 20–23]. Three-AB is a well-recognized competitive inhibitor of PARP that helps to reduce the degree of tissue injury caused by myocardial infarction  and laryngeal injury  in rats. Three-AB has also been shown to preserve mitochondrial respiration, NAD+, and ATP . This preservative effect suggests that PARP inhibition may improve cellular energy homeostasis and prohibit cell dysfunction and death. Our results prove that 3-AB prevents metabolic acidosis, a typical sign of lactic acidosis and acute kidney injury. PARP inhibition is thus confirmed to preserve cellular energy homeostasis and maintain kidney function during inflammatory cell injury.
Our experiment was designed without a control group given 3-AB alone. We know, however, that 3-AB administration to rabbits brings about no changes in the mean arterial pressure, central venous pressure, pulmonary arterial pressure, cardiac output, or alveolar-arterial oxygen difference (AaDO2) after 4 h . Pulmonary edema and hemodynamic changes thus seem unlikely to develop in response to 3-AB, as an increase of AaDO2 is associated with edema formation. Changes were similarly absent in the gene expressions of TNF-α, IL-1β, and IL-6 in lungs and in the number of PARP-positive epithelial cells in mice receiving 3-AB without intratracheal LPS administration . Taken together, these findings suggest that 3-AB alone confers no effects on hemodynamics, pulmonary edema, cytokine gene expression or PARP activation.
The significantly high plasma creatinine and potassium levels, the biochemical parameters for renal function, at 4 h in our LPS group indicated that intratracheal LPS induced injury and inflammation not only in the lung, but also in the kidney via organ crosstalk between the lung and kidney. Along with this line, the LPS-treated rats exhibited significantly higher expressions of PARP and NF-κB in the lung and kidney and significantly higher mRNA expression of the NF-κB-dependent proinflammatory cytokines TNF-α, IL-1β and IL-6 in both organs. The enhanced expression levels partly implicate PARP activation as a cause of renal inflammation in LPS-induced lung inflammation and demonstrate PARP’s effect as a mediator of the transcriptional activation of NF-κB-dependent cytokines. The present study focused little on meaningful specific targets of downstream NF-κB signaling, but earlier studies have shown the LPS-induced NF-κB signaling pathway through PARP activation. LPS enhanced the binding of PARP-1 with the NF-κB subunit p65 (RelA) and poly(adenosine diphosphate-ribosyl)ation of p65, which in turn upregulated the transcriptional activity of the NF-κB and mRNA expressions of IL-1β in murine macrophages . The extracellular signal-regulated kinase (ERK)-dependent phosphorylation of PARP-1 also regulated PARP-1 activity and NF-κB activation . PARP inhibitor activated the phosphatidylinositol 3-kinase/AKT pathway and inactivated the ERK1/2and p38 mitogen-activated protein kinase in LPS-induced inflammation in mice, which resulted in the inactivation of NF-κB .
Renal dysfunction occurs as a consequence of ventilator-induced lung injury superimposed with LPS via the peroxynitrite-induced PARP activation, and pretreatment with PARP inhibitors confers beneficial effects on lung and kidney injury [12, 17]. Moreover, PARP over-activation has been observed in acute renal dysfunction induced by LPS, and PARP inhibition has been identified as a potential target for AKI caused by LPS .
The renal inflammation secondary to LPS-induced acute lung inflammation was mediated via the activation of PARP and NF-κB in the present study. Regarding lung-kidney crosstalk, acute lung inflammation and the associated mechanical ventilation induced biotrauma by releasing proinflammatory cytokines into the systemic circulation and distant organs such as the kidney. Furthermore, acute lung inflammation with subsequent blood gas changes had adverse effects on renal hemodynamics and function. Treatment with 3-AB, a pharmacological inhibitor of PARP, attenuated the lung and kidney inflammation by inhibiting NF-κB-dependent proinflammatory cytokines. The 3-AB treatment appeared to promptly block the initiation of the vicious cycle between the lung and kidney. We thus conclude that 3-AB attenuates lung-kidney crosstalk, one of the mechanisms of multiple organ dysfunction syndrome.
No measurements of plasma endotoxin levels were taken in the present study. Another study has shown, however, that pulmonary-to-systemic translocation of endotoxin can occur . Specifically, plasma endotoxin levels were significantly increased over a period from 40 min to 3 h after intratracheal instillation of LPS (500 μg) in mechanically ventilated rabbits . In our study we administered a larger dose of LPS (16 mg/kg) intratracheally. Hence, the lung-to-kidney combined response in our animals may have been due to LPS escaped into the pulmonary circulation together with a secondary response by the kidney to soluble mediators released by the distressed lung.
PARP also takes part in the regulation of the expression of various proteins implicated in inflammatory and immune responses at the transcriptional level. NF-κB, the key transcriptional factor, plays a critical role in the regulation of these proteins, while PARP has been shown to act as a co-activator in the NF-κB mediated transcription [6, 8]. The functional association between PARP-1 and NF-κB has been observed in association with the transcriptional activation of NF-κB and a systemic inflammatory response . LPS induced PARP and NF-κB staining in the lungs and kidneys of our animals, and PARP inhibition markedly attenuated NF-κB staining in both organs. This result is consistent with the LPS-conferred rises in the mRNA expressions of TNF-α, IL-1β and IL-6 in these organs and the action of PARP inhibitor in attenuating the same. The lung PARP staining was relatively strong in the present study, while the kidney PARP staining was relatively weak. The lung NF-κB staining, meanwhile, was relatively weak, while the kidney NF-κB was relatively strong. We have no clear explanation for the different staining intensity between the lung and kidney. Many factors can influence the intensity of positive signals during color development with DAB solution. To control for this, we stained samples from each organ at the same time under identical conditions. The difference in staining intensity between the two organs may have been caused by a discrepancy in the time performance of the immunostaining between the organs. The difference is very unlikely to have stemmed from a translational component.
Three-AB competitively inhibits PARP by blocking PARP’s ability to ribosylate adenosine diphosphate without affecting its enzymatic (DNA-binding) activity. We administered 3-AB after LPS instillation in the present study, hence the expression of PARP might have been upregulated in the lung and kidney after the LPS-treatment. In experiments with rat astroglial cell cultures, the time course of PARP expression showed an up-regulation (about +90%) after only 1 h of LPS and interferon γ treatment and a progressive decrease hours later (18 h) . We thus speculate that 3-AB may not have totally blocked PARP activity in the present study. We believe, instead, that 3-AB acted as a blockade of PARP transcription. Overall, our findings suggest that the PARP inhibitor 3-AB reduces lung and renal inflammation by inhibiting NF-κB stimulation.