Role of the renin-angiotensin system in the development and treatment of acute respiratory distress syndrome and ventilator-induced lung injury

• Main Authors: Jih-Shuin Jerng, 鄭之勛
• Other Authors: Pan-Chyr Yang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/11570053438667971561

博士 === 臺灣大學 === 臨床醫學研究所 === 95 === Respiratory failure remains an important clinical problem despite substantial medical progress during the past decades and the emergence of various new modalities of mechanical ventilation as well as intensive care. Among a variety of conditions that may result in severe respiratory failure, the acute lung injury and acute respiratory failure remain the most challenging for intensivists and pulmonologists. The acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by an acute onset of bilateral pulmonary haziness resulting in severely impaired gas exchange functions and poor compliance of the lungs, leading to virtually all patients requiring mechanical ventilation to overcome life-threatening respiratory failure. Despite substantial progress in critical care, the mortality rate of ARDS remains high, and studies have shown that the majority of deaths result from multiple organ failure rather than refractory hypoxemia. Most of the available treatments todate have shown no benefit to patient survival. Based on current problems and clinical requirement, we interested and engaged in the studies of acute lung injury and ARDS, trying to provide further data and experience in the understanding of these devastating syndromes. First we performed a retrospective analysis of mecical records of ARDS patients treated in a tertiary referred medical center. The clinical picture of patients with ARDS in Taiwan has seldom been reported, although new definitions of ARDS have been introduced over the past years. Therefore we wished to investigate the clinical characteristics, modalities of management, and outcomes in patients with ARDS. Case records were selected trough a computerized search of diagnosis codified at discharge. Patients who met the criteria of the American-European Consensus Conference definition of ARDS were included and their medical records were reviewed. We identified a total of 145 patients (91 men; aged 58 years). Malignancy (n=53) and diabetes mellitus (n=23) were the most common co-morbid conditions. Pneumonia (n=90) was the most common risk factor. The lung injury score at the time of ARDS diagnosis was 2.89 ± 0.40, while the worst value of PaO2/FIO2 was 86.8 ± 3.8 mmHg. Among the 145 patients, 130 received mechanical ventilation and 118 were treated in the intensive care unit. The in-hospital mortality was 87%, while in 70 patients receiving intensive treatment for ARDS, the mortality rate was 74%, with the most common causes of death being multiple organ failure (54%) and respiratory failure (23%). We found that the mortality in patients with ARDS was high in the tertiary referral institution. Our findings suggested that aggressive ventilatory, pharma- cologic, and supportive therapy may be important to achieve a higher survival rate. Subsequently we conducted a human study to investigate the genetic association and predisposition in the development and evolution of ARDS. During the past decades, the mortality of ARDS has improved to some extent, mainly due to progress of the intensive care, but still remained relatively high; therefore the possibility of prevention had been the mainstay of investigation. However, based on epidemiologic data and current practice, the attempt to prevent the development of ARDS remains difficult because there is a relatively low rate of ARDS in the general population. Several risk factors for ARDS, most notable of which are sepsis and pneumonia, have been identified, but only a small portion of patients with such risk factors subsequently develop ARDS. Therefore the identification of this small portion of patients at risk is currently not possible. It is speculated that genetic factors may play a certain role in development and progression of ARDS; therefore studies examining the polymorphisms of the genes involved in ARDS are a reasonable approach. Among the pathophysiolic mechanisms and pathways of acute lung injury and ARDS, the putative involvement of angiotensin II has gained increasing interests, and the renin-angiotensin system (RAS) has become one of the study interests in ARDS. Angiotensin-converting enzyme (ACE) is a key enzyme that converts angiotensin to angiotensin II. While the intron 16 of the ACE gene contains a restriction fragment length polymorphism consisting of the presence (insertion, I) or absence (deletion, D) of a 287-bp alu repeat sequence, the I/D polymorphism has been reported to account for 47% of the variance in plasma ACE level, whereas the serum ACE levels correspond to ACE insertion/deletion (I/D) genotypes in the order: II < DI < DD. It has been shown that the DD frequency was increased in ARDS patients compared with the non-ARDS ICU patients, coronary bypass grafting patients, and general population control groups and was significantly associated with mortality. There was no report of ACE genotypes in the Chinese patients with ARDS. For further understanding of the genetic predisposition for ARDS, we wished to study the role of the renin-angiotensin system, and therefore hypothesized that polymorphism of the ACE gene affects the risk and outcome of ARDS in the Chinese population. We performed a prospective observational study of ARDS patients were >18 yrs of age, who fulfilled the American-European Consensus Committee criteria for ARDS: a) acute onset, b) bilateral pulmonary infiltrates, c) severely impaired oxygenation (i.e., Pao2/Fio2 of <200 mm Hg), and d) pulmonary artery occlusion pressure of <18 mm Hg or no evidence of left atrial hypertension. In addition, two control groups, consisting of “at-risk” patients and “non–at-risk” patients, respectively, were recruited for comparison. The patients in the at-risk group were admitted to the ICU due to acute respiratory failure but did not progress to ARDS throughout the hospital course. The non–at-risk group had no history of respiratory failure or admission to the ICU for any reason. Clinical data were collected at admission to the ICU, including co-morbidities, Acute Physiology and Chronic Health Evaluation (APACHE II) scores, sex, age, and reason for admission, lung injury scores, and major organ functions. The primary outcome of this study was the survival at the 28th day of ARDS onset. The secondary outcome was the survival at hospital discharge. Peripheral blood samples were obtained, genomic DNA was extracted, and the ACE I/D genotypes were determined by polymerase chain reaction (PCR) amplification of the respective fragments for the D and I alleles from intron 16 of the ACE gene, using primers: 5''CTGGAGACCACTCCCATCCTTTCT3'' and 5''GATGTGGCCA- TCACATTCGTCAGAT3''. Because of the concerns about mistyping ID as DD, all samples found to have the DD genotype were subjected to a second, independent PCR amplification with a primer pair recognizing insertion-specific sequence (5''TGGGACCACAGCGCCCGCCACTAC''3 and 5''TCGCCAGCCCTCCCATGCCCATAA''3). Statistical analyses were performed with SPSS software package. Continuous data were expressed as mean ± SD. Comparisons of the continuous data were performed by analysis of variance or two-sample Student''s t-tests. The chi-square tables were used to compare the observed number of each genotype with those expected for a population in Hardy-Weinberg equilibrium and to compare the genotype frequencies between the ARDS population and the control groups. Survival curves were estimated by the Kaplan-Meier method, and the statistical significances were tested using the log-rank test. Multivariate analyses for outcomes were performed by the Cox proportional hazard methods. A p value less than 0.05 was considered statistical significant. For the results of this part of study, we included 101 ARDS patients (age, 60 ± 21 yrs; 68 men), 138 at-risk control group patients (age, 71 ± 16 yrs; 86 men), and 210 non–at-risk control group subjects (age, 57 ± 13 yrs; 146 men). The incidence of ARDS was approximately 1.75% among medical ICU patients. Lung Injury Scores of the ARDS group and the at-risk group were different (p < 0.001), although APACHE II scores were similar (p=0.10). None of them deviated from the Hardy-Weinberg equilibrium. In all groups, the D allele accounted for <0.3 of the allele frequency. There was no significant difference in the genotype and allele frequ- encies between the ARDS patients and the at-risk group and the non–at-risk group. Sex (p=0.87), age (p=0.995), and underlying diseases were similar in the three genotype groups of ARDS patients. The APACHE II scores (p=0.16) and Lung Injury Scores (p=0.96) were also similar between the three genotypes. The 28-day mortality rate of the ARDS patients was 54%, but of them the mortality rates were significantly diff- erent between the three ACE genotypes (p=0.036), with the II genotype having a higher possibility of 28-day survival. Regrouping as II and non-II also showed a survival benefit of the II group than the non-II group. Survival analysis showed that the II genotype (p=0.007) and hospital-acquired pneumonia (p=0.008) were independent prognostic factors for 28-day survival. Patients with an APACHE II score >25 tended to have a higher risk for 28-day mortality (p=0.082). The in-hospital mortality rate for ARDS was 71%, which was different between ACE genotypes (p=0.038). The II genotype (p=0.012) and hospital-acquired pneumonia (p=0.006) were independent prognostic factors for the outcome at hospital discharge. Patients with an APACHE II score >25 tended to have a higher risk for in-hospital mortality (p=0.10). This part of study suggested that the polymorphism of the ACE gene affects the outcome rather than risk for ARDS in the Chinese population. In the western countries the D allele frequency is usually higher than that of I allele, accounting for 0.51 to 0.56 in the general population, whereas in the Asian countries, the frequency of the D allele is lower, with the D allele frequency among ethnic Chinese people being 0.3~0.45. This may result in a smaller proportion of people carrying the DD genotype, ranging from 0.09 to 0.16 in the ethnic Chinese populations. The findings here may confirm the effect of ACE polymorphism on the outcome of ARDS, but due to a relatively small proportion of people carrying the DD genotype in the general population among Asian people, more cases may be required to assess the risk of ARDS. Although the case numbers in this series are not large, the proportions of the ACE genotypes are similar to those previously reported in Chinese ethnic groups. Because the genotype distribution did not deviate from the Hardy-Weinberg Equilibrium, the possibility of genotyping error is low. There are still no data concerning the estimated incidence of ARDS in Taiwan. Therefore, despite the possibility of an association of the D allele with the risk of ARDS, there is still a lack of data concerning the comparison of incidences of ARDS among Asian and white people. As the majority of ARDS patients died of multiple organ failure, this study has not explained why the ACE gene polymorphism can affect the outcome. We do not know whether there is another pathway triggered due to ACE polymorphism. This study also has some limitations. The levels of angiotensin II were not determined in the ARDS patients, therefore we lack direct functional evidence of the contribution of gene polymorphism to the clinical presentation. However, correlation between the ACE geno- types and the serum ACE levels has been shown, and in the ethnic Chin- ese population, a report has shown that the plasma ACE activity is highest in the DD genotype, followed by the ID, and lowest in the II genotype. The ACE activity in the ID genotype has been shown to be intermediate. In our study, the inclusion criteria were similar to other reports, and all patients received a positive end-expiratory pressure level of no less than 10 cm H2O, but the possibility of inclusion of the “transient ARDS,” as described by Ferguson et al., cannot be excluded. It is also difficult to find adequate control groups as the study was not a cohort-based one, and all subjects in the control groups still have the chance of developing ARDS in face of some risks, although the non–at-risk control groups were apparently older. A population-based cohort study should be more powerful in confirming the hypothesis that ACE genotype determines the risk and outcome for ARDS. It is postulated that patients at risk for developing ARDS appear as candidates for preventive treatment with these agents. The relevance of treatment with these agents and the clinical course of the patients require further clinical trials. In summary, we found that the ACE I/D polymorphism is a significant prognostic factor for the outcome of ARDS in the Chinese population. Patients with the II genotype have a significantly better chance of 28-day survival and at-discharge survival than those with the non-II genotypes. In the third part, we performed animal studies on the effect of injurious mechanical ventilation on the rat lungs in an in vivo model. Although an indispensable in the management of critically ill patients with respiratory failure, mechanical ventilation (MV) can also subject the lungs to substantial abnormal stretching stress, resulting in structural changes, impaired gas exchange and activation of the inflammatory process and leading to ventilator-induced lung injury (VILI) and significant risk to the patients. Inflammatory cells and pro-inflammatory mediators are considered to play an important role in VILI pathogenesis. However, the exact mechanism by which MV triggers the inflammatory process remains unclear. Based on our previous clinical study, we were interested in the involvement of the renin-angiotensin system (RAS) in the development of ventilator-induced lung injury. The involvement of the RAS in inflammatory responses has received attention. The RAS is considered to be a key mediator of inflammation. Moreover, angiotensin II, the key factor of the RAS, has been shown in several in vitro studies to activate an inflammatory process by up-regulation of the synthesis of pro-inflammatory cytokines and chemokines via the type 1 (AT1) and type 2 (AT2) angiotensin II receptors and subsequent activation of the NF-kappaB pathway. Thus it is probable that the RAS is actively involved in the development of lung injury. In conditions that can lead to lung inflammation, such as high-volume ventilation, the RAS may play an important role in the development of lung inflammation. We therefore investigated the involvement of the RAS in VILI. We conducted a series of animal experiments regarding mechanical ventilation and lug injury. Male Sprague-Dawley rats weighing 200-250 g were anesthetized and tracheostomized, and subsequently subjected to mechanical ventilation with a small animal ventilator according to design protocol. Animals were divided into different groups: 1) non-ventilated controls; 2) treated with MV with a high tidal volume (40 ml/kg tidal volume, 3 cmH2O of positive end-expiratory pressure [PEEP], 20 breaths/min, room air); 3) treated with MV with a low tidal volume (7 ml/kg tidal volume, 3 cmH2O of PEEP, 100 breaths/min, room air). MV was applied for 4 hours and the peak airway pressure was monitored throughout. Additional groups of rats received captopril pre-treatment before MV or were treated with losartan or PD123319 during MV. For the captopril-treated group, 50 mg/kg of captopril was added to the drinking water (500 mg/l) for the three days preceding mechanical ventilation. For the losartan- or PD123319-treated, losartan (10 mg/kg) or PD123319 (10 mg/kg) was injected intravenously via a pump during the 4 hours of MV. The lungs were removed and fixed, and sections were prepared and stained with hematoxylin and eosin, and scored for lung injury. Myeloperoxidase (MPO) activity, a marker enzyme for neutrophil infil- tration into the lung, was also assessed. We also performed bronchiolo- alveolar lavage (BAL), and the total protein level in BAL fluid was determined using a commercialized BCA Protein Assay Kit. Total RNA from the lung was isolated, and TNF-alpha and macrophage inflammatory protein (MIP)-2 mRNA levels were assayed by RT-PCR. Moreover, real-time RT-PCR was also used to quantitatively measure mRNA levels for RAS components. Oligonucleotide primers for rat angiotensinogen, ACE, ACE2, and the AT1 and AT2 receptors were designed from the GenBank databases (NM 012544 and NM 134432) using Primer Express ®, and real-time RT-PCR was performed in an ABI PRISM 7500 Sequence Detector. Lung tissue levels of angiotensin II were determined with EIA. To investigate the involvement of the NF-kappaB pathway in VILI, we performed Western blotting to assess the amounts of NF-kappaB, I-kappaB and phosphorylated I-kappaB in the rat lungs. Antibodies to proliferating cell nuclear antigen (PCNA) or c-Jun N-terminal kinase 1 (JNK1) were used to detect these proteins, used as loading controls for the nuclear and cytosolic samples, respectively. Protein levels of the RAS components in the rat lung were determined by Western blotting. Assay for MIP-2 protein level was performed using a rat MIP-2 ELISA kit (Biosource International, Camarillo, CA). Continuous data were expressed as mean + SD. Comparisons of continuous variables between groups were performed using the t test and one-way analysis of variance using SPSS 10 software. A p value of less than 0.05 was considered significant. In this part of study, we found that the peak airway pressure was higher in the high-volume (HV) than the low-volume (LV) group, but did not change significantly throughout the MV course. The mean arterial pressure was markedly decreased in the rats pre-treated with captopril. Arterial blood gas data were similar between groups at the beginning of MV and throughout the course. Histological studies showed that there was no significant inflammatory cell infiltration in the lungs of the control or LV groups, whereas HV ventilation resulted in mild lung injury and mild neutrophil infiltrations, but the alveolar architecture was preserved. The neutrophil infiltration in the HV group was attenuated by captopril pre-treatment. The pathologic lung injury scores were compatible with the histological data. HV significantly increased MPO activity and this effect was significantly attenuated by captopril. The MPO activities were similar between control and LV groups. HV ventilation also increases protein leak into the alveolar space, and this was reduced by captopril. In the HV group, mRNA levels of TNF-alpha and MIP-2 increased progressively during MV, and then decreased gradually after cessation of MV. The HV group had higher levels of TNF-alpha and MIP-2 mRNA; these could be significantly attenuated by captopril. Same trend is also shown by lung MIP-2 and serum MIP-2 protein levels. The nuclear fraction of NF-kappaB was markedly increased in the HV and LPS-treated groups, suggesting nuclear translocation of this factor. In addition, translocation of NF-kappaB in the HV group was significantly, but not completely, attenuated by captopril. On the cytosolic protein blots, the amount of I-kappaB was decreased by HV, with increase in phosphorylated I-kappaB level, which could also be attenuated by captopril. The lung tissue angiotensin II level was increased in the HV group, but not in the LV group. Real-time RT-PCR of the lung tissue showed that HV increased mRNA levels for angiotensinogen and the AT1 and AT2 receptors, but had no significant effect on ACE mRNA levels. The protein levels of the ACE, AT1 and AT2 were similar between groups. We also assessed the mRNA and protein levels of ACE2 in the lungs. The mRNA expression of ACE2 was significantly decreased by HV, but not by LV. The lung levels of ACE2 protein of the rats were very low as compared with the level in the kidney or heart. Difference between groups was not significant. During mechanical ventilation, the blood pressure decrease became more profound in the losartan-treated group, but not in the PD123319-treated rats. Concomitant infusion of either losartan or PD123319 during mechanical ventilation attenuated the protein leak into the BALF. The increase in lung tissue MIP-2 mRNA levels induced by HV was attenuated by the concomitant infusion of either losartan or PD123319. The increase of lung tissue myeloperoxidase activity by HV was also attenuated by either losartan or PD123319. In this part of study using rat in vivo model, there are two novel findings. Firstly, the RAS is activated by HV ventilation in the in vivo animal model. Over-distension of lung units by HV ventilation resulted in up-regulated expression of RAS components and the AT1 and AT2 receptors and increased lung angiotensin II production. Secondly, the RAS plays an important role in VILI. Treatment with an ACE inhibitor or angiotensin receptor antagonist attenuated VILI, with suppression of cytokine expression and NF-kappaB activity in the lungs. We therefore believe that the RAS is actively involved in the pathogenesis of VILI. The RAS has been considered a mediator of inflammation and may therefore play an important pathogenic role in the inflammatory process associated with VILI. Our findings may provide further in vivo evidence of the involvement of the RAS in VILI. The pathogenic role of RAS in the inflammatory process and VILI is strongly supported by the fact that captopril pre-treatment attenuated lung inflammation suppressed TNF-alpha and MIP-2 expression and NF-kappaB activity and reduced blood levels of angiotensin II. Based on these findings, we believe that ACE inhibition may be beneficial in animals ventilated with injurious high tidal volumes. Our findings also provide support for the NF-kappaB pathway being the down-stream response pathway for the action of angiotensin II in the inflammation process, as ACE inhibition attenuated the nuclear translocation of NF-kappaB. However, the RAS may also be influenced by NF-kappaB, as inhibition of NF-kappaB also attenuates RAS activity. Together with our findings, these results suggest that a positive feedback system may be present, and this possibly explains why high-volume MV triggers the RAS and why RAS-mediated ventilator-induced lung inflammation developed rapidly in our animal model study. Our finding that captopril treatment attenuated VILI and the inflammation process in the animal model may have important clinical implications. Therapeutic agents that block the production (ACE inhibitors) or action (angiotensin II receptor antagonist) of angiotensin II may be used as anti-inflammatory agents for the treatment or prevention of VILI. The main limitation of this part of study is that it only focused on the short-term effects of injurious MV. However, since we have shown that active inflammation in the lungs can develop early in injurious MV, we believe that if these injurious settings were used for a longer period, the inflammation and injury would be further aggravated. We do not know the long-term consequence of drug treatment, especially in the clinical setting in which treatment with these agents can cause hypotension, which is clearly undesirable in critically ill patients. This study involved injurious MV as a pure insult to originally healthy lungs, which may differ from clinical scenarios in which the patients may suffer from other initial insults, such as pneumonia, sepsis, or even ARDS, before receiving MV. The local ACE and ACE2 activities were also not measured in this study. Further investigations are needed to elucidate the detailed mechanism of involvement of RAS in VILI. In summary, the renin-angiotensin system plays an important role in the pathogenesis of the inflammatory process in ventilator-induced lung injury. Treatment with an ACE inhibitor or angiotensin receptor antagonist can attenuate ventilator-induced lung injury in the animal model. In the future studies, we hope to further investigate the epidemiologic and clinical features of ARDS, and basic mechanisms of ventilator- induced lung injury. There has been no prospective study for the incidence of ARDS in Taiwan. One of the main difficulties in the epidemiologic study is that there is no specific code for ARDS. For many patients the diagnosis was coded as acute respiratory failure or pneumonia, as it has been in our institution. Therefore screening for ARDS from a large population with respiratory failure is considered very time comsuming. One of the possible ways to solve this problem is to report or register cases with ARDS prospectively, as advocated by the Acute Respiratory Distress Syndrome Network (ARDS Net). However, the main advantage for this registry system so far is the feasibility of prospective clinical trials and observational study for management and outcome, but not for studying incidence. With established databank, we hope to investigate the association of genetic variations and clinical manifestations of ARDS patients further in detail. Additional complex studies such as genomic analysis might also be possible. Despite that the animal model of ventilator-induced lung injury, such as our model, has been widely accepted in the literature, it still has some limitation. The basic and underlying molecular mechanisms for the triggering events and signaling pathways may not be clearly studied by this model with heterogenous cell types. Specificlly labeled markers, as might be used in the molecular imaging methods, e.g., MRI or PET, might be candidated for in vitro investigations for specific labeled cell types or molecules. We would also further investigate the effect of deletrous effects of mechanical ventilation on systemic function of other organs. One of the potential targets is the skeletal muscles. We heve shown that injurious ventilation setting might increased pulmonary as well as systemic expression of proinflammatory mediators, therefore studies on their effects on systemic skeletal muscles might provide some speculation. As respiratory muscle weakness is one of the main causes of ventilator dependence in patients with respiratory failure, this further study may give us further understanding of the mechanism. Evaluation of the potential adverse effects of new modalities for ventilatory support is important. Based one current data and the literature, it is likely that further minimization of the tidal volumes during mechanical ventilation might be helpful for patients with ARDS. Available modalities include high-frequency oscillation ventilation and extracorporeal membrane oxygenation. Despite that some reports have shown benefit to the patients, while others have not; further investigation of possible injury by these modalities requires evaluation. There has been much advance in the field of critical care medicine, both clinical and basic, especially in inflammation and its related conditions, such as sepsis, shock, and aute lung injury. These conditions are far more closely related as we had previously percepted, as more understanding has been obtained by a growing body of research evidence. The respiratory system is a non-dispensible part for human vitality that maintenance of adequate respiratory function is of prime importance in the clinical medicine, both in diagnosis and treatment. Advances in basic as well as physiologic researches might be very beneficial to clinical management of patients suffering from respiratory insufficiency, which might result in a massive burden both on patients and the medical resource. Together with the speculation that the problems caused by lung injury are not limited to the lung itself, and systemic organ dysfunction might ensue, extensive studies for the basic mechanisms of acute lung injury, acute respiratory distress syndrome, and ventilator-induced lung injury might have great clinical implications. We hope to extend our scope and depth of studies, both clinical and basic, for better understanding as well as better patient care.