Among inflammatory illnesses, pneumonia often presents as sepsis, which is a potentially fatal whole-body inflammation in response to invading pathogens.1 The current incidence of sepsis in the United States is at least 240 patients/100,000 people, with the mortality rate from 25% to 30% for severe sepsis and up to 40%–70% for septic shock.2 A significant number of these patients have pneumonia.3 With advances in medical care, many patients with pneumonia do not need hospitalization and most who admitted to the hospital survive to discharge. Nevertheless, pneumonia remains common and is still one of the leading causes of hospital admission and death through the world.4–6 The pneumonia-related mortality remains at 10% to 15%.6
Calcium (Ca2+), as a major second messenger in cellular signaling events, has a pivotal role in severe infections and sepsis. Altered Ca2+ homeostasis has been found to be one of the underlying pathophysiological processes in sepsis. Attenuating the disrupted Ca2+ homeostasis has been shown to decrease overall mortality in various animal septic models.7–11 All types of calcium channel blockers (CCBs), by inhibiting the flow of extracellular Ca2+ influx through voltage-gated Ca2+ channels such as L-type Ca2+ channels (LTCCs),12 have been found to decrease sepsis related end organ damages and mortality in animal models.7,9,13–16 However, to our best knowledge, no study had been conducted in human subjects to investigate the potential beneficial effect(s) of CCBs in sepsis. As a result, the role of CCBs in severe infections like pneumonia in human remains unclear. Here, we hypothesized preadmission CCB use is associated with better outcomes in pneumonia/sepsis.
Study setting and patients
We conducted this retrospective study at Cooper University Hospital, a tertiary academic medical center in Southern New Jersey. The study was approved by Cooper University Hospital's Institutional Review Board. Patients older than 40 years with a discharge diagnostic International classification of diseases, Ninth Edition code of 481, 482, 485, or 486 from January, 2012, to December, 2014, were included into the initial screening pool. We did not include patients younger than 40 years because they are less likely to have hypertension and be prescribed with a CCB. Chest radiography and clinical presentation were reviewed for the presence of clinically compatible pneumonia at the first 72 hours of admission. Our exclusion criteria were (1) patients who were not on a CCB but started on any CCB during hospitalization; (2) patients who were initially admitted to outside facilities and had stayed there more than 72 hours; (3) patients who were admitted for surgical procedure(s) or to trauma service; (4) patients who were transferred to Cooper University Hospital for procedures only, including, but are not limited to, cardiac catheterization, bronchoscopy, endoscopic retrograde cholangiopancreatography, or endobronchial ultrasound; (5) patients presented with cardiac arrest; (6) patients who expired in less than 24 hours after admission or left the hospital against the medical advice; and (7) pregnant women. Patient data were extracted from the EPIC electronic database, including demographics, hemodynamics, medications (including statin), laboratory values and comorbidities, including history of diabetes, ischemic heart disease, congestive heart failure, arrhythmias, and renal, liver, and gastrointestinal disease. Although data on the specific type of CCBs used were collected, for the purpose of analysis, patients on any type of preadmission CCB therapy will be grouped as “CCB users.” Other covariate data to be collected include the length of hospital stays (LOS); the Pneumonia Severity Index (PSI) on arrival; the need for intensive care unit (ICU) admission; whether there was an associated bacteremia; severe sepsis or acute respiratory insufficiency. Severe sepsis is defined as sepsis-induced tissue hypoperfusion or organ dysfunction, including sepsis-induced hypotension; lactate above upper limits of laboratory normal; creatine greater than 2.0; platelet count <100,000; or coagulopathy with international normalized ratio >1.5 for patients who were not taking any anticoagulation agent.17,18 Respiratory insufficiency was arbitrarily defined as oxygen requirement greater than 6 L of nasal cannula oxygen. Acute renal injury is defined by Acute Kidney Injury Network criteria with creatine increasing more than 0.3 mg per deciliter from the baseline.19 We also retrieved the data for statin and immunosuppressant use, as statins could potentially confound clinical effects of CCBs.20 The immunosuppressant is defined as a chronic steroid, biological products for autoimmune or rheumatologic conditions, and concurrent chemotherapy. Age-adjusted Charlson Comorbidity Index (CCI) was used as a measure of comorbidities. CCI is a system for classification of severity that uses recorded data on secondary diagnoses to assign a weight to each morbidity, thereby generating the patient's risk for death.21,22 The final CCI score is the sum of weight assigned to 19 predetermined clinical conditions.22 The age-adjusted complete CCI we used is to add one point for each additional 10 years to the initial score.22 CCI has been shown be a reliable tool for measurement of comorbidities and risk adjustment in population-based studies.23,24 The index has been validated for its ability to predict mortality in various disease subgroups, including cancer, renal disease, stroke, liver disease, and intensive care.25,26
Statistical analyses were conducted using SAS v9.4 software (SAS Institute, Cary, NC). Continuous data are expressed as means with SDs and categorical variables are reported as frequencies and percentages. The primary analysis compared patients who were receiving a CCB with controls who were not receiving a CCB as outpatient. Propensity score matching was used to control for potential confounding and selection bias. The CCB patient group was matched to the non-CCB patient group using propensity scores based on the following variables: age, gender, CCI, statin and immunosuppressant use. Logistic regression was then used to estimate the odds ratio (OR) of the effect of CCB on the clinical outcomes. The results are expressed as ORs with 95% confidence interval (CI). A P-value <0.05 was considered statistically significant. The association between prehospital CCB use and continuous outcomes, including the LOS and PSI on arrival, were analyzed by the Student t test. Kaplan–Meier analysis was used to relate risk of sepsis to age. The log-rank test was used to compare groups.
During the 3-year study period, there were 2214 eligible patients hospitalized due to pneumonia who were older than 40 years. One thousand three hundred sixty-nine patients met inclusion and exclusion criteria and then were included into the study. Among these 1369 patients, 411 were on CCBs (30.02%) and 958 were non-CCB users before hospitalization (Figure 1).
The patient characteristics classified by CCB users or non-CCB users before propensity score matching are shown in Table 1. The mean age was 69.35 years for CCB users and 65.02 years for non-CCB users. The CCB users were more likely to take statins at home (47.69% vs. 39.46%). Moreover, CCB users had higher CCI scores at the baseline (CCI, mean ± SD: CCB users 6.78 ± 3.143 vs. non-CCB users 4.88 ± 2.930, P < 0.05). In this baseline comparison, the age of CCB user group was significantly older than that of non-CCB group (CCB users: 69.35 ± 13.145 vs. non-CCB users 65.02 ± 13.687, P < 0.05). Similarly, CCB users had statistically higher predicted 1 year mortality, based on the CCI scores. We did not further test the statistical significance for any specific comorbidity, statin or immunosuppressant usage at this phase because we would balance all these covariates through propensity score matching in the CCB user and non-user group. The most common comorbidity was hypertension (CCB users: 97.8% vs. non-CCB users 61.4%), followed by smoking (CCB users 72.50% vs. non-CCB users 70.46%) and diabetes (CCB users 45.50% vs. non-CCB users 33.19%). The most common CCB prescribed was amlodipine. Three hundred twenty-five patients were on amlodipine (79.0%), followed in frequency by diltiazem (55, 13.4%), verapamil (13, 3.2%), nifedipine (13, 3.2%), and felodipine (5, 1.2%).
Propensity score-matched analysis
Propensity score matching of the study population yielded 387 patients in each of the matched group of patients (Figure 1). The CCB group and non-CCB group did not differ in terms of their baseline characteristics in matched patients (Table 2). Severe sepsis developed during the hospitalization in 42 (5.43%) and 80 (10.35%) patients in CCB users and non-CCB users, respectively (OR, 0.466; 95% CI, 0.311–0.697; P = 0.002). Using Kaplan–Meier analysis based on the patient age, the risk of having severe sepsis was significantly lower in CCB users than in non-CCB users (control) (Figure 2; P = 0.0005). CCB use was associated with a lower risk of having bacteremia (OR, 0.498; 95% CI, 0.262–0.99; P = 0.0327). Fifteen patients (1.94%) developed bacteremia in CCB user group and 29 patients (2.75%) had such complication in the non-CCB group. The risk of developing acute respiratory insufficiency was inversely associated with prehospital use of CCBs (OR, 0.573; 95% CI, 0.412–0.798; P = 0.0010). Our study also showed a significant association between CCB use and a reduced risk of ICU admission (OR, 0.602; 95% CI, 0.432–0.840; P = 0.0028). However, there was no such statistically significant association in terms of CCB use and in-hospital mortality or acute renal injury by Acute Kidney Injury Network criteria (ORs of 0.726 and 0.961, respectively; P > 0.05) (Table 3).
Furthermore, there were significant differences in the LOS and severity of illness by the PSI on arrival. The mean LOS for CCB users and non-CCB users were 6.05 and 8.12, respectively (6.05 ± 5.12 vs. 8.12 ± 7.85, P < 0.0001). Prehospital CCB use was associated with less severe pneumonia based on the PSI on arrival (mean 95.09 ± 29.81 vs. 105.74 ± 33.63, P < 0.0001) (Table 4).
In sepsis, the host response to pathogens is mediated through innate and adaptive immune systems. After being triggered by an initial stimulus, the cells of the innate immune system release plenteous amounts of cytokines, chemokines, complement-activation products, and intracellular alarmins during the early and late phase of sepsis, such as tumor necrosis factor-α, nitric oxide (NO).27–29 NO, in concert with cytokines, stimulates the expression of enzymes and then in consequence; it enhances formation of vasoactive mediators and contributes to the development of hypotension.30,31 Elevated levels of NO are thought to contribute to the propagation of multiple organ failure during sepsis.31 Calcium (Ca2+), the major second messenger in cellular signaling events, is required for the generation of inducible NO synthase (iNOS).10
Ca2+ is a highly versatile intracellular second messenger and regulates many complicated cellular processes.32,33 Influx of Ca2+ from the extracellular fluid is required for sustained elevation of the cytosolic Ca2+ concentration and full activation of Ca2+-dependent processes.34 Altered Ca2+ homeostasis had been found to have a pivotal role in severe infections and sepsis. In sepsis, the intracellular Ca2+ concentration in aortic smooth muscle was found to increase more than 2-folds, and this effect could be ablated by sodium dantrolene, potentially by decreasing release of Ca2+ from the endoplasmic reticulum/sarcoplasmic reticulum (SR),8 and inhibiting Ca2+ influx from extracellular space.35 Similar results were seen in mouse model of sepsis induced by cecal ligation and puncture, where the mean intracellular Ca2+ concentration in cardiomyocytes was found to increase more than 21% at 24 hours.36 Uncontrolled Ca2+ leaks from SR/endoplasmic reticulum and extracellular space cause cytosolic Ca2+ overload to a toxic level,37 which in turn, is associated with various adverse effects, such as a compromised force generating capacity37; mitochondria Ca2+ overload38; activation of Ca2+-dependent calpain, which promotes muscle proteolysis39 and muscle weakness,40 all of these could lead to vasodilatation, tissue hypoperfusion, and cardiovascular dysfunction.41,42 Attenuating or even ablating such disrupted Ca2+ homeostasis could potentially work as a therapeutic target for sepsis.
CCBs inhibit the flow of extracellular Ca2+ influx through ion-specific channels that span the cell wall. It is widely accepted that Ca2+ release-activated Ca2+ channels (or stored operated Ca2+ entry) are the major routes of Ca2+ influx in electrically nonexcitable cells, including hematopoietic cells, whereas voltage-gated Ca2+ channels such as LTCCs serve as the principal routes of Ca2+ entry into electrically excitable cells such as neurons and myocytes. However, recent pharmacological and molecular genetic studies have revealed the existence of functional LTCCs and/or LTCC-like channels in a variety of immune cells including mast cells.43 Although several types of calcium channels have been identified on plasma membrane, currently available CCBs all inhibit LTCC and block Ca2+ influx from extracellular space.12
The interests for the protective effects of CCBs in sepsis can be traced back to 1980s. Lee and Lum7 found that in rats, CCBs (verapamil, nitrendipine, and nilvadipine), administered intravenously 15 minutes before endotoxin, produced a dose-dependent reduction in mortality. Since then, multiple pathways had been suggested to be underlying mechanisms of such effect.
Evidences have shown that lipopolysaccharide (LPS) activates the nuclear factor kappa β (NF-κβ) pathway through phosphorylation of inhibitor of kappa β-a (Iκβa) through Ik kinase (Ik K). Degradation of Iκβ-a unmasks the NF-κβ nucleus translocation sequences, allowing NF-κβ p65 subunit to enter the nucleus to directly transcript the target genes.44,45 It has been well documented that Iκβa degradation is dependent on elevation of intracellular Ca2+.9,46,47 Verapamil and other CCBs had been found to have an inhibitory effect on NF-κβ activation by decreasing the nuclear translocation of p65 subunit10,48 and further cytokine synthesis, such as tumor necrosis factor α.11,15
It is widely assumed that the products released by activated neutrophils play an important role in the generation of tissue injury induced by sepsis. These products include proteolytic enzymes and reactive oxygen species. It is a prevalent concept that reactive oxygen species production is enhanced and that antioxidant defenses are depressed during septic shock.49 Two decades ago, NO emerged as a potentially important player in the pathogenesis of sepsis. It was reported that inducible iNOS can be induced by endotoxin, oxidative stress, and proinflammatory cytokines.47,50,51 Several CCBs, including amlodipine, have been demonstrated to protect against NOS induction,14,52 and prolong survival times in endotoxin shock in various animal models.50,53 Further studies had showed the mechanism of prevention NO induction by CCBs is due to an inhibition of LPS stimulated cytokines release.9
After all, these pathways are interdependent, and Ca2+ homeostasis plays a pivotal role in these processes. Attenuating the disrupted Ca2+ homeostasis helps CCBs exerting their protective effects.10 It has been reported that diltiazem treatment of endotoxic rats restored the cytosolic Ca2+ level to that found in controls.13 The SR Ca2+ handling dysfunction is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction.38 In rat sepsis models, rats were subjected to LPS for 6 hours. The serum alanine aminotransferase level was elevated significantly; this response was accompanied by an increase in inducible iNOS mRNA formation in the intact liver. Pretreatment of rats with CCBs (ie, diltiazem, nifedipine, or verapamil, which are structurally unrelated) before LPS exposure attenuated the serum alanine aminotransferase level and iNOS mRNA expression in the liver. This inhibitory effect is linked to decreasing nuclear levels of the p65 subunit of NF-κβ,10 which is one of the most important mediators in sepsis. However, it has been well documented that Iκβa degradation is dependent on elevation of intracellular Ca2+.9,46,47 By blocking the extracellular Ca2+ influx and further Ca2+ release from SR, CCBs have been shown to attenuate the LPS-dependent elevation in intracellular Ca2+ in cultured Kupffer cells.54 Furthermore, treatment of Kupffer cells with CCBs before stimulation with LPS resulted in a preservation of cytoplasmic Iκβa protein, supportive of the theory of the essential requirement of Ca2+ for Iκβ degradation from the NF-κβ–Iκβ complex. More recently, using cultured neonatal mice cardiomyocytes as well as cecal ligation and puncture mice model, Celes et al. had showed that there is striking increment in cytosolic Ca2+ in cardiomyocytes after exposed to serum from septic mice. This increment of Ca2+ is associated with elevated expression of calpain, a Ca2+-dependent protease involving muscle proteolysis in ICU acquired weakness. Treatment of verapamil and dantrolene prevented the increase in calpain-1 level, preserved cardiac contractile function, and striking improved survival rate of septic mice.36 Taking together, evidence speak clearly a protective role of CCBs in sepsis. However, there is only one clinical study in literature reports that CCBs protect renal transplant patients from sepsis related complications.55 To our knowledge, no study has been conducted in general human subjects to explore the beneficial role of CCBs in sepsis.
Here, we conducted the very first clinical study to investigate the association between CCBs and outcomes in severe infections and sepsis. Our study supports the compelling evidence from animal studies that CCBs are beneficial in sepsis. We screened more than 2200 patients who were 40 years older and with a diagnosis of pneumonia for hospitalization. To avoid potential confounding and selection biases, we used propensity score matching by the closest scores on age, gender, CCI, statin and immunosuppressant usage status. As shown in Table 2, our study showed a good balance in these covariates in the CCB user group and the non-CCB user group.
In our study, preadmission CCB use was associated with a lesser disease severity by PSI (P < 0.0001) on arrival, which indicates CCBs are beneficial primarily in the early phase of infection, possibly by attenuating the toxic elevation of cytosolic Ca2+ concentration.13 Preadmission CCB use was also associated with better clinical outcomes in our study, including a lower risk of severe sepsis, bacteremia and acute respiratory insufficiency (Tables 3 and 4, P < 0.05). In contrast, preadmission CCB use did not affect the risk of concomitant acute renal injury or the in-hospital mortality.
From resource utilization purpose, our study showed preadmission use was associated with a shorter LOS (Table 3, P < 0.0001) and a lower chance of requiring ICU level of care. Given pneumonia is such a common disease and one of the leading causes of hospital admission and death, even a minor decrease in LOS could have major resource-saving implications.
Although we initially intended to study the association between CCB use and the benefit on the short-term (etc 30 days') or long-term (etc, 1 year's) mortality in patients with pneumonia and sepsis, a significant portion of study subjects lost follow-ups on discharge, based on their admission or outpatient appointment data in EPIC database. We were unable to collect such information. However, our study showed no significant association between preadmission CCB use and all cause in-hospital mortality (Table 3).
The credibility and general applicability of main findings of our study are supported by several factors. First, we utilized a propensity score matching technique in an attempt to balance factors affecting the exposure to control for confounders affecting outcomes. Second, we used CCI to minimize the potential confounding effect of underlying comorbidities. As we mentioned early, CCI included 19 comorbidities and had been well tested in population-based study for various disease processes.26 Third, the results are biologically plausible and supported by compelling evidences from various animal septic models. As discussed above, CCBs could attenuate toxic cytosolic Ca2+ elevation, reduce cytokine production, and preserve muscle contractility with favorable hemodynamic outcomes.36
Our study has several limitations. First, it is a single-center retrospective study; thus, our findings are potentially subjected to selection or confounding bias. The propensity score matching of this study showed excellent balance between the CCB users and non-CCB users and minimized the baseline differences, such as age, gender, statin and immunosuppressant usage, and baseline comorbidities. However, unaccounted (unobserved) factors could have biased the outcomes. For example, body mass index has been found to be associated with the adverse outcomes in severe infections and sepsis.56 The potential imbalance of body mass index in our study could confound our outcomes. Second, we grouped patients on any type of preadmission CCB therapy grouped as “CCB users.” Although all CCBs inhibit LTCC and block Ca2+ influx from extracellular space,12 there could be a difference in such capacity, and this effect could be dosage dependent. Third, we included patients who developed hypotension or septic shock. In those patients, the CCBs, as antihypertensive agents, were apparently discontinued after the presence of hypotension. The outcomes of this portion of patients could potentially confound the overall outcomes.
In conclusion, this is the first study to investigate the association of preadmission CCB use and outcomes in pneumonia in human subjects. The results reported here add credence to the data from animal septic models that CCBs are associated with better outcomes in severe infections and sepsis. Prospective observational cohort and/or randomized trials are needed to examine such association found in this retrospective observational study. Given CCBs' wide use and excellent safety profiles, positive results of CCB treatment would have substantial clinical and public health implications.
The authors thank Dr. Philip Dellinger, the Chief and Chairman of Department of Medicine, and Dr. Stephen Trzeciak, Division Head of Critical Care Medicine, Department of Medicine at Cooper Medical School of Rowan University for their wisdom, support, inspiration, and valuable comments during writing this essay.
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