In Vitro Studies
To determine whether SPIR is antifibrotic in vitro, human colonic myofibroblasts (CCD-18co) were stimulated with TGF-β to induce a fibrotic phenotype characterized by increased actin stress fiber accumulation, αSMA protein expression, and increased expression of profibrotic genes including Acta2, Col1a1, and Ctgf (data not shown). SPIR repressed TGF-β induction of αSMA protein expression in a dose-dependent manner (Fig. 2A). SPIR repressed actin stress fiber formation (data not shown). Both SPIR and its active metabolite, canrenone, dramatically repressed TGF-β-induced αSMA protein expression (data not shown). SPIR repressed the expression of fibrotic genes Acta2, Col1a1, and Ctgf to levels indistinguishable from untreated controls (Fig. 2B). Canrenone significantly repressed Acta2 mRNA expression, but its reduction in Col1a1 and Ctgf mRNA expression were not statistically significant (Fig. 2B).
Figure 2. Results of in vitro studies. (A) SPIR inhibits TGF-β induction of αSMA protein expression in colonic myofibroblasts. A representative western blot of αSMA expression in protein extracts from CCD-18co colonic myofibroblasts stimulated for 24 hours with TGF-β is shown. Increasing amounts of SPIR from 10 μM to 100 μM reduce αSMA expression to levels comparable to unstimulated cells (no Tx). GAPDH expression serves as the loading control for the amount of protein. (B) Treatment of TGF-β stimulated CCD-18co cells with SPIR represses expression of Acta2, Col1a1, and Ctgf. A metabolite of SPIR, canrenone (CAN), partially represses profibrotic gene expression. (C) The role of the RAAS pathway in fibrosis and the relationship of pathway inhibitors. (D) Inhibitors of the RAAS pathway aliskiren (ALK), enalaprilat (ENT), and losartan (LOR) partially repress TGF-β induction of fibrotic genes with partial repression of Acta2 expression but have minimal effect on Col1a1 expression (E). Results are from nine independent experiments. Asterisks denote statistically significant comparisons between untreated control cells (no Tx) and the treatment groups. Brackets denote comparisons between TGF-β treated and other treatment groups. *P < 0.05, ***P < 0.001.
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Given the antifibrotic effect of SPIR, we investigated whether blocking upstream components of the RAAS pathway (Fig. 2C) would block fibrosis in vitro. In TGF-β-stimulated myofibroblasts, other RAAS inhibitors aliskiren (renin inhibitor), enalaprilat (ACE inhibitor), and losartan (angiotensin II type I receptor blocker [ARB]) partially repressed profibrotic gene expression (Fig. 2D,E), suggesting a role for the RAAS pathway in intestinal fibrosis.
Rodent Colitis Studies
Unexpectedly, in two animal models of intestinal inflammation and fibrosis, treatment with SPIR caused significant and rapid mortality. In the rat TNBS chronic colitis model, 20 mg/kg/day SPIR (10-fold lower than well-tolerated doses in other rat models18) produced 67% mortality (95% confidence interval [CI]: 0.046–0.68) (Fig. 3A). A dose–response experiment in the rat TNBS model demonstrated increased survival with decreasing SPIR dose. Mortality was 33% at 10 mg/kg/day, and 0% at 2.5 or 0.5 mg/kg/day (Fig. 3B). However, lower doses of SPIR did not reduce the development of fibrosis, as determined by gross pathology, histopathology, fibrotic gene expression, and protein expression (αSMA) (data not shown). Similar experiments with losartan resulted in no mortality and no improvement in fibrosis (data not shown).
Figure 3. Results of rodent colitis studies. (A) Increased mortality in rats with chronic TNBS colitis treated with SPIR. Kaplan–Meier mortality estimates of rats with chronic TNBS and 20 mg/kg/day SPIR (TNBS+SP) compared to no mortality with TNBS alone and in untreated rats (no Tx). Data are from three rats per experimental group. (B) Increased mortality in rats with chronic TNBS colitis treated with SPIR is dose-dependent. The Kaplan–Meier curve demonstrates increased mortality in rats with chronic TNBS colitis treated with doses of 10 or 20 mg/kg/day SPIR (T+SP 20, T+SP 10) compared to low doses of SPIR (T+SP 0.5, T+SP 2.5), TNBS alone, or rats with no treatment (no Tx). Data are from three rats per experimental group. (C) SPIR treatment increases mortality in the S. typhimurium mouse model of colitis. Mortality occurred in mice with S. typhimurium-induced colitis treated with 0.7 mg/kg/day SPIR (SP+St), compared with mice infected with S. typhimurium without SPIR (St), uninfected mice (no Tx), or mice receiving 0.7 mg/kg/day SPIR alone (SP). Uninfected mice which received SPIR treatment over 15 days had 0% mortality until subsequent S. typhimurium induction of colitis (SPSP + St) at day 16 (vertical arrow), which produced 100% mortality by day 5 postinfection. Data are from five mice per experimental group.
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In the mouse S. typhimurium infection model of intestinal fibrosis, treatment with 0.7 mg/kg/day of SPIR produced 80% mortality (95% CI: 0.69–1.00) by day 9 of colitis compared to 20% mortality in the S. typhimurium-infected group (Fig. 3C). No mortality occurred in uninfected mice receiving 0.7 mg/kg/day of SPIR. However, when the SPIR-treated cohort was later challenged with S. typhimurium, 100% mortality occurred by day 5 postinfection.
Retrospective Study of C. difficile Colitis Clinical Outcomes
To determine whether SPIR use was associated with increased mortality in humans with intestinal inflammation, we identified a total of 4008 inpatients with a discharge diagnosis of CDI in University of Michigan Hospital admissions from January 1, 2000 through December 31, 2009. These 4008 patients accounted for a total of 5166 CDI-associated hospitalizations in that 10-year period. There were 352 patients on SPIR during at least one hospitalization; these patients were hospitalized 391 times. The average time between hospitalizations for SPIR users was not significantly different from nonusers, while the average number of hospitalizations for the two groups did differ (1.4 vs. 1.3, P = 0.02, n = 4,008) (Table 1). In patients on SPIR therapy, the average dose was 79.9 mg (±71.5). The average age, gender ratio, and race did not differ between SPIR users and nonusers.
Table 1. Characteristics of Patients with C. difficile Colitis on Spironolactone Therapy
|Characteristic||Spironolactone Therapy (N = 352) Mean (± SD), Mean [Range] or n (%)||No Spironolactone Therapy (N = 3656) Mean (± SD), Mean [Range] or n (%)||P value (N)|
|Age (years)||57.1 (± 14.2)||57.6 (± 17.9)||0.53|
|Male||181 (51.4%)||1859 (50.9%)||0.84|
|Race|| || ||0.29|
| Caucasian||282 (80.1%)||3025 (82.7%)|| |
| African-American||37 (10.5%)||369 (10.1%)|| |
| Other||33 (9.4%)||262 (7.2%)|| |
|Hospitalizations (individual)||391||4775|| |
|No. of hospitalizations per patient||1.4 [1 – 6]||1.3 [1 – 12]||0.02|
|Time between hospitalizations (days)||132.5 [1 – 1483]||129.2 [1 -2383]||0.90|
|Heart failure||137 (38.9%)||549 (15.0%)||<0.0001|
|Liver disease||151 (42.9%)||273 (7.5%)||<0.0001|
|Diabetes||90 (25.6%)||835 (22.8%)||0.25|
|Baseline measures (per hospitalization)|| || || |
|Sodium (mmol/L)||134.9 (± 5.6)||137.4 (± 4.8)||<0.0001 (5121)|
|Potassium (mmol/L)||4.2 (± 0.6)||4.1 (± 0.5)||0.0004 (4582)|
|Creatinine (mg/dL)||1.3 (± 0.7)||1.5 (± 1.6)||<0.0001 (5118)|
|BUN (mg/dL)||27.5 (± 18.3)||25.3 (± 20.4)||0.03 (5119)|
|INR||1.6 (± 1.0)||1.4 (± 1.0)||0.007 (3763)|
|Albumin (g/dL)||3.1 (± 0.7)||3.1 (± 0.7)||<0.0001 (4432)|
|Total bilirubin (mg/dL)||3.1 (± 5.3)||1.3 (± 3.5)||<0.0001 (4479)|
|MELD||6.6 (± 9.0)||2.5 (± 10.1)||<0.0001 (3416)|
|Charlson-Deyo Comorbidity Index||4.1 (± 2.7)||3.9 (± 2.7)||0.25 (3741)|
|Outcomes|| || || |
|Mortality (by individual)||56 (15.9%)||334 (9.1%)||<0.0001|
|Mortality (by hospitalization)||49 (12.5%)||341 (7.1%)||0.0001|
|Length of each hospitalization (days)||25.0 [1 – 171]||17.1 [1- 851]||<0.0001|
|Cost per hospitalization (in $1000′s)||197.9 [0 – 2489.4]||110.4 [0 – 6388.0]||<0.0001|
As expected, the group on SPIR therapy had significantly more patients with a diagnosis of heart failure (38.9% vs. 15.0%, P < 0.0001) and liver disease (42.9% vs. 7.5%, P < 0.0001) compared to nonusers of SPIR. The mortality in those patients with heart failure was 77 of 686 (11.2%), in those with liver disease was 71 of 424 (16.7%), and in patients with neither heart failure nor liver disease was 242 of 2898 (8.4%). The majority of patients taking SPIR without a history of heart failure or liver disease were doing so due to hypertension or evidence of edema.
Laboratory values affected by heart or liver dysfunction (creatinine, sodium, international normalized ratio [INR], total bilirubin, potassium, albumin) were significantly different between users and nonusers of SPIR (Table 1), and were used as proxies for disease severity in a predictive model. Bilirubin and INR were higher in the SPIR group while sodium, albumin, and creatinine were lower. Discharge day potassium was higher in the SPIR group. The MELD17 score showed a significant difference between the two groups, while there was no significant difference in Charlson-Deyo comorbidity index.19, 20 The clinical outcomes in the two groups demonstrated significant differences, with longer length of stay, higher mortality, and higher costs per hospitalization in the SPIR users.
Figure 4 shows the single predictor ORs for inpatient mortality in all patients after adjustment for hospitalization number. In this analysis (n = 5166, patients on SPIR had 1.84 [95% CI: 1.34–2.53] times the odds of inpatient mortality of those not on the medication). For each additional 25 mg of SPIR dose, the OR for mortality was increased by 13% (95% CI: 6%–20%). Liver disease, higher MELD scores, and heart failure were associated with higher inpatient mortality, while Charlson-Deyo scores were not. ACE-I (angiotensin converting enzyme inhibitor) and ARB use were both associated with decreased mortality.
Figure 4. Single predictor odds ratios for inpatient mortality in patients with C. difficile colitis. For each predictor, the odds ratio from a logistic model for inpatient mortality was adjusted for hospitalization number (≤2 vs. >2) and is presented with 95% CIs. A vertical dashed line at 1 represents the point of no effect.
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These predictors were combined in a multivariate logistic regression model (n = 4415) to predict inpatient mortality (Fig. 5A). We found that SPIR use remained a significant predictor of mortality, and found a statistically significant interaction between SPIR dose and liver disease, described below. Testing the other univariate predictors, we found that blood urea nitrogen (BUN), total bilirubin, albumin, and use of ACE-I and ARB medications were significant contributors to our model of inpatient mortality. No significant interaction between heart failure and SPIR use was found. In all, 4415 hospitalizations without missing values were included in this model. Hospitalization number was also included for each individual in the model; patients were more likely to die during one of the first two hospitalizations than in subsequent hospitalizations (OR 1.52, 95% CI: 0.92–2.50, P = 0.10). This model had fairly high explanatory power, with a c statistic of 0.71. The linear form of SPIR dosing in the model was statistically superior to a quadratic or logarithmic transformation. The exclusion of high doses of SPIR (>100 mg) did not substantially alter the model.
Figure 5. Adjusted odds ratios for and predicted probabilities of inpatient mortality in patients with C. difficile colitis. (A) The adjusted ORs for inpatient mortality in a multivariate model containing SPIR, liver disease, heart failure, the interaction between SPIR and liver disease, ACE-I use, ARB use, total bilirubin, albumin, BUN, and hospitalization number (≤2 vs. >2). The OR for liver diseases with and without SPIR and the OR of liver disease with SPIR are compared to patients without liver disease not taking SPIR (Reference group). (B) The predicted probability (with 95% CI) of inpatient mortality in patients with liver disease, heart failure, and neither liver disease nor heart failure while using three possible doses of SPIR for that disease state. For each disease state the average total bilirubin, albumin, and BUN were used in conjunction with the proportion of patients on ACE-I and ARB medications. Details for each disease state are in the Supporting Materials. In all groups the probability is shown for hospitalization number ≤2. This demonstrates that SPIR use is associated with increased mortality in patients without liver disease.
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Because of the interaction between liver disease and SPIR, patients with liver disease had a (nonsignificant) protective effect from SPIR of 29% (OR 0.71, 95% CI: 0.47–1.07), while patients without liver disease had significantly worse outcomes with SPIR. Compared to the reference group (patients without liver disease not taking SPIR), patients with liver disease taking the average dose of SPIR (80 mg) had an adjusted OR for inpatient mortality of 1.07 (95% CI: 0.70–1.64). In patients without liver disease, the adjusted OR for inpatient mortality associated with 80 mg SPIR was 1.99 (95% CI: 1.51–2.63) (Fig. 5A) compared to the reference group. We show the direct effects of this interaction term on mortality in Figure 5B, where the probabilities of inpatient mortality in patients with liver disease, heart failure, and with neither of these comorbidities are illustrated. To show the dose–response effect, we modeled the predicted probability of inpatient mortality for each disease state for patients on high-dose SPIR, low-dose SPIR, and those not taking the drug. Predicted probabilities are based on the average values of total bilirubin, albumin, and BUN, ACE-I/ARB use, and hospitalization number ≤2. Patients with liver disease had a lower probability of inpatient mortality if they were using SPIR. In contrast, in patients with heart failure, who are typically on lower doses of SPIR, we demonstrate an increase in inpatient mortality with SPIR use. The patients without liver disease or heart failure were on higher average doses of SPIR, and this is reflected in a significantly increased risk of inpatient mortality.