Clinical trial NCT05362474
Conditions: Diabetic Kidney Disease
Sponsor: University of Colorado, Denver
Start date: 2022-07-01; Completion date: 2023-06-20
Enrollment: 6 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Diabetic kidney disease (DKD) is associated with significant morbidity and mortality. Identifying new treatments for DKD to be used alone or in combination with other therapies is a high priority. Inflammation plays a key role in DKD and targeting pro-inflammatory lipid mediators called leukotrienes may represent a promising therapy for DKD. The current proposal will investigate whether montelukast, a leukotriene antagonist, reduces proteinuria and improves vascular function and arterial stiffness in patients with DKD.

Detailed description:

Arms: arm 1: montelukast 10mg orally once a day
Interventions: intervention 1: Montelukast
Intervention descriptions: intervention 1: 10mg orally once a day

Protocol documents:
Prot_SAP_001.pdf: 
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w 
COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Protocol #: 22-0187 
Project Title: Targeting Leukotrienes in Kidney Disease: A Pilot Study 
 
Principal Investigator:  Jessica Kendrick MD  
Version Date: 9/23/2022  
 
 
 
 
 
  
 
I. Hypotheses and Specific Aims:  Chronic kidney disease (CKD) is highly prevalent in the 
United States and is associated with significant morbidity.  Diabetic kidney disease (DKD), 
is  pathologically characterized by renal fibrosis, tubular epithelial atrophy, and intra-renal 
inflammation.  Progression of DKD to end stage kidney disease (ESKD) leads to a 
dramatic worsening in patients’ quality of life as well to a high rate of cardiovascular 
disease (CVD) and mortality.  Due to the high disease burden of DKD, therapies that 
prevent kidney disease progression are urgently needed.  Equally important are therapies 
that improve cardiovascular risk, the leading cause of mortality, in patients with DKD. 
 
Currently approved therapies to prevent DKD progression include agents that block the 
renin angiotensin aldosterone system (RAAS) and sodium-glucose cotransport 2 inhibitors 
(SGLT2i).  While both RAAS antagonists and SGLT2i are effective treatments for patients 
with DKD, residual risks for CKD progression remain. Additionally, SGLT inhibitors may 
not be safe in all DKD groups. Hence, identifying new treatments for CKD to be used 
alone or in combination with other therapies is a high priority. Since inflammation is 
a unifying pathway in most forms of CKD, targeting intrarenal macrophages and 
lymphocytes may represent a promising target.  Experimental CKD models have shown 
that macrophages are crucial mediators of renal epithelial damage, fibroblast activation, 
and CKD progression. 
 
Our earlier studies have shown that macrophages promote tubular damage and renal 
fibrosis through activation of the 5-lipoxygenase (5-LO) pathway.  The 5-LO pathway is 
responsible for the synthesis of pro-inflammatory lipid mediators, known as leukotrienes. 
Leukotrienes have been implicated in the etiology of CKD and CVD. We showed that 
leukotrienes promote chronic inflammation and maladaptive renal tubular repair in several 
mouse models of renal fibrosis.  We have recently found that short term inhibition of 
cysteinyl leukotrienes with montelukast (a leukotriene antagonist) in mice with acute 
kidney injury potently prevents CKD. This is associated with decreased chronic 
inflammation and improved tubuloepithelial repair. Leukotrienes also cause endothelial 
dysfunction which increases glomerular permeability to albumin and causes vascular 
dysfunction leading to CVD. Short-term treatment with a 5-LO inhibitor reduces proteinuria 
in patients with glomerulonephritis. Additionally, montelukast use is associated with 
reduced cardiovascular events in the general population.  However, to date, no 
interventional studies have examined the effect of montelukast on kidney or 
cardiovascular outcomes in patients with DKD. Montelukast is currently used for 
asthma treatment and has an excellent safety profile for long-term use in both pediatric 
and adult patients, making it an attractive and safe option for chronic therapy in patients 
with DKD. 
 
Our overall hypothesis is that cysteinyl leukotrienes promote DKD progression and 
vascular disease by increasing inflammation, tubular damage, endothelial dysfunction and 


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fibrosis. We will conduct a prospective, open-label pilot clinical trial examining the effect of 
oral montelukast for 3 months  in 20 patients with DKD  (eGFR 30-59 ml/min/1.73m2) with 
urine albumin to creatinine ratio of 200-5000 mg/g and use of a RAAS inhibitor (may also 
be on stable dose of SGLT2i) to test the following aims: 
 
Hypothesis 1: Treatment with montelukast will reduce proteinuria in patients with DKD. 
Specific Aim One: To determine changes in 24-hour urine proteinuria before and after 3 
months of montelukast therapy. 
 
Hypothesis 2: Treatment with montelukast will improve vascular endothelial function and 
arterial stiffness in patients with DKD. 
Specific Aim 2: To determine vascular endothelial function (measured by EndoPat) and 
large elastic artery stiffness (measured by the gold standard aortic pulse wave velocity) 
before and after 3 months of montelukast therapy. 
 
Hypothesis 3: Treatment with montelukast will reduce kidney and vascular inflammation 
in patients with DKD. 
Specific Aim 3: To determine kidney macrophage infiltration (measured by urinary 
sCD163) and cytokine production (measured by urinary MCP-1 and plasma TNFR-1) 
before and after 3 months of montelukast therapy. 
 
This novel study will be conducted by experienced PIs and investigative team with 
expertise in cysteinyl leukotrienes, vascular function and clinical trials. Results from this 
study will inform the design of a larger, randomized controlled trial examining whether 
montelukast may be a safe and promising option to use for treatment of kidney and 
vascular disease in patients with CKD. 
 
 
II. Background and Significance:  
1. Critical need for novel therapeutic strategies in chronic kidney disease (CKD). 
CKD is highly prevalent in the United States and is associated with significant 
morbidity and mortality. In the US, almost 15% of Medicare beneficiaries have 
significant CKD.1 Patients with advanced CKD have dramatically higher mortality rates 
compared to the general population. Preservation of kidney function should therefore 
improve health outcomes. Blood pressure control and antagonists of the renin 
angiotensin system (RAAS) are the recommended therapies to preserve kidney 
function. Recently, inhibitors of the sodium glucose cotransporter (SGLT2) have been 
shown to reduce disease progression in patients with diabetes and CKD.2,3 However, 
residual albuminuria remains present during SGLT2 inhibitor treatment and is 
associated with adverse kidney and heart outcomes.4 Despite the availability of 
SGLT2 inhibitors, over 120,000 patients progress to end stage kidney disease (ESKD) 
yearly. Additionally, the adoption of SGLT2 inhibitors in patients with diabetic and 
nondiabetic CKD remains low.5 Furthermore, SGLT2 inhibitors may not be safe in all 
CKD groups. For example, in type 1 diabetes there may be an increased risk of 
euglycemic diabetic ketoacidosis with SGLT2 inhibitors.6 Data from animal models 
suggests an increased risk of cyst burden in animals with polycystic kidney disease 
treated with SGLT2 inhibitors.7 Finally, SGLT2 inhibitors may also be associated with 
an increased risk of infections in kidney transplant recipients.8 Identifying new 
treatments for CKD to be used alone or in combination with other therapies is a 
high priority.  
 


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2. Inflammation plays a key role in CKD progression and cardiovascular disease. 
Most forms of CKD are characterized pathologically by inflammation in the kidney.9 
Renal macrophage and lymphocyte infiltration are seen in CKD and macrophage 
number correlates with the severity of CKD.10 Experimental models have shown that 
macrophages promote CKD progression through induction of chronic inflammation, 
tubular injury, and fibrosis.10,11 Studies in diabetic kidney disease have shown 
increasing proinflammatory cytokine levels with kidney disease progression.12 In the 
Chronic Renal Insufficiency Cohort, elevated levels of inflammatory markers such as 
TNF-, are independent predictors of CKD progression and are linked with rapid loss 
of kidney function.13 Atherosclerosis is also driven by chronic inflammation. Significant 
inflammatory cell infiltrates have been found in atherosclerotic plaques.14 
Proinflammatory cytokines released by macrophages lead to endothelial dysfunction 
and microcalcification.15 Systemic markers of inflammation (e.g. C-reactive protein) 
are strong predictors of future cardiovascular events.16 CVD is the leading cause of 
death in patients with CKD.17 Two of the greatest contributors to CVD in CKD are the 
development of vascular endothelial dysfunction, most commonly assessed as 
brachial artery flow mediated dilation (FMDBA) and stiffening of the large elastic 
arteries (aorta and carotid arteries).18 Hence, treatments that target chronic 
inflammation may have a significant impact on CVD outcomes in CKD. Despite 
widespread treatment with RAAS antagonists and SGLT2 inhibitors, there remains an 
unacceptably high rate of CKD progression and cardiovascular complications of CKD. 
Treatments that specifically target inflammatory pathways may improve 
outcomes in patients with CKD and may synergistically decrease progression 
with RAAS antagonists and SGLT2 inhibitors.   
 
3. Leukotrienes are potent lipid mediators of chronic inflammatory diseases. Lipid 
mediators produced through the action of 5-lipoxygenase (5-LO) play a critical role in 
inflammation and disease progression in many settings.19,20  In many tissues, 
macrophages and neutrophils exert biological effects through 5-lipoxygenase (Figure 
1). These macrophage-derived leukotrienes promote chronic inflammation and 
fibrosis. After arachidonic acid is liberated from membrane phospholipids by group IVA 
cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO) and 5-lipoxygenase-
activating protein (FLAP) catalyze the synthesis of a class of eicosanoids, known as 
leukotrienes. After synthesis of leukotriene A4 by 5-LO, LTA4 hydrolase (LTA4H) 
catalyzes production of leukotriene B4 (LTB4) while LTC4 synthase (LTC4S) is 
responsible for the synthesis of leukotriene C4, D4, and E4 (LTC4, LTD4, LTE4), 
collectively known as the cysteinyl leukotrienes.  
 
LTB4 binds to the cell surface receptors BLT1 and BLT2 while cysteinyl leukotrienes 
bind to separate cell surface receptors (CysLTR1, CysLTR2, and GPR99).  Cysteinyl 
leukotrienes are sequentially metabolized from LTC4 to LTD4 to LTE4.  After activating 
the specific cell surface receptors, leukotrienes regulate a wide range of biological 
processes, including inflammation and epithelial damage. In animal models, 
leukotrienes promote allergic diseases, pulmonary fibrosis, and liver fibrosis.21-23 
 


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Figure 1. The 5-lipoxygenase pathway.  After 
activation of cytosolic phospholipase A2 (cPLA2) 
membrane phospholipids are released as arachidonic 
acid, a 20-carbon lipid.  Leukocytes predominantly 
express 5-lipoxygenase and synthesize the short-lived 
LTA4.  LTA4 hydrolase and LTC4 synthase are 
expressed in many cell types, including renal tubular 
epithelial cells and catalyze production of LTB4 and the 
cysteinyl leukotrienes (LTC4, LTD4, and LTE4), 
respectively. 
 
4. Leukotrienes play a key role in the pathogenesis of CKD. Elevated levels of LTB4 
are associated with several models of kidney disease as it is a potent chemoattractant 
for neutrophils.24,25 Cysteinyl leukotrienes trigger vasoconstriction resulting in reduced 
renal blood flow (RBF) and glomerular filtration rate (GFR).26 Humans with CKD have 
increased tubulointerstitial expression of 5-LO.27 The increased expression of 5-LO 
correlates with the severity of proteinuria and with serum creatinine levels.27 In animal 
models of glomerulonephritis, inhibition of 5-LO results in reduced proteinuria and 
preservation of GFR.28,29 As shown in our preliminary data, inhibition of 5-LO 
strongly reduces renal fibrosis in animal models of CKD.25 Additionally, we have 
shown that inhibition of leukotrienes with montelukast (a leukotriene antagonist) in 
mice with acute kidney injury prevents progression to CKD (preliminary data section 
1). In a small study of 11 adult patients with glomerulonephritis, short-term (4 days) 
administration of a 5-LO antagonist (MK-591) resulted in a significant decrease in 
proteinuria.30 In children with steroid dependent minimal change disease, treatment 
with montelukast for 12 months significantly reduced relapse rates.31 However, to 
date, no randomized trials examining the effect of montelukast on kidney 
outcomes in patients with CKD have been performed and thus is the primary 
aim of the current study. 
 
5. Leukotrienes mediate cardiovascular disease. Increased expression of 5-LO, LTB4 
and cysteinyl leukotrienes are found in human atherosclerotic lesions.32 This 
expression increases as lesions progress from early to late stage of atherosclerosis.33 
5-LO expression is mostly localized to macrophages.32,33 In patients with chronic 
obstructive pulmonary disease, high levels of cysteinyl leukotrienes correlate strongly 
with cardiovascular disease.34,35 Increased intracoronary production of cysteinyl 
leukotrienes has also been shown in patients undergoing coronary angioplasty.36 In 
animal models, montelukast has beneficial effects on vascular endothelial cell function 
and myocardial remodeling.37,38 In a randomized, placebo-controlled crossover trial of 
a 5-LO inhibitor in patients with myocardial infarction, 4 weeks of treatment resulted in 
significant reduction is LTB4 and myeloperoxidase.39 Patients with asthma treated with 
montelukast for 6 months had lower levels of C-reactive protein and lipid levels 
compared to patients treated with placebo.40 A recent observational study of 800 adult 
asthmatic patients found that montelukast use was associated with a significant 
independent reduction in cardiovascular events.41 Thus, targeting the 5-LO pathway 
may be a strategy for reducing cardiovascular events. To date, no randomized 
controlled trials have been performed examining the effect of montelukast on 
surrogate markers of CVD and thus represents an aim of the current study.  
 


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6. Rigor 
of 
Prior 
Research: 
Leukotrienes play a 
significant 
role 
in 
kidney 
disease 
progression 
and 
cardiovascular 
disease in preclinical 
models. 
Observational 
studies in humans 
suggest a significant 
role for leukotrienes 
in cardiovascular and kidney disease. Small studies show that montelukast, a 
leukotriene antagonist) results in reduced proteinuria, less relapse of nephrotic 
syndrome and lower levels of LTB4 and inflammatory markers. However, no 
randomized placebo-controlled trials have been performed in CKD examining the 
effect of montelukast on proteinuria, renal hemodynamics or surrogate markers of 
cardiovascular disease and inflammation. While SGLT2 inhibitors are becoming 
standard of care for patients with CKD, these medications may not be safe in all 
patients with CKD. Additionally, these medications can be cost prohibitive. This 
highlights the need for novel, inexpensive treatments for CKD progression and CVD in 
CKD, such as montelukast. Montelukast has been used extensively for the treatment 
of other diseases for decades and has an excellent safety profile in both adults and 
children. We hypothesize that montelukast will reduce cytokine production, pro-fibrotic 
factors and vasoconstriction leading to improvements in albuminuria, endothelial 
function and arterial stiffness (Figure 2). Positive results from the proposed study 
would provide evidence needed for a larger, multicenter, randomized controlled trial 
examining the effect of montelukast on hard clinical outcomes. Results from this study 
may also improve our ability to offer personalized treatments for patients with CKD.  If 
montelukast does effectively decrease intrarenal inflammation in patients, that would 
support further study of montelukast in subgroups of patients with inflammatory CKD 
(high urinary biomarkers of inflammation or LTE4).  
 
III. Preliminary Studies/Progress Report:   
1. 
Preliminary Data: 
a. Montelukast 
decreases 
inflammation 
and 
CKD 
progression in a mouse model of 
CKD. We have previously showed 
that inhibition of 5-LO and LTC4 
synthase reduced fibrosis in two 
mouse models of renal fibrosis.25 
We next tested whether targeted 
inhibition of the cysteinyl leukotriene 
receptor 
1 
(CysLTR1) 
with 
montelukast would also reduce renal 
injury in mice using the folate 
nephropathy model.   In the folate 
nephropathy model, mice develop 
CysLTR1
Renal Macrophages
CysLTR1
Cysteinyl LTs
Vascular Macrophages
Pro-fibrotic factors
Cytokine Production
Vasconstriction
Chronic Inflammation
Glomerular Endothelial
Injury/Proteinuria
Cytokine Production
Vasoconstriction
Vessel Fibrosis
Endothelial dysfunction
Arterial Stiffness
Montelukast
Montelukast
CKD 
progression
CVD
 
Figure 2. Proposed Mechanism of Action  
Figure 3. Montelukast decreases renal fibrosis and CKD 
progression.  A. Renal fibrosis was assessed using second 
harmonic generation microscopy.  Montelukast improved 
tubular architecture post-folate (green) and decreased collagen 
deposition (red). B. Montelukast was associated with improved 
plasma BUN. * p <0.05 


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acute kidney injury after administration of high dose folic acid.  This is followed by 
a period of renal inflammation, inadequate renal tubular repair, and eventually 
development of CKD.  After mice had established AKI, we began treatment with 
montelukast daily for two weeks. After an additional 75 days, mice were collected 
to assess the effects on CKD progression.  We hypothesized that short-term 
inhibition of CysLTR1 during the critical period of renal inflammation and renal 
repair would prevent chronic inflammation and CKD progression. 
 
We first found that mice treated with a 
short-term course of montelukast had 
significantly less renal fibrosis and 
improved renal function (Figure 3).  To 
determine 
which 
pathways 
were 
regulated by montelukast in CKD, we 
next performed transcriptomic analysis 
of injured kidneys using RNA-Seq.  
Gene 
ontology 
enrichment 
analysis 
showed 
that 
montelukast 
regulated 
several pathways involved in immune function (Figure 4). To test whether this was 
associated 
with 
diminished 
chronic 
inflammation, 
we 
performed 
immunofluorescence for the macrophage marker CD68 in injured kidneys. 75 days 
after stopping montelukast, we found that montelukast had dramatically reduced 
the number of interstitial macrophages in the kidney (Figure 5). In summary, 
these data show that short-term, low dose montelukast exerts very potent 
anti-inflammatory and anti-fibrotic effects in a mouse model of CKD. 
 
b.  Feasibility of the proposed study is demonstrated by the MPI Dr. Kendrick’s 
experience in the NIH sponsored Vitamin D and Arterial Function in CKD 
Trial42 and Bicarbonate Administration in CKD Trial (NCT0291560). The 
Vitamin D trial conducted by Dr. Kendrick (Co-PI) was a single-center study 
examining the effects of oral cholecalciferol vs. calcitriol for 6 months on vascular 
function in 128 CKD stage 3b-4 patients. Recruitment was accomplished in 36 
months with an attrition rate of 10%.42 The Bicarbonate Administration in CKD Trial 
is an ongoing 12 month trial of patients with CKD stage 3-4 examining the effect of 
oral sodium bicarbonate on vascular function and left ventricular mass with a 
current attrition rate of 11.0%. 24-hour urines were collected at 3 time points in the 
Bicarbonate Administration in CKD showing that it is feasible to collect 24-hour 
urines for albuminuria in patients with CKD. 
 
c. Feasibility of recruitment is demonstrated by the large pool of patients 
available for enrollment. At the University of Colorado Hospital and Denver 
Health Medical Center, we have access to over 4,000 patients with CKD. 
Figure 5. Montelukast decreases 
macrophage infiltration in injured 
kidneys.  Immunofluorescence for 
CD68 showed that short term 
montelukast treatment significantly 
reduced CD68+ cells.  * p< 0.05. 
 
Figure 4.  Montelukast 
prevents inflammatory gene 
transcription post-folate. 
Homogenized kidneys were 
analyzed from vehicle- and 
montelukast-treated mice using 
RNA-Seq.  Gene set enrichment 
analysis (GSEA) using 
Hallmark-Inflammatory 
Responses is shown. 


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Additionally, we have a recruitment database of patients with CKD stage 3 willing 
to participate in clinical trials. Feasibility of recruitment is demonstrated by prior 
recruitment experience of the investigators in several CKD interventional trials.   
 
 
IV. Research Methods 
Subjects. After obtaining their written informed consent, 20 patients with DKD (defined as 
estimated GFR (eGFR) 30-59 ml/min/1.73m2) with albuminuria of 200-5000 mg/g will 
serve as subjects. This range of albuminuria was chosen as macroalbuminuria is 
associated with more adverse outcomes in patients with CKD and a previous small study 
found a greater reduction in albuminuria with a 5-LO inhibitor in participants with greater 
albuminuria at baseline.30 Relevant biological variables: Men and women age 30-70 
years of all races/ethnicities will be included. Major inclusion/exclusion criteria are 
presented in the table below (Table 1). Participants with GFR <30 ml/min/1.73m2 are 
excluded as this is advanced kidney disease when complications become more common 
and vascular damage may not be reversible. Participants will undergo screening for eGFR 
at the Kidney Disease Research Center (KDRC) at the University of Colorado Anschutz 
Medical Campus. Estimated GFR will be calculated using the new 2021 Chronic Kidney 
Disease Epidemiology Collaboration (CKD-EPI) prediction equation.43 Participants will be 
recruited from nephrology clinics at the University of Colorado and Denver Health Medical 
Center. Additionally, patients will be recruited from our CKD database, which contains 
over 300 CKD patients willing to participate in clinical trials. Feasibility of recruitment is 
demonstrated by prior recruitment experience of the investigative team in several CKD 
interventional trials42 (NCT02915601, NCT04600323). For this study  
 
Table 1 
Inclusion Criteria 
Exclusion Criteria 
• 
Age 30-70 years 
• 
Urine albumin to creatinine ratio 200-5000 mg/g on 
first morning void 
• 
CKD stage 3 at time of screening (eGFR 30-59 
ml/min/1.73m2)  
• 
Blood pressure <140/90 mm Hg prior to baseline 
visit 
• 
Use of angiotensin converting enzyme inhibitor or 
angiotensin receptor blocker or mineralocorticoid 
receptor antagonist with stable dose for 4 weeks  
• 
History of diabetes type 1 or type 2 
• 
BMI < 40 kg/m2 (FMD measurements can be 
inaccurate in severely obese patients).  
• 
Stable anti-hypertensive regimen for at least one 
month prior to enrollment 
• 
Stable diabetes regiment for at least one month 
prior to enrollment 
• 
Sedentary or recreationally active (≤2 days of 
vigorous aerobic exercise as vigorous exercise 
may affect vascular function measurements) 
• 
Able to provide consent 
 
• 
Significant comorbid conditions that lead the 
investigator to conclude that life expectancy is 
less than 1 year 
• 
Uncontrolled hypertension 
• 
Factors 
judged 
to 
limit 
adherence 
to 
interventions 
(e.g. 
history 
of 
medication 
noncompliance, noncompliance with follow up 
visits, significant cognitive impairment, etc.) 
• 
Anticipated initiation of dialysis or kidney 
transplantation within 3 months 
• 
Current participation in another research study 
• 
Pregnancy or planning to become pregnant or 
currently breastfeeding 
• 
Allergy to aspirin 
• 
Severe hepatic impairment (Child-Pugh Class 
C) 
• 
History of major psychiatric disorder 
• 
Use of inhaled or systemic corticosteroids or 
long-acting beta agonists (higher risk of 
neuropsychiatric reaction) 
• 
Current use of phenobarbital, rifampin or 
carbamazepine. 
 
 
Rigorous Experimental Design. A 3-month open-label, prospective pilot study with 
oral montelukast will be conducted. The study will include 3phases: screening, baseline, 
and follow-up.  


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• 
Screening: Subjects will undergo screening for inclusion/exclusion criteria during a 
1-2 week period. Participants will have urine albumin/creatinine performed and 
basic metabolic panel to determine eligibility.  
• 
Baseline: During the baseline phase the following will be measured: 1) 24-hour 
albuminuria; 2) vascular function (EndoPat, aPWV); 2) serum chemistry panel; 3) 
urine MCP-1, sCD163 and LTE4; and 4) plasma TNFR-1.  
• 
Follow-up: The follow-up schedule includes assessments at 3 months with monthly 
safety visits (either via phone, in person, or survey link). An additional safety check 
will be performed via phone, in person, or survey link 2 weeks after the last study 
visit.   
 
Study Drug Dosing: Study drug will be received from the University of Colorado 
Hospital Pharmacy. Each bottle will contain 90 tablets of montelukast 10mg. 
Participants will receive 1 tablet daily (10mg) for the entire 3 months. The tablet will be 
taken in the evening per manufacturer recommendations. The dose of montelukast 
may be adjusted.  
 
Rationale for study duration: From the study team’s experience with other 
interventional studies evaluating albuminuria and vascular endothelial function 
changes can be seen in 4-6 weeks. Changes in aPWV can be seen after 12 weeks. 
Changes in patients with CKD stage 3 may require longer periods of time for these 
endpoints and published data use a period of 3-6 months.42,44 Hence, we are 
proposing a 3-month intervention for this pilot study. 
 
Data Collection 
• 
Demographics, medical history and physical examination will be performed at 
screening or baseline visit.  
• 
Circulating measures: Subjects will report fasted for standard blood chemistry analysis 
at baseline and 3 months. A urine pregnancy test will be performed if clinically 
indicated at the screening visit. 
• 
Outcome measures: 
o 24-hour albuminuria will be measured at baseline and 3 months 
o EndoPat and aPWV will be measured at baseline and 3 months.  
o Urinary LTE4, MCP-1, and CD163 will be measured at baseline and 3 months. 
o Plasma TNFR-1 and basic metabolic panel will be measured at baseline and 3 
months. 
• 
Resting BP: Arterial BP will be measured in triplicate while seated at rest using an 
automated oscillometric machine (Dinamap) at all in-person study visits. 
• 
Measures of adherence and safety: 
o Pill counts will be assessed at the 3-month visit to document subject 
adherence. 
o A neuropsychiatric assessment will be performed monthly to assess for any 
changes in mood, depression or anxiety symptoms via phone, in person, or 
survey link. We will utilize the Patient Health Questionnaire-9 (PHQ-9) and the 
General Anxiety Disorder-7 (GAD-7). These questionnaires will assess 
depression and anxiety symptoms, as well as any suicidal ideations and are 
routinely used in patients with CKD.45,46 We chose these questionnaires as 
they are validated in patients with CKD and can be completed in 10 
minutes.45,46 Any participant that has a significant change in score from the 


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previous month or that reports suicidal ideations, will be referred immediately 
to Dr. Ritchie, Co-I. Dr. Ritchie is a clinical psychologist. She will contact the 
patient and do a risk assessment to determine if the participant needs urgent 
mental health treatment. Participants that require urgent psychiatric care will be 
discontinued on study drug and removed from the study. We will also assess 
sleep including insomnia, sleepwalking etc. Finally, we will assess whether the 
participant is experiencing any memory problems, tremor or uncontrolled 
muscle movements. Participants that report these issues will be instructed to 
stop the study drug immediately. Participants will also be informed at study 
initiation to stop the drug and call immediately should they experience any 
change in mood, depression, aggression, agitation, sleep disturbances, or 
suicidal thoughts.  
o Adverse event assessment will be performed monthly either via telephone, in 
person, or survey link. Additional telephone calls and PRN follow-up visits will 
occur at the discretion of the PIs. 
 
Safety monitoring and expected adverse events: Reporting of Side Effects. Subjects 
will be instructed to report serious or worrisome side effects to the investigators and their 
primary care physician (who will have been informed of their patient’s participation in and 
the nature of the study). A subject experiencing side effects during off hours will contact 
Dr. Kendrick, who is available 24 hours a day. Dr. Furgeson will be on call during any 
period when Dr. Kendrick is not available. Thus, the physicians on the study team will 
work together to provide 24/7 medical coverage to subjects. Any unexpected adverse 
events will be reported immediately to the Colorado Institutional Review Board. 
 
1. Montelukast is well tolerated and has been used in pediatric and adult patients for 
many years. The most common adverse reactions (incidence ≥ 5% and greater 
than placebo) include upper respiratory infection, fever, headache, pharyngitis, 
cough, abdominal pain, diarrhea, otitis media, influenza, rhinorrhea, sinusitis and 
otitis. These side effects are minor and will be assessed on a monthly basis via 
phone, in person, or survey link. A serious side effect of montelukast is a 
neuropsychiatric adverse reaction. This reaction has been reported in patients with 
and without a history of psychiatric illness. This has been added as a Black Box 
Warning by the FDA. Reactions include agitation, aggressive behavior, anxiety, 
depression, restlessness, suicidal ideations or tendencies. Some patients also 
develop sleep disturbances (abnormal dreams, insomnia, sleep walking). 
Additionally disturbances in attention or memory impairment, tremor or involuntary 
movements may occur (up to 1 in 1,000 people). Sleep disturbances, depression 
and agitation may affect up to 1 in 100 people taking montelukast. Suicidal 
behavior and hallucinations occur very rarely (up to 1 in 10,000 people). We will 
assess for these serious adverse events monthly through the use of 
questionnaires regarding depression, anxiety and sleep via phone, in person, or 
survey link. We will also assess for any new tremors or involuntary movements. 
Dr. Ritchie, Co-Investigator, is a clinical psychologist and researcher and will 
immediately do a risk assessment on any participant presenting with symptoms to 
determine if urgent psychiatric treatment is required and will arrange for treatment. 
Additionally, study drug will be stopped immediately. Most of these reactions 
resolve with stopping montelukast. We will exclude patients with a history of major 
depressive disorder, or that are on corticosteroids (inhaled or systemic) or long-
acting beta-agonists as this may increase the risk of neuropsychiatric reactions. 


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2. Fasting: There is a small risk that blood sugars will be lower than normal in 
participants with diabetes while fasting. Participants will also be asked to check 
their blood sugars while fasting. If participants are unable to be fasting for 12 
hours, they will be allowed to have a small snack prior to the study measurements. 
The fasting instructions for this study is similar to what we have prescribed in our 
other studies in participants with CKD with and without diabetes.  
 
3. Blood pressure, heart rate and oxygen saturation: There are no known risks 
associated with collecting these vitals.  
 
4. Physical exam and 24 hour urine samples: There are no known risks 
associated with the physical exam or urine collections. The 24-hour urine 
collections may be cumbersome for participants but the protocol used in this study 
is similar to what we have done in our other studies in participants with CKD. 
 
5. Venous blood draw: discomfort associated with insertion of the needle; local 
bleeding and a small hematoma (~10% of cases); risk of infection of a hematoma 
or significant external blood loss (<1 in 1000), risk of fainting. Participants with 
CKD may have plans in place for future vascular access for hemodialysis and thus 
we will draw blood in the arm not designated for future vascular access.  
 
6. Vascular function measurements: inflating the blood pressure cuff below the 
elbow during this procedure may cause a mild to moderate intensity “pins and 
needles or numbing” sensation that goes away as soon as the cuff is deflated. If 
participants have a fistula or graft, EndoPat will be performed in the opposite arm. 
 
7. Confidentiality: There is a risk that people outside of the research team will see 
the research information. While we will do all that we can to protect participant 
information, it cannot be guaranteed. Our study will follow HIPAA guidelines to 
protect anyone who participates in this research study. All study procedures will be 
conducted in a private room with study-related personnel only. 
 
8. Pregnancy: There are no adequate and well-controlled studies in pregnant 
women. Montelukast should be used during pregnancy only if clearly needed. No 
teratogenicity was observed in rats and rabbits at doses approximately 100 and 
110 times, respectively, the maximum recommended daily oral dose in adults 
based on AUCs. Congenital limb defects have been rarely reported in the offspring 
of women being treated with montelukast during pregnancy. A causal relationship 
between these events and montelukast has not been established. 
 
Outcome Measures 
a) Primary Outcome 
1. 24-hour albuminuria:  Participants will perform a 24-hour urine collection and 
return it the day of their visit at baseline and 3 months. Participants will be given 
the containers to collect the 24-hour urine samples at the screening visits.   
 
2. Justification for primary outcome: Albuminuria is a marker of kidney damage 
and is an early and sensitive marker in many types of kidney disease.47 The gold 


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standard for assessing albuminuria is a 24-hour urine collection.48 Dr. Kendrick, 
MPI, has extensive experience in collections of 24-hour urine in participants with 
CKD (NCT0291560). 
 
b) Secondary Outcome 
Vascular endothelial function.  All measurements will be made after a 12 hour fast 
(water allowed), 12 hours after abstaining from tobacco and caffeine, and 24 hours after 
abstaining from alcohol and exercise. No new over the counter medications within 48 
hours. To control for menstrual cycle, all premenopausal women will be tested in the early 
follicular phase (days 1-6 of the menstrual cycle). Measurements will be made at baseline 
and 3 months. 
 
Vascular endothelial function will be measured using EndoPat 2000. Testing will be 
performed in a quiet room with the patient in a supine position. EndoPat bio sensor is 
placed on the patient’s right and left index fingers. A blood pressure cuff is wrapped 
around the non-dominant arm and records baseline data for 5 minutes. The cuff then 
inflates and occludes the brachial artery for another 5 minutes. The cuff is then released 
to measure the dilatory response. EndoPat’s software performs an automatic analysis of 
the data obtained from both fingers and provides a post-occlusion to pre-occlusion ratio. 
The RHI score is provided in a logarithmic scale. A score less than 7 indicates endothelial 
dysfunction. Testing can be completed in 20 minutes. EndoPat has been used in 
numerous studies accessing endothelial function. 
 
Large Elastic Artery Stiffness and Compliance: Aortic PWV, the gold standard 
measurement of large elastic artery stiffness, will be determined using the SphygmoCor 
device at baseline and 3 months.49 Measurement of pulse wave analysis: The cuff of the 
SphygmoCor device will be placed on the non-dominant arm. BP will be measured 
involving an automatic recording of brachial BP immediately followed by reinflation of the 
cuff. The cuff is held at inflation for a period of 5 seconds during which time volumetric 
waveforms are recorded. Measurement of aPWV: Briefly, a transcutaneous custom 
tonometer will be positioned at the carotid artery and a cuff will be placed at the femoral 
artery to measure PWV. The distance between the carotid and the femoral cuff are 
measured as is the distance between the femoral artery and the femoral cuff. These 
distances are entered into the SphygmoCor software to determine the carotid-femoral 
distance used for calculation of PWV.49 The tonomoter is positioned over the marked 
location with the strongest carotid pulse detected by the operator. The device 
automatically captures the pulse wave recording and will be attempted for a full cuff 
inflation/deflation cycle, pluse the first 20 s of the second cuff inflation. SphygmoCor 
software will perform the velocity calculation.49  
 
Laboratory Measurements: 
a) Plasma comprehensive metabolic panel (CMP): A CMP will be performed by 
the University of Colorado Hospital Clinical laboratory at UC-AMC using standard 
procedures at baseline and 3 months. 
b) Urinary sCD163: Urinary sCD163 levels will be assayed using Human CD163 
DuoSet ELISA kit in Dr. Furgeson’s lab as has been previously described.50   
c) Urinary MCP-1: Urinary MCP-1 will be measured using the R&D Systems Human 
CCL2/MCP-1 Quantikine ELISA kit as has been previously described.51  
d) Plasma TNFR-1: Plasma TNFR-1 will be measured using Human 
TNFR1/TNFRSF1A Quantikine ELISA kit as has been described.52  


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e) Urinary LTE4: After lipid extraction from urine samples, urinary LTE4 will be 
measured by liquid chromatography/mass spectrometry.  
 
Justification for secondary outcomes: Vascular endothelial dysfunction and arterial 
stiffness are key pathophysiological antecedents to CVD in patients with CKD.53,54 
EndoPat ia a common, non-invasive technique used to assess vascular endothelial 
function. Aortic PWV is the gold standard measurement of arterial stiffness and is non-
invasive. Dr. Kendrick, MPI and the University of Colorado investigative team have 
expertise in these vascular function measurements.42,55 3) Increased inflammation may 
play a critical role in leukotriene activation and subsequent kidney and vascular 
dysfunction. The effect of montelukast on urinary markers of inflammation and 
macrophage activation and endovascular markers of inflammation has not been 
evaluated. Plasma levels of TNFR-1 (a biomarker of systemic inflammation) exhibit a 
robust association with CKD progression in a variety of patient populations, including 
those with diabetic kidney disease and hypertensive nephrosclerosis.56,57 Urinary MCP-1, 
a biomarker of systemic and renal inflammation, is also strongly associated with CKD 
progression in patients with acute kidney injury and hypertension.58,59 Although there are 
limited non-invasive methods to measure macrophage number within kidneys; urinary 
soluble CD163 has been shown to correlate well with inflammation on renal biopsies.50,60 
Since LTC4 and LTD4 are rapidly metabolized to LTE4, urinary LTE4 measurements are a 
very sensitive and specific way to measure changes in total body cysteinyl leukotrienes.61 
Dr. Furgeson, MPI has experience with these laboratory measurements. 
 
Data Management: Data will be collected and managed using Research Electronic Data 
Capture (REDCap), a secure web application designed to support data capture for 
research studies. The database is hosted at the University of Colorado Development and 
Informatics Service Center (DISC), which will be used as a central location for data 
processing and management. Data quality checks will be ongoing, but will formally occur 
(e.g., missing data, duplicate entries) at least annually when progress reports are 
prepared. Blood and urine samples that are not assayed immediately will be stored for 
later analysis in locked -80˚ freezers managed by the PI and study staff. All samples will 
be labeled with the least amount of information necessary to accurately identify the 
samples (e.g., study number, subject identification number, visit date, and test time-point). 
Participants who drop from the study may request destruction of samples.   
 
Power Calculations: The goal of this pilot study is to provide parameters to allow for a 
more accurate estimation of sample size for a larger clinical trial. Our sample size was 
determined based on feasibility and availability, not a specified statistical power.  
 
Statistical Analysis: Descriptive statistics will be calculated for all variables. For 
example, mean and standard deviation will be calculated for a continuous variable, and 
count and proportion will be provided for a categorical or ordinal variable. For a 
continuous variable that is not normally distributed, median and interquartile range will be 
provided, and natural log transformation will be performed before further analysis. The 
95% confidence interval will be calculated as well if appropriate.  
 
For the primary outcome 24-hour albuminuria, its log value will be used in analysis. 
Analysis with a linear regression model will be performed to assess the montelukast effect 


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by regressing the albuminuria at end of study on baseline albuminuria. Furthermore, 
analysis with adjustment for age, sex will be performed. For the secondary outcomes for 
the linear regression model also is applicable, and the same analysis will be performed.   
 
Potential Problems and Alternative Strategies 
• 
Although subject recruitment and retention are always challenging, we should be 
able to complete the study in the proposed timeline given our group’s excellent 
track record in recruiting, enrolling and retaining participants in clinical trials. The 
investigative team has access to over 4,000 patients in the CKD clinics at the 
University of Colorado and the Denver Health Medical Center.  
• 
We should have few difficulties with the proposed experimental procedures and 
protocols as they are already establised in Dr. Kendrick’s vascular laboratories and 
Dr. Furgeson’s laboratory at the UC-AMC. The investigative team has extensive 
experience with the proposed study measurements. 
• 
Systolic BP is an important determinant of proteinuria and endothelial function and 
could be a theoretical confounder when interpreting the mechanisms by which 
montelukast improves proteinuria and vascular function. All participants must have 
their BP controlled prior to entering the study. 
• 
We recognize that ACEi/ARB reduce proteinuria and kidney disease progression 
in patients with CKD. It is possible montelukast has a synergistic effect with these 
medications. Participants must be on an ACEi/ARB to participate in the study. 
• 
We acknowledge there are other markers of inflammation and it possible that 
urinary CD163 levels are low in our patient population. Additionally, we 
acknowledge that measurement of leukotrienes and other inflammatory cytokines 
in the blood may also be important. For this reason, we are creating a biobank and 
blood obtained at visits will be stored for future research purposes. 
 
Potential Benefits of the Proposed Research to the Subjects and Others: 
Participants will receive benefits associated with overall knowledge of their health from the 
extensive testing performed for screening purposes and the established subject 
characteristics (i.e. blood pressure, blood chemistries, etc.). The findings we will generate 
will answer important questions about montelukast administration in patients with DKD 
and help investigators design future clinical trials in this population. Thus, the new 
knowledge generated from this proposal will yield potential benefit to DKD patients in 
general and to society as a whole.  
 
4. Importance of the Knowledge to be Gained: The new knowledge generated from this 
proposal will facilitate planning of future clinical trials in DKD patients that will aim to 
reduce the morbidity and mortality of DKD. 
 
 
 
 
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