Effects of an intravenous infusion of diltiazem on glomerular filtration rate, electrolyte excretion, and urine output in healthy dogs

2.1 Animals

A prospective, randomized, unmasked, crossover study was used. This protocol was approved by the Institutional Animal Care and Use Committee at Kansas State University. Informed consent was obtained from all clients before enrollment. The target cohort was apparently healthy, client-owned dogs >1 year of age and weighing >10 kg. Dogs were enrolled between April 2021 and August 2021. Dogs were deemed to be normal based on history provided by the client and physical examination findings performed by 1 of the investigators. Those determined to be normal were further screened for underlying renal disease with a CBC, serum biochemical profile, urinalysis, indirect systemic blood pressure measurement, and GFR measurement.

2.2 Screening diagnostics

Screening took place approximately 30 days before study interventions were initiated. Study participants were fasted for 10 to 12 hours before obtaining biosamples, but access to water was not restricted. Briefly, the CBC, biochemical profile, and urinalysis were performed inhouse by the Kansas State University Veterinary Health Center clinical pathology laboratory. GFR was measured by iohexol clearance reported in mL/min/kg. Iohexol at a dose of 300 mg/kg was administered IV to each patient, and blood was collected at timepoints 120, 180, and 240 min after the injection. Serum was submitted to the Michigan State University Veterinary Diagnostic Laboratory for determination of iohexol clearance using standard methods (inductively coupled plasma-mass spectrometry).²² This GFR measurement also served as the study participant's baseline. Indirect systolic blood pressure was measured by Doppler sphygmomanometry in accordance with the American College of Veterinary Internal Medicine guidelines to determine if blood pressure was normal (systolic blood pressure between 90 and 160 mm Hg).²³ Echocardiographic and electrocardiographic data was also collected on this cohort and is reported separately. Dogs diagnosed with underlying disease based on these screening diagnostics were excluded.

2.3 CRI administration

Dogs were randomized to receive either diltiazem (diltiazem hydrochloride injection, 0.5%, Akorn, Inc., Lake Forest, Illinois, USA) or the same volume of 5% dextrose in water (D5W) (5% dextrose injection USP, B. Braun Medical Inc., Bethlehem, Pennsylvania, USA). Half of the dogs received diltiazem first which was determined by simple randomization (first half of names pulled out of a container). Diltiazem was diluted with D5W to a concentration of 1 mg/mL according to the manufacturer's instructions and administered as an IV bolus of 240 μg/kg followed by a CRI at 6 μg/kg/min for 300 min. An IV loading dose of 240 μg/kg was chosen as this dose should quickly achieve steady state concentrations of 120 ng/mL based on the volume of distribution of diltiazem in dogs.²⁴ A diltiazem CRI of 6 μg/kg/min should maintain this steady state concentration and was not expected to cause hypotension.^{25, 26} Since hypotension can be seen with a diltiazem IV bolus,²⁶ the loading dose was administered slowly over 10 min. For the dogs randomized to receive diltiazem, blood pressure was monitored every other minute during the loading dose to monitor for diltiazem-induced hypotension. Hypotension was defined as a systolic blood pressure <90 mm Hg.²⁷ If hypotension developed but blood pressure remained >80 mm Hg, the rescue protocol was to discontinue the diltiazem for 5 min and recheck a blood pressure. If blood pressure was >90 mm Hg, diltiazem administration was resumed at a rate 50% lower than the original rate. If blood pressure was still <90 mm Hg or if the original measurement is <80 mm Hg, a crystalloid fluid at a dose of 10 mL/kg IV was to be administered as needed until systolic blood pressure is >90 mm Hg, and the experiment would be discontinued. After the loading dose, diltiazem was infused at a rate of 6 μg/kg/min for a total of 300 min. Blood pressure and heart rate was continually monitored every 15 min during this CRI whereas those patients in the D5W infusion treatment group had blood pressure and heart rate monitored every 60 min.

2.4 Data collection

Dogs were fasted for 10 to 12 hours before administration of each treatment. Access to water was not restricted before or during the CRI. A sterile IV peripheral catheter and urinary catheter (5 or 6 French Foley catheter) attached to a closed collection system were placed in each dog. If needed, butorphanol at a dose of 0.2 to 0.4 mg/kg IV was given to facilitate placement of the urinary catheter since this medication has minimal effects on renal function.²⁸ The urinary bladder was emptied at the time of urinary catheter placement. UOP was quantified for 1 hour before administering the diltiazem or D5W bolus. After starting each CRI, urine production was quantified hourly until completion.

At the time of placement of the catheters, blood and urine were collected and submitted to the Kansas State University Veterinary Health Center clinical pathology laboratory in order to obtain values for calculation of FENa. Immediately after completion of the diltiazem and D5W CRIs, blood and urine were collected again for after infusion evaluation of FENa. FENa was calculated based on the following formula: FENa = 100 × ([Na_urine × creatinine_serum]/[Na_serum × creatinine_urine]).

GFR was measured again by iohexol clearance from serum samples submitted to the Michigan State University Veterinary Diagnostic Laboratory. After diltiazem had reached steady state for 60 min, iohexol was administered IV at a dose of 300 mg/kg to all dogs followed by blood sample collection at 120, 180, and 240 min after the injection. Both diltiazem and D5W infusions were continued during sample collection for GFR measurement.

After a 7-day washout period, dogs returned and received the opposite treatment, allowing for each of them to serve as their own control. This timeframe allowed for elimination of both diltiazem²⁴ and iohexol²⁹ given during the first treatment. Data collection was similar to the first treatment.

2.5 Statistical analysis

Sample size calculation was conducted using commercially available software (G*Power 3.1, Düsseldorf, Germany). Based on healthy dogs receiving a CRI of D5W, a total of 10 dogs with each serving as their own control provided 80% power to detect, as significant, a 30% change in GFR.¹⁰ Variables were checked for normal distribution with the D'Agostino-Pearson normality test (Prism 8, GraphPad Software, Inc. San Diego, California, USA). Since most results were not normally distributed, nonparametric tests were used. The Wilcoxon matched-pairs signed rank test was used to compare before diltiazem infusion and before D5W infusion GFR and FENa, to those taken after diltiazem infusion and after D5W infusion, respectively. The after diltiazem infusion GFR and FENa, were also be compared to their respective after D5W infusion variables with a Wilcoxon matched-pairs signed rank test. UOP measurements were compared using the Freidman test. Statistical significance was set at P < .05.

3.1 Study cohort

Ten client-owned dogs were originally screened for the study including 6 spayed females and 4 castrated males. Five of the females were excluded because of an inability to place an indwelling urinary catheter with sedation with butorphanol alone. Consequently, 5 additional male dogs were recruited for this study. A total of 10 dogs completed the study which included 9 castrated males and 1 spayed female. None of the dogs that completed the study received any sedation. The median age was 3 years (range, 1-6). There were 6 breeds represented, including Border Collie (n = 2), Goldendoodle (n = 2), Pembroke Welsh Corgi (n = 1), and Golden Retriever (n = 1). The remaining 4 dogs were of mixed breeding. Median weight was 25.3 kg (range, 14.8-31.6). Screening diagnostics were within normal limits for all enrolled dogs. None of the dogs were hypotensive at any point of the study.

3.2 Glomerular filtration rate

Three GFR values were obtained for each patient during the study which consisted of a baseline, GFR after administration of diltiazem, and GFR after administration of D5W. Results are presented in Figure 1. GFR did not significantly increase from baseline (median = 2.371 mL/min/kg, range = 1.605-4.359) with either administration of diltiazem (median = 2.305 mL/min/kg, range = 1.629-4.387; median difference = 0.080 mL/min/kg, 95% confidence interval [CI] = −0.417 to 0.757; P = .85) or D5W (median = 2.389 mL/min/kg, range = 1.600-3.557; median difference = 0.014 mL/min/kg, 95% CI = −0.426 to 0.189; P = .49). Furthermore, there was no statistical difference found between GFR after administration of diltiazem and GFR after administration of D5W (median difference = −0.036 mL/min/kg, 95% CI = −0.241 to 1.112; P = .69).

3.3 Fractional excretion of sodium

FENa did not significantly increase from baseline with diltiazem (baseline median = 0.038%, range = 0-0.5; diltiazem median = 0.045%, range = 0-0.9; median difference = 0%, 95% CI = −0.1 to 0.1; P = .81) or with D5W (baseline median = 0.1%, range = 0-0.3; D5W median = 0.17%, range = 0.1-0.4; median difference = 0%, 95% CI = −0.1 to 0.2; P = .44) (Figure 2). Additionally, FENa after diltiazem did not differ significantly from FENa after D5W (median difference = 0.1%, 95% CI = −0.1 to 0.2; P = .26). The baseline FENa results obtained before each CRI were not significantly different from each other (P > .99).

3.4 Urine output

Median baseline UOP before diltiazem administration was 0.12 mL/kg/hour (range, 0-1.37), and median baseline UOP before D5W administration was 0.22 mL/kg/hour (range, 0-1.88). Baseline UOP between the 2 groups was not significantly different (P = .74). UOP did not vary significantly over time with either diltiazem (P = .06) or D5W (P = .06) (Figure 3).