Evaluating Hormonal Mechanisms of Vitamin D Receptor Agonist Therapy in Diabetic Kidney Disease: The VALIDATE-D Study

Jenifer M Brown; Kristina Secinaro; Jonathan S Williams; Anand Vaidya

Disclosures

BMC Endocr Disord. 2013;13(33) 

In This Article

Methods/Design

Study Population and Recruitment

The VALIDATE-D study will enroll 40 adults, aged 18–70 years, with Type 2 diabetes (T2DM), microalbuminuria, and without known CKD (estimated glomerular filtration rate > 60 mL/min). Subjects will be recruited from the local community and from the outpatient clinics of the Division of Endocrinology, Diabetes, and Hypertension within the Department of Medicine at Brigham and Women's Hospital, Boston, USA. Eligibility will be assessed at an in-person screening visit involving detailed history, physical examination, and laboratory assessment.

Inclusion criteria include T2DM that is controlled with diet, oral hypoglycemic agents, incretin analogues, or a single basal insulin injection; microalbuminuria; normal blood pressure or stage 1 hypertension treated with 0–1 antihypertensive agents; and normal blood cell counts, liver function, and electrocardiogram.

Exclusion criteria include CKD (glomerular filtration rate < 60 mL/min); poorly controlled T2DM (defined as HbA1c > 8.5% or the use of >1 daily insulin injection or a history or retinopathy); stage 2–3 hypertension or the use of more than one antihypertensive agent; kidney stones, parathyroid or granulomatous disorders, liver or heart failure, or coronary artery disease or congestive heart failure or cerebrovascular accidents. All subjects provide informed consent, and all study procedures have been approved by the Brigham & Women's Hospital institutional and ethics board as well as the Brigham and Women's Hospital Center for Clinical Investigation. Recruitment was initiated in September 2012 and is expected to be completed by September 2016.

Study Protocol Overview

A detailed study schema is shown in Figure 2. All study visits will occur in the Clinical Research Center (CRC) at Brigham and Women's Hospital. Studies will be performed in the CRC in order to control posture, time of day, dietary intake, and medication administration, since all of these factors are known to influence RAS activity. In brief, detailed profiling of the RAS will be performed at baseline before any intervention (Visits 2 & 3), again after double-blinded randomization to calcitriol or placebo (Visits 5 & 6) to assess whether calcitriol lowers RAS activity, and again after the addition of lisinopril to the blinded study drug to assess for an additive or synergistic relationship between VDR-agonist and ACE inhibitor therapy on the RAS and proteinuria (Visits 8 & 13).

Figure 2.

VALIDATE-D study schema : subjects will be screened and then may undergo a medication washout to withdraw interfering anti-hypertensive medications (when applicable). Aim 1: The circulating RAS will be assessed on LS diet before and after randomization to calcitriol/placebo (visits 2 & 5). Aim 2: The renal-vascular tissue-RAS will be assessed on HS diet before and after calcitriol/placebo (visits 3 & 6). Aim 3: All subjects will then receive lisinopril 5 mg/d in addition to calcitriol/placebo to assess the impact of combination therapy (calcitriol + lisinopril vs. placebo + lisinopril) on the renal-vascular tissue-RAS (visits 6 & 8) and proteinuria (visits 6, 8, 13). Serum calcium and phosphate will be monitored weekly throughout the study for safety, at each visit and at designated safety checks (visits 4, 7, 9–12).

Washout. It is well known that many medications interfere with physiologic RAS responses or with the assays that measure the RAS. To optimize the reliability and accuracy of RAS measurements, medications that interfere with RAS assessments will be withdrawn. Subjects using RAS inhibitors (such as angiotensin converting enzyme [ACE] inhibitors, angiotensin receptor blockers, or mineralocorticoid receptor antagonists) will stop their medications for 2 months prior to study. Subjects using beta-blockers or diuretics will stop their medications for 1 month prior to study, whereas those on calcium channel blockers will stop for 2 weeks prior to study. Medication washout will be supervised by a study staff based on previously described safety protocols[5] after providing and instructing each subject on how to use a home sphygmomanometer. As previously described, in cases where hypertension (159/99 mmHg) results following withdrawal of an antihypertensive medication, amlodipine will be used to lower blood pressure as it has a neutral effects on RAS activity.

Study Diets. All study procedures will be conducted using one of two specifically designed study diets.

Low-salt Diet

The low salt (LS) diet includes 10 mEq of sodium, 100 mEq of potassium, and <600 mg of calcium per day. The purpose of this diet is to induce maximal stimulation of RAS components to allow for adequate measurements of the circulating RAS. Subjects will consume the LS diet for 1 week prior to study visits where the circulating RAS will be measured (Visits 2 and 5). All daily meals and food intake during the LS dietary phase will be prepared and provided to study participants by the CRC dietary kitchen staff.

High-salt Diet

The high sat (HS) diet includes 200 mEq of sodium, 50 mEq of potassium, and <600 mg of calcium per day. The purpose of this diet is to induce maximal suppression of the circulating RAS components in order to most effectively measure the renal-vascular tissue-RAS, as previously described.[5,17] Subjects will consume the HS diet for 1 week prior to study visits where the renal-vascular tissue-RAS will be evaluated (Visits 3, 6, and 8). The HS diet involves consumption of an ad lib diet, with supplements provided to achieve the sodium and potassium contents.

Low Calcium Diet

Subjects will be instructed and taught to restrict dietary calcium intake during the LS and HS dietary phases (<600 mg/day), and also during the duration of the study in between study visits (<1000 mg/day). The purpose of calcium restriction is to minimize the risk of hypercalcemia and hyperphosphatemia associated with calcitriol administration; this risk is negligible in the context of low dietary calcium intake.[27–29] A registered dietician will coach subjects throughout the study on restricting calcium intake and will provide materials to guide dietary intake at home (Dietary Calcium Intake Recommendations Additional file 1). Compliance with study diets will be confirmed by 24-hour urine collections obtained at every study visit.

Baseline Assessments of the Circulating and Renal-vascular Tissue-RAS (Visits 2 & 3). After a screening visit (Visit 1) and a potential antihypertensive washout phase, all subjects will undergo baseline assessments to characterize the circulating and renal-vascular tissue-RAS.

Circulating RAS on LS Diet (Visit 2)

Study visit 2 is designed to measure the baseline circulating RAS under conditions designed to induce maximal stimulation of the circulating RAS: restricted dietary sodium intake and upright posture. After one week of LS diet consumption, subjects are studied at 8 AM in the morning (Figure 2). Subjects will maintain an upright standing posture for at least 1 hour. Blood samples are obtained at the end of the postural phase to measure circulating RAS components (plasma renin activity, angiotensn II, aldosterone).

Renal-vascular Tissue-RAS on HS Diet (Visit 3)

Study visit 3 is designed to assess baseline renal-vascular tissue-RAS activity by measuring changes in renal plasma flow (RPF) in response to an infusion of exogenous angiotensin II. Angiotensin II is a vasopressor that specifically interacts with angiotensin receptors to induce renal-vascular vasoconstriction resulting in a decrease in RPF. The magnitude of the decline in RPF to angiotensin II is inversely related to renal-vascular tissue-RAS activity (Figure 3); greater renal-vascular responses to angiotensin II signify lower local tissue-RAS activity.[5,30,31] Using this physiologic methodology, measurement of RPF responses to angiotensin II provide an indirect method to assess the human renal-vascular tissue-RAS.

Figure 3.

Reciprocal relationship between renal-vascular tissue-RAS activity and the renal-vascular response to angiotensin II. A blunted response to angiotensin II reflects a high level of local, endogenous, renal-vascular tissue-RAS activity (the smaller the decline in renal plasma flow in response to an infusion of angiotensin II, the greater the activity of the local renal-vascular tissue-RAS). Conversely, a robust response to angiotensin II is observed when tissue-RAS activity is low.

To adequately measure the renal-vascular tissue-RAS, subjects are studied on a HS diet and in overnight supine posture to suppress endogenous circulating RAS components, thereby amplifying measurable RPF responses to angiotensin II.[32] After completing one week of HS diet, subjects will be admitted to the CRC and maintained supine overnight and throughout the study procedures on the following morning. In the morning, two intravenous lines will be placed to facilitate infusion of study drugs and frequent blood sampling. Weight-based para-aminohippurate (PAH) will then be infused and its clearance used to measure RPF throughout the duration of the study visit.[5] After allowing PAH concentrations to reach a steady state over 60 minutes, blood will be sampled to establish the baseline RPF. Angiotensin II will then be infused at 0.3 ng/kg/min for 45 minutes and escalated to 1 ng/kg/min for an additional 45 minutes to assess dose–response characteristics. PAH will be measured during and after each angiotensin II infusion to measure RPF dynamics. Blood pressure will be monitored closely throughout the infusions based on standardized safety protocols[5] that we have extensive experience performing in our CRC. The study visit will end upon completion of the angiotensin II infusions, and subjects will be discharged to undergo randomization to the study drug.

Randomization to Study Drug. Following study visit 3, subjects will be randomized to either calcitriol 0.75 mcg/day or placebo arm using a 2 block randomization. Double-blinded randomization will be performed by the investigational drug pharmacy at our institution, and neither the subjects nor the investigators will know the identity of the randomized study drug.

The Influence of Calcitriol on the RAS (Visits 5 & 6). Circulating RAS on LS Diet (Visit 5): After 2 weeks of daily calcitriol or placebo, subjects will return on LS diet and complete visit 5, which is identical to visit 2. Aim 1 of this study (to assess whether direct VDR activation lowers circulating RAS activity in human diabetes) is executed in study visits 2 and 5. Anticipated Results: It is anticipated that calcitriol therapy will lower circulating RAS components (plasma renin activity and angiotensin II) when compared to placebo, demonstrating for the first time that VDR agonism can inhibit the circulating RAS in human diabetes.

Renal-vascular Tissue-RAS on HS Diet (Visit 6)

After a third week on the study drug, subjects will return on HS diet for an overnight CRC study visit 6, that is identical to Visit 3. This visit will permit assessment of study Aim 2 (to assess whether direct VDR activation lowers renal-vascular tissue-RAS activity in human diabetes) by comparing the renal-vascular tissue-RAS activity before and after calcitriol/placebo. Anticipated Results: It is anticipated that calcitriol therapy will raise basal RPF and increase the RPF sensitivity to angiotensin II, thereby demonstrating that VDR activation improves renal-vascular hemodynamics by lowering local renal-vascular RAS activity in human diabetics without CKD.

The Influence of Combination VDR-agonist and ACE Inhibitor Therapy on the Renal-vascular Tissue RAS (Visit 8). Upon completing study visit 6, all subjects will receive lisinopril (initially 2.5 mg daily, which will be increased to 5 mg daily after one week, unless blood pressures are below 90/50 mmHg or subjects experience orthostatic symptoms) in addition to their randomized study drug (Figure 2). After 2 weeks of daily combined therapy (calictirol + lisinopril or placebo + lisinopril), subjects will return on the HS diet for a final inpatient CRC visit that is identical to study visits 3 & 6. Comparing the outcomes of study visits 6 and 8 will permit evaluation of Aim 3 of this study (to assess whether direct VDR activation in combination with ACE inhibition exerts an additive or synergistic effect on the renal-vascular tissue-RAS and proteinuria). Anticipated Results: Combination therapy (calcitriol + lisinopril) will additively or synergistically raise RPF and increase the RPF sensitivity to angioensin II infusion, reflecting decreased renal-vascular tissue-RAS activity when compared to placebo + lisinopril.

The Influence of Chronic Combination Therapy on Proteinuria (Visit 13). Following completion of study visit 8, subjects will be discharged home to continue both the study drug and lisinopril for three months of long-term therapy (Figure 2). For the final visit of the study, subjects will return after one week of HS diet with a completed 24-hour urine collection, which will be analyzed for protein. The 24-hour urine protein measured at this visit will be compared using repeated measures analyses to the urine protein levels obtained from 24-hour urine collections completed at each inpatient visit on HS dietary balance (visits 3, 6, 8). Anticipated Results: Combination therapy (calcitriol + lisinopril) will additively or synergistically lower proteinuria when compared to placebo + lisinopril, thereby paralleling prior animal studies that showed that the combination of VDR agonist + conventional RAS inhibitor therapy induced synergistic renoprotective benefits in mouse models of diabetes.[19–21]

Safety & Monitoring

Calcium/Phosphate. To minimize the risk of hypercalcemia due to calcitriol, all subjects will undergo laboratory evaluation of serum calcium and phosphate at 1–3 week intervals throughout the study duration, including at the aforementioned study visits and additionally at designated safety visits (visits 4, 7, 9–12). If calcium or phosphate rises above the upper limit of normal, the test will be repeated in 3 days and subjects will be counseled by our dedicated study dietician on adherence to a low calcium diet (Dietary Calcium Intake Recommendations Additional file 1). If calcium or phosphate levels remain elevated, or the calcium-phosphate product exceeds 50, the dose of study drug will be reduced by one third. Based on several prior studies that have employed chronic VDR-agonist therapy in combination with lowered dietary calcium intake, our dosing and study duration are unlikely to invoke hypercalcemia or hyperphosphatemia.[27–29]

Blood Pressure. During PAH and angiotensin II infusion, blood pressure (BP) will be monitored every 2 minutes using an automated sphyngomanometer. An increase in BP is an expected effect of angiotensin II infusion, and the following protocol has been used safely in over 1500 subjects.[5] The 0.3 ng/kg/min dose was selected because it does not induce a pressor effect but still influences renal-vascular hemodynamics,[30] and the 1 ng/kg/min dose was selected because it has previously been shown to induce minimal BP elevations well within a safety range.[5,30] A study physician will supervise all angiotensin II infusions as previously described.[5]

All subjects will monitor their BP daily with a home sphygmomanometer while on the washout phase (when applicable) and while on lisinopril and will communicate their readings to study staff twice weekly. Study staff will monitor for unsafe high BP during the washout period (>159/99 mmHg) or unsafe hypotension during lisinopril treatment (<90/50 mmHg). Subjects who develop high BPs during the washout period may be treated with amlodipine which has a neutral effect on RAS components. Subjects who develop low BPs on lisinopril may have their dose reduced 50%, however, if a 2.5 mg daily dose is not tolerated, subjects will be removed from the study.

Blood Glucose. Subjects will be asked to check their home blood glucose daily, and study staff will monitor their glycemic control throughout the study. If fasting morning blood glucose is consistently >150 mg/dL, medications will be adjusted by a study physician. Such adjustments include increasing the dose of a home medication or adding a second oral hypoglycemic agent.

Lipids. Any subject who has a screening LDL cholesterol greater than 100 mg/dL, will either be prescribed simvastatin 20-40 mg daily, or if already taking a statin medication, have their pre-existing statin dose increased, to remain consistent with clinical standards of care for diabetes.

Laboratory Measurements

25(OH)D (Diasorin, Inc., Stillwater, MN), 1,25(OH)2D (Diasorin), parathyroid hormone (Beckman Coulter, Fullerton, CA), and serum and urine electrolytes (including calcium and phosphate) will be measured at baseline at all visits. Components of the circulating RAS, including plasma renin activity (Diasorin) and aldosterone (Siemens, Los Angeles, CA), will be measured from samples drawn in upright and supine posture (Visits 2, 5, 13) and before and during angiotensin II infusion (Visits 3, 6, 8).[5] For assessment of RPF, para-aminohippurate will be measured in triplicate at steady-state and during angiotensin II infusion and will then be adjusted by body surface area in all RPF calculations (Visits 3, 6, 8).

Statistical Analyses

Sample Size Calculations. Sample size calculations were made using our prior data evaluating the impact of vitamin D on the systemic[23] and renal-vascular tissue-RAS[5] by paired analyses with an alpha-error of 0.05. For Aim 1, prior data suggest that therapy with a VDR agonist could reduce plasma renin activity, a measure of the circulating RAS, by 1 ng/mL/hr (SD = 0.54). A minimum of 5 subjects would be required in each study arm for a paired comparison to detect this difference with a power of 0.9. In our pilot intervention study, we demonstrated an effect of vitamin D therapy on the renal-vascular tissue-RAS (Aims 2 & 3) in a sample of 14 subjects. To account for underestimation of sample-size, variability in measurements, and to permit non-paired comparisons, we intend to study 20 subjects in each intervention group.

Outcomes. Paired and repeated measures analyses will be used to evaluate intra-individual changes in the circulating RAS, RPF responses to angiotensin II infusion (i.e. renal-vascular tissue-RAS), and urine protein levels. Non-paired comparisons will be used to evaluate changes in these parameters between the calcitriol and placebo groups. A two-tailed P-value < 0.05 will be considered statistically significant.

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