Study Design and Setting
This is a 24-month prospective cohort study of 260 men and women attending outpatient renal clinics at the University Health Network (UHN), and at St. Michael's Hospital in Toronto, Ontario, Canada. Other sites will be recruited as necessary. Subjects will undergo all testing (described below) upon study entry and then at 12 and 24 months with the exception of bone biopsy, which will be performed only once at 12 months. Subjects who progress to ESRD will have assessments at the time of dialysis initiation or transplantation, and then 12 months later. Subjects will also be contacted by telephone every 4 months, at which time they will be asked about the occurrence of falls and fractures since their last study contact.
This study has been reviewed by the local Research Ethics Boards, and has received ethics approval at all participating institutions.
Inclusion Criteria: Men and women aged 18 years and older, with a glomerular filtration rate of ≤60 mL/min/1.73 m2 at study entry are eligible for recruitment.
Exclusion Criteria: Patients who are unable to give informed consent, unable to have spinal radiographs or BMD measurements; those taking bisphosphonates, calcitonin, hormone replacement therapy, or the oral contraceptive pill are excluded. Subjects allergic to tetracycline are excluded from the bone biopsy component of the study.
All assessments (below), with the exception of bone biopsy, will be obtained at a single site (UHN) using standard protocols.
i. Bone Mineral Density
BMD will be measured at the lumbar spine (L1 to L4), the total hip, femoral neck and proximal radius using a bone densitometer (Hologic). To reduce variability, BMD will be measured using a standard protocol (including regular use of a phantom spine). BMD tests will be reported according to the International Society for Clinical Densitometry (ISCD) protocol by a single ISCD certified clinician blinded to the study hypotheses.
ii. High Resolution Peripheral Quantitative Computed Tomography
HR-pQCT measurements will be obtained at the non-dominant radius (non-weight bearing site), and also at the tibia (weight bearing site) using the Xtreme CT device (Scanco Medical AG, Basserdorf, Switzerland). Measurements that can be obtained from HR-pQCT include: total volumetric density, cortical volumetric density, trabecular volumetric density, cortical thickness, trabecular thickness, trabecular separation, and trabecular number. The reproducibility of density measurements is <0.3% CV (phantom) .
iii. Markers of Bone Turnover
Blood samples will be collected, and then centrifuged at 4°C for 15 minutes. Serum will be aliquotted and immediately stored in microtubes at -80°C for future measurement of bone turnover markers and bone cytokines.
iv. Bone biopsy
Bone histomorphometry will be performed in a subgroup of 50 subjects. To ensure adequate power, 25 subjects with confirmed prevalent fractures will be included, as well as 25 without confirmed fracture. We will obtain a bone biopsy of the anterior superior iliac crest at 12 month follow up only.
Subjects will be given tetracycline labels administered over ~2 week period as follows: Two days of tetracycline (250 mg, by mouth, four times per day), and then given a 12 day intermission (days 3-14). On day 15, subjects will be given the same tetracycline (250 mg, by mouth, four times per day) for 4 days (days 15-18). Anterior iliac crest bone biopsies will be obtained one to two days later. By labeling twice with tetracycline standard histomorphometry can determine the mineral apposition rate and mineralizing perimeter for the two separate labeling periods and thereby calculate the BFR.
Bone biopsy technique:
The iliac biopsy will be obtained following the protocol of Meunier , using a Bordier trephine, 8 mm in internal diameter, to avoid fracture and compression of the core of bone and minimize sampling error. The procedure is performed under local anesthesia, supplemented by parenteral sedation if needed to achieve proper muscle relaxation.
Samples will be processed at 4°C according to standardized techniques. The primary histomorphometric variable to be studied is the BFR. The classification of bone disease associated with ESRD is based on the BFR and it has been purported that either an increased BFR (e.g. >500 μm/mm tissue area/day; as seen in hyperparathyroidism) or a decreased BFR (e.g. <108 μm/mm tissue area/day; as seen in adynamic bone disease), may be the strongest predictor of fractures. We will determine if the BFR is a prognostic factor for fracture by first classifying turnover into low, normal or high, based on the reference values noted above.
v. Muscle Strength, Balance and Falls
Muscle strength and balance will be assessed using three tests: The Timed Up and Go (TUG), Hand Grip, and in a subgroup of subjects, the 6-Minute Walk Test (6MW). These tests allow determination of muscle strength and balance across a wide range of functional abilities, has been validated in populations with renal disease and has been shown to be associated with fractures in cross sectional studies of patients with ESRD .
Primary Outcome Variable
i. Spine and Nonspine Fractures
Subjects will be questioned about a history of non-spine fractures at baseline and every 4 months during the follow up. Fractures will be classified as either traumatic or "low trauma" (osteoporotic) based on World Health Organization criteria; an osteoporotic fracture is defined as a fracture from one's standing height or less. Data from a prospective study demonstrate that self-report of fractures is accurate for osteoporotic fractures . Subjects will have anterior and lateral radiographs of the thoracic and lumbar spine at study entry to evaluate the presence of baseline (prevalent) vertebral fractures and then at 12 and 24 months to assess the development of new (incident) vertebral fractures. All radiographs will be obtained and reviewed using a standardized methodology, vertebral morphometry , by a muscular-skeletal radiologist with expertise in the radiographic diagnosis of vertebral fractures.
Withdrawal from Study
If requested, subjects will be withdrawn from the study without prejudice, as documented and explained at the time of consenting.
i. Sample Size Estimations
Based on our prior experience with recruiting subjects with ESRD, and the natural history of CKD the study anticipates recruitment of 260 subjects over 2 years. It is estimated that of these 260 subjects, 60 will have renal transplants, progress to ESRD or die, leaving 200 subjects. The power calculations were based on comparisons of mean baseline values between 30% of patients with prevalent fractures and 70% without prevalent fractures. This 30% is a conservative estimate given prior data that demonstrates that 50% of CKD patients have sustained a fracture prior to starting dialysis [2–5], and data from the Canadian Multicentre Osteoporosis Study demonstrates that among a population based sample of healthy men and women 50 years and older 38% of men and 41% of women have had a fracture . A sample size of 200 subjects is adequate to meet all our hypotheses. Sample size calculations for each of the primary hypotheses are detailed below:
1. BMD at cortical sites (the mid radius) will be associated with fractures
Based on a review of the literature, a well accepted minimal clinically important difference (MCID) in BMD between fractured and non-fractured patients is 3%, which is associated with standard deviations (SD) that range from 4.5% to 6.8% [25–32]. We will have 81% (at a SD of 6.8%) to 99% power (at a SD of 4.5%) to detect a 3% difference in mid radius BMD.
2. Volumetric BMD at the forearm by HR-pQCT will be associated with fractures
The limited published data suggest that an MCID in volumetric BMD ranges from 1 to 3 with a SD that varies from 1 to 2 . The most conservative assumption is a MCID of 1 with an SD 2. We will have 89% power to detect this MCID.
3. There will be a positive correlation between the "TUG" and presence of fractures
Our data demonstrates a MCID of 5 seconds that can be associated with a SD of 10 to 12 seconds. We will have 89 to 76% power to detect this difference.
4. There will be a positive association between fractures and the presence of either: adynamic bone disease, or hyperparathyroid bone disease, as defined by the BFR
Power calculations for this hypothesis were based on the assumption that of the 60 patients with prevalent fractures (30% of 200) just under half (40%) would agree to biopsy (25 subjects) and we would recruit another 25 non-fractured subjects for a total of 50. If we assume that 85% of patients with fracture would have an abnormal BFR, we have 80% power to detect an abnormal BFR rate of 45% or lower in the non-fractured group. We anticipate that approximately 45% of subjects will have incident fractures in the two-year follow-up period.
ii. Statistical Analyses
We will use linear regression models to assess relationships, with BMD, volumetric BMD (by HR-pQCT), TUG and BFR (from bone biopsy) as predictor variables and presence or absence of prevalent fracture as the main outcome variable. Unadjusted analyses will be performed to determine the association between each predictor variable of interest and fracture outcome. Those with a positive association will be adjusted for potentially confounding covariates such as gender, age, weight, serum calcium, diabetes, and intact serum PTH. Goodness-of-fit will be evaluated for all models by inspecting plots of residuals, as well as use of Hosmer and Lemeshow Goodness of fit test; variables will be transformed as necessary. Our primary analyses will not be stratified by gender as earlier work by our group has not demonstrated a significant difference in fractures by gender among patients with renal disease. However supplementary analyses will be performed separately by gender and by the presence of type II diabetes. We will consider a p value of less than 0.05 statistically significant and we will not adjust for multiple comparisons. Our study statistician will perform our analyses using STATA version 10 (STATA Corp. College Stn. TX) and R 2.7 (R Development Core Team (2008). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria)
Anticipated Challenges and Solutions
We have anticipated some potential challenges and have offered corresponding solutions:
1. Subjects drop out after enrollment
Based on chart review of the Renal Management Clinic at the UHN, approximately 15 to 20% of subjects progress to ESRD, receive a transplant, transfer care or die every year, with very few patients lost to follow up. To account for these potential "drop outs" after enrollment, we will recruit 260 subjects, in anticipation that 200 will continue to be followed.
2. Unable to recruit patients from single site
The clinic at UHN has a potential pool of over 500 subjects, however, we have established collaborative links with St. Michael's Hospital in Toronto as well as with other Toronto area institutions, which will assist us in recruiting and meeting our sample size goals.
3. Failure to obtain adequate samples for bone biopsy component
It is possible, due to the invasive nature of the bone biopsy procedure that few patients will consent. To maximize the number of biopsies and to limit drop outs, the protocol was designed so that we obtain a single bone biopsy at one year follow up, rather than obtaining repeat bone biopsy samples. Note that there is a paucity of data on bone histomorphometry and stages of CKD - thus at a minimum our findings will serve as pilot data for a larger study.