Conceived and designed the experiments: TAR MT LØ ALL USN. Performed the experiments: DJ HB EJE BLL. Analyzed the data: TAR ALL LØ HB. Contributed reagents/materials/analysis tools: EJE HB DJ BLL. Wrote the paper: TAR ALL HB BLL.
The study was supported by a grant of US$ 9,100 from GlaxoSmithKline (
Our objective was to compare the bone and renal effects among HIV-infected patients randomized to abacavir or tenofovir-based combination anti-retroviral therapy.
In an open-label randomized trial, HIV-infected patients were randomized to switch from zidovudine/lamivudine (AZT/3TC) to abacavir/lamivudine (ABC/3TC) or tenofovir/emtricitabine (TDF/FTC). We measured bone mass density (BMD) and bone turnover biomarkers (osteocalcin, osteocalcin, procollagen type 1 N-terminal propeptide (P1NP), alkaline phosphatase, type I collagen cross-linked C-telopeptide (CTx), and osteoprotegerin). We assessed renal function by estimated creatinine clearance, plasma cystatin C, and urinary levels of creatinine, albumin, cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL). The changes from baseline in BMD and renal and bone biomarkers were compared across study arms.
Of 40 included patients, 35 completed 48 weeks of randomized therapy and follow up. BMD was measured in 33, 26, and 27 patients at baseline, week 24, and week 48, respectively. In TDF/FTC-treated patients we observed significant reductions from baseline in hip and lumbar spine BMD at week 24 (−1.8% and −2.5%) and week 48 (−2.1% and −2.1%), whereas BMD was stable in patients in the ABC/3TC arm. The changes from baseline in BMD were significantly different between study arms. All bone turnover biomarkers except osteoprotegerin increased in the TDF/FTC arm compared with the ABC/3TC arm, but early changes did not predict subsequent loss of BMD. Renal function parameters were similar between study arms although a small increase in NGAL was detected among TDF-treated patients.
Switching to TDF/FTC-based therapy led to decreases in BMD and increases in bone turnover markers compared with ABC/3TC-based treatment. No major difference in renal function was observed.
Clinicaltrials.gov
Highly active anti-retroviral therapy (HAART) has substantially reduced mortality and morbidity from human immunodeficiency virus (HIV) infection. With effective HAART HIV-infected patients have a life expectancy approaching that of the general population
At the initiation of this study fixed-dose combinations of tenofovir (TDF)/emtricitabine (FTC) or abacavir (ABC)/lamivudine (3TC) were recommended for initial treatment of HIV infection
Previous studies have indicated an increased prevalence of osteopenia and osteoporosis
Case reports of renal dysfunction associated with TDF therapy
The Efficacy and Safety of Switching from Zidovudine to Tenofovir or Abacavir in HIV-infected Patients (SWAP) study was a randomized trial comparing switching from zidovudine (AZT)/3TC to ABC/3TC or TDF/FTC. We have previously published a report on cardiovascular biomarkers from this trial
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
The SWAP study was an open-label, parallel-group, randomized clinical trial assessing the safety and efficacy of switching from AZT to ABC- or TDF-based therapy. The study design and patient group have been described previously
DXA scans (DXA machine Hologic Discovery A (S/N 82962)) of the lumbar spine and hip were performed at baseline, week 24 and 48 at The Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Denmark. Serum and plasma samples were collected at baseline and week 4, 12, 24, and 48. Samples were stored at −80°C until analyses. We measured 2 biomarkers of bone formation, osteocalcin (OC) and procollagen type 1 N-terminal propeptide (P1NP;)
Twenty-four hour urine collection was done at baseline and week 12, 24, and 48, but was incomplete or omitted in several patients at various time points. Instead, we measured plasma creatinine and phosphate levels at baseline and week 4, 8, 12, 24, and 48 (Vitros FS 5.1 Ortho-clinical Diagnostics) and used the Cockcroft-Gault formula to estimate creatinine clearance (eCrCl). Plasma cysC was measured at baseline and week 48. In addition, we determined urinary levels of creatinine, albumin, cysC, and neutrophil gelatinase-associated lipocalin (NGAL) among patients with urine samples from at least 2 time points and used the ratios of albumin, cysC, and NGAL levels relative to urine creatinine concentration to identify signs of renal dysfunction. The change in ratio from baseline to the second measurement was compared across study arms regardless of which time the latter was performed. Plasma and urinary cysC was determined by a particle enhanced nephelometric assay using the N Latex cysC assay on the Behring Nephelometer II (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) as described previously
Baseline data were compared using χ2-test for categorical variables and two-sample t-test or Wilcoxon rank sum test for continuous variables. Percentage changes from baseline to week 24 and 48 in total hip and lumbar spine BMD were computed and compared within and between study arms using one- and two-sample t-tests, respectively. The changes from baseline to the various time points in levels of bone and renal biomarkers were compared between study arms using two-sample t-test or Wilcoxon rank sum test, as appropriate. Data distribution was assessed through visual inspection of frequency histograms and normal probability plots; logarithmic transformation was explored on skewed data to allow parametric hypothesis testing whenever possible. Linear regression was used to explore whether changes in bone turnover biomarkers at early time points (baseline to week 4 and 12, respectively) predicted subsequent reductions in BMD at week 48. Statistical analyses were per-protocol as pre-specified in the study protocol.
Details of study treatment and the patient group have been described previously
None of the study participants received systemic corticosteroids, bisphosphonates, vitamin D, calcium or other therapies known to affect bone metabolism at enrolment or during the study. Baseline characteristics of the patients are shown in
Parameter | TDF/FTC (n = 20) | ABC/3TC (n = 20) |
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Mean age (95% CI), years | 45.9 (41.8–49.9) | 49.6 (44.0–55.1) |
Male sex, n (%) | 14 (70.0) | 11 (55.0) |
Active smokers, n (%) | 9 (45.0) | 6 (30.0) |
Mean weight (95% CI), kg | 74.9 (67.3–82.5) | 73.2 (67.6–78.9) |
Mean BMI (95% CI), kg/m2 | 25.0 (23.0–27.0) | 25.2 (24.2–26.1) |
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Zidovudine/lamivudine at baseline, n (%) | 20 (100) | 20 (100) |
PI at baseline, n (%) | 6 (30.0) | 6 (30.0) |
Efavirenz at baseline, n (%) | 9 (45.0) | 11 (55.0) |
Nevirapine at baseline, n (%) | 5 (25.0) | 3 (15.0) |
Mean CD4+ cell count (95% CI), cells/mm3, | 540 (444–636) | 567 (447–687) |
Median duration of HAART exposure (IQR), years | 8.9 (4.2–10.8) | 7.2 (4.5–10.4) |
Median time since HIV-diagnosis (IQR), years | 10.2 (7.4–14.9) | 10.6 (8.0–11.7) |
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Mean lumbar spine BMD (95% CI), g/cm2 | 0.99 (0.93–1.04) | 1.00 (0.95–1.05) |
Mean hip BMD (95% CI), g/cm2 | 0.92 (0.86–0.98) | 0.90 (0.84–0.97) |
Median plasma osteocalcin (IQR), mikg/L | 17.9 (15.3–22.5) | 21.0 (17.5–27.2) |
Median plasma P1NP (IQR), mikg/L | 33.6 (29.8–48.2) | 51.5 (33.8–62.6) |
Median plasma CTx (IQR), ng/mL | 0.25 (0.18–0.32) | 0.34 (0.22–0.49) |
Median plasma osteoprotegerin (IQR), pmol/L | 3.4 (2.6–3.8) | 3.1 (2.3–3.9) |
Median plasma alkaline phosphatase (IQR), U/L | 74.5 (65.5–90.5) | 82.0 (68.5–99.5) |
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Mean systolic blood pressure (95% CI), mmHg | 135 (127–142) | 133 (122–143) |
Mean diastolic blood pressure (95% CI), mmHg | 84 (80–89) | 85 (79–90) |
Mean fasting plasma glucose (95% CI), mmol/L | 5.4 (5.2–5.5) | 5.4 (5.2–5.6) |
Median eCrCl a.m. Cockcroft-Gault (IQR), mL/min | 130 (118–140) | 120 (92–154) |
Median urine NGAL/creatinine ratio (IQR) | 0.42 (0.08–1.72) | 0.28 (0.00–0.71) |
Median urine cystatin C/creatinine ratio (IQR) | 0.92 (0.55–1.54) | 1.05 (0.54–1.15) |
Median urine albumine/creatinine ratio (IQR) | 0.39 (0.23–0.88) | 0.47 (0.32–1.20) |
BMI = body mass index; PI = protease inhibitor; HAART = highly active antiretroviral therapy; HIV = human immunodeficiency virus; BMD = bone mass density; P1NP = procollagen type 1 N-terminal propeptide; CTx = type I collagen cross-linked C-telopeptide; eCrCl = estimated creatinine clearance; NGAL = neutrophil gelatinase-associated lipocalin. Normally distributed data are presented as means with 95% confidence interval (95% CI) in parenthesis. Skewed data are presented as medians with interquartile range (IQR) in parenthesis.
As DXA scan could not be conducted in all patients, changes from baseline in BMD was only evaluated for 26 (16 TDF/FTC arm and 10 ABC/3TC arm) and 27 (15 TDF/FTC arm and 12 ABC/3TC arm) patients at week 24 and 48, respectively. In 7 cases (4 TDF/FTC arm and 3 ABC/3TC arm) the week 4 measurements of OPG was omitted due to lack of plasma.
We observed significant reductions from baseline in both hip and lumbar spine BMD at week 24 and 48 among TDF/FTC-treated patients, whereas BMD was stable in patients in the ABC/3TC arm. The mean percentage change at week 24 in hip and lumbar spine BMD was −1.8% (95% CI −2.6 to −1.1;
Percentage and absolute mean changes from baseline in lumbar spine (a, c) and hip (b, d) bone mass density as measured by dual energy x-ray absorptiometry (DXA). BMD = bone mass density; TDF/FTC = tenofovir/emtricitabine; ABC/3TC = abacavir/lamivudine. Error bars show 95% confidence intervals.
All biomarkers of bone turnover except OPG increased in the TDF/FTC arm compared with the ABC/3TC arm (
Baseline levels of 25-OH vitamin D (a) and percentage changes from baseline in biomarkers of bone turnover (b–f). P1NP = Procollagen type 1 N-terminal propeptide; CTx = Type I collagen cross-linked C-telopeptide (CTx); OPG = Osteoprotegerin; TDF/FTC = tenofovir/emtricitabine; ABC/3TC = abacavir/lamivudine. Mean values are depicted for normally distributed data and median values are depicted for skewed data. Accordingly, error bars show 95% confidence intervals or interquartile ranges.
A decrease in eCrCl from baseline was observed in both study arms that reached statistical significance at week 12, 24, and 48 in the TDF/FTC arm, and at week 12 and 48 in the ABC/3TC arm. In contrast, no significant change was observed from baseline to week 48 in the plasma levels of cysC in any of the study arms. There was no difference between study arms in the change from baseline, neither in eCrCl nor plasma cysC at any time point (
Changes from baseline to the indicated time points in estimated creatinine clearance (a), levels of plasma cystatin C at baseline and week 48 (b), changes from baseline to week 48 in plasma cystatin C (c), and changes from baseline to the last measurement in ratios of urinary biomarkers relative to urine creatinine concentration (d, e, f). Changes in urinary biomarkers (d, e, f) were evaluated in 29 patients (15 in the TDF/FTC arm and 14 in the ABC/3TC arm); of these, 24 were followed from baseline to week 48. Error bars in (a) show 95% confidence intervals; the horizontal line in (c) represents the mean value, while the horizontal lines in (d, e, f) represent median values. eCrCl = estimated creatinine clearance; NGAL = neutrophil gelatinase-associated lipocalin TDF/FTC = tenofovir/emtricitabine; ABC/3TC = abacavir/lamivudine.
There was no difference in blood pressure between study arms at baseline or at the end of the study (data not shown).
In this randomized clinical trial among virologically suppressed HIV-infected adults, the introduction of fixed-dose treatment with TDF/FTC lead to decreases in hip and lumbar spine BMD compared with fixed-dose treatment with ABC/3TC. In addition, we detected increases in the majority of measured bone turnover biomarkers in TDF/FTC- compared with ABC/3TC-treated patients. There was no difference in renal function between the two study arms as evaluated by eCrCl or plasma cysC, but a minor increase in urinary NGAL/creatinine ratio was observed in the TDF/FTC arm compared with the ABC/3TC arm.
The strength of the study is the randomized design combined with exploratory analyses of several biomarkers of bone and renal function. Concurrent inflammation was assessed at baseline and did not differ between study groups. Furthermore, the entry criteria required that patients be negative for the HLAB5701 allele, had not previously been exposed to ABC or TDF, and had been virologically suppressed for at least 3 months prior to randomization.
Only one previous randomized clinical trial (STEAL) has evaluated BMD in virologically suppressed HIV-infected patients switching to a TDF- or ABC-based regimen
Our results support the findings of previous studies
Whether the observed changes in BMD in our study and those of others are clinically relevant is a very interesting but difficult to answer question. The observed loss of BMD is quite small and not likely to confer a consequent increase in fracture risk in the majority of HIV-infected patients. At least there is no data to suggest that patients with higher risk of fractures should not receive TDF, but alternatives may be considered for those with previous fractures or known osteoporosis
It has been suggested that the initial loss of BMD may stabilize after 1 year of treatment and therefore may be caused by a continued effect of viral replication on bone turnover until complete suppression, even though other explanations have also been put forward
We measured several bone turnover markers to provide additional information on the effects of these treatments on bone remodeling and potentially the mechanisms underlying the changes seen in BMD. Bone turnover markers increased in the TDF/FTC arm within the first 12 weeks after treatment switch and remained stably elevated hereafter. This indicate that loss of BMD is associated with an increased rate of bone resorption and formation that can be detected as early as 12 weeks after initiation of TDF, and possibly already after 4 weeks. The early time point evaluated in this study is particularly interesting, as comparable studies have not provided data on bone turnover biomarkers so shortly after treatment shift to a TDF-based regimen. Both OC and OPG showed initial decreases in the TDF/FTC arm followed by increase or stabilization, which raises the question if certain signaling events relevant for the bone remodeling effects of TDF are taking place within the first few weeks of treatment switch. However, this remains speculative and the meaning of these finding are uncertain. Whether the changes in markers of bone turnover are reflecting direct effects of the treatment on the osteoclasts or osteoblasts or both
We did not detect any differences between study arms in the changes in eCrCl or plasma cysC, but still observed a minor increase in 1 of 3 measured biomarkers of renal dysfunction among patients in the TDF/FTC arm. This is comparable to the findings in the HAART-initiation ASSERT-study
The primary limitation of our study is the failure to reach the intended number of enrolled patients, which reduced the statistical power to detect differences between study arms. In addition, DXA was not performed in all patients and we only have 48 weeks of follow-up, which is too short to acknowledge the long-term effect on BMD reductions that appear to occur primarily within the first 24–48 weeks with stabilization hereafter. Interestingly, while eCrCl decreased over time in both study arms, no change in plasma cysC was observed indicating a stable GFR. We did not include GFR measurement by an exogenous marker and thus cannot compare these two estimates with more precise GFR-measurements. However, the eCrCl estimate based on plasma creatinine is dependent on factors other than GFR, including changes in muscle mass and tubular secretion of creatinine, and is possible that either the HIV infection or the anti-viral treatment may influence such factors. Previous studies comparing eCrCl and cysC based GFR estimates on HIV patients with normal renal function and on anti-viral treatment have generated mixed results
Long-term effects of HAART, including effects on bone and renal function, will continue to be outcomes of interest in future clinical trials. Beside this, more prospective studies will be needed to evaluate potential interventions to prevent or reverse loss of BMD. While several clinical trials have shown that bisphosphonate, vitamin D and calcium, either separate or in combination, are safe and effective in the treatment of decreased BMD among HIV-infected patients on HAART
In conclusion, we observed decreases in hip and lumbar spine BMD, in conjunction with early increases in bone turnover markers, in patients randomized to TDF/FTC-based treatment compared with ABC/3TC. Changes in renal function were not different between study arms although a very small increase in NGAL was detected among TDF-treated patients. The clinical significance of the latter is unclear.
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We thank the study participants for their involvement in the study and the study nurses, Iben Rose Loftsheim and Inge Vejlgaard Arbs, for an excellent effort as trial site coordinators. We thank the medical laboratory technicians Erik Hagen Nielsen and Lene Svinth Jønke for technical assistance in processing and handling of blood samples and analyses.