EPIDEMIOLOGY

Relationship Between Homocysteine and Mortality in Chronic Kidney Disease

Vandana Menon, MD; Mark J. Sarnak, MD; Tom Greene, PhD; Xuelei Wang, MS;
Arema A. Pereira, MD; Gerald J. Beck, PhD; John W. Kusek, PhD; Jacob Selhub,
PhD; Allan J. Collins, MD; Andrew S. Levey, MD; Michael G. Shlipak, MD

BackgroundThe relationship between total homocysteine (tHcy) and outcomes has

not been investigated in patients with chronic kidney disease stages 3 to 4.
Methods and ResultsThe Modification of Diet in Renal Disease Study was a
randomized, controlled trial of 840 patients. Serum tHcy was measured in frozen
samples collected at baseline (n=804). Survival status and cause of death were
obtained from the National Death Index. To evaluate its association with all-cause
and cardiovascular disease (CVD) mortality, tHcy was evaluated both as tertiles
(<14.7, 14.7 to 19.5, >19.6 jumol/L) and as a continuous variable (per 10/ju,mol/L).
Participants had a mean age of 5212 years and glomerular filtration rate (GFR) of 33
12 mL/min per 1.73 m2; 60% were male, and 85% were white. During a median
follow-up of 10 years, 195 (24%) died from any cause, and 118 (15%) from CVD.
The level of GFR was lower and proteinuria higher in the highest tHcy tertile. There
was no association between the highest tertile of tHcy and all-cause (hazard ratio
[HR]; 95% confidence interval [CI[, 1.32, 0.94 to 1.85) or CVD (HR; 95% CI, 1.50,
0.96 to 2.34) mortality in univariate analyses; this association was further attenuated
by adjustment for GFR (HR; 95% CI all-cause, 1.04, 0.72 to 1.51; CVD, 1.20, 0.73 to
1.95). There was no association between tHcy as a continuous variable and all-cause
(0.98, 0.83 to 1.16) or CVD (1.04, 0.85 to 1.27) mortality.
ConclusionsHyperhomocystinemia does not appear to be a risk factor for all-cause
or CVD mortality in the Modification of Diet in Renal Disease Study. Prior studies
demonstrating an association between tHcy and CVD risk may have inadequately
adjusted for the confounding effects of kidney function. (Circulation. 2006;113:1572-
1577.)
Key Words: kidney mortality epidemiology

The high prevalence of hyperhomocystinemia in patients with chronic kidney disease

(CKD)1-2 has generated interest in a potential role for total homocysteine (tHcy) as a
risk factor for the excess risk of cardiovascular disease (CVD) evident in this patient
population.3-4 Hyperhomocystinemia appears to be a predictor of future CVD in
patients with established coronary artery disease5 and in patients with type 2 diabetes
mellitus.6 However, studies in the general population examining the association
between levels of tHcy and CVD have yielded inconsistent results. Results from a
few studies have demonstrated a strong association,7 whereas others have found no
association8 or were only able to detect a relationship in certain subgroups.9 Notably,
these studies either have not adjusted for kidney function or have relied on serum
creatinine, a less sensitive marker of kidney function, as a proxy for glomerular
filtration rate (GFR). Thus, given that reduced GFR is an established risk factor for
CVD,10 variations in adjustment for

Clinical Perspective p 1577

kidney function may explain the heterogeneity of study results.
Levels of tHcy are correlated with GFR, proteinuria, and nutritional status, as well as
with traditional CVD risk factors.'" Given these correlations and the inconsistency in
the literature, it remains unclear as to whether tHcy is an independent risk factor for
CVD or rather a marker of severity of kidney disease in patients with CKD. The
objectives of this study were to evaluate whether tHcy was an independent risk factor
for all-cause and CVD mortality in a cohort of patients with CKD stages 3 to 4 who
had precise measures of kidney function.

Methods
The Modification of Diet in Renal Disease (MDRD) Study, conducted from 1989 to
1993, was a randomized, controlled trial to study the effect of dietary protein
restriction and blood pressure control on the progression of kidney disease. Details of
the study have been published previously.12 In brief, 585 patients with a baseline
GFR of 25 to 55 mL/min per 1.73 m2 were randomized in study A, and 255 patients
with a baseline GFR of 13 to 24 mL/min per 1.73 m2 were randomized in study B.
Patients in study A and study B were combined for the current analyses.
As described previously, levels of total Hey were measured in frozen samples
collected at baseline from 804 participants of the MDRD Study cohort.13 Samples
were assayed for tHcy by using high-performance liquid chromatography with
fluorometric detection. GFR was assessed by the kidney clearance of I25l-
iothalamate.14
Survival status and cause of death were ascertained from the National Death Index. A
death was ascribed to CVD if the primary cause of death was International
Classification of Diseases, 9th Revision (ICD-9) code 390 to 459 (n=96) or if kidney
disease was listed as the primary cause of death and CVD was the secondary cause
(n=22). Survival time was defined as time from randomization to death or end of
follow-up (December 31, 2000). Data collection procedures were approved by the
Cleveland Clinic and Tufts-New England Medical Center Institutional Review
Boards.

Statistical Analysis
Demographic, CVD risk factors, and kidney disease factors were compared across
tertiles of tHcy through the use of the \~ test for categoric data, ANOVA for
approximately normally distributed continuous variables, and the Kruskall-Wallis test
for skewed continuous variables.
Incidence rates for mortality (per 1000 person-years) were calculated for tertiles of
tHcy. Unadjusted and 3 adjusted Cox proportional hazards models were conducted in
sequence to evaluate the association of tHcy with all-cause and CVD mortality.
Model 1 adjusted for GFR only; model 2 added age, gender, race, and random
assignments to protein diets and blood pressure targets; model 3 added proteinuria,
cause of kidney disease, history of coronary artery disease and diabetes, and body
mass index. We calculated the proportional change in the Cox regression coefficients
for tHcy after addition of GFR to quantify its attenuation.
To maximize statistical power to examine the relationship between tHcy and
mortality, continuous variable analyses were conducted with hazard ratios (HR)
presented per tHcy higher by 10 jamol/L. Given the non-normal distribution of tHcy,
we repeated the analyses by using log-transformed values for tHcy. Because the
results from these analyses were very similar to the ones using untransformed tHcy,
we present the latter for ease of interpretation. We also evaluated interactions between
tHcy and study (study A versus study B) and between tHcy and GFR to test for any
potential differential effects of severity of kidney disease on the relationships
examined. Proportional hazards assumptions were tested by using log-minus-log
survival plots and plots of Schoenfeld residuals versus survival time.

Additional Analyses
The fully adjusted Cox models (model 3) were repeated for all-cause and CVD
mortality after the exclusion of individuals with prevalent CVD (defined as history of
coronary artery disease, n = 77).
Because dietary protein intake may modify tHcy levels and use of random
assignments to protein diet to represent protein intake may not fully account for this
effect, we repeated the fully adjusted Cox model (model 3) for all-cause and CVD
mortality using adjusted protein intake estimated from urine urea nitrogen excretion
at 1 year and with tHcy as a continuous variable.
C-reactive protein (CRP) and albumin may be related to mortality in CKD15;
therefore, we repeated model 3 with the addition of CRP and albumin to assess any
potential effect of these covariates on the association between tHcy as a continuous
variable and mortality.
Homocysteine may be related to progression of kidney disease, and kidney failure
may modify the relationship between tHcy and mortality outcomes. Therefore, Cox
models (corresponding to model 3) were repeated with tHcy as a continuous variable
and using two additional outcomes. Cox regression was performed with the out-come
of death before reaching kidney failure (need for renal replacement therapy with
dialysis or transplantation) with censoring at kidney failure or end of follow-up and
using a composite outcome of death or kidney failure.
The authors had full access to the data and take full responsibility for its integrity. All
authors have read and agree to the manuscript as written.
 Results
Baseline Characteristics
The study cohort had an average age of 52 12 years, GFR of 3312 mL/min per
1.73m2, and median (interquartile range) tHcy of 16.8 (8) /Limol/L. The sample was
predominantly white (91%); 60% were male, and 15% were current smokers. Higher
tHcy was associated with male gender, history of coronary artery disease, lower body
mass index, and lower prevalence of diabetes (Table 1). Glomerular filtration rate was
lower and level of proteinuria higher in the higher tHcy groups; however, there was
no difference in the cause of kidney disease.

Outcomes
Median follow-up for survival analyses was 10 years (range, 3 to 140 months). All-
cause mortality rate was 24% (n= 195), and CVD mortality rate was 15% (n=118).
There were 60 (22%), 58 (22%), and 77 (29%) deaths from all causes and 33 (12%),
37 (14%) and 48 (18%) deaths caused by CVD in the respective tertiles. Incidence
rates for all-cause and CVD mortality, per tertile, are presented in the Figure.
There was a nonsignificant positive association between the highest tertile of tHcy
and both all-cause and CVD mortality in unadjusted analyses that was largely
attenuated by adjustment for GFR (Table 2). Additional adjustment for random
assignments, demographic, CVD, and other kidney disease factors had little
incremental effect on attenuating the association between the high tHcy tertile and
each mortality outcome.
After adjustment for GFR, the Cox regression coefficient for high tHcy and all-cause
mortality decreased from 0.278 to 0.037, a proportional change of 87%. Similarly, for
CVD mortality adjusting for GFR decreased the Cox regression coefficient from
0.406 to 0.179, a proportional change of 56%.
The Cox model was repeated evaluating tHcy as a continuous variable (Table 3).
Again, there was no association between tHcy and either all-cause or CVD mortality
in unadjusted or adjusted analyses. In models examining all-cause and CVD
mortality, respectively, interaction terms between study (study A versus study B) and
tHcy (P=0.70; f=0.69) and GFR and tHcy (P=0.73, P=0.93) were not significant.

Additional Analyses
There was no association between tHcy and all-cause (0.89, 0.69 to 1.15) or CVD
(0.82, 0.57 to 1.18) mortality in the subgroup of participants without history of
prevalent CVD. Substituting adjusted protein intake at 1 year for random assignments
to low protein diet in model 3 did not alter the results for all-cause (1.00, 0.85 to
1.18) or CVD mortality (1.05,0.86 to 1.29). The addition of CRP and albumin also as
covariates did not appreciably alter the HR for tHcy in models examining all-cause
(1.00, 0.85 to 1.17) or CVD mortality (1.05, 0.87 to 1.27).
Sixty-five participants died before reaching kidney failure. There was no association
between tHcy and death before kidney failure in a univariate Cox model (1.06, 0.81
to 1.39). We did not perform multivariate analyses because of limited power, given
the small number of events.
Five hundred ninety-three participants died or reached kidney failure by December
31, 2000. There was no association between tHcy and the composite outcome of
kidney failure or death in the fully adjusted Cox model (1.02, 0.94 to 1.11).

Discussion

In this cohort of patients with CKD stages 3 to 4, tHcy does not appear to be an
independent predictor of all-cause or CVD mortality. In unadjusted analysis, a
nonsignificant positive association was observed between elevated tHcy and the
mortality outcomes that was largely attenuated with adjustment for GFR. These
findings raise the question of whether prior studies that found tHcy to be a CVD risk
factor adequately adjusted for kidney function.
The results of prospective cohort studies investigating hyperho-mocystinemia as an
independent risk factor for the development of CVD have been conflicting. A recent
study found that hyperhomo-cystinemia, defined as tHcy levels >15 jumol/L, was an
independent predictor of CVD mortality in a large cohort of patients with type 2
diabetes.6 In a population-based cohort of women, participants in the highest quintile
for Hey were at increased risk for fatal and nonfatal acute myocardial infarctions.16
Homocysteine was an independent risk factor for all-cause mortality in Jewish men
and women aged 50 years and older,7 in patients with angiographically confirmed
coronary artery disease,5 and in the Physicians Health Study.17 However, other
studies suggest that the relationship between Hey and CVD exists only in specific
subgroups such as those with a previous history of CVD,9-18 in the elderly,9 in
patients with type 2 diabetes,19 and in patients with hypertension.

None of the studies that demonstrated an association between tHcy and outcomes
adjusted for level of kidney function with the gold standard of GFR as in this study.
One study used estimated creatinine clearance,6 some used serum creatinine,5-7-16
and others did not adjust for any marker of kidney function.9-1SJ9 The prevalence of
CKD may have been high in several of these studies because they included the
elderly, hypertensive patients, and patients with heart disease and type 2 diabetes, all
populations at high risk for kidney disease. Underlying kidney dysfunction may thus
have confounded the association of tHcy and cardiovascular outcomes in these
studies because serum creatinine is a less sensitive marker of kidney function.
In data from the Hoorn Study, tHcy was associated with increased risk of coronary
events in diabetic but not nondiabetic individuals.22 The association in diabetics
persisted after adjustment for estimated GFR, calculated using the MDRD Study
formula, but was attenuated by adjustment for history of cardiovascular disease. In a
study of patients with type 2 diabetes, tHcy was a significant predictor of all-cause
but not CVD mortality after adjustment for albumin excretion rate and estimated
creatinine clearance as measures of kidney function.
In contrast to the previously cited studies, other studies have failed to demonstrate an
independent relationship between Hey and CVD. An analysis from the
Atherosclerosis Risk in Communities Study8 and a population-based study from
Australia did not find an association between Hey and CVD.24 Investigators from the
Multiple Risk Factor Intervention Trial found no association between Hey and fatal
and nonfatal CVD events.2"5 Of these studies with a null result, two adjusted for
serum creatinine,2024 whereas two others did not include markers of kidney function
in their multivar-iate analyses.8-25 Of note, these studies finding no association were
conducted in relatively healthy cohorts that were likely to have low prevalence of
CKD.
Two meta-analyses have examined the associations between Hey levels and ischemic
heart disease. A publication from the Homocysteine Studies Collaboration using 30
prospective and retrospective studies found a 3-jianol/L lower Hey level was
associated with an 11% lower risk of ischemic heart disease and 19% lower risk of
stroke.27 In another meta-analysis that included 20 prospective studies, a 5-/xmol/L
increase in Hey was associated with a 32% increase in the risk of ischemic heart
disease.28 Neither of these analyses adjusted for level of kidney function.
There are limited data evaluating Hey as a risk factor for CVD in patients with CKD
before reaching kidney failure. In a cohort of 147 patients with creatinine clearances
between 20 and 55 mL/min per 1.73 m2, high Hey was an independent risk factor for
incident CVD.29 However, tHcy was only available in a subset of 93 patients, and
estimated creatinine clearance was the proxy for kidney function. In a cohort of 227
stable kidney transplant recipients (average serum creatinine, 1.90.8 mg/dL),
Ducloux et aP found an independent association between Hey and a composite
outcome of fatal and nonfatal CVD events. However, serum creatinine was used as
the marker of kidney function in this analysis, leaving the possibility that actual GFR
may still confound the association. One prospective study of 733 kidney transplant
recipients using a body surface area-adjusted measure of kidney function estimated
using the Cockroft-Gault formula (mean=55.8 mL/min per 1.73 m2) found an
association between Hey levels and all-cause mortality that was independent of
kidney function.31 Differences in the results from this study compared with our study
may be attributable to cohort differences including different kidney disease severity
and burden of preexisting CVD. Alternatively, Hey may have a differential impact as
a risk factor for CVD in patients who have received transplantation.
Several studies have examined the association between elevated levels of tHcy and
outcomes in dialysis patients, with contradictory findings. Although a few studies
have found high tHcy to be a risk factor for outcomes,32^34 others have failed to
demonstrate this association,35 and some have described an inverse relationship
between tHcy levels and outcomes.36^38 However, it must be acknowledged that the
dialysis population is very different from patients with stage 3 to 4 CKD, and,
moreover, it is not possible to study the effect of kidney function per se in this
population.
We and other investigators have reported that GFR is a major determinant of Hey
levels in the general population as well as in patients with CKD.' ''39 40 In the
present study, adjustment for GFR attenuated most of the relationship between tHcy
and mortality, suggesting that GFR was the primary confounder for the observed
association of high tHcy levels and the mortality end points. Reduced GFR is a
known important risk factor for CVD10; thus, the varied measurement and
adjustment for kidney function in prior literature may largely explain the
heterogeneity of study results.
It is also possible that tHcy may be a risk factor for progression of kidney disease and
thereby promote the development of CVD. In support of this hypothesis, in a
population-based cohort, tHcy was found to be a predictor of the development of
microalbuminuria in nondiabetic individuals, and this relationship was independent of
GFR.41 Conversely, studies in patients with kidney disease, including data from the
MDRD Study, have failed to demonstrate any association between tHcy and
progression. 13<42-43 In our study, we did not find a relationship between tHcy and
a composite outcome of death or kidney failure.
Another possibility is that our null findings could be specific to the population we
studied. The MDRD Study cohort consists of nondiabetic, predominantly white
patients, an overrepresentation of patients with polycystic kidney disease, with few
older adults and a low burden of CVD. In addition, we were unable to assess nonfatal
CVD events, which may have different associations with Hey than mortality end
points. As described in a previous publication,13 a 2-hour delay in sample processing
may in theory result in a systematic measurement error resulting in elevated tHcy
levels caused by release of Hey from red blood cells. However, we have previously
demonstrated that the range of tHcy levels, prevalence of hyperhomocystinemia, and
magnitude of associations with known variables such as folic acid and GFR in our
sample are very similar to those previously reported.1 We also acknowledge that the
relatively wide confidence intervals surrounding the hazard ratios preclude
definitively ruling out an association between tHcy and outcomes. Finally, it must be
recognized that the majority of the study participants reached kidney failure requiring
dialysis during follow-up. Kidney failure is known to be associated with increased
tHcy levels, and this may have influenced the associations studied. However, the lack
of association between tHcy and mortality in participants who died before reaching
kidney failure suggests that baseline tHcy is not a major determinant of outcomes in
this cohort of patients with stage 3 to 4 CKD.
The major strength of this study is the inclusion of a large cohort of patients with
precise measures of kidney function. In addition, this study sample has a wide range
of tHcy levels, includes a large proportion individuals with abnormal tHcy values,1
and a high number of outcomes during follow-up. This is a relatively healthy CKD
population; however, this makes it an ideal population to assess a potentially
independent effect of tHcy on mortality in the absence of powerful confounders such
as diabetes and preexisting CVD.
In summary, tHcy does not appear to be a risk factor for all-cause or CVD mortality
in the MDRD Study cohort. Homo-cysteine may be a marker for severity of kidney
disease rather than a mediator for the development of CVD, in patients with CKD
stages 3 to 4. Prior studies demonstrating an association between tHcy and CVD risk
may have been confounded by their less precise measurements of GFR.

Acknowledgments
This study was supported by grants NIDDK K23 DK067303, K23 DK02904, and
UO1 DK 35073; National Kidney Foundation of Massachusetts, Rhode Island, New
Hampshire, and Vermont, Inc; and the Dialysis Clinic Incorporated. Dr. Shlipak is
funded by R01 HL073208-01 and RO1 DK066488-01, the American Federation for
Aging Research and National Institute on Aging (Paul Beeson Scholars Program),
and the Robert Wood Johnson Foundation (Generalist Faculty Scholars Program).
This project was supported in part by the US Department of Agriculture under
cooperative agreement No. 58-1950-001. Any opinions, findings, conclusions, or
recommendations expressed in this publication are those of the author(s) and do not
necessarily reflect the view of the US Department of Agriculture. We acknowledge
the help of Frederick Van Lente, PhD, Cleveland Clinic, in splitting the frozen
samples.

Disclosures
None.

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CLINICAL PERSPECTIVE
Previous studies in the general population have suggested an association between
high homocysteine levels and adverse outcomes such as cardiovascular disease and
mortality. There is a close correlation between kidney function and levels of
homocysteine, and kidney disease is associated with high prevalence of
hyperhomocystinemia. Reduced kidney function is recognized as an independent risk
factor for cardiovascular disease and mortality. Although many of the previous
studies demonstrating an association between high homocysteine levels and outcomes
were conducted in populations at high risk for kidney disease, such as the elderly,
patients with diabetes, and patients with preexisting cardiovascular disease, few of
them adequately adjusted for kidney function. A few used imprecise measures of
kidney function such as serum creatinine or estimated creatinine clearance. In this
study, we examined the association between homocysteine levels and outcomes in a
cohort of patients with chronic kidney disease stages 3 to 4 (before reaching kidney
failure), using precise measures of glomerular filtration rate (GFR), the gold standard
for measurement of kidney function. There was a modest but nonsignificant
association between the highest tertile of homocysteine and all-cause and
cardiovascular disease mortality in unadjusted analyses. This relationship was largely
attenuated by adjustment for GFR. The observed relationship between homocysteine
and outcomes may be accounted for by its association with kidney function. Prior
studies demonstrating an association between homocysteine and cardiovascular
disease risk may have inadequately adjusted for the confounding effects of kidney
function.