an estimated 10 million us adults have peripheral artery disease ( pad ) , a manifestation of systemic atherosclerosis that has been linked to reduced functional capacity and increased risk of cardiovascular morbidity and mortality .
preventable or treatable risk factors in the initiation and development of pad include cigarette smoking , type 2 diabetes , hypertension , and hypercholesterolemia , which together may account for 75% of the excess pad risk .
besides these  traditional  risk factors , kidney function has also been associated with incident pad risk in a number of studies and is recognized as a pad risk factor .
the clinical standard for the assessment of kidney function is the creatininebased estimated glomerular filtration rate ( egfr ) .
however , creatinine ( molecular weight of 113 da ) is a byproduct of muscle breakdown and tends to overestimate the egfr in individuals with lower creatinine generation due to loss of muscle mass ( such as those with pad ) .
in addition , renal tubules actively secrete creatinine , further reducing the value of creatinine to estimate gfr . both 2microglobulin , a subunit of the major histocompatibility class i complex , and cystatin c , a cysteine protease inhibitor , have been proposed as alternative markers for the detection of renal impairment . owing to their relatively low molecular weight compared with , for example , albumin ( 66 kda ) , 2microglobulin ( 12 kda ) and cystatin c ( 13 kda ) pass freely through the glomerular filtration barrier .
furthermore , these filtration markers are produced at a relatively constant rate and are neither affected by muscle mass nor subject to tubular secretion . in previous observational studies , both cystatin c and 2microglobulin have been shown to predict cardiovascular events and mortality more strongly than creatinine .
however , evidence for an association between cystatin c and future risk of pad is sparse and conflicting . also ,
although circulating 2microglobulin has previously been identified as a factor strongly linked to the presence and severity of pad in crosssectional studies , there are no studies that have prospectively examined this association .
we , therefore , investigated the associations between 3 markers of kidney function ( ie , 2microglobulin , cystatin c , and creatinine ) with future risk of the development of pad in 2 large and wellcharacterized cohorts of men and women .
the nurses health study ( nhs ) is a prospective cohort study of 121 701 female nurses aged 30 to 55 years at recruitment in 1976 .
the health professionals followup study ( hpfs ) is a prospective cohort study of 51 529 male dentists , optometrists , pharmacists , podiatrists , osteopathic physicians , and veterinarians aged 40 to 75 years at recruitment in 1986 . of these individuals ,
32 826 women provided a blood sample in 1990 and 18 224 men provided blood specimens in 1994 .
we excluded individuals who had a history of cardiovascular disease , including myocardial infarction ; surgical / percutaneous revascularization of the coronary , carotid , or peripheral beds ; confirmed pad ; stroke ; and transient ischemic attack .
the casecontrol analytic datasets include all incident symptomatic pad cases , for a total of 144 cases and 432 controls in the nhs and 143 cases and 429 controls in the hpfs .
controls were matched 3:1 to cases on age , race , smoking history , month of blood draw ( within 3 months ) , and fasting status , following the nested casecontrol design described by prentice and breslow .
we selected controls at random , conditional on the matching factors , from participants free of cardiovascular diseases at the time the incident case occurred ( risk set sampling ) .
we restricted all cases and controls to those free of cardiovascular diseases at baseline to ensure identification of only incident cases .
this included angina , myocardial infarction , surgical / percutaneous revascularization of the coronary , carotid , or peripheral beds , claudication , stroke , and transient ischemic attack , which are all assessed biennially .
because older age categories had fewer participants , we relaxed the age match range yearbyyear if necessary to a maximum of within 3 years .
the institutional review boards of the brigham and women 's hospital , harvard school of public health , and beth israel deaconess medical center approved the study protocol
blood samples were shipped overnight with a cold pack to the central laboratory , centrifuged on arrival , aliquotted , and stored in liquid nitrogen at 130c to 196c .
nhs specimens were anticoagulated with heparin , and hpfs were anticoagulated with edta . of all samples , 97% of those from the nhs and 95% of those from the hpfs
were received within 24 hours ; cystatin c and creatinine levels are known to remain stable in whole blood even when stored at room temperature up to 48 hours before separation .
all analyses were performed in a laboratory certified by the centers for disease control and prevention / national heart , lung , and blood institute lipid standardization program with commercially available analytic systems .
measurements of 2microglobulin , cystatin c , and creatinine were performed on the roche p modular system ( roche diagnostics ) via an immunoturbidimetric technique ( 2microglobulin and cystatin c ) or enzymatic method ( creatinine ) , using reagents and calibrators from roche ( 2microglobulin and creatinine ) or from genzyme ( cystatin c ) .
interassay coefficients of variation for 2microglobulin , cystatin c , and creatinine were 5.9% , 3.0% , and 2.2% for nhs and 5.8% , 8.1% , and 6.5% for hpfs , respectively .
the determination of highdensity lipoprotein cholesterol , lowdensity lipoprotein cholesterol , highsensitivity creactive protein ( crp ) , triglycerides , and hemoglobin a1c has been described in greater detail elsewhere .
participants reported the occurrence of diagnosed medical conditions , including claudication and revascularization for arterial disease of the leg , during the previous 2 years on biennial mailed questionnaires .
we collected medical records from treating physicians and hospitals for those who reported either condition .
we defined pad as arterial disease below the aortic bifurcation ( ie , excluding abdominal aortic aneurysm and renal artery stenosis ) . confirmed pad required 1 of the following ( in order of severity / certainty ) in the medical record : ( 1 ) report of amputation , bypass , or other revascularization procedure for occlusive artery disease , ( 2 ) angiogram showing 50% stenosis of 1 artery with congruent symptoms in the ipsilateral limb , ( 3 ) anklebrachial index < 0.90 at rest , or ( 4 ) physician 's diagnosis . questionnaires provided information about anthropometric data and lifestyle , including weight , detailed information about smoking , parental history of myocardial infarction , alcohol consumption , physical activity per week , medical history concerning medication use , and selfreported history of diabetes , hypertension , and hypercholesterolemia .
participants reported past smoking , time since quitting , and the average number of cigarettes smoked per day .
packyears were calculated as years of smoking multiplied by the average number of packs smoked per day and updated biennially .
we calculated body mass index ( bmi ) by dividing weight ( kg ) by squared height ( m ) .
physical activity was expressed as metabolic equivalent taskhours based on selfreported types and durations of activities over the previous year .
previous studies demonstrated that the validity and reproducibility of all these selfreported measures are high .
we calculated the egfr based both on the creatininebased chronic kidney disease epidemiology collaboration ( egfrckdepi ) equation and on a more recent , combined creatinine 
continuous data are summarized as either meansd for normally distributed variables or median and interquartile range for nonnormally distributed variables .
we used generalized linear mixed models and cochran  mantel  haenszel tests to compare continuous variables and categorical variables by casecontrol status , respectively , accounting for clustering by matching status .
associations between 2microglobulin , cystatin c , and creatinine and pad risk factors were examined using ageadjusted spearman partial correlation coefficients . to examine the prospective association of 2microglobulin , cystatin c , and creatinine with subsequent pad risk , we treated these markers as both a categorical ( in quartiles ) and a continuous ( per 1sd increase in logtransformed marker ) variable .
logtransformation of the markers led to a superior model fit . to test for a linear trend in the categorical analysis
we also examined the possibility of nonlinear associations between the 3 biomarkers and pad nonparametrically with restricted cubic splines . in tests for nonlinearity
, we used the likelihood ratio test , comparing the model with only the linear term to the model with the linear and the cubic spline terms .
we used conditional logistic regression to estimate the odds ratio ( or ) and 95% ci for pad associated with increasing concentrations of these markers . because the controls were selected using risk set sampling , the ors derived from conditional logistic regression provide unbiased estimates of the incidence rate ratio as a measure of relative risk ( rr ) .
we included covariables in our models as linear variables if appropriate or as categorical if they were discrete or their association with pad was nonlinear .
analyses that defined hypertension based on either a physician 's diagnosis or a patient 's antihypertensive medication use ( thiazides , blockers , calcium channel blockers , and angiotensinconverting enzyme or angiotensin ii receptor antagonists ) yielded similar results . to pool the rr estimates for women and men , we used metaanalyses with fixed effects or random effects when the test for heterogeneity was significant .
we tested interactions with crossproduct terms between ( continuous ) logtransformed concentrations of the kidney function markers and selected variables in multivariableadjusted conditional or unconditional logistic regression models with additional adjustment for the matching factors .
all analyses were performed using sas statistical software ( version 9.3 ; sas institute ) .
the nurses health study ( nhs ) is a prospective cohort study of 121 701 female nurses aged 30 to 55 years at recruitment in 1976 .
the health professionals followup study ( hpfs ) is a prospective cohort study of 51 529 male dentists , optometrists , pharmacists , podiatrists , osteopathic physicians , and veterinarians aged 40 to 75 years at recruitment in 1986 . of these individuals ,
32 826 women provided a blood sample in 1990 and 18 224 men provided blood specimens in 1994 .
we excluded individuals who had a history of cardiovascular disease , including myocardial infarction ; surgical / percutaneous revascularization of the coronary , carotid , or peripheral beds ; confirmed pad ; stroke ; and transient ischemic attack .
the casecontrol analytic datasets include all incident symptomatic pad cases , for a total of 144 cases and 432 controls in the nhs and 143 cases and 429 controls in the hpfs .
controls were matched 3:1 to cases on age , race , smoking history , month of blood draw ( within 3 months ) , and fasting status , following the nested casecontrol design described by prentice and breslow .
we selected controls at random , conditional on the matching factors , from participants free of cardiovascular diseases at the time the incident case occurred ( risk set sampling ) .
we restricted all cases and controls to those free of cardiovascular diseases at baseline to ensure identification of only incident cases .
this included angina , myocardial infarction , surgical / percutaneous revascularization of the coronary , carotid , or peripheral beds , claudication , stroke , and transient ischemic attack , which are all assessed biennially .
because older age categories had fewer participants , we relaxed the age match range yearbyyear if necessary to a maximum of within 3 years .
the institutional review boards of the brigham and women 's hospital , harvard school of public health , and beth israel deaconess medical center approved the study protocol
blood samples were shipped overnight with a cold pack to the central laboratory , centrifuged on arrival , aliquotted , and stored in liquid nitrogen at 130c to 196c .
nhs specimens were anticoagulated with heparin , and hpfs were anticoagulated with edta . of all samples , 97% of those from the nhs and 95% of those from the hpfs
were received within 24 hours ; cystatin c and creatinine levels are known to remain stable in whole blood even when stored at room temperature up to 48 hours before separation .
all analyses were performed in a laboratory certified by the centers for disease control and prevention / national heart , lung , and blood institute lipid standardization program with commercially available analytic systems .
measurements of 2microglobulin , cystatin c , and creatinine were performed on the roche p modular system ( roche diagnostics ) via an immunoturbidimetric technique ( 2microglobulin and cystatin c ) or enzymatic method ( creatinine ) , using reagents and calibrators from roche ( 2microglobulin and creatinine ) or from genzyme ( cystatin c ) .
interassay coefficients of variation for 2microglobulin , cystatin c , and creatinine were 5.9% , 3.0% , and 2.2% for nhs and 5.8% , 8.1% , and 6.5% for hpfs , respectively .
the determination of highdensity lipoprotein cholesterol , lowdensity lipoprotein cholesterol , highsensitivity creactive protein ( crp ) , triglycerides , and hemoglobin a1c has been described in greater detail elsewhere .
participants reported the occurrence of diagnosed medical conditions , including claudication and revascularization for arterial disease of the leg , during the previous 2 years on biennial mailed questionnaires .
we collected medical records from treating physicians and hospitals for those who reported either condition .
we defined pad as arterial disease below the aortic bifurcation ( ie , excluding abdominal aortic aneurysm and renal artery stenosis ) . confirmed pad required 1 of the following ( in order of severity / certainty ) in the medical record : ( 1 ) report of amputation , bypass , or other revascularization procedure for occlusive artery disease , ( 2 ) angiogram showing 50% stenosis of 1 artery with congruent symptoms in the ipsilateral limb , ( 3 ) anklebrachial index < 0.90 at rest , or ( 4 ) physician 's diagnosis .
questionnaires provided information about anthropometric data and lifestyle , including weight , detailed information about smoking , parental history of myocardial infarction , alcohol consumption , physical activity per week , medical history concerning medication use , and selfreported history of diabetes , hypertension , and hypercholesterolemia .
participants reported past smoking , time since quitting , and the average number of cigarettes smoked per day .
packyears were calculated as years of smoking multiplied by the average number of packs smoked per day and updated biennially .
we calculated body mass index ( bmi ) by dividing weight ( kg ) by squared height ( m ) .
physical activity was expressed as metabolic equivalent taskhours based on selfreported types and durations of activities over the previous year .
previous studies demonstrated that the validity and reproducibility of all these selfreported measures are high .
we calculated the egfr based both on the creatininebased chronic kidney disease epidemiology collaboration ( egfrckdepi ) equation and on a more recent , combined creatinine 
continuous data are summarized as either meansd for normally distributed variables or median and interquartile range for nonnormally distributed variables .
we used generalized linear mixed models and cochran  mantel  haenszel tests to compare continuous variables and categorical variables by casecontrol status , respectively , accounting for clustering by matching status .
associations between 2microglobulin , cystatin c , and creatinine and pad risk factors were examined using ageadjusted spearman partial correlation coefficients . to examine the prospective association of 2microglobulin , cystatin c , and creatinine with subsequent pad risk , we treated these markers as both a categorical ( in quartiles ) and a continuous ( per 1sd increase in logtransformed marker ) variable .
logtransformation of the markers led to a superior model fit . to test for a linear trend in the categorical analysis
we also examined the possibility of nonlinear associations between the 3 biomarkers and pad nonparametrically with restricted cubic splines . in tests for nonlinearity
, we used the likelihood ratio test , comparing the model with only the linear term to the model with the linear and the cubic spline terms .
we used conditional logistic regression to estimate the odds ratio ( or ) and 95% ci for pad associated with increasing concentrations of these markers . because the controls were selected using risk set sampling , the ors derived from conditional logistic regression provide unbiased estimates of the incidence rate ratio as a measure of relative risk ( rr ) .
we included covariables in our models as linear variables if appropriate or as categorical if they were discrete or their association with pad was nonlinear .
analyses that defined hypertension based on either a physician 's diagnosis or a patient 's antihypertensive medication use ( thiazides , blockers , calcium channel blockers , and angiotensinconverting enzyme or angiotensin ii receptor antagonists ) yielded similar results . to pool the rr estimates for women and men , we used metaanalyses with fixed effects or random effects when the test for heterogeneity was significant .
we tested interactions with crossproduct terms between ( continuous ) logtransformed concentrations of the kidney function markers and selected variables in multivariableadjusted conditional or unconditional logistic regression models with additional adjustment for the matching factors .
all analyses were performed using sas statistical software ( version 9.3 ; sas institute ) .
baseline characteristics of pad cases and controls are shown in table 1 . as expected , both female and male cases had higher levels of crp , triglycerides , and hemoglobin a1c and were more likely to have a history of diabetes , hypertension , or hypercholesterolemia than were matched controls . in men , 2microglobulin and cystatin c levels
were significantly higher among cases than among controls , but in women , 2microglobulin and cystatin c levels did not differ between cases and controls .
creatinine was higher in male cases but lower in female cases compared to their matched controls .
based on egfr categories of the kidney disease : improving global outcomes ( kdigo ) clinical practice guideline , 66% of the women and 50% of the men had a normal or high kidney function ( egfrckdepi 90 ml min per 1.73 m ) and 33% and 46% , respectively , had a mildly reduced kidney function ( egfrckdepi between 60 and 89 ml min per 1.73 m ) .
baseline characteristics of women and men who developed symptomatic peripheral artery disease during followup ( cases ) and matched controls matching factors , besides age , race , and smoking status , included date of blood draw and fasting status .
data are expressed as mean ( sd ) , median ( interquartile range ) , or number ( percentage ) .
p values are derived from mixed linear models for continuous variables and cochran  mantel  haenszel tests for categorical variables controlling for matched sets .
egfr indicates estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; ldl , lowdensity lipoprotein ; meth , metabolic equivalent hours .
cystatin c and 2microglobulin were strongly correlated with each other ( =0.74 , p<0.001 for women ; =0.77 , p<0.001 for men ) ( table 2 ) and highly correlated with creatinine , although not as strongly as with each other .
both 2microglobulin and cystatin c were correlated with age and modestly with bmi , crp , and triglycerides but not with packyears of smoking in both men and women .
besides an inverse correlation with packyears of smoking , no consistent correlations were observed between creatinine and cardiovascular risk factors in men and women .
both unadjusted spearman 's rank correlation coefficients and pearson productmoment correlation coefficients are shown in table 3 .
spearman partial correlation coefficients between 2microglobulin , cystatin c , and creatinine and selected cardiovascular risk factors among 431 control women and 429 control men correlations are ageadjusted except age .
hdl indicates highdensity lipoprotein ; ldl , lowdensity lipoprotein . * p<0.05 , p<0.01 , p<0.001 .
spearman 's rank correlation coefficients and pearson productmoment correlation coefficients between 2microglobulin , cystatin c , and creatinine and selected cardiovascular risk factors among 431 control women and 429 control men hdl indicates highdensity lipoprotein ; ldl , lowdensity lipoprotein . *
p<0.05 , p<0.01 , p<0.001 . during a median followup of 15 years ( interquartile range 11 to 17 years )
, pad was detected in 144 women , and during a median followup of 7 years ( interquartile range 4 to 10 years ) pad was detected in 143 men .
of the 144 pad cases among women , 74 ( 51% ) cases were confirmed by medical records indicating surgery or revascularization procedures , 24 ( 17% ) cases by angiogram , 39 ( 27% ) cases by anklebrachial index , and 7 ( 5% ) cases by physician diagnosis .
of the 143 pad cases among men , medical reports of amputation , bypass , or other revascularization procedure confirmed 87 ( 61% ) pad cases ; angiogram or doppler ultrasound , 15 ( 10% ) cases ; anklebrachial index < 0.90 , ( 16% ) 23 cases ; and physician diagnosis , 18 ( 13% ) cases . because there was no evidence for nonlinear associations between markers of kidney function and future risk of pad , we conducted analyses per 1sd increase in logtransformed markers .
higher circulating levels of 2microglobulin , cystatin c , and creatinine were associated with increased risk of symptomatic pad in men but not in women ( table 4 ) .
rrs among women tended to be higher for 2microglobulin than the other 2 measures , and the difference between men and women was significant for cystatin c but not for 2microglobulin ( figure ) . although expected given the limited variation in age , sex , and ethnicity ( the main determinants of egfrckdepi besides creatinine ) between cases and matched controls , the associations with risk of pad were similar if kidney function was estimated using the egfrckdepi formula rather than using circulating creatinine ( table 5 ) .
associations were also similar when we modeled the inverse of 2microglobulin or cystatin c ( table 5 ) , as surrogate of egfr , compared with the logtransformed values of 2microglobulin or cystatin c , respectively .
relative risks for peripheral artery disease according to 2microglobulin , cystatin c , and creatinine in women and men hdl indicates highdensity lipoprotein ; ldl , lowdensity lipoprotein .
matching factors included age , race , smoking status , month of blood draw , and fasting status .
adjusted for creactive protein , hdl cholesterol , ldl cholesterol , hemoglobin a1c , triglycerides , packyears of smoking , type 2 diabetes , hypertension , hypercholesterolemia , parental history of myocardial infarction before age 60 years , body mass index , physical activity , and alcohol consumption .
relative risks for peripheral artery disease according to egfrckdepi , egfrcreatcys , inverse of 2microglobulin , and inverse of cystatin c in women and men ckdepi indicates chronic kidney disease epidemiology collaboration ; creatcys , creatinine  cystatin c ; egfr , estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; ldl , lowdensity lipoprotein .
p for trend based on median of quartiles . matching factors included age , race , smoking status , month of blood draw , and fasting status .
adjusted for hdl cholesterol , ldl cholesterol , creactive protein , hemoglobin a1c , triglycerides , packyears of smoking , type 2 diabetes , hypertension , hypercholesterolemia , parental history of myocardial infarction before age 60 years , body mass index , physical activity , and alcohol consumption .
adjusted relative risks ( 95% ci ) for peripheral artery disease per 1sd increment in 2microglobulin , cystatin c , and creatinine .
relative risks are adjusted for creactive protein ; hdl and ldl cholesterol ; hemoglobin a1c ; triglycerides ; creatinine ( except creatinine , which is adjusted for 2microglobulin instead ) ; packyears of smoking ; history of type 2 diabetes , hypertension , and hypercholesterolemia ; parental history of myocardial infarction before age 60 years ; body mass index ; physical activity ; and alcohol consumption .
matching factors are age , race , smoking status , month of blood draw , and fasting status . to calculate the pooled estimates , we used metaanalyses with fixed effects or random effects in case of significant heterogeneity ( p<0.05 ) .
hdl indicates highdensity lipoprotein ; hpfs , health professionals followup study ; ldl , lowdensity lipoprotein ; nhs , nurses health study .
although the rrs increased slightly after further accounting for circulating creatinine , 2microglobulin and cystatin c remained unassociated with pad risk among women ( figure ) . for comparison ,
the corresponding multivariableadjusted rr for each 1sd increment in crp was 1.48 ( 95% ci 1.14 to 1.94 ) in women and 1.39 ( 95% ci 1.08 to 1.79 ) in men .
in exploratory analyses when models were mutually adjusted for both 2microglobulin and cystatin c , the rrs per each 1sd increment were 1.44 ( 95% ci 0.94 to 2.22 ) for 2microglobulin and 0.73 ( 0.48 to 1.12 ) for cystatin c in women and 1.31 ( 95% ci , 0.84 to 2.03 ) for 2microglobulin and 1.26 ( 0.77 to 2.06 ) for cystatin c in men . in further analyses , we found no interactions of 2microglobulin or cystatin c with creatinine , conventional cardiovascular risk factors , or duration of followup in women and men ( table 6 ) .
p for interaction values between 2microglobulin and cystatin c and selected risk factors in the association with symptomatic pad hdl indicates highdensity lipoprotein ; ldl , lowdensity lipoprotein . in pooled analyses of men and women ,
the rrs per each 1sd increment were 1.31 ( 95% ci 1.04 to 1.64 ) for 2microglobulin and 1.19 ( 95% ci 0.74 to 1.93 ) for cystatin c in multivariableadjusted models including adjustment for creatinine ( figure ) .
when we adjusted for egfrckdepi instead of circulating creatinine , the pooled rrs for 2microglobulin ( rr 1.28 , 95% ci 1.02 to 1.60 ) and cystatin c ( rr 1.13 , 95% ci 0.89 to 1.43 ) remained essentially the same .
similar results were obtained for 2microglobulin ( rr 1.33 , 95% ci 1.05 to 1.70 ) and for cystatin c ( rr 1.20 , 95% ci 0.73 to 1.96 ) when we excluded subjects with an egfrckdepi < 60 ml min per 1.73 m. the pooled multivariableadjusted estimate for creatinine ( rr 1.09 , 95% ci 0.73 to 1.64 ) decreased further after additionally accounting for either 2microglobulin ( rr 0.94 , 95% ci 0.77 to 1.15 ) or cystatin c ( rr 0.98 , 95% ci 0.79 to 1.21 ) .
in 2 large nested casecontrol studies , circulating 2microglobulin and cystatin c and , to a lesser extent , creatinine were associated with incident pad in men , although creatinine was no longer associated with risk after adjustment for either 2microglobulin or cystatin c. circulating cystatin c and creatinine were completely unassociated with risk of pad among women . in pooled analyses among men and women ,
the association of 2microglobulin with pad risk remained after accounting for circulating creatinine and for other established cardiovascular risk factors .
moreover , 2microglobulin was also associated with pad risk in individuals with egfrs above the clinical cutoff for chronic kidney disease ( egfrckdepi 60 ml min per 1.73 m ) , extending the possible range over which kidney function is related to risk .
using highthroughput proteomic profiling , a crosssectional study that included both men and women demonstrated that 2microglobulin correlated strongly with pad severity , independent of other risk factors .
our present study complements and extends this finding by showing a prospective association between 2microglobulin and future risk of symptomatic pad among subjects free of diagnosed cardiovascular or kidney disease . this increased risk of systemic atherosclerosis concords with positive associations between 2microglobulin and risk of coronary heart disease in postmenopausal women and in the general population . among japanese
our data indicate that the association of 2microglobulin with incident symptomatic pad is largely independent of circulating creatinine or creatininebased egfr .
this may suggest that 2microglobulin represents kidney function that is not well captured by circulating creatinine .
indeed , 2microglobulin is correlated more strongly with gfr measured by inulin than is serum creatinine .
another possibility that can not be excluded by the present data is that 2microglobulin reflects other , nongfr determinants of pad risk . given the modest correlations with crp , 2microglobulin may be a marker of local or systemic inflammation , but the observed association with pad remained after adjustment for crp .
in addition , it is possible that circulating 2microglobulin reflects amyloid deposition and aggregation in the vessel wall as part of the atherosclerotic process . perhaps most intriguingly , the repetitive ischemia
 reperfusion injury of the limbs that occurs as pad develops may promote 2microglobulin shedding from endothelial cell surfaces due to its noncovalent binding with the major histocompatibility complex class i chain without direct attachment to the cell membrane .
few prospective studies have investigated the association of cystatin c with risk of incident pad among apparently healthy subjects . in > 4000 communitydwelling elderly subjects of the cardiovascular health study , cystatin c was associated with an increased risk of lower extremity pad procedures ( bypass surgery , angioplasty , or amputation ) only among subjects in the highest quintile ( threshold for cystatin c >
1.27 mg / l ) . in a nested casecontrol study with 133 symptomatic pad cases in the physicians health study ,
no evidence was found for an increased risk with elevated cystatin c levels . however , the 95th cutoff percentile for cystatin c based on the distribution of the controls was < 1.08 mg / l .
although we did not find evidence for a nonlinear association , the higher pad risk in our study among men appeared to be statistically significant when comparing the first ( < 0.83 mg / l ) cystatin c quartile with the fourth ( > 1.10 mg / l ) , but no such association was observed in women .
these results suggest that only particularly high levels of cystatin c may be associated with increased risk of pad , although differences between studies hamper direct comparisons .
first , determination of 2microglobulin is already widely available and routinely measured in clinical settings for hematological and lymphoproliferative diseases ( eg , multiple myeloma and lymphoma ) .
measurement of 2microglobulin for the early detection of pad may thus be easily incorporated into routine care cardiovascular risk prediction . of note , measurement of cystatin c
second , these findings suggest that ( preservation of ) kidney function may be an additional target to reduce the risk of pad . it can not be excluded that lower kidney function may be a consequence rather than a cause of atherosclerosis , particularly if renal arteries occlude in parallel with arteries of the lower extremities .
the observation , however , that kidney function is associated with pad risk even after extensive adjustment for cardiovascular risk factors suggests a role for kidney function itself as pad risk promoter .
relative risks of all 3 markers of kidney function tended to be higher in the hpfs ( men ) than in the nhs ( women ) . besides sex
first , plasma was stored in heparin tubes for nhs samples versus in edta tubes for hpfs samples , which may have created differential measurement of these markers .
this is unlikely , though , because baseline correlations between these markers with each other and with other confounding variables were almost exactly the same for the women in nhs as for the men in hpfs . also , the coefficients of variation of the measurements for all 3 markers were relatively low for both hpfs and nhs .
it is also unlikely that differences in end point definitions may explain the stronger associations because definitions were similar between the 2 studies and positive associations of comparable magnitude between wellknown pad risk factors , such as for crp and lipoprotein(a ) , were observed in both studies .
third , egfr was systematically higher in women , and in these analyses , only 2microglobulin tended to be positively associated with risk .
this suggests that it may provide more useful discrimination at high levels of egfr than other markers of kidney function .
nevertheless , the stronger associations among men could reflect differences in pad pathophysiology between sexes , although a recent metaanalysis indicated that there is no reason to assume that kidney function is a less important risk factor for other forms of cardiovascular disease in women than in men .
future studies should address whether markers of kidney function universally perform better in men or whether 2microglobulin is a superior marker to assess risk of the development of pad .
strengths of the current study are the large number of biochemical and traditional cardiovascular risk factors for which we could adjust in our analyses , the prospective design , the longterm followup , the use of risk set sampling to select controls , the homogeneity of the 2 study populations , and the use of adjudicated events with a pronounced atherosclerotic origin . besides these strengths , several potential limitations warrant to be mentioned .
subclinical or asymptomatic pad , which may have otherwise been detected through an abnormal anklebrachial index or pulse examination , is likely to have been missed .
however , outcomes included in this analysis were confirmed by medical records , reducing the likelihood of falsepositive cases at the expense of a risk of a falsely low incidence rate .
second , participants in both cohorts were predominantly white ; extrapolation of our results to other race / ethnicity subgroups should therefore be done with caution .
blacks , for instance , have higher circulating creatinine but not 2microglobulin or cystatin c levels and are at higher risk of the development of pad .
therefore , it is impossible to know if the differences in significance between men and women were due to differences in study design or true sex differences . fourth , although 2microglobulin and cystatin c clearly outperformed creatinine in assessing pad risk , particularly among the men , we can not directly determine whether that reflects better estimation of gfr or simply nonrenal effects .
fifth , although crp was strongly associated with risk in both cohorts , we adjusted for only crp , which may be an imperfect measure of inflammation .
finally , we did not have data on albuminuria , which could potentially have captured other aspects of chronic kidney disease .
the present study identified an association between circulating 2microglobulin and future risk of symptomatic pad in pooled analyses of men and women . when the 2 sexes were studied separately , 2microglobulin and cystatin c were associated with risk of symptomatic pad in men but not in women .
the associations of these emerging markers of kidney function with pad risk appeared to be independent of circulating creatinine or creatininebased egfr , which is the current clinical standard for the assessment of kidney function and wellknown cardiovascular risk factors .
cystatin c and particularly 2microglobulin may have greater potential than creatinine for the early detection of systemic atherosclerotic diseases .
appendix beta2-microglobulin , cystatin c , and creatinine and risk of symptomatic peripheral artery disease .
 background2microglobulin and cystatin c may have advantages over creatinine in assessing risk associated with kidney function . 
 we therefore investigated whether emerging filtration markers , 2microglobulin and cystatin c , are prospectively associated with risk of the development of peripheral artery disease ( pad).methods and resultswe conducted nested casecontrol studies among women within the nurses health study ( 19902010 ) and among men within the health professionals followup study ( 19942008 ) with the use of archived blood samples collected before pad diagnosis . during followup , symptomatic pad was confirmed in 144 women and 143 men . 
 controls were matched 3:1 based on age , race , smoking status , fasting status , and date of blood sampling . 
 conditional logistic regression models were used to estimate relative risks ( rrs ) and were adjusted for plasma creatinine and cardiovascular risk factors . in women , 
 the rrs ( 95% ci ) per 1sd ) increment were 1.16 ( 0.85 to 1.58 ) for 2microglobulin and 0.94 ( 0.69 to 1.28 ) for cystatin c. corresponding rrs in men were 1.50 ( 1.08 to 2.09 ) for 2microglobulin and 1.54 ( 1.07 to 2.22 ) for cystatin c. there was no association between creatinine and pad risk in women , whereas the association in men ( rr 1.41 , 95% ci 1.10 to 1.81 ) disappeared after adjustment for either 2microglobulin or cystatin c. in pooled analyses of men and women , only 2microglobulin was associated with pad risk ( rr 1.31 , 95% ci 1.04 to 1.64).conclusionsin pooled analyses , 2microglobulin was associated with an increased risk of symptomatic pad ; a similar association with cystatin c was observed only in men . 
 the findings suggest that 2microglobulin may capture the atherosclerosispromoting or atherosclerosisrelated elements of kidney dysfunction better than creatinine . 