cardiovascular disease ( cvd ) and diabetic nephropathy ( dn ) are the major causes of mortality in individuals with type 1 diabetes ( 13 ) , and a growing body of evidence from basic and clinical studies ( 47 ) supports the relationship between cvd and renal function in health and disease .
cvd and dn have been proposed to be manifestations of the same underlying pathology and also exist as interrelated risk factors ( 8,9 ) .
the increased mesangial matrix associated with dn is similar to the pathophysiology of coronary atherosclerosis ( 8) .
while atherosclerosis tends to remain subclinical until adulthood in individuals with type 1 diabetes ( 10 ) , adolescents with type 1 diabetes demonstrate reduced peak exercise capacity and decreased cardiac function compared with nondiabetic adolescents ( 11 ) .
although the mechanisms underlying the reduced peak exercise capacity are poorly understood , we have previously shown that insulin resistance and elevated ldl cholesterol ( ldl - c ) were associated with low peak volume of oxygen ( vo2peak ) ( 11 ) . elevated albumin - to - creatinine ratio ( acr ) and glomerular filtration rate ( gfr ) , which are early manifestations of dn , are also increasingly recognized in children and adolescents with type 1 diabetes ( 1,12 ) . increased gfr was shown to be associated with increased cardiovascular mortality in adults ( 13 ) .
little is known , however , about the possible associations between early renal health and exercise capacity in adolescents with type 1 diabetes , and whether this relationship is independent of insulin sensitivity .
accordingly , we sought to examine the associations between markers of renal health and peak exercise capacity in adolescents with type 1 diabetes .
we hypothesized that cardiopulmonary fitness would be directly associated with acr and inversely related to estimated gfr ( egfr ) in adolescents with type 1 diabetes .
pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements .
control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado .
sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels .
screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) .
type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement .
the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test .
inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects .
study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) .
pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) .
all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) .
participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study .
the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained .
a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents .
for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise .
arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise .
the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio .
subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st .
paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) .
vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) .
blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute )
was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu  m  min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) .
samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) .
the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays .
serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2  ( serum creatinine/96 )  ( serum cystatin c/1.2 )  ( weight/45 )  ( age/14 ) ( 19 ) .
this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) .
spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated .
analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) .
differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the  test .
egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr
anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups .
pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level .
pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements .
control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado .
sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels .
screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) .
type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement .
the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test .
inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects .
study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) .
pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) .
all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) .
participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study .
the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained .
a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents .
measurements were made during bicycle ergometer testing using a metabolic cart . for all bicycle tests ,
vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise .
arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise .
the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio .
subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants ,
a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st .
paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) .
vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) .
blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl .
insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu  m  min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) .
samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) .
the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays .
serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2  ( serum creatinine/96 )  ( serum cystatin c/1.2 )  ( weight/45 )  ( age/14 ) ( 19 ) .
this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) .
spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated .
analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) .
differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the  test .
egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr ( low < 5.50 , middle range 5.509.13 , and high > 9.13 mg / g ) were stratified into tertiles .
anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups .
pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level .
adolescents with type 1 diabetes had reduced peak exercise capacity compared with nondiabetic control subjects ( vo2peak 31.5  6.3 vs. 36.2  7.9 ml / kg  min , p = 0.046 ; vo2peak / lean kg 43.7  7.0 vs. 51.0  8.6 ml / lean kg  min , p = 0.007 ) , despite similar age , tanner stage , bmi , and habitual level of physical activity . when adjusting for egfr , the differences in tanner stage and sex - adjusted means for vo2peak / kg ( lsms  se 32.3  1.0 vs. 34.2  1.5 , p = 0.30 ) and vo2peak / lean kg ( lsms  se 49.0  2.1 vs. 45.0  1.4 , p = 0.09 ) between adolescents with and without type 1 diabetes lost significance .
characteristics for adolescents with type 1 diabetes data are reported as lsms  se , unless otherwise indicated .
data are reported as the median ( q25q75 ) . in adolescents with type 1 diabetes , egfr correlated strongly with vo2peak ( r = 0.55 , r = 30.22% , p = 0.002 ) and vo2peak / lean kg ( r = 0.44 , r = 19.58% , p = 0.02 ) , and remained significantly associated with vo2peak and vo2peak / lean kg after adjusting for sex and tanner stage ( tables 2 and 3 ) .
additional adjustments for insulin sensitivity , hba1c level , sbp , or ldl - c level did not attenuate the significance of the associations among egfr , vo2peak , and vo2peak / lean kg ; however , the association between egfr and vo2peak / lean kg became less significant ( p = 0.048 ; table 3 ) . in contrast , the associations between egfr and vo2peak / kg and vo2peak / lean kg in adolescent control subjects were not significant in fully adjusted models ( data not shown ) .
in contrast to egfr , lnacr did not correlate with vo2peak (   se 1.42  1.49 , p = 0.35 ) and vo2peak / lean kg (   se 2.24  1.71 , p = 0.20 ) when adjusted for sex and tanner stage .
associations between egfr and vo2peak / kg data are reported as   se ; p value .
age was not adjusted for because it is part of the bouvet egfr equation . *
adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level .
associations between egfr and vo2peak / lean kg data are reported as   se ; p value .
adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . when stratifying egfrs into tertiles , subjects in the highest egfr tertile had significantly lower vo2peak ( lsm  se 26.91  1.52 vs. 35.53  1.88 , p = 0.002 ) and vo2peak / lean kg ( lsm  se 39.65  2.07 vs. 46.18  2.55 , p = 0.048 ) adjusted for sex and tanner stage , compared with those in the lowest egfr tertile ( fig .
1 ) . stratifying acr into tertiles demonstrated no differences in vo2peak or vo2peak / lean kg among the three groups ( data not shown ) .
tanner stage and sex - adjusted means for vo2peak / kg and vo2peak / lean kg stratified by tertiles of egfr .
our major observation in this study was that in adolescents with type 1 diabetes , a disease characterized by reduced exercise capacity , egfr was independently and negatively associated with cardiopulmonary fitness , independent of insulin sensitivity and other important risk factors .
we previously demonstrated reduced peak exercise capacity , and cardiac and vascular function in otherwise healthy , nonobese adolescents with type 1 diabetes , compared with well - matched nondiabetic control subjects of similar bmi , pubertal stage , and habitual level of physical activity ( 11 ) .
furthermore , we found that insulin sensitivity correlated strongly with vo2peak in youths with type 1 diabetes , as did ldl - c level to a lesser degree ( 11 ) . in this report , we present for the first time an independent relationship between renal health and cardiopulmonary fitness that is independent of insulin sensitivity , ldl - c level , and other important covariates .
this association may represent a novel pathway of a cardiorenal connection in type 1 diabetes separate from atherosclerosis .
type 1 diabetes is associated with shortened life span , and cardiac and vascular dysfunction , independent of coronary artery disease ( 20 ) .
in fact , low fitness levels in adults with and without diabetes are associated with cvd mortality and decreased longevity ( 21,22 ) .
control of conventional risk factors such as glucose level , cholesterol level , and blood pressure is important , but does not abolish the cardiovascular risk in subjects with type 1 diabetes ( 2,23 ) . although not without controversy , the finnish diabetic nephropathy study ( 24 ) and the pittsburgh epidemiology of diabetes complications study ( 3 ) demonstrated no increase in mortality in adults with type 1 diabetes compared with nondiabetic control subjects in the absence of dn .
in contrast , the coronary artery calcification in type 1 diabetes study ( 9 ) demonstrated strong associations among egfr , acr , and progression of coronary artery calcification in adults with type 1 diabetes in the absence of established dn , suggesting that renal health may be associated with early cardiovascular pathophysiology that may take years to manifest clinically .
the specific mechanisms underlying the association between renal and cardiovascular health , however , remain unclear , but increasing evidence supports the importance of shared risk factors and pathogenic pathways , including atherosclerosis ( 6,7,11,2528 ) . reduced exercise capacity and its association with renal health highlighted in this article , taken together with the established relationship between fitness and premature cvd mortality , may provide an alternative and novel pathway explaining the premature mortality of subjects with type 1 diabetes ( 21,22 ) .
there is currently no generally accepted definition for renal hyperfiltration ( 29 ) , but it is increasingly recognized that elevated gfr is an early hemodynamic abnormality seen in diabetes that is linked with an increased risk of dn ( 1 ) .
the meta - analysis from the chronic kidney disease prognosis consortium ( 30,31 ) demonstrated significantly greater all - cause mortality and cardiovascular mortality in individuals with the highest gfr values ( > 105 ml / min/1.73 m ) .
similarly , the renal iohexol clearance survey in tromso 6 ( renis - t6 ) recently reported ( 13 ) significantly greater odds of both carotid atherosclerosis and left ventricular hypertrophy in participants in the highest gfr quartile ( > 101.2 ml / min/1.73 m ) .
it is , however , also plausible that the mechanisms underlying the association between renal health and exercise capacity in adolescents with type 1 diabetes differ from the link seen with atherosclerosis .
the mechanisms responsible for the relationship between renal health and exercise capacity are not yet clear .
adults with type 1 diabetes and elevated gfr demonstrate cardiovascular dysfunction , including increases in arterial stiffness and altered flow - mediated dilatation ( 32,33 ) .
we previously demonstrated reduced forearm blood flow in youths with type 1 diabetes ( 11 ) as evidence of vascular dysfunction , which correlated with reduced vo2peak .
moreover , alterations in arterial stiffness have also been reported to be present in youths with type 1 diabetes ( 34 ) .
elevated gfr is associated with endothelial dysfunction via cyclooxygenase-2 activity and nitric oxide synthase inhibition , and relatively higher blood pressures ( 3537 ) , both of which could directly impact exercise capacity .
elevated gfr is also associated with increased carotid intimal medial thickness and left ventricular hypertrophy in nondiabetic individuals , further suggesting that increased gfr potentially reflects a generalized change in vascular function rather than simply reflecting intrarenal abnormalities ( 13 ) .
endothelial dysfunction could also contribute to elevated gfr , thus the relationships could be bidirectional .
we did not observe a significant association between acr and peak exercise capacity , potentially because increasing evidence supports the distinction of albuminuria as a risk factor for dn and not an early phenotype of dn ( 38 ) .
furthermore , the prevalence of microalbuminuria ( n = 9 ) was low due to the young age of our population .
. however , to minimize the effect of sample size , we used careful and detailed physiological measurements , including gold standard fitness testing and hyperinsulinemic - euglycemic clamp studies .
we also chose groups similar in habitual physical activity , pubertal stage , sex , and bmi , and controlled for prestudy diet and physical activity .
another limitation to the current study included the cross - sectional design , which prevents the determination of causality , and whether the association holds true longitudinally ; for that reason , the data should be viewed as hypothesis generating .
the direction of the association between renal health and exercise capacity ( i.e. , whether renal health leads to reduced exercise capacity or vice versa ) is also difficult to determine with the available data .
results from this study may also not be generalizable to older people with type 1 diabetes or to cohorts with a different ethnic distribution . finally , we used an estimate of gfr to assess renal function ; however , to estimate the gfr we used the bouvet equation , which was shown to have the best performance in estimating gfr in children and adolescents with normal to elevated gfrs ( 18 ) .
the mean egfr in our lean nondiabetic control subjects is slightly lower than gfrs measured by insulin level in adolescents in some studies ( 39 ) , but is consistent with gfrs estimated by creatinine and cystatin c levels in adolescents in large - scale studies ( 40 ) . in conclusion ,
in adolescents with type 1 diabetes , a disease characterized by reduced peak exercise capacity compared with nondiabetic adolescents , there was a strong relationship between egfr and cardiopulmonary fitness , independent of directly measured insulin sensitivity and other important risk factors .
this observation provides further evidence for the broad interactions observed between renal and cardiovascular function in type 1 diabetes .
further research is needed to investigate the longitudinal relationships between renal and cardiopulmonary health in adolescents with type 1 diabetes , and to elucidate the specific mechanisms underlying this relationship as potential therapeutic targets .
 objectivediabetic nephropathy and cardiovascular disease are strongly related in adults with type 1 diabetes , yet little is known about this relationship in adolescents prior to the onset of detectable clinical disease . 
 we hypothesized that cardiopulmonary fitness would be directly associated with albumin - to - creatinine ratio ( acr ) and inversely related to estimated glomerular filtration rate ( egfr ) in adolescents with type 1 diabetes.research design and methodssixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects of similar pubertal stage and bmi had insulin sensitivity ( glucose infusion rate [ gir ] ) , measured by hyperinsulinemic - euglycemic clamp , and lean body mass , measured by dexa . 
 cardiopulmonary fitness was measured by cycle ergometry to obtain peak volume of oxygen ( vo2peak ) , and renal function was measured by egfr using the bouvet equation ( measuring creatinine and cystatin c levels ) and acr.resultsadolescents ( 15.5  2.2 years of age ) with type 1 diabetes ( 6.3  3.8 years diabetes duration ) had reduced vo2peak ( 31.5  6.3 vs. 36.2  7.9 ml / kg  min , p = 0.046 ) and vo2peak / lean kg ( 43.7  7.0 vs. 51.0  8.6 ml / lean kg  min , p = 0.007 ) compared with nondiabetic control subjects . 
 egfr was inversely associated with vo2peak and vo2peak / lean kg after adjusting for sex , tanner stage , gir , hba1c level , systolic blood pressure , and ldl cholesterol level (   se , vo2peak : 0.19  0.07 , p = 0.02 ; vo2peak / lean kg : 0.19  0.09 , p = 0.048 ) . 
 moreover , participants in the highest tertile for egfr had significantly lower sex- and tanner - adjusted vo2peak and vo2peak / lean kg compared with participants in the lowest tertile.conclusionsadolescents with type 1 diabetes had reduced exercise capacity , which was strongly associated with renal health , independent of insulin sensitivity . 
 future studies should examine the underlying interrelated pathophysiology in order to identify probable targets for treatment to reduce cardiovascular and renal complications . 
Introduction
Research Design and Methods
Subjects
Activity Questionnaires and Body Composition
Exercise Testing
Insulin Sensitivity
Renal Measurements
Statistical Analysis
Results
Conclusions