the study subjects were type 2 diabetic patients enrolled in the diabetes management through an integrated delivery system ( dmids ) project ( nct00288678 , clinicaltrials.gov ) ( 6 ) . the patient recruitment period was between august 2003 and december 2005 , with the project carried out through december 2009 . all type 2 diabetic subjects were eligible to be recruited unless they met any of the following exclusion criteria : 1 ) those aged < 30 or > 70 years ; 2 ) type 1 diabetic patients ; 3 ) pregnant women ; and 4 ) those who had major diabetes complications including leg amputation , uremia , or hospitalization in the previous year as the result of acute myocardial infarction , heart failure , or stroke . of these enrollees , 22 subjects receiving insulin injection , 323 with a urine albumin - to - creatinine ratio ( acr ) 30 mg / g in at least one of the first two urine tests , and 126 subjects with missing fasting insulin data at baseline were excluded . the remaining 738 subjects , who had normoalbuminuria ( acr < 30 mg / g in the first two consecutive urine tests ) , were selected for further investigation . written informed consent was obtained from all enrollees . the institutional review board at the national health research institutes reviewed and approved this study . fasting ( overnight for at least 8 h ) venous blood and spot urine specimens were collected every 6 months . glycated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography ( variant ii ; bio - rad laboratories , hercules , ca ) . fasting glucose , triglycerides , and hdl cholesterol were measured by an automatic analyzer ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) . insulin was measured by a chemiluminescent immunometric assay ( immulite 1000 ; diagnostic products , los angeles , ca ) . urinary albumin was measured by the immunoturbidimetric method ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) . dipstick urinalysis was performed by an automated chemical analyzer ( clinitek 500 ; bayer , elkhart , in ) . all blood and urine samples were kept in temperature - proof containers at 2c to 8c , transported by express delivery to a central laboratory , and measured within 8 h. waist circumference was measured at the level of the midpoint between the lowest rib and the iliac crest . a mercury sphygmomanometer was used to measure blood pressure after subjects sat for 5 min . blood pressure was measured three times separated by 1 min ; the mean of these three measurements was recorded . we used the homa of insulin resistance ( homa - ir ) to assess insulin resistance ( homa - ir = { insulin [ mu / l ]  fasting glucose [ mmol / l]}/22.5 ) ( 7 ) . those who had acr 30 mg / g in two consecutive urine tests were defined as having microalbuminuria . the urine samples were excluded from analysis if microscopic urinalysis showed erythrocytes > 5/high - power field ( hpf ) , white blood cells > 5/hpf , epithelial cells > 5/hpf , and appearance of casts or bacteria . those who had smoked fewer than 100 cigarettes in their lifetime were defined as nonsmokers . for those who had smoked > 100 cigarettes , ex - smokers were defined as having stopped smoking completely at least 1 month prior to recruitment and current smokers as having a daily or occasional smoking habit at the time of recruitment . data were expressed as mean  sd for continuous variables , or as counts and proportions for categorical variables . student t tests and  analyses were used for continuous and categorical variables , respectively . the incidence rate of microalbuminuria in the follow - up period was estimated by the number of observed new microalbuminuria cases per 1,000 person - years . the person - years were calculated as the time elapsed from the date of recruitment until the date of death , microalbuminuria development , or the end of follow - up , whichever came first . the calculation of a 95% ci for the incidence rate was based on the assumption that the observed incident cases followed a poisson distribution . we estimated the incidence rate of microalbuminuria in different homa - ir quartiles not only for all study subjects but also for different subgroups according to their glycemic control ( hba1c 8 vs. > kaplan - meier analyses and univariate cox proportional hazards models were used to explore the association between homa - ir and microalbuminuria development . the predictors used in cox proportional hazards models  including baseline demographic and metabolic profiles ( age at diabetes onset , sex , education , diabetes duration , smoking status , bmi , waist circumference , triglycerides and hdl cholesterol , urine acr , hba1c , and hypertension status ) . multivariable cox proportional hazards modeling was used to determine the independent effects of homa - ir on microalbuminuria development . observations were censored at the end of the study or the date that patients died or dropped out of the study , whichever occurred first . results were expressed as hazard ratios ( hrs ) and 95% cis compared with the reference group . to eliminate residual confounding effects , for the survival analysis they were those who met the following criteria at baseline : no acei ( angiotensin - converting enzyme inhibitor ) and arb ( angiotensin ii receptor blocker ) use , bmi < 24 kg / m , triglycerides < 150 mg / dl , waist circumference < 80 cm ( female ) or < 90 cm ( male ) , hdl cholesterol > 40 mg / dl ( male ) or > 50 mg / dl ( female ) , and blood pressure < 130/80 mmhg . the proportional hazards assumption , the constant hr over time , was evaluated by comparing estimated log - log survival curves for all covariates . all assessed log - log survival plots graphically showed two parallel lines , indicating no violation of the assumption . a test for trend analyses were performed with sas software , version 9.1 ( sas institute , cary , nc ) . fasting ( overnight for at least 8 h ) venous blood and spot urine specimens were collected every 6 months . glycated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography ( variant ii ; bio - rad laboratories , hercules , ca ) . fasting glucose , triglycerides , and hdl cholesterol were measured by an automatic analyzer ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) . insulin was measured by a chemiluminescent immunometric assay ( immulite 1000 ; diagnostic products , los angeles , ca ) . urinary albumin was measured by the immunoturbidimetric method ( hitachi 7060 ; hitachi high technologies , tokyo , japan ) . dipstick urinalysis was performed by an automated chemical analyzer ( clinitek 500 ; bayer , elkhart , in ) . all blood and urine samples were kept in temperature - proof containers at 2c to 8c , transported by express delivery to a central laboratory , and measured within 8 h. waist circumference was measured at the level of the midpoint between the lowest rib and the iliac crest . a mercury sphygmomanometer was used to measure blood pressure after subjects sat for 5 min . blood pressure was measured three times separated by 1 min ; the mean of these three measurements was recorded . we used the homa of insulin resistance ( homa - ir ) to assess insulin resistance ( homa - ir = { insulin [ mu / l ]  fasting glucose [ mmol / l]}/22.5 ) ( 7 ) . those who had acr 30 mg / g in two consecutive urine tests were defined as having microalbuminuria . the urine samples were excluded from analysis if microscopic urinalysis showed erythrocytes > 5/high - power field ( hpf ) , white blood cells > 5/hpf , epithelial cells > 5/hpf , and appearance of casts or bacteria . those who had smoked fewer than 100 cigarettes in their lifetime were defined as nonsmokers . for those who had smoked > 100 cigarettes , ex - smokers were defined as having stopped smoking completely at least 1 month prior to recruitment and current smokers as having a daily or occasional smoking habit at the time of recruitment . data were expressed as mean  sd for continuous variables , or as counts and proportions for categorical variables . student t tests and  analyses were used for continuous and categorical variables , respectively . the incidence rate of microalbuminuria in the follow - up period was estimated by the number of observed new microalbuminuria cases per 1,000 person - years . the person - years were calculated as the time elapsed from the date of recruitment until the date of death , microalbuminuria development , or the end of follow - up , whichever came first . the calculation of a 95% ci for the incidence rate was based on the assumption that the observed incident cases followed a poisson distribution . we estimated the incidence rate of microalbuminuria in different homa - ir quartiles not only for all study subjects but also for different subgroups according to their glycemic control ( hba1c 8 vs. > kaplan - meier analyses and univariate cox proportional hazards models were used to explore the association between homa - ir and microalbuminuria development . the predictors used in cox proportional hazards models  including baseline demographic and metabolic profiles ( age at diabetes onset , sex , education , diabetes duration , smoking status , bmi , waist circumference , triglycerides and hdl cholesterol , urine acr , hba1c , and hypertension status ) . multivariable cox proportional hazards modeling was used to determine the independent effects of homa - ir on microalbuminuria development . observations were censored at the end of the study or the date that patients died or dropped out of the study , whichever occurred first . results were expressed as hazard ratios ( hrs ) and 95% cis compared with the reference group . to eliminate residual confounding effects , for the survival analysis they were those who met the following criteria at baseline : no acei ( angiotensin - converting enzyme inhibitor ) and arb ( angiotensin ii receptor blocker ) use , bmi < 24 kg / m , triglycerides < 150 mg / dl , waist circumference < 80 cm ( female ) or < 90 cm ( male ) , hdl cholesterol > 40 mg / dl ( male ) or > 50 mg / dl ( female ) , and blood pressure < 130/80 mmhg . the proportional hazards assumption , the constant hr over time , was evaluated by comparing estimated log - log survival curves for all covariates . all assessed log - log survival plots graphically showed two parallel lines , indicating no violation of the assumption . a test for trend analyses were performed with sas software , version 9.1 ( sas institute , cary , nc ) . table 1 displays demographic and biochemical characteristics of study subjects . compared with those who retained a status of normoalbuminuria , patients who developed microalbuminuria were more likely to have lower education , higher homa - ir , and higher baseline acr . baseline demographic and biochemical characteristics of type 2 diabetic patients with and without progression to microalbuminuria data are n ( % ) or mean  sd unless otherwise indicated . sbp , systolic blood pressure ; dbp , diastolic blood pressure ; ccb , calcium channel blocker . the kaplan - meier survival curves in fig . 1 indicate that the probability of remaining normoalbuminuric for those in the 2nd to 4th homa - ir quartiles was significantly lower than for subjects in the lowest quartile . the significant adjusted hrs for those in 3rd and 4th quartiles in the multivariable cox proportional hazards model , shown in table 2 , confirm the independent association between insulin resistance and microalbuminuria development . figure 2 shows the adjusted hr and trend test for those with good metabolic profiles were similar to what was observed for overall subjects . kaplan - meier estimates of probability of normoalbuminuria according to 4 quartiles of homa - ir at baseline . cox proportional hazards models for progression to microalbuminuria * data are hr ( 95% ci ) . * adjusted hr of microalbuminuria development in type 2 diabetic patients for overall subjects and those with good metabolic profiles at baseline . type 2 diabetic patients with good metabolic profiles were those who had no acei / arb use , bmi < 24 kg / m , triglycerides < 150 mg / dl , waist circumference < 80 cm ( female ) or < 90 cm ( male ) , hdl cholesterol > 40 mg / dl ( male ) or > 50 mg / dl ( female ) , and blood pressure < 130/80 mmhg . the controlled covariates in the survival analyses included demographics ( baseline age , sex , education , smoking status , and diabetes duration ) and baseline biomedical profiles ( waist circumference , bmi , triglycerides , acr , hdl cholesterol , hba1c , and mean arterial pressure ) . the reference group for each model was those who were in the lowest quartile of the corresponding homa - ir index . the mean arterial pressure ( map ) was calculated by the formula : mean arterial pressure = diastolic blood pressure + 1/3 ( systolic blood pressure  diastolic blood pressure ) . in addition to overall subjects , this dose - response relationship remained true for type 2 diabetic patients with poor glycemic or blood pressure control . furthermore , we classified the homa - ir score based on its tertile instead of quartile . the incidence rate ratios of tertile 3 , in comparison with tertile 1 , were 1.60 ( 95% ci 1.002.55 ) , 2.71 ( 1.804.15 ) , 1.92 ( 1.163.27 ) , and 1.70 ( 1.142.54 ) for subgroups with hba1c 8% , hba1c > 8% , blood pressure 130/80 mmhg and blood pressure > 130/80 mmhg , respectively . their p values ( 0.041 , 0.022 , 0.013 , and 0.007 , respectively ) for dose - response trend tests were also significant ( data not shown ) . incidence of microalbuminuria in subjects in different homa - ir quartiles , according to their glycemic and blood pressure control bp , blood pressure ; rr , rate ratio . * test for trend for incidence rate ratio from homa - ir q1 to q4 . insulin resistance is shown in this longitudinal study to be a strong predictor in determining the development of microalbuminuria within 5 years for the type 2 diabetic patients with acr < 30 mg / g at enrollment . compared with those with a homa - ir score in the lowest quartile , those in the highest quartile had increased incidences of microalbuminuria by about 17% ( for those with poor blood pressure control ) to 92% ( for those with good blood pressure control ) . furthermore , the adjusted dose - response effect of insulin resistance on development of microalbuminuria was significant in overall subjects and the subgroup with good metabolic profiles . overall , this prospective study provides new epidemiological evidence linking elevated risk of diabetic nephropathy with insulin resistance . a growing body of studies has demonstrated that insulin resistance is associated with hemodynamic alteration in the kidney . hyperinsulinemia has been reported to be able to raise glomerular hydrostatic pressure , increase renal vascular permeability , aggravate glomerular hyperfiltration , and enhance renal sodium reabsorption ( 810 ) . altogether , the steady state of renal endothelial functions and hemodynamic harmonization may be changed because of cascading reactions caused by insulin resistance . this could explain underlying mechanisms for the significant association between the homa - ir score and microalbuminuria development observed in this study . similar to previous studies ( 3,11,12 ) , our study found that those with a higher homa - ir were more likely to have metabolic syndrome ( data not shown ) and higher urine acr at baseline . metabolic syndrome and urine acr have been well recognized as risk factors associated with microalbuminuria ( 2,4,5,1315 ) . because of this intertwining feature in the cluster of insulin resistance , metabolic syndrome , and microalbuminuria , we could not completely exclude the possibility that insulin resistance is a biochemical marker rather than a causal factor in the process of microalbuminuria development . in order to ensure the effects of insulin resistance , we calculated the incidence rate ratio and performed multivariable survival analyses for subjects stratified by different baseline characteristics . the causal association is considered to be truly independent because the significant dose - response effect of homa - ir on microalbuminuria development was demonstrated ( fig . 2 ) not only in overall subjects but also in type 2 diabetic subjects with no acei / arb use and good metabolic profiles . we found that the less educated were also more likely to have higher hba1c and to be current smokers ( data not shown ) , both of which are risk factors for the development of microalbuminuria . these findings are in accordance with previous studies in both western and oriental countries ( 16,17 ) . this universal evidence implies that those with lower educational attainment are at higher risk of developing chronic kidney disease . first , we used the homa - ir index instead of the hyperinsulinemic euglycemic glucose clamp ( 18 ) to measure insulin resistance . although some previous papers argued that the homa - ir index might have limitations to assess insulin resistance in certain circumstances ( 19 ) , it is the most frequently used index to evaluate insulin resistance in the general population and diabetic patients ( 20 ) . the uk prospective diabetes study also used the homa - ir index to monitor insulin sensitivity in its type 2 diabetic cohort ( 21 ) . in line with other epidemiologic studies , we believe the classification of insulin resistance by quartiles in this study is reliable . second , we did nt measure fat deposition by using standardized dual - energy x - ray absorptiometry or computed tomography to evaluate association between abdominal adiposity and nephropathy ( 22 ) . instead , abdominal adiposity was estimated by waist circumference , which is suggested as a predictor of insulin resistance and albuminuria ( 23,24 ) . although we adjusted for waist circumference in all of our multivariable survival analyses , the homa - ir score remained a significant predictor of microalbuminuria development , either in the entire cohort ( table 2 ) or in a subgroup of individuals with good metabolic profiles ( fig . 2 ) . moreover , small sample size in subgroups in quartiles 14 of glycemic control or blood pressure control in table 3 could affect statistical significance . if we classified homa - ir based on its tertile , the incidence rate ratios of tertile 3 for subgroups with hba1c 8% , hba1c > 8% , blood pressure 130/80 mmhg , and blood pressure > 130/80 mmhg were all significant as compared with those in tertile 1 . these results indicate the dose - response effects of homa - ir on microalbuminuria were robust . in conclusion , this is the first prospective study investigating an association between insulin resistance and development of microalbuminuria in type 2 diabetic patients . we reconfirm that type 2 diabetic patients predisposed to higher insulin resistance are more likely to develop microalbuminuria . future research should aim at stopping renal function deterioration by improving insulin sensitivity for type 2 diabetes with high insulin resistance .