this was a prospective observational study of patients attending the department of endocrinology at pusan national university hospital . the study was conducted with the approval of the institutional review board of pusan national university hospital . a total of 264 korean type 2 diabetic patients were consecutively enrolled at the outpatient clinics between may 2008 and december 2009 . all patients fulfilled the following inclusion criteria : age 18 years and estimated gfr ( egfr ) 30 ml / min/1.73 m. we excluded patients with thyroid disorders or who had been medicated within 6 months prior to the study because thyroid function could affect the cystatin c level ( 19 ) . additional exclusion criteria were 1 ) active urinary tract infection , 2 ) renal disease other than diabetic nephropathy , 3 ) neoplastic disorders , 4 ) severe liver dysfunction , 5 ) active or chronic infection or inflammatory disorders , 6 ) pregnancy , and 7 ) a recent ( within 6 months ) history of acute myocardial infarction , stroke , or occlusive peripheral vascular disease . a random spot urine sample and a blood sample were obtained from each patient at the clinic visit . medical histories and anthropometric measurements the egfr level was calculated using the modification of diet in renal disease ( mdrd ) study formula for the korean population : mdrd = 107.904  ( serum creatinine [ mg / dl ] )  age ( 20 ) . a correction factor of 0.667 the serum and urinary cystatin c levels were measured by the latex agglutination test ( modular p800 ; roche diagnostics , mannhein , germany ) . the interassay and intra - assay coefficients of variations of cystatin c in our laboratory were as follows : < 4.2 and < 3.4% , respectively , for serum and < 7.9 and < 10.1% , respectively , for urine . urine specimens with cystatin c levels < 0.01 mg / l were assumed to have a concentration of 0.01 mg / l . the data on urinary cystatin c was also expressed as ratios of urinary cystatin c to urinary creatinine in order to assess different hydration states and renal functions of the patients . the ratio between urine mass concentrations of cystatin c and creatinine in micrograms per millimole was calculated and designated as the urinary cystatin c - to - creatinine ratio ( ccr ) . since we obtained total proteinuria and albuminuria values from each patient at baseline , we were able to estimate the amount of nonalbumin proteinuria through the following calculation : nap - to - creatinine ratio ( napcr ) = protein - to - creatinine ratio ( pcr )  albumin - to - creatinine ratio ( acr ) . the lowest detectable level and the coefficient of variation in our laboratory were as follows : for total proteinuria , 0.7 mg / dl and < 4.8% , respectively ; for albuminuria , 0.2 mg / dl and < 7.4% , respectively . they were managed to give priority to the best treatment according to standard guidelines at each outpatient clinic of two endocrinologists ( i.j.k . and s.s.k . ) . thirteen patients were excluded during follow - up as follows : four patients died of other causes , six patients were hospitalized for acute myocardial infarction and active infections , such as pneumonia , and three patients were diagnosed with additional malignancies during the follow - up period . finally , a total of 237 patients with type 2 diabetes were enrolled for this study . the two tubular damage markers were measured at intervals of 12  1 ( mean  sd ) months at the outpatient clinic during the follow - up period . serum creatinine was routinely measured for the estimation of gfr at intervals of 6  1 ( mean  sd ) months during the follow - up period using the same methods . the latest egfr calculations were used in the assessment of the annual decline in egfr . chronic kidney disease ( ckd ) stage 3 or greater was defined as having egfr < 60 ml / min/1.73 m in two consecutive measurements from the last follow - up visit . the data are presented as mean  sd for normally distributed variables and the medians ( interquartile range ) for nonnormally distributed variables . the distribution of continuous variables was examined for skewness and kurtosis , and logarithm - transformed values of nonnormally distributed variables were used for analysis . we conducted multivariate regression analyses with the annual rates of decline in egfr as dependent variables and urinary ccr and napcr as independent variables , respectively . multivariate logistic analysis , using a backward procedure on the basis of likelihood ratios , was conducted to determine whether both tubular markers would be predictive factors for ckd stage 3 or greater . the odds ratios ( ors ) of urinary levels of cystatin c and nap were calculated with reference to the lowest tertile of each variable . a p value of < 0.05 derived from the two - tailed student t test was considered statistically significant . this was a prospective observational study of patients attending the department of endocrinology at pusan national university hospital . the study was conducted with the approval of the institutional review board of pusan national university hospital . a total of 264 korean type 2 diabetic patients were consecutively enrolled at the outpatient clinics between may 2008 and december 2009 . all patients fulfilled the following inclusion criteria : age 18 years and estimated gfr ( egfr ) 30 ml / min/1.73 m. we excluded patients with thyroid disorders or who had been medicated within 6 months prior to the study because thyroid function could affect the cystatin c level ( 19 ) . additional exclusion criteria were 1 ) active urinary tract infection , 2 ) renal disease other than diabetic nephropathy , 3 ) neoplastic disorders , 4 ) severe liver dysfunction , 5 ) active or chronic infection or inflammatory disorders , 6 ) pregnancy , and 7 ) a recent ( within 6 months ) history of acute myocardial infarction , stroke , or occlusive peripheral vascular disease . a random spot urine sample and a blood sample were obtained from each patient at the clinic visit . medical histories and anthropometric measurements the egfr level was calculated using the modification of diet in renal disease ( mdrd ) study formula for the korean population : mdrd = 107.904  ( serum creatinine [ mg / dl ] )  age ( 20 ) . a correction factor of 0.667 the serum and urinary cystatin c levels were measured by the latex agglutination test ( modular p800 ; roche diagnostics , mannhein , germany ) . the interassay and intra - assay coefficients of variations of cystatin c in our laboratory were as follows : < 4.2 and < 3.4% , respectively , for serum and < 7.9 and < 10.1% , respectively , for urine . urine specimens with cystatin c levels < 0.01 mg / l were assumed to have a concentration of 0.01 mg / l . the data on urinary cystatin c was also expressed as ratios of urinary cystatin c to urinary creatinine in order to assess different hydration states and renal functions of the patients . the ratio between urine mass concentrations of cystatin c and creatinine in micrograms per millimole was calculated and designated as the urinary cystatin c - to - creatinine ratio ( ccr ) . since we obtained total proteinuria and albuminuria values from each patient at baseline , we were able to estimate the amount of nonalbumin proteinuria through the following calculation : nap - to - creatinine ratio ( napcr ) = protein - to - creatinine ratio ( pcr )  albumin - to - creatinine ratio ( acr ) . the lowest detectable level and the coefficient of variation in our laboratory were as follows : for total proteinuria , 0.7 mg / dl and < 4.8% , respectively ; for albuminuria , 0.2 mg / dl and < 7.4% , respectively . they were managed to give priority to the best treatment according to standard guidelines at each outpatient clinic of two endocrinologists ( i.j.k . and s.s.k . ) . thirteen patients were excluded during follow - up as follows : four patients died of other causes , six patients were hospitalized for acute myocardial infarction and active infections , such as pneumonia , and three patients were diagnosed with additional malignancies during the follow - up period . finally , a total of 237 patients with type 2 diabetes were enrolled for this study . the two tubular damage markers were measured at intervals of 12  1 ( mean  sd ) months at the outpatient clinic during the follow - up period . serum creatinine was routinely measured for the estimation of gfr at intervals of 6  1 ( mean  sd ) months during the follow - up period using the same methods . the latest egfr calculations were used in the assessment of the annual decline in egfr . chronic kidney disease ( ckd ) stage 3 or greater was defined as having egfr < 60 ml / min/1.73 m in two consecutive measurements from the last follow - up visit . the data are presented as mean  sd for normally distributed variables and the medians ( interquartile range ) for nonnormally distributed variables . the distribution of continuous variables was examined for skewness and kurtosis , and logarithm - transformed values of nonnormally distributed variables were used for analysis . we conducted multivariate regression analyses with the annual rates of decline in egfr as dependent variables and urinary ccr and napcr as independent variables , respectively . multivariate logistic analysis , using a backward procedure on the basis of likelihood ratios , was conducted to determine whether both tubular markers would be predictive factors for ckd stage 3 or greater . the odds ratios ( ors ) of urinary levels of cystatin c and nap were calculated with reference to the lowest tertile of each variable . a p value of < 0.05 derived from the two - tailed student t test was considered statistically significant . the mean age of the patients was 58.5  11.1 years ( range , 1880 years ) , and there were 115 males and 122 females . the patients were categorized into three groups according to acr : those with acr < 30 mg / g creatinine ( normoalbuminuria group , n = 149 ) , those with acr 30299 mg / g creatinine ( microalbuminuria group , n = 58 ) , and those with acr 300 mg / g creatinine ( macroalbuminuria group , n = 30 ) . age , duration of diabetes , systolic blood pressure ( sbp ) , hba1c , hdl cholesterol , and triglycerides were significantly different between the groups . estimated gfr tended to decrease with increasing degrees of albuminuria ( p value for trend < 0.001 ) . more antihypertensive and lipid - lowering agents were administered in the macroalbuminuria group than in the normo- and microalbuminuria groups . baseline characteristics of metabolic and laboratory parameters in patients with type 2 diabetes the urinary cystatin c level and ccr were significantly higher in the macroalbuminuria group than in the normoalbuminuria and microalbuminuria groups ( both p < 0.001 ) , whereas they were not significantly different between the normoalbuminuria and microalbuminuria groups ( table 1 ) . urinary napcr was significantly higher in the macroalbuminuria group than in the normo- and microalbuminuria groups ( both p < 0.001 ) , and they were also significantly different between the normo- and microalbuminuria groups ( p < 0.001 ) . urinary acr positively correlated with urinary ccr ( r = 0.450 , p < 0.001 ) and urinary napcr ( r = 0.699 , p < 0.001 ) . in addition , at baseline , urinary ccr and napcr , both markers of tubular damage , positively correlated with each other ( r = 0.597 , p < 0.001 ) . the median follow - up period was 29.0 months ( range , 13.044.0 months ) . there was a median annual decline in egfr of 1.31 ml / min/1.73 m / year during the follow - up period . the values of annual egfr decline significantly correlated with both baseline urinary ccr ( r = 0.272 , p < 0.001 ) ( fig . 1b ) in univariate regression analysis ( supplementary table 2 ) . after adjusting for age and significant clinical factors affecting the decline of egfr , both urinary ccr and napcr remained significantly associated with a decline in egfr ( table 2 ) . after additionally adjusting for baseline egfr and serum cystatin c ( only analysis for urinary ccr ) , both urinary ccr and napcr remained significantly associated with the annual decline in egfr in the final model ( r = 0.160 , p = 0.021 ; r = 0.412 , p < 0.001 , respectively ) . single regression analysis of the annual rate of the decline in egfr by using urinary ccr ( a ) and napcr ( b ) . multiple regression analysis of the annual rate of decline in egfr as a dependent variable to test whether urinary ccr and napcr might have prognostic values in early diabetic nephropathy , we analyzed patients with baseline egfr 60 ml / min/1.73 m or with normoalbuminuria . in patients with egfr 60 ml / min/1.73 m , both urinary ccr and napcr showed positive correlations with a decline in egfr in the final model after adjusting for several clinical parameters ( r = 0.144 , p = 0.031 ; r = 0.282 , p = 0.001 , respectively ) . in the normoalbuminuria group , the urinary napcr remained significantly associated with a decline in egfr ( r = 0.261 , p = 0.002 ) in the final model , whereas urinary ccr did not ( r = 0.097 , p = 0.199 ) . in addition , urinary napcr showed a significant association with a decline in egfr ( r = 0.201 , p = 0.018 ) in patients with both egfr 60 ml / min/1.73 m and normoalbuminuria . in univariate logistic regression analysis , the number of patients who progressed to ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) was higher in those in the upper tertiles of both the urinary levels of cystatin c and nap than in those in the lower tertiles ( or 7.26 for urinary cystatin c ; 48.71 for napcr ; p < 0.001 in both ) ( table 3 ) . the increased levels of both urinary ccr and napcr remained significantly associated with progression to ckd stage 3 or greater after adjusting for several clinical factors in each multivariate model . logistic regression of development of ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) at last follow - up the mean age of the patients was 58.5  11.1 years ( range , 1880 years ) , and there were 115 males and 122 females . the patients were categorized into three groups according to acr : those with acr < 30 mg / g creatinine ( normoalbuminuria group , n = 149 ) , those with acr 30299 mg / g creatinine ( microalbuminuria group , n = 58 ) , and those with acr 300 mg / g creatinine ( macroalbuminuria group , n = 30 ) . age , duration of diabetes , systolic blood pressure ( sbp ) , hba1c , hdl cholesterol , and triglycerides were significantly different between the groups . estimated gfr tended to decrease with increasing degrees of albuminuria ( p value for trend < 0.001 ) . more antihypertensive and lipid - lowering agents were administered in the macroalbuminuria group than in the normo- and microalbuminuria groups . baseline characteristics of metabolic and laboratory parameters in patients with type 2 diabetes the urinary cystatin c level and ccr were significantly higher in the macroalbuminuria group than in the normoalbuminuria and microalbuminuria groups ( both p < 0.001 ) , whereas they were not significantly different between the normoalbuminuria and microalbuminuria groups ( table 1 ) . urinary napcr was significantly higher in the macroalbuminuria group than in the normo- and microalbuminuria groups ( both p < 0.001 ) , and they were also significantly different between the normo- and microalbuminuria groups ( p < 0.001 ) . urinary acr positively correlated with urinary ccr ( r = 0.450 , p < 0.001 ) and urinary napcr ( r = 0.699 , p < 0.001 ) . in addition , at baseline , urinary ccr and napcr , both markers of tubular damage , positively correlated with each other ( r = 0.597 , p < 0.001 ) . the median follow - up period was 29.0 months ( range , 13.044.0 months ) . there was a median annual decline in egfr of 1.31 ml / min/1.73 m / year during the follow - up period . the values of annual egfr decline significantly correlated with both baseline urinary ccr ( r = 0.272 , p < 0.001 ) ( fig . 1b ) in univariate regression analysis ( supplementary table 2 ) . after adjusting for age and significant clinical factors affecting the decline of egfr , both urinary ccr and napcr remained significantly associated with a decline in egfr ( table 2 ) . after additionally adjusting for baseline egfr and serum cystatin c ( only analysis for urinary ccr ) , both urinary ccr and napcr remained significantly associated with the annual decline in egfr in the final model ( r = 0.160 , p = 0.021 ; r = 0.412 , p < 0.001 , respectively ) . single regression analysis of the annual rate of the decline in egfr by using urinary ccr ( a ) and napcr ( b ) . multiple regression analysis of the annual rate of decline in egfr as a dependent variable to test whether urinary ccr and napcr might have prognostic values in early diabetic nephropathy , we analyzed patients with baseline egfr 60 ml / min/1.73 m or with normoalbuminuria . in patients with egfr 60 ml / min/1.73 m , both urinary ccr and napcr showed positive correlations with a decline in egfr in the final model after adjusting for several clinical parameters ( r = 0.144 , p = 0.031 ; r = 0.282 , p = 0.001 , respectively ) . in the normoalbuminuria group , the urinary napcr remained significantly associated with a decline in egfr ( r = 0.261 , p = 0.002 ) in the final model , whereas urinary ccr did not ( r = 0.097 , p = 0.199 ) . in addition , urinary napcr showed a significant association with a decline in egfr ( r = 0.201 , p = 0.018 ) in patients with both egfr 60 ml / min/1.73 m and normoalbuminuria . in univariate logistic regression analysis , the number of patients who progressed to ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) was higher in those in the upper tertiles of both the urinary levels of cystatin c and nap than in those in the lower tertiles ( or 7.26 for urinary cystatin c ; 48.71 for napcr ; p < 0.001 in both ) ( table 3 ) . the increased levels of both urinary ccr and napcr remained significantly associated with progression to ckd stage 3 or greater after adjusting for several clinical factors in each multivariate model . logistic regression of development of ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) at last follow - up in this study , urinary cystatin c and nap , both clinical tubular damage markers , positively correlated with each other at baseline . both markers were significantly associated with the annual decline in egfr in type 2 diabetic nephropathy . in particular , both tubular damage makers affected a decline in egfr at the early stage of nephropathy in type 2 diabetic patients ( egfr 60 ml / min/1.73 m ) . urinary nap affected egfr decline in patients with both egfr 60 ml / min/1.73 m and normoalbuminuria , although urinary cystatin c did not reach statistical significance . in addition , the increased levels of the two markers were also associated with the progression of ckd stage 3 or greater at the last follow - up . in general , unlike healthy subjects , diabetic patients are continuously exposed to the various metabolic and hemodynamic risks associated with this disease ( 21 ) . recent studies have mainly focused on tubular damage , which is known to correlate with acute kidney injury in patients with diabetic nephropathy ( 410 ) . some cross - sectional studies have reported that several tubular markers increase more in diabetic patients than in healthy controls , and this correlated with the severity of albuminuria ( 48 ) . in our study , nap correlated with the severity of baseline albuminuria , which is consistent with the results of previous studies . however , urinary cystatin c mainly increased in the macroalbuminuria group and was not significantly different between the microalbuminuria and normoalbuminuria groups . microalbuminuria has generally been considered the earliest marker for the development of diabetic nephropathy in clinical settings ( 22 ) . microalbuminuria is diagnosed when significant glomerular damage has occurred ( 23 ) and does not necessarily lead to renal impairment because nephropathy sometimes occurs in the normoalbuminuric patients ( 24,25 ) . in our study , although urinary cystatin c was not associated with a decline in egfr in normoalbuminuric patients , urinary nap was significantly associated with a decline in egfr independent of baseline albuminuria and egfr . it is necessary to assess tubular damage independent of albuminuria in patients with early development and progression of diabetic nephropathy because tubular damage may play a significant role in the normoalbuminuric renal insufficiency . serum cystatin c is the most valid marker to estimate the gfr , rather than serum creatinine , and to predict progression of renal dysfunction ( 26 ) . an increase in urinary cystatin c , independent of serum cystatin c , is suggestive of renal tubular damage ( 13 ) . in our study , urinary cystatin c was a predictor of renal impairment independent of serum cystatin c. thus , urinary cystatin c also plays some role in predicting renal decline independent of serum cystatin c , although serum cystatin c itself , an indicator for the estimation of gfr , is very important for predicting renal decline . naps include -1 microglobulin , -2 macroglobulin , igg , cystatin c , transferrin nephrin , matrix metalloproteinase-9 , and tissue inhibitor of metalloproteinases-1 ( 27 ) . it is well known that each nap is related to renal damage in various chronic renal diseases , including diabetic nephropathy ( 28 ) . because there is little evidence to support the role of nap in diabetic nephropathy , it is worthwhile to investigate this issue . interestingly , in our type 2 diabetic patients , nap has been shown to be a stronger predictor for renal impairment than urinary cystatin c. at normal levels of protein loss , albumin is a minor component of total urinary protein , although albumin becomes the most significant single protein present as protein loss increases ( 29 ) . this supports our result that indicates the clinical value of nap in the prediction of renal impairment in normoalbuminuric patients . a recent study on tubular damage markers in acute kidney injury has demonstrated that unnormalized values are also useful for predicting ongoing injury ( 30 ) . the unnormalized values had similar patterns as the normalized values in baseline characteristics and univariate analysis results ( table 1 and supplementary table 2 ) . the statistical significance of unnormalized values was lost in multivariate analysis , whereas that of normalized values was not ( data not shown ) . interestingly , serum uric acid was found to be associated with renal decline in the most analyzed model in this study and has recently been shown to be related to diabetic nephropathy ( 31,32 ) . uric acid might induce renal microvascular disease independent of blood pressure through stimulation of the renin - angiotensin system ( ras ) and inhibition of endothelial nitric oxide ( 33 ) . in addition , lowering therapy in a diabetic animal model significantly reduced albuminuria and ameliorated tubulointerstitial inflammation , suggesting a role for uric acid in diabetic nephropathy ( 34 ) . first , the follow - up period of this study was relatively short , even though the development and progression of diabetic nephropathy require a longer time frame . however , it is considered that the occurrence of tubular damage , as expressed by biomarkers , could be a significant predictor for relatively rapid decline in renal function during a short period of time . second , we measured the urinary levels of cystatin c , albumin , and protein with single random spot urine samples , although urine samples were collected at the outpatient clinic from patients without illness or renal diseases other than diabetic nephropathy . despite these limitations , it is noteworthy that the two aforementioned tubular damage markers can easily be checked and used to assess the development and progression of diabetic nephropathy in clinical settings . third , although there have been a few studies reporting that urinary cystatin c and nap mainly increase due to tubular rather than glomerular damage , it is unclear whether urinary cystatin c and nap originate from tubular damage alone in clinical conditions involving both tubular and glomerular damage , such as diabetic nephropathy . further studies are needed to investigate whether both urinary cystatin c and nap are biomarkers of tubular damage in clinical conditions with a massive glomerular protein load , such as diabetic nephropathy . in conclusion , it is suggested that urinary cystatin c and nap , along with albuminuria , may be sensitive and specific markers for predicting renal impairment in type 2 diabetic patients and may help to further elucidate the role of tubular damage in the pathophysiological mechanisms of the development and progression of type 2 diabetic nephropathy .