diabetic nephropathy ( dn ) is an important health problem in the worldwide adult population , and approximately 2040% of dn patients inevitably progress to end stage renal disease ( esrd ) .
albuminuria is an important marker to diagnosis and predicts the progression of dn [ 2 , 3 ] , but some studies have shown that only 3045% of diabetic patients with microalbuminuria have progressed to macroalbuminuria in 10-year follow - up ; 30% of them regressed to normoalbuminuria , and 3040% remained at microalbuminuria level .
furthermore , 20% of type 2 diabetic ( t2 dm ) patients had their glomerular filtration rate ( gfr ) decline before detecting albuminuria .
all the renal cellular elements including the glomerular endothelium , mesangial cells , podocytes , and tubular epithelium are affected in setting of chronic hyperglycemia .
furthermore , renal tubular and interstitial compartments have been increasingly reported to play an integral role in the pathogenesis of dn and they correlate well with progressive renal function decline and represent a final common pathway of chronic kidney disease ( ckd ) . in t2 dm ,
novel tubular biomarkers that relate to renal tubular injury could improve risk stratification and prediction .
neutrophil gelatinase associated lipocalin ( ngal ) is expressed in the renal tubular epithelium , and a rise in urinary concentrations may provide a potential biomarker of a chronically injured kidney .
kidney injury molecule-1 ( kim-1 ) is a type 1 membrane protein expressed on the apical membrane of proximal tubule cells .
its ectodomain is cleaved and released in the lumen of the tubule and finally appears in urine , which is stable .
cystatin - c is produced from nucleated cells in the body and easily filtered by the glomeruli and is reabsorbed and catabolized by the proximal tubule .
previous study showed that the urine cystatin - c ( ucys - c ) level represented an indicator of renal tubular dysfunction .
finally , changes in angiotensinogen could influence ras activity and increases in intrarenal ras components , in parallel with the severity of fibrotic renal damage , have been demonstrated in chronic progressive nephropathy [ 9 , 10 ] .
thus , all tubular biomarkers serve as a promising biomarker for tubule damage . starting from renal tubular injury playing a role in the pathogenesis of dn and progressive decline of renal function
, we hypothesized that baseline urinary tubular injury biomarkers are an important determinant of lower gfr in a wider cohort of t2 dm patients .
however , inconsistent results have been produced with regard to predictors of all biomarkers in type 2 dm with nephropathy .
age , sex , and diabetes duration matched subjects with various stages of normoalbuminuria ( urine albumin creatinine ratio ( uacr ) < 30  mg albumin / g creatinine , n = 94 ) , microalbuminuria ( uacr 30300  mg albumin / g creatinine , n = 95 ) , and macroalbuminuria t2 dm ( uacr > 300  mg albumin / g creatinine and/or persistent proteinuria , n = 114 ) were recruited in february 2014 and march 2015 and followed up for a least 12 months at the outpatient clinic , department of internal medicine , phramongkutklao hospital .
the study was approved by the ethics committee of the institute review board at the royal thai army medical department and all patients gave written informed consent .
exclusion criteria included acute kidney injury ( aki ) episode , pregnancy , unspecified type of dm , and patient life expectancy < 1 year .
all patient histories were carefully recorded by interview and confirmed by checking patient records and recording drug prescriptions . clinical examination , including assessment of body mass index ( bmi ) , systolic and diastolic blood pressure ( bp ) , fasting plasma glucose , and other basic laboratory data , was conducted .
bp was measured three times , and the average value was used to analyze data .
common biochemical parameters including urea , creatinine , hemoglobin a1c , serum lipids and electrolytes , albumin , hemoglobin , and proteinuria were measured at baseline in all patients , according to standard methods in a routine clinical laboratory .
estimated gfr was assessed using the chronic kidney disease epidemiology collaboration ( ckd - epi ) equation .
urine albumin was measured on a nephelometric analyzer and urine creatinine was measured on a multiple analyzer ( modular p chemistry analyzer ; roche diagnostics ) .
urine albumin and creatinine for urine samples collected from participants and albuminuria were reported as albumin creatinine ratio ( uacr ) .
thirty milliliters of fresh urine was centrifuged at 4,000  rpm for 10 minutes and then stored at 80c until assayed .
all specimens were diluted often to obtain concentration at the optimal density according to the elisa kit instruction .
coefficients of variation for urine tubular biomarkers assays were < 10% , for intra - assay and interassay variation .
, usa and canada ) levels were expressed as nanograms per gram of creatinine ( ungal and ukim-1 ) .
cystatin - c ( r&d systems china co. , ltd ) levels were expressed as micrograms per gram of creatinine ( uccr ) .
urine angiotensinogen ( r&d systems china co. , ltd ) by solid phase elisa technique was expressed as nanograms per gram of creatinine ( uang ) .
after the baseline assessments , patients were followed up prospectively until the end of the observation period .
the latter was defined by the combined outcomes of percentage changes of gfr decline from baseline and rapid renal progression was defined by decreased estimated gfr 25% from baseline in one year .
patients were personally contacted in case they missed any appointment and at the end of the study , to avoid eventual loss during follow - up .
data were presented as mean  sd , median , or percentage frequency , as appropriate .
receiver operating characteristic ( roc ) analysis was used to calculate the area under the curve ( auc ) for ungal , ukim-1 , uccr , uang , and uacr , and to find the best cut - off values of other tubular biomarkers to identify the progression to renal endpoint .
kaplan - meier curves were generated to assess renal survival in subjects with each cut - off point values of tubular biomarkers above and below the optimal roc - derived cut - off levels .
for examination of associations with decline gfr and quartiles of urinary biomarkers , we first examined the unadjusted relationships and then adjusted the models for bmi , systolic bp , anemia , raas blocker , serum creatinine , and uacr by multivariate cox proportional hazard regression analysis .
a total of 303 subjects ( 96% ) at the second visit of the study were recruited for analysis .
mean age of patients was 66.4  11.4 years , and more than half were male ( 55.4% ) .
onset of t2 dm was 12.2  9.2 years , all patients had follow - up time of 12.3  4 months . regarding clinical and laboratory data ,
mean estimated gfr was 50  29.7  ml / min/1.73  m , mean uacr was 887  210  mg / gcr , and hba1c was 7.3  1.5% . during the observational period ( median follow - up of 12.3  4 months ) ,
41 patients ( 13.5% ) had gfr decline 25% yearly from baseline as rapid renal progression .
the patients with rapid renal progression had a higher prevalence of anemia ; raas blocker usage increased systolic bp and decreased baseline estimated gfr and baseline uacr ( table 1 ) .
urine tubular biomarker levels of uccr , uang , ukim-1 , and ungal were significantly higher in the rapid renal progression group when compared with nonrapid renal progression group , represented by median ( interquartile range at 25%75% ) value of uccr 3.01 [ 1.0417.15 ] versus 7.52 [ 4.7416.49 ]  mcg / gm ( p < 0.001 ) , uang 2.41 [ 0.3110.73 ] versus 14.26 [ 3.2924.48 ]  mcg / gm ( p < 0.001 ) , ukim-1 67.5 [ 32.1132.2 ] versus 133.1 [ 81.9255.6 ]  ng / gm ( p < 0.001 ) , and ungal 751.3 [ 413.91350.5 ] versus 1058.3 [ 702.11693.3 ]  ng / gm ( p = 0.004 ) as shown in figure 1 . macroalbuminuric and microalbuminuric
t2 dm patients had higher levels of all urine tubular biomarkers as compared to normoalbuminuric patients .
however , in subgroup of albuminuria , there were no significant differences in the urine tubular biomarkers between rapid and nonrapid renal progression groups .
the relative small sample size and few reported end points were detected in each subgroup ( table 2 ) .
roc analysis showed an auc for uccr , uang , ukim-1 , ungal , and uacr of 0.72 ( 95% ci : 0.64 to 0.79 ) , 0.71 ( 95% ci : 0.63 to 0.79 ) , 0.71 ( 95% ci : 0.63 to 0.79 ) , 0.64 ( 95% ci : 0.560.72 ) , and 0.76 ( 95% ci : 0.680.84 ) , respectively , as shown in figure 2 .
for uccr , the best cut - off level was 3.43  mcg / g ( sensitivity 80.5% , specificity 56.9% ) , for uang was 4.52  mcg / g ( sensitivity 73.2% , specificity 63.4% ) , for ukim-1 was 95  ng / g ( sensitivity 70.7% , specificity 63.4% ) , and for ungal was 772  ng / g ( sensitivity 68.3% , specificity 51.1% ) .
all tubular biomarkers demonstrated intermediate performance to predict rapid renal progression in t2 dm patients .
kaplan - meier survival curves in patients with uccr , uang , ukim-1 , and ungal levels above and below the optimal cut - off are presented in figure 3 .
subjects with uccr , uang , ukim-1 , and ungal values above 3.43  mcg / g , 4.52  mcg / g , 95  ng / g , and 772  ng / g , respectively , experienced a significantly faster evolution to endpoint ( p < 0.05 ) with a mean follow - up time to progression of 12 months . to identify putative risk factors
associated with the progression of ckd , we performed a cox regression analysis in the model for all variables that differed at baseline in patients who reached the endpoint during the whole follow - up period ( anemia , systolic bp , raas blocker usage , baseline serum creatinine , and uacr ) .
univariate analysis showed significantly increased risk for rapid ckd progression in the group of upper quartiles of all urine tubular biomarkers when compared with lower quartiles ( table 3 ) .
multiple cox regression analysis after adjusting potential risks for ckd progression revealed that all urine tubular biomarkers including uccr , uang , ukim-1 , and ungal were significantly associated with rapid ckd progression at the end of the study .
findings from the present study clearly indicate that all novel tubular biomarkers , that is , uccr , uang , ukim-1 , and ungal , represent novel risk markers of dn progression .
all novel urinary tubular biomarkers showed a most impressive predictive power in such a contest even after adjusting for conventional risk factors ( baseline bmi , systolic bp , anemia , raas blocker used , serum creatinine , and uacr ) .
this suggests that all novel tubular biomarkers would not be simple surrogate indexes of baseline estimated gfr , but markers on their own , predicting dn progression beyond the information provided by serum creatinine and other conventional risk factors . in recent years , however , different studies have underlined the crucial role played by the renal tubule in the genesis of progressive acute and chronic kidney disease and its evolution to terminal stage .
importantly , in diabetic with renal pathology , renal function correlated better with the degree of tubule - interstitial lesions than with that of the glomerular lesions .
new biomarkers of the processes that induce these tubulointerstitial changes may ultimately prove to be better predictors of disease progression and long - term prognosis than our current markers .
cystatin - c , angiotensinogen , ngal , and kim-1 are known markers of acute kidney injury .
all of these markers are upregulated in renal tubules after renal injury [ 1518 ] .
cystatin - c , angiotensinogen , and kim-1 markers located in the proximal tubules following renal injury , whereas ngal was defined in the distal tubules .
recent literature suggests that all of these may be a marker to detect progression of ckd .
prior cross - sectional studies examining the increased urine tubular biomarkers include cystatin , kim-1 , ngal , and angiotensinogen in early ckd and early dn [ 1922 ] .
similarly , in one - year observational follow - up study in 74 t2 dm patients with nephropathy , high urine ngal levels at baseline correlated with declined levels of estimated gfr and increased serum creatinine .
one study in t2 dm demonstrated that kim-1 predicted the decline of gfr in unadjusted analysis   and two studies in t1 dm also showed that kim-1 levels were significantly higher in the patients who progressed from nonmacroalbuminuria to ckd stage 3   and macroalbuminuria to late stage of ckd .
in addition , urine cystatin - c as reported from a prospective study predicted the ckd progression in the largest study reported to date , with 237 patients with t2 dm . therefore , our study measured all of promising urinary tubular biomarkers at the same time in t2 dm patients .
our results are consistent with those in additional studies , and we provide more validation for these biomarkers associated with the progressive course of the disease . in this study , we found that t2 dm with rapid renal progression had significantly increased levels of urinary biomarkers when compared with the nonrapid renal progression group . from roc curves
( auc 0.640.72 ) each tubular biomarker presented an intermediate accuracy performance in predicting rapid renal progression and all tubular biomarkers had an auc less than the auc of uacr ( 0.76 ) possibly affecting the level of proteinuria in our population .
cox proportional hazard models , representing all novel tubular biomarkers , were independent predictors of ckd progression in t2 dm .
our study integrated novel tubular biomarkers at the same time and compared the performance of each tubular biomarker with the standard urinary biomarker , uacr .
two studies in early ckd and short - term ( less than five years ) duration of t2 dm showed that only increased urine ngal , but not urine kim-1 , was significantly associated with gfr decline [ 28 , 29 ] . as opposed to previous studies in participants with ckd   and early stage of dn , all tubular biomarkers levels in our study were significantly correlated with gfr decline and albuminuria .
a significant rise in urinary tubular levels may indicate severe tubular cells damage in setting of established nephropathy in t2 dm population .
study limitations included being a single center study and having a short follow - up period of 12 months .
however , in our study , urine biomarkers were measured in a cohort of both early and advanced dn patients who might have a more rapid decline in renal function in limited time of follow - up .
our primary endpoint was also reached by fifteen percent of the patients , and the statistical model was powerful enough to establish independent relationships between biomarkers and gfr decline in t2 dm .
our study did not collect data of main renal outcomes for demonstrating the doubling of serum creatinine and initiating long - term dialysis .
the relatively small sample size of our study limits the precision and power to detect associations of moderate strength .
follow - up was based on a single serum creatinine and gfr calculation for detecting a decrease in gfr of > 25% from baseline ; it may have some potential bias due to imprecision in estimated gfr .
finally , our data suggest that all the biomarkers measured in the urine are derived mostly or entirely from a renal tubular source .
however , the data do not definitively exclude the possibility that some fraction of the excreted biomarkers especially plasma kim-1 may be derived from glomerular filtration .
animal experimental study addressed that plasma kim-1 was highly increased in diabetic animals compared to nondiabetic animals .
another study also indicated that plasma kim-1 level predicted rate of gfr decline and incidence of esrd in a cohort of t1 dm patients with advanced nephropathy . in conclusion ,
t2 dm patients with high levels of urine tubular biomarkers ( urine cystatin - c , angiotensinogen , kim-1 , and ngal ) presented more rapid decline in renal function .
all urine tubular biomarkers were independent predictors of rapid renal progression among t2 dm patients . for future study , in early stage t2 dm in the normo- or microalbuminuria group , novel tubular biomarkers may possibly have a role in early detection compared to the conventional marker to predict ckd progression .
 
 background . 
 tubulointerstitial injury is both a key feature of diabetic nephropathy and an important predictor of renal dysfunction . 
 novel tubular biomarkers related to renal injury in diabetic nephropathy could improve risk stratification and prediction . 
 methods . a total of 303 type 2 diabetic patients were followed up . 
 the baseline urine values of cystatin - c to creatinine ratio ( uccr ) , angiotensinogen to creatinine ratio ( uang ) , ngal to creatinine ratio ( ungal ) , and kim-1 to creatinine ratio ( ukim-1 ) were measured . 
 the primary outcome was a decline in estimated gfr of 25% yearly from baseline . 
 results . 
 urine tubular biomarkers of uccr , uang , ungal , and ukim-1 were significantly higher according to the degree of albuminuria and all were significantly higher among patients with rapid decline in estimated gfr of 25% yearly from baseline . 
 all biomarkers predicted primary outcomes with roc for uccr of 0.72 ; 95% ci 0.640.79 , for uang of 0.71 ; 95% ci 0.630.79 , for ungal of 0.64 ; 95% ci 0.560.72 , and for ukim-1 of 0.71 ; 95% ci 0.630.79 . using multivariate cox regression analysis , 
 the number of patients with rapid renal progression was higher among those in the upper quartiles of all biomarkers than in those in the lower quartiles . 
 conclusions . 
 type 2 diabetic patients with high levels of urine tubular biomarkers had a more rapid decline in renal function . 