diabetic nephropathy ( dn ) is a common complication of type 2 diabetes mellitus ( dm ) and is the leading cause of end - stage renal disease ( esrd ) .
microalbuminuria is an early sign of dn , which correlates with and can predict the progression of renal damage and cardiovascular morbidity [ 15 ] . the discovery of biomarkers for the earlier stages of dn would enable early intervention to reduce the impact of this chronic vascular complication .
diabetic nephropathy is associated with altered vascular structure , endothelial dysfunction , and disrupted homeostasis and angiogenesis [ 4 , 6 , 7 ] .
abnormal glomerular angiogenesis in patients with dn is associated with glomerular hypertrophy and results in glomerular capillary injury and urinary albumin excretion [ 8 , 9 ] .
thus , the mechanisms for the development of abnormal angiogenesis in dn involve a complicated interplay between pro- and antiangiogenic factors .
two families of growth factors , angiopoietin / tie-2 and vascular endothelial growth factor ( vegf)/vegf receptor ( vegfr ) , are thought to be associated with the development of dn .
vegf increases vascular permeability and is mitogenic for endothelial cells , acting early and at most points of the angiogenic cascade . within the angiopoietin family ,
angiopoietin-1 ( ang-1 ) and angiopoietin-2 ( ang-2 ) are the best - studied ligands for tie-2 receptors .
ang-1 signaling via tie-2 is involved in capillary sprouting , endothelial cell survival , and vascular remodeling .
several studies suggest that ang-2 signaling , in combination with vegf , leads to sprouting angiogenesis , while ang-2 signaling in the absence of vegf causes vessels to regress .
hence , selective upregulation of vegf and ang-2 may lead to aberrant proliferation of leaky , friable vessels .
emerging evidence suggests that vegf and angiopoietin are critical in glomerular physiology and in the pathogenesis of glomerular disease in dm [ 1417 ] .
previous studies have reported upregulated plasma levels of vegf and ang-2 in human and animal dn [ 10 , 16 , 1820 ] .
notably , circulating ang-2 levels associated with albuminuria have been reported in chronic kidney disease ( ckd )   and systemic lupus erythematosus ( sle ) .
transgenic mice with inducible overexpression of ang-2 in podocytes have been shown to develop significant increases in albuminuria , which , in turn , correlates with and predicts the progression of renal damage in dm . increased ang-2 levels in patients with dm
, little is known about the urinary levels of these angiogenic factors in different stages of dn , and potential correlations between them and albuminuria have not yet been studied . in the present study , we measured urinary ang-2 , ang-1 , and vegf levels to elucidate the possible correlation between urinary angiogenic factors and renal damage in patients with various phases of type 2 dm .
the retrospective study included 113 insulin - dependent patients with type 2 dm recruited from the department of endocrinology and nephrology at union hospital ( wuhan , china ) between december 2012 and march 2014 .
type 2 dm was defined according to established who criteria   ( i.e. , fasting blood glucose ( fbg )  7.0  mmol / l , postprandial blood glucose  11.1  mmol / l , or symptoms of dm with random blood glucose  11.1  mmol / l ) .
all patients maintained a stable body weight for at least 3 months before beginning the study .
patients with acute vascular events or hospitalization ( defined as stroke , myocardial infarction , unstable angina , or coronary or peripheral revascularization within the last 3 months ) ; high - range hypertension ( 160/100  mmhg ) ; current infection ; or evidence of neoplastic , hepatic , or significant renal disease ( requiring dialysis ) within 3 months prior to enrollment were excluded from the study .
diagnosis of dn was made according to the criteria of kidney disease outcomes quality initiative ( kdoqi ) .
based on the urinary albumin excretion rate ( uaer ) at baseline , patients were classified as having normoalbuminuria ( dn1 group : uaer < 20  g / min ) , microalbuminuria ( dn2 group : uaer 20200  g / min ) , or macroalbuminuria ( dn3 group : uaer > 200  g / min ) .
thirty subjects undergoing routine health checks were recruited into the control group ( nc ) .
the study protocol was approved by the medical ethics committee of union hospital , tongji medical college , huazhong university of science and technology , and was conducted according to the principles of the declaration of helsinki .
morning preprandial levels of fasting blood sugar ( fbg ) , serum creatinine , triglyceride ( tg ) , high - density lipoprotein cholesterol ( hdl - ch ) , low - density lipoprotein cholesterol ( ldl - ch ) , and total cholesterol were measured with a full automatic biochemical analyzer ( hitachi 7150 , tokyo , japan ) .
glycosylated hemoglobin ( hba1c ) was measured with a d10 hemoglobin testing system ( bio - rad laboratories , hercules , ca , usa ) , using a cation exchange hplc and an immunoturbidimetric assay method ( roche / hitachi 902 cobas system ) .
twenty - four  h urine samples were collected for the determination of ang-1 , ang-2 , and vegf levels and the estimation of uaer .
urinary protein quantitative measurements ( 24  h upqm ) were obtained for all patients .
renal function was assessed by glomerular filtration rate ( gfr ) detection with single photon emission computed tomography ( spect ) ( ge millennium mg dualhead , ge healthcare , milwaukee , wi , usa , usa ) .
levels of angiogenic factors ( ang-1 , ang-2 , and vegf ) in serum and urine were measured by enzyme - linked immunosorbant assay ( elisa ) in frozen serum samples and in urine samples . in brief ,
monoclonal antibodies specific for ang-1 ( raybiotech , norcross , ga , usa ) , ang-2 ( raybiotech ) , and vegf ( neobioscience technology co. , shenzhen , china ) were precoated onto microplates .
ang-1 , ang-2 , or vegf present in a sample was bound to the wells by the immobilized antibody .
the wells were washed , and biotinylated anti - human antibody specific for ang-1 , ang-2 , or vegf was added . after the removal of unbound antibodies ,
the wells were washed , and a 3,3,5,5-tetramethylbenzidine ( tmb ) substrate solution was added . the tmb solution changed in color from blue to yellow , in proportion to the bound concentration of ang-1 , ang-2 , or vegf .
the absorbance of the solution in each well was measured by a microplate reader ( bio - tek elx800 ; vt , usa ) at a wavelength of 450  nm . all samples were examined in duplicate , and mean values were used for statistical analysis .
statistical analysis was performed using a commercially available statistical software package ( spss for windows , version 18.0 ; spss , chicago , il , usa ) .
one - way analysis of variance ( anova ) with tukey 's post hoc test was used to compare groups of normally distributed data .
categorical data were analyzed using the chi - square test , and pearson 's or spearman 's correlation coefficients were used to test associations between variables .
a stepwise multiple regression analysis was performed to identify the predictors of ang-2 ( dependent variable ) .
all tests were two tailed , and values of p < 0.05 were considered statistically significant .
no significant differences were found in age , gender , bmi , diastolic blood pressure ( dbp ) , total cholesterol , ldl - ch , hdl - ch , or tg among the three diabetic groups and the control group . however , systolic blood pressure ( sbp ) , hba1c , and fbs were higher in the diabetic patients than in control subjects .
hba1c appeared to be lower in the dn3 group than in the dn1 and dn2 groups after long - term treatment with hypoglycemic drugs ; however , no significant differences in hba1c and fbs were found among the three diabetic groups .
serum creatinine was significantly higher , and gfr was significantly lower , in the dn3 group than in the dn1 and dn2 groups ( p < 0.001 ) .
both uaer and 24  h upqm increased progressively from the dn1 to the dn3 groups ( p < 0.001 ) .
serum levels of ang-2 were markedly increased in diabetic patients compared with values in the control group ( p < 0.001 ; figure 1(a ) ) .
moreover , serum ang-2 was significantly higher in patients with macroalbuminuria ( dn3 ) than those in the dn1 and dn2 groups ( p < 0.001 ; figure 1(a ) ) .
diabetic patients exhibited higher levels of urinary ang-2 than controls ( p < 0.001 ; figure 1(b ) ) , and urinary ang-2 increased in a stepwise manner with increasing degrees of albuminuria in the three diabetic groups ( p < 0.001 ; figure 1(b ) ) .
no significant difference was found in serum ang-1 levels between any of the four groups ( data not shown ) .
however , urinary ang-1 levels were significantly higher in the dn1 group than in the control group ( p < 0.05 ; figure 1(c ) ) and lower in the dn3 group than in the control group ( p < 0.001 ) .
in addition , patients in the dn1 and dn2 groups had significantly higher urinary ang-1 levels than those in the dn3 group ( p < 0.001 ; figure 1(c ) ) .
no significant difference was found in serum vegf among the groups ( data not shown ) ; however , subjects with dm exhibited significantly higher urinary vegf levels than the control subjects ( p < 0.001 ; figure 1(d ) ) .
moreover , urinary vegf was significantly higher in patients with macroalbuminuria ( dn3 ) than in the dn1 and dn2 groups ( p < 0.001 ; figure 1(d ) ) .
no difference was observed in urinary vegf in diabetic patients with or without microalbuminuria . 

table 2 and figure 2 summarize the results of the analyses undertaken in patients with dn .
serum levels of ang-2 were significantly positively correlated with urinary ang-2 and vegf levels ( all p < 0.001 ) , as well as with uaer , 24  h upqm ( both p < 0.001 ) , and serum creatinine ( p < 0.001 ) .
in addition , serum ang-2 was negatively correlated with gfr ( p < 0.001 ) .
similarly , urinary ang-2 was strongly correlated with urinary vegf , uaer , 24  h upqm , and serum creatinine ( all p < 0.001 ) .
a negative correlation was found between gfr and urinary ang-2 ( p < 0.001 ) .
no significant correlations were found between serum or urinary ang-2 and age , bmi , dbp , sbp , hba1c , fbs , hdl - ch , ldl - ch , total cholesterol , or triglyceride ( data not shown ) . in these subjects ,
stepwise multiple regression analyses including uaer , gfr , age , bmi , dbp , sbp , fbs , hba1c , and serum ang-2 identified that uaer ( p < 0.001 ) and gfr ( p = 0.001 ) are significant predictors of urinary ang-2 .
this study is the first to investigate urinary levels of ang-1 , ang-2 , and vegf in human type 2 dm with varying uaers .
( 1 ) urinary ang-2 increased in a stepwise manner in type 2 dm patients with various degrees of kidney damage ( normoalbuminuria , microalbuminuria , and macroalbuminuria ) .
this alteration was accompanied by increased urinary vegf , as well as early increased and later decreased urinary ang-1 .
( 2 ) urinary ang-2 levels in normoalbuminuria patients increased prior to changes in albumin levels .
( 3 ) among the angiogenic growth factors , urinary ang-2 was strongly associated with degree of albuminuria and gfr in type 2 dm patients .
alterations in the vegf and ang-1/ang-2 system have been reported to play an important role in the pathobiology of glomerular disease in dm [ 2 , 1417 , 20 , 26 ] and chronic kidney disease ( ckd ) [ 5 , 2729 ] .
most of those studies focus on circulating levels of angiogenic growth factors . in this study
, we evaluated simultaneously the levels of ang-1 , ang-2 , and vegf in both serum and urine .
we found that urinary ang-2 increased stepwise with albuminuria levels than ang-1 and vegf at a greater rate .
this observation is likely due to the tubular pathophysiological changes , which occur before the glomerular stage of disease .
this suggests that the serum and urinary ang-2 are related to subclinical tubular impairment and may be an earlier measurable marker of renal involvement before the onset of albuminuria .
ang-2 has been linked to increased microvascular permeability , and podocyte overexpression of ang-2 was shown to produce albuminuria in transgenic mice .
circulating ang-2 levels correlating positively with proteinuria have been reported in human sle   and ckd . in our type 2
dm patients , we found that albuminuria is a significant predictor for urinary ang-2 levels after adjustments were made for those possible confounders .
the association between urinary ang-2 and albuminuria suggests that upregulation of ang-2 may destabilize glomerular endothelial cells and directly or indirectly affect podocytes , leading to the deterioration of glomerular filtration barrier function [ 5 , 31 ] .
abundant ang-2 protein was detected by immunohistochemical staining in glomeruli  in endothelial cells alongside capillary loops  in renal biopsies from patients with dm ( data not shown ) . taken together
, these findings suggest that high glucose - induced glomerular endothelial damage may lead to secretion of ang-2 , and elevated expression of ang-2 in the glomerular endothelium may further increase albuminuria through the damaged glomerular filtration barrier .
the positive correlation with serum creatinine and the negative correlation with gfr suggest that increased ang-2 may be associated with the development of renal impairment .
blood ang-2 levels rise in line with the decline in renal function in type 2 dm   and ckd , and this inverse correlation may predict long - term mortality in patients with ckd [ 27 , 33 ] . in our study
these observations suggest that urinary ang-2 levels increase in parallel to the deterioration of renal function . in our study , elevated urinary ang-2 was correlated with serum ang-2 , suggesting that urinary levels of ang-2 may be representative of local production and release of ang-2 into the circulation in patients with dn .
alternatively , as dn progresses , decreased gfr leads to higher serum ang-2 , which allows greater penetration of the glomerular barrier and leads to proteinuria .
however , the multivariate analysis showed that serum ang-2 is not a predictor for urinary ang-2 .
based on the correlation between serum ang-2 and hba1c , accumulation of advanced glycation end product ( age ) in endothelial cells subjected to hyperglycemia may upregulate serum levels of ang-2 and vegf [ 34 , 35 ] .
however , no correlation was found between urinary and/or serum ang-2 and hba1c in our study .
however , the possibility can be not excluded that increased urinary ang-2 levels are a consequence of mechanisms that are unassociated with the accumulation of ages in the glomerular endothelium .
we unexpectedly found that urinary ang-1 was significantly higher in dm patients with normoalbuminuria and lower in those with macroalbuminuria than in control subjects .
this observation , also reported by rizkalla , showed upregulation of ang-1 in the early phase of the disease and progressive downregulation of renal ang-1 expression in experimental dm [ 17 , 26 ] .
ang-1 is produced by glomerular podocytes [ 36 , 37 ] and plays an important role in maintaining the structure and integrity of the glomerular filtration barrier .
further studies will seek to determine whether this increase is a short - term response of podocytes to hyperglycemia or will reduce the vascular permeability through endothelial cell glycocalyx layer   modifications or other mechanisms .
the decrease in ang-1 in subjects with macroalbuminuria suggests that ang-1 production is attenuated at the later stage of dn , which may be associated with a decreased number or function of podocytes , or both .
, we found no differences in serum ang-1 levels in groups with varying degrees of albuminuria .
however , dessapt - baradez reported decreased ang-1 levels in mice with streptozotocin - induced type 1 dm , which was accompanied by marked albuminuria , nephromegaly , hyperfiltration , glomerular ultrastructural alterations , and aberrant angiogenesis
. the differential expression pattern of ang-1 in serum may be due to the different subjects and varying stage of dn .
urinary vegf was increased in our study in all diabetic groups  even at the normoalbuminuric stage  though serum vegf was unaltered .
these findings are in agreement with previously published findings [ 10 , 14 , 18 , 40 ] .
however , some centers have reported an increase in plasma vegf in type 2 dm [ 19 , 41 , 42 ] .
these differences likely reflect the different populations being studied , as well as variations in study design .
the results of this study show that urinary ang-2 increases in a stepwise manner in type 2 diabetic patients with varying degrees of kidney damage .
ang-2 measurement in urine is a useful , noninvasive tool for the evaluation of renal involvement in the course of dm , especially in normoalbuminuric patients .
further investigations with a larger sample size and a prospective design are required to confirm the potential application of ang-2 as a useful biomarker for the early detection of diabetic nephropathy .
 
 aims . to evaluate the levels of angiopoietin-1 ( ang-1 ) , ang-2 , and vascular endothelial growth factor ( vegf ) in serum and urine , and their association with albuminuria in patients with type 2 diabetes mellitus 
 . methods . in 113 type 2 diabetic patients with normoalbuminuria , microalbuminuria , and macroalbuminuria and 30 healthy controls , 
 the levels of ang-1 , ang-2 , and vegf in serum and urine were measured by enzyme - linked immunosorbent assay ( elisa ) . results . 
 urinary and serum levels of ang-2 were significantly higher in diabetic patients with normoalbuminuria than in healthy controls . increased urinary ang-2 level was positively associated with the degree of albuminuria . 
 urinary ang-1 levels were significantly higher in normoalbuminuria patients and lower in macroalbuminuria patients than in controls . 
 the levels of urinary vegf increased in the albuminuria subgroup , though serum levels of ang-1 and vegf did not change . 
 urinary ang-2 levels were correlated positively with albuminuria and negatively with glomerular filtration rate ( gfr ) . 
 stepwise multiple regression analysis identified albuminuria ( p < 0.001 ) and gfr ( p = 0.001 ) as significant predictors of urinary ang-2 . conclusions . 
 our data suggest that urinary ang-2 is stepwise increased with renal damage in patients with type 2 diabetes mellitus and is associated with albuminuria . 