diabetic nephropathy ( dn ) represents an important cause of chronic kidney disease ( ckd ) that frequently leads to end stage renal disease ( esrd ) .
diabetes mellitus ( dm ) is a frequent disease and dn is one of its main complications .
it is appreciated that up to 40% of the patients with type i and type ii dm present dn . in western countries , diabetes is a leading cause of chronic kidney disease frequently leading to chronic renal replacement therapy ( rrt ) due to esrd . taking into account the increased incidence of both dm and of dn , the detection of early dn is of paramount importance , in order to provide appropriate therapy that prevents or slows evolution towards esrd .
, microalbuminuria represents a marker of the generalized endothelial dysfunction present in dm , linking renal involvement with cardiovascular and cerebral impairment . in time
, it has been demonstrated that microalbuminuria reflects not only glomerular injury but also tubular lesions , filtered albumin being reabsorbed at tubular level .
additionally , new biomarkers have been studied in order to identify tubular lesions in dm .
the new tubular biomarkers have been detected in both type 1 and type 2 dm early renal dysfunction that precedes microalbuminuria . at present , the assessment of early dn involves numerous biomarkers .
they span the period of normoalbuminuria that precedes microalbuminuria but also the evolution of renal involvement during microalbuminuria and macroalbuminuria .
until they are universally accepted they are analyzed in relationship with the levels of albuminuria , especially of microalbuminuria . at present , markers of inflammatory and oxidative processes accompanying dm and dn are also being assessed . since literature abounds in studies on markers highlighting renal dysfunction in different stages of the evolution of dm , we decided to restrict our study to the early phase of dn .
an update of the urinary biomarkers used in early dn is useful for establishing their role in the early diagnosis of this disease , with subsequent prophylactic and therapeutic implications .
we insist on urinary biomarkers because they are easily drawn , which allows population screening , and because they can detect tubular lesions , which occur very early in dm .
some of the biomarkers are constitutive elements of the nephron , such as markers atepithelial cell ( podocyte ) level , for example , nephrine and podocalyxin ; glomerular basement membrane level : collagen and laminin ; endothelial ( vegf ) ; tubular cell level , for example , ngal , nag , and kim .
epithelial cell ( podocyte ) level , for example , nephrine and podocalyxin ; glomerular basement membrane level : collagen and laminin ; endothelial ( vegf ) ; tubular cell level , for example , ngal , nag , and kim .
some have mixed origin ; they can originate both in tubular cells and in podocytes , for example , angiotensinogen [ 7 , 8 ] .
some are derived from the circulation , for example , transferrin , ceruloplasmin , and immunoglobulins g and m. they pass into the urine because of glomerular lesions which result in increased permeability for plasma proteins .
matheson classifies the biomarkers according to both their origin and the pathologic processes impairing the nephron : kidney damage , oxidative stress , and inflammation : biomarkers of renal dysfunction , inflammatory biomarkers ( cytokines and chemokines),oxidative stress biomarkers .
biomarkers of renal dysfunction , inflammatory biomarkers ( cytokines and chemokines ) , oxidative stress biomarkers .
another classification belongs to hong and chia who present 3 categories of biomarkers : glomerular , tubular , other proteins .it should be noted that products of metabolism in dm are also eliminated in the urine , and they can trigger toxic effects , for example , advanced glycation end products ( age ) . since we will frequently refer to microalbuminuria in presenting other biomarkers used in studying lesions of the nephron , namely , of the glomerulus and of the tubules , we will first present the main observations regarding microalbuminuria in diagnosing dn .
recent literature uses new terms , like moderately increased albuminuria for microalbuminuria and severely increased albuminuria for macroalbuminuria .
however , the classical terms of microalbuminuria and microalbuminuria continue to be in wide use , as they are more practical .
kidney biopsy examination in patients with type 1 dm and microalbuminuria most frequently finds normal histological aspects .
however , dn lesions were found in a small number of patients . according to mckenna and thompson , microalbuminuria is predictive element of the future development of end stage renal disease .
microalbuminuria can regress towards normoalbuminuria , it can persist as such , or it can progress towards albuminuria [ 14 , 15 ] . the evolution of microalbuminuria towards macroalbuminuria is usually related to arterial hypertension and reduced gfr , an important part being generally played by risk factors .
persistent microalbuminuria is related to future development of end stage renal disease and to cardiovascular risk .
it should be noted that diminution of gfr usually occurs after the development of microalbuminuria , but there are situations when even normoalbuminuria is accompanied by diminution of the gfr .
microalbuminuria is an important biomarker in type 2 dm , being frequently used in population - based screenings .
regarding the prevalence of microalbuminuria in type 2 dm patients we highlight a review of newman et al .
they found microalbuminuria in 26% of the patients with ten - year duration of the disease . a study on 24,000 patients found that asian and hispanic patients with type 2 dm present more often microalbuminuria ( 43% ) than whites ( 33% ) . in china , shanghai , microalbuminuria has a prevalence of 41% among patients with type 2 dm .
it can regress towards normal values , it can progress towards macroalbuminuria , or it can remain unchanged . in a study on 216 patients , araki et al
. found , after a 6-year follow - up , regression of microalbuminuria in 51% cases and progression to severely increased albuminuria in 28% cases .
the risk of progression to severely increased albuminuria is higher in patients with microalbuminuria as compared to patients with normoalbuminuria .
the diminution of gfr is also higher in patients with severely increased albuminuria than in those with microalbuminuria .
glycemic control , ace inhibitors , and arbs for blood pressure control play an important role in the evolution of microalbuminuria .
it should be mentioned that microalbuminuria has been considered a glomerular biomarker . to date , emerging data point to the role of the tubules in producing microalbuminuria [ 24 , 25 ] . as such
we did not include this marker among glomerular biomarkers but approached it separately , according to its potential role as both a glomerular and tubular biomarker .
transferrin is a protein with a molecular weight of 76.5  kda . because of its low molecular weight and its less ionic load it filters easily through the glomerular barrier .
as increased urinary transferrin was found in type 2 dm normoalbuminuric patients , concomitantly with urinary ceruloplasmin and immunoglobulin g , preceding microalbuminuria , it could be considered a biomarker of early dn . in microalbuminuric patients the levels of urinary transferrin increase .
they also increase in patients with type 2 dm with vascular complications : coronary artery disease , diabetic retinopathy , and so forth .
patients with initial high levels of urinary transferrin excretion will develop microalbuminuria more frequently than those with normal levels .
it is an anionic plasma protein with a molecular weight of 150  kda that crosses the glomerular barrier with difficulty .
as presented above , urinary igg can be secreted before the stage of microalbuminuria , concomitantly with increased values of urinary transferrin , urinary ceruloplasmin , and urinary orosomucoid .
it was also found in some type 2 dm patients with normoalbuminuria , arguing in favour of its use for early detection of renal lesions , even prior to albuminuria : ceruloplasmin could have in type 2 dm patients a dn predictive effect , similar to urinary transferrin and urinary immunoglobulin g . according to yamazaki et al .
the urinary ceruloplasmin excretion rate ( cer ) and clearance of ceruloplasmin increase in parallel with the progression of albuminuria . in fact , in type 2 dm patients there could exist a parallelism between increased values of urinary transferrin , urinary immunoglobulin g , and urinary ceruloplasmin .
type iv collagen is a component of the glomerular basement membrane and of the mesangial matrix . in dn , lesions are produced both at glomerular capillary level and at mesangial level .
its excretion in urine might serve as early indicator of renal injury associated with dn .
increased levels of type iv urinary collagen are reported for normoalbuminuric patients with type 1 dm .
other authors also report increased excretion of type iv collagen and of laminin in patients with type 1 dm .
high urinary type iv collagen excretion was also reported in normoalbuminuric patients with impaired glucose tolerance .
urinary type iv collagen could reflect morphological renal alterations in patients with type 2 dm .
a relationship between the severity of histological lesions and urinary type iv collagen was reported in patients with type 2 dm .
it could also allow both detection of early dn in patients with type 2 dm and differential diagnosis with glomerulonephritis , where its levels are low .
its urinary excretion is increased in normoalbuminuric type 2 dm patients , being correlated with nag ( n - acetyl - beta - d - glucosaminidase ) and alpha-1-microglobulin excretion .
glycosaminoglycans are components of the glomerular basement membrane as well as of the extracellular matrix . in dm alterations of these
urinary glycosaminoglycans are associated with other tubular biomarkers , for example , tamm - horsfall protein , which expresses a distal tubular dysfunction in diabetic patients .
it is a biomarker related to lesions of the glomerular capillary walls and reflects their increased permeability .
it is mainly considered to predict renal lesions , being less relevant as an early marker of dn .
these were studied only sporadically , without sufficient data to support their use as markers of early dn .
although the use of urinary glomerular biomarkers has not become current practice yet , glomerular biomarkers have been reported in some normoalbuminuric patients , leading to the conclusion that albuminuria might not represent the most sensitive glomerular biomarker .
however , their clinical applicability needs to be confirmed in high - quality validation studies .
tubular biomarkers have shown that tubular dysfunction can be present early in dn , sometimes preceding glomerular injury .
these biomarkers have proven highly sensitive as compared to microalbuminuria , which is considered the gold standard biomarker of dn .
in fact , presently , microalbuminuria is regarded not only as a glomerular biomarker , but also as a tubular one .
neutrophil gelatinase - associated lipocalin  is a glycoprotein present in the kidneys at tubular cell level and is considered to be protective against renal damage .
urinary ngal is a biomarker used in assessing tubular lesions in dm , its increased values being present even in the initial phases of the disease , namely , in normoalbuminuric patients .
thus , in type 1 dm high urinary ngal can precede microalbuminuria [ 50 , 51 ] .
urinary ngal had high values in type 2 dm patients with normoalbuminuria , increasing progressively in patients with microalbuminuria and macroalbuminuria .
the values of kim-1 ( kidney injury molecule-1 ) increased in parallel , indicating an early and progressive lesion . however , fu et al .
reported in type 2 dm patients who present hyperfiltration and increased values of urinary ngal , as well as of urinary kim-1 , as compared to the values of patients with normal gfr .
urinary ngal in type 2 dm patients could have a role in predicting the evolution of disease .
urinary alpha-1-microglobulin is a serum protein with low molecular weight ( 27-kda ) , which allows it to get easily filtered through the glomerular capillary wall .
tubular dysfunction leads to alteration of reabsorption with increased excretion in the urine . in a cross - sectional study , hong et al .
analyzed 590 type 2 dm patients and found that 33.6% patients with normoalbuminuria presented increased values of urinary alpha-1-microglobulin , a fact that could be explained by tubular injury that precedes the occurrence of microalbuminuria , being a more sensitive and an earlier urinary biomarker .
this is why assessments of alpha-1-microglobulin are associated with the assessment of other urinary biomarkers , urinary albumin included .
reported high values of urinary alpha-1-microglobulin in normoalbuminuric patients , a fact pleading for an early tubular injury in type 2 dm in this stage .
they did not find correlations between urinary alpha-1-microglobulin , beta-2 microglobulin , and the albumin / creatinine ratio with plasma asymmetric dimethyl - arginine .
reported in normoalbuminuric type 2 dm patients high values of urinary kim-1 , which indicates lesions of the proximal tubule in early stages of the disease .
de carvalho et al . reported in type 2 dm normoalbuminuric patients high values of kim-1 , these values increasing progressively in patients with microalbuminuria and macroalbuminuria .
moreover , kim-1 presents higher elimination in type 2 dm patients with hyperfiltration than in patients with normal glomerular filtration .
these biomarkers  kim-1 and ngal  could plead for a deleterious lesional effect of hyperfiltration on the proximal tubule .
. however could not demonstrate a value of urinary kim-1 that could be predictive of the evolution of glomerular function ( gfr ) in patients with type 1 dm . according to nielsen et al .
nag is an enzyme located in the lysosomes of proximal tubular cells . in case of dysfunction , namely , of injury of proximal tubular cells
, nag is eliminated into the urine in higher quantities , being a sensitive tubular biomarker .
elevated serum cys c levels and urinary nag activities were found only in normoalbuminurics , not in controls .
, did not find that urinary nag has clinical significance as an early biomarker of dn . in type 2
consider that urinary nag is the most sensitive biomarker for detecting early damage in diabetic patients .
the renin angiotensin aldosterone system ( raas ) is involved in the pathogenesis of dn .
the constitutive elements of raas are present at kidney level , defining a local raas .
urinary angiotensinogen in normoalbuminuric type 2 dm patients is higher than in controls and it increases progressively in microalbuminuric and especially in macroalbuminuric patients .
kim et al . did not confirm these observations in a study on type 2 dm patients .
they found that the values of urinary angiotensinogen are not different from those of the controls , in normoalbuminuric and microalbuminuric type 2 dm patients , but higher values were described in macroalbuminuric patients .
these observations point to the need of further studies necessary for the validation of this biomarker . increased urinary angiotensinogen could represent a risk factor in renal and cardiovascular complications . since activation of raas could intervene in the evolution of dn , administration of ace - i is recommended .
at the same time , urinary angiotensinogen could be a marker for assessing the renoprotective effects of alogliptin to type 2 dm patients .
assessment of gfr by means of cystatin c is considered to be a method that is not influenced by body mass , being comparable and even better than methods using serum creatinine .
patients with microalbuminuria present higher values of urinary cystatin c than those without microalbuminuria , urinary cystatin c having a predictive role for the progression of diabetic nephropathy ( dn ) .
urinary cystatin c level could be an independent factor for identifying renal dysfunction in type 2 dm patients with normoalbuminuria , including patients with gfr < 60/ml / min/1.73  m .
find a significant positive correlation between serum cystatin c , urinary nag , lacticodehydrogenase , alkaline phosphatase activities , and serum creatinine levels .
serum and urinary cystatin c are useful biomarkers for assessing early nephropathy in type 2 dm .
urinary l - fabp is a protein with low molecular weight expressed in the cytoplasm of human proximal tubular cells .
was found in type 1 dm patients who presented normoalbuminuria , having a predictive role regarding the progression towards microalbuminuria and of microalbuminuria towards macroalbuminuria .
patients with type 2 dm with normoalbuminuria also presented high levels of urinary l - fabp , this protein being considered as a useful biomarker for diagnosing early diabetic nephropathy .
in fact , urinary l - fabp has been confirmed as a tubular biomarker by the ministry of health and welfare in japan .
the values of urinary l - fabp increase with the decline of renal function . although some authors , like chou et al . , do not ascribe a predictive role to urinary l - fabp in type 2 dm patients , others , like panduru et al .
, consider that urinary l - fabp is an independent predictor of the progression of dn .
nephrine is a transmembrane protein in the structure of the slit diaphragm . in dm podocyte dysfunction
nephrinuria was also reported in some normoalbuminuric type 2 dm patients [ 62 , 90 ] .
dysregulation of nephrine in podocytes in dn could lead to nephrinuria in normoalbuminuric patients , preceding microalbuminuria . in albuminuric patients ,
nephrinuria is positively correlated with albuminuria and negatively correlated with gfr , being a biomarker of dn in other phases of dm as well .
podocyte impairment in dm involves not only nephrine but also other podocyte elements , for example , vegf .
they manage to show , in most cases , that microalbuminuria does not represent a reliable biomarker for diagnosing incipient lesions of dn .
however , up to now , none of these biomarkers has been established as gold standard for the identification of early dn .
dm is accompanied by chronic inflammatory processes affecting the whole body , the kidneys included .
urinary tnf alpha presents in type 2 dm patients with microalbuminuria and macroalbuminuria higher values than in patients with normoalbuminuria .
cherney et al . analyzed in a complex study on normoalbuminuric type 1 dm patients forty - two urinary cytokines / chemokines .
they found that the urinary level of il6 and il8 , the platelet - derived growth factor , and rantes are not altered in patients with normal albumin - creatinine ratio .
consider that these markers could have a role in assessing the risk of dn in patients with type 1 dm . in type 1
dm patients , renal hyperfiltration is related to increased excretion of inflammatory cytokines / chemokines .
tashiro et al . found in type 2 dm patients that il8 is high in early stages of dn and mcp-1 increases in advanced stages .
a study on type 2 dm patients with normoalbuminuria and microalbuminuria found higher values of il8 , ip10 , mcp-1 , g - csf , eotaxin , and rantes in patients with microalbuminuria than in normoalbuminurics or in controls .
ibrahim and rached also found that urinary mcp-1 is higher in patients with microalbuminuria than in normoalbuminurics or healthy controls .
urinary orosomucoid has higher values in type 1 dm patients with normoalbuminuria than in controls .
type 2 dm patients presented increased excretion of orosomucoid in the urine , in parallel with the excretion of immunoglobulin g , ceruloplasmin , and transferrin .
appreciate that orosomucoid is a significant independent factor for diabetic microvascular complications and can be considered as an early marker of renal injury .
urinary markers of inflammation are useful for assessing inflammatory processes in dn , even in early stages .
it is eliminated into the urine without being metabolized . at present , it is considered a marker for oxidative stress . after a 5-year follow - up , hinokio et al .
find that 8-oxodg in urine is a useful clinical marker to predict the development of diabetic nephropathy in diabetic patients .
there was a significant progression of diabetic nephropathy in the patients with higher excretion of 8-oxodg in urine compared with the patients with moderate or lower excretion of 8-oxodg .
reported increased excretion of 8-ohdg in type 1 dm patients 9 years after the onset of disease , mainly related to poor glycemic control .
heart fatty acid binding protein ( h - fabp ) is a marker of distal tubular damage . in a study on a cohort of type 1 and type 2 dm patients and an assessment of their markers of glomerular lesions ( igg ) , markers of proximal tubular lesions ( urinary kim-1 , nag , ngal , and cystatin ) , and a marker of distal tubular lesions ( urinary h - fabp ) in relationship with albuminuria and gfr , nauta et al .
reported higher values of urinary nag , ngal , and h - fabp in normoalbuminurics than in controls . on the other hand ,
age eliminated in the urine induce a toxic tubular effect producing tubular dysfunction . in type 2
dm patients with normoalbuminuria high values of urinary alpha-1-microglobulin and of urinary kim-1 were found secondary to tubular dysfunction prior to the onset of microalbuminuria . at the same time , urinary age were high , being correlated with these markers .
found in type 2 dm patients high values of urinary age in 50% of the patients with normoalbuminuria and in 85% of those with microalbuminuria .
pentosidine , a component of age , is a biomarker for their formation and accumulation .
podocyte lesions appear during dm and dn , respectively , the disease being considered a podocytopathy as mentioned above .
the assessment of podocyte injury can be accomplished by monitoring the number of podocyte cells in the urine or , more precisely , by means of using podocyte urinary biomarkers ( podocalyxin and nephrine ) .
a study on dm patients found that the values of the number of urinary podocytes in normoalbuminuric patients are not significantly different from those of controls . in patients with microalbuminuria and nephrotic syndrome ,
urinary podocalyxin originates in the podocyte apical surface , occurring in vesicle form . in dm patients ,
the podocalyxin level presented higher levels in patients with microalbuminuria than in patients with normoalbuminuria .
another study on dm patients found high values of urinary podocalyxin in more than half of the patients with normoalbuminuria , these values being higher in patients with microalbuminuria and macroalbuminuria .
urinary podocalyxin is correlated with the values of urinary nag and of urinary beta 2 microglobulin .
consider that urinary podocalyxin can be an early biomarker for detecting early podocyte injury in patients with dm .
zheng assessed the urinary microrna profile of podocyte - associated molecules ( synaptopodin , podocalyxin , cd2-ap , -actin4 , and podocin ) as biomarkers in patients with normoalbuminuria , microalbuminuria , and macroalbuminuria and they reported its increase during the progression of dn .
urinary vegf was detected in type 2 dm patients , being correlated in these patients with urinary alpha-1-microglobulin , a biomarker for proximal tubular lesions .
kim et al . found that vegf is excreted at higher values than controls in normoalbuminuric type 2 dm patients .
fetuin a is glycosylated glycoprotein was considered an inhibitor for ectopic calcium deposition and promoter of insulin resistance .
it was found that elevated urinary fetuin a excretion is a risk for development of diabetic nephropathy .
urinary retinol - binding protein is a low molecular weight protein that was found to have high urinary values ( together with nag ) in normoalbuminuric patients , reflecting tubular dysfunction in early dn .
the value of serum retinol - binding protein 4 as a biomarker in assessing the severity of coronary artery disease is to be mentioned .
urinary retinol - binding protein 4 as a biomarker in assessing dn needs further studies .
urinary heme oxygenase-1 was found in type 2 dm patients before the onset of significant albuminuria , thus being a possible biomarker of early dn .
periostin is a cell adhesion molecule which is not normally present in kidneys . in tubulointerstitial lesions it is however expressed in the kidneys , being eliminated in the urine .
this is why urinary periostin could be used as a marker of injury at this level . since high levels of periostin
can be identified in dm patients before significant albuminuria , periostin could represent a marker of diabetic renal injury .
urinary alpha klotho presents higher values in normoalbuminuric type 2 dm patients than in controls .
it can also be a marker of diabetic injury . analyzing a group of normoalbuminuric , microalbuminuric , and macroalbuminuric type 2 dm patients , sun et al .
noted that the urinary level of microvesicle - bound dipeptidyl peptidase - iv is related to the severity of dn .
recent studies point to the usage of urine - specific microrna as a biomarker for early stages of dn . analyzing the studies in the literature , yang et al .
issued the hypothesis that urine - specific microrna would be a marker that can be used in the early stages of dn .
recently , argyropoulos highlighted the predictive role of urinary microrna regarding microalbuminuria in type 1 dm .
adipokine zinc - alpha-2 glycoprotein is assigned to the major histocompatibility complex class i of proteins .
also appreciate adipokine zinc - alpha-2 glycoprotein as a novel urinary biomarker for normoalbuminuric diabetic nephropathy . at present proteomic investigations
are engaged in identification of new urinary biomarkers to be used in the early diagnosis of dn .
in fact , proteomics studies noted the fact that microalbuminuria is not a perfect biomarker for early detection of dn [ 130 , 131 ] .
urinary proteomics begins to stand out as a noninvasive method of detecting early dn . among proteomics studies on diagnosing dn
, who reported that collagen fragments were a prominent biomarker 35 years before the onset of microalbuminuria .
a potential role is also attributed to exosome proteomics for identifying new biomarkers for dn .
showed a panel of 3 proteins which is differentially present in urinary exosomes from dn patients .
urinary proteomic analysis can have an important role in the implementation of new biomarkers in dn . at present , the prospect of discovering new biomarkers in dm and dn respectively is incumbent both on proteomics and on genomics , transcriptomics , and metabolomics .
microalbuminuria , although frequently contested as a biomarker of early dn , is used so far as reference biomarker in assessing other urinary biomarkers in early dn . until present there is no other biomarker that can substitute in practice microalbuminuria , the new biomarkers being sustained by limited studies and requiring validation .
the concomitant assessment of several urinary biomarkers in relationship with microalbuminuria could represent at present a method of diagnosing early dn .
the great progress in discovering new biomarkers could lead to the development of an  ideal  urinary biomarker to detect early diabetic dn in the future .
progresses in the field of urinary biomarkers in dn , promising both in proteomics and in other modern techniques , develop remarkably at present .
 diabetic nephropathy ( dn ) is a frequent and severe complication of diabetes mellitus ( dm ) . 
 its diagnosis in incipient stages may allow prompt interventions and an improved prognosis . towards this aim , biomarkers for detecting early dn can be used . 
 microalbuminuria has been proven a remarkably useful biomarker , being used for diagnosis of dn , for assessing its associated condition  
 mainly cardiovascular ones  and for monitoring its progression . 
 new researches are pointing that some of these biomarkers ( i.e. , glomerular , tubular , inflammation markers , and biomarkers of oxidative stress ) precede albuminuria in some patients . 
 however , their usefulness is widely debated in the literature and has not yet led to the validation of a new  gold standard  
 biomarker for the early diagnosis of dn . 
 currently , microalbuminuria is an important biomarker for both glomerular and tubular injury . 
 other glomerular biomarkers ( transferrin and ceruloplasmin ) are under evaluation . 
 tubular biomarkers in dn seem to be of a paramount importance in the early diagnosis of dn since tubular lesions occur early . 
 additionally , biomarkers of inflammation , oxidative stress , podocyte biomarkers , and vascular biomarkers have been employed for assessing early dn . 
 the purpose of this review is to provide an overview of the current biomarkers used for the diagnosis of early dn . 