Cystatin C CysC based assays in estimating GFR for clinical trials in DN offer an alternative approach due to the complexity and time-consuming nature of other reference test methods.

This Numerous studies have validated CysC as a marker of renal function[ 26 - 28 ]. Its levels are well correlated with GFR and unlike serum creatinine, are unaffected by muscle mass. These benefits should, however, be taken into consideration alongside the higher cost of the immunoassay and the greater intraindividual variability[ 28 ] compared to serum creatinine Formulae for estimating GFR including both creatinine and CysC have been proposed but to date have not been proven to enhance precision in identifying and monitoring early stages of GFR decline in diabetes[ 32 ].

Glomerular damage increases permeability to plasma proteins resulting in their excretion in the urine. In addition, abnormalities of extracellular matrix synthesis and degradation in kidney disease can lead to increased urinary excretion of matrix proteins, reflecting glomerular injury.

Although albumin excretion remains the current gold standard marker of glomerular damage in the clinical setting, a number of other proteins have been proposed as useful indicators of early glomerular damage. Transferrin is a plasma protein with a slightly greater molecular weight It is also less ionic than glycosylated albumin and thus less easily repelled by glomerular basement membrane polyanion[ 34 ].

Elevated urinary transferrin excretion has been demonstrated in patients with diabetes compared with healthy controls, even in in the absence of albuminuria[ 35 ]. Transferrinuria has been shown to correlate with UAE and to increase in parallel with it[ 36 ].

In a 24 mo follow up study it has been demonstrated that increased urinary transferrin excretion predicted development of MA in a cohort of normoalbuminuric type 2 diabetic patients independent of age, diabetes duration, blood pressure, HbA1c and baseline lipid levels[ 33 ].

Elsewhere it has also been shown that transferrinuria predicted development of MA at 5 years follow up[ 36 ]. Transferrin has also been proposed as a mediator of tubular toxicity, as its reabsorption results in release of reactive iron in proximal tubular cells promoting formation of hydroxyl radicals[ 37 , 38 ].

Studies have reported correlations between urinary transferrin excretion and other microvascular diabetic complications such as retinopathy[ 38 ]. Taken together, the above data suggest that transferrinuria may serve as a sensitive indicator of early proteinuria and increased vascular permeability.

Accumulation and altered distribution of basement membrane components is one of the structural hallmarks of DN and these changes precede the development of MA[ 39 ]. Type IV collagen is a normal constituent of mesangial matrix as well as tubular and glomerular basement membranes, with molecular weight of kDa.

Both serum and urine levels have been shown to be elevated in patients with diabetes[ 40 ]. Urinary type IV collagen excretion has been shown to correlate closely with degree of UAE, as well as diabetes duration, blood pressure and serum creatinine[ 41 , 42 ].

Significantly higher excretion of type IV collagen has been found even in normoalbuminuric diabetic patients as well as patients with impaired glucose tolerance, suggesting that this may serve as an early indicator of DN, preceding the onset of MA[ 42 , 43 ].

In addition, type IV collagen excretion has been found to decrease with improved glycaemic control, suggesting that this marker is also reversible in early disease[ 44 ].

Type IV collagen may also play a role in differentiating DN from other non-diabetic kidney diseases, as the ratio of type IV collagen to albumin has been found to be significantly higher in DN in comparison to other glomerulopathies[ 40 ].

Ceruloplasmin is a kDa acute phase protein with well characterised functions in the metabolism of copper and iron[ 36 ]. It has been suggested that ceruloplasmin may leak through glomerular capillary walls in DN and evidence confirms increased excretion in both impaired glucose tolerance and diabetes compared with healthy controls[ 36 , 45 ].

Increased urinary ceruloplasmin excretion has also been demonstrated in normoalbuminuric patients with diabetes[ 45 ]. In addition, urinary ceruloplasmin excretion appears to parallel UAE[ 31 , 46 ]. In a 5 year follow up study, it was demonstrated that increased urinary ceruloplasmin excretion predicted development of MA in normoalbuminuric type 2 diabetic patients[ 36 ].

Improved glycaemic control appears to reverse this increase[ 46 ]. Fibronectin is a high molecular weight kDa plasma glycoprotein mainly produced by endothelial cells and fibroblasts which plays a role in cell adhesion to vascular endothelium[ 35 ]. Fibronectin biosynthesis is increased in patients with diabetes and studies have suggested that plasma levels correlate with retinopathy and MA[ 47 ].

Increased urinary levels of fibronectin have been found in type 2 diabetic patients in comparison with healthy controls, as well as in subjects with MA compared to normoalbuminuric subjects[ 47 ].

However, there is only a weak positive correlation between plasma fibronectin and urinary albumin levels perhaps limiting its potential usefulness as an early marker of DN[ 47 ], and there is no published evidence comparing urinary fibronectin with UAE in terms of predictive value for diabetic nephropathy.

Plasma proteins of low molecular weight are excreted in increased quantities in the urine due to deficient tubular reabsorption or increased secretion by tubular epithelial cells.

Similarly, urinary enzymes are thought to be sensitive markers of tubular damage as they are not filtered at the glomerulus due to their high molecular weight[ 31 , 36 ].

Neutrophil Gelatinase-Associated Lipocalin NGAL is a small molecule of 25 kDa belonging to the lipocalin superfamily. These proteins play a role in binding and transporting small hydrophobic molecules, apoptosis and immune regulation. NGAL is stored mainly in the specific granules of neutrophils and also expressed at low levels in several other human tissues[ 48 , 49 ].

NGAL shows significant promise in the diagnostic and clinical setting as a marker of acute kidney injury[ 48 ] and is thought to also play a renoprotective role as a mediator of tubular cell proliferation[ 49 ].

Studies have confirmed an association between NGAL and obesity, insulin resistance and hyperglycaemia in human subjects[ 49 ]. Urinary NGAL concentration has been found to be increased in diabetic subjects compared with healthy controls[ 50 ] and to correlate negatively with eGFR, and positively with CysC, serum creatinine and urea in patients with type 2 diabetes[ 48 ].

Significant increases in urinary NGAL concentration have been demonstrated from normo- to micro- to macroalbuminuric groups of patients with type 1 diabetes[ 51 ]. Similar results have been published in a study of type 2 diabetic patients[ 52 ].

Urinary NGAL correlates positively with glomerular hyperfiltration early in the clinical course of diabetes[ 53 ] and higher values have been found to be associated with enhanced decline in eGFR in type 2 diabetes patients with proteinuria, although this correlation was no longer statistically significant after adjustment for factors including systolic blood pressure, HbA1c and diabetes duration[ 53 ].

However, other prospective studies have not confirmed these associations[ 54 , 55 ] and further investigation of the role of urinary NGAL in DN is required.

Kidney injury molecule 1 KIM1 has been shown to be a marker of tubular damage in various chronic kidney diseases[ 56 , 57 ]. This type 1 cell membrane glycoprotein is expressed on the apical membrane of proximal tubule cells and is involved in the phagocytosis of damaged cells in the proximal tubules[ 52 ].

Expression is undetectable in normal healthy kidneys but mRNA and protein are markedly upregulated in acute kidney injury[ 58 ]. In a cross-sectional study urinary KIM1 excretion has been found to be increased in diabetic patients compared to healthy controls.

A weak but significant increase of urinary KIM1 concentration was noted with increasing degree of UAE[ 50 ]. Increased urinary KIM1 excretion has also been shown in type 2 diabetics with glomerular hyperfiltration[ 52 ].

In a 3 year prospective interventional study, high baseline levels of urinary KIM1 were found to be associated with faster decline in GFR in type 1 diabetes with DN; an association no longer significant after adjustment for traditional risk markers including blood pressure and glycaemic control[ 58 ].

Similar findings have been described in type 2 diabetes populations[ 55 ]. Studies have shown that treatment with renin angiotensin system RAAS blocking agents reduced urinary KIM1 excretion in parallel to reductions in blood pressure and UAE[ 59 ].

In addition, low baseline urinary KIM1 excretion is strongly associated with regression of MA during a 2 year follow up period, independent of clinical characteristics[ 57 ]. This supports the hypothesis that KIM1 is a good marker of active tubular damage, rather than pre-existing scarring[ 58 ].

N-acetyl-b-d-glucosaminidase NAG is a lysosomal enzyme which is predominantly located in the renal tubules. It cannot be filtered from blood through an intact glomerular membrane due to its high molecular weight kDa , thus its activity detected in urine reflects tubular dysfunction.

Urinary NAG activity is increased in a variety of tubulointerstitial diseases. It is elevated in populations with diabetes compared to controls, even in normoalbuminuric patients[ 33 , 53 ].

It correlates with the degree of UAE and excretion of transferrin and creatinine[ 60 - 62 ]. Although no significant association has been found between urinary NAG and glomerular hyperfiltration[ 52 ], prospective follow up studies have shown that higher levels of NAG at baseline are predictive of subsequent DN[ 63 ].

In addition, lower baseline NAG levels are significantly associated with regression of MA at follow up[ 57 ]. Finally, significant increases in NAG excretion have been reported in type 2 diabetic patients with both micro- and macrovascular complications[ 63 - 65 ] and in fact NAG levels have been attributed comparable diagnostic value to UAE in this regard[ 65 ].

Liver-type fatty acid binding protein L-FABP is a low molecular weight 15 kDa intracellular carrier protein that is expressed in the proximal tubule and liver[ 66 , 67 ]. It is produced in response to tubulointerstitial compromise, and thus has potential as a marker of structural and functional renal tubular damage[ 67 ].

In a cross sectional study of patients with type 1 diabetes and varying degrees of UAE, urinary L-FABP levels were significantly higher compared to healthy controls. The levels increased with increasing degree of albumin excretion.

Intervention with Lisinopril was associated with significant reductions in UAE and urinary L-FABP excretion in those with diabetes[ 68 ]. However, there is no correlation between L-FABP and rate of change of eGFR in patients with type 2 diabetes[ 54 ]; therefore further studies are needed to elicudate its value as a predictive marker for DN.

Low molecular weight proteins are freely filtered at the glomerulus and some have been used as markers of tubular damage in various renal diseases[ 36 ].

β2-microglobulin β2MG is a Urinary β2MG excretion is known to be elevated in patients with reduced GFR and some evidence links β2MG with tubular injury[ 69 ].

β2MG has also been associated with macrovascular complications in type 2 diabetes[ 63 ]. However, its diagnostic utility is limited by its poor stability at acidic pH[ 70 ].

The stable microprotein αmicroglobulin A1M may offer an alternative means of evaluating tubular function. This 26 kDa glycoprotein is freely filtered at the glomerulus and almost completely reabsorbed in the proximal tubules, thus even minor degrees of proximal tubular dysfunction lead to increased urinary A1M excretion[ 71 , 72 ].

Urinary A1M excretion has been shown to be greater in patients with type 2 diabetes compared to healthy controls[ 33 , 42 ]. A1M levels have also been found to correlate with diabetes duration and degree of diabetes control[ 63 , 71 ].

There is evidence that urinary A1M excretion significantly increases with degree of MA in type 2 diabetes[ 71 - 73 ]. However, Hong et al [ 72 ] found in a cross-sectional study that although UAE and A1M were directly related, in some patients one could be present in the absence of the other, suggesting that urinary A1M as a measure of tubular function may be complementary to MA as a measure of glomerular function in assessment of early DN.

Retinol binding protein RBP is another low molecular weight protein 21 kDa which is freely filtered at the glomerulus and almost completely reabsorbed in the proximal tubule; as such its presence in the urine is indicative of even very minor degrees of tubular dysfunction[ 33 ].

Increased urinary RBP excretion has been described in diabetic patients compared to controls, even in patients with normal UAE[ 16 , 70 , 73 ]. RBP levels have also been found to correlate with both micro- and macrovascular complications in type 2 diabetic patients[ 64 , 74 ].

RBP, therefore, may also have a complementary role in early detection of DN together with biomarkers of glomerular damage such as UAE or transferrin.

Immunoglobulin free light chains FLCs kappa and lambda undergo similar glomerular filtration and near complete tubular reabsorption[ 36 ]; consequently their presence in the urine can also be indicative of proximal tubular dysfunction[ 75 ]. However, as yet there is little further published evidence regarding use of FLCs as a predictive tool for early detection of DN.

Oxidative stress is thought to be one of the key mediators of vascular complications of diabetes. Generation of reactive oxygen species ROS as a result of hyperglycaemia contributes to development of diabetes complications through sorbitol accumulation, formation of advanced glycation end products AGE and activation of protein kinase C[ 77 , 78 ].

Since it is excreted into urine without being further metabolised its urinary concentration serves as an index of oxidative stress[ 79 ]. Increased concentrations of 8-OHdG have been described in both urine and mononuclear cells of diabetic patients[ 80 ], and urinary excretion appears to correlate closely with the severity of DN and retinopathy as well as HbA1c[ 81 ].

In a prospective longitudinal study of Japanese diabetic patients, urinary 8-OHdG excretion at baseline was associated with later development of DN after 5 years of follow up[ 81 ], indicating its potential as a clinical predictive marker.

AGE have been associated with the pathogenesis of diabetes complications[ 82 ]. AGE-modified proteins generally undergo glomerular filtration and subsequent catabolism at the proximal tubule, thus it seems intuitive that the presence of AGE-modified protein fragments in urine may also herald early tubular dysfunction.

Pentosidine is one of the major molecular structural components of AGEs and acts as a marker of their formation and accumulation[ 83 ].

Urinary excretion of Pentosidine has been shown to be higher in patients with diabetes compared to healthy controls[ 84 ]. Increased urinary and plasma Pentosidine levels have been demonstrated in patients with DN[ 85 ].

More recently its potential as a marker of microvascular complications of diabetes has been shown with associations between serum Pentosidine levels and diabetic retinopathy, hypertension and hyperlipidaemia in addition to DN[ 86 ]. Although initially no correlation between Pentosidine levels and UAE were reported[ 84 ], recent publications have challenged this finding; one study reported significantly increased serum Pentosidine levels in diabetes patients with MA compared to normoalbuminuric controls[ 87 ] and another study found increased median urinary Pentosidine excretion in diabetes patients with macroalbuminuria compared to controls[ 62 ].

Evidence is accumulating that immune and inflammatory mechanisms also play a role in the pathogenesis of DN[ 88 ], as cause rather than consequence of disease[ 89 ]. Individuals who progress to DN appear to display features of low grade inflammation for years before clinically detectable disease[ 90 , 91 ].

As a result, cytokines and other components involved in the process of inflammation and endothelial damage have attracted attention as potential markers of DN. Orosomucoid, or αacid glycoprotein AGA is a single chain polypeptide produced mainly by the liver.

It is released in response to inflammation under the stimulation of cytokines such as interleukin-6 IL-6 and tumour necrosis factor-α TNF-α [ 92 ].

AGA levels have been found to be associated with ischaemic heart disease, lung cancers and diabetes[ 92 , 93 ]. It has been suggested that high AGA levels may predict the development of type 2 diabetes[ 94 ].

In a cross sectional study of outpatients with type 2 diabetes and no known cardiovascular disease, serum AGA levels were found to correlate significantly with UAE[ 95 ]. In addition, proteomic work has identified urinary AGA as an independent risk factor for DN[ 96 , 97 ].

Urinary AGA excretion appears to increase in parallel with UAE and data indicate that urinary AGA is elevated in the early stages of DN[ 95 ].

The potential predictive value of urinary AGA in DN has been shown[ 98 ] but further work is needed to determine whether AGA could be used as a biomarker of disease development and treatment response. TNF-α and IL-6 are two major pro-inflammatory cytokines that stimulate the acute phase response by triggering production of other proteins such as CRP and AGA[ 89 , 93 ].

Patients with DN have higher serum and urinary concentrations of TNF-α than healthy controls or normoalbuminuric subjects[ 99 , ]. Urinary TNF-α excretion has also been shown to correlate with NAG excretion, a marker of severity of tubular damage[ 99 ].

TNF-α mediates its effects via two distinct receptors, TNF receptor 1 TNFR1 and TNFR2, which are both membrane bound and also can be found in serum in soluble form[ ].

Serum levels of both these receptors have been shown to correlate with GFR in diabetic patients independently of albuminuria status[ ]. More recent data suggest that serum concentrations of TNFR1 and TNFR2 have potential as predictors of progressive renal disease in diabetes[ , ].

Patients with TNFR levels in the highest quartile show significantly elevated cumulative incidence of reaching stage CKD over 12 years of follow up compared with those in the lower quartiles.

This has been shown in both type 1 and type 2 diabetes, in the presence or absence of proteinuria[ , ]. Serum IL-6 has been shown to be elevated in patients with diabetes compared to control subjects, as well as between normo-, macroalbuminuric and overtly proteinuric patient groups[ , ].

In addition, IL-6 has been linked to glomerular basement membrane thickening[ ]. Furthermore, association has been demonstrated between circulating levels of both TNF-α and IL-6 and micro- and macrovascular complications of diabetes[ ]. Vascular endothelial growth factor VEGF is a potent cytokine that induces angiogenesis and increases endothelial permeability[ ].

It adversely affects the glomerular filtration barrier by enhancing its permeability to macromolecules and exacerbating proteinuria[ ].

Urinary VEGF excretion appears to be elevated in patients with diabetes, even at the normoalbuminuric stage[ , ]. A significant increased urinary excretion of VEGF in micro- and macroalbuminuric type 1 diabetic patients has been demonstrated[ ].

Work in type 2 diabetes demonstrated that urinary VEGF concentration increases with DN stage. This has not been demonstrated in plasma[ ]. However, baseline serum VEGF level did appear to be predictive of subsequent DN in a follow up study of children with type 1 diabetes[ ].

In addition, both serum and urinary VEGF levels have been shown to be elevated in patients with diabetic retinopathy, although the sensitivity of urinary detection was poor[ ]. Taken together, these findings led to the proposal that plasma VEGF is a reliable marker of generalised vascular dysfunction and retinopathy, whereas urinary concentration may serve as a sensitive predictor of risk of subsequent MA[ ] Table 1.

In Seaquist et al [ ] demonstrated strong familial clustering of DN, triggering a search for associated genetic variants. However, identifying gene variants that predispose to DN is complex as susceptibility is likely to be determined by a large number of common allelic variants, each of which may confer a modest increase in relative risk.

In addition, overall risk of developing DN is a result of a combination of both genetic and environmental influences.

Advances in genotyping technology have led to use of genome wide association scans GWAS for studying disease susceptibility across the entire genome. In relation to DN the creation of groups such as Family Investigation of Nephropathy and Diabetes FIND and Genetics of Kidneys in Diabetes GoKinD have facilitated such research.

The FIND group is a large multicentre consortium making use of family based linkage analyses in multi-ethnic groups to identify genes with significance in type 2 DN[ ].

Further publications by the group have shown a significant contribution of chromosomes 1q43, 8q GoKinD group have accumulated a collection of DNA for genetic association studies of DN in the context of type 1 diabetes[ ].

This group have identified genetic associations for DN susceptibility at candidate loci near the FRMD3 and CARS genes[ ]. In addition, variants in the ELMO1 gene on chromosome 7p have previously been linked with DN in Japanese and African-American populations with type 2 diabetes[ ].

GWAS data from the GoKinD collection confirmed this association in a Caucasian population[ ]. A genome wide linkage scan in Diabetes Heart Study families detected significant evidence for linkage with eGFR on chromosomes 2p16, 7q21 and 13q Evidence for linkage to UAE however was far weaker[ ].

In addition, genome wide DNA methylation analysis in a case control study of Irish patients with type 1 diabetes identified 19 prospective CpG sites associated with risk of DN[ ].

In the Genetics of Nephropathy: an International Effort consortium undertook a meta-analysis of GWAS of DN in type 1 diabetes. They identified signals in an intron in the AFF3 gene on chromosome 15 and linked this to DN mechanistically by providing evidence that AFF3 expression is linked to transforming growth factor beta-driven fibrosis in cultured epithelial cells[ , ].

Although this locus technically did not replicate, the potential for misclassification through identifying cases using clinical rather than histological criteria may have led to reduced statistical power[ ]. These methods have attracted attention in recent years as a potentially important tool for early, pre-clinical disease detection as they allow simultaneous examination of the patterns of multiple urinary and plasma proteins.

In view of the complex pathogenesis of type 2 diabetes, it is perhaps simplistic to expect that a single biomarker will provide sufficient sensitivity and specificity for disease prediction, detection and treatment monitoring, and therefore such multimarker approaches are appealing.

Both urinary and plasma proteome analysis have identified a number of biomarkers which are significantly associated with DN, such as specific collagen fragments[ , ], cytokines[ , ] and RBP[ ].

A panel of 65 urinary biomarkers DN65 have been identified which distinguished normoalbuminuric patients with diabetes from those with DN. This panel proved sensitive and specific for distinguishing DN from other causes of CKD in both single and multicentre settings[ , ].

CKD is a panel of urinary peptides which shows promise as a tool for early detection of DN. First described in , the panel was initially shown to distinguish between CKD of any aetiology and healthy controls with It has also recently been shown to predict adverse outcomes including death or end-stage renal disease in CKD patients[ ].

Two further studies have demonstrated the predictive power of CKD in identifying diabetic patients at risk of progression to overt DN. In longitudinal samples from a small cohort of 35 diabetic patients Zürbig et al [ ] showed that application of the classifier to samples from normoalbuminuric subjects up to 5 years prior to detection of macroalbuminuria enabled early identification of those at risk of progression area under the curve 0.

Similarly, Roscioni et al [ ] applied the classifier to samples from the Prevention of REnal and Vascular ENd-stage Disease PREVEND cohort. Results showed that classifier score at baseline was independently associated with progression of albuminuria[ ].

Further to this CKD has recently been validated in a multicentre setting. In urine samples obtained from 87 cases of DN and 78 controls at 9 centres worldwide the classifier distinguished cases from controls with high consistency across all centres areas under the curve ranging from 0.

A classification factor cut-off of 0. Metabolomics involves the measurement of low molecular weight intermediate and end-products of cellular functions in a biological sample, and has recently emerged as a tool with potential in novel biomarker discovery. The metabolome combines biological information from the genome, transcriptome and proteome, allowing identification of physiological and pathological changes in response to disease processes.

As with proteomics, a variety of sample types including serum, plasma, tissue and urine can be analysed in this way[ ]. A number of studies have explored the application of metabolomics approaches in kidney disease[ ].

For example, in a cross sectional analysis of plasma metabolites using samples from 30 non-diabetic male subjects with CKD stage , major differences were identified in arginine metabolism, carboxylate anion transport and coagulation pathways with increasing CKD stage[ ].

However, this study did not include patients with diabetes and in fact there are a limited number of such studies focussing on diabetic kidney disease.

In serum samples from 78 type 2 diabetic participants, a panel of 19 metabolites was identified which could differentiate DN from normoalbuminuria, all of which correlated significantly with albumin creatinine ratio. A model comprising the five best performing markers including γ-butyrobetaine and symmetric dimethylarginine resulted in AUC value of 0.

Another study using serum samples from patients with DN, normoalbuminuric diabetic patients and healthy volunteers showed significant changes in amino acid and phospholipid metabolism between study categories, as evidenced by alterations in leucine, as well as the sphingolipids dihydrosphingosine and phytosphingosine[ ].

Additionally, the application of metabolomics methods to renal cortex samples from streptozocin induced diabetic rats identified an increase in intrarenal organic toxins, including glucuronides, uraemic toxins and others associated with glucotoxicity, which were significantly correlated with 24 h urinary protein levels.

Furthermore, treatment with the ACE-inhibitor Fosinopril appeared to block the accumulation of these toxins[ ]. There is little published evidence from longitudinal studies to determine the predictive power of these methods for detection of individuals at risk of DN.

One such paper published earlier this year described the application of metabolomics methods to urine and plasma samples from the PREVEND study over a median follow up period of 2. Differences were seen in plasma histidine and butenoylcarnitine, as well as urine hexose, glutamine and tyrosine between individuals who transitioned in albuminuria stage compared to control sample who did not.

Adding these metabolites to a predictive model including baseline albuminuria and eGFR appeared to improve risk estimation for transition to macroalbuminuria[ ]. However, the complexity of the human metabolome remains perhaps the biggest challenge in translating these techniques into everyday clinical practice Figure 1.

DN is a leading cause of end stage renal disease and in combination with the increasing worldwide prevalence of diabetes poses an enormous burden to healthcare systems. UAE is currently the gold standard for detection and monitoring of nephropathy and cardiovascular risk in diabetes; however its predictive powers have limitations and research is focussing on biomarkers which may offer greater sensitivity and earlier detection to facilitate earlier intervention.

A degree of caution should, however, be exercised in relation to aggressive early intervention as to date there is little evidence of benefit from these strategies and more intensive RAAS blockade can result in a high incidence of unwanted adverse effects[ , ].

The Randomised Olmesartan and Diabetes MA Prevention study confirmed a significant delay in onset of MA with olmesartan therapy in normoalbuminuric type 2 diabetes patients, but caused controversy regarding increased fatal cardiovascular events in the treatment group[ ].

It could be argued that perhaps these studies have not targeted recruitment towards a population at particularly high risk of developing DN and focussing efforts in the direction of these individuals may yield more positive results. Identification of biomarkers to stratify patients according to DN risk may allow randomised controlled trials to focus on the population most likely to derive benefit from early, aggressive intervention.

Markers of glomerular damage show some promise for this purpose. In particular transferrin and type IV collagen appear to detect glomerular dysfunction at the normoalbuminuric stage although head to head comparative data are lacking.

Similarly, given that tubular damage can precede glomerular pathology, markers such as NAG, KIM1 and NGAL are interesting.

Evidence also points towards the role of oxidative stress in the pathogenesis of DN, meaning markers such as 8-OHdG and pentosidine merit further investigation. Low grade inflammation and endothelial damage is detectable in the pre-clinical stages of DN, leading to heightened interest in markers such as cytokines and AGA.

These too appear to be potentially useful tools in the earlier detection of DN, although again comparative work in relation to UAE would strengthen the case for their use.

The development of new technologies has led to exciting possibilities in the search for ideal biomarkers for DN but, despite the vast number that have been studied, none has so far demonstrated superiority to albuminuria.

While biomarker research in the preclinical setting is advancing, none of those biomarkers described above have been validated or are available commercially for clinical use. In addition, none have been described in relation to nonalbuminuric DN, which may reflect a separate disease process.

All such potentially interesting markers require further large scale validation in prospective clinical studies to determine whether they can make the transition from bench to bedside. org study which is currently recruiting, may help to redress this balance. As the complexities of the biochemical mechanisms underpinning DN continue to be unravelled it is perhaps simplistic to expect that a single biomarker will be sufficient for risk stratification as we move towards predictive and personalised medicine, and as such the shift towards systems biology integrating different technologies into multimarker strategies might provide greater sensitivity and specificity.

A number of biomarkers show promise as tools for early detection of DN, yet to date none have out-performed microalbumin in larger scale, prospective longitudinal studies. Multimarker approaches such as metabolomic or proteomic methods are particularly appealing as they also offer an insight into the multiple complex pathophysiological processes underlying DN.

In order to advance these efforts, cross-omics profiling, large scale biobanking and extended clinical phenotyping will be necessary to derive disease-stage specific models.

It should be borne in mind that nonalbuminuric DN is not uncommon and may reflect an alternative underlying disease process, therefore longitudinal studies investigating the performance of biomarkers to identify these individuals early may also be of interest.

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Advanced Search. About the Journal Submit a Manuscript Current Issue Search All Articles. This Article. Curr Opin Nephrol Hypertens. Currently, the studies focused on the podocyte-specific protein products because it was difficult to detect urinary podocytes directly.

One study by Wang et al. Messenger RNA expression of podocyte-associated molecules in the urinary sediment of patients with diabetic nephropathy. Nephron Clin Pract.

showed that urinary mRNA levels of podocin, synaptopodin and nephrin in DN patients were extremely higher than those found in control subjects by real-time quantitative PCR. These results were also proved by renal biopsy.

Also, synaptopodin level was positively correlated with urinary albumin excretion and serum creatinine concentration while negatively correlated with GFR. Patients, however, were not divided into different subgroups according to their average level of urinary protein.

The validation of these podocyte-specific protein products in early stages of DN was not confirmed in this study. Further research performed by Hara et al. revealed that urinary synaptopodin level of type 2 diabetes mellitus patients complicated by nephropathy was higher when compared to control subjects, even before the occurrence of proteinuria and associated with the level of urinary albumin and HbA 1c , indicating that synaptopodin was a biomarker with high sensitivity to podocyte injury in diabetic patients.

Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin.

Another report by Jim et al. In addition, urinary level of nephrin showed a strong association with UACR so that it might be a useful biomarker for nephropathic patients in preclinical stage. Dysregulated nephrin in diabetic nephropathy of type 2 diabetes: a cross sectional study.

PLoS One. Type IV collagen is the main component of the glomerular basement membrane and extracellular matrix, and does not pass through glomerular filtration barrier under normal circumstances. Therefore, type IV collagen could be used as a biomarker of basement membrane injury.

The study found that urinary type IV collagen levels were higher before microalbuminuria and associated with urinary albumin and serum creatinine, suggesting that urinary type IV collagen may be a promising biomarker for early diagnosis of DN.

Asian multicenter trials on urinary type IV collagen in patients with diabetic nephropathy. J Clin Lab Anal. Endothelial cells injury can directly affect the permeability of the glomerular filtration membrane.

Generally, von Willebrand factor vWF is mostly synthesized by endothelial cells. Plasma vWF levels increase when endothelial cells are stimulated or damaged.

Jensen 45 45 Jensen T. Increased plasma concentration of von Willebrand factor in insulin dependent diabetics with incipient nephropathy. first discovered that plasma levels of vWF are higher in type 1 diabetes mellitus patients, indicating that there is endothelial cell dysfunction in diabetic patients.

Subsequently, a number of studies have shown that plasma vWF levels in patients with DN are significantly higher than those in patients without kidney disease and control subjects, indicating that plasma vWF may contribute to the early diagnosis of diabetic nephropathy.

Insulin resistance and endothelial dysfunction in type 2 diabetes patients with or without microalbuminuria. Vascular endothelial markers, von Willebrand factor and thrombomodulin index, are specifically elevated in type 2 diabetic patients with nephropathy: comparison of primary renal disease.

Di Yi Jun Yi Da Xue Xue Bao. Hyperglycemia does aggravate vascular endothelial injury by up-regulating the expression of adhesion molecules by endothelial cells. Mechanism underlying up-regulation of ICAM-1 and VCAM-1 expressions induced by high glucose in endothelial cells. Chinese J Cardiovasc Med.

The study about type 2 diabetes mellitus patients from Malaysia discovered that plasma levels of intercellular adhesion molecule-1 ICAM-1 are elevated in DN patients. Genetic, epigenetic and protein analyses of intercellular adhesion molecule 1 in Malaysian subjects with type 2 diabetes and diabetic nephropathy.

J Diabetes Complications. Vascular endothelial growth factor VEGF can affect the filtration of large molecular weight proteins through glomerular filtration barrier by promoting endothelial cell proliferation and increasing vascular permeability.

Researchers have found that plasma and urinary levels of VEGF in DN patients were both elevated. Especially in type 2 diabetes mellitus subjects, urinary VEGF level was higher in normoalbuminuric patients than in control subjects and gradually increased along with the DN stages.

These findings suggested that VEGF may be an effective biomarker for early diagnosis in DN patients. Elevated vascular endothelial growth factor in type 1 diabetic patients with diabetic nephropathy. Kidney Int Suppl. Plasma and urinary vascular endothelial growth factor and diabetic nephropathy in Type 2 diabetes mellitus.

Diabet Med. Fibrosis is one of the pathological features of diabetic complications caused by extracellular matrix alterations and mesangial expansion.

Hyperglycemia up-regulates the expression of transforming growth factor-β1 TGF-β1 , which is considered to be the most crucial cytokine in glomerulosclerosis and tubulointerstitial fibrosis. Role of TGF-beta in the progression of renal fibrosis.

Contrib Nephrol. Data by Xie showed that serum TGF-β1 level of patients with microalbuminuria was significantly higher than that of patients with normoalbuminuria and control subjects.

Interestingly, urinary levels of TGF-β1 are already elevated in normoalbuminuria subjects and gradually increase along with DN progression, so that TGF-β1 was considered a sensitive biomarker in the early phase of diabetic nephropathy. Significance of serum and urinary TGF-β1 to the early diagnosis of diabetic nephropathy.

Strait Pharmaceutical J. Pigment epithelial-derived factor PEDF is a member of the serine protease superfamily and is involved in the formation of extracellular matrix and vascular endothelial growth factor. PEDF levels were found to be decreased in the kidney of diabetic mice, suggesting that it may have a protective effect in diabetic microvascular lesions.

Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy. Researchers also found that urinary PEDF levels in DN patients are significantly higher than in control patients, indicating that PEDF is probably an effective biomarker of DN.

Urinary pigment epithelium-derived factor as a marker of diabetic nephropathy. These biomarkers were summarized in Table 1. The level of proteinuria in the early stages of DN can tell us whether there is glomerular damage or not and the extent of the damage.

Investigation of proteinuria continues to be the gold standard for diagnosis and staging of DN. Urinary markers of glomerular injury in diabetic nephropathy. Int J Nephrol. In addition, albumin, transferrin TRF , ceruloplasmin CER and immunoglobulin G IgG in urine can also reflect functional changes in glomerular filtration.

Narita et al. found that urinary levels of TRF, CER and IgG in normoalbuminuric patients were significantly higher than those in control subjects and they strongly correlated with each other, indicating that TRF, CER and IgG may be more sensitive makers for changes in filtration function than albuminuria in the early stages of DN.

Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients. Tubulointerstitial injury plays an important role in DN development process and even prior to glomerular injury.

In addition, about one third of the patients with diabetes mellitus have decreased renal function prior to proteinuria.

Therefore, we should pay more attention to the biomarkers of tubulointerstitial injury, which can contribute to the early diagnosis and treatment of DN patients. Progression of chronic kidney disease: too much cellular talk causes damage. Kidney Int. Regression of microalbuminuria in type 1 diabetes.

a1-Microglobulin, retinol-binding protein 4 RBP4 and other low molecular weight proteins can freely pass through the glomerular filtration membrane and then be reabsorbed in the tubules.

These were early biomarkers of tubular injury because of their increase in urine after renal tubular damage. Researchers found that urinary levels of a1-microglobulin and RBP4 in patients with normoalbuminuria were significantly higher than those in control subjects and were both associated with the levels of HbA 1c , so that detection of two biomarkers may be helpful for early diagnosis of diabetic nephropathy.

Urinary alpha1-microglobulin as a marker of nephropathy in type 2 diabetic Asian subjects in Singapore. Urinary excretion of n-acetyl-beta-D-glucosaminidase and retinol binding protein as alternative indicators of nephropathy in patients with type 1 diabetes mellitus. Some other biomarkers of tubular injury, such as neutrophil gelatinase-associated lipocalin NGAL , N-acetyl-β-D-glucosidase NAG , kidney injury molecule-1 KIM-1 and heart-type fatty acid binding protein H-FABP applied only to predict acute kidney injury.

Early diagnosis of acute kidney injury. Curr Opin Crit Care. Urinary biomarkers in the early detection of acute kidney injury after cardiac surgery. Clin J Am Soc Nephrol. A recent study discovered that urinary levels of NGAL, NAG, KIM-1, H-FABP of patients with normoalbuminuria were significantly higher than those of control subjects and increased gradually along with the DN stages.

In addition, they all significantly correlated with urinary albumin levels, indicating that they might be early biomarkers for DN diagnosis. Glomerular and tubular damage markers are elevated in patients with diabetes. These biomarkers were summarized in Table 2.

In recent years, there has been an important achievement regarding the finding of associated biomarkers in all aspects of diabetic nephropathy. These research findings contribute greatly to our understanding of disease mechanisms. So far, there is no biomarker that can replace proteinuria.

We believe that advances in research methods based on genomics, proteomics and metabolomics will provide much more convenience in future.

However, we should also take all the questions into our consideration, such as the fact that there is no universally accepted standard for subject inclusion and staging, not all researches made adjustment for significant parameters and few studies have discussed the effectiveness of multi-biomarker detection.

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table of contents « previous current next ». Text EN Text English. PDF Download PDF English. Summary Diabetic nephropathy is the main cause of chronic kidney disease, and represents the most common and serious complication of diabetes.

Keywords: Diabetes Mellitus; Diabetic Nephropathies; Biomarkers. Introduction Diabetes mellitus DM is an endocrine and metabolic disease that has serious impact on human health. Biomarkers associated with DN pathogenesis A large number of prospective studies confirm that hyperglycemia is the most important risk factor for DN.

Biomarkers of oxidative stress The occurrence and progression of DN is closely related with oxidative stress.

Biomarkers of DNA injury 8-hydroxy-2'-deoxyguanine 8-OHdG is a sensitive biomarker of DNA damage to assess oxidative stress in the human body. Biomarkers of protein and lipid injury Biomarkers associated with protein injury comprise pentosidine, 2,4-dinitrophenylhydrazine DNPH and advanced oxidation protein product AOPP.

Biomarkers of glutathione antioxidant system and lipid peroxidation A growing number of studies reported that DM and its complications were closely related to oxidative stress, so we supposed that the biomarkers related to antioxidant defense system and lipid peroxidation LPO induced by free radicals may be potential biomarkers of kidney damage in diabetic patients.

Biomarkers of inflammation Inflammatory response could be activated by biochemical, metabolic or hemodynamic disorders when a large number of white blood cells gather in the kidney. Biomarkers of RAAS activation Renin-angiotensin-aldosterone system RAAS plays an important role in regulating blood pressure by producing aldosterone in human body.

FIGURE 1 Summary of biomarkers associated with DN pathogenesis. Table 1 Summary of biomarkers associated with DN pathology. Table 2 Summary of biomarkers associated with renal function changes.

Study conducted at Shengjing Hospital of China Medical University, Shenyang, Liaoning, China. References 1 Shaw JE, Sicree RA, Zimmet PZ. Gross JL, Azevedo MJ, Silveiro SP, Canani LH, Caramori ML, Zelmanovitz T.

Tabaei BP, Al-Kassab AS, Ilag LL, Zawacki CM, Herman WH. Diabetes Control and Complications Trial Research Group, Nathan DM, Genuth S, Lachin J, Cleary P, Crofford O, Davis M, et al.

Kim et al. Urinary cystatin C and albuminuria may be sensitive and specific markers for predicting kidney impairment. Age, HbA1c, systolic blood pressure, uric acid, albuminuria, baseline eGFR, use of RAS inhibitors and lipid-lowering agents, serum cystatin C.

Biomarkers of inflammation. Niewczas et al. Elevated circulating TNFR levels are strong predictors of progression to ESKD in subjects with and without proteinuria. Skupien et al.

Circulating TNFR2 is a major determinant of kidney function decline. Lopes-Virella et al. High levels of E-selectin and soluble TNFR1 and TNFR2 levels were important predictors of incident albuminuria.

Treatment, albuminuria, use of RAS inhibitors, baseline retinopathy, sex, age, HbA1c, diabetes duration. Biomarkers of oxidative stress. Xu et al. Individuals with T2DM have higher levels of 8-OHdG compared to healthy individuals.

Sanchez et al. Higher levels of 8-OHdG were associated with increased risk of kidney disease. Age, sex, cohort, duration of diabetes, HbA1c, insulin therapy, systolic blood pressure, use of antihypertensive drugs, RAS inhibitors, diabetic retinopathy stage, lipid-lowering drugs, eGFR, albuminuria.

Serdar et al. Although urinary 8-OHdG levels increase in diabetic patients, their levels do not improve prediction of progressive DKD over and above measuring albuminuria. Omics-based novel biomarkers. Bhensdadia et al.

The haptoglobin to creatinine ratio may be useful to predict risk of DKD before the development of albuminuria or kidney function decline.

Zurbig et al. CKD predicted progression to macroalbuminuria 5 years prior to actual onset. Age, sex, DM type, albuminuria, eGFR, systolic and diastolic blood pressure, HbA1c, glucose.

Roscioni et al. CKD predicted development of albuminuria independent of other kidney biomarkers used to predict DKD development or progression. Albuminuria, eGFR, use of RAS inhibitors. Age, baseline eGFR, systolic and diastolic blood pressure.

Tofte et al. High-risk patients defined by CKD were more likely to develop microalbuminuria. Age, sex, HbA1c, systolic blood pressure, retinopathy, albuminuria, eGFR.

Lindhardt et al. CKD predicted development of albuminuria. Treatment group, age, sex, systolic blood pressure, albuminuria, eGFR, HbA1c, diabetes duration.

Hyperglycemia induces upregulation of SGLT2, leading to increased glucose reabsorption, which predisposes proximal epithelial cells to a hypoxic injury and causes advanced glycation end-product AGE production. Similarly, increased albumin reabsorption by proximal tubules culminates into proximal epithelial cell toxicity [ 10 ].

Thus, high diabetic-milieu and AGE facilitate pro-inflammatory and apoptosis cascade leading to tubulointerstitial injury and fibrosis. Reactive oxygen species ROS and reactive nitrogen species RNS are responsible for oxidative stress, leading to disease progression.

Mitochondria and the nicotinamide adenine dinucleotide phosphate oxidase NOX family are the primary sources of reactive oxygen stress in the kidney.

It primarily gets produced by enzymatic reactions, from mitochondrial aerobic respiration ETC and a lesser amount from endoplasmic reticulum and peroxisomes [ 11 ].

Oxidative stress is also contributed from hyperglycemia-induced AGEs production in the later stages of non-enzymatic glycation of sugar and protein, by the process called Maillard reaction [ 11 ].

Hyperglycemia-induced oxidative stress mediates DNA damage, lipid peroxidation, mitochondrial dysfunction, and infiltration of inflammatory cells, progressing to renal cell damage [ 8 ].

DKD progression is accelerated by the activation of the nuclear factor-kappa light chain enhancer for B cells Nf-kB by an activated immune system and inflammation. Macrophages, dendritic cells, and mast cells make up the renal mononuclear phagocytic cells MNPs which consist of macrophages, dendritic cells, and mast cells are involved from the innate immune system.

Macrophage infiltrations are prominent in glomeruli and interstitium of DKD patients [ 12 ]. Its accumulation in glomeruli leads to glomerulosclerosis and in interstitium predicts loss of GFR. Glomerular infiltration of dendritic cells is proportional to proteinuria during the progression of DKD.

While mast cell infiltration of the interstitium is correlated with serum creatinine, but not with proteinuria. These mast cell degranulation and IL-1β from macrophages together stimulate renal fibroblast proliferation, whereas IL-1 stimulates mesangial cell proliferation [ 12 ].

The cellular arm of adaptive immunity involved in DKD is T cells Th1, Th17 and limited involvement of B cells, which are associated with proteinuria [ 12 ].

Albuminuria and eGFR are the commonly used legacy markers of renal function decline in routine clinical practice, although they lack specificity and sensitivity in predicting the DKD progression in diabetic patients. Proteinuria does not always precede renal function decline, suggesting early involvement of tubulointerstitial compartment rather glomerular [ 4 , 13 ].

On the other hand, eGFR estimation also has some downsides as a biomarker in diagnosing and stratifying DKD progression, since its calculation using serum creatinine interferes with the patient's muscle mass and meat diet.

eGFR estimation using the CKD-EPI equation also gives underestimated values in type 2 diabetic patients [ 14 ]. Understanding these limitations and exploring potential biomarkers is necessary for both clinical applications in future research for improved diagnostic and prognostic tools.

Over the past decades, immense efforts in research have been carried out to validate alternative biomarkers. Numerous biomarkers were identified for this purpose, and initial findings from many studies have been promising.

These novel biomarkers can be classified according to the pathological effects on renal structure, as shown in Table 1. Biomarkers linked to glomerular injury would be a significant tool in guiding early diagnosis and identifying patients with rapid renal deterioration. Multiple glomerular biomarkers provide great evidence in representing glomerular injury as urine protein estimation alone cannot predict the progression of DKD.

Structurally Type 1 V Collagen is a protein with three polypeptide α-chains in triple helix form which serves as the main basement membrane constituent of the glomerulus, tubules, and mesangial matrix [ 15 ]. Mesangial expansion score and tubulointerstitial injury score were statistically correlated with urinary type IV collagen, suggesting the pathogenic processes of DKD reflected in the elevation of this protein [ 15 ].

Tomino et al. Ijima et al. studied the urinary type IV collagen in the normo-microalbuminuric group, excluding overt proteinuria, and after 1-year follow-up, the normoalbuminuric group with a higher level of urinary type IV collagen excretion had developed microalbuminuria [ 17 ].

The findings of the above studies suggest the importance of type IV collagen as a biomarker in diagnosing the onset of microalbuminuria. Additionally, Morita et al.

argue with their findings among the T1DM population that type IV collagen was independently associated with microalbuminuria [ 18 ]. However, Araki S et al. While, serum type IV collagen was found higher in diabetic retinopathy, indicating its involvement in predicting microvascular complications [ 20 ].

From the above-mentioned studies, urinary type 1V collagen is an indicator of early onset and disease progression in both type-1 and type-2 diabetic patients, and its higher concentration in the serum indicates the onset of diabetic nephropathy. Fibronectin is a fibrillar protein on the cell surface, and its soluble form in plasma is associated with constriction of the glomerular extracellular matrix.

It is primarily synthesized in fibroblast and endothelial cells, and its upregulation in capillary and mesangium of the glomerulus in diabetic patients has been reported [ 21 ]. Plasma fibronectin was found progressively increasing from normoalbuminuric to microalbuminuric patients [ 21 ].

Marked elevation of urinary fibronectin U-FN was associated with overt proteinuria in both T1DM and T2DM [ 22 , 23 ]. Urinary and plasma fibronectin were found to be linked with micro- and macrovascular complications such as retinopathy, neuropathy, and cardiovascular incidence among diabetic patients [ 22 , 24 ].

All these studies demonstrated predictive performance of FN for microalbuminuria and overt proteinuria, in addition to micro-macrovascular complications in type 1 and type 2 diabetic patients. Although a great number of researchers found increased U-FN in DKD, the exact origin of FN remains unclear, as it was synthesized from multiple sources other than renal cells.

Further clinical research is necessary to compare it to albuminuria and to determine its significance. Laminin is an adhesive and non-collagenous component of glomerular basement membranes and mesangium.

To date, 15 laminin isoforms are identified in which A2 laminin reflects mesangial matrix expansion in DKD [ 25 ]. Banu et al. have demonstrated a higher level of laminin in normoalbuminuric patients, suggesting that it would be a marker for predicting albuminuria, and found a positive correlation with tubular dysfunction markers including NAG and α- 1microglobulin [ 26 ].

Rashed et al. have observed progressively increasing serum laminin with In aggregate, serum laminin would be a marker for identifying the onset and progression of diabetic kidney disease as well as diabetic microangiopathy.

CysC is a low-molecular-weight protein that acts as an endogenous cysteine proteinase and is identified as a potential surrogate indicator for GFR estimation, because, unlike serum creatinine, it does not influence extrarenal factors [ 29 ] which leads to the increased diagnostic utility of serum CysC to evaluate kidney damage, reflecting directly to GFR.

Diagnostic utility of serum CysC in normoalbuminuric patients has also been well documented in a cohort of T1DM, indicating the predictive performance of CysC before the renal dysfunction appears [ 30 ] which is well supported by Qamar et al. in type 2 diabetic patients with a sensitivity of In multiple studies done among CKD patients with T1DM and T2DM, showed a significant role of serum CysC as a predictor of progression to ESRD [ 32 ].

Clinical research has observed the positive association of serum CysC with retinopathy [ 33 ] and cardiovascular risk [ 34 ] in type 2 DM. Nevertheless, as recommended in current KDIGO guidelines, CysC would result in increased health care costs [ 35 ].

The above studies displayed that serum cystatin C could be a promising biomarker for early diagnosis and for predicting the progression of DKD, as it is a strong predictor of microvascular and macrovascular complications of diabetes.

GAGs are mucopolysaccharides 13 and 30 kDa. The most prevalent types are chondroitin and dermatan sulfate, keratan sulfate, heparan sulfate, and heparin. Heparan sulfate gives negative potential to the glomerular basement membrane GBM to control the perm selectivity of the glomerulus.

In DKD, endothelial dysfunction leads to loss of these functional groups which gives rise to hyperfiltration resulting in albuminuria [ 36 ].

The Steno hypothesis postulates a defect in heparan sulfate regulation in the glomerulus, which determines the high susceptibility of diabetic patients to develop proteinuria and eventually induces the excretion of heparan sulfate in the urine [ 37 ]. Many experimental studies supporting this hypothesis observed a rise in urinary GAG excretion, especially heparan sulfate in T1DM and T2DM patients with microalbuminuria and macroalbuminuria than the normoalbuminuric group, suggesting that GAG is a vital screening predictor of microalbuminuria [ 38 ].

The functional role of GAG in permeability properties of retinal basement membrane has also been documented. Kahaly et al. have evaluated high urinary GAG concentration in diabetic nephropathy patients with retinopathy complications [ 39 ]. Linked with these findings, Budak et al.

have also reported a positive correlation of GAG with diabetic retinopathy [ 40 ]. Urinary GAG evaluation could be a vital marker for predicting the onset of microalbuminuria in type 1 and type 2 diabetic patients and microvascular complications of diabetes in the progressive stages.

IgG is a kDa anionic immunoprotein in serum [ 41 ]. The urinary excretion of this high-molecular-weight protein indicates increased GBM porosity with large shunt-like pores and podocyte deficiency and effacement of foot processes.

Thus, IgG appears in progressive DKD when severe irreversible kidney lesions occur [ 41 ]. Yashima et al. have reported a significant elevation of urinary IgG in normoalbuminuric DKD patients among the T2DM population and also observed a correlation with progressive diffuse glomerular lesions [ 42 ].

Multiple studies in type 2 diabetic patients have suggested that urinary level of IgG predicts the onset of microalbuminuria [ 41 , 43 ].

Apart from using total IgG level, subtype levels and their ratio has been used as a marker of glomerular charge selectivity impairment. Altogether, these studies suggest that measurement of urinary IgG could be an effective marker for predicting the onset of microalbuminuria in T2DM.

Ceruloplasmin is a copper carrier and acts as a pro-oxidant in severe oxidant stress conditions. It has been studied as an independent risk factor for the incidence of cardiovascular diseases and insulin resistance [ 45 ].

Jung Lee et al. have found T2DM with a higher incidence of serum ceruloplasmin level in progressors than those in non-progressors, indicating an independent factor for progression [ 45 ]. Furthermore, urinary ceruloplasmin was also found elevated in T2DM before albuminuria appears [ 43 ]. A similar observation was found in T1DM [ 46 ].

This evidence suggests parallel evaluation of both serum and urine ceruloplasmin would be an independent predictive marker in DKD progression in T2DM. However, further studies require in the type 1 population, which is limited so far.

L-PGDS is a lipocalin secretory protein that synthesizes prostaglandin D2. It is primarily produced in the choroid plexus in the brain and discharged readily to circulating blood with chemical features like albumin, such as anionic charge, and can move quickly through the glomerular capillary due to its small molecular weight 20—31 kDa.

Thus, urine L-PGDS reflect minor changes in the permeability of glomerular capillary walls [ 47 ]. Researchers have studied its utility in predicting the early stages of DKD by observing the elevated urinary L-PGDS in diabetic patients with normoalbuminuria [ 48 ]. There was a significant increase in urine L-PGDS than serum level in normo- or macroalbuminuria in parallelly in advanced DKD [ 49 ].

The findings of these studies suggest that evaluation of L-PGDS in urine could identify the early onset of DKD. Transferrin is a glycoprotein with two iron-binding domains, which is primarily produced in the liver.

It is involved in multiple functions like iron transportation and immune regulation against micro-organisms [ 50 ]. Gonzalez et al. Iron liberated from transferrin contributes to oxidative stress and insulin resistance in T2DM patients, and thus, dysregulation of iron homeostasis is associated with the development of DKD [ 50 ].

Kanauchi et al. in their study observed the correlation of urinary transferrin with progressive changes such as interstitial fibrosis, atrophic renal tubular cells, and infiltration of renal interstitium with inflammatory cells [ 52 ].

The renal accumulation of iron and excretion of transferrin in urine leads to a lower serum level, which indicates renal cell toxicity resulting from accumulated free iron [ 50 ]. Excretion of transferrin in urine has been reported in normoalbuminuric T2DM, indicating prediction at an early stage than albuminuria in DKD [ 43 ].

High excretion of urinary transferrin in normoalbuminuric and microalbuminuric patients among T1DM was reported by previous researchers [ 53 ].

These findings were supported by a retrospective cohort study which suggests that low serum transferrin was associated with ESRD in diabetic patients [ 50 ]. Additionally, studies have also been reported transferrinuria in predicting micro- and macrovascular complications of diabetes which leads to retinopathy [ 54 ] and cardiovascular disease [ 55 ].

These findings suggest that the increased urinary and lower serum level of transferrin in all diabetic patients would predict the development of microalbuminuria and significant indicator for complications of diabetes.

The renal tubules and interstitial compartments play a significant role in the development of DKD [ 56 ]. The extent of tubulointerstitial damage may determine renal function decline in diabetes, even in normoalbuminuric renal insufficiency [ 56 ].

Hence, the tubular indicators of kidney injury have a pivotal role to measure the degree of long-term kidney impairment in DKD patients. NGAL is a neutrophil granular constituent belonging to the lipocalin protein family. In renal injury, the distal tubules and collecting duct signify the higher expression of NGAL.

It has been validated as an acute kidney injury AKI biomarker extensively. NGAL is involved in antimicrobial defense mechanisms and anti-apoptosis. It is a definite marker of acute renal damage, because a burnt-out nephron does not generate NGAL [ 57 ].

Evidences have been postulated on the significant role of NGAL in CKD and later in DKD [ 58 ] which has been supported by the findings of S. Hwang et al. Urinary NGAL uNGAL -to-creatinine ratio was found useful to differentiate DKD from non-diabetic kidney disease with high specificity Kaul et al.

observed that the significant rise of serum NGAL sNGAL and uNGAL from normo-micro-macroalbuminuria in T2DM [ 57 ]. Peng He et al. Growing evidences have depicted a positive correlation of NGAL with albuminuria and other tubular markers, including RBP4, Cystatin C, and KIM-1, whereas it is negatively correlated with eGFR, suggesting the involvement of NGAL in DKD progression [ 61 , 62 , 63 ].

In contrast, Kim et al. reported that no significant difference in NGAL was found in normoalbuminuric and macroalbuminuric patients [ 64 ].

In summary, these clinical studies suggest that sNGAL and uNGAL could be valuable markers for diagnosing the onset of DKD and stratifying the disease into different stages. However, large-scale prospective studies are necessary to implement NGAL as a biomarker into routine clinical use.

Cystatin C is a Normally, it is not present in urine significantly. Previous studies have demonstrated uCysC as a promising biomarker of tubular dysfunction in AKI [ 65 ], and it has been extensively studied in DKD [ 66 ].

Xian et al. have evaluated the diagnostic performance of uCysC in DKD and the onset of microalbuminuria among T2DM. With respect to DKD diagnosis, the area under the ROC curve was 0. These studies suggest uCysC as a sensitive biomarker mirroring tubular impairment, which can be determined before the onset of microalbuminuria.

In response to injury, KIM-I is predominantly expressed in the apical membrane of proximal tubular cells. Palmitic acid bounded albumin uptake by proximal tubules is being enhanced by KIM-1, leading to further tubulointerstitial damage [ 67 ].

Van Timmeren et al. demonstrated that KIM-1 mainly expresses the luminal side of dedifferentiated proximal tubular areas, which had more fibrosis and inflammation [ 67 ]. Several studies have documented an estimation of KIM-1 in urine uKIM-1 as a predictive indicator for AKI as it appears well before serum creatinine increases [ 68 ].

Fourth, Ali et al. reported uKIM-1 with more specificity and sensitivity than urine albumin in diagnosing early stages of DKD [ 69 ], and Gohda et al.

reported a significant association of serum KIM-1 with a lower GFR rate. Based on these studies, uKIM-1 appears to be a promising marker for diagnosing early onset and predicting the different stages of disease progression in type 1 and type 2 diabetic patients. Upon this, serum level estimation might be a sensitive marker for progression as GFR declines.

RBP4 is a low-molecular-weight protein associated with the lipocalin family, predominantly synthesized in the liver and adipose tissue. The main function of RBP-4 is to transfer small hydrophobic molecules to the cell membrane. In diabetes, an association of RBP4 concentration has been documented with the magnitude of insulin resistance, suggesting increased levels of RBP4 predicts insulin resistance [ 72 ].

Increased plasma and urinary RBP4 concentration have been reported with low eGFR [ 72 ] [ 73 ]. A longitudinal study among T1DM has reported an increased level of urinary RBP4 in microalbuminuric patients [ 74 ] and the diagnostic utility of urinary RBP4 in T2DM patients with an AUC of 0.

The serum level of RBP-4 was found to be associated with proliferative diabetic retinopathy and coronary cerebrovascular or peripheral vascular diseases among type 2 diabetes [ 72 , 75 ]. Discordant results were shown by E Akbay's study, indicating that diabetic retinopathy and cardiovascular complications do not exhibit any change in serum RBP4 in T2DM patients [ 76 ].

RBP4 could be a valid marker for identifying the early onset of DKD and predicting renal function impairment in progressive stages in T1DM and T2DM. In addition, this marker could be a predictor for microvascular and macrovascular complications of diabetes. L-FABP is a 14 kDa protein produced mainly in the cytoplasm of proximal tubules and is involved in the metabolism of the long-chain fatty acids.

Uncontrolled reabsorption of free fatty acids to tubular cells by L-FABP leads to tubulointerstitial damage [ 77 ]. According to Kamijo et al. Higher levels of L-FABP in the normoalbuminuric group suggest that it could be a risk factor for disease progression [ 78 ].

Corroborating these findings, a year follow-up study by Araki S et al. Additionally, the urinary level of L-FABP offers statistical significance with urine albumin level and inversely correlates with GFR [ 78 ]. Thus, evaluation of urinary L-FABP in T2 DM serves as a risk factor for DKD progression and could be considered as a promising tubular marker in predicting the incidence of cardiovascular disease and renal function impairment.

Evidence from epidemiological and mechanistic research suggests that oxidative stress plays a key role in mediating progression and complications. Thereby, markers linked to ROS production have considerable potential to stratify DKD stages.

Numerous evidence has indicated urinary 8-oxodG is a risk factor for cancer, atherosclerosis, and diabetes [ 80 ].

Xu et al. Clinical research with 5 years of follow-up reported significant progression of diabetic kidney disease in patients with higher urinary 8-oxodG [ 82 ].

Urinary 8-oxodG has been proposed as a characteristic pathogenic component in diabetic retinopathy development in T1DM and T2DM [ 83 , 84 ]. Etiane et al. found diagnostic ability of 8-oxodG with an AUC of 0. This marker has also been observed with macrovascular complications in T2DM [ 86 ].

These above-mentioned findings conclude that excretion of urinary 8-oxodG could be an independent predictor for disease progression and development of microvascular and macrovascular complications of diabetes. Pentosidine is an advanced glycoxidation product formed by the covalent binding of amino groups with glucose moiety [ 87 ].

Miura et al. demonstrated a serum pentosidine level more marked progressively in microalbuminuria and advanced stages of nephropathy [ 88 ]. Bruce A et al. found higher excretion in urine among patients with microalbuminuria and early decline of GFR [ 89 ]. Diabetic patients with a high level of pentosidine were found to be an independent predictor of diabetic retinopathy, cardiovascular disease, and all-cause mortality [ 90 , 91 ].

Lines to this evidence, measurement of pentosidine level in urine and serum may provide the basis for identifying patients at risk of early GFR decline and could be a promising biomarker for diabetic microvascular and macrovascular complications.

Uric acid is produced by purine metabolism and has been shown to play an independent function in predicting DKD progression and many clinical studies have been focused targeting its level in the prognosis of DKD. Bartakova et al. found initial hyperuricemia is a strong determinant of DKD progression [ 92 ].

Zoppini et al. analyzed that the cumulative incidence of CKD with GFR decline among T2DM was significantly higher in those who had hyperuricemia, considered as an independent risk factor in disease progression and as a strong predictor of GFR decline [ 93 ]; furthermore, T1DM with higher serum uric acid levels were developed persistent macroalbuminuria [ 94 ].

These evidences suggest that serum uric acid could be an independent predictor of later development of macroalbuminuria in type 1 and type 2 diabetic patients. Recent researchers have reported the potential role of local and systemic inflammatory pathways in the progression of DKD with chronic inflammation and subsequent extracellular matrix expansion [ 95 ].

TNF-α expresses in glomerular and tubular cells in all stages of diabetes, mainly monocyte-produced cytokines, and predisposes in all the stages of the pathogenesis of DKD progression by inducting and infiltrating inflammatory cells to the kidney and activation of apoptosis system.

Thereby elevated level of TNF-α has been noted with hypertrophy, hyperfiltration, and alterations of intra-glomerular blood flow, resulting in reduced renal function [ 95 ]. A meta-analysis by Qiao et al. reported T1DM patients have significantly increased TNF-α as compared to healthy controls [ 96 ].

Furthermore, Navarro JF et al. documented that serum TNF-α is elevated with advanced renal dysfunction and correlates with urinary protein excretion, suggesting that this cytokine has an intensive role in the onset of proteinuria in these patients [ 97 ].

While Stangou et al. reported a significant positive correlation of urinary TNF-α, but not serum TNF-α with the severity of microalbuminuria in T2DM [ 98 ]. An experimental animal, study corroborated the key role of TNF-α in mediating the pathogenesis of diabetic peripheral neuropathy [ 99 ].

Elevated TNF-α is also associated with microvascular and macrovascular complications in diabetic patients [ , ] and in the prediction of diabetic retinopathy in T2DM with an AUC of 0.

The above studies suggest that serum and urine TNF-α could be a potential biomarker to predict the degree of microalbuminuria in T1DM and T2DM. TNF-α receptors are type1 transmembrane proteins with cysteine-rich motifs seen in glomerular and tubular cells.

These are of two types, TNF-α receptor 1 55 kDa and TNF -α receptor 2 75 kDa. TNF-α binds to these respective receptors and induces inflammatory pathways and apoptosis [ ].

Current studies have appreciated the contribution of TNF-α receptors on the magnitude of DKD development through the TNF α —TNFR2 inflammatory pathways [ ]. Sharad et al. found a strong correlation of serum TNF-α receptors with microalbuminuria among T1DM, suggesting the crucial role in disease progression [ ].

These findings have been supported by evidence of a marked stepwise increase from normo-micro-macroalbuminuria in T2DM [ ]. Multiple studies have reported that TNFRs are associated independently with declined renal function and ESRD [ ].

Furthermore, it has been described that serum TNF receptors are predictors of diabetic retinopathy in T1DM [ ]. Therefore, circulating TNF-α receptors are associated with the progression of DKD and could be a predictor of microalbuminuria and advanced renal impairment. MCP-1 is a pro-inflammatory cytokine produced by mononuclear leukocytes, cortical tubular epithelial cells, and podocytes that has been linked to renal inflammation, glomerular injury, tubular atrophy, and fibrosis via nuclear factor-kappa B [ ].

Renal expression of MCP-1 was also correlated with the quantity of infiltrated macrophages, interstitial lesions, and the degree of albuminuria [ ]. Fufaa et al.

demonstrated a substantial correlation of urine MCP-1 uMCP-1 levels with cortical interstitial expansion and disease progression in T1DM who had normoalbuminuria [ ]. When comparing DKD patients to healthy controls, Wada [ ] and Banba [ ] discovered elevated urinary excretion of MCP-1 in DKD patients.

Shoukry et al. Early progressive GFR decline has a positive correlation with high uMCP-1 [ ] also associated with young-onset of T2DM with diabetic retinopathy [ ]. These observations suggest that MCP-1 could be a promising inflammatory marker in diagnosing early progressive renal decline and diabetic microvascular complications.

TGF-β activates fibrogenesis and thereby progression of DKD by the increased extracellular matrix deposition and glomerular mesangial hypertrophy [ ]. Flores et al. have shown raised urinary and plasma TGF- β in clinical onset of T1DM [ ].

Similarly, patients with T2DM who had microalbuminuria had been reported elevated serum and urinary TGF-β [ ]. On the other hand, Rivarola et al. Supporting this, a study conducted among T2DM resulted in increased serum and urine TGF-β, being more pronounced in macroalbuminuria compared to microalbuminuria and normoalbuminuria group [ ], suggesting that this biomarker could be a good candidate in predicting macroalbuminuria in type 2 DM.

CTGF is a secretory protein in renal cells induced by hyperglycemia. It stimulates extracellular matrix synthesis, cell migration, and interstitial matrix deposition by the epithelial-to-mesenchymal transition in diabetic patients [ ].

T1DM patients showed increased urinary CTGF with the severity of renal function deterioration in terms of albumin excretion and GFR decline [ ] and could be a predictor for ESRD with an AUC under ROC of 0.

Its expression has also been reported in diabetic retinopathy [ ]. CTGF could be an independent predictor of ESRD and mortality in DKD and it is also a predictor of diabetic retinopathy and looks promising.

It is a major immunoregulatory cytokine in mesangial expansion. Sangoi et al. observed higher serum IL-6 even before the onset of albuminuria [ ]. Multiple studies support these findings with evidence that serum and urinary IL-6 were increasing with disease progression in T1DM [ ] and T2DM [ ] and have a pronounced association with macrovascular complications [ ].

Thus, it could be a marker of the onset of microalbuminuria and early progressive renal decline, and it strongly predicts the macrovascular complications of diabetes. The classification based on pathogenesis and utilization of these biomarkers can be the future in predicting the early onset of microalbuminuria and progressive renal function decline in both T1DM and T2DM.

These biomarkers are relevant to not only predicting the progression of DKD but also diabetic microvascular and macrovascular complications, as shown in Table 2. Few of the biomarkers have been studied in the type 2 diabetic population on their diagnostic utility for DKD and established cut-off value with the area under the ROC curve from 0.

These are 0. Under hyperglycaemic injury, renal cells are activated to release MPs into plasma and urine before the onset of DKD [ ]. Therefore, these have emerged as biomarkers in DKD. A review by Sheyu Li et al. reported a higher level of circulating MPs reported with T2DM and as independent predictors for microvascular complications of diabetes [ ].

MPs can be easily isolated from body fluids via non-invasive methods. These properties facilitate the use as a potential non-invasive biomarker in the progression of DKD.

More large-scale studies are needed for further relevance in this regard. Urinary exosomes, 40— nm originate as internal vesicles, and that contain protein indicators of renal failure and structural damage. It has turned out to be a potential non-invasive biomarker source.

However, exosome isolation was challenging. Using liquid chromatography and mass spectrometry, the scope of existing approaches has enlarged to evaluate more urinary exosome-associated proteins [ ].

In recent years, microRNA is reported to be involved in the DKD progression via inflammation, hypertrophy, autophagy, endoplasmic reticulum ER stress, oxidative stress, insulin resistance, and podocyte injury.

Jia et al. observed a positive correlation of miRNA expression for TGF beta stimulator with albuminuria and reported good diagnostic efficiency [ ].

Based on the evidence mentioned above, urine mRNA has prognostic significance as a non-invasive, early indicator of renal impairment. The pathophysiology of DKD and its progression is multifactorial.

Therefore, assessment of development and progression of DKD cannot be relied solely on albuminuria and creatinine. The identification of novel biomarkers based on pathogenesis of DKD involving various renal structures looks promising.

In this review, we have summarized the potential 22 novel biomarkers with respect to the pathogenesis of DKD development. Each biomarker has its role in either identifying DKD early or predicting progression of DKD over and above clinical history and standardized markers like albuminuria and creatinine.

Few of them appear to be useful for predicting other micro- and macrovascular complications like retinopathy and cardiovascular disease.

This panel of biomarkers now warrants further validation on large-scale longitudinal studies involving type 1 and type 2 diabetes populations before the transition to clinical routine.

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Biomarkers of inflammation. Niewczas et al. Elevated circulating TNFR levels are strong predictors of progression to ESKD in subjects with and without proteinuria. Skupien et al. Circulating TNFR2 is a major determinant of kidney function decline. Lopes-Virella et al. High levels of E-selectin and soluble TNFR1 and TNFR2 levels were important predictors of incident albuminuria.

Treatment, albuminuria, use of RAS inhibitors, baseline retinopathy, sex, age, HbA1c, diabetes duration. Biomarkers of oxidative stress. Xu et al. Individuals with T2DM have higher levels of 8-OHdG compared to healthy individuals.

Sanchez et al. Higher levels of 8-OHdG were associated with increased risk of kidney disease. Age, sex, cohort, duration of diabetes, HbA1c, insulin therapy, systolic blood pressure, use of antihypertensive drugs, RAS inhibitors, diabetic retinopathy stage, lipid-lowering drugs, eGFR, albuminuria.

Serdar et al. Although urinary 8-OHdG levels increase in diabetic patients, their levels do not improve prediction of progressive DKD over and above measuring albuminuria. Omics-based novel biomarkers.

Bhensdadia et al. The haptoglobin to creatinine ratio may be useful to predict risk of DKD before the development of albuminuria or kidney function decline. Zurbig et al. CKD predicted progression to macroalbuminuria 5 years prior to actual onset. Age, sex, DM type, albuminuria, eGFR, systolic and diastolic blood pressure, HbA1c, glucose.

Roscioni et al. CKD predicted development of albuminuria independent of other kidney biomarkers used to predict DKD development or progression. Albuminuria, eGFR, use of RAS inhibitors.

Age, baseline eGFR, systolic and diastolic blood pressure. Tofte et al. High-risk patients defined by CKD were more likely to develop microalbuminuria. Age, sex, HbA1c, systolic blood pressure, retinopathy, albuminuria, eGFR.

Lindhardt et al. CKD predicted development of albuminuria. Treatment group, age, sex, systolic blood pressure, albuminuria, eGFR, HbA1c, diabetes duration.

Han et al. Non-esterified and esterified fatty acid discriminated albuminuria stages. Hirayama et al. Combination of 19 serum metabolites enabled accurate discrimination of DKD. Serum leucine, dihydrosphingosine, phytosphingosine.

Zhang et al. Serum metabolite levels of leucine, dihydrosphingosine, and phytosphingosine were significantly different in patients with T2DM and healthy controls. Urine hexose, glutamine, tysorine, plasma butenoylcarnitine, histidine. Pena et al. Urine hexose, glutamine, tyrosine, plasma butenoylcarnitine and histine predicted development of albuminuria.

Looker et al. A panel of 14 biomarkers that included the symmetric to asymmetric dimethylarginine ratio, and Cacylcarnitine increased the predictive ability of rapid progression. Age, sex, baseline eGFR, albuminuria, HbA1c, use of RAS inhibitors.

Urinary 3-hydroxy-isobutyrate, 3-methyl-crotonyglycine, aconitic acid, citric acid. Kwan et al. Age, race, sex, smoking, body mass index, HbA1c, mean arterial pressure, albuminuria, baseline eGFR.

Urinary leucine, valine, isoleucine, pseudouridine, threonine, citrate, 2-hydroxyiso- butyrate, pyroglutamate, tyrosine, alanine. Mutter et al.